Perelman School of Medicine at the University of Pennsylvania

McKay Orthopaedic Research Laboratory

Mauck Laboratory

Readings for Discussion

TransAm – April 2015

Mesenchymal Stem Cell Implantation in Osteoarthritic Knees: Is Fibrin Glue Effective as a Scaffold?

PubMed Article

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Kim YS, Choi YJ, Suh DS, Heo DB, Kim YI, Ryu JS, Koh YG.
BACKGROUND:

The cell-based tissue engineering approach that uses mesenchymal stem cells (MSCs) has addressed the issue of articular cartilage repair in osteoarthritic (OA) knees. However, to improve outcomes, an advanced surgical procedure with tissue-engineered scaffolds may be needed to treat patients with large cartilage lesions.
PURPOSE:

To investigate the clinical and second-look arthroscopic outcomes of the implantation of MSCs loaded in fibrin glue as a scaffold in patients with OA knees and to compare these outcomes with those of MSC implantation without a scaffold.
STUDY DESIGN:

Cohort study; Level of evidence, 3.
METHODS:

This study retrospectively evaluated 54 patients (56 knees) who were examined with second-look arthroscopy after MSC implantation for cartilage lesions in their OA knees. Patients were divided into 2 groups: 37 patients (39 knees) were treated with MSC implantation without a scaffold (group 1), and 17 patients (17 knees) underwent implantation of MSCs loaded in fibrin glue as a scaffold (group 2). Clinical outcomes were evaluated according to the International Knee Documentation Committee (IKDC) score and the Tegner activity scale, and cartilage repair was assessed with the International Cartilage Repair Society (ICRS) grade. Statistical analyses were performed to identify various prognostic factors associated with the clinical and second-look arthroscopic outcomes.
RESULTS:

At final follow-up (mean, 28.6 months; range, 24-34 months), the mean IKDC score and Tegner activity scale in each group significantly improved: group 1, from 38.1±7.7 to 62.0±11.7 (IKDC) and from 2.5±0.9 to 3.5±0.8 (Tegner); group 2, from 36.1±6.2 to 64.4±11.5 (IKDC) and from 2.2±0.8 to 3.8±0.8 (Tegner) (P<.001 for all). According to the overall ICRS cartilage repair grades, 9 of the 39 lesions (23%) in group 1 and 12 of the 17 lesions (58%) in group 2 achieved a grade of I or II. There was a significant difference in ICRS grades between the groups (P=.028). Overweight (body mass index≥27.5 kg/m2) and large lesion size (≥5.7 cm2) were significant predictors of poor clinical and arthroscopic outcomes in group 1 (P<.05 for both). There was a similar trend in group 2, but the differences were not significant, possibly owing to the smaller sample size.
CONCLUSION:

Clinical and arthroscopic outcomes of MSC implantation were encouraging for OA knees in both groups, although there were no significant differences in outcome scores between groups. However, at second-look arthroscopy, there were better ICRS grades in group 2.

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Regulation of dendrimer/dextran material performance by altered tissue microenvironment in
inflammation and neoplasia.

PubMed Article

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Nuria Oliva, Maria Carcole, Margarita Beckerman, Sivan Seliktar, Alison Hayward, James Stanley, Nicola Maria Anne Parry, Elazer R. Edelman, and Natalie Artzi.

A "one material fits all" mindset ignores profound differences in target tissues that affect their responses and reactivity. Yet little attention has been paid to the role of diseased tissue on material performance, biocompatibility, and healing capacity. We assessed material-tissue interactions with a prototypical adhesive material based on dendrimer/dextran and colon as a model tissue platform. Adhesive materials have high sensitivity to changes in their environment and can be exploited to probe and quantify the influence of even subtle modifications in tissue architecture and biology. We studied inflammatory colitis and colon cancer and found not only a difference in adhesion related to surface chemical interactions but also the existence of a complex interplay that determined the overall dendrimer/dextran biomaterial compatibility. Compatibility was contextual, not simply a constitutive property of the material, and was related to the extent and nature of immune cells in the diseased environment present before material implantation. We then showed how to use information about local alterations of the tissue microenvironment to assess disease severity. This in turn guided us to an optimal dendrimer/dextran formulation choice using a predictive model based on clinically relevant conditions.

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TransAm – March 2015

Adverse effects of stromal vascular fraction during regenerative treatment of the intervertebral disc: observations in a goat model.

PubMed Article

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Detiger SE, Helder MN, Smit TH, Hoogendoorn RJ.
Stromal vascular fraction (SVF), an adipose tissue-derived heterogeneous cell mixture containing, among others, multipotent adipose stromal cells (ASCs) and erythrocytes, has proved beneficial for a wide range of applications in regenerative medicine. We sought to establish intervertebral disc (IVD) regeneration by injecting SVF intradiscally during a one-step surgical procedure in an enzymatically (Chondroitinase ABC; cABC) induced goat model of disc degeneration. Unexpectedly, we observed a severe inflammatory response that has not been described before, including massive lymphocyte infiltration, neovascularisation and endplate destruction. A second study investigated two main suspects for these adverse effects: cABC and erythrocytes within SVF. The same destructive response was observed in healthy goat discs injected with SVF, thereby eliminating cABC as a cause. Density gradient removal of erythrocytes and ASCs purified by culturing did not lead to adverse effects. Following these observations, we incorporated an extra washing step in the SVF harvesting protocol. In a third study, we applied this protocol in a one-step procedure to a goat herniation model, in which no adverse responses were observed either. However, upon intradiscal injection of an identically processed SVF mixture into our goat IVD degeneration model during a fourth study, the adverse effects surprisingly occurred again. Despite our quest for the responsible agent, we eventually could not identify the mechanism through which the observed destructive responses occurred. Although we cannot exclude that the adverse effects are species-dependent or model-specific, we advertise caution with the clinical application of autologous SVF injections into the IVD until the responsible agent(s) are identified.

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TransAm – February 2015

Protein-releasing polymeric scaffolds induce fibrochondrocytic differentiation of endogenous cells for knee meniscus regeneration in sheep.

PubMed Article

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Chang H Lee, Scott A Rodeo, Lisa Ann Fortier, Chuanyong Lu, Cevat Erisken, Jeremy J Mao.
Regeneration of complex tissues, such as kidney, liver, and cartilage, continues to be a scientific and translational challenge. Survival of ex vivo cultured, transplanted cells in tissue grafts is among one of the key barriers. Meniscus is a complex tissue consisting of collagen fibers and proteoglycans with gradient phenotypes of fibrocartilage and functions to provide congruence of the knee joint, without which the patient is likely to develop arthritis. Endogenous stem/progenitor cells regenerated the knee meniscus upon spatially released human connective tissue growth factor (CTGF) and transforming growth factor-β3 (TGFβ3) from a three-dimensional (3D)-printed biomaterial, enabling functional knee recovery. Sequentially applied CTGF and TGFβ3 were necessary and sufficient to propel mesenchymal stem/progenitor cells, as a heterogeneous population or as single-cell progenies, into fibrochondrocytes that concurrently synthesized procollagens I and IIα. When released from microchannels of 3D-printed, human meniscus scaffolds, CTGF and TGFβ3 induced endogenous stem/progenitor cells to differentiate and synthesize zone-specific type I and II collagens. We then replaced sheep meniscus with anatomically correct, 3D-printed scaffolds that incorporated spatially delivered CTGF and TGFβ3. Endogenous cells regenerated the meniscus with zone-specific matrix phenotypes: primarily type I collagen in the outer zone, and type II collagen in the inner zone, reminiscent of the native meniscus. Spatiotemporally delivered CTGF and TGFβ3 also restored inhomogeneous mechanical properties in the regenerated sheep meniscus. Survival and directed differentiation of endogenous cells in a tissue defect may have implications in the regeneration of complex (heterogeneous) tissues and organs.

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TransAm – January 2015

A Comparison of the Functionality and In Vivo Phenotypic Stability of Cartilaginous Tissues Engineered from Different Stem Cell Sources.

PubMed Article

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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-β3. 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.

 

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TransAm – December 2014

Developing functional musculoskeletal tissues through hypoxia and lysyl oxidase-induced collagen cross-linking.

PubMed Article

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Makris EA, Responte DJ, Paschos NK, Hu JC, Athanasiou KA.

The inability to recapitulate native tissue biomechanics, especially tensile properties, hinders progress in regenerative medicine. To address this problem, strategies have focused on enhancing collagen production. However, manipulating collagen cross-links, ubiquitous throughout all tissues and conferring mechanical integrity, has been underinvestigated. A series of studies examined the effects of lysyl oxidase (LOX), the enzyme responsible for the formation of collagen cross-links. Hypoxia-induced endogenous LOX was applied in multiple musculoskeletal tissues (i.e., cartilage, meniscus, tendons, ligaments). Results of these studies showed that both native and engineered tissues are enhanced by invoking a mechanism of hypoxia-induced pyridinoline (PYR) cross-links via intermediaries like LOX. Hypoxia was shown to enhance PYR cross-linking 1.4- to 6.4-fold and, concomitantly, to increase the tensile properties of collagen-rich tissues 1.3- to 2.2-fold. Direct administration of exogenous LOX was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOX was found to enhance native tissue tensile properties 1.9-fold. LOX concentration- and time-dependent increases in PYR content (∼ 16-fold compared with controls) and tensile properties (approximately fivefold compared with controls) of neocartilage were also detected, resulting in properties on par with native tissue. Finally, in vivo subcutaneous implantation of LOX-treated neocartilage in nude mice promoted further maturation of the neotissue, enhancing tensile and PYR content approximately threefold and 14-fold, respectively, compared with in vitro controls. Collectively, these results provide the first report, to our knowledge, of endogenous (hypoxia-induced) and exogenous LOX applications for promoting collagen cross-linking and improving the tensile properties of a spectrum of native and engineered tissues both in vitro and in vivo.

 

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TransAm – November 2014

Establishment of a Herniation Model and Experiments With an Anular Closure Device.

