Perelman School of Medicine at the University of Pennsylvania

McKay Orthopaedic Research Laboratory

Mauck Laboratory

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.

PubMed Article

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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.

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TransAm – March 25, 2013

Meeting the need for regenerative therapies I: target-based incidence and its relationship to U.S. spending, productivity, and innovation.

PubMed Article

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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.

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TransAm – December 17, 2012

Concentrated bone marrow aspirate improves full-thickness cartilage repair compared with microfracture in the equine model.

PubMed Article

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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.

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Temporal growth factor release from platelet-rich plasma, trehalose lyophilized platelets, and bone marrow aspirate and their effect on tendon and ligament gene expression.

PubMed Article

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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.

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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.

PubMed Article

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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.

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TransAm – October 22, 2012

Osteochondritis Dissecans Knee Histology Studies Have Variable Findings and Theories of Etiology.

PubMed Article

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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.

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Does Extracorporeal Shock Wave Therapy Enhance Healing of Osteochondritis Dissecans of the Rabbit Knee?: A Pilot Study.

PubMed Article

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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.

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CMD – October 10, 2012

ECM stiffness primes the TGFB pathway to promote chondrocyte differentiation.

PubMed Article

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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.

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TransAm – October 8, 2012

Tissue engineering for total meniscal substitution: animal study in sheep model— results at 12 months.

PubMed Article

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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.

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Isolation, characterization, and differentiation of stem cells for cartilage regeneration.

PubMed Article

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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.

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CDM – September 26, 2012

Cell Mechanics, Structure, and Function Are Regulated by the Stiffness of the three-Dimensional Microenvironment.

PubMed Article

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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.

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