Readings for Discussion
TransAm: Translational Animal Models Journal Club
CMD: Cell Mechanobiology and Differentiation Journal Club
ECM: Engineering Contructs and Materials Journal Club
TransAm - April 22, 2013
Nucleus Pulposus Cells Expressing hBMP7 Can Prevent the Degeneration of Allogenic IVD in a Canine Transplantation Model.Chaofeng W, Chao Z, Deli W, Jianhong W, Yan Z, Cheng X, Hongkui X, Qing H, Dike R.
We have previously explored the possibilities of allogenic intervertebral disc (IVD) curing disc degeneration disease in clinical practice. The results showed that the motion and stability of the spinal unit was preserved after transplantation of allogenic IVD in human beings at 5-year follow-up. However, mild degeneration was observed in the allogenic transplanted IVD cases. In this study, we construct the biological tissue engineering IVD by injecting the nucleus pulposus cells (NPCs) expressing human bone morphogenetic protein 7 (hBMP7) into cryopreserved IVD, and transplant the biological tissue engineering IVD into a beagle dog to investigate whether NPCs expressing hBMP7 could prevent the degeneration of the transplanted allogenic IVDs. At 24 weeks after transplantation, MRI scan showed that IVD allografts injected NPCs expressing hBMP7 have a slighter signs of degeneration than IVD allografts with NPCs or without NPCs. The range of motion of left-right rotation in the group without NPCs was bigger than that of two cells injection group. PKH-26-labeled cells were identified at IVD allograft. The study demonstrated that NPCs expressing hBMP7 could survive at least 24 weeks and prevent the degeneration of the transplanted IVD. This solution might have a potential role in preventing the IVD allograft degeneration in long time follow-up.
TransAm - March 25, 2013
Meeting the need for regenerative therapies I: target-based incidence and its relationship to U.S. spending, productivity, and innovation.Parenteau N, Hardin-Young J, Shannon W, Cantini P, Russell A.
Regenerative therapies possess high theoretical potential for medical advance yet their success as commercial therapeutics is still open to debate. Appropriate data on target opportunities that provide perspective and enable strategic decision making is necessary for both efficient and effective translation. Up until now, this data have been out of reach to research scientists and many start-up companies-the very groups currently looked to for the critical advance of these therapies. The target-based estimate of opportunity presented in this report demonstrates its importance in evaluating medical need and technology feasibility. In addition, analysis of U.S. research spending, productivity, and innovation reveals that U.S. basic research in this field would benefit from greater interdisciplinarity. Overcoming the barriers that currently prevent translation into high value therapies that are quickly clinically adopted requires simultaneous integration of engineering, science, business, and clinical practice. Achieving this integration is nontrivial.
TransAm - December 17, 2012
Concentrated bone marrow aspirate improves full-thickness cartilage repair compared with microfracture in the equine model.Fortier LA, Potter HG, Rickey EJ, Schnabel LV, Foo LF, Chong LR, Stokol T, Cheetham J, Nixon AJ.
BACKGROUND: The purpose of this study was to compare the outcomes of treatment with bone marrow aspirate concentrate, a simple, one-step, autogenous, and arthroscopically applicable method, with the outcomes of microfracture with regard to the repair of full-thickness cartilage defects in an equine model. METHODS: Extensive (15-mm-diameter) full-thickness cartilage defects were created on the lateral trochlear ridge of the femur in twelve horses. Bone marrow was aspirated from the sternum and centrifuged to generate the bone marrow concentrate. The defects were treated with bone marrow concentrate and microfracture or with microfracture alone. Second-look arthroscopy was performed at three months, and the horses were killed at eight months. Repair was assessed with use of macroscopic and histological scoring systems as well as with quantitative magnetic resonance imaging. RESULTS: No adverse reactions due to the microfracture or the bone marrow concentrate were observed. At eight months, macroscopic scores (mean and standard error of the mean, 9.4 + or - 1.2 compared with 4.4 + or - 1.2; p = 0.009) and histological scores (11.1 + or - 1.6 compared with 6.4 + or - 1.2; p = 0.02) indicated improvement in the repair tissue in the bone marrow concentrate group compared with that in the microfracture group. All scoring systems and magnetic resonance imaging data indicated that delivery of the bone marrow concentrate resulted in increased fill of the defects and improved integration of repair tissue into surrounding normal cartilage. In addition, there was greater type-II collagen content and improved orientation of the collagen as well as significantly more glycosaminoglycan in the bone marrow concentrate-treated defects than in the microfracture-treated defects. CONCLUSIONS: Delivery of bone marrow concentrate can result in healing of acute full-thickness cartilage defects that is superior to that after microfracture alone in an equine model. CLINICAL RELEVANCE: Delivery of bone marrow concentrate to cartilage defects has the clinical potential to improve cartilage healing, providing a simple, cost-effective, arthroscopically applicable, and clinically effective approach for cartilage repair.
Temporal growth factor release from platelet-rich plasma, trehalose lyophilized platelets, and bone marrow aspirate and their effect on tendon and ligament gene expression.
McCarrel T, Fortier L.
Platelet-rich plasma (PRP) has generated substantial interest for tendon and
ligament regeneration because of the high concentrations of growth factors in
platelet alpha-granules. This study compared the temporal release of growth
factors from bone marrow aspirate (BMA), PRP, and lyophilized platelet product (PP),
and measured their effects on tendon and ligament gene expression. Blood and BMA were
collected and processed to yield PRP and plasma. Flexor digitorum superficialis tendon
(FDS) and suspensory ligament (SL) explants were cultured in 10% plasma in DMEM (control),
BMA, PRP, or PP. TGF-beta1 and PDGF-BB concentrations were determined at 0, 24, and 96 h
of culture using ELISA. Quantitative RT-PCR for collagen types I and III (COL1A1, COL3A1),
cartilage oligomeric matrix protein (COMP), decorin, and matrix metalloproteinases-3 and 13 (MMP-3, MMP-13) was performed.
TGF-beta1 and PDGF-BB concentrations were highest in PRP and PP. Growth factor quantity
was unchanged in BMA, increased in PRP, and decreased in PP over 4 days. TGF-beta1 and
platelet concentrations were positively correlated. Lyophilized PP and PRP resulted in
increased COL1A1:COL3A1 ratio, increased COMP, and decreased MMP-13 expression. BMA
resulted in decreased COMP and increased MMP-3 and MMP-13 gene expression. Platelet
concentration was positively correlated with COL1A1, ratio of COL1A1:COL3A1, and COMP,
and negatively correlated with COL3A1, MMP-13, and MMP-3. White blood cell
concentration was positively correlated with COL3A1, MMP3, and MMP13, and negatively
correlated with a ratio of COL1A1:COL3A1, COMP, and decorin. These findings support
further in vivo investigation of PRP and PP for treatment of tendonitis and desmitis.
TransAm - November 19, 2012
Evaluation of thiol-modified hyaluronan and elastin-like polypeptide composite augmentation in early-stage disc degeneration: comparing 2 minimally invasive techniques.Leckie AE, Akens MK, Woodhouse KA, Yee AJ, Whyne CM.
STUDY DESIGN: An in vitro biomechanical and imaging study generated from an in vivo porcine model of early stage degenerative disc disease was used to evaluate mechanical property restoration, comparing 2 minimally invasive injection techniques. OBJECTIVE: To evaluate the ability of an injectable hydrogel to restore the mechanical properties of spinal motion segments with early stage disc degeneration, comparing 2 minimally invasive injection techniques. SUMMARY OF BACKGROUND DATA: Treatment of early-stage disc degeneration may benefit from a combination of tissue engineering and minimally invasive therapeutic approaches. A recently developed hydrogel, thiol-modified hyaluronan elastin-like polypeptide (TMHA/EP) composite, has demonstrated potential as an injectable nucleus replacement. METHODS: From a total of thirteen 35-kg Yorkshire boars, early-stage lumbar disc degeneration was introduced into 10 pigs via injection of chondroitinase ABC. After degeneration, 8 pigs received TMHA/EP augmentation; 1 disc via direct needle injection and a second using a modified kyphoplasty approach. High-resolution magnetic resonance images were acquired of the excised spinal motion segments, followed by biomechanical testing in axial compression, flexion-extension, lateral bending, and torsion. RESULTS: The degenerate control motion segments were generally less stiff and more flexible than healthy controls. The injection of TMHA/EP into the degenerated nucleus produced similar mechanical stiffness to healthy controls. The direct-injected discs showed a dispersive pattern of TMHA/EP within the nucleus, whereas the modified kyphoplasty method yielded a bolus of hydrogel. Yet, mechanical behavior was comparable considering the 2 minimally invasive augmentation techniques. CONCLUSION: The TMHA/EP composite can restore initial mechanical behavior in early-stage disc degeneration. Although both augmentation methods yielded mechanical properties comparable with healthy controls, direct injection represents a simpler technique, uses a smaller-gauge needle, does not introduce air into the disc, and yields a dispersive pattern that may be beneficial for future delivery of cells or growth factors.
TransAm - October 22, 2012
Osteochondritis Dissecans Knee Histology Studies Have Variable Findings and Theories of Etiology.Shea KG, Jacobs JC Jr, Carey JL, Anderson AF, Oxford JT.
BACKGROUND: Although many etiological theories have been proposed for osteochondritis dissecans (OCD), its etiology remains unclear. Histological analysis of the articular cartilage and subchondral bone tissues of OCD lesions can provide useful information about the cellular changes and progression of OCD. Previous research is predominantly comprised of retrospective clinical studies from which limited conclusions can be drawn. QUESTIONS/PURPOSES: The purposes of this study were threefold: (1) Is osteonecrosis a consistent finding in OCD biopsy specimens? (2) Is normal articular cartilage a consistent finding in OCD biopsy specimens? (3) Do histological studies propose an etiology for OCD based on the tissue findings? METHODS: We searched the PubMed, Embase, and CINAHL databases for studies that conducted histological analyses of OCD lesions of the knee and identified 1560 articles. Of these, 11 met our inclusion criteria: a study of OCD lesions about the knee, published in the English language, and performed a histological analysis of subchondral bone and articular cartilage. These 11 studies were assessed for an etiology proposed in the study based on the study findings. RESULTS: Seven of 11 studies reported subchondral bone necrosis. Four studies reported normal articular cartilage, two studies reported degenerated or irregular articular cartilage, and five studies found a combination of normal and degenerated or irregular articular cartilage. Five studies proposed trauma or repetitive stress and two studies proposed poor blood supply as possible etiologies. CONCLUSIONS: We found limited research on histological analysis of OCD lesions of the knee. Future studies with consistent methodology are necessary to draw major conclusions about the histology and progression of OCD lesions. Inconsistent histologic findings have resulted in a lack of consensus regarding the presence of osteonecrosis, whether the necrosis is primary or secondary, the association of cartilage degeneration, and the etiology of OCD. Such studies could use a standardized grading system to allow better comparison of findings.
Does Extracorporeal Shock Wave Therapy Enhance Healing of Osteochondritis Dissecans of the Rabbit Knee?: A Pilot Study.
Lyon R, Liu XC, Kubin M, Schwab J.
BACKGROUND: Severe osteochondritis dissecans (OCD) in children and adolescents often necessitates surgical interventions (ie, drilling, excision, or débridement). Since extracorporeal shock wave therapy (ESWT) enhances healing of long-bone nonunion fractures, we speculated ESWT would reactivate the healing process in OCD lesions.
QUESTIONS/PURPOSES: We asked whether ESWT would enhance articular cartilage quality, bone and cartilage density, and histopathology of osteochondral lesions compared to nontreated controls in an OCD rabbit model.
METHODS: We harvested a 4-mm-diameter plug of the weightbearing osteochondral surface on the medial femoral condyle of each knee in 20 skeletally immature (8-week-old) female rabbits. We placed a piece of acellular collagen-glycosaminoglycan matrix into the cavity and then replaced the plug. Two weeks after surgery, we sedated each rabbit and treated the right knee in a single setting with shock waves: 4000 impulses at 4 Hz and 18 kV. The left knee was a sham control. Ten weeks after surgery, we assessed cartilage morphology of the lesion using a modified Outerbridge Grading System, bone and cartilage density using histologic imaging, bone and cartilage morphology using the histopathology assessment system, and radiographic bone density and union and compared these parameters between ESWT-treated and control knees.
RESULTS: Histologically, we observed more mature bone formation and better healing (1.1 versus 3.4) and density of the cartilage (60 versus 49) on the treated side. Radiographically, we noted an increase in bony density (154 versus 138) after ESWT.
CONCLUSIONS: ESWT accelerated the healing rate and improved cartilage and subchondral bone quality in the OCD rabbit model.
CLINICAL RELEVANCE: This therapeutic modality may be applicable in OCD treatment in the pediatric population. Future research will be necessary to determine whether it may play a role in healing of human osteochondral defects.
CMD - October 10, 2012
ECM stiffness primes the TGFB pathway to promote chondrocyte differentiation.Allen JL, Cooke ME, Alliston T.
Cells encounter physical cues such as extracellular matrix (ECM) stiffness in a microenvironment replete with biochemical cues. However, the mechanisms by which cells integrate physical and biochemical cues to guide cellular decision making are not well defined. Here we investigate mechanisms by which chondrocytes generate an integrated response to ECM stiffness and transforming growth factor ß (TGFß), a potent agonist of chondrocyte differentiation. Primary murine chondrocytes and ATDC5 cells grown on 0.5-MPa substrates deposit more proteoglycan and express more Sox9, Col2a1, and aggrecan mRNA relative to cells exposed to substrates of any other stiffness. The chondroinductive effect of this discrete stiffness, which falls within the range reported for articular cartilage, requires the stiffness-sensitive induction of TGFß1. Smad3 phosphorylation, nuclear localization, and transcriptional activity are specifically increased in cells grown on 0.5-MPa substrates. ECM stiffness also primes cells for a synergistic response, such that the combination of ECM stiffness and exogenous TGFß induces chondrocyte gene expression more robustly than either cue alone through a p38 mitogen-activated protein kinase-dependent mechanism. In this way, the ECM stiffness primes the TGFß pathway to efficiently promote chondrocyte differentiation. This work reveals novel mechanisms by which cells integrate physical and biochemical cues to exert a coordinated response to their unique cellular microenvironment.
TransAm - October 8, 2012
Tissue engineering for total meniscal substitution: animal study in sheep model--results at 12 months.Kon E, Filardo G, Tschon M, Fini M, Giavaresi G, Marchesini Reggiani L, Chiari C, Nehrer S, Martin I, Salter DM, Ambrosio L, Marcacci M.
The aim of the study was to investigate the use of a hyaluronic acid/polycaprolactone material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with expanded autologous chondrocytes. Eighteen skeletally mature sheep were treated. The animals were divided into three groups: cell-free scaffold, scaffold seeded with autologous chondrocytes, and meniscectomy alone. The implant was sutured to the capsule and to the meniscal ligament. At a 12-month gross assessment, histology and histomorphometry were used to assess the meniscus implant, knee joint, and osteoarthritis development. All implants showed excellent capsular ingrowth at the periphery. The implant gross assessment showed significant differences between cell-seeded and cell-free groups (p=0.011). The histological analysis indicated a cellular colonization throughout the implanted constructs. Avascular cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. Joint gross assessment showed that sheep treated with scaffold implantation achieved a significant higher score than those underwent meniscectomy (p<0.0005), and the Osteoarthritis Research Society International score showed that osteoarthritic changes were significantly less in the cell-seeded group than in the meniscectomy group (p=0.047), even though results were not significantly superior to those of the cell-free scaffold. Seeding of the scaffold with autologous chondrocytes increases its tissue regeneration capacity, providing a better fibrocartilaginous tissue formation. The study suggests the potential of the novel hyaluronic acid/polycaprolactone scaffold for total meniscal substitution, although this approach has to be further improved before being applied into clinical practice.
Isolation, characterization, and differentiation of stem cells for cartilage regeneration.
Beane OS, Darling EM.
The goal of tissue engineering is to create a functional replacement for tissues
damaged by injury or disease. In many cases, impaired tissues cannot provide
viable cells, leading to the investigation of stem cells as a possible alternative.
Cartilage, in particular, may benefit from the use of stem cells since the tissue has
low cellularity and cannot effectively repair itself. To address this need, researchers
are investigating the chondrogenic capabilities of several multipotent stem cell sources,
including adult and extra-embryonic mesenchymal stem cells (MSCs), embryonic stem cells
(ESCs), and induced pluripotent stem cells (iPSCs). Comparative studies indicate that
each cell type has advantages and disadvantages, and while direct comparisons are
difficult to make, published data suggest some sources may be more promising for
cartilage regeneration than others. In this review, we identify current approaches for
isolating and chondrogenically differentiating MSCs from bone marrow, fat, synovium,
muscle, and peripheral blood, as well as cells from extra-embryonic tissues, ESCs, and
iPSCs. Additionally, we assess chondrogenic induction with growth factors, identifying
standard cocktails used for each stem cell type. Cell-only (pellet) and scaffold-based
studies are also included, as is a discussion of in vivo results.
CMD - September 26, 2012
Cell Mechanics, Structure, and Function Are Regulated by the Stiffness of the three-Dimensional Microenvironment.Chen J, Irianto J, Inamdar S, Pravincumar P, Lee DA, Bader DL, Knight MM.
This study adopts a combined computational and experimental approach to determine the mechanical, structural, and metabolic properties of isolated chondrocytes cultured within three-dimensional hydrogels. A series of linear elastic and hyperelastic finite-element models demonstrated that chondrocytes cultured for 24 h in gels for which the relaxation modulus is less than 5 kPa exhibit a cellular Young's modulus of 5 kPa. This is notably greater than that reported for isolated chondrocytes in suspension. The increase in cell modulus occurs over a 24-h period and is associated with an increase in the organization of the cortical actin cytoskeleton, which is known to regulate cell mechanics. However, there was a reduction in chromatin condensation, suggesting that changes in the nucleus mechanics may not be involved. Comparison of cells in 1% and 3% agarose showed that cells in the stiffer gels rapidly develop a higher Young's modulus of ~20 kPa, sixfold greater than that observed in the softer gels. This was associated with higher levels of actin organization and chromatin condensation, but only after 24 h in culture. Further studies revealed that cells in stiffer gels synthesize less extracellular matrix over a 28-day culture period. Hence, this study demonstrates that the properties of the three-dimensional microenvironment regulate the mechanical, structural, and metabolic properties of living cells.
CMD - September 12, 2012
Actin cap associated focal adhesions and their distinct role in cellular mechanosensing.Kim DH, Khatau SB, Feng Y, Walcott S, Sun SX, Longmore GD, Wirtz D.
The ability for cells to sense and adapt to different physical microenvironments plays a critical role in development, immune responses, and cancer metastasis. Here we identify a small subset of focal adhesions that terminate fibers in the actin cap, a highly ordered filamentous actin structure that is anchored to the top of the nucleus by the LINC complexes; these differ from conventional focal adhesions in morphology, subcellular organization, movements, turnover dynamics, and response to biochemical stimuli. Actin cap associated focal adhesions (ACAFAs) dominate cell mechanosensing over a wide range of matrix stiffness, an ACAFA-specific function regulated by actomyosin contractility in the actin cap, while conventional focal adhesions are restrictively involved in mechanosensing for extremely soft substrates. These results establish the perinuclear actin cap and associated ACAFAs as major mediators of cellular mechanosensing and a critical element of the physical pathway that transduce mechanical cues all the way to the nucleus.
CMD - August 1, 2012
Role of nesprin-1 in nuclear deformation in endothelial cells under static and uniaxial stretching conditions.Anno T, Sakamoto N, Sato M.
The linker of nucleus and cytoskeleton (LINC) complex, including nesprin-1, has been suggested to be crucial for many biological processes. Previous studies have shown that mutations in nesprin-1 cause abnormal cellular functions and diseases, possibly because of insufficient force transmission to the nucleus through actin filaments (F-actin) bound to nesprin-1. However, little is known regarding the mechanical interaction between the nucleus and F-actin through nesprin-1. In this study, we examined nuclear deformation behavior in nesprin-1 knocked-down endothelial cells (ECs) subjected to uniaxial stretching by evaluating nuclear strain from lateral cross-sectional images. The widths of nuclei in nesprin-1 knocked-down ECs were smaller than those in wild-type cells. In addition, nuclear strain in nesprin-1 knocked-down cells, which is considered to be compressed by the actin cortical layer, increased compared with that in wild-type cells under stretching condition. These results indicate that nesprin-1 knockdown releases the nucleus from the tension of F-actin bound to the nucleus, thereby increasing allowance for deformation before stretching, and that F-actin bound to the nucleus through nesprin-1 causes sustainable force transmission to the nucleus.
TransAm - August 27, 2012
A novel in vivo murine model of cartilage regeneration. Age and strain-dependent outcome after joint surface injury.Eltawil NM, De Bari C, Achan P, Pitzalis C, Dell'accio F.