PubMed Article

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Wilke HJ, Ressel L, Heuer F, Graf N, Rath S.
STUDY DESIGN:

Biomechanical in vitro study.
OBJECTIVE:

To establish a reliable in vitro herniation model with human cadaver spines that enables evaluation of anular closure devices.
SUMMARY OF BACKGROUND DATA:

Biomechanically, it is desirable to close anulus defects after disc herniation to preserve as much nucleus as possible. Multiple anular closure options exist to prevent reherniation. A reliable test procedure is needed to evaluate the efficacy and reliability of these implants.
METHODS:

Two groups of human lumbar segments (n = 6 per group) were tested under cyclic loading until herniation occurred or 100,000 load cycles were applied. One group contained moderate/severe degenerated discs. A second group had mild degenerated discs. Intradiscal pressure was measured in the intact state to confirm disc quality.If herniation occurred, the extruded material was reinserted into the disc and the anulus defect was treated with the Barricaid anular closure device (Intrinsic Therapeutics, Inc., Woburn, MA). Disc height and 3-dimensional flexibility of the specimens in the intact, defect, and implanted states were measured under pure moments in each principal motion plane. Afterwards, provocation of reherniation was attempted with additional 100,000 load cycles.
RESULTS:

Likelihood of herniation was strongly linked to disc degeneration and supported by the magnitude of intradiscal pressure. In moderate/severe degenerated discs, only 1 herniation was created. In mild degenerated discs, herniations were reliably created in all specimens. Using this worst-case model, herniation caused a significant reduction of disc height, which was nearly restored with the implant. In no case was reherniation or implant migration visible after 100,000 load cycles after Barricaid implantation.
CONCLUSION:

We established a human herniation model that reliably produced nucleus extrusion during cyclic loading by selecting specimens with low disc degeneration. The Barricaid seems to prevent nucleus from reherniating. The reliability of this method suggests the opportunity to investigate other anulus closure devices and nucleus replacement techniques critically.

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TransAm – October 2014

Bone Plug Versus Suture-Only Fixation of Meniscal Grafts: Effect on Joint Contact Mechanics During Simulated Gait.

PubMed Article

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Wang H, Gee AO, Hutchinson ID, Stoner K, Warren RF, Chen TO, Maher SA.
BACKGROUND:

Meniscus allograft transplantation (MAT) is primarily undertaken to relieve the symptoms associated with meniscal deficiencies. However, its ability to restore normal knee joint contact mechanics under physiological loads is still unclear.
PURPOSE:

To quantify the dynamic contact mechanics associated with 2 commonly used fixation techniques in MAT of the medial compartment: transosseous suture fixation via bone plugs and suture-only fixation at the horns.
STUDY DESIGN:

Controlled laboratory study.
METHODS:

Physiological loads to mimic gait were applied across 7 human cadaveric knees on a simulator. A sensor placed on the medial tibial plateau recorded dynamic contact stresses under the following conditions: (1) intact meniscus, (2) MAT using transosseous suture fixation via bone plugs at the anterior and posterior horns, (3) MAT using suture-only fixation, and (4) total medial meniscectomy. A "remove-replace" procedure was performed to place the same autograft for both MAT conditions to minimize the variability in graft size, geometry, and material property and to isolate the effects of the fixation technique. Contact stress, contact area, and weighted center of contact stress (WCoCS) were quantified on the medial plateau throughout the stance phase.
RESULTS:

Knee joint contact mechanics were sensitive to the meniscal condition primarily during the first half of the gait cycle. After meniscectomy, the mean peak contact stress increased from 4.2 ± 1.2 MPa to 6.2 ± 1.0 MPa (P = .04), and the mean contact area decreased from 546 ± 132 mm2 to 192 ± 122 mm2 (P = .01) compared with the intact meniscus during early stance (14% of the gait cycle). After MAT, the mean contact stress significantly decreased with bone plug fixation (5.0 ± 0.7 MPa) but not with suture-only fixation (5.9 ± 0.7 MPa). Both fixation techniques partially restored the contact area, but bone plug fixation restored it closer to the intact condition. The location of WCoCS in the central cartilage region (not covered by the meniscus) shifted peripherally throughout the stance phase. Bone plug fixation exhibited correction to this peripheral offset, but suture-only fixation did not.
CONCLUSION:

Under dynamic loading, transosseous fixation at the meniscal horns provides superior load distribution at the involved knee compartment after meniscal transplantation compared with suture-only fixation. Particular attention should be directed to the ability of medial MAT to function during the early stance phase.
CLINICAL RELEVANCE:

Transosseous fixation via bone plugs provides superior load distribution of a transplanted meniscal allograft compared with suture fixation alone at time zero.

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Three-dimensional characterization of in vivo intervertebral disc degeneration using EPIC-mCT.

PubMed Article

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Maerz T, Newton MD2 Kristof K, Motovylyak O, Fischgrund JS, Park DK, Baker KC.
OBJECTIVE:

Small animal models are commonly employed to study progression of and potential treatment techniques for degenerative disc disease (DDD), but assessment using conventional imaging techniques is challenging due to resolution. The objective of this study was to employ equilibrium partitioning of an ionic contrast agent micro computed tomography (EPIC - µCT) to map three-dimensional (3D) degenerative changes in the rabbit intervertebral disc (IVD).
MATERIALS AND METHODS:

In vivo degeneration was induced surgically in 12 New Zealand White rabbits via percutaneous annular puncture and percutaneous nucleotomy. IVDs were harvested after 3 and 6 weeks. EPIC-µCT imaging was performed on fresh, IVDs before and after formalin fixation, and 3D IVD volumes were segmented. IVDs were histologically stained with Safranin-O/Fast-Green and Hematoxylin & Eosin (H&E). EPIC-µCT attenuation and 3D morphological measurements were assessed in healthy and degenerate IVDs and compared to qualitative grading and disc height measurement from histology.
RESULTS:

EPIC-µCT caused pronounced contrast enhancement of the IVD. Annular puncture and nucleotomy produced mild and severe degenerative changes, respectively. IVD attenuation following contrast enhancement increased significantly in nucleotomized discs at 3 and 6 weeks. IVD attenuation correlated significantly with histologic score and disc height measurements. Disc height decreased most extensively in the posterior and lateral aspects of the IVD. 3D morphological measurements correlated strongly to IVD attenuation and were more sensitive to degenerative changes than histologic measurements. Formalin fixation reduced the attenuation of IVDs by ∼10%.
CONCLUSION:

EPIC-µCT is sensitive to in vivo DDD induced by nucleotomy and provides a high resolution 3D method for mapping degenerative changes in rabbit IVDs.

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TransAm – September 2014

Clinical, Radiographic, and Histological Outcomes After Cartilage Repair With Particulated Juvenile Articular Cartilage: A 2-Year Prospective Study.

PubMed Article

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Farr J, Tabet SK, Margerrison E, Cole BJ.
BACKGROUND:

Biological repair of cartilage lesions remains a significant clinical challenge because of the lack of natural regeneration and limited treatment options.
HYPOTHESIS:

Treatment of articular cartilage lesions in the knee with particulated juvenile articular cartilage (PJAC) will result in an improvement in patient symptoms of pain and function and magnetic resonance imaging (MRI) findings at 2 years compared with baseline.
STUDY DESIGN:

Case series; Level of evidence, 4.
METHODS:

Patients with symptomatic articular cartilage lesions on the femoral condyles or trochlear groove of the knee were identified for treatment with PJAC. There were 25 patients with a mean age of 37.0 ± 11.1 years and a mean lesion size of 2.7 ± 0.8 cm2. All patients were assessed preoperatively (baseline) with a knee examination and surveys including the International Knee Documentation Committee (IKDC) subjective knee form, 100-mm visual analog scale (VAS) for pain, and Knee injury and Osteoarthritis Outcome Score (KOOS). Patients were followed at predetermined time points postoperatively through 2 years. Also, MRI was performed at baseline and at 3, 6, 12, and 24 months. At 2 years, patients were given the option of undergoing voluntary diagnostic arthroscopic surgery with cartilage biopsy to assess the histological appearance of the cartilage repair including safranin O staining for proteoglycans and immunostaining for type I and II collagen.
RESULTS:

Clinical outcomes demonstrated statistically significant increases at 2 years after surgery compared with baseline, with improvements seen as early as 3 months. Over the 24-month follow-up period, the IKDC score increased from a mean of 45.7 to 73.6, KOOS-pain score from 64.1 to 83.7, KOOS-symptoms score from 64.6 to 81.4, KOOS-activities of daily living score from 73.8 to 91.5, KOOS-sports and recreation score from 44.6 to 68.3, and KOOS-quality of life score from 31.8 to 59.9. The MRI results suggested that T2-weighted scores were returning to a level approximating that of normal articular cartilage by 2 years. Histologically, the repair tissue in biopsy samples from 8 patients was composed of a mixture of hyaline and fibrocartilage; immunopositivity for type II collagen was generally higher than for type I collagen, and there appeared to be excellent integration of the transplanted tissue with the surrounding native articular cartilage. Other than elective biopsies, there were no reoperations, although 1 graft delamination was reported at 24 months.
CONCLUSION:

This study demonstrates a rapid, safe, and effective treatment for cartilage defects. For the patient population investigated, the clinical outcomes of the PJAC technique showed a significant improvement over baseline, with histologically favorable repair tissue 2 years postoperatively.

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Engineered autologous cartilage tissue for nasal reconstruction after tumour resection: an observational first-in-human trial.

PubMed Article

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Ilario Fulco, MD, Sylvie Miot, PhD, Dr Martin D Haug, MD, Andrea Barbero, PhD, Anke Wixmerten, MSc, Sandra Feliciano, BSc, Francine Wolf, BSc, Prof Gernot Jundt, MD, Anna Marsano, PhD, Jian Farhadi, MD, Prof Michael Heberer, MD, Prof Marcel Jakob, MD, Prof Dirk J Schaefer, MD, Prof Ivan Martin, PhD.