OBJECTIVES: To generate and validate a murine model of joint surface repair following acute mechanical injury.METHODS: Full thickness defects were generated in the patellar groove of C57BL/6 and DBA/1 mice by microsurgery. Control knees were either sham-operated or non-operated. Outcome was evaluated by histological scoring systems. Apoptosis and proliferation were studied using TUNEL and Phospho-Histone H3 staining, respectively. Type II collagen neo-deposition and degradation were evaluated by immunostaining using antibodies to the CPII telopeptide and C1,2C (Col2-3/4Cshort), respectively. Aggrecanases and matrix metalloproteinases (MMPs) activity were assessed by immunostaining for TEGE(373) and VDIPEN neo-epitopes. RESULTS: Young 8-week-old DBA/1 mice displayed consistent and superior healing of the articular cartilage defect. Age-matched C57BL/6 mice repaired poorly and developed features of osteoarthritis (OA). Compared to C57BL/6, DBA/1 mice displayed a progressive decline of chondrocyte apoptosis, cell proliferation within the repair tissue, persistent type II collagen neo-deposition, less type II collagen degradation, less aggrecanases and more MMP-induced aggrecan degradation. Eight-month-old DBA/1 mice failed to repair, but, in contrast to age-matched C57BL/6 mice, developed no signs of OA. CONCLUSION: We have generated and validated a murine model of cartilage regeneration in which the outcome of joint surface injury is strain and age dependent. This model will allow, for the first time, the dissection of different pathways involved in joint surface regeneration in adult mammals using the powerful technology of mouse genetics.
Functional mesenchymal stem cell niches in adult mouse knee joint synovium in vivo.
Kurth TB, Dell'accio F, Crouch V, Augello A, Sharpe PT, De Bari C.
OBJECTIVE: We previously reported that human synovium contains cells that, after culture
expansion, display properties of mesenchymal stem cells (MSCs). The objective of this
study was to identify MSCs in native synovium in vivo. METHODS: To identify stem cells
in the synovium in vivo, a double nucleoside analog cell-labeling scheme was used in a
mouse model of joint-surface injury. For labeling of slow-cycling cells, mice received
iododeoxyuridine (IdU) for 30 days, followed by a 40-day washout period. For labeling of
cells that proliferate after injury, mice underwent knee surgery to produce an articular
cartilage defect and received chlorodeoxyuridine (CIdU) for 4 days, starting at multiple
time points after surgery. Unoperated and sham-operated joints served as controls. Knee
joint paraffin sections were analyzed by double and triple immunostaining to detect
nucleoside analogs, conventional MSC markers, and chondrocyte-lineage markers. RESULTS:
Long-term-retaining, slow-cycling IdU-positive cells were detected in the synovium. At 4
days and 8 days after injury, there was marked proliferation of IdU-positive cells, which
costained for CIdU. IdU-positive cells were nonhematopoietic, nonendothelial stromal
cells, were distinct from pericytes, and stained positive for MSC markers. MSCs were
phenotypically heterogeneous and located in topographically distinct niches in the lining
layer and the subsynovial tissue. Twelve days after injury, double nucleoside-labeled
cells within synovium were embedded in cartilage-specific metachromatic extracellular
matrix and costained positive for the chondrocyte-lineage markers Sox9 and type II
collagen. CONCLUSION: Our findings provide the first evidence of the existence of
resident MSCs in the knee joint synovium that undergo proliferation and chondrogenic
differentiation following injury in vivo.
TransAm - July 16, 2012
Regenerating nucleus pulposus of the intervertebral disc using biodegradable nanofibrous polymer scaffolds.Feng G, Zhang Z, Jin X, Hu J, Gupte MJ, Holzwarth JM, Ma PX.
Low back pain is a leading health problem in the United States, which is most often resulted from nucleus pulposus (NP) degeneration. To date, the replacement of degenerated NP relies entirely on mechanical devices. However, a biological NP replacement implant is more desirable. Here, we report the regeneration of NP tissue using a biodegradable nanofibrous (NF) scaffold. Rabbit NP cells were seeded on the NF scaffolds to regenerate NP-like tissue both in vitro and in a subcutaneous implantation model. The NP cells on the NF scaffolds proliferated faster than those on control solid-walled (SW) scaffolds in vitro. Significantly more extracellular matrix (ECM) production (glycosaminoglycan and type II collagen) was found on the NF scaffolds than on the control SW scaffolds. The constructs were then implanted in the caudal spine of athymic rats for up to 12 weeks. The tissue-engineered NP could survive, produce functional ECM, remain in place, and maintain the disc height, which is similar to the native NP tissue.
Spine degeneration in a murine model of chronic human tobacco smokers.
Wang D, Nasto LA, Roughley P, Leme AS, Houghton AM, Usas A, Sowa G, Lee J, Niedernhofer L, Shapiro S, Kang J, Vo N.
OBJECTIVE: To investigate the mechanisms by which chronic tobacco smoking promotes intervertebral disc degeneration (IDD) and vertebral degeneration in mice.
METHODS: Three month old C57BL/6 mice were exposed to tobacco smoke by direct inhalation
(4 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans.
Total disc proteoglycan (PG) content [1,9-dimethylmethylene blue (DMMB) assay], aggrecan
proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry)
were analyzed. PG and collagen syntheses ((35)S-sulfate and (3)H-proline incorporation,
respectively) were measured using disc organotypic culture. Vertebral osteoporosity was
measured by micro-computed tomography. RESULTS: Disc PG content of smoke-exposed mice was 63% of unexposed control, while new PG and collagen syntheses were 59% and 41% of those of untreated mice, respectively. Exposure to tobacco smoke dramatically increased metalloproteinase-mediated proteolysis of disc aggrecan within its interglobular domain (IGD). Cellular senescence was elevated two-fold in discs of smoke-exposed mice. Smoke exposure increased vertebral endplate porosity, which closely correlates with IDD in humans.
CONCLUSIONS: These findings further support tobacco smoke as a contributor to spinal
degeneration. Furthermore, the data provide a novel mechanistic insight, indicating
that smoking-induced IDD is a result of both reduced PG synthesis and increased
degradation of a key disc extracellular matrix protein, aggrecan. Cleavage of
aggrecan IGD is extremely detrimental as this results in the loss of the entire
glycosaminoglycan-attachment region of aggrecan, which is vital for attracting water
necessary to counteract compressive forces. Our results suggest identification and
inhibition of specific metalloproteinases responsible for smoke-induced aggrecanolysis
as a potential therapeutic strategy to treat IDD.
TransAm - July 2, 2012
Bioinspired nanofibers support chondrogenesis for articular cartilage repair.Coburn JM, Gibson M, Monagle S, Patterson Z, Elisseeff JH.
Articular cartilage repair remains a significant and growing clinical challenge with the aging population. The native extracellular matrix (ECM) of articular cartilage is a 3D structure composed of proteinaceous fibers and a hydrogel ground substance that together provide the physical and biological cues to instruct cell behavior. Here we present fibrous scaffolds composed of poly(vinyl alcohol) and the biological cue chondroitin sulfate with fiber dimensions on the nanoscale for application to articular cartilage repair. The unique, low-density nature of the described nanofiber scaffolds allows for immediate cell infiltration for optimal tissue repair. The capacity for the scaffolds to facilitate cartilage-like tissue formation was evaluated in vitro. Compared with pellet cultures, the nanofiber scaffolds enhance chondrogenic differentiation of mesenchymal stems cells as indicated by increased ECM production and cartilage specific gene expression while also permitting cell proliferation. When implanted into rat osteochondral defects, acellular nanofiber scaffolds supported enhanced chondrogenesis marked by proteoglycan production minimally apparent in defects left empty. Furthermore, inclusion of chondroitin sulfate into the fibers enhanced cartilage-specific type II collagen synthesis in vitro and in vivo. By mimicking physical and biological cues of native ECM, the nanofiber scaffolds enhanced cartilaginous tissue formation, suggesting their potential utility for articular cartilage repair.
Hyaluronic Acid-Binding Scaffold for Articular Cartilage Repair.
Unterman SA, Gibson M, Lee JH, Crist J, Chansakul T, Yang EC, Elisseeff JH.
Hyaluronic acid (HA) is an extracellular matrix molecule with multiple physical and biological functions found in many
tissues, including cartilage. HA has been incorporated in a number of biomaterial and scaffold systems. However, HA in
the material may be difficult to control if it is not chemically modified and chemical modification of HA may negatively
impact biological function. In this study, we developed a poly(ethylene glycol) hydrogel with noncovalent HA-binding
capabilities and evaluated its ability to support cartilage formation in vitro and in an articular defect model.
Chondrogenic differentiation of mesenchymal stem cells encapsulated in the HA-interactive scaffolds containing various
amounts of exogenous HA was evaluated. The HA-binding hydrogel without exogenous HA produced the best cartilage as
determined by biochemical content (glysocaminoglycan and collagen), histology (Safranin O and type II collagen staining),
and gene expression analysis for aggrecan, type I collagen, type II collagen, and sox-9. This HA-binding formulation was then translated to an osteochondral defect model in the rat knee. After 6 weeks, histological analysis demonstrated improved cartilage tissue production in defects treated with the HA-interactive hydrogel compared to noninteractive control scaffolds and untreated defects. In addition to the tissue repair in the defect space, the Safranin O staining in cartilage tissue surrounding the defect was greater in treatment groups where the HA-binding scaffold was applied. In sum, incorporation of a noncovalent HA-binding functionality into biomaterials provides an ability to interact with local or exogenous HA, which can then impact tissue remodeling and ultimately new tissue production.
TransAm - June 4, 2012
Biological and biomechanical effects of fibrin injection into porcine intervertebral discsBuser Z, Kuelling F, Liu J, Liebenberg E, Thorne KJ, Coughlin D, Lotz JC.
Surgically denucleated porcine intervertebral discs (IVD) were injected with BIOSTAT BIOLOGX Fibrin Sealant (FS), and the in vivo effects were assessed over time by histological, biochemical, and mechanical criteria. The objectives were to test whether the intradiscal injection of FS stimulates disc healing. Disc avascularity prevents the deposition of a provisional fibrin scaffold that typically facilitates soft tissue repair. Poor disc wound healing leads to disc damage accumulation and chronic inflammation characterized by overproduction of proinflammatory cytokines and proteolytic enzymes. Four lumbar IVDs from each of 31 Yucatan minipigs were randomized to untreated controls; degenerative injury (nucleotomy); and nucleotomy plus FS injection. Animals were killed at 1, 2, 3, 6, and 12 weeks postsurgery. IVDs were harvested to quantify (1) architecture using morphological and histological grading; (2) proteoglycan composition using DMMB assay; (3) cytokine content using ELISA; and (4) mechanical properties using quantitative pressure/volume testing. There was progressive invasion of annular tissue into the nucleus of nucleotomy discs and concomitant reduction in proteoglycan content. By contrast, FS supplementation inhibited nuclear fibrosis and facilitated proteoglycan content recovery over time. FS discs synthesized significantly less TNF-? than degenerate discs (66% vs. 226%, P < 0.05) and had upregulation of IL-4 (310% vs. 166%) and TGF-? (400% vs. 117%) at 2 to 3 weeks posttreatment. At the third week postsurgery, the denucleated discs were less stiff than controls (pressure modulus 779.9 psi vs. 2754.8 psi; P < 0.05) and failed at lower pressures (250.5 psi vs. 492.5 psi; P < 0.05). The stiffness and leakage pressure of the FS-treated discs recovered to control values after 6 and 12 weeks, respectively. FS facilitated structural, compositional, and mechanical repair of the surgically damaged IVD. These FS-derived benefits are likely due to its conductive scaffold properties and metabolically active constituents such as thrombin, factor XIII, and aprotinin acetate.Surgically denucleated porcine intervertebral discs (IVD) were injected with BIOSTAT BIOLOGX Fibrin Sealant (FS), and the in vivo effects were assessed over time by histological, biochemical, and mechanical criteria. The objectives were to test whether the intradiscal injection of FS stimulates disc healing. Disc avascularity prevents the deposition of a provisional fibrin scaffold that typically facilitates soft tissue repair. Poor disc wound healing leads to disc damage accumulation and chronic inflammation characterized by overproduction of proinflammatory cytokines and proteolytic enzymes. Four lumbar IVDs from each of 31 Yucatan minipigs were randomized to untreated controls; degenerative injury (nucleotomy); and nucleotomy plus FS injection. Animals were killed at 1, 2, 3, 6, and 12 weeks postsurgery. IVDs were harvested to quantify (1) architecture using morphological and histological grading; (2) proteoglycan composition using DMMB assay; cytokine content using ELISA; and mechanical properties using quantitative pressure/volume testing. There was progressive invasion of annular tissue into the nucleus of nucleotomy discs and concomitant reduction in proteoglycan content. By contrast, FS supplementation inhibited nuclear fibrosis and facilitated proteoglycan content recovery over time. FS discs synthesized significantly less TNF-? than degenerate discs (66% vs. 226%, P < 0.05) and had upregulation of IL-4 (310% vs. 166%) and TGF-? (400% vs. 117%) at 2 to 3 weeks posttreatment. At the third week postsurgery, the denucleated discs were less stiff than controls (pressure modulus 779.9 psi vs. 2754.8 psi; P < 0.05) and failed at lower pressures (250.5 psi vs. 492.5 psi; P < 0.05). The stiffness and leakage pressure of the FS-treated discs recovered to control values after 6 and 12 weeks, respectively. FS facilitated structural, compositional, and mechanical repair of the surgically damaged IVD. These FS-derived benefits are likely due to its conductive scaffold properties and metabolically active constituents such as thrombin, factor XIII, and aprotinin acetate.
Nucleus pulposus replacement and regeneration/repair technologies: present status and future prospects.
Lewis G.
Degenerative disc disease is implicated in the pathogenesis of many painful conditions of the back, chief among
which is low back pain. Acute and/or chronic low back pain (A/CLBP) afflicts a large number of people, thus making
it a major healthcare issue with concomitant cost ramifications. When conservative treatments for A/CLBP, such as bed
rest, anti-inflammatory medications, and physical therapy, prove to be ineffectual, surgical options are recommended.
The most popular of these is discectomy followed by fusion. Although there are many reports of good to excellent outcomes
with this method, there are concerns, such as long-term adverse biomechanical consequences to adjacent functional spinal
unit(s). A surgical option that has been attracting much attention recently is replacement or regeneration/repair of the
nucleus pulposus, an approach that holds the prospect of not compromising either mobility or function and causing no
adjacent-level injury. There is a sizeable body of literature highlighting this option, comprising in vitro biomechanical
studies, finite element analyses, animal-model studies, and limited clinical evaluations. This work is a review of this body of literature and is organized into four parts, with the focus being on replacement technologies, regeneration/repair technologies, and detailed expositions on 14 areas for future study. This review ends with a summary of the salient points made.
TransAm - May 21, 2012
A new histology scoring system for the assessment of the quality of human cartilage repair: ICRS II.Mainil-Varlet P, Van Damme B, Nesic D, Knutsen G, Kandel R, Roberts S.
A reliable and reproducible method is needed to assess cartilage repair. This study was undertaken to test the reproducibility of 2 established histological scoring systems, the Modified O'Driscoll Scale (MODS) and International Cartilage Research Society (ICRS) Visual Assessment Scale (ICRS I), and subsequently to develop and evaluate a new grading system for cartilage repair.Cohort study; Level of evidence, 2. A total of 107 cartilage biopsy specimens were graded using MODS and ICRS I, and the reader variability was measured. The new grading system, ICRS II, was developed and the inter- and intrareader variability determined by 3 independent readers. Collagen type II deposition was assessed immunohistochemically. The MODS and ICRS I demonstrated high interreader variability, with MODS also showing high intrareader variability. A new histological scoring system, ICRS II, was developed comprising 14 criteria to assess parameters related to chondrocyte phenotype and tissue structure. The ICRS II demonstrated lower inter- and intrareader variability compared with MODS or ICRS I. The overall assessment and matrix staining scores had the best correlation coefficients for inter- and intrareader variability (r = .81 and .82, respectively). The extent of collagen type II in cartilage, considered a marker of differentiation toward hyaline cartilage, could represent a measure of good cartilage repair. A correlation coefficient of .56 was obtained between the extent of collagen type II staining and the overall assessment score. The ICRS II represents an improvement over current histological cartilage repair grading systems in terms of reader reproducibility. The clinical relevance and its ability to predict long-term repair durability will be assessed once long-term clinical data become available.
Histological assessment of cartilage repair: a report by the Histology Endpoint Committee of the International Cartilage Repair Society (ICRS).
Mainil-Varlet P, Aigner T, Brittberg M, Bullough P, Hollander A, Hunziker E, Kandel R, Nehrer S, Pritzker K, Roberts S, Stauffer E.
TransAm - May 7, 2012
Influence of in vitro maturation of engineered cartilage on the outcome of osteochondral repair in a goat model.Miot S, Brehm W, Dickinson S, Sims T, Wixmerten A, Longinotti C, Hollander AP, Mainil-Varlet P, Martin I.
This study was designed to determine if the maturation stage of engineered cartilage implanted in a goat model of cartilage injury influences the repair outcome. Goat engineered cartilage was generated from autologous chondrocytes cultured in hyaluronic acid scaffolds using 2 d, 2 weeks or 6 weeks of pre-culture and implanted above hydroxyapatite/hyaluronic acid sponges into osteochondral defects. Control defects were left untreated or treated with cell-free scaffolds. The quality of repair tissues was assessed 8 weeks or 8 months post implantation by histological staining, modified O'Driscoll scoring and biochemical analyses. Increasing pre-culture time resulted in progressive maturation of the grafts in vitro. After 8 weeks in vivo, the quality of the repair was not improved by any treatment. After 8 months, O'Driscoll histology scores indicated poor cartilage architecture for untreated (29.7 ± 1.6) and cell-free treated groups (24.3 ± 5.8). The histology score was improved when cellular grafts were implanted, with best scores observed for grafts pre-cultured for 2 weeks (16.3 ± 5.8). As compared to shorter pre-culture times, grafts cultured for 6 weeks (histology score: 22.3 ± 6.4) displayed highest type II/I collagen ratios but also inferior architecture of the surface and within the defect, as well as lower integration with native cartilage. Thus, pre-culture of engineered cartilage for 2 weeks achieved a suitable compromise between tissue maturity and structural/integrative properties of the repair tissue. The data demonstrate that the stage of development of engineered cartilage is an important parameter to be considered in designing cartilage repair strategies.
A comparison of the functionality and in vivo phenotypic stability of cartilaginous tissues engineered from different stem cell sources.
Vinardell T, Sheehy EJ, Buckley CT, Kelly DJ.
Joint-derived stem cells are a promising alternative cell source for cartilage repair therapies that may overcome many of the problems associated with the use of primary chondrocytes (CCs). The objective of this study was to compare the in vitro functionality and in vivo phenotypic stability of cartilaginous tissues engineered using bone marrow-derived stem cells (BMSCs) and joint tissue-derived stem cells
following encapsulation in agarose hydrogels. Culture-expanded BMSCs, fat pad-derived stem cells (FPSCs), and synovial membrane-derived stem cells (SDSCs) were encapsulated in agarose and maintained in a chondrogenic medium supplemented with transforming growth factor-ß3. After 21 days of culture, constructs were either implanted subcutaneously into the back of nude mice for an additional
28 days or maintained for a similar period in vitro in either chondrogenic or hypertrophic media formulations. After 49 days of in vitro culture in chondrogenic media, SDSC constructs accumulated the highest levels of sulfated glycosaminoglycan (sGAG) (~2.8% w/w) and collagen (~1.8% w/w) and were mechanically stiffer than constructs engineered using other cell types. After subcutaneous
implantation in nude mice, sGAG content significantly decreased for all stem cell-seeded constructs, while no significant change was observed in the control constructs engineered using primary CCs, indicating that the in vitro chondrocyte-like phenotype generated in all stem cell-seeded agarose constructs was transient. FPSCs and SDSCs appeared to undergo fibrous dedifferentiation or resorption,
as evident from increased collagen type I staining and a dramatic loss in sGAG content. BMSCs followed a more endochondral pathway with increased type X collagen expression and mineralization of the engineered tissue. In conclusion, while joint tissue-derived stem cells possess a strong intrinsic chondrogenic capacity, further studies are needed to identify the factors that will lead to the generation of a
more stable chondrogenic phenotype.
TransAm - April 23, 2012
Accelerated versus nonaccelerated rehabilitation after anterior cruciate ligament reconstruction: a prospective, randomized, double-blind investigation evaluating knee joint laxity using roentgen stereophotogrammetric analysis.Beynnon BD, Johnson RJ, Naud S, Fleming BC, Abate JA, Brattbakk B, Nichols CE.