Background

Autologous native cartilage from the nasal septum, ear, or rib is the standard material for surgical reconstruction of the nasal alar lobule after two-layer excision of non-melanoma skin cancer. We assessed whether engineered autologous cartilage grafts allow safe and functional alar lobule restoration.
Methods

In a first-in-human trial, we recruited five patients at the University Hospital Basel (Basel, Switzerland). To be eligible, patients had to be aged at least 18 years and have a two-layer defect (≥50% size of alar subunit) after excision of non-melanoma skin cancer on the alar lobule. Chondrocytes (isolated from a 6 mm cartilage biopsy sample from the nasal septum harvested under local anaesthesia during collection of tumour biopsy sample) were expanded, seeded, and cultured with autologous serum onto collagen type I and type III membranes in the course of 4 weeks. The resulting engineered cartilage grafts (25 mm × 25 mm × 2 mm) were shaped intra-operatively and implanted after tumour excision under paramedian forehead or nasolabial flaps, as in standard reconstruction with native cartilage. During flap refinement after 6 months, we took biopsy samples of repair tissues and histologically analysed them. The primary outcomes were safety and feasibility of the procedure, assessed 12 months after reconstruction. At least 1 year after implantation, when reconstruction is typically stabilised, we assessed patient satisfaction and functional outcomes (alar cutaneous sensibility, structural stability, and respiratory flow rate).
Findings

Between Dec 13, 2010, and Feb 6, 2012, we enrolled two women and three men aged 76–88 years. All engineered grafts contained a mixed hyaline and fibrous cartilage matrix. 6 months after implantation, reconstructed tissues displayed fibromuscular fatty structures typical of the alar lobule. After 1 year, all patients were satisfied with the aesthetic and functional outcomes and no adverse events had been recorded. Cutaneous sensibility and structural stability of the reconstructed area were clinically satisfactory, with adequate respiratory function.
Interpretation

Autologous nasal cartilage tissues can be engineered and clinically used for functional restoration of alar lobules. Engineered cartilage should now be assessed for other challenging facial reconstructions.
Funding

Foundation of the Department of Surgery, University Hospital Basel; and Krebsliga beider Basel.

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TransAm – August 2014

ISSLS Prize Winner: Adaptations to the Multifidus Muscle in Response to Experimentally Induced
Intervertebral Disc Degeneration.

PubMed Article

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Stephen H. M. Brown, PhD, Diane E.Gregory, PhD, J. Austin Carr, MS, Samuel R. Ward, PhD, Koichi Masuda, MD, and Richard L. Lieber, PhD

Study Design. Basic science study of the rabbit multifidus muscle response to intervertebral disc degeneration.
Objective.To assess changes in passive mechanical properties, associated protein structure, and histology of multifidus in response to disc degeneration produced by experimental needle puncture.

Summary of Background Data.Relationships have been reported between muscle dysfunction and low back injury; however, little is known about the cause and effect of such relationships.
Methods.Twelve rabbits were studied; 4 in each of 3 groups:
control, 4-weeks postintervertebral disc injury (4-week disc
degeneration), and 12-weeks postintervertebral disc injury
(12-week disc degeneration). Single multifidus fibers and bundles of fibers were isolated and tested for slack sarcomere length and elastic modulus. Titin isoform mass, myosin heavy chain distribution, and
muscle histology were also examined.

Results. Compared to control, individual muscle fibers were 34% stiffer and fiber bundle 107% stiffer in the 12-week disc degeneration group. No changes were detected at 4-week disc degeneration. No statistically significant change was found for MHC distribution in the 12-week disc degeneration group when
compared to control, whereas titin isoforms were larger (P<0.05) in the 12-week disc degeneration group. Histology revealed select regions of multifidus, at 12-week disc degeneration, with increased
space between bundles of fibers, which in some instances was partly occupied by adipose tissue.

Conclusion. Multifidus becomes stiffer, both in individual fibers and fiber bundles, in response to experimentally induced intervertebral disc degeneration. This cannot be explained by change in fiber-type due to reduced muscle use, nor by the increased size of the protein titin (which would reduce stiffness). We hypothesize that fiber bundles become stiffer by proliferation and/or reorganization of collagen content within the muscle but the basis for fiber stiffening is not known.

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TransAm – July 2014

Atomic force microscopy reveals age-dependent changes in nanomechanical properties of the extracellular matrix of native human menisci: Implications for joint degeneration and osteoarthritis
.

PubMed Article

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Kwok J, Grogan S, Meckes B, Arce F, Lal R, D'Lima D.
With aging, the menisci become more susceptible to degeneration due to sustained mechanical stress accompanied by age-related changes in the extracellular matrix (ECM). However, the mechanistic relationship between age-related meniscal degeneration and osteoarthritis (OA) development is not yet fully understood. We have examined the nanomechanical properties of the ECM of normal, aged, and degenerated human menisci using atomic force microscopy (AFM). Elasticity maps of the ECM revealed a unique differential qualitative nanomechanical profile of healthy young tissue: prominent unimodal peaks in the elastic moduli distribution in each region (outer, middle, and inner). Healthy aged tissue showed similar regional elasticity but with both unimodal and bimodal distributions that included higher elastic moduli. In contrast, degenerated OA tissue showed the broadest distribution without prominent peaks indicative of substantially increased mechanical heterogeneity in the ECM. AFM analysis reveals distinct regional nanomechanical profiles that underlie aging-dependent tissue degeneration and OA.
FROM THE CLINICAL EDITOR:

The authors of this study used atomic force microscopy to determine the nanomechanical properties of the extracellular matrix in normal and degenerated human menisci, as well as in menisci undergoing healthy aging. Comparison of these properties help to understand the relationship between healthy ageing, and age-dependent joint degeneration and osteoarthritis.

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Effect of Autologous Platelet Rich Fibrin on the Healing of Experimental Articular Cartilage Defects of the Knee in an Animal Model.

PubMed Article

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Davoud Kazemi, Ashraf Fakhrjou, Vahid Mirzazadeh Dizaji, and Majid Khanzadeh Alishahi.
The effect of autologous platelet rich fibrin (PRF), a second generation platelet product, on the healing of experimental articular cartilage lesions was evaluated in an animal model. Full thickness cartilage lesions with a diameter of 6 mm and depth of 5 mm were created in the weight bearing area of femoral condyles of both hind limbs in 12 adult mixed breed dogs. Defects in the left hind limb of each dog were repaired by PRF implantation whereas those in the right hind limb were left empty. The animals were euthanized at 4, 16, and 24 weeks following surgery and the resultant repair tissue was investigated macroscopically and microscopically. The results of macroscopic and histological evaluations indicated that there were significant differences between the PRF treated and untreated defects. In conclusion, the present study indicated that the use of platelet rich fibrin as a source of autologous growth factors leads to improvement in articular cartilage repair.

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TransAm – June 2014

Bioengineering a Multicomponent Spinal Motion Segment Construct—A 3D Model for Complex Tissue Engineering.

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Chik TK, Chooi WH, Li YY, Ho FC, Cheng HW, Choy TH, Sze KY, Luk KK, Cheung KM, Chan BP.
Intervertebral disc degeneration is an important clinical problem but existing treatments have significant drawbacks. The ability to bioengineer the entire spinal motion segment (SMS) offers hope for better motion preservation strategies but is extremely challenging. Here, fabrication of a multicomponent SMS construct with complex hierarchical organization from mesenchymal stem cells and collagen-based biomaterials, using a module-based integrative approach, is reported. The construct consists of two osteochondral subunits, a nucleus pulposus (NP-)-like core and a multi-lamellae annulus fibrosus (AF-)-like component. Chondrogenic medium is crucial for stabilizing the osteochondral subunits, which are shown to allow passive nutrient diffusion, while cyclic compression is necessary for better fiber matrix organization. Cells adhere, survive, and interact with the NP-like core. Cyclic torsional loading stimulates cell alignment in the AF-like lamellae and the number of lamellae affects the mechanical properties of the construct. This work represents an important milestone in SMS tissue engineering and provides a 3D model for studying tissue maturation and functional remodeling.

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pCMV–BMP-2–Transfected Cell–Mediated Gene Therapy in Anterior Cruciate Ligament Reconstruction in Rabbits.

PubMed Article

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Wang CJ, Weng LH, Hsu SL, Sun YC, Yang YJ, Chan YS, Yang YL.
PURPOSE:

This study investigated the effect of plasmid cytomegalovirus (pCMV)-bone morphogenetic protein 2 (BMP-2) gene therapy on the healing of the tendon-bone interface after anterior cruciate ligament (ACL) reconstruction in rabbits.
METHODS:

The pCMV-BMP-2 was synthesized from full-length human BMP-2 complementary deoxyribonucleic acid, followed by cloning into pCMV Script vector (Clontech Laboratories, Inc., San Jose, CA), and was delivered by a xenogeneic (rat kidney) cell line. The ACL was reconstructed by the transfer of extensor digital tendon in the proximal tibia. In the study group the pCMV-BMP-2 gene-transfected normal rat kidney cells mixed with calcium alginate gel were placed at the tendon-bone interface, whereas no pCMV-BMP-2 was used in the control group. The evaluations included radiography, bone mineral density, magnetic resonance imaging, biomechanical study, histologic examination, and immunohistochemical analysis.
RESULTS:

Bone mineral density showed no significant difference between the groups (P > .05). Magnetic resonance imaging showed significantly better contact between tendon and bone in the study group compared with the control group (P < .0001). In the biomechanical study, significantly higher failure load and maximal graft tension were noted in the study group compared with the control group (P = .034). The modes of graft failure were rupture of the tendon proper in 78% and graft pullout from the bone tunnel in 22% of specimens in the study group versus graft rupture in 22% and graft pullout in 78% in the control group (P = .018). On histologic examination, the study group showed significantly better integration between tendon and bone, as well as more bone tissue around the tendon graft, than the control group (P = .0004). On immunohistochemical analysis, the study group showed significantly higher expressions of von Willebrand factor, vascular endothelial growth factor, proliferation cell nuclear antigen, and BMP-2 than the control group (P < .05).
CONCLUSIONS:

The pCMV-BMP-2 gene therapy significantly improved the healing of tendon to bone and promoted angiogenesis and osteogenesis at the tendon-bone interface after ACL reconstruction in the rabbit model.
CLINICAL RELEVANCE:

Application of pCMV-BMP-2 gene therapy may be an effective adjunct therapy in ACL reconstruction.

 

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TransAm – May 2014

Disc degeneration reduces the delamination strength of the anulus fibrosis in the
rabbit anular disc puncture model.

PubMed Article

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Gregory DE, Bae WC, Sah RL, Masuda K.
BACKGROUND CONTEXT:

Degenerative disc disease is a common pathologic disorder accompanied by both structural and biochemical changes. Changes in stress distribution across the disc can lead to annulus fibrosus (AF) damage that can affect the strength and integrity of the disc. Given that some present degeneration therapies incorporate biological regrowth of the nucleus pulposus (NP), it is crucial that the AF remains capable of containing this newly grown material.
PURPOSE:

To examine the resistance of AF to delamination using an adhesive peel test in experimentally degenerated rabbit discs.
STUDY DESIGN:

Experimentally induced disc degeneration; excised AF tissue study.
METHODS:

Disc degeneration was induced in eight New Zealand white rabbits by annular puncture; four additional rabbits served as controls. In experimental rabbits, an 18-gauge needle was inserted into the anterolateral AF region of levels L2-L3 and L4-L5, and disc height was monitored by X-ray. Animals were sacrificed at 4 and 12 weeks postsurgery and magnetic resonance images and X-rays were taken. Four discs were excised from the experimental animals; two punctured (L2-L3 and L4-L5) and two controls (L3-L4 and L6-L7). The same four discs were also excised from the age-matched control animals and served as nonpunctured control discs. To determine resistance to delamination, AF samples were dissected from each disc and subjected to a mechanical peel test at 0.5 mm/s.
RESULTS:

Magnetic resonance imaging and X-ray images confirmed dehydration of the NP and reduced disc height, similar to that found in clinical degeneration. Resistance to delamination was significantly lower in punctured/degenerated discs compared with both the nonpunctured discs from the same animal (27% lower) and the nonpunctured control discs (30% lower) (p=.024).
CONCLUSIONS:

The findings of this study suggest that degeneration increases the potential for delamination between AF layers. Given this substantial change to the integrity of the AF after degeneration, clinical treatments should not only target rehydration or regrowth of the NP, but should also target repair and strengthening of the AF to confine the NP.

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Matrix-Applied Characterized Autologous Cultured Chondrocytes Versus Microfracture: Two-Year Follow-up of a Prospective Randomized Trial.

PubMed Article

Full Reading

Saris D, Price A, Widuchowski W, Bertrand-Marchand M, Caron J, Drogset JO, Emans P, Podskubka A, Tsuchida A, Kili S, Levine D, Brittberg M; on behalf of the SUMMIT study group.
BACKGROUND:

Randomized controlled trials studying the efficacy and safety of matrix-applied characterized autologous cultured chondrocytes (MACI) versus microfracture (MFX) for treating cartilage defects are limited.
PURPOSE:

To compare the clinical efficacy and safety of MACI versus MFX in the treatment of patients with symptomatic cartilage defects of the knee.
STUDY DESIGN:

Randomized controlled clinical trial; Level of evidence, 1.
METHODS:

Patients enrolled in the SUMMIT (Demonstrate the Superiority of MACI implant to Microfracture Treatment) trial had ≥1 symptomatic focal cartilage defect (Outerbridge grade III or IV; ≥3 cm2) of the femoral condyles or trochlea, with a baseline Knee Injury and Osteoarthritis Outcome Score (KOOS) pain value <55. The co-primary efficacy endpoint was the change in the KOOS pain and function subscores from baseline to 2 years. Histological evaluation and magnetic resonance imaging (MRI) assessments of structural repair tissue, treatment failure, the remaining 3 KOOS subscales, and safety were also assessed.
RESULTS:

Of the 144 patients treated, 137 (95%) completed the 2-year assessment. Patients had a mean age of 33.8 years and a mean lesion size of 4.8 cm2. The mean KOOS pain and function subscores from baseline to 2 years were significantly more improved with MACI than with MFX (pain: MACI, 37.0 to 82.5 vs MFX, 35.5 to 70.9; function: MACI, 14.9 to 60.9 vs MFX, 12.6 to 48.7; P = .001). A significant improvement in scores was also observed on the KOOS subscales of activities of daily living (MACI, 43.5 to 87.2 vs MFX, 42.6 to 75.8; P < .001), knee-related quality of life (MACI, 18.8 to 56.2 vs MFX, 17.2 to 47.3; P = .029), and other symptoms (MACI, 48.3 to 83.7 vs MFX, 44.4 to 72.2; P < .001) for patients treated with MACI compared with MFX. Repair tissue quality was good as assessed by histology/MRI, but no difference was shown between treatments. A low number of treatment failures (nonresponders: MACI, 12.5% vs MFX, 31.9%; P = .016) and no unexpected safety findings were reported.
CONCLUSION:

The treatment of symptomatic cartilage knee defects ≥3 cm2 in size using MACI was clinically and statistically significantly better than with MFX, with similar structural repair tissue and safety, in this heterogeneous patient population. Moreover, MACI offers a more efficacious alternative than MFX with a similar safety profile for the treatment of symptomatic articular cartilage defects of the knee.

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TransAm – April 2014

Repair of meniscal lesions using a scaffold-free tissue-engineered construct derived from allogenic synovial MSCs in a miniature swine model.

PubMed Article

Full Reading

Moriguchi Y, Tateishi K, Ando W, Shimomura K, Yonetani Y, Tanaka Y, Kita K, Hart DA, Gobbi A, Shino K, Yoshikawa H, Nakamura N.

The menisci of the knee are fibro-cartilaginous tissues and play important roles in the joint, and the loss of the meniscus predisposes the knee to degenerative changes. However, the menisci have limited healing potential due to the paucity of vascularity. The purpose of the present study was to test the feasibility of a scaffold-free tissue-engineered construct (TEC) derived from synovial mesenchymal stem cells (MSCs) to repair incurable meniscal lesions. Porcine synovial MSCs were cultured in monolayers at high density in the presence of ascorbic acid followed by the suspension culture to develop a three-dimensional cell/matrix construct (TEC). A 4-mm cylindrical defect was created bilaterally in the medial meniscus of skeletally mature miniature pigs. The defects were implanted with an allogenic TEC or were left empty. After 6 months, the TEC-treated defects were consistently repaired by a fibro-cartilaginous tissue with good tissue integration to the adjacent host meniscal tissue, while the untreated were either partially or not repaired. The ratio of Safranin O positive area within the central body of the meniscus adjacent to the original defect was significantly higher in the TEC-treated group than in the control group. Moreover, TEC treatment significantly reduced the size and severity of post-traumatic chondral lesions on the tibial plateau. These results suggest that the TEC could be a promising stem cell-based implant to repair meniscal lesions with preventive effects from meniscal body degeneration and the development of post-traumatic arthritis.

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TransAm – March 2014

Assessment of Intervertebral Disc Degeneration Based on Quantitative MRI Analysis: an in vivo study.

PubMed Article

Full Reading

Grunert P, Hudson KD, Macielak MR, Aronowitz E, Borde BH, Alimi M, Njoku I, Ballon D, Tsiouris AJ, Bonassar LJ, Härtl R.
STUDY DESIGN:

Animal experimental study.
OBJECTIVE:

To evaluate a novel quantitative imaging technique for assessing disc degeneration.
SUMMARY OF BACKGROUND DATA:

T2-relaxation time (T2-RT) measurements have been used to assess disc degeneration quanti-tatively. T2 values correlate with the water content of intervertebral disc tissue and thereby allow for the indirect measurement of nucleus pulposus (NP) hydration.
METHODS:

We developed an algorithm to subtract out magnetic resonance imaging (MRI) voxels not representing NP tissue on the basis of T2-RT values. Filtered NP voxels were used to measure nuclear size by their amount and nuclear hydration by their mean T2-RT. This technique was applied to 24 rat-tail intervertebral discs (IVDs), which had been punctured with an 18-gauge needle according to different techniques to induce varying degrees of degeneration. NP voxel count and average T2-RT were used as parameters to assess the degeneration process at 1 and 3 months postpuncture. NP voxel counts were evaluated against radiograph disc height measurements and qualitative MRI studies on the basis of the Pfirrmann grading system. Tails were collected for histology to correlate NP voxel counts to histological disc degeneration grades and to NP cross-sectional area measurements.
RESULTS:

NP voxel count measurements showed strong correlations to qualitative MRI analyses (R = 0.79, P < 0.0001), histological degeneration grades (R = 0.902, P < 0.0001), and histological NP cross-sectional area measurements (R = 0.887, P < 0.0001).In contrast to NP voxel counts, the mean T2-RT for each punctured group remained constant between months 1 and 3. The mean T2-RTs for the punctured groups did not show a statistically significant difference from those of healthy IVDs (63.55 ms ± 5.88 ms mo 1 and 62.61 ms ± 5.02 ms) at either time point.
CONCLUSION:

The NP voxel count proved to be a valid parameter to assess disc degeneration quantitatively in a needle puncture model. The mean NP T2-RT does not change significantly in needle-puncture-induced degenerated IVDs. IVDs can be segmented into different tissue components according to their innate T2-RT.

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Tissue-engineered intervertebral discs: MRI results and histology in the rodent spine.