The relationship between the biomechanical dose of rehabilitation exercises administered after anterior cruciate ligament (ACL) reconstruction and the healing response of the graft and knee is not well understood. After ACL reconstruction, rehabilitation administered with either accelerated or nonaccelerated programs produces the same change in the knees' 6 degrees of freedom, or envelope, laxity values. Randomized controlled trial; Level of evidence. Patients who underwent ACL reconstruction with a bone-patellar tendon-bone autograft were randomized to rehabilitation with either accelerated (19 week) or nonaccelerated (32 week) programs. At the time of surgery, and then 3, 6, 12, and 24 months later, the 6 degrees of freedom knee laxity values were measured using roentgen stereophotogrammetric analysis and clinical, functional, and patient-oriented outcome measures. Eighty-five percent of those enrolled were followed through 2 years. Laxity of the reconstructed knee was restored to within the limits of the contralateral, normal side at the time of surgery (baseline) in all participants. Patients in both programs underwent a similar increase in the envelope of knee laxity over the 2-year follow-up interval (anterior-posterior translation 3.2 vs 4.5 mm, and coupled internal-external rotations 2.6° vs 1.9° for participants in the accelerated and nonaccelerated programs, respectively). Those who underwent accelerated rehabilitation experienced a significant improvement in thigh muscle strength at the 3-month follow-up compared with those who participated in nonaccelerated rehabilitation, but no differences between the programs were seen after this time interval. At the 2-year follow-up, the groups were similar in terms of clinical assessment, patient satisfaction, function, proprioception, and isokinetic thigh muscle strength. Rehabilitation with the accelerated and nonaccelerated programs administered in this study produced the same increase in the envelope of knee laxity. A majority of the increase in the envelope of knee laxity occurred during healing when exercises were advanced and activity level increased. Patients in both programs had the same clinical assessment, functional performance, proprioception, and thigh muscle strength, which returned to normal levels after healing was complete. For participants in both treatment programs, the Knee Injury and Osteoarthritis Outcome Score (KOOS) assessment of quality of life did not return to preinjury levels.
The "ligamentization" process in anterior cruciate ligament reconstruction: what happens to the human graft? A systematic review of the literature.
Claes S, Verdonk P, Forsyth R, Bellemans J.
Surgical anterior cruciate ligament reconstruction using tendon grafts has become the standard to treat the functionally unstable anterior cruciate ligament-deficient knee. Although tendons clearly differ biologically from ligaments, multiple animal studies have shown that the implanted tendons indeed seem to remodel
into a ligamentous "anterior cruciate ligament-like" structure.
The goal of this study was to systematically review the current literature on the "ligamentization" process in human anterior cruciate ligament reconstruction.
The key finding of this systematic review is that a free tendon graft replacing a ruptured human anterior cruciate ligament undergoes a series of biologic processes termed "ligamentization." The graft seems to remain viable at any time during this course. Histologically, the mature grafts may resemble the normal human
anterior cruciate ligament, but ultrastructural differences regarding collagen fibril distribution do persist. Different stages of the ligamentization process are described, but no agreement exists on their time frame. Problematic direct transmission of animal data to the human situation, the limited number of reports considering the ligamentization process in
humans, and the potential biopsy sampling error attributable to superficial graft biopsies necessitate further human studies on anterior cruciate ligament graft ligamentization.
Local administration of autologous synovium-derived cells improve the structural properties of anterior cruciate ligament autograft reconstruction in sheep.
Kondo E, Yasuda K, Katsura T, Hayashi R, Azuma C, Tohyama H.
The structural properties of a tendon autograft deteriorate during the remodeling phase after anterior cruciate ligament (ACL) reconstruction.
Fifty-two mature sheep were used. In each animal, the right knee underwent ACL reconstruction using the semitendinosus tendon autograft; then, the animals were divided into 5 groups of 10. No additional treatments were applied in group 1, whereas fibrin sealant was applied around the graft in group 2. In group 3, autologous synovium-derived cells cultured in standard medium were applied around the graft with fibrin sealant, whereas autologous synovium-derived cells cultured in TGFß-supplemented medium were applied with fibrin sealant in group 4. In group 5, fibrin sealant containing 20 ng of TGFß was applied around the graft. Each animal was sacrificed at 12 weeks after the surgery. In each group, 7 and 3 sheep were used for biomechanical and histologic evaluations, respectively. The remaining 2 sheep were used to confirm whether the applied cells infiltrated the graft at 1 week after surgery.
Confocal microscope observations showed that the applied cells that were labeled before implantation infiltrated into the superficial portion of the graft at 1 week. Biomechanically, the maximum load and the stiffness of groups 4 and 5 were significantly greater than those of groups 1, 2, and 3. Histologically, necrotic lesions were observed in the core portion of the midsubstance in groups 1 and 2. In groups 3, 4, and 5, no necrotic lesions were found in the midsubstance.
A local application of autologous synovium-derived cells cultured in TGFß-supplemented medium or a fibrin sealant containing TGFß significantly inhibits the natural deterioration of the structural properties of the tendon graft after ACL reconstruction.
CMD - April 11, 2012
ROCK1 feedback regulation of the upstream small GTPase RhoA.Tang AT, Campbell WB, Nithipatikom K.
Rho-associated coiled-coil containing protein kinase 1 (ROCK1) is a key downstream effector of the small GTPase RhoA. Targeting ROCK1 has shown promising clinical potential in cancer, cardioprotection, hypertension, diabetes, neuronal regeneration, and stem cell biology. General working hypothesis in previous studies has centered on the function of ROCK1 as a downstream sequence in the RhoA signaling pathway. In this study, the effects of the direct inhibition of ROCK1 on the activity of upstream RhoA and Rac1 were examined using a combined pharmacological and genetic approach. We report an intriguing mechanism by which the inhibition of ROCK1 indirectly diminishes the activity of upstream RhoA through the stimulation of Tiam1-induced Rac1 activity. This novel feedback mechanism, in which ROCK1 mediates upstream Rac1 and RhoA activity, offers considerable insight into the diverse effects of ROCK1 on the functional balance of the Rho family of small GTPases, which regulates actin cytoskeleton reorganization processes and the resulting overall behavior of cells.
TransAm - April 9, 2012
Bioactive electrospun scaffold for annulus fibrosus repair and regeneration.Vadalà G, Mozetic P, Rainer A, Centola M, Loppini M, Trombetta M, Denaro V.
Annulus fibrosus (AF) tissue engineering is gathering increasing interest for the development of strategies to reduce recurrent disc herniation (DH) rate and to increase the effectiveness of intervertebral disc regeneration strategies. This study evaluates the use of a bioactive microfibrous poly(L: -lactide) scaffold releasing Transforming Growth Factor (TGF)-ß1 (PLLA/TGF) for the repair and regeneration of damaged AF. PLLA/TGF scaffold induced an anabolic stimulus on AFCs, mimicking the ECM three-dimensional environment of AF tissue. This bioactive scaffold showed encouraging results that allow envisaging an application for AF tissue engineering strategies and AF repair after discectomy for the prevention of recurrent DH.
Intervertebral Disk Disease With Spinal Cord Penetration in a Yucatan Pig.
Lapointe JM, Summers BA.
A 9-month-old female Yucatan pig was euthanized after acute onset of paraplegia. Gross and microscopic examination revealed dorsal dissection of the nucleus
of the L2-L3 intervertebral disk through the annulus fibrosus, extrusion of nucleus pulposus material through the overlying dura mater and into the spinal cord,
and associated acute spinal hemorrhage and necrosis. This is to the authors' knowledge the first reported case of intervertebral disk disease in swine.
Transplantation of Human Adipose-Derived Stem Cells in a Rabbit Model of Traumatic Degeneration of Lumbar Discs.
Chun HJ, Kim YS, Kim BK, Kim EH, Kim JH, Do BR, Hwang SJ, Hwang JY, Lee YK.
The purpose of the present study is to assess the possibility of disc regeneration by treatment with adipose-derived stem cells (ADSCs) in a rabbit model of degenerative
disc disease, and to evaluate the efficacy of a percutaneous technique for constructing a model of degenerative disc disease in rabbits.
The study sample consisted of 20 mature male New Zealand white rabbits. Intervertebral discs were injured in each rabbit by a percutaneous technique at L2-3, L3-4, and
L4-5 under C-arm guidance with a 19-gauge spinal needle. Magnetic resonance images (MRI) were checked at 6, 9, 12, and 15 weeks after injury to evaluate disc degeneration. Nineteen
weeks after injury, ADSCs were injected into the L4-5 disc space, with saline injected into the L3-4 disc as a control, using a 21-gauge spinal needle. Histologic confirmations of degenerated
discs were performed at 10 and 18 weeks after injury with safranin O and trichrome stains.
MRI revealed intervertebral disc degeneration from 9 weeks after injury, and full degeneration at 15 weeks after injury, when compared with uninjured control discs. We confirmed the
proliferation of ADSCs at the L4-5 level in 10-week rabbits after cell injection. Histologically, the ADSC-injected discs exhibited elevated extracellular matrix secretion and little ossification
of damaged cartilage in the nucleus pulposus compared with degenerative control discs.
These results suggest that the injection of ADSCs into injured lumbar discs could be an effective treatment for degenerative disc disease by promoting the cartilage regeneration.
TransAm - March 26, 2012
The maturity of tissue-engineered cartilage in vitro affects the repairability for osteochondral defect.Jin CZ, Cho JH, Choi BH, Wang LM, Kim MS, Park SR, Yoon JH, Oh HJ, Min BH.
Cartilage tissue engineering using cells and biocompatible scaffolds has emerged as a promising approach to repair of cartilage damage. To date, however, no engineered cartilage has proven to be equivalent to native cartilage in terms of biochemical and compression properties, as well as histological features. An alternative strategy for cartilage engineering is to focus on the in vivo regeneration potential of immature engineered cartilage. Here, we used a rabbit model to evaluate the extent to which the maturity of engineered cartilage influenced the remodeling and integration of implanted extracellular matrix scaffolds containing allogenous chondrocytes. Full-thickness osteochondral defects were created in the trochlear groove of New Zealand white rabbits. Left knee defects were left untreated as a control (group 1), and right knee defects were implanted with tissue-engineered cartilage cultured in vitro for 2 days (group 2), 2 weeks (group 3), or 4 weeks (group 4). Histological, chemical, and compression assays of engineered cartilage in vitro showed that biochemical composition became more cartilagenous, and biomechanical property for compression gradually increased with culture time. In an in vivo study, gross imaging and histological observation at 1 and 3 months after implanting in vitro-cultured engineered cartilage showed that defects in groups 3 and 4 were repaired with hyaline cartilage-like tissue, whereas defects were only partially filled with fibrocartilage after 1 month in groups 1 and 2. At 3 months, group 4 showed striking features of hyaline cartilage tissue, with a mature matrix and a columnar arrangement of chondrocytes. Zonal distribution of type II collagen was most prominent, and the International Cartilage Repair Society score was also highest at this time. In addition, the subchondral bone was well ossified. In conclusion, in vivo engineered cartilage was remodeled when implanted; however, its extent to maturity varied with cultivation period. Our results showed that the more matured the engineered cartilage was, the better repaired the osteochondral defect was, highlighting the importance of the in vitro cultivation period.
Meniscal Repair Versus Partial Meniscectomy: A Systematic Review Comparing Reoperation Rates and Clinical Outcomes.
Paxton ES, Stock MV, Brophy RH.
PURPOSE:The aim of this investigation was to compare reoperation rates and clinical outcomes after meniscal repair and partial meniscectomy.
CONCLUSIONS: Whereas meniscal repairs have a higher reoperation rate than partial meniscectomies, they are associated with better long-term outcomes.
CMD - March 14, 2012
Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy.Mak KK, Kronenberg HM, Chuang PT, Mackem S, Yang Y.
Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP(-/-) limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP(-/-) embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP(-/-) embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP(-/-) embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/beta-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy
Human articular chondrocytes secrete parathyroid hormone-related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis.
Fischer J, Dickhut A, Rickert M, Richter W.
The use of bone marrow-derived mesenchymal stem cells (MSCs) has shown promise in cell-based cartilage regeneration. A yet-unsolved problem, however, is the unwanted up-regulation of markers of hypertrophy, such as alkaline phosphatase (AP) and type X collagen, during in vitro chondrogenesis and the formation of unstable
calcifying cartilage at heterotopic sites. In contrast, articular chondrocytes produce stable, nonmineralizing cartilage. The aim of this study was to address whether coculture of MSCs with human articular chondrocytes (HACs) can suppress the undesired hypertrophy in differentiating MSCs.
MSCs were differentiated in chondrogenic medium that had or had not been conditioned by parallel culture with HAC pellets, or MSCs were mixed in the same pellet with the HACs (1:1 or 1:2 ratio) and cultured for 6 weeks. Following in vitro differentiation, the pellets were transplanted into SCID mice.
The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced by differentiation in HAC-conditioned medium, and less type X collagen protein was deposited relative to type II collagen. AP activity was significantly lower in the cells that had been differentiated in conditioned medium,
and transplants showed significantly reduced calcification in vivo. In mixed HAC/MSC pellets, suppression of AP was dose-dependent, and in vivo calcification was fully inhibited. Chondrocytes secreted parathyroid hormone-related protein (PTHrP) throughout the culture period, whereas PTHrP was down-regulated in favor of IHH up-regulation in control
MSCs after 2-3 weeks of chondrogenesis. The main inhibitory effects seen with HAC-conditioned medium were reproducible by PTHrP supplementation of unconditioned medium.
HAC-derived soluble factors and direct coculture are potent means of improving chondrogenesis and suppressing the hypertrophic development of MSCs. PTHrP is an important candidate soluble factor involved in this effect
CMD - February 29, 2012
Chromatin modifications and their function.Kouzarides T.
The surface of nucleosomes is studded with a multiplicity of modifications. At least eight different classes have been characterized to date and many different sites have been identified for each class. Operationally, modifications function either by disrupting chromatin contacts or by affecting the recruitment of nonhistone proteins to chromatin. Their presence on histones can dictate the higher-order chromatin structure in which DNA is packaged and can orchestrate the ordered recruitment of enzyme complexes to manipulate DNA. In this way, histone modifications have the potential to influence many fundamental biological processes, some of which may be epigenetically inherited.
Biophysical regulation of histone acetylation in mesenchymal stem cells.
Li Y, Chu JS, Kurpinski K, Li X, Bautista DM, Yang L, Sung KL, Li S.
Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions.
However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape,
HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 µm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on
an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were
subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied
by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate
can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical
signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.
CMD - January 18, 2012
Magnetic manipulation of nanorods in the nucleus of living cells.Celedon A, Hale CM, Wirtz D.
The organization of chromatin in the cell nucleus is crucial for gene expression regulation. However, physically probing the nuclear interior is challenging because high forces have to be applied using minimally invasive techniques. Here, magnetic nanorods embedded in the nucleus of living cells are subjected to controlled rotational forces, producing micron-sized displacements in the nuclear interior. The resulting time-dependent rotation of the nanorods is analyzed in terms of viscoelastic parameters of the nucleus, in wild-type and Lamin A/C deficient cells. This method and analysis reveal that Lamin A/C knockout, together perhaps with other changes that result from the knockout, induce significant decreases in the nuclear viscosity and elasticity.
Lab Meeting - December 16, 2011
Parsing the early cytoskeletal and nuclear organizational cues that demarcate stem cell lineagesLiu E, Gordonov S, Treiser MD, Moghe PV
Our recent report suggests that subtle changes in early cytoskeletal protein-level organization correlate with long-term stem cell lineage commitment. In this extra-view, we dissect changes in the expression of both cytoskeletal and nuclear-regulating genes that may precede and, possibly, govern the formative lineage-specific organizational cues. Human mesenchymal stem cells cultured on glass under basal, osteogenic, and adipogenic induction media were analyzed for gene expression profiles within the first 24 hours. Several key actin organization regulating genes and nuclear and cell cycle regulatory genes were found to be upregulated in osteogenic media compared to adipogenic and basal conditions. Given the role of both cytoskeletal and nuclear genes, we examined the possibility of classifying stem cell subpopulations using high content imaging approaches based on the organization of both actin, as previously proposed, as well as nuclear organization and distribution of a nuclear organizational protein, the nuclear mitotic apparatus (NuMA). A pool of combined cytoskeletal and nuclear descriptors were merged into a composite feature space via dimensionality reduction, data fusion, and classification methodologies. This composite approach enabled feature-based identification of specific lineage committed as well as non-differentiating cell populations. Using the improved classification of this high-content imaging-based profiling tool, we demonstrate that MSCs induced to differentiate to either osteogenic or adipogenic lineages are discernable within the first 24 hours from each other and from non-differentiating cells.
Lab Meeting - December 2, 2011
Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trialBolli R, Chugh AR, D'Amario D, Loughran JH, Stoddard MF, Ikram S, Beache GM, Wagner SG, Leri A, Hosoda T, Sanada F, Elmore JB, Goichberg P, Cappetta D, Solankhi NK, Fahsah I, Rokosh DG, Slaughter MS, Kajstura J, Anversa P
BACKGROUND: c-kit-positive, lineage-negative cardiac stem cells (CSCs) improve post-infarction left ventricular (LV) dysfunction when administered to animals. We undertook a phase 1 trial (Stem Cell Infusion in Patients with Ischemic cardiOmyopathy [SCIPIO]) of autologous CSCs for the treatment of heart failure resulting from ischaemic heart disease.
Lab Meeting - November 4, 2011
Cells keep a memory of their tissue origin during axolotl limb regenerationKragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein HH, Tanaka EM
During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine. Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip
Rinkevich Y, Lindau P, Ueno H, Longaker MT, Weissman IL
The regrowth of amputated limbs and the distal tips of digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation. Taking tissue engineering to heart
Dolgin E.
TransAm - October 24, 2011
Hydrogel meniscal replacement in the sheep knee: preliminary evaluation of chondroprotective effects.Kelly BT, Robertson W, Potter HG, Deng XH, Turner AS, Lyman S, Warren RF, Rodeo SA.
BACKGROUND:Meniscal allograft transplantation has become a viable surgical alternative for a select group of patients with deficient or irreparable menisci. Subjective results are encouraging; long-term success, durability, and safety of allograft meniscal transplantation are uncertain. CONCLUSION: Although promising preliminary results for hydrogel meniscal replacement were seen at early time points, significant cartilage degeneration and implant failure were seen at 1 year, and overall performance was worse than was allograft transplantation. Improvements in hydrogel material properties and surface characteristics and more accurate size matching may improve outcomes.
Meniscal Repair Versus Partial Meniscectomy: A Systematic Review Comparing Reoperation Rates and Clinical Outcomes.
Paxton ES, Stock MV, Brophy RH.
PURPOSE:The aim of this investigation was to compare reoperation rates and clinical outcomes after meniscal repair and partial meniscectomy.
CONCLUSIONS: Whereas meniscal repairs have a higher reoperation rate than partial meniscectomies, they are associated with better long-term outcomes.
CMD - October 12, 2011
Transcriptome-wide noise controls lineage choice in mammalian progenitor cellsChang HH, Hemberg M, Barahona M, Ingber DE, Huang S.
Phenotypic cell-to-cell variability within clonal populations may be a manifestation of 'gene expression noise', or it may reflect stable phenotypic variants. Such 'non-genetic cell individuality' can arise from the slow fluctuations of protein levels in mammalian cells. These fluctuations produce persistent cell individuality, thereby rendering a clonal population heterogeneous. However, it remains unknown whether this heterogeneity may account for the stochasticity of cell fate decisions in stem cells. Here we show that in clonal populations of mouse haematopoietic progenitor cells, spontaneous 'outlier' cells with either extremely high or low expression levels of the stem cell marker Sca-1 (also known as Ly6a; ref. 9) reconstitute the parental distribution of Sca-1 but do so only after more than one week. This slow relaxation is described by a gaussian mixture model that incorporates noise-driven transitions between discrete subpopulations, suggesting hidden multi-stability within one cell type. Despite clonality, the Sca-1 outliers had distinct transcriptomes. Although their unique gene expression profiles eventually reverted to that of the median cells, revealing an attractor state, they lasted long enough to confer a greatly different proclivity for choosing either the erythroid or the myeloid lineage. Preference in lineage choice was associated with increased expression of lineage-specific transcription factors, such as a >200-fold increase in Gata1 (ref. 10) among the erythroid-prone cells, or a >15-fold increased PU.1 (Sfpi1) (ref. 11) expression among myeloid-prone cells. Thus, clonal heterogeneity of gene expression level is not due to independent noise in the expression of individual genes, but reflects metastable states of a slowly fluctuating transcriptome that is distinct in individual cells and may govern the reversible, stochastic priming of multipotent progenitor cells in cell fate decision.
TransAm - October 10, 2011
Matrix-Associated Implantation of Predifferentiated Mesenchymal Stem Cells Versus Articular ChondrocytesMarquass B, Schulz R, Hepp P, Zscharnack M, Aigner T, Schmidt S, Stein F, Richter R, Osterhoff G, Aust G, Josten C, Bader A.
BACKGROUND: The use of predifferentiated mesenchymal stem cells (MSC) leads to better histological results compared with undifferentiated MSC in sheep. This raises the need for a longer term follow-up study and comparison with a clinically established method. HYPOTHESIS: We hypothesized that chondrogenic in vitro predifferentiation of autologous MSC embedded in a collagen I hydrogel leads to better structural repair of a chronic osteochondral defect in an ovine stifle joint after 1 year. We further hypothesized that resulting histological results would be comparable with those of chondrocyte-seeded matrix-associated autologous chondrocyte transplantation (MACT). CONCLUSION: Repair of chronic osteochondral defects with collagen hydrogels composed of chondrogenically predifferentiated MSC shows no signs of degradation after 1 year in vivo. In addition, pre-MSC gels lead to partially superior histological results compared with articular chondrocytes.
CMD - September 28, 2011
Individual fates of mesenchymal stem cells in vitroKrinner A, Hoffmann M, Loeffler M, Drasdo D, Galle J.