PubMed Article

Full Reading

Grunert P, Gebhard HH, Bowles RD, James AR, Potter HG, Macielak M, Hudson KD, Alimi M, Ballon DJ, Aronowitz E, Tsiouris AJ, Bonassar LJ, Härtl R.
OBJECT:

Tissue-engineered intervertebral discs (TE-IVDs) represent a new experimental approach for the treatment of degenerative disc disease. Compared with mechanical implants, TE-IVDs may better mimic the properties of native discs. The authors conducted a study to evaluate the outcome of TE-IVDs implanted into the rat-tail spine using radiological parameters and histology.
METHODS:

Tissue-engineered intervertebral discs consist of a distinct nucleus pulposus (NP) and anulus fibrosus (AF) that are engineered in vitro from sheep IVD chondrocytes. In 10 athymic rats a discectomy in the caudal spine was performed. The discs were replaced with TE-IVDs. Animals were kept alive for 8 months and were killed for histological evaluation. At 1, 5, and 8 months, MR images were obtained; T1-weighted sequences were used for disc height measurements, and T2-weighted sequences were used for morphological analysis. Quantitative T2 relaxation time analysis was used to assess the water content and T1ρ-relaxation time to assess the proteoglycan content of TE-IVDs.
RESULTS:

Disc height of the transplanted segments remained constant between 68% and 74% of healthy discs. Examination of TE-IVDs on MR images revealed morphology similar to that of native discs. T2-relaxation time did not differ between implanted and healthy discs, indicating similar water content of the NP tissue. The size of the NP decreased in TE-IVDs. Proteoglycan content in the NP was lower than it was in control discs. Ossification of the implanted segment was not observed. Histological examination revealed an AF consisting of an organized parallel-aligned fiber structure. The NP matrix appeared amorphous and contained cells that resembled chondrocytes.
CONCLUSIONS:

The TE-IVDs remained viable over 8 months in vivo and maintained a structure similar to that of native discs. Tissue-engineered intervertebral discs should be explored further as an option for the potential treatment of degenerative disc disease.

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TransAm – February 2014

Adult Human Mesenchymal Stem Cells Delivered via Intra-Articular Injection to the Knee Following
Partial Medial Meniscectomy.

PubMed Article

Full Reading

Vangsness CT Jr, Farr J 2nd, Boyd J, Dellaero DT, Mills CR, LeRoux-Williams M.
BACKGROUND:

There are limited treatment options for tissue restoration and the prevention of degenerative changes in the knee. Stem cells have been a focus of intense preclinical research into tissue regeneration but limited clinical investigation. In a randomized, double-blind, controlled study, the safety of the intra-articular injection of human mesenchymal stem cells into the knee, the ability of mesenchymal stem cells to promote meniscus regeneration following partial meniscectomy, and the effects of mesenchymal stem cells on osteoarthritic changes in the knee were investigated.
METHODS:

A total of fifty-five patients at seven institutions underwent a partial medial meniscectomy. A single superolateral knee injection was given within seven to ten days after the meniscectomy. Patients were randomized to one of three treatment groups: Group A, in which patients received an injection of 50 × 10⁶ allogeneic mesenchymal stem cells; Group B, 150 × 10⁶ allogeneic mesenchymal stem cells; and the control group, a sodium hyaluronate (hyaluronic acid/hyaluronan) vehicle control. Patients were followed to evaluate safety, meniscus regeneration, the overall condition of the knee joint, and clinical outcomes at intervals through two years. Evaluations included sequential magnetic resonance imaging (MRI).
RESULTS:

No ectopic tissue formation or clinically important safety issues were identified. There was significantly increased meniscal volume (defined a priori as a 15% threshold) determined by quantitative MRI in 24% of patients in Group A and 6% in Group B at twelve months post meniscectomy (p = 0.022). No patients in the control group met the 15% threshold for increased meniscal volume. Patients with osteoarthritic changes who received mesenchymal stem cells experienced a significant reduction in pain compared with those who received the control, on the basis of visual analog scale assessments.
CONCLUSIONS:

There was evidence of meniscus regeneration and improvement in knee pain following treatment with allogeneic human mesenchymal stem cells. These results support the study of human mesenchymal stem cells for the apparent knee-tissue regeneration and protective effects.

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Attenuation of osteoarthritis via blockade of the SDF-1/CXCR4 signaling pathway.

PubMed Article

Full Reading

Wei F, Moore DC, Wei L, Li Y, Zhang G, Wei X, Lee JK, Chen Q.
INTRODUCTION:

This study was performed to evaluate the attenuation of osteoarthritic (OA) pathogenesis via disruption of the stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) signaling with AMD3100 in a guinea pig OA model.
METHODS:

OA chondrocytes and cartilage explants were incubated with SDF-1, siRNA CXCR4, or anti-CXCR4 antibody before treatment with SDF-1. Matrix metalloproteases (MMPs) mRNA and protein levels were measured with real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The 35 9-month-old male Hartley guinea pigs (0.88 kg ± 0.21 kg) were divided into three groups: AMD-treated group (n = 13); OA group (n = 11); and sham group (n = 11). At 3 months after treatment, knee joints, synovial fluid, and serum were collected for histologic and biochemical analysis. The severity of cartilage damage was assessed by using the modified Mankin score. The levels of SDF-1, glycosaminoglycans (GAGs), MMP-1, MMP-13, and interleukin-1 (IL-1β) were quantified with ELISA.
RESULTS:

SDF-1 infiltrated cartilage and decreased proteoglycan staining. Increased glycosaminoglycans and MMP-13 activity were found in the culture media in response to SDF-1 treatment. Disrupting the interaction between SDF-1 and CXCR4 with siRNA CXCR4 or CXCR4 antibody attenuated the effect of SDF-1. Safranin-O staining revealed less cartilage damage in the AMD3100-treated animals with the lowest Mankin score compared with the control animals. The levels of SDF-1, GAG, MMP1, MMP-13, and IL-1β were much lower in the synovial fluid of the AMD3100 group than in that of control group.
CONCLUSIONS:

The binding of SDF-1 to CXCR4 induces OA cartilage degeneration. The catabolic processes can be disrupted by pharmacologic blockade of SDF-1/CXCR4 signaling. Together, these findings raise the possibility that disruption of the SDF-1/CXCR4 signaling can be used as a therapeutic approach to attenuate cartilage degeneration.

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Arthroscopic Partial Meniscectomy versus Sham Surgery for a Degenerative Meniscal Tear.

PubMed Article

Full Reading

Sihvonen R, Paavola M, Malmivaara A, Itälä A, Joukainen A, Nurmi H, Kalske J, Järvinen TL; Finnish Degenerative Meniscal Lesion Study (FIDELITY) Group.
BACKGROUND:

Arthroscopic partial meniscectomy is one of the most common orthopedic procedures, yet rigorous evidence of its efficacy is lacking.
METHODS:

We conducted a multicenter, randomized, double-blind, sham-controlled trial in 146 patients 35 to 65 years of age who had knee symptoms consistent with a degenerative medial meniscus tear and no knee osteoarthritis. Patients were randomly assigned to arthroscopic partial meniscectomy or sham surgery. The primary outcomes were changes in the Lysholm and Western Ontario Meniscal Evaluation Tool (WOMET) scores (each ranging from 0 to 100, with lower scores indicating more severe symptoms) and in knee pain after exercise (rated on a scale from 0 to 10, with 0 denoting no pain) at 12 months after the procedure.
RESULTS:

In the intention-to-treat analysis, there were no significant between-group differences in the change from baseline to 12 months in any primary outcome. The mean changes (improvements) in the primary outcome measures were as follows: Lysholm score, 21.7 points in the partial-meniscectomy group as compared with 23.3 points in the sham-surgery group (between-group difference, -1.6 points; 95% confidence interval [CI], -7.2 to 4.0); WOMET score, 24.6 and 27.1 points, respectively (between-group difference, -2.5 points; 95% CI, -9.2 to 4.1); and score for knee pain after exercise, 3.1 and 3.3 points, respectively (between-group difference, -0.1; 95% CI, -0.9 to 0.7). There were no significant differences between groups in the number of patients who required subsequent knee surgery (two in the partial-meniscectomy group and five in the sham-surgery group) or serious adverse events (one and zero, respectively).
CONCLUSIONS:

In this trial involving patients without knee osteoarthritis but with symptoms of a degenerative medial meniscus tear, the outcomes after arthroscopic partial meniscectomy were no better than those after a sham surgical procedure. (Funded by the Sigrid Juselius Foundation and others; ClinicalTrials.gov number, NCT00549172.).

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Intra-Articular Injection of Human Meniscus Stem/Progenitor Cells Promotes Meniscus Regeneration and Ameliorates Osteoarthritis Through Stromal Cell-Derived Factor-1/CXCR4-Mediated Homing.

PubMed Article

Full Reading

Shen W, Chen J, Zhu T, Chen L, Zhang W, Fang Z, Heng BC, Yin Z, Chen X, Ji J, Chen W, Ouyang HW.
Meniscus injury is frequently encountered in clinical practice. Current surgical therapy involving partial or complete meniscectomy relieves pain in the short-term but often leads to osteoarthritis (OA) in the long-term. In this study, we report a new strategy of articular cartilage protection by intra-articular injection of novel human meniscus stem/progenitor cells (hMeSPCs). We found that hMeSPCs displayed both mesenchymal stem cell characteristics and high expression levels of collagen II. In the rat meniscus injury model, hMeSPC transplantation not only led to more neo-tissue formation and better-defined shape but also resulted in more rounded cells and matured extracellular matrix. Stromal cell-derived factor-1 (SDF-1) enhanced the migration of hMeSPCs, whereas AMD3100 abolished the chemotactic effects of SDF-1 on hMeSPCs, both in vitro and in vivo. In an experimental OA model, transplantation of hMeSPCs effectively protected articular cartilage, as evidenced by reduced expression of OA markers such as collagen I, collagen X, and hypoxia-inducible factor 2α but increased expression of collagen II. Our study demonstrated for the first time that intra-articular injection of hMeSPCs enhanced meniscus regeneration through the SDF-1/CXCR4 axis. Our study highlights a new strategy of intra-articular injection of hMeSPCs for meniscus regeneration.

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TransAm – January 2014

Regeneration of a goat femoral head using a tissue-specific, biphasic scaffold fabricated with CAD/CAM technology.

PubMed Article

Full Reading

Ding C, Qiao Z, Jiang W, Li H, Wei J, Zhou G, Dai K.
Tissue engineering is considered as a promising approach for the regeneration of biological joint theoretically and thus provides a potential treatment option for advanced osteoarthritis. However, no significant progresses so far have been made in regenerating biological joint. In this study, a biphasic scaffold, which was consisted of polylactic acid-coated polyglycolic acid (PGA/PLA) scaffold and poly-ε-caprolactone/hydroxyapatite (PCL/HA) scaffold, was designed and used for regeneration of goat femoral head. The content of PLA and HA was optimized to a proper ratio, thus the scaffolds could achieve appropriate stiffness which was more conducive to articular cartilage and bone regeneration respectively. Furthermore, computer-aided design and manufacturing (CAD/CAM) technology was employed to fabricate the biphasic scaffolds into the desired shape and structure. The biphasic scaffolds with fine cell biocompatibility matched perfectly. Chondrocytes and bone marrow stromal cells (BMSCs) were seeded into the scaffolds for cartilage and bone regeneration respectively. After 10 weeks of implantation in nude mice subcutaneously, the cell-scaffold constructs successfully regenerated goat femoral heads. The regenerated femoral heads presented a precise appearance in shape and size similar to that of native goat femoral heads with a smooth, continuous, avascular, and homogeneous cartilage layer on the surface and stiff bone-like tissue in the microchannels of PCL/HA scaffold. Additionally, histological examination of the regenerated cartilage and bone showed typical histological structures and biophysical properties similar to that of native ones with specific matrix deposition and a well-integrated osteochondral interface. The strategy established in the study provides a promising approach for regenerating a biological joint which could be used to reconstruct the impaired joint.

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Quantitative T2 mapping to characterize the process of intervertebral disc degeneration in a
rabbit model.

PubMed Article

Full Reading

Sun W, Zhang K, Zhao CQ, Ding W, Yuan JJ, Sun Q, Sun XJ, Xie YZ, Li H, Zhao J.
BACKGROUND:

To investigate the potential of T2 mapping for characterizing the process of intervertebral disc degeneration (IDD) in a rabbit model.
METHODS:

Thirty-five rabbits underwent an annular stab to the L4/5 discs (L5/6 discs served as internal normal controls). Degenerative changes were graded according to the modified Thompson classification and quantified in T2 respectively at pre-operation, 1, 3, 6, 12 and 24 weeks postoperatively. After MRI analysis, expression analysis of aggrecan and type II collagen gene in nucleus pulposus (NP) was performed using real time polymerase chain reaction (real-time PCR). The longitudinal changes in NP T2 and gene expressions were studied by repeated measures and ANOVA, linear regression was performed for their correlations through the process of IDD. The reliability analysis of method of measurement of NP T2 was also performed.
RESULTS:

There was a strong inverse correlation between NP T2 and Thompson grades (r = -0.85). The decline of L4/5 NP T2 through 24 weeks was nonlinear, the most significant decrease was observed in 3 weeks postoperatively (P<0.05). The tendency was confirmed at gene expression levels. NP T2 correlated strongly with aggrecan (R² = 0.85, P<0.01) and type II collagen (R² = 0.78, P<0.01) gene expressions. The intraclass correlation coefficients for interobserver and intraobserver reliability were 0.963 and 0.977 respectively.
CONCLUSIONS:

NP T2 correlates well with aggrecan and type II collagen gene expressions. T2 mapping could act as a sensitive, noninvasive tool for quantitatively characterizing the process of IDD in longitudinal study, help better understanding of the pathophysiology of IDD, assist us to detect the degenerative cascade, and develop a T2-based quantification scale for evaluation of IDD and efficacy of therapeutic interventions.

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TransAm – December 2013

Immunoselected STRO-3+ mesenchymal precursor cells and restoration of the extracellular matrix of degenerate intervertebral discs.

PubMed Article

Full Reading

Ghosh P, Moore R, Vernon-Roberts B, Goldschlager T, Pascoe D, Zannettino A, Gronthos S, Itescu S.
OBJECT:

Chronic low-back pain of discal origin is linked strongly to disc degeneration. Current nonsurgical treatments are palliative and fail to restore the disc extracellular matrix. In this study the authors examined the capacity of ovine mesenchymal precursor cells (MPCs) to restore the extracellular matrix of degenerate discs in an ovine model.
METHODS:

Three adjacent lumbar discs of 24 adult male sheep were injected intradiscally with chondroitinase-ABC (cABC) to initiate disc degeneration. The remaining lumbar discs were used as normal controls. Three months after cABC injection, the L3-4 discs of all animals were injected with either a high dose (4 × 10(6) cells, in 12 sheep) or low dose (0.5 × 10(6) cells, in 12 sheep) of MPCs suspended in hyaluronic acid (HA). The adjacent L4-5 degenerate discs remained untreated; the L5-6 discs were injected with HA only. The animals were euthanized at 3 or 6 months after MPC injections (6 sheep from each group at each time point), and histological sections of the lumbar discs were prepared. Radiographs and MR images were obtained prior to cABC injection (baseline), 3 months after cABC injection (pretreatment), and just prior to necropsy (posttreatment).
RESULTS:

Injection of cABC decreased the disc height index (DHI) of target discs by 45%-50%, confirming degeneration. Some recovery in DHI was observed 6 months after treatment in all cABC-injected discs, but the DHI increased to within baseline control values only in the MPC-injected discs. This improvement was accompanied by a reduction in MRI degeneration scores. The histopathology scores observed at 3 months posttreatment for the high-dose MPC-injected discs and at 6 months posttreatment for the low-dose MPC-injected discs were significantly different from those of the noninjected and HA-injected discs (p <0.001) but not from the control disc scores.
CONCLUSIONS:

On the basis of the findings of this study, the authors conclude that the injection of MPCs into degenerate intervertebral discs can contribute to the regeneration of a new extracellular matrix.

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Implantation of Allogenic Synovial Stem Cells Promotes Meniscal Regeneration in a Rabbit
Meniscal Defect Model.

PubMed Article

Full Reading

Horie M, Driscoll MD, Sampson HW, Sekiya I, Caroom CT, Prockop DJ, Thomas DB.
BACKGROUND:

Indications for surgical meniscal repair are limited, and failure rates remain high. Thus, new ways to augment repair and stimulate meniscal regeneration are needed. Mesenchymal stem cells are multipotent cells present in mature individuals and accessible from peripheral connective tissue sites, including synovium. The purpose of this study was to quantitatively evaluate the effect of implantation of synovial tissue-derived mesenchymal stem cells on meniscal regeneration in a rabbit model of partial meniscectomy.
METHODS:

Synovial mesenchymal stem cells were harvested from the knee of one New Zealand White rabbit, expanded in culture, and labeled with a fluorescent marker. A reproducible 1.5-mm cylindrical defect was created in the avascular portion of the anterior horn of the medial meniscus bilaterally in fifteen additional rabbits. Allogenic synovial mesenchymal stem cells suspended in phosphate-buffered saline solution were implanted into the right knees, and phosphate-buffered saline solution alone was placed in the left knees. Meniscal regeneration was evaluated histologically at four, twelve, and twenty-four weeks for (1) quantity and (2) quality (with use of an established three-component scoring system). A similar procedure was performed in four additional rabbits with use of green fluorescent protein-positive synovial mesenchymal stem cells for the purpose of tracking progeny following implantation.
RESULTS:

The quantity of regenerated tissue in the group that had implantation of synovial mesenchymal stem cells was greater at all end points, reaching significance at four and twelve weeks (p < 0.05). Tissue quality scores were also superior in knees treated with mesenchymal stem cells compared with controls at all end points, achieving significance at twelve and twenty-four weeks (3.8 versus 2.8 at four weeks [p = 0.29], 5.7 versus 1.7 at twelve weeks [p = 0.008], and 6.0 versus 3.9 at twenty-four weeks [p = 0.021]). Implanted cells adhered to meniscal defects and were observed in the regenerated tissue, where they differentiated into type-I and II collagen-expressing cells, at up to twenty-four weeks.
CONCLUSIONS:

Synovial mesenchymal stem cells adhere to sites of meniscal injury, differentiate into cells resembling meniscal fibrochondrocytes, and enhance both quality and quantity of meniscal regeneration.

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TransAm – April 22, 2013

Nucleus Pulposus Cells Expressing hBMP7 Can Prevent the Degeneration of Allogenic IVD in a Canine Transplantation Model.

PubMed Article

Full Reading

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

Full Reading

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

Full Reading

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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TransAm – October 8, 2012

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

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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.

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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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MechDiff– April 2015

Cell mechanosensitivity to extremely low magnitude signals is enabled by a LINCed nucleus.

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Uzer G, Thompson WR, Sen B, Xie Z, Yen SS, Miller S, Bas G, Styner M, Rubin CT, Judex S, Burridge K, Rubin J.
A cell's ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSC), high magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of FAK/mTORC2/Akt signaling generated at focal adhesions [1]. Physiologic systems also rely on a persistent barrage of low level signals to regulate behavior [2]. Exposing MSC to extremely low magnitude mechanical signals (LMS) suppresses adipocyte formation [3] despite the virtual absence of substrate strain(<0.001%) [2], suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating focal adhesion kinase (FAK) and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the focal adhesions, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting LINC (Linker of Nucleoskeleton and Cytoskeleton) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by high magnitude strain (HMS) was unaffected by LINC decoupling, consistent with signal initiation at the focal adhesion (FA) mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with "outside-in" signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent "inside-inside" signaling.

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Rac1-Dependent Phosphorylation and Focal Adhesion Recruitment of Myosin IIA Regulates Migration and Mechanosensing.