In vitro cultivated stem cell populations are in general heterogeneous with respect to their expression of differentiation markers. In hematopoietic progenitor populations, this heterogeneity has been shown to regenerate within days from isolated subpopulations defined by high or low marker expression. This kind of plasticity has been suggested to be a fundamental feature of mesenchymal stem cells (MSCs) as well. Here, we study MSC plasticity on the level of individual cells applying a multi-scale computer model that is based on the concept of noise-driven stem cell differentiation. By simulation studies, we provide detailed insight into the kinetics of MSC organisation. Monitoring the fates of individual cells in high and low oxygen culture, we calculated the average transition times of individual cells into stem cell and differentiated states. We predict that at low oxygen the heterogeneity of a MSC population with respect to differentiation regenerates from any selected subpopulation in about two days. At high oxygen, regeneration becomes substantially slowed down. Simulation results on the composition of the functional stem cell pool of MSC populations suggest that most of the cells that constitute this pool originate from more differentiated cells. Individual cell-based models are well-suited to provide quantitative predictions on essential features of the spatio-temporal organisation of MSC in vitro. Our predictions on MSC plasticity and its dependence on the environment motivate a number of in vitro experiments for validation. They may contribute to a better understanding of MSC organisation in vitro, including features of clonal expansion, environmental adaptation and stem cell ageing.
TransAm - September 26, 2011
Experimental Intervertebral Disc Regeneration with Tissue-Engineered Composite in a Canine ModelRuan DK, Xin H, Zhang C, Wang C, Xu C, Li C, He Q.
The aims of this study were to construct the composite of poly (L-lactic-co-glycolic acid) (PLGA) scaffold-nucleus pulposus (NP) cells using tissue engineering methods and to investigate the in vivo performance of the composite in a canine model. NP cells were isolated from the lumbar intervertebral discs of a beagle dog. NP cells were cultured, expanded in vitro, and seeded onto a three-dimensional porous PLGA scaffold. The composite was tested in 18 beagle dogs that were randomly divided into three groups: nucleotomy alone (A), nucleotomy with PLGA implantation (B), and nucleotomy with PLGA scaffold/NP cells composite implantation (C). X-ray and magnetic resonance imaging were performed pre- and postoperatively. Evaluation of disc height, segment stability, and biomechanics and immunohistochemical analysis were performed. Dog NP cells attached and showed proliferation activity within the PLGA scaffold in vitro and in vivo. Disc height, segmental stability, and T2-weighted signal intensity on magnetic resonance imaging scans were well preserved in group C dogs with the engineered composite. PHK-26-positive cells were found within the area of the NP 8 weeks postoperatively. The NP cell-PLGA scaffold composite can prevent or delay the degeneration process after nucleotomy in the canine model. This hybrid composite might be a promising construct for intervertebral disc regeneration. Nucleus pulposus replacement: basic science and indications for clinical use
Di Martino A, Vaccaro AR, Lee JY, Denaro V, Lim MR.
STUDY DESIGN: A critical review of available and emerging nucleus pulposus replacement implants. OBJECTIVES: To review the biomechanics, design, and clinical data of currently available and developing nucleus pulposus replacement technologies. The interest in minimally invasive treatment of degenerative disc disease has grown as the technology for intervertebral motion-sparing devices continues to improve. Replacement of nucleus pulposus without anular obliteration represents a tempting alternative to spinal fusion procedures. The aim in nucleus pulposus replacement is to slow adjacent level degeneration, restore normal loads to the diseased level, and restore segmental spinal biomechanics. METHODS: A literature review of currently available biomaterials, biomechanics, and available preclinical and clinical data on nucleus pulposus replacement implants. RESULTS: New synthetic biomaterials have recently been developed to closely mimic native biomechanics during compressive loading cycles of the intervertebral disc. This, in conjunction with improved understanding of global spine biomechanics, has allowed the development of novel nucleus replacement implants. These implants are currently at different stages of preclinical and clinical investigations. CONCLUSIONS: Although some of the newly designed prosthesis have shown some promising results in preclinical studies, rigorous short- and long-term clinical evaluations will be critical in evaluating their true efficacy.
Lab Meeting - September 23, 2011
Directed blood vessel growth using an angiogenic microfiber/microparticle composite patchDeVolder RJ, Bae H, Lee J, Kong H
An angiogenic microfiber patch that releases angiogenic growth factors along aligned fibers and subsequently directs the spacing and orientation of mature and functional blood vessel formation is presented. The fiber patches are prepared by electrostatically binding electrosprayed vascular endothelial growth factor (VEGF)-encapsulating poly(lactide-co-glycolide) (PLGA) microparticles with electrospun poly(lactide) (PLA) microfibers.
TransAm - September 12, 2011
Follow-up of collagen meniscus implant patients: clinical, radiological, and magnetic resonance imaging results at 5 yearsBulgheroni P, Murena L, Ratti C, Bulgheroni E, Ronga M, Cherubino P.
This study investigated at medium term follow-up the clinical outcomes and any progression of knee osteoarthritis in a population of patients that underwent arthroscopic placement of a collagen meniscus implant. Thirty-four patients underwent arthroscopic placement of a collagen meniscus implant for a symptomatic deficiency of medial meniscal tissue. Follow-up evaluation included Lysholm II score and Tegner activity scores and MR arthrography of the knee at 2 and 5 years after surgery. Plain radiographs were also obtained at 5 years. Six patients were excluded. In eight cases arthroscopic second look evaluation was performed. Lysholm and Tegner activity scores at 2 and 5 years after surgery improved significantly compared to the preoperative score. These patients showed good to excellent clinical results after 5 years from a CMI placement. The chondral surfaces of the medial compartment had not degenerated further since placement of the CMI. MR signal had continued to mature between 2 and 5 years after implant, progressively decreasing signal intensity but in any case comparable to the low signal of a normal meniscus. In most of cases the CMI-new tissue complex had a slight reduction in size, compared to a normal medial meniscus, but the new tissue had no apparent negative effects. Translational models for studying meniscal repair and replacement: what they can and cannot tell us
Arnoczky SP, Cook JL, Carter T, Turner AS.
Advances in clinical practice often have their roots in basic science investigations that provide the proof of principle of the treatment concept in question. However, if these concepts are to become reality, they first must be tested in translational animal models to confirm both safety and efficacy. The need to identify appropriate translational models in musculoskeletal tissue engineering and regenerative repair is of critical importance. This is especially true in meniscal research, where the functional anatomy of the structure is directly related to its contribution to the complex biomechanics of the joint and its role in chondroprotection. While no one animal model has established itself as the most appropriate for all aspects of meniscal research, several species have been used to successfully test specific hypotheses. A careful and comprehensive comparison must always be done to validate the utility of a specific animal model. Therefore, it is the purpose of this article to provide an overview of the considerations involved when selecting a translational model for meniscal research. The knee meniscus: structure-function, pathophysiology, current repair techniques, and prospects for regeneration
Makris EA, Hadidi P, Athanasiou KA
Extensive scientific investigations in recent decades have established the anatomical, biomechanical, and functional importance that the meniscus holds within the knee joint. As a vital part of the joint, it acts to prevent the deterioration and degeneration of articular cartilage, and the onset and development of osteoarthritis. For this reason, research into meniscus repair has been the recipient of particular interest from the orthopedic and bioengineering communities. Current repair techniques are only effective in treating lesions located in the peripheral vascularized region of the meniscus. Healing lesions found in the inner avascular region, which functions under a highly demanding mechanical environment, is considered to be a significant challenge. An adequate treatment approach has yet to be established, though many attempts have been undertaken. The current primary method for treatment is partial meniscectomy, which commonly results in the progressive development of osteoarthritis. This drawback has shifted research interest toward the fields of biomaterials and bioengineering, where it is hoped that meniscal deterioration can be tackled with the help of tissue engineering. So far, different approaches and strategies have contributed to the in vitro generation of meniscus constructs, which are capable of restoring meniscal lesions to some extent, both functionally as well as anatomically. The selection of the appropriate cell source (autologous, allogeneic, or xenogeneic cells, or stem cells) is undoubtedly regarded as key to successful meniscal tissue engineering. Furthermore, a large variation of scaffolds for tissue engineering have been proposed and produced in experimental and clinical studies, although a few problems with these (e.g., byproducts of degradation, stress shielding) have shifted research interest toward new strategies (e.g., scaffoldless approaches, self-assembly). A large number of different chemical (e.g., TGF-?1, C-ABC) and mechanical stimuli (e.g., direct compression, hydrostatic pressure) have also been investigated, both in terms of encouraging functional tissue formation, as well as in differentiating stem cells. Even though the problems accompanying meniscus tissue engineering research are considerable, we are undoubtedly in the dawn of a new era, whereby recent advances in biology, engineering, and medicine are leading to the successful treatment of meniscal lesions.
Lab Meeting - August 26, 2011
Tissue-engineered intervertebral discs produce new matrix, maintain disc height, and restore biomechanical function to the rodent spineBowles RD, Gebhard HH, Hartl R, Bonassar LJ
Lower back and neck pain are leading physical conditions for which patients see their doctors in the United States. The organ commonly implicated in this condition is the intervertebral disc (IVD), which frequently herniates, ruptures, or tears, often causing pain and limiting spinal mobility. To date, approaches for replacement of diseased IVD have been confined to purely mechanical devices designed to either eliminate or enable flexibility of the diseased motion segment. Here we present the evaluation of a living, tissue-engineered IVD composed of a gelatinous nucleus pulposus surrounded by an aligned collagenous annulus fibrosus in the caudal spine of athymic rats for up to 6 mo. When implanted into the rat caudal spine, tissue-engineered IVD maintained disc space height, produced de novo extracellular matrix, and integrated into the spine, yielding an intact motion segment with dynamic mechanical properties similar to that of native IVD. These studies demonstrate the feasibility of engineering a functional spinal motion segment and represent a critical step in developing biological therapies for degenerative disc disease.
TransAM - August 15, 2011
Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges.Bron JL, Helder MN, Meisel HJ, Van Royen BJ, Smit TH
Lumbar discectomy is a very effective therapy for neurological decompression in patients suffering from sciatica due to hernia nuclei pulposus. However, high recurrence rates and persisting post-operative low back pain in these patients require serious attention. In the past decade, tissue engineering strategies have been developed mainly targeted to the regeneration of the nucleus pulposus (NP) of the intervertebral disc. Accompanying techniques that deal with the damaged annulus fibrous are now increasingly recognised as mandatory in order to prevent re-herniation to increase the potential of NP repair and to confine NP replacement therapies. In the current review, the requirements, achievements and challenges in this quickly emerging field of research are discussed. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration
Mwale F, Masuda K, Pichika R, Epure LM, Yoshikawa T, Hemmad A, Roughley PJ, Antoniou J
Intervertebral disc (IVD) degeneration is associated with proteolytic degradation of the extracellular matrix, and its repair requires both the production of extracellular matrix and the downregulation of proteinase activity. These properties are associated with several growth factors. However, the use of growth factors in clinical practice is limited by their high cost. This cost can be circumvented using synthetic peptides, such as Link N, which can stimulate the synthesis of proteoglycan and collagen by IVD cells in vitro. The purpose of the present study was to evaluate the effect of Link N in vivo in a rabbit model of IVD degeneration. Genipin-crosslinked fibrin hydrogels as a potential adhesive to augment intervertebral disc annulus repair
Schek RM, Michalek AJ, Iatridis JC
Treatment of damaged intervertebral discs is a significant clinical problem and, despite advances in the repair and replacement of the nucleus pulposus, there are few effective strategies to restore defects in the annulus fibrosus. An annular repair material should meet three specifications: have a modulus similar to the native annulus tissue, support the growth of disc cells, and maintain adhesion to tissue under physiological strain levels. We hypothesized that a genipin crosslinked fibrin gel could meet these requirements. Our mechanical results showed that genipin crosslinked fibrin gels could be created with a modulus in the range of native annular tissue. We also demonstrated that this material is compatible with the in vitro growth of human disc cells, when genipin:fibrin ratios were 0.25:1 or less, although cell proliferation was slower and cell morphology more rounded than for fibrin alone. Finally, lap tests were performed to evaluate adhesion between fibrin gels and pieces of annular tissue. Specimens created without genipin had poor handling properties and readily delaminated, while genipin crosslinked fibrin gels remained adhered to the tissue pieces at strains exceeding physiological levels and failed at 15-30%. This study demonstrated that genipin crosslinked fibrin gels show promise as a gap-filling adhesive biomaterial with tunable material properties, yet the slow cell proliferation suggests this biomaterial may be best suited as a sealant for small annulus fibrosus defects or as an adhesive to augment large annulus repairs. Future studies will evaluate degradation rate, fatigue behaviors, and long-term biocompatibility. Animal models of intervertebral disc degeneration: lessons learned
Lotz JC
STUDY DESIGN: A literature review of intervertebral disc degeneration animal models. OBJECTIVES: Focus is placed on those models that suggest degeneration mechanisms relevant to human. SUMMARY OF BACKGROUND DATA: Medical knowledge from observational epidemiology and intervention studies suggest many etiologic causal factors in humans. Animal models can provide basic science data that support biologic plausibility as well as temporality, specificity, and dose-response relationships.
CMD - August 1, 2011
RhoA/ROCK signaling regulates Sox9 expression and actin organization during chondrogenesisWoods A, Wang G, Beier F
Endochondral ossification is initiated by the differentiation of mesenchymal precursor cells to chondrocytes (chondrogenesis). This process is characterized by a strong interdependence of cell shape, cytoskeletal organization, and the onset of chondrogenic gene expression, but the molecular mechanisms mediating these interactions are not known. Here we investigated the role of the RhoA/ROCK pathway, a well characterized regulator of cytoskeletal organization, in chondrogenesis. We show that pharmacological inhibition of ROCK signaling by Y27632 resulted in increased glycosaminoglycan synthesis and elevated expression of the chondrogenic transcription factor Sox9, whereas overexpression of RhoA in the chondrogenic cell line ATDC5 had the opposite effects. Suppression of Sox9 expression by ROCK signaling was achieved through repression of Sox9 promoter activity. These molecular changes were accompanied by reorganization of the actin cytoskeleton, where RhoA/ROCK signaling suppressed cortical actin organization, a hallmark of differentiated chondrocytes. This led us to analyze the regulation of Sox9 expression by drugs affecting cytoskeletal dynamics. Both inhibition of actin polymerization by cytochalasin D and stabilization of existing actin filaments by jasplakinolide resulted in increased Sox9 mRNA levels, whereas inhibition of microtubule polymerization by colchicine completely blocked Sox9 expression. In conclusion, our data suggest that RhoA/ROCK signaling suppresses chondrogenesis through the control of Sox9 expression and actin organization.
TransAM - August 1, 2011
Preclinical Studies for Cartilage Repair Recommendations from the International Cartilage Repair SocietyMark B. Hurtig, Michael D. Buschmann, Lisa A. Fortier, Caroline D. Hoemann, Ernst B. Hunziker, Jukka S. Jurvelin, Pierre Mainil-Varlet, C. Wayne McIlwraith, Robert L. Sah, and Robert A. Whiteside
Investigational devices for articular cartilage repair or replacement are considered to be significant risk devices by regulatory bodies. Therefore animal models are needed to provide proof of efficacy and safety prior to clinical testing. The financial commitment and regulatory steps needed to bring a new technology to clinical use can be major obstacles, so the implementation of highly predictive animal models is a pressing issue. Until recently, a reductionist approach using acute chondral defects in immature laboratory species, particularly the rabbit, was considered adequate; however, if successful and timely translation from animal models to regulatory approval and clinical use is the goal, a step-wise development using laboratory animals for screening and early development work followed by larger species such as the goat, sheep and horse for late development and pivotal studies is recommended. Such animals must have fully organized and mature cartilage. Both acute and chronic chondral defects can be used but the later are more like the lesions found in patients and may be more predictive. Quantitative and qualitative outcome measures such as macroscopic appearance, histology, biochemistry, functional imaging, and biomechanical testing of cartilage, provide reliable data to support investment decisions and subsequent applications to regulatory bodies for clinical trials. No one model or species can be considered ideal for pivotal studies, but the larger animal species are recommended for pivotal studies. Larger species such as the horse, goat and pig also allow arthroscopic delivery, and press-fit or sutured implant fixation in thick cartilage as well as second look arthroscopies and biopsy procedures. In situ crosslinking elastin-like polypeptide gels for application to articular cartilage repair in a goat osteochondral defect model
Nettles DL, Kitaoka K, Hanson NA, Flahiff CM, Mata BA, Hsu EW, Chilkoti A, Setton LA
The objective of this study was to evaluate an injectable, in situ crosslinkable elastin-like polypeptide (ELP) gel for application to cartilage matrix repair in critically sized defects in goat knees. One cylindrical, osteochondral defect in each of seven animals was filled with an aqueous solution of ELP and a biocompatible, chemical crosslinker, while the contralateral defect remained unfilled and served as an internal control. Joints were sacrificed at 3 (n = 3) or 6 (n = 4) months for MRI, histological, and gross evaluation of features of biomaterial performance, including integration, cellular infiltration, surrounding matrix quality, and new matrix in the defect. At 3 months, ELP-filled defects scored significantly higher for integration by histological and gross grading compared to unfilled defects. ELP did not impede cell infiltration but appeared to be partly degraded. At 6 months, new matrix in unfilled defects outpaced that in ELP-filled defects and scored significantly better for MRI evidence of adverse changes, as well as integration and proteoglycan-containing matrix via gross and histological grading. The ELP-crosslinker solution was easily delivered and formed stable, well-integrated gels that supported cell infiltration and matrix synthesis; however, rapid degradation suggests that ELP formulation modifications should be optimized for longer-term benefits in cartilage repair applications. Short-term retention of labeled chondrocyte subpopulations in stratified tissue-engineered cartilaginous constructs implanted in vivo in mini-pigs
Chawla K, Klein TJ, Schumacher BL, Jadin KD, Shah BH, Nakagawa K, Wong VW, Chen AC, Masuda K, Sah RL
It is likely that effective application of cell-laden implants for cartilage defects depends on retention of implanted cells and interaction between implanted and host cells. The objectives of this study were to characterize stratified cartilaginous constructs seeded sequentially with superficial (S) and middle (M) chondrocyte subpopulations labeled with fluorescent cell tracking dye PKH26 (*) and determine the degree to which these stratified cartilaginous constructs maintain their architecture in vivo after implantation in mini-pigs for 1 week. Alginate-recovered cells were seeded sequentially to form stratified S*/M (only S cells labeled) and S*/M* (both S and M cells labeled) constructs. Full-thickness defects (4 mm diameter) were created in the patellofemoral groove of adult Yucatan mini-pigs and filled with portions of constructs or left empty. Constructs were characterized biochemically, histologically, and biomechanically, and stratification visualized and quantified, before and after implant. After 1 week, animals were sacrificed and implants retrieved. After 1 week in vivo, glycosaminoglycan and collagen content of constructs remained similar to that at implant, whereas DNA content increased. Histological analyses revealed features of an early repair response, with defects filled with tissues containing little matrix and abundant cells. Some implanted (PKH26-labeled) cells persisted in the defects, although constructs did not maintain a stratified organization. Of the labeled cells, 126 +/- 38% and 32 +/- 8% in S*/M and S*/M* constructs, respectively, were recovered. Distribution of labeled cells indicated interactions between implanted and host cells. Longer-term in vivo studies will be useful in determining whether implanted cells are sufficient to have a positive effect in repair.
Lab Meeting - July 15, 2011
Role of YAP/TAZ in mechanotransductionDupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S, Elvassore N, Piccolo S
Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.
Lab Meeting - July 1, 2011
Conversion of Mechanical Force into TGF-B-Mediated Biochemical SignalsMaeda T, Sakabe T, Sunaga A, Sakai K, Rivera AL, Keene DR, Sasaki T, Stavnezer E, Iannotti J, Schweitzer R, Ilic D, Baskaran H, Sakai T
Mechanical forces influence homeostasis in virtually every tissue [1, 2]. Tendon, constantly exposed to variable mechanical force, is an excellent model in which to study the conversion of mechanical stimuli into a biochemical response [3-5]. Here we show in a mouse model of acute tendon injury and in vitro that physical forces regulate the release of active transforming growth factor (TGF)-B from the extracellular matrix (ECM). The quantity of active TGF-B detected in tissue exposed to various levels of tensile loading correlates directly with the extent of physical forces. At physiological levels, mechanical forces maintain, through TGF-B/Smad2/3-mediated signaling, the expression of Scleraxis (Scx), a transcription factor specific for tenocytes and their progenitors. The gradual and temporary loss of tensile loading causes reversible loss of Scx expression, whereas sudden interruption, such as in transection tendon injury, destabilizes the structural organization of the ECM and leads to excessive release of active TGF-B and massive tenocyte death, which can be prevented by the TGF-B type I receptor inhibitor SD208. Our findings demonstrate a critical role for mechanical force in adult tendon homeostasis. Furthermore, this mechanism could translate physical force into biochemical signals in a much broader variety of tissues or systems in the body.
FRiTE - April 28, 2011
Biophysical regulation of histone acetylation in mesenchymal stem cellsLi Y, Chu JS, Kurpinski K, Li X, Bautista DM, Yang L, Paul Sung KL, Li S
Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions. However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape, HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 µm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.