PubMed Article

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Ana M. Pasapera, Sergey V. Plotnikov, Robert S. Fischer, Lindsay B. Case, Thomas T. Egelhoff, Clare M. Waterman.
Background

Cell migration requires coordinated formation of focal adhesions (FAs) and assembly and contraction of the actin cytoskeleton. Nonmuscle myosin II (MII) is a critical mediator of contractility and FA dynamics in cell migration. Signaling downstream of the small GTPase Rac1 also regulates FA and actin dynamics, but its role in regulation of MII during migration is less clear.
Results

We found that Rac1 promotes association of MIIA with FA. Live-cell imaging showed that, whereas most MIIA at the leading edge assembled into dorsal contractile arcs, a substantial subset assembled in or was captured within maturing FA, and this behavior was promoted by active Rac1. Protein kinase C (PKC) activation was necessary and sufficient for integrin- and Rac1-dependent phosphorylation of MIIA heavy chain (HC) on serine1916 (S1916) and recruitment to FA. S1916 phosphorylation of MIIA HC and localization in FA was enhanced during cell spreading and ECM stiffness mechanosensing, suggesting upregulation of this pathway during physiological Rac1 activation. Phosphomimic and nonphosphorylatable MIIA HC mutants demonstrated that S1916 phosphorylation was necessary and sufficient for the capture and assembly of MIIA minifilaments in FA. S1916 phosphorylation was also sufficient to promote the rapid assembly of FAs to enhance cell migration and for the modulation of traction force, spreading, and migration by ECM stiffness.
Conclusions

Our study reveals for the first time that Rac1 and integrin activation regulates MIIA HC phosphorylation through a PKC-dependent mechanism that promotes MIIA association with FAs and acts as a critical modulator of cell migration and mechanosensing.

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MechDiff – February 2015

Rac1 nucleocytoplasmic shuttling drives nuclear shape changes and tumor invasion.

PubMed Article

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Navarro-Lérida I, Pellinen T, Sanchez SA, Guadamillas MC, Wang Y, Mirtti T, Calvo E, Del Pozo MA.
Nuclear membrane microdomains are proposed to act as platforms for regulation of nuclear function, but little is known about the mechanisms controlling their formation. Organization of the plasma membrane is regulated by actin polymerization, and the existence of an actin pool in the nucleus suggests that a similar mechanism might operate here. We show that nuclear membrane organization and morphology are regulated by the nuclear level of active Rac1 through actin polymerization-dependent mechanisms. Rac1 nuclear export is mediated by two internal nuclear export signals and through its interaction with nucleophosmin-1 (B23), which acts as a Rac1 chaperone inside the nucleus. Rac1 nuclear accumulation alters the balance between cytosolic Rac1 and Rho, increasing RhoA signaling in the cytoplasm and promoting a highly invasive phenotype. Nuclear Rac1 shuttling is a finely tuned mechanism for controlling nuclear shape and organization and cell invasiveness.

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Actin stress in cell reprogramming.

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Jun Guo, Yuexiu Wang, Frederick Sachs, and Fanjie Meng.
Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin–cpstFRET–actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFP-tagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals.

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MechDiff – January 2015

Molecular adhesion between cartilage extracellular matrix macromolecules.

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Rojas FP, Batista MA, Lindburg CA, Dean D, Grodzinsky AJ, Ortiz C, Han L.
In this study, we investigated the molecular adhesion between the major constituents of cartilage extracellular matrix, namely, the highly negatively charged proteoglycan aggrecan and the type II/IX/XI fibrillar collagen network, in simulated physiological conditions. Colloidal force spectroscopy was applied to measure the maximum adhesion force and total adhesion energy between aggrecan end-attached spherical tips (end radius R ≈ 2.5 µm) and trypsin-treated cartilage disks with undamaged collagen networks. Studies were carried out in various aqueous solutions to reveal the physical factors that govern aggrecan-collagen adhesion. Increasing both ionic strength and [Ca(2+)] significantly increased adhesion, highlighting the importance of electrostatic repulsion and Ca(2+)-mediated ion bridging effects. In addition, we probed how partial enzymatic degradation of the collagen network, which simulates osteoarthritic conditions, affects the aggrecan-collagen interactions. Interestingly, we found a significant increase in aggrecan-collagen adhesion even when there were no detectable changes at the macro- or microscales. It is hypothesized that the aggrecan-collagen adhesion, together with aggrecan-aggrecan self-adhesion, works synergistically to determine the local molecular deformability and energy dissipation of the cartilage matrix, in turn, affecting its macroscopic tissue properties.

 

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MechDiff – December 2014

Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit survival.

PubMed Article

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Harada T, Swift J, Irianto J, Shin JW, Spinler KR, Athirasala A, Diegmiller R, Dingal PC, Ivanovska IL, Discher DE.
Cell migration through solid tissue often involves large contortions of the nucleus, but biological significance is largely unclear. The nucleoskeletal protein lamin-A varies both within and between cell types and was shown here to contribute to cell sorting and survival in migration through constraining micropores. Lamin-A proved rate-limiting in 3D migration of diverse human cells that ranged from glioma and adenocarcinoma lines to primary mesenchymal stem cells (MSCs). Stoichiometry of A- to B-type lamins established an activation barrier, with high lamin-A:B producing extruded nuclear shapes after migration. Because the juxtaposed A and B polymer assemblies respectively conferred viscous and elastic stiffness to the nucleus, subpopulations with different A:B levels sorted in 3D migration. However, net migration was also biphasic in lamin-A, as wild-type lamin-A levels protected against stress-induced death, whereas deep knockdown caused broad defects in stress resistance. In vivo xenografts proved consistent with A:B-based cell sorting, and intermediate A:B-enhanced tumor growth. Lamins thus impede 3D migration but also promote survival against migration-induced stresses.

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A mechanical checkpoint controls multicellular growth thought YAP/TAZ regulation by actin-processing factors.

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Aragona M, Panciera T, Manfrin A, Giulitti S, Michielin F, Elvassore N, Dupont S, Piccolo S.
Key cellular decisions, such as proliferation or growth arrest, typically occur at spatially defined locations within tissues. Loss of this spatial control is a hallmark of many diseases, including cancer. Yet, how these patterns are established is incompletely understood. Here, we report that physical and architectural features of a multicellular sheet inform cells about their proliferative capacity through mechanical regulation of YAP and TAZ, known mediators of Hippo signaling and organ growth. YAP/TAZ activity is confined to cells exposed to mechanical stresses, such as stretching, location at edges/curvatures contouring an epithelial sheet, or stiffness of the surrounding extracellular matrix. We identify the F-actin-capping/severing proteins Cofilin, CapZ, and Gelsolin as essential gatekeepers that limit YAP/TAZ activity in cells experiencing low mechanical stresses, including contact inhibition of proliferation. We propose that mechanical forces are overarching regulators of YAP/TAZ in multicellular contexts, setting responsiveness to Hippo, WNT, and GPCR signaling.

 

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MechDiff – October 2014

Bone Morphogenetic Protein-2-Induced Signaling and Osteogenesis Is Regulated by Cell Shape, RhoA/ROCK, and Cytoskeletal Tension.

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Yang-Kao Wang, Xiang Yu Daniel M. Cohen, Michele A. Wozniak, Michael T. Yang, Lin Gao, Jeroen Eyckmans, and Christopher S. Chen.
Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is classically thought to be mediated by different cytokines such as the bone morphogenetic proteins (BMPs). Here, we report that cell adhesion to extracellular matrix (ECM), and its effects on cell shape and cytoskeletal mechanics, regulates BMP-induced signaling and osteogenic differentiation of hMSCs. Using micropatterned substrates to progressively restrict cell spreading and flattening against ECM, we demonstrated that BMP-induced osteogenesis is progressively antagonized with decreased cell spreading. BMP triggered rapid and sustained RhoA/Rho-associated protein kinase (ROCK) activity and contractile tension only in spread cells, and this signaling was required for BMP-induced osteogenesis. Exploring the molecular basis for this effect, we found that restricting cell spreading, reducing ROCK signaling, or inhibiting cytoskeletal tension prevented BMP-induced SMA/mothers against decapentaplegic (SMAD)1 c-terminal phosphorylation, SMAD1 dimerization with SMAD4, and SMAD1 translocation into the nucleus. Together, these findings demonstrate the direct involvement of cell spreading and RhoA/ROCK-mediated cytoskeletal tension generation in BMP-induced signaling and early stages of in vitro osteogenesis, and highlight the essential interplay between biochemical and mechanical cues in stem cell differentiation.

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MechDiff – July 2014

A FAK-Cas-Rac-lamellipodin signaling module transduces extracellular matrix stiffness into mechanosensitive cell cycling.

PubMed Article

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Bae YH, Mui KL, Hsu BY, Liu SL, Cretu A, Razinia Z, Xu T, Puré E, Assoian RK.
Tissue and extracellular matrix (ECM) stiffness is transduced into intracellular stiffness, signaling, and changes in cellular behavior. Integrins and several of their associated focal adhesion proteins have been implicated in sensing ECM stiffness. We investigated how an initial sensing event is translated into intracellular stiffness and a biologically interpretable signal. We found that a pathway consisting of focal adhesion kinase (FAK), the adaptor protein p130Cas (Cas), and the guanosine triphosphatase Rac selectively transduced ECM stiffness into stable intracellular stiffness, increased the abundance of the cell cycle protein cyclin D1, and promoted S-phase entry. Rac-dependent intracellular stiffening involved its binding partner lamellipodin, a protein that transmits Rac signals to the cytoskeleton during cell migration. Our findings establish that mechanotransduction by a FAK-Cas-Rac-lamellipodin signaling module converts the external information encoded by ECM stiffness into stable intracellular stiffness and mechanosensitive cell cycling. Thus, lamellipodin is important not only in controlling cellular migration but also for regulating the cell cycle in response to mechanical signals.

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The regulation of gene expression during onset of differentiation by nuclear mechanical heterogeneity.