CarTE - April 27, 2011
Chondrocyte repopulation of the zone of death induced by osteochondral harvestMcGregor AJ, Amsden BG, Waldman SD
OBJECTIVE: Harvesting osteochondral grafts results in a zone of chondrocyte death (ZCD) in and around the periphery of the graft, creating a barrier for chondrocytes to migrate to the graft periphery, thus limiting cartilage-to-cartilage healing. The purpose of this study was to repopulate the induced ZCD through the combined effects of collagenase treatment and delivery of a chemotactic agent. DESIGN: In bovine cartilage, the ZCD induced by the OATS™ osteochondral harvesting system was determined, followed by a corresponding collagenase treatment to penetrate the developed ZCD. The chemotactic potential of platelet derived growth factor (PDGF-bb), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (bFGF) (2.5-100 ng/mL) was then assessed using a modified Boyden chamber assay to select an appropriate agent to induce chondrocyte migration. Afterwards, the combined effects of collagenase treatment and chondrocyte chemotaxis on the repopulation of an induced ZCD were examined in cartilage explants over a 4-week-period. RESULTS: The OATS™ osteochondral harvesting system induced a significant ZCD (173 µm, 95% CI: [72-274 µm]) in the grafts. Chondrocyte chemotaxis was induced by all agents investigated at concentrations greater than 25 ng/mL. After 4 weeks in culture, collagenase treatment alone reduced the ZCD by approximately 40% relative to untreated explants. Coupling the collagenase treatment with 25 ng/mL IGF-I reduced the ZCD by approximately 80% relative to untreated explants, and 65% relative to explants treated only with collagenase. CONCLUSION: Treating cartilage explants with collagenase and 25 ng/mL IGF-I resulted in a decreased ZCD after a 4-week-period, and increased chondrocyte density within the induced ZCD.
Lab Meeting - April 22, 2011
Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cellsWang N, Tolic-Nørrelykke IM, Chen J, Mijailovich SM, Butler JP, Fredberg JJ, Stamenovic D
The tensegrity hypothesis holds that the cytoskeleton is a structure whose shape is stabilized predominantly by the tensile stresses borne by filamentous structures. Accordingly, cell stiffness must increase in proportion with the level of the tensile stress, which is called the prestress. Here we have tested that prediction in adherent human airway smooth muscle (HASM) cells. Traction microscopy was used to measure the distribution of contractile stresses arising at the interface between each cell and its substrate; this distribution is called the traction field. Because the traction field must be balanced by tensile stresses within the cell body, the prestress could be computed. Cell stiffness (G) was measured by oscillatory magnetic twisting cytometry. As the contractile state of the cell was modulated with graded concentrations of relaxing or contracting agonists (isoproterenol or histamine, respectively), the mean prestress ((t)) ranged from 350 to 1,900 Pa. Over that range, cell stiffness increased linearly with the prestress: G (Pa) = 0.18(t) + 92. While this association does not necessarily preclude other interpretations, it is the hallmark of systems that secure shape stability mainly through the prestress. Regardless of mechanism, these data establish a strong association between stiffness of HASM cells and the level of tensile stress within the cytoskeleton.
FRiTE - April 14, 2011
Tissue ingrowth after implantation of a novel, biodegradable polyurethane scaffold for treatment of partial meniscal lesionsVerdonk R, Verdonk P, Huysse W, Forsyth R, Heinrichs EL
BACKGROUND: A novel, biodegradable, aliphatic polyurethane scaffold was designed to fulfill an unmet clinical need in the treatment of patients with irreparable partial meniscal lesions. HYPOTHESIS: Treatment of irreparable partial meniscal lesions with an acellular polyurethane scaffold supports new tissue ingrowth. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Fifty-two patients (with 34 medial and 18 lateral lesions) were recruited into a prospective, single-arm, multicenter, proof-of-principle study and treated with the polyurethane scaffold. The scaffold was implanted after partial meniscectomy using standard surgeon-preferred techniques for suturing. Tissue ingrowth was assessed at 3 months by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and at 12 months by gross examination during second-look arthroscopy, in the course of which a biopsy sample from the inner free edge of the scaffold meniscus was taken for qualitative histologic analysis. RESULTS: Tissue ingrowth at 3 months was demonstrated on DCE-MRI in 35 of 43 (81.4%) patients. All but one 12-month second-look (43 of 44 [97.7%]) showed integration of the scaffold with the native meniscus and all biopsy specimens (44) showed fully vital material, with no signs of cell death or necrosis. Three distinct layers were observed based on morphologic structure, vessel structure presence or absence, and extracellular matrix composition. CONCLUSION: The DCE-MRI demonstrated successful early tissue ingrowth into the scaffold. The biopsy findings demonstrated the biocompatibility of the scaffold and ingrowth of tissue with particular histologic characteristics suggestive of meniscus-like tissue. In conclusion, these data show for the first time consistent regeneration of tissue when using an acellular polyurethane scaffold to treat irreparable partial meniscus tissue lesions.
CarTE - April 13, 2011
Cell-ECM traction force modulates endogenous tension at cell-cell contactsMaruthamuthu V, Sabass B, Schwarz US, Gardel ML
Cells in tissues are mechanically coupled both to the ECM and neighboring cells, but the coordination and interdependency of forces sustained at cell-ECM and cell-cell adhesions are unknown. In this paper, we demonstrate that the endogenous force sustained at the cell-cell contact between a pair of epithelial cells is approximately 100 nN, directed perpendicular to the cell-cell interface and concentrated at the contact edges. This force is stably maintained over time despite significant fluctuations in cell-cell contact length and cell morphology. A direct relationship between the total cellular traction force on the ECM and the endogenous cell-cell force exists, indicating that the cell-cell tension is a constant fraction of the cell-ECM traction. Thus, modulation of ECM properties that impact cell-ECM traction alters cell-cell tension. Finally, we show in a minimal model of a tissue that all cells experience similar forces from the surrounding microenvironment, despite differences in the extent of cell-ECM and cell-cell adhesion. This interdependence of cell-cell and cell-ECM forces has significant implications for the maintenance of the mechanical integrity of tissues, mechanotransduction, and tumor mechanobiology.
Lab Meeting - April 8, 2011
Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-γ agonistsShimono K, Tung WE, Macolino C, Chi AH, Didizian JH, Mundy C, Chandraratna RA, Mishina Y, Enomoto-Iwamoto M, Pacifici M, Iwamoto M
Heterotopic ossification consists of ectopic bone formation within soft tissues after surgery or trauma. It can have debilitating consequences, but there is no definitive cure. Here we show that heterotopic ossification was essentially prevented in mice receiving a nuclear retinoic acid receptor-γ (RAR-γ) agonist. Side effects were minimal, and there was no significant rebound effect. To uncover the mechanisms of these responses, we treated mouse mesenchymal stem cells with an RAR-γ agonist and transplanted them into nude mice. Whereas control cells formed ectopic bone masses, cells that had been pretreated with the RAR-γ agonist did not, suggesting that they had lost their skeletogenic potential. The cells became unresponsive to rBMP-2 treatment in vitro and showed decreases in phosphorylation of Smad1, Smad5 and Smad8 and in overall levels of Smad proteins. In addition, an RAR-γ agonist blocked heterotopic ossification in transgenic mice expressing activin receptor-like kinase-2 (ALK2) Q207D, a constitutively active form of the receptor that is related to ALK2 R206H found in individuals with fibrodysplasia ossificans progressiva. The data indicate that RAR-γ agonists are potent inhibitors of heterotopic ossification in mouse models and, thus, may also be effective against injury-induced and congenital heterotopic ossification in humans.
Regenerative medicine: a snapshot of the current regulatory environment and standards
Messenger MP, Tomlins PE
There has been an immense amount of research activity in the emerging field of regenerative medicine which dates back to the early 1980s and beyond, with many examples of products that have made the difficult transition from the laboratory to commercially available goods. This process, like many product developments, is fraught with issues that range from identifying and realizing the market needs to the challenge of raising capital. It can be argued that this transition process is even more challenging for regenerative medicine products, since, in many cases, the existing infrastructural network, i.e., regulations and standards that support medicines and medical devices, are insufficient. Indeed, this non-compliance with the existing framework is often cited as a barrier to commercialization. Here, we review the current regulatory framework and standards portfolio and discuss their role in commercializing potential regenerative medicine products. The broader issue of regenerative medicine research itself and the economics of commercialization would exceed the scope of this Essay and are not covered.
Lab Meeting - March 25, 2011
Bioartificial matrices for therapeutic vascularizationPhelps EA, Landázuri N, Thulé PM, Taylor WR, García AJ
Therapeutic vascularization remains a significant challenge in regenerative medicine applications. Whether the goal is to induce vascular growth in ischemic tissue or scale up tissue-engineered constructs, the ability to induce the growth of patent, stable vasculature is a critical obstacle. We engineered polyethylene glycol-based bioartificial hydrogel matrices presenting protease-degradable sites, cell-adhesion motifs, and growth factors to induce the growth of vasculature in vivo. Compared to injection of soluble VEGF, these matrices delivered sustained in vivo levels of VEGF over 2 weeks as the matrix degraded. When implanted subcutaneously in rats, degradable constructs containing VEGF and arginine-glycine-aspartic acid tripeptide induced a significant number of vessels to grow into the implant at 2 weeks with increasing vessel density at 4 weeks. The mechanism of enhanced vascularization is likely cell-demanded release of VEGF, as the hydrogels may degrade substantially within 2 weeks. In a mouse model of hind-limb ischemia, delivery of these matrices resulted in significantly increased rate of reperfusion. These results support the application of engineered bioartificial matrices to promote vascularization for directed regenerative therapies.
FRiTE - March 17, 2011
Cell shape and substrate rigidity both regulate cell stiffnessTee SY, Fu J, Chen CS, Janmey PA
Cells from many different tissues sense the stiffness and spatial patterning of their microenvironment to modulate their shape and cortical stiffness. It is currently unknown how substrate stiffness, cell shape, and cell stiffness modulate or interact with one another. Here, we use microcontact printing and microfabricated arrays of elastomeric posts to independently and simultaneously control cell shape and substrate stiffness. Our experiments show that cell cortical stiffness increases as a function of both substrate stiffness and spread area. For soft substrates, the influence of substrate stiffness on cell cortical stiffness is more prominent than that of cell shape, since increasing adherent area does not lead to cell stiffening. On the other hand, for cells constrained to a small area, cell shape effects are more dominant than substrate stiffness, since increasing substrate stiffness no longer affects cell stiffness. These results suggest that cell size and substrate stiffness can interact in a complex fashion to either enhance or antagonize each other's effect on cell morphology and mechanics.
Lab Meeting - March 11, 2011
Environmental sensing through focal adhesionsGeiger B, Spatz JP, Bershadsky AD
Recent progress in the design and application of artificial cellular microenvironments and nanoenvironments has revealed the extraordinary ability of cells to adjust their cytoskeletal organization, and hence their shape and motility, to minute changes in their immediate surroundings. Integrin-based adhesion complexes, which are tightly associated with the actin cytoskeleton, comprise the cellular machinery that recognizes not only the biochemical diversity of the extracellular neighbourhood, but also its physical and topographical characteristics, such as pliability, dimensionality and ligand spacing. Here, we discuss the mechanisms of such environmental sensing, based on the finely tuned crosstalk between the assembly of one type of integrin-based adhesion complex, namely focal adhesions, and the forces that are at work in the associated cytoskeletal network owing to actin polymerization and actomyosin contraction.
Patterning network structure to spatially control cellular remodeling and stem cell fate within 3-dimensional hydrogels
Khetan S, Burdick JA
The spatially directed 3-dimensional (3D) remodeling of synthetic materials may be useful to regionally control cell behavior. In this work, we developed a process to synthesize hyaluronic acid hydrogels using multiple modes of crosslinking applied sequentially; a primary addition reaction to introduce protease degradable peptide crosslinks, then a UV light-induced secondary radical reaction (enabling spatial control) to introduce non-degradable kinetic chains. These differential network structures either permitted (primary crosslinking only, "-UV") or inhibited (sequential crosslinking, "+UV") cellular remodeling. This behavior was validated by controlling the outgrowth from chick aortic arches or the spreading of encapsulated mesenchymal stem cells (MSCs), where only -UV regions permitted arch outgrowth and MSC spreading. Additionally, network structures dictated adipogenic/osteogenic MSC fate decisions, with spatial control, by controlling encapsulated MSC spreading. This manipulation of microenvironmental cues may be valuable for advanced tissue engineering applications requiring the spatial control of cells in 3D.
FRiTE - March 3, 2011
Local treatment of meniscal lesions with vascular endothelial growth factorKopf S, Birkenfeld F, Becker R, Petersen W, Stärke C, Wruck CJ, Tohidnezhad M, Varoga D, Pufe T
BACKGROUND: The healing potential in the avascular regions of the meniscus is very limited, and improving the vascularity might be a reasonable way to improve healing. Vascular endothelial growth factor (VEGF) is one of the most potent proangiogenetic factors. We hypothesized that the local application of VEGF(165) would (1) improve the healing of a lesion in the avascular region of the meniscus, (2) induce angiogenesis in both the avascular and vascular regions, and (3) increase the amounts of VEGF mRNA and VEGF. METHODS: In eighteen sheep, the medial menisci were cut longitudinally in the avascular region and were sutured. Three groups were established depending on the suture material: (1) uncoated Ethibond, (2) Ethibond coated with VEGF(165) and its carrier Poly(D,L-Lactide) (PDLLA), and (3) Ethibond coated with PDLLA. The contralateral medial menisci served as a control group. Each of the three suture type groups included six animals. After eight weeks, the sheep were killed, and the menisci were examined macroscopically. Immunohistochemistry of Factor VIII and VEGF and real-time reverse-transcription polymerase chain reaction (RT-PCR) of VEGF mRNA were performed. Additionally, the VEGF release kinetics from the VEGF/PDLLA-coated suture were evaluated in vitro. RESULTS: In this model, VEGF did not improve meniscal healing. It did not increase angiogenesis in the avascular or vascular region, the VEGF concentration, or the amount of VEGF mRNA. VEGF release from the coated suture peaked on Day 3 and was nearly zero on Day 9. CONCLUSIONS: The local application of VEGF(165) as eluted from suture did not increase meniscal angiogenesis or improve meniscal healing. In addition, there was no effect on the amount of VEGF mRNA and VEGF. The VEGF carrier (PDLLA) may have been inadequate because of the short duration of VEGF supply.
CarTE - March 2, 2011
Glucose concentration and medium volume influence cell viability and glycosaminoglycan synthesis in chondrocyte-seeded alginate constructsHeywood HK, Bader DL, Lee DA
Increasing the thickness of tissue-engineered cartilage is associated with loss of chondrocyte viability and biosynthetic activity at the tissue center. Exceptionally high volumes of culture medium, however, can maintain cellularity and glycosaminoglycan synthesis throughout 4-mm-thick constructs. We hypothesized that glucose supplementation could replicate the augmentation of tissue formation achieved by medium volume. Chondrocyte-alginate constructs (40x10(6) cells/mL) were cultured for 14 days in 0.4-6.4 mL/10(-6) cells of either low- (5.1 mM) or high- (20.4 mM) glucose medium. Glucose was critical to chondrocyte viability, and glucose uptake increased significantly (P < .001) with both medium volume and glucose supplementation. After 14 days, constructs cultured in 0.4 mL/10(-6) cells of low-glucose medium had a mass of 172 +/- 6.1 mg and glycosaminoglycan (GAG) content of 0.32 +/- 0.03 mg (mean +/- standard deviation). A 4-fold increase in medium volume increased the final construct mass by 44% and GAG content by 207%. An equivalent increase in glucose supply in the absence of volume change increased these parameters by just 10% and 73%, respectively. A similar trend was observed from 0.8 to 3.2 mL/10(-6) cells, when maximal values of construct GAG content and mass were obtained. Therefore, medium volume remains an important consideration for the optimal culture of tissue-engineered cartilage.
FRiTE - February 17, 2011
MRI-based characterization of bone anatomy in the human knee for size matching of a medial meniscal implantElsner JJ, Portnoy S, Guilak F, Shterling A, Linder-Ganz E
Allograft or synthetic menisci have been suggested as a means to restore contact pressures following meniscectomy. However, when the natural meniscus is severely damaged/absent, the necessary size cannot be determined according to the recipient size and there is a need to estimate it from magnetic resonance imaging (MRI) of the contralateral knee or the injured knee bones. The use of the contralateral-knee for size matching is problematic due to economic and practical reasons. Hence, there are significant advantages for a sizing algorithm based only on the candidate knee geometry. The aim of this study is to characterize midrange values and variability of knee dimensions and to develop a set of mathematical relations representing knee dimensions using a minimum of imaging-based bone measurements. Tibia, femur, and meniscus measurements were taken in 118 MRI scans and used to develop a representative parametric knee model in which all dimensions are expressed using tibia plateau width. The model was verified by comparing the predicted values to direct MRI measurements for 20 additional subjects by means of the Pearson correlation and Bland and Altman (1986, "Statistical Methods for Assessing Agreement Between Two Methods of Clinical Measurement," Lancet, 1, pp. 307-310) plot. Anatomical parameters in the male knee were significantly larger (~17%) compared with corresponding female measurements. However, most relations between tibia, femur, and meniscus measurements (43/56) were not significantly different between male and female populations (p = 0.05), indicating that differences between male and female joints are generally related to scaling and not shape. Dimensions predicted by the knee model were in a good agreement with dimensions measured directly from the MRI (R(2)>0.96) and the Bland and Altman plot indicated that ~95% of data points were well within the ± 2 standard deviation lines of agreement. The model proposed in this study is advantageous in being able to describe typical knee proportions for a given tibial width and can be used to predict the dimensions of a candidate knee based on a single measurement. The anatomical/anthropometric data presented in the study can be utilized in a sizing algorithm for artificial meniscal implants or in the design of artificial meniscus prostheses.
CarTE - February 16, 2011
Autologous engineering of cartilageEmans PJ, van Rhijn LW, Welting TJ, Cremers A, Wijnands N, Spaapen F, Voncken JW, Shastri VP
Treatment of full-thickness damage to hyaline cartilage is hampered by the limited availability of autologous healthy cartilage and the lengthy, cost-prohibitive cell isolation and expansion steps associated with autologous cartilage implantation (ACI). Here we report a strategy for de novo engineering of ectopic autologous cartilage (EAC) within the subperiosteal space (in vivo bioreactor), through the mere introduction of a biocompatible gel that might promote hypoxia-mediated chondrogenesis, thereby effectively overcoming the aforementioned limitations. The EAC is obtained within 3 wk post injection of the gel, and can be press-fit into an osteochondral defect where it undergoes remodeling with good lateral and subchondral integration. The implanted EAC showed no calcification even after 9 mo and attained an average O'Driscoll score of 11 (versus 4 for controls). An "on demand" autologous source of autologous cartilage with remodeling capacity is expected to significantly impact the clinical options in repair of trauma to articular cartilage.
Lab Meeting - January 28, 2011
The elusive nature and function of mesenchymal stem cellsNombela-Arrieta C, Ritz J, Silberstein LE
Mesenchymal stem cells (MSCs) are a diverse subset of multipotent precursors present in the stromal fraction of many adult tissues and have drawn intense interest from translational and basic investigators. MSCs have been operationally defined by their ability to differentiate into osteoblasts, adipocytes and chondrocytes after in vitro expansion. Nevertheless, their identity in vivo, heterogeneity, anatomical localization and functional roles in adult tissue homeostasis have remained enigmatic and are only just starting to be uncovered.
Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture
Gilbert PM, Havenstrite KL, Magnusson KE, Sacco A, Leonardi NA, Kraft P, Nguyen NK, Thrun S, Lutolf MP, Blau HM
Stem cells that naturally reside in adult tissues, such as muscle stem cells (MuSCs), exhibit robust regenerative capacity in vivo that is rapidly lost in culture. Using a bioengineered substrate to recapitulate key biophysical and biochemical niche features in conjunction with a highly automated single-cell tracking algorithm, we show that substrate elasticity is a potent regulator of MuSC fate in culture. Unlike MuSCs on rigid plastic dishes (approximately 10(6) kilopascals), MuSCs cultured on soft hydrogel substrates that mimic the elasticity of muscle (12 kilopascals) self-renew in vitro and contribute extensively to muscle regeneration when subsequently transplanted into mice and assayed histologically and quantitatively by noninvasive bioluminescence imaging. Our studies provide novel evidence that by recapitulating physiological tissue rigidity, propagation of adult muscle stem cells is possible, enabling future cell-based therapies for muscle-wasting diseases.
FRiTE - January 20, 2011
The limb bud Shh-Fgf feedback loop is terminated by expansion of former ZPA cellsScherz PJ, Harfe BD, McMahon AP, Tabin CJ
Vertebrate limb outgrowth is driven by a positive feedback loop involving Sonic Hedgehog (Shh), Gremlin, and Fgf4. By overexpressing individual components of the loop at a time after these genes are normally down-regulated in chicken embryos, we found that Shh no longer maintains Gremlin in the posterior limb. Shh-expressing cells and their descendants cannot express Gremlin. The proliferation of these descendants forms a barrier separating the Shh signal from Gremlin-expressing cells, which breaks down the Shh-Fgf4 loop and thereby affects limb size and provides a mechanism explaining regulative properties of the limb bud.
FRiTE - December 9, 2010
Cyclic tensile culture promotes fibroblastic differentiation of marrow stromal cells encapsulated in poly(ethylene glycol)-based hydrogelsDoroski DM, Levenston ME, Temenoff JS
To inform future efforts in tendon/ligament tissue engineering, our laboratory has developed a well-controlled model system with the ability to alter both external tensile loading parameters and local biochemical cues to better understand marrow stromal cell differentiation in response to both stimuli concurrently. In particular, the synthetic, poly(ethylene glycol)-based hydrogel material oligo(poly(ethylene glycol) fumarate) (OPF) has been explored as a cell carrier for this system. This biomaterial can be tailored to present covalently incorporated bioactive moieties and can be loaded in our custom cyclic tensile bioreactor for up to 28 days with no loss of material integrity. Human marrow stromal cells encapsulated in these OPF hydrogels were cultured (21 days) under cyclic tensile strain (10%, 1? Hz, 3? h of strain followed by 3? h without) or at 0% strain. No difference was observed in cell number due to mechanical stimulation or across time (n?=?4), with cells remaining viable (n?=?4) through 21 days. Cyclic strain significantly upregulated all tendon/ligament fibroblastic genes examined (collagen I, collagen III, and tenascin-C) by day 21 (n?=?6), whereas genes for other pathways (osteogenic, chondrogenic, and adipogenic) did not increase. After 21 days, the presence of collagen I and tenascin-C was observed via immunostaining (n?=?2). This study demonstrates the utility of this hydrogel/bioreactor system as a versatile, yet well-controlled, model environment to study marrow stromal cell differentiation toward the tendon/ligament phenotype under a variety of conditions.
CarTE - December 8, 2010
Unique biomaterial compositions direct bone marrow stem cells into specific chondrocytic phenotypes corresponding to the various zones of articular cartilageNguyen LH, Kudva AK, Guckert NL, Linse KD, Roy K
Numerous studies have reported generation of cartilage-like tissue from chondrocytes and stem cells, using pellet cultures, bioreactors and various biomaterials, especially hydrogels. However, one of the primary unsolved challenges in the field has been the inability to produce tissue that mimics the highly organized zonal architecture of articular cartilage; specifically its spatially varying mechanical properties and extra-cellular matrix (ECM) composition. Here we show that different combinations of synthetic and natural biopolymers create unique niches that can "direct" a single marrow stem cell (MSC) population to differentiate into the superficial, transitional, or deep zones of articular cartilage. Specifically, incorporating chondroitin sulfate (CS) and matrix metalloproteinase-sensitive peptides (MMP-pep) into PEG hydrogels (PEG:CS:MMP-pep) induced high levels of collagen II and low levels of proteoglycan expression resulting in a low compressive modulus, similar to the superficial zone. PEG:CS hydrogels produced intermediate-levels of both collagen II and proteoglycans, like the transitional zone, while PEG:hyaluronic acid (HA) hydrogels induced high proteoglycan and low collagen II levels leading to high compressive modulus, similar to the deep zone. Additionally, the compressive moduli of these zone-specific matrices following cartilage generation showed similar trend as the corresponding zones of articular cartilage, with PEG:CS:MMP-pep having the lowest compressive modulus, followed by PEG:CS while PEG:HA had the highest modulus. These results underscore the potential for composite scaffold structures incorporating these biomaterial compositions such that a single stem-progenitor cell population can give rise to zonally-organized, functional articular cartilage-like tissue.
Lab Meeting - December 3, 2010
The epigenetic mechanism of mechanically induced osteogenic differentiationArnsdorf EJ, Tummala P, Castillo AB, Zhang F, Jacobs CR
Epigenetic regulation of gene expression occurs due to alterations in chromatin proteins that do not change DNA sequence, but alter the chromatin architecture and the accessibility of genes, resulting in changes to gene expression that are preserved during cell division. Through this process genes are switched on or off in a more durable fashion than other transient mechanisms of gene regulation, such as transcription factors. Thus, epigenetics is central to cellular differentiation and stem cell linage commitment. One such mechanism is DNA methylation, which is associated with gene silencing and is involved in a cell's progression towards a specific fate. Mechanical signals are a crucial regulator of stem cell behavior and important in tissue differentiation; however, there has been no demonstration of a mechanism whereby mechanics can affect gene regulation at the epigenetic level. In this study, we identified candidate DNA methylation sites in the promoter regions of three osteogenic genes from bone marrow derived mesenchymal stem cells (MSCs). We demonstrate that mechanical stimulation alters their epigenetic state by reducing DNA methylation and show an associated increase in expression. We contrast these results with biochemically induced differentiation and distinguish expression changes associated with durable epigenetic regulation from those likely to be due to transient changes in regulation. This is an important advance in stem cell mechanobiology as it is the first demonstration of a mechanism by which the mechanical micro-environment is able to induce epigenetic changes that control osteogenic cell fate, and that can be passed to daughter cells. This is a first step to understanding that will be vital to successful bone tissue engineering and regenerative medicine, where continued expression of a desired long-term phenotype is crucial.
FRiTE - November 11, 2010
Nuclear mechanotransduction: response of the lamina to extracellular stress with implications in agingPhilip JT, Dahl KN
Mechnotransduction, the phenomenon by which cells respond to applied force, is necessary for normal cell processes and is implicated in the pathology of several diseases including atherosclerosis. The exact mechanisms which govern how forces can affect gene expression have not been determined, but putative direct force effects on the genome would require transduction through the nuclear lamina. In this study we show that nuclei in cells exposed to shear stress significantly change shape, upregulate nuclear lamins and move lamins from the nuclear interior to the nuclear periphery. We hypothesize that the augmentation of the nuclear lamina at the nuclear periphery protects the nuclear interior from the force and allows a nuclear adaptation to shear stress. We also investigate the shear stress response of nuclei in cells that have been transfected with lamin A Delta50, which significantly stiffens nuclei. Lamin A Delta50 causes the premature aging syndrome Hutchinson-Gilford progeria syndrome (HGPS) and models many aspects of normal aging. We find that the presence of lamin A Delta50 in only 30% of cells greatly reduces the response of the nuclear lamina in all cells in the flow field. We suggest that cells expressing lamin A Delta50 lack the ability to adapt to flow and may prevent neighboring cells from adapting as well. These results provide insight into the development of cardiovascular disease both in patients with HGPS and in normal aging.
Lab Meeting - November 5, 2010
Cells transmit spatial information by orienting collagen fibersKlebe RJ, Caldwell H, Milam S
Parallel orientation of large cell populations occurs when cells are cultured on thin collagen films if and only if a gel is restrained at two or more edges. The direction in which cell bodies orient is determined by the geometry of the gel. It can be shown that tension exerted by cells on a collagen gel leads to reorientation of collagen fibers in a direction determined by the initial geometry of the collagen gel. Hence, tension generated by cells leads to collagen fiber orientation which, in turn, determines the spatial orientation and morphology of cells. Thus, without cell-to-cell contact, cells can communicate with each other by applying tension to collagen fibers in their extracellular matrix. Information concerning the shape of the environment can be transmitted via such long range forces.
FRiTE - October 28, 2010
Mechanical activation of ß-catenin regulates phenotype in adult murine marrow-derived mesenchymal stem cellsCase N, Xie Z, Sen B, Styner M, Zou M, O'Conor C, Horowitz M, Rubin J
Regulation of skeletal remodeling appears to influence the differentiation of multipotent mesenchymal stem cells (MSC) resident in the bone marrow. As murine marrow cultures are contaminated with hematopoietic cells, they are problematic for studying direct effects of mechanical input. Here we use a modified technique to isolate marrow-derived MSC (mdMSC) from adult mice, yielding a population able to differentiate into adipogenic and osteogenic phenotypes that is devoid of hematopoietic cells. In pure mdMSC populations, a daily strain regimen inhibited adipogenic differentiation, suppressing expression of PPAR? and adiponectin. Strain increased ß-catenin and inhibition of adipogenesis required this effect. Under osteogenic conditions, strain activated ß-catenin signaling and increased expression of WISP1 and COX2. mdMSC were also generated from mice lacking caveolin-1, a protein known to sequester ß-catenin: caveolin-1((-/-)) mdMSC exhibited retarded differentiation along both adipogenic and osteogenic lineages but retained mechanical responses that involved ß-catenin activation. Interestingly, caveolin-1((-/-)) mdMSC failed to express bone sialoprotein and did not form mineralized nodules. In summary, mdMSC from adult mice respond to both soluble factors and mechanical input, with mechanical activation of ß-catenin influencing phenotype. As such, these cells offer a useful model for studies of direct mechanical regulation of MSC differentiation and function.
CarTE - October 27, 2010
Hierarchically structured, hyaluronic acid-based hydrogel matrices via the covalent integration of microgels into macroscopic networksJha AK, Malik MS, Farach-Carson MC, Duncan RL, Jia X
We aimed to develop biomimetic hydrogel matrices that not only exhibit structural hierarchy and mechanical integrity, but also present biological cues in a controlled fashion. To this end, photocrosslinkable, hyaluronic acid (HA)-based hydrogel particles (HGPs) were synthesized via an inverse emulsion crosslinking process followed by chemical modification with glycidyl methacrylate (GMA). HA modified with GMA (HA-GMA) was employed as the soluble macromer. Macroscopic hydrogels containing covalently integrated hydrogel particles (HA-c-HGP) were prepared by radical polymerization of HA-GMA in the presence of crosslinkable HGPs. The covalent linkages between the hydrogel particles and the secondary HA matrix resulted in the formation of a diffuse, fibrilar interface around the particles. Compared to the traditional bulk gels synthesized by photocrosslinking of HA-GMA, these hydrogels exhibited a reduced sol fraction and a lower equilibrium swelling ratio. When tested under uniaxial compression, the HA-c-HGP gels were more pliable than the HA-p-HGP gels and fractured at higher strain than the HA-GMA gels. Primary bovine chondrocytes were photoencapsulated in the HA matrices with minimal cell damage. The 3D microenvironment created by HA-GMA and HA HGPs not only maintained the chondrocyte phenotype but also fostered the production of cartilage specific extracellular matrix. To further improve the biological activities of the HA-c-HGP gels, bone morphogenetic protein 2 (BMP-2) was loaded into the immobilized HGPs. BMP-2 was released from the HA-c-HGP gels in a controlled manner with reduced initial burst over prolonged periods of time. The HA-c-HGP gels are promising candidates for use as bioactive matrices for cartilage tissue engineering.
Donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering
Siddappa R, Licht R, van Blitterswijk C, de Boer J
The use of multipotent human mesenchymal stem cells (hMSCs) for tissue engineering has been a subject of extensive research. The donor variation in growth, differentiation and in vivo bone forming ability of hMSCs is a bottleneck for standardization of therapeutic protocols. In this study, we isolated and characterized hMSCs from 19 independent donors, aged between 27 and 85 years, and investigated the extent of heterogeneity of the cells and the extent to which hMSCs can be expanded without loosing multipotency. Dexamethasone-induced ALP expression varied between 1.2- and 3.7-fold, but no correlation was found with age, gender, or source of isolation. The cells from donors with a higher percentage of ALP-positive cells in control and dexamethasone-induced groups showed more calcium deposition than cells with lower percentage of ALP positive cells. Despite the variability in osteogenic gene expression among the donors tested, ALP, Collagen type 1, osteocalcin, and S100A4 showed similar trends during the course of osteogenic differentiation. In vitro expansion studies showed that hMSCs can be effectively expanded up to four passages (approximately 10-12 population doublings from a P0 culture) while retaining their multipotency. Our in vivo studies suggest a correlation between in vitro ALP expression and in vivo bone formation. In conclusion, irrespective of age, gender, and source of isolation, cells from all donors showed osteogenic potential. The variability in ALP expression appears to be a result of sampling method and cellular heterogeneity among the donor population.
Lab Meeting - October 22, 2010
Decreased muscle loading delays maturation of the tendon enthesis during postnatal developmentThomopoulos S, Kim HM, Rothermich SY, Biederstadt C, Das R, Galatz LM
Physical environment influences the development and maintenance of musculoskeletal tissues. The current study uses an animal model to explore the role of the physical environment on the postnatal development of the supraspinatus tendon enthesis. A supraspinatus intramuscular injection of botulinum toxin A was used to paralyze the left shoulders of mice at birth. The supraspinatus muscles of right shoulders were injected with saline to serve as contralateral controls. The supraspinatus enthesis was examined after 14, 21, 28, and 56 days of postnatal development. Histologic assays were used to examine fibrocartilage morphology and percentage osteoclast surface. Micro-computed tomography was used to examine muscle geometry and bone architecture. At 14 days there were no differences between groups in fibrocartilage formation, muscle geometry, bone architecture, or osteoclast surface. When comparing groups at 21, 28, and 56 days, muscle volume was decreased, fibrocartilage development was delayed, mineralized bone was decreased, and osteoclast surface was higher at each timepoint in the botulinum group compared to the contralateral saline control group. Our results indicate that the development of the tendon enthesis is sensitive to its mechanical environment. A reduction in muscle loading delayed the development of the tendon-to-bone insertion site by impeding the accumulation of mineralized bone. Physical factors did not play a significant role in enthesis maturation in the first 14 days postnatally, implying that biologic factors may drive early postnatal development.
FRiTE - October 14, 2010
"Aligned-to-random" nanofiber scaffolds for mimicking the structure of the tendon-to-bone insertion siteXie J, Li X, Lipner J, Manning CN, Schwartz AG, Thomopoulos S, Xia Y
We have demonstrated the fabrication of "aligned-to-random" electrospun nanofiber scaffolds that mimic the structural organization of collagen fibers at the tendon-to-bone insertion site. Tendon fibroblasts cultured on such a scaffold exhibited highly organized and haphazardly oriented morphologies, respectively, on the aligned and random portions.
Nanofiber scaffolds with gradations in mineral content for mimicking the tendon-to-bone insertion site
Li X, Xie J, Lipner J, Yuan X, Thomopoulos S, Xia Y
We have demonstrated a simple and versatile method for generating a continuously graded, bonelike calcium phosphate coating on a nonwoven mat of electrospun nanofibers. A linear gradient in calcium phosphate content could be achieved across the surface of the nanofiber mat. The gradient had functional consequences with regard to stiffness and biological activity. Specifically, the gradient in mineral content resulted in a gradient in the stiffness of the scaffold and further influenced the activity of mouse preosteoblast MC3T3 cells. This new class of nanofiber-based scaffolds can potentially be employed for repairing the tendon-to-bone insertion site via a tissue engineering approach.
CarTE - October 13, 2010
In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitmentRussell KC, Phinney DG, Lacey MR, Barrilleaux BL, Meyertholen KE, O'Connor KC
In regenerative medicine, bone marrow is a promising source of mesenchymal stem cells (MSCs) for a broad range of cellular therapies. This research addresses a basic prerequisite to realize the therapeutic potential of MSCs by developing a novel high-capacity assay to quantify the clonal heterogeneity in potency that is inherent to MSC preparations. The assay utilizes a 96-well format to (1) classify MSCs according to colony-forming efficiency as a measure of proliferation capacity and trilineage potential to exhibit adipo-, chondro-, and osteogenesis as a measure of multipotency and (2) preserve a frozen template of MSC clones of known potency for future use. The heterogeneity in trilineage potential of normal bone marrow MSCs is more complex than previously reported: all eight possible categories of trilineage potential were detected. In this study, the average colony-forming efficiency of MSC preparations was 55-62%, and tripotent MSCs accounted for nearly 50% of the colony-forming cells. The multiple phenotypes detected in this study infer a more convoluted hierarchy of lineage commitment than described in the literature. Greater cell amplification, colony-forming efficiency, and colony diameter for tri- versus unipotent clones suggest that MSC proliferation may be a function of potency. CD146 may be a marker of multipotency, with approximately 2-fold difference in mean fluorescence intensity between tri- and unipotent clones. The significance of these findings is discussed in the context of the efficacy of MSC therapies. The in vitro assay described herein will likely have numerous applications given the importance of heterogeneity to the therapeutic potential of MSCs.
CarTE - September 29, 2010
The local matrix distribution and the functional development of tissue engineered cartilage, a finite element studySengers BG, Van Donkelaar CC, Oomens CW, Baaijens FP
Assessment of the functionality of tissue engineered cartilage constructs is hampered by the lack of correlation between global measurements of extra cellular matrix constituents and the global mechanical properties. Based on patterns of matrix deposition around individual cells, it has been hypothesized previously, that mechanical functionality arises when contact occurs between zones of matrix associated with individual cells. The objective of this study is to determine whether the local distribution of newly synthesized extracellular matrix components contributes to the evolution of the mechanical properties of tissue engineered cartilage constructs. A computational homogenization approach was adopted, based on the concept of a periodic representative volume element. Local transport and immobilization of newly synthesized matrix components were described. Mechanical properties were taken dependent on the local matrix concentration and subsequently the global aggregate modulus and hydraulic permeability were derived. The transport parameters were varied to assess the effect of the evolving matrix distribution during culture. The results indicate that the overall stiffness and permeability are to a large extent insensitive to differences in local matrix distribution. This emphasizes the need for caution in the visual interpretation of tissue functionality from histology and underlines the importance of complementary measurements of the matrix's intrinsic molecular organization.
Lab Meeting - September 24, 2010
Efficient cell migration requires global chromatin condensationGerlitz G, Bustin M
Cell migration is a fundamental process that is necessary for the development and survival of multicellular organisms. Here, we show that cell migration is contingent on global condensation of the chromatin fiber. Induction of directed cell migration by the scratch-wound assay leads to decreased DNaseI sensitivity, alterations in the chromatin binding of architectural proteins and elevated levels of H4K20me1, H3K27me3 and methylated DNA. All these global changes are indicative of increased chromatin condensation in response to induction of directed cell migration. Conversely, chromatin decondensation inhibited the rate of cell migration, in a transcription-independent manner. We suggest that global chromatin condensation facilitates nuclear movement and reshaping, which are important for cell migration. Our results support a role for the chromatin fiber that is distinct from its known functions in genetic processes.
CarTE - September 15, 2010
The restoration of full-thickness cartilage defects with BMSCs and TGF-beta 1 loaded PLGA/fibrin gel constructsWang W, Li B, Yang J, Xin L, Li Y, Yin H, Qi Y, Jiang Y, Ouyang H, Gao C
Poly(lactide-co-glycolide) (PLGA) sponge was filled with fibrin gel, bone marrow mesenchymal stem cells (BMSCs) and transforming growth factor-ß1 (TGF-ß1) to obtain a construct for cartilage restoration in vivo. The PLGA sponge lost its weight steadily in vitro, but degraded much faster in the construct of PLGA/fibrin gel/BMSCs implanted in the full-thickness cartilage defects. The in vivo degradation of the fibrin gel inside the construct was prolonged to 12 wk too. The CM-DiI labeled allogenic BMSCs were detectable after transplantation (implantation) into the defects for 12 wk by small animal in vivo fluorescence imaging and confocal laser scanning microscopy. In vivo repair experiments were firstly performed by implantation of the PLGA/fibrin gel/BMSCs and PLGA/BMSCs constructs into full-thickness cartilage defects (3 mm in diameter and 4 mm in depth) of New Zealand white rabbits for 12 wk. The defects implanted with the PLGA/fibrin gel/BMSCs constructs were filled with cartilage-like tissue containing collagen type II and glycosaminoglycans (GAGs), while those by the PLGA/BMSCs constructs were filled with fibrous-like tissues. To repair the defects of larger size (4 mm in diameter), addition of growth factors was mandatory as exemplified here by further loading of TGF-ß1. Implantation of the PLGA/fibrin gel/BMSCs/TGF-ß1 constructs into the full-thickness cartilage defects for 12 wk resulted in full restoration of the osteochondral tissue. The neo-cartilage integrated well with its surrounding cartilage and subchondral bone. Immunohistochemical and GAGs staining confirmed the similar distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that the cartilage special genes were significantly up-regulated compared with those of the TGF-ß1 absent constructs.
CarTE - September 1, 2010
Impact of growth factors and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cellsWeiss S, Hennig T, Bock R, Steck E, Richter W
Common in vitro protocols for chondrogenesis of mesenchymal stem cells (MSCs) induce an inadequate, hypertrophic differentiation cascade reminiscent of endochondral bone formation. We aimed to modify chondrogenic protocols in order to identify potent inducers, promotors, and inhibitors to achieve better chondrogenesis. Nine factors suspected to stimulate or inhibit chondrogenesis were used for chondrogenic in vitro induction of MSC. Differentiation was assessed by immunohistochemistry, alcian-blue staining, qRT-PCR, and quantification of alkaline phosphatase (ALP) activity. Pre-differentiated pellets were transplanted subcutaneously into SCID mice to investigate stable cartilage formation. Transforming growth factor (TGF)-beta was always required for chondrogenic differentiation and deposition of a collagen-type-II-positive extracellular matrix, while bone morphogenetic protein (BMP)-2, -4, -6, -7, aFGF, and IGF-I (10 ng/ml) were alone not sufficiently inductive. Each of these factors allowed differentiation in combination with TGF-beta, however, without preventing collagen type X expression. bFGF or parathyroid hormone-like peptide (PTHrP) inhibited the TGF-beta-responsive COL2A1 and COL10A1 expression and ALP induction when added from day 0 or 21. In line with a reversible ALP inhibition, in vivo calcification of pellets was not prevented. Late up-regulation of PTH1R mRNA suggests that early PTHrP effects may be mediated by a receptor-independent pathway. While TGF-beta was a full inducer, bFGF and PTHrP were potent inhibitors for early and late chondrogenesis, seemed to induce a shift from matrix anabolism to catabolism, but did not selectively suppress COL10A1 expression. Within a developmental window of collagen type II(+)/collagen type X(-) cells, bFGF and PTHrP may allow inhibition of further differentiation toward hypertrophy to obtain stable chondrocytes for transplantation purposes.