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Talwar S, Jain N, Shivashankar GV.
Embryonic stem (ES) cells exhibit plasticity in nuclear organization as well as variability in gene expression. Although such physicochemical features are important in lineage commitment, mechanistic insights coupling nuclear plasticity and gene expression have not been elucidated. To probe this, we developed single cell micro-patterned assay to map nuclear deformation and its correlation with gene expression. We found an inherent heterogeneity in nuclear pliability of ES cells. Softer nuclei deformed to the underlying substrate geometry while the stiffer ones remained spherical. Stiffer nuclei were strongly correlated with decreased global histone (H3) acetylation and an increase in Lamin A/C expression. Interestingly, these cells also have higher nuclear accumulation of the transcription cofactor MRTF-A (myocardin-related transcription factor A) and an upregulation of its downstream target genes. Taken together, our results provide compelling evidence to show that the mechanical heterogeneity of stem cell nucleus can regulate transcriptional programs during onset of cellular differentiation.

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MechDiff– June 2014

Mechanical stretching stimulates smooth muscle cell growth, nuclear protein import, and nuclear pore expression through mitogen-activated protein kinase activation.

PubMed Article

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Richard MN, Deniset JF, Kneesh AL, Blackwood D, Pierce GN.
Although it is known that mechanical stretching of cells can induce significant increases in cell growth and shape, the intracellular signaling pathways that induce this response at the level of the cell nucleus is unknown. The transport of molecules from the cell cytoplasm to the nucleoplasm through the nuclear pore is a key pathway through which gene expression can be controlled in some conditions. It is presently unknown if mechanical stimuli can induce changes in nuclear pore expression and/or function. The purpose of the present investigation was to determine if mechanical stretching of a cell will alter nuclear protein import and the mechanisms that may be responsible. Vascular smooth muscle cells that were mechanically stretched exhibited an increase in proliferating cell nuclear antigen expression, cell number, and cell size within 24-48 h. Cells were microinjected with marker proteins for nuclear import. Nuclear protein import was significantly stimulated in stretched cells when compared with control. This was associated with an increase in the expression of nuclear pore proteins as detected by Western blots. Inhibition of the MAPK pathway blocked the stretch-induced stimulation of both cell proliferation and nuclear protein import. We conclude that nuclear protein import and nuclear pore density can adapt to mechanical stimuli during the process of cell growth through a MAPK-mediated mechanism.

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MechDiff– May 2014

Rigidity sensing and adaptation through regulation of integrin types.

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Alberto Elosegui-Artola, Elsa Bazellières, Michael D. Allen, Ion Andreu, Roger Oria, Raimon Sunyer, Jennifer J. Gomm, John F. Marshall, J. Louise Jones, Xavier Trepat & Pere Roca-Cusachs.

Tissue rigidity regulates processes in development, cancer and wound healing. However, how cells detect rigidity, and thereby modulate their behaviour, remains unknown. Here, we show that sensing and adaptation to matrix rigidity in breast myoepithelial cells is determined by the bond dynamics of different integrin types. Cell binding to fibronectin through either α5β1 integrins (constitutively expressed) or αvβ6 integrins (selectively expressed in cancer and development) adapts force generation, actin flow and integrin recruitment to rigidities associated with healthy or malignant tissue, respectively. In vitro experiments and theoretical modelling further demonstrate that this behaviour is explained by the different binding and unbinding rates of both integrin types to fibronectin. Moreover, rigidity sensing through differences in integrin bond dynamics applies both when integrins bind separately and when they compete for binding to fibronectin.

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Pleiotrophin commits human bone marrow mesenchymal stromal cells towards hypertrophy during chondrogenesis.

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Bouderlique T, Henault E, Lebouvier A, Frescaline G, Bierling P, Rouard H, Courty J, Albanese P, Chevallier N.
Pleiotrophin (PTN) is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC) are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC) under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.

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MechDiff– March 2014

Three-dimensional cell migration does not follow a random walk.

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Pei-Hsun Wua, Anjil Giria, Sean X. Sunb, and Denis Wirtza.
Cell migration through 3D extracellular matrices is critical to the normal development of tissues and organs and in disease processes, yet adequate analytical tools to characterize 3D migration are lacking. Here, we quantified the migration patterns of individual fibrosarcoma cells on 2D substrates and in 3D collagen matrices and found that 3D migration does not follow a random walk. Both 2D and 3D migration features a non-Gaussian, exponential mean cell velocity distribution, which we show is primarily a result of cell-to-cell variations. Unlike in the 2D case, 3D cell migration is anisotropic: velocity profiles display different speed and self-correlation processes in different directions, rendering the classical persistent random walk (PRW) model of cell migration inadequate. By incorporating cell heterogeneity and local anisotropy to the PRW model, we predict 3D cell motility over a wide range of matrix densities, which identifies density-independent emerging migratory properties. This analysis also reveals the unexpected robust relation between cell speed and persistence of migration over a wide range of matrix densities.

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Involvement of fibroblast growth factor 18 in dedifferentiation of cultured human chondrocytes.

PubMed Article

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Yamaoka H, Nishizawa S, Asawa Y, Fujihara Y, Ogasawara T, Yamaoka K, Nagata S, Takato T, Hoshi K.
OBJECTIVE:

Chondrocytes inevitably decrease production of cartilaginous matrices during long-term cultures with repeated passaging; this is termed dedifferentiation. To learn more concerning prevention of dedifferentiation, we have focused here on the fibroblast growth factor (FGF) family that influences chondrocyte proliferation or differentiation.
MATERIALS AND METHODS:

We have compared gene expression between differentiated cells in passage 3 (P3) and dedifferentiated ones in P8 of human cultured chondrocytes. We also performed ligand administration of the responsive factor or its gene silencing, using small interfering RNA (siRNA).
RESULTS:

FGFs 1, 5, 10, 13 and 18 were higher at P8 compared to P3, while FGFs 9 and 14 were lower. Especially, FGF18 showed a 10-fold increase by P8. Ligand administration of FGF18 in the P3 cells, or its gene silencing using siRNA in the P8 cells, revealed dose-dependent increase and decrease respectively in type II collagen/type I collagen ratio. Exogenous FGF18 also upregulated expression of transforming growth factor beta (TGF-beta), the anabolic factor of chondrocytes, in P3 chondrocytes, but P8 cells maintained a low level of TGF-beta expression, suggesting a decrease in responsiveness of TGF-beta to FGF18 stimulation in the dedifferentiated chondrocytes.
CONCLUSION:

FGF18 seems to play a role in maintenance of chondrocyte properties, although its expression was rather high in dedifferentiated chondrocytes. Upregulation of FGF18 in dedifferentiated chondrocytes implied that it may be a marker of dedifferentiation.

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MechDiff– February 2014

Integrin activation and internalization on soft ECM as a mechanism of induction of stem cell differentiation by ECM elasticity

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Jing Du, Xiaofei Chen, Xudong Liang, Guangyao Zhang, Jia Xu, Linrong He, Qingyuan Zhan, Xi-Qiao Feng, Shu Chien, and Chun Yanga.
The mechanism by which ECM elasticity induces lineage specification of stem cells has not been clearly understood. Integrins are well-documented mechanosensors that are positioned at the beginning of the sensing pathway. By using an antibody specifically recognizing the active conformation of β1 integrin, we observed that β1 integrin activation in bone marrow mesenchymal stem cells (BMMSCs) was induced by soft substrate to a significantly greater degree than by stiff substrate. In contrast, however, the level of cell surface integrin on soft substrate was significantly lower than that on stiff substrate. Soft substrate markedly enhanced the internalization of integrin, and this internalization was mediated mainly through caveolae/raft-dependent endocytosis. The inhibition of integrin internalization blocked the neural lineage specification of BMMSCs on soft substrate. Furthermore, soft substrate also repressed the bone morphogenetic protein (BMP)/Smad pathway at least partially through integrin-regulated BMP receptor endocytosis. A theoretical analysis based on atomic force microscopy (AFM) data indicated that integrin–ligand complexes are more easily ruptured on soft substrate; this outcome may contribute to the enhancement of integrin internalization on soft substrate. Taken together, our results suggest that ECM elasticity affects integrin activity and trafficking to modulate integrin BMP receptor internalization, thus contributing to stem cell lineage specification.

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Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage.

PubMed Article

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Joos H, Wildner A, Hogrefe C, Reichel H, Brenner RE.
NTRODUCTION:

The repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints.
METHODS:

We characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1β) as well as tumor necrosis factor alpha (TNF-α) were tested with a modified Boyden chamber assay. The influence of IL-1β and TNF-α was additionally examined by scratch assays and outgrowth experiments.
RESULTS:

A comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1β and TNF-α significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC.
CONCLUSION:

These results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1β and TNF-α inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.

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MechDiff– January 2014

Actomyosin-dependent formation of the mechanosensitive talin-vinculin complex reinforces actin anchoring.

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Ciobanasu C, Faivre B, Le Clainche C.
The force generated by the actomyosin cytoskeleton controls focal adhesion dynamics during cell migration. This process is thought to involve the mechanical unfolding of talin to expose cryptic vinculin-binding sites. However, the ability of the actomyosin cytoskeleton to directly control the formation of a talin-vinculin complex and the resulting activity of the complex are not known. Here we develop a microscopy assay with pure proteins in which the self-assembly of actomyosin cables controls the association of vinculin to a talin-micropatterned surface in a reversible manner. Quantifications indicate that talin refolding is limited by vinculin dissociation and modulated by the actomyosin network stability. Finally, we show that the activation of vinculin by stretched talin induces a positive feedback that reinforces the actin-talin-vinculin association. This in vitro reconstitution reveals the mechanism by which a key molecular switch senses and controls the connection between adhesion complexes and the actomyosin cytoskeleton.

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MechDiff– October 10, 2012

ECM stiffness primes the TGFB pathway to promote chondrocyte differentiation.

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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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MechDiff– September 26, 2012

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

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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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