Enhanced biochemical and biomechanical properties of scaffolds generated by flock technology for cartilage tissue engineering
Steck E, Bertram H, Walther A, Brohm K, Mrozik B, Rathmann M, Merle C, Gelinsky M, Richter W
Natural cartilage shows column orientation of cells and anisotropic direction of collagen fibers. However, matrices presently used in matrix assisted autologous chondrocyte implantation do not show any fiber orientation. Our aim was to develop anisotropic scaffolds with parallel fiber orientation that were capable to support a cellular cartilaginous phenotype in vitro. Scaffolds were created by flock technology and consisted of a membrane of mineralized collagen-type-I as substrate, gelatine as adhesive, and parallel oriented polyamide flock fibers vertically to the substrate. Confocal laser-scan microscopy demonstrated that mesenchymal stem cells (MSC) adhered and proliferated well in the scaffolds and cell vitality remained high over time. Articular chondrocytes seeded in a collagen-type-I gel into flock scaffolds deposited increasing amounts of proteoglycans and collagen-type-II over time. MSC-seeded flock scaffold constructs under chondrogenic conditions deposited significantly more proteoglycans and collagen-type-II compared to MSC collagen-type-I-gel-constructs only. Biomechanical testing revealed higher initial hardness of flock scaffolds compared to a clinically applied collagen-type-I/III scaffold combined with superior relaxation and an increasing hardness in MSC loaded flock biocomposites during chondrogenesis. In conclusion, flock technology allows fabrication of scaffolds with anisotropic fiber orientation which mediate superior biomechanical and biochemical composition of tissue engineering constructs for cartilage repair.
Lab Meeting - July 16, 2010
Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fateHuebsch N, Arany PR, Mao AS, Shvartsman D, Ali OA, Bencherif SA, Rivera-Feliciano J, Mooney DJ
Stem cells sense and respond to the mechanical properties of the extracellular matrix. However, both the extent to which extracellular-matrix mechanics affect stem-cell fate in three-dimensional microenvironments and the underlying biophysical mechanisms are unclear. We demonstrate that the commitment of mesenchymal stem-cell populations changes in response to the rigidity of three-dimensional microenvironments, with osteogenesis occurring predominantly at 11-30 kPa. In contrast to previous two-dimensional work, however, cell fate was not correlated with morphology. Instead, matrix stiffness regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction dependent and correlated with osteogenic commitment of mesenchymal stem-cell populations. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion-ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feed back to manipulate their fate.
FRiTE - July 8, 2010
Cytoskeleton-based forecasting of stem cell lineage fatesTreiser MD, Yang EH, Gordonov S, Cohen DM, Androulakis IP, Kohn J, Chen CS, Moghe PV
Stem cells that adopt distinct lineages cannot be distinguished based on traditional cell shape. This study reports that higher-order variations in cell shape and cytoskeletal organization that occur within hours of stimulation forecast the lineage commitment fates of human mesenchymal stem cells (hMSCs). The unique approach captures numerous early (24 h), quantitative features of actin fluororeporter shapes, intensities, textures, and spatial distributions (collectively termed morphometric descriptors). The large number of descriptors are reduced into "combinations" through which distinct subpopulations of cells featuring unique combinations are identified. We demonstrate that hMSCs cultured on fibronectin-treated glass substrates under environments permissive to bone lineage induction could be readily discerned within the first 24 h from those cultured in basal- or fat-inductive conditions by such cytoskeletal feature groupings. We extend the utility of this approach to forecast osteogenic stem cell lineage fates across a series of synthetic polymeric materials of diverse physicochemical properties. Within the first 24 h following stem cell seeding, we could successfully "profile" the substrate responsiveness prospectively in terms of the degree of bone versus nonbone predisposition. The morphometric methodology also provided insights into how substrates may modulate the pace of osteogenic lineage specification. Cells on glass substrates deficient in fibronectin showed a similar divergence of lineage fates, but delayed beyond 48 h. In summary, this high-content imaging and single cell modeling approach offers a framework to elucidate and manipulate determinants of stem cell behaviors, as well as to screen stem cell lineage modulating materials and environments.
FRiTE - June 10, 2010
A mechanical model of actin stress fiber formation and substrate elasticity sensing in adherent cellsWalcott S, Sun SX
Tissue cells sense and respond to the stiffness of the surface on which they adhere. Precisely how cells sense surface stiffness remains an open question, though various biochemical pathways are critical for a proper stiffness response. Here, based on a simple mechanochemical model of biological friction, we propose a model for cell mechanosensation as opposed to previous more biochemically based models. Our model of adhesion complexes predicts that these cell-surface interactions provide a viscous drag that increases with the elastic modulus of the surface. The force-velocity relation of myosin II implies that myosin generates greater force when the adhesion complexes slide slowly. Then, using a simple cytoskeleton model, we show that an external force applied to the cytoskeleton causes actin filaments to aggregate and orient parallel to the direction of force application. The greater the external force, the faster this aggregation occurs. As the steady-state probability of forming these bundles reflects a balance between the time scale of bundle formation and destruction (because of actin turnover), more bundles are formed when the cytoskeleton time-scale is small (i.e., on stiff surfaces), in agreement with experiment. As these large bundles of actin, called stress fibers, appear preferentially on stiff surfaces, our mechanical model provides a mechanism for stress fiber formation and stiffness sensing in cells adhered to a compliant surface.
Lab Meeting - June 4, 2010
Lamin A/C deficiency causes defective nuclear mechanics and mechanotransductionLammerding J, Schulze PC, Takahashi T, Kozlov S, Sullivan T, Kamm RD, Stewart CL, Lee RT
Mutations in the lamin A/C gene (LMNA) cause a variety of human diseases including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. The tissue-specific effects of lamin mutations are unclear, in part because the function of lamin A/C is incompletely defined, but the many muscle-specific phenotypes suggest that defective lamin A/C could increase cellular mechanical sensitivity. To investigate the role of lamin A/C in mechanotransduction, we subjected lamin A/C-deficient mouse embryo fibroblasts to mechanical strain and measured nuclear mechanical properties and strain-induced signaling. We found that Lmna-/- cells have increased nuclear deformation, defective mechanotransduction, and impaired viability under mechanical strain. NF-kappaB-regulated transcription in response to mechanical or cytokine stimulation was attenuated in Lmna-/- cells despite increased transcription factor binding. Lamin A/C deficiency is thus associated with both defective nuclear mechanics and impaired mechanically activated gene transcription. These findings suggest that the tissue-specific effects of lamin A/C mutations observed in the laminopathies may arise from varying degrees of impaired nuclear mechanics and transcriptional activation.
FRiTE - May 27, 2010
Nanoconfinement controls stiffness, strength and mechanical toughness of beta-sheet crystals in silkKeten S, Xu Z, Ihle B, Buehler MJ
Silk features exceptional mechanical properties such as high tensile strength and great extensibility, making it one of the toughest materials known. The exceptional strength of silkworm and spider silks, exceeding that of steel, arises from beta-sheet nanocrystals that universally consist of highly conserved poly-(Gly-Ala) and poly-Ala domains. This is counterintuitive because the key molecular interactions in beta-sheet nanocrystals are hydrogen bonds, one of the weakest chemical bonds known. Here we report a series of large-scale molecular dynamics simulations, revealing that beta-sheet nanocrystals confined to a few nanometres achieve higher stiffness, strength and mechanical toughness than larger nanocrystals. We illustrate that through nanoconfinement, a combination of uniform shear deformation that makes most efficient use of hydrogen bonds and the emergence of dissipative molecular stick-slip deformation leads to significantly enhanced mechanical properties. Our findings explain how size effects can be exploited to create bioinspired materials with superior mechanical properties in spite of relying on mechanically inferior, weak hydrogen bonds.
FRiTE - May 13, 2010
Restriction of receptor movement alters cellular response: physical force sensing by EphA2Salaita K, Nair PM, Petit RS, Neve RM, Das D, Gray JW, Groves JT
Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction were observed. EphA2 transport can be blocked by physical barriers nanofabricated onto the underlying substrate. This physical reorganization of EphA2 alters the cellular response to ephrin-A1, as observed by changes in cytoskeleton morphology and recruitment of a disintegrin and metalloprotease 10. Quantitative analysis of receptor-ligand spatial organization across a library of 26 mammary epithelial cell lines reveals characteristic differences that strongly correlate with invasion potential. These observations reveal a mechanism for spatio-mechanical regulation of EphA2 signaling pathways.
CarTE - May 12, 2010
HIF1 alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesisAmarilio R, Viukov SV, Sharir A, Eshkar-Oren I, Johnson RS, Zelzer E
During early stages of limb development, the vasculature is subjected to extensive remodeling that leaves the prechondrogenic condensation avascular and, as we demonstrate hereafter, hypoxic. Numerous studies on a variety of cell types have reported that hypoxia has an inhibitory effect on cell differentiation. In order to investigate the mechanism that supports chondrocyte differentiation under hypoxic conditions, we inactivated the transcription factor hypoxia-inducible factor 1alpha (HIF1alpha) in mouse limb bud mesenchyme. Developmental analysis of Hif1alpha-depleted limbs revealed abnormal cartilage and joint formation in the autopod, suggesting that HIF1alpha is part of a mechanism that regulates the differentiation of hypoxic prechondrogenic cells. Dramatically reduced cartilage formation in Hif1alpha-depleted micromass culture cells under hypoxia provided further support for the regulatory role of HIF1alpha in chondrogenesis. Reduced expression of Sox9, a key regulator of chondrocyte differentiation, followed by reduction of Sox6, collagen type II and aggrecan in Hif1alpha-depleted limbs raised the possibility that HIF1alpha regulation of Sox9 is necessary under hypoxic conditions for differentiation of prechondrogenic cells to chondrocytes. To study this possibility, we targeted Hif1alpha expression in micromass cultures. Under hypoxic conditions, Sox9 expression was increased twofold relative to its expression in normoxic condition; this increment was lost in the Hif1alpha-depleted cells. Chromatin immunoprecipitation demonstrated direct binding of HIF1alpha to the Sox9 promoter, thus supporting direct regulation of HIF1alpha on Sox9 expression. This work establishes for the first time HIF1alpha as a key component in the genetic program that regulates chondrogenesis by regulating Sox9 expression in hypoxic prechondrogenic cells.
Lab Meeting - May 7, 2010
Traction dynamics of filopodia on compliant substratesChan CE, Odde DJ
Cells sense the environment's mechanical stiffness to control their own shape, migration, and fate. To better understand stiffness sensing, we constructed a stochastic model of the "motor-clutch" force transmission system, where molecular clutches link F-actin to the substrate and mechanically resist myosin-driven F-actin retrograde flow. The model predicts two distinct regimes: (i) "frictional slippage," with fast retrograde flow and low traction forces on stiff substrates and (ii) oscillatory "load-and-fail" dynamics, with slower retrograde flow and higher traction forces on soft substrates. We experimentally confirmed these model predictions in embryonic chick forebrain neurons by measuring the nanoscale dynamics of single-growth-cone filopodia. Furthermore, we experimentally observed a model-predicted switch in F-actin dynamics around an elastic modulus of 1 kilopascal. Thus, a motor-clutch system inherently senses and responds to the mechanical stiffness of the local environment.
Lab Meeting - April 23, 2010
Multilineage potential of adult human mesenchymal stem cellsPittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR
Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.
FRiTE - April 15, 2010
Repair of meniscal cartilage white zone tears using a stem cell/collagen-scaffold implantPabbruwe MB, Kafienah W, Tarlton JF, Mistry S, Fox DJ, Hollander AP
Injuries to the avascular region of knee meniscal cartilage do not heal spontaneously. To address this problem we have developed a new stem cell/collagen-scaffold implant system in which human adult bone marrow mesenchymal stem cells are seeded onto a biodegradable scaffold that allows controlled delivery of actively dividing cells to the meniscus surface. Sandwich constructs of two white zone ovine meniscus discs with stem cell/collagen-scaffold implant in between were cultured in vitro for 40 days. Histomorphometric analysis revealed superior integration in the stem cell/collagen-scaffold groups compared to the cell-free collagen membrane or untreated controls. The addition of TGF-beta1 to differentiate stem cells to chondrocytes inhibited integration. Biomechanical testing demonstrated a significant 2-fold increase in tensile strength in all constructs using the stem cell/collagen-scaffold compared to control groups after 40 days in culture. Integration was significantly higher when collagen membranes were used that had a more open/spongy structure adjacent to both meniscal cartilage surfaces, whereas a collagen scaffold designed for osteoinduction failed to induce any integration of meniscus. In conclusion, the stem cell/collagen-scaffold implant is a potential therapeutic treatment for the repair of white zone meniscal cartilage tears. Copyright 2009 Elsevier Ltd. All rights reserved.
CARTE - April 14, 2010
Self-assembling peptide hydrogels modulate in vitro chondrogenesis of bovine bone marrow stromal cellsKopesky PW, Vanderploeg EJ, Sandy J, Kurz B, Grodzinsky A
Our objective was to test the hypothesis that self-assembling-peptide hydrogel scaffolds provide cues which enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs). BMSCs were encapsulated within two unique peptide-hydrogel sequences and chondrogenesis was compared to that in agarose hydrogels. BMSCs in all three hydrogels underwent TGF-beta1-mediated chondrogenesis as demonstrated by comparable gene expression and biosynthesis of ECM molecules. Expression of an osteogenic marker was unchanged and an adipogenic marker was suppressed by TGF-beta1 in all hydrogels. Cell proliferation occurred only in the peptide hydrogels, not in agarose; resulting in higher glycosaminoglycan content and more spatially uniform proteoglycan and collagen type II deposition. The G1-positive aggrecan produced in peptide hydrogels was predominantly the full-length species, whereas that in agarose was predominantly the aggrecanase product G1-NITEGE. Unique cell morphologies were observed for BMSCs in each peptide-hydrogel sequence, with extensive cell-cell contact present for both, whereas BMSCs in agarose remained rounded over 21 days in culture. Differences in cell morphology within the two peptide scaffolds may be related to sequence-specific cell adhesion. Taken together, this study demonstrates that self-assembling-peptide hydrogels enhance chondrogenesis compared to agarose as shown by ECM production, DNA content and aggrecan molecular structure.
Lab Meeting - April 9, 2010
Emergence of patterned stem cell differentiation within multicellular structuresRuiz SA, Chen CS
The ability of stem cells to differentiate into specified lineages in the appropriate locations is vital to morphogenesis and adult tissue regeneration. Although soluble signals are important regulators of patterned differentiation, here we show that gradients of mechanical forces can also drive patterning of lineages. In the presence of soluble factors permitting osteogenic and adipogenic differentiation, human mesenchymal stem cells at the edge of multicellular islands differentiate into the osteogenic lineage, whereas those in the center became adipocytes. Interestingly, changing the shape of the multicellular sheet modulated the locations of osteogenic versus adipogenic differentiation. Measuring traction forces revealed gradients of stress that preceded and mirrored the patterns of differentiation, where regions of high stress resulted in osteogenesis, whereas stem cells in regions of low stress differentiated to adipocytes. Inhibiting cytoskeletal tension suppressed the relative degree of osteogenesis versus adipogenesis, and this spatial patterning of differentiation was also present in three-dimensional multicellular clusters. These findings demonstrate a role for mechanical forces in linking multicellular organization to spatial differentials of cell differentiation, and they represent an important guiding principle in tissue patterning that could be exploited in stem cell-based therapies. Disclosure of potential conflicts of interest is found at the end of this article.
FRiTE - April 1, 2010
Modulation of anisotropy in electrospun tissue-engineering scaffolds: Analysis of fiber alignment by the fast Fourier transformAyres C, Bowlin GL, Henderson SC, Taylor L, Shultz J, Alexander J, Telemeco TA, Simpson DG
We describe the use of the fast Fourier transform (FFT) in the measurement of anisotropy in electrospun scaffolds of gelatin as a function of the starting conditions. In electrospinning, fiber alignment and overall scaffold anisotropy can be manipulated by controlling the motion of the collecting mandrel with respect to the source electrospinning solution. By using FFT to assign relative alignment values to an electrospun matrix it is possible to systematically evaluate how different processing variables impact the structure and material properties of a scaffold. Gelatin was suspended at varying concentrations (80, 100, 130, 150 mg/ml) and electrospun from 2,2,2 trifluoroethanol onto rotating mandrels (200-7000 RPM). At each starting concentration, fiber diameter remained constant over a wide range of mandrel RPM. Scaffold anisotropy developed as a function of fiber diameter and mandrel RPM. The induction of varying degrees of anisotropy imparted distinctive material properties to the electrospun scaffolds. The FFT is a rapid method for evaluating fiber alignment in tissue-engineering materials.
CARTE - March 17, 2010
Donor sex and age influence the chondrogenic potential of human femoral bone marrow stem cellsPayne KA, Didiano DM, Chu CR
OBJECTIVE: Damaged articular cartilage does not heal well and can progress to osteoarthritis (OA). Human bone marrow stem cells (BMC) are promising cells for articular cartilage repair, yet age- and sex-related differences in their chondrogenesis have not been clearly identified. The purpose of this study is to test whether the chondrogenic potential of human femoral BMC varies based on the sex and/or age of the donor. DESIGN: BMC were isolated from 21 males (16-82years old (y.o.)) and 20 females (20-77y.o.) during orthopaedic procedures. Cumulative population doubling (CPD) was measured and chondrogenesis was evaluated by standard pellet culture assay in the presence or absence of transforming growth factor beta 1 (TGFbeta1). Pellet area was measured, and chondrogenic differentiation was determined by Toluidine blue and Safranin O-Fast green histological grading using the Bern score and by glycosaminoglycan (GAG) content. RESULTS: No difference in CPD was observed due to donor sex or age. The increase in pellet area with addition of TGFbeta1 and the Bern score significantly decreased with increasing donor age in male BMC, but not in female BMC. A significant reduction in GAG content per pellet was also observed with increasing donor age in male BMC. This was not observed in female BMC. CONCLUSIONS: This study showed an age-related decline in chondroid differentiation with TGFbeta1 stimulation in male BMC, but not in female BMC. Understanding the mechanisms for these differences will contribute to improved clinical use of autologous BMC for articular cartilage repair, and may lead to the development of customized age- or sex-based treatments to delay or prevent the onset of OA.
CARTE - March 3, 2010
Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP profile and is overcome by BMP-6Hennig T, Lorenz H, Thiel A, Goetzke K, Dickhut A, Geiger F, Richter W
Recent interest has focused on mesenchymal stem cells (MSC) for tissue engineering and regenerative therapy of cartilage defects. MSC originating from adipose tissue (ATSC) are attractive as they are easily available and abundant. They have similar properties like bone marrow derived MSC (BMSC), except for a reduced chondrogenic potential under standard culture conditions driven by TGFbeta. Aim of this study was to search for possible differences explaining the reduced differentiation capacity of ATSC and to eliminate it by adaptation of induction protocols. Expanded MSC were analyzed for their growth factor and related receptor repertoire and ATSC spheroid cultures were supplemented with BMP-2,-4,-6,-7, TGFbeta, FGFa, FGFb, IGF-1, and PTHrP alone or in combination with TGFbeta. In contrast to BMSC, ATSC showed reduced expression of BMP-2, -4, and -6 mRNA and did not express TGFbeta-receptor-I protein. Consistent with this, increased concentrations of TGFbeta did not improve chondrogenesis of ATSC. BMP6 treatment induced TGFbeta-receptor-I expression and combined application of TGFbeta and BMP-6 eliminated the reduced chondrogenic potential of ATSC inducing a gene expression profile similar to differentiated BMSC. Like in BMSC, chondrogenesis of ATSC was associated with hypertrophy according to premature collagen Type X expression, upregulation of alkaline-phosphatase activity and in vivo calcification of spheroids after ectopic transplantation in SCID mice. In conclusion, a distinct BMP and TGFbeta-receptor repertoire may explain the reduced chondrogenic capacity of ATSC in vitro, which could be compensated by exogenous application of lacking factors. Further studies should now be directed to induce chondrogenesis in the absence of hypertrophy.
FRiTE - February 18, 2010
Cooperative effects of Rho and mechanical stretch on stress fiber organizationKaunas R, Nguyen P, Usami S, Chien S
The small GTPase Rho regulates the formation of actin stress fibers in adherent cells through activation of its effector proteins Rho-kinase and mDia. We found in bovine aortic endothelial cells that inhibitions of Rho, Rho-kinase, and mDia (with C3, Y27632, and F1F2Delta1, respectively) suppressed stress fiber formation, but fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency). In contrast to the predominately perpendicular alignment of stress fibers to the stretch direction in normal cells, the stress fibers in cells with Rho pathway inhibition became oriented parallel to the stretch direction. In cells with normal Rho activity, the extent of perpendicular orientation of stress fibers depended on the magnitude of stretch. Expressing active RhoV14 plasmid in these cells enhanced the stretch-induced stress fiber orientation by an extent equivalent to an additional approximately 3% stretch. This augmentation of the stretch-induced perpendicular orientation by RhoV14 was blocked by Y27632 and by F1F2Delta1. Thus, the activity of the Rho pathway plays a critical role in determining both the direction and extent of stretch-induced stress fiber orientation in bovine aortic endothelial cells. Our results demonstrate that the stretch-induced stress fiber orientation is a function of the interplay between Rho pathway activity and the magnitude of stretching.
CARTE - February 17, 2010
Zonal changes in the three-dimensional morphology of the chondron under compression: the relationship among cellular, pericellular, and extracellular deformation in articular cartilageChoi JB, Youn I, Cao L, Leddy HA, Gilchrist CL, Setton LA, Guilak F
The pericellular matrix (PCM) is a narrow region of tissue that completely surrounds chondrocytes in articular cartilage. Previous theoretical models of the "chondron" (the PCM with enclosed cells) suggest that the structure and properties of the PCM may significantly influence the mechanical environment of the chondrocyte. The objective of this study was to quantify changes in the three-dimensional (3D) morphology of the chondron in situ at different magnitudes of compression applied to the cartilage extracellular matrix. Fluorescence immunolabeling for type-VI collagen was used to identify the boundaries of the cell and PCM, and confocal microscopy was used to form 3D images of chondrons from superficial, middle, and deep zone cartilage in explants compressed to 0%, 10%, 30%, and 50% surface-to-surface strain. Lagrangian tissue strain, determined locally using texture correlation, was highly inhomogeneous and revealed depth-dependent compressive stiffness and Poisson's ratio of the extracellular matrix. Compression significantly decreased cell and chondron height and volume, depending on the zone and magnitude of compression. In the superficial zone, cellular-level strains were always lower than tissue-level strains. In the middle and deep zones, however, tissue strains below 25% were amplified at the cellular level, while tissue strains above 25% were decreased at the cellular level. These findings are consistent with previous theoretical models of the chondron, suggesting that the PCM can serve as either a protective layer for the chondrocyte or a transducer that amplifies strain, such that cellular-level strains are more homogenous throughout the tissue depth despite large inhomogeneities in local ECM strains.
Lab Meeting - February 12, 2010
Muscle contraction is necessary to maintain joint progenitor cell fateKahn J, Shwartz Y, Blitz E, Krief S, Sharir A, Breitel DA, Rattenbach R, Relaix F, Maire P, Rountree RB, Kingsley DM, Zelzer E
During embryogenesis, organ development is dependent upon maintaining appropriate progenitor cell commitment. Synovial joints develop from a pool of progenitor cells that differentiate into various cell types constituting the mature joint. The involvement of the musculature in joint formation has long been recognized. However, the mechanism by which the musculature regulates joint formation has remained elusive. In this study, we demonstrate, utilizing various murine models devoid of limb musculature or its contraction, that the contracting musculature is fundamental in maintaining joint progenitors committed to their fate, a requirement for correct joint cavitation and morphogenesis. Furthermore, contraction-dependent activation of beta-catenin, a key modulator of joint formation, provides a molecular mechanism for this regulation. In conclusion, our findings provide the missing link between progenitor cell fate determination and embryonic movement, two processes shown to be essential for correct organogenesis.
FRiTE - February 4, 2010
Inhibition of matrix metalloproteinases enhances in vitro repair of the meniscusMcNulty AL, Weinberg JB, Guilak F
Damage or injury of the meniscus is associated with onset and progression of knee osteoarthritis (OA). The intrinsic repair capacity of the meniscus is inhibited by inflammatory cytokines, such as interleukin-1 (IL-1). Using an in vitro meniscal repair model system, we examined the hypothesis that inhibition of matrix metalloproteinases (MMPs) in the presence of IL-1 will enhance repair of meniscal lesions. Integrative repair of the meniscus was examined between two concentric explants cultured with IL-1 and various MMP inhibitors for 14 days. Throughout the culture period, we assessed total specific MMP activity in the media. At harvest, biomechanical testing to assess the strength of repair and histologic staining were performed. IL-1 decreased the shear strength of repair, as compared with control explants. In the presence of IL-1, the broad-spectrum MMP inhibitor GM 6001 decreased the MMP activity in the media, increased the shear strength of repair, and enhanced tissue repair in the interface. However, individual MMP inhibitors did not alter the shear strength of repair in either the presence or absence of IL-1. These findings suggest IL-1 may inhibit meniscal repair through upregulation of MMPs, but inhibition of multiple MMPs may be necessary to promote integrative meniscal repair.
CARTE - February 3, 2010
The role of hydrogel structure and dynamic loading on chondrocyte gene expression and matrix formationNicodemus GD, Bryant SJ
Crosslinked poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for cartilage tissue engineering because of their ability to mimic the aqueous environment and mechanical properties of native cartilage. In this study, hydrogel crosslinking density was varied to study the influence of gel structure and the application of dynamic loading (continuous, 1 Hz, 15% amplitude strain) on chondrocyte gene expression over approximately 1 week culture. Gene expression was quantified using real-time RT-PCR for collagen II and aggrecan, the major cartilage extracellular matrix (ECM) components, and collagen I, an indicator of chondrocyte de-differentiation. When chondrocytes were encapsulated in PEG gels with low or high crosslinking, a high collagen II expression compared to collagen I expression (1000 or 100,000:1, respectively) indicated the native chondrocyte phenotype was retained. In the absence of loading, relative gene expression for collagen II and aggrecan was significantly higher (e.g., 2-fold and 4-fold, respectively, day 7) in the low crosslinked gels compared to gels with higher crosslinking. Dynamic loading, however, showed little effect on ECM gene expression in both crosslinked systems. To better understand the cellular environment, ECM production was qualitatively assessed using an in situ immunofluorescent technique and standard histology. A pericellular matrix (PCM) was observed as early as day 3 post-encapsulation and the degree of formation was dependent on gel crosslinking. These results suggest the PCM may protect the cells from sensing the applied loads. This study demonstrates that gel structure has a profound effect on chondrocyte gene expression, while dynamic loading has much less of an effect at early culture times.
Lab Meeting - January 29, 2010
Photodegradable hydrogels for dynamic tuning of physical and chemical propertiesKloxin AM, Kasko AM, Salinas CN, Anseth KS
We report a strategy to create photodegradable poly(ethylene glycol)-based hydrogels through rapid polymerization of cytocompatible macromers for remote manipulation of gel properties in situ. Postgelation control of the gel properties was demonstrated to introduce temporal changes, creation of arbitrarily shaped features, and on-demand pendant functionality release. Channels photodegraded within a hydrogel containing encapsulated cells allow cell migration. Temporal variation of the biochemical gel composition was used to influence chondrogenic differentiation of encapsulated stem cells. Photodegradable gels that allow real-time manipulation of material properties or chemistry provide dynamic environments with the scope to answer fundamental questions about material regulation of live cell function and may affect an array of applications from design of drug delivery vehicles to tissue engineering systems.
FRiTE - January 21, 2010
Material properties of the cell dictate stress-induced spreading and differentiation in embryonic stem cellsChowdhury F, Na S, Li D, Poh YC, Tanaka TS, Wang F, Wang N
Growing evidence suggests that physical microenvironments and mechanical stresses, in addition to soluble factors, help direct mesenchymal-stem-cell fate. However, biological responses to a local force in embryonic stem cells remain elusive. Here we show that a local cyclic stress through focal adhesions induced spreading in mouse embryonic stem cells but not in mouse embryonic stem-cell-differentiated cells, which were ten times stiffer. This response was dictated by the cell material property (cell softness), suggesting that a threshold cell deformation is the key setpoint for triggering spreading responses. Traction quantification and pharmacological or shRNA intervention revealed that myosin II contractility, F-actin, Src or cdc42 were essential in the spreading response. The applied stress led to oct3/4 gene downregulation in mES cells. Our findings demonstrate that cell softness dictates cellular sensitivity to force, suggesting that local small forces might have far more important roles in early development of soft embryos than previously appreciated.
CARTE - January 20, 2010
Developmental and osteoarthritic changes in Col6a1-knockout mice: Biomechanics of type VI collagen in the cartilage pericellular matrixAlexopoulos LG, Youn I, Bonaldo P, Guilak F
OBJECTIVE: Chondrocytes, the sole cell type in articular cartilage, maintain the extracellular matrix (ECM) through a homeostatic balance of anabolic and catabolic activities that are influenced by genetic factors, soluble mediators, and biophysical factors such as mechanical stress. Chondrocytes are encapsulated by a narrow tissue region termed the "pericellular matrix" (PCM), which in normal cartilage is defined by the exclusive presence of type VI collagen. Because the PCM completely surrounds each cell, it has been hypothesized that it serves as a filter or transducer for biochemical and/or biomechanical signals from the cartilage ECM. The present study was undertaken to investigate whether lack of type VI collagen may affect the development and biomechanical function of the PCM and alter the mechanical environment of chondrocytes during joint loading. METHODS: Col6a1(-/-) mice, which lack type VI collagen in their organs, were generated for use in these studies. At ages 1, 3, 6, and 11 months, bone mineral density (BMD) was measured, and osteoarthritic (OA) and developmental changes in the femoral head were evaluated histomorphometrically. Mechanical properties of articular cartilage from the hip joints of 1-month-old Col6a1(-/-), Col6a1(+/-), and Col6a1(+/+) mice were assessed using an electromechanical test system, and mechanical properties of the PCM were measured using the micropipette aspiration technique. RESULTS: In Col6a1(-/-) and Col6a1(+/-) mice the PCM was structurally intact, but exhibited significantly reduced mechanical properties as compared with wild-type controls. With age, Col6a1(-/-) mice showed accelerated development of OA joint degeneration, as well as other musculoskeletal abnormalities such as delayed secondary ossification and reduced BMD. CONCLUSION: These findings suggest that type VI collagen has an important role in regulating the physiology of the synovial joint and provide indirect evidence that alterations in the mechanical environment of chondrocytes, due to either loss of PCM properties or Col6a1(-/-)-derived joint laxity, can lead to progression of OA.
Lab Meeting - December 4, 2009
Transcriptome-wide noise controls lineage choice in mammalian progenitor cellsChang HH, Hemberg M, Barahona M, Ingber DE, Huang S
Phenotypic cell-to-cell variability within clonal populations may be a manifestation of 'gene expression noise', or it may reflect stable phenotypic variants. Such 'non-genetic cell individuality' can arise from the slow fluctuations of protein levels in mammalian cells. These fluctuations produce persistent cell individuality, thereby rendering a clonal population heterogeneous. However, it remains unknown whether this heterogeneity may account for the stochasticity of cell fate decisions in stem cells. Here we show that in clonal populations of mouse haematopoietic progenitor cells, spontaneous 'outlier' cells with either extremely high or low expression levels of the stem cell marker Sca-1 (also known as Ly6a) reconstitute the parental distribution of Sca-1 but do so only after more than one week. This slow relaxation is described by a gaussian mixture model that incorporates noise-driven transitions between discrete subpopulations, suggesting hidden multi-stability within one cell type. Despite clonality, the Sca-1 outliers had distinct transcriptomes. Although their unique gene expression profiles eventually reverted to that of the median cells, revealing an attractor state, they lasted long enough to confer a greatly different proclivity for choosing either the erythroid or the myeloid lineage. Preference in lineage choice was associated with increased expression of lineage-specific transcription factors, such as a >200-fold increase in Gata1 among the erythroid-prone cells, or a >15-fold increased PU.1 (Sfpi1) expression among myeloid-prone cells. Thus, clonal heterogeneity of gene expression level is not due to independent noise in the expression of individual genes, but reflects metastable states of a slowly fluctuating transcriptome that is distinct in individual cells and may govern the reversible, stochastic priming of multipotent progenitor cells in cell fate decision.
Lab Meeting - October 30, 2009
Mechanism of mRNA transport in the nucleusVargas DY, Raj A, Marras SA, Kramer FR, Tyagi S
The mechanism of transport of mRNA-protein (mRNP) complexes from transcription sites to nuclear pores has been the subject of many studies. Using molecular beacons to track single mRNA molecules in living cells, we have characterized the diffusion of mRNP complexes in the nucleus. The mRNP complexes move freely by Brownian diffusion at a rate that assures their dispersion throughout the nucleus before they exit into the cytoplasm, even when the transcription site is located near the nuclear periphery. The diffusion of mRNP complexes is restricted to the extranucleolar, interchromatin spaces. When mRNP complexes wander into dense chromatin, they tend to become stalled. Although the movement of mRNP complexes occurs without the expenditure of metabolic energy, ATP is required for the complexes to resume their motion after they become stalled. This finding provides an explanation for a number of observations in which mRNA transport appeared to be an enzymatically facilitated process.
FT-IR imaging of native and tissue-engineered bone and cartilage
Boskey A, Pleshko Camacho N
Fourier transform infrared (FT-IR) imaging and microspectroscopy have been extensively applied to the analyses of tissues in health and disease. Spatially resolved mid-IR data has provided insights into molecular changes that occur in diseases of connective or collagen-based tissues, including, osteoporosis, osteogenesis imperfecta, osteopetrosis and pathologic calcifications. These techniques have also been used to probe chemical changes associated with load, disuse, and micro-damage in bone, and with degradation and repair in cartilage. This review summarizes the applications of FT-IR microscopy and imaging for analyses of bone and cartilage in healthy and diseased tissues, and illustrates the application of these techniques for the characterization of tissue-engineered bone and cartilage.
FRiTE - October 22, 2009
Measurement of local strains in intervertebral disc anulus fibrosus tissue under dynamic shear: contributions of matrix fiber orientation and elastin contentMichalek AJ, Buckley MR, Bonassar LJ, Cohen I, Iatridis JC
Shear strain has been implicated as an initiator of intervertebral disc anulus failure, however a clear, multi-scale picture of how shear strain affects the tissue microstructure has been lacking. The purposes of this study were to measure microscale deformations in anulus tissue under dynamic shear in two orientations, and to determine the role of elastin in regulating these deformations. Bovine AF tissue was simultaneously shear loaded and imaged using confocal microscopy following either a buffer or elastase treatment. Digital image analysis was used to track through time local shear strains in specimens sheared transversely, and stretch and rotation of collagen fiber bundles in specimens sheared circumferentially. The results of this study suggest that sliding does not occur between AF plies under shear, and that interlamellar connections are governed by collagen and fibrilin rather than elastin. The transverse shear modulus was found to be approximately 1.6 times as high in plies the direction of the collagen fibers as in plies across them. Under physiological levels of in-plane shear, fiber bundles stretched and re-oriented linearly. Elastin was found to primarily stiffen plies transversely. We conclude that alterations in the elastic fiber network, as found with IVD herniation and degeneration, can therefore be expected to significantly influence the AF response to shear making it more susceptible to micro failure under bending or torsion loading.
Lab Meeting - October 5, 2009
Physical plasticity of the nucleus in stem cell differentiationPajerowski JD, Dahl KN, Zhong FL, Sammak PJ, Discher DE
Cell differentiation in embryogenesis involves extensive changes in gene expression structural reorganization within the nucleus, including chromatin condensation and nucleoprotein immobilization. We hypothesized that nuclei in naive stem cells would therefore prove to be physically plastic and also more pliable than nuclei in differentiated cells. Micromanipulation methods indeed show that nuclei in human embryonic stem cells are highly deformable and stiffen 6-fold through terminal differentiation, and that nuclei in human adult stem cells possess an intermediate stiffness and deform irreversibly. Because the nucleo-skeletal component Lamin A/C is not expressed in either type of stem cell, we knocked down Lamin A/C in human epithelial cells and measured a deformability similar to that of adult hematopoietic stem cells. Rheologically, lamin-deficient states prove to be the most fluid-like, especially within the first approximately 10 sec of deformation. Nuclear distortions that persist longer than this are irreversible, and fluorescence-imaged microdeformation with photobleaching confirms that chromatin indeed flows, distends, and reorganizes while the lamina stretches. The rheological character of the nucleus is thus set largely by nucleoplasm/chromatin, whereas the extent of deformation is modulated by the lamina.
FRiTE - September 24, 2009
Composite polymer systems with control of local substrate elasticity and their effect on cytoskeletal and morphological characteristics of adherent cellsChou SY, Cheng CM, LeDuc PR
At the interface between extracellular substrates and biological materials, substrate elasticity strongly influences cell morphology and function. The associated biological ramifications comprise a diversity of critical responses including apoptosis, differentiation, and motility, which can affect medical devices such as stents. The interactions of the extracellular environment with the substrate are also affected by local properties wherein cells sense and respond to different physical inputs. To investigate the effects of having localized elasticity control of substrate microenvironments on cell response, we have developed a method to control material interface interactions with cells by dictating local substrate elasticity. This system is created by generating a composite material system with alternating, linear regions of polymers that have distinct stiffness characteristics. This approach was used to examine cytoskeletal and morphological changes in NIH 3T3 fibroblasts with emphasis on both local and global properties, noting that cells sense and respond to distinct material elasticities. Isolated cells sense and respond to these local differences in substrate elasticity by extending processes along the interface. Also, cells grown on softer elastic regions at higher densities (in contact with each other) have a higher projected area than isolated cells. Furthermore, when using chemical agents such as cytochalasin-D to disrupt the actin cytoskeleton, there is a significant increase in projected area for cells cultured on softer elastic regions This method has the potential to promote understanding of biomaterial-affected responses in a diversity of areas including morphogenesis, mechanotransduction, stents, and stem cell differentiation.
Lab Meeting - September 18, 2009
Microfabricated tissue gauges to measure and manipulate forces from 3D microtissuesLegant WR, Pathak A, Yang MT, Deshpande VS, McMeeking RM, Chen CS
Physical forces generated by cells drive morphologic changes during development and can feedback to regulate cellular phenotypes. Because these phenomena typically occur within a 3-dimensional (3D) matrix in vivo, we used microelectromechanical systems (MEMS) technology to generate arrays of microtissues consisting of cells encapsulated within 3D micropatterned matrices. Microcantilevers were used to simultaneously constrain the remodeling of a collagen gel and to report forces generated during this process. By concurrently measuring forces and observing matrix remodeling at cellular length scales, we report an initial correlation and later decoupling between cellular contractile forces and changes in tissue morphology. Independently varying the mechanical stiffness of the cantilevers and collagen matrix revealed that cellular forces increased with boundary or matrix rigidity whereas levels of cytoskeletal and extracellular matrix (ECM) proteins correlated with levels of mechanical stress. By mapping these relationships between cellular and matrix mechanics, cellular forces, and protein expression onto a bio-chemo-mechanical model of microtissue contractility, we demonstrate how intratissue gradients of mechanical stress can emerge from collective cellular contractility and finally, how such gradients can be used to engineer protein composition and organization within a 3D tissue. Together, these findings highlight a complex and dynamic relationship between cellular forces, ECM remodeling, and cellular phenotype and describe a system to study and apply this relationship within engineered 3D microtissues.
FRiTE - July 16, 2009
Tension is required for fibripositor formationKapacee Z, Richardson SH, Lu Y, Starborg T, Holmes DF, Baar K, Kadler KE
Embryonic tendon cells (ETCs) have actin-rich fibripositors that accompany parallel bundles of collagen fibrils in the extracellular matrix. To study fibripositor function, we have developed a three-dimensional cell culture system that promotes and maintains fibripositors. We show that ETCs cultured in fixed-length fibrin gels replace the fibrin during ~6 days in culture with parallel bundles of narrow-diameter collagen fibrils that are uniaxially aligned with fibripositors, thereby generating a tendon-like construct. Fibripositors occurred simultaneously with onset of parallel collagen fibrils. Interestingly, the constructs have a tendon-like crimp. In initial experiments to study the effects of tension, we showed that cutting the constructs resulted in loss of tension, loss of fibripositors and the appearance of immature fibrils with no preferred orientation.
CARTE - July 14, 2009
Catabolic responses of chondrocyte-seeded peptide hydrogel to dynamic compressionKisiday JD, Lee JH, Siparsky PN, Frisbie DD, Flannery CR, Sandy JD, Grodzinsky AJ
This study investigated the role of matrix metalloproteases and aggrecanases during dynamic compression-induced aggrecan catabolism in chondrocyte-seeded self-assembling peptide hydrogel. One- to two-week-old bovine chondrocytes were encapsulated into peptide hydrogel and cultured for 14 days prior to the application of an alternate day loading protocol. Dynamic compression-induced aggrecan catabolism was explored by evaluating GAG loss to the culture medium, zymography for matrix metalloproteases (MMPs), gene expression of MMPs and ADAMTS proteases, and Western blot analysis for aggrecan fragments. The application of loading over 4 days increased GAG loss to the medium three- to four-fold relative to free-swelling controls. Zymogram analysis detected increased concentrations of latent MMP-9 and MMP-3 in the culture medium relative to free-swelling culture. Real-time PCR showed expression levels of MMPs and ADAMTS proteases in loaded samples that ranged from 2.5- to 95-fold higher than free-swelling culture. Aggrecan fragment analysis did not detect small (50-80 kDa) molecular weight fragments in free-swelling culture; however, dynamic compression samples contained 60-80 kDa fragments that were detected by both anti-G1 and NITEGE probes, demonstrating ADAMTS but not MMP degradation. These data suggest that partially mature cartilage tissue engineering constructs may be susceptible to catabolic degradation.