Mauck Laboratory

TransAm: Translational Animal Models Journal Club

June 2018

Low-oxygen conditions promote synergistic increases in chondrogenesis during co-culture of human osteoarthritic stem cells and chondrocytes
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May 2018

Effect of intra-articular administration of superparamagnetic iron oxide nanoparticles (SPIONs) for MRI assessment of the cartilage barrier in a large animal model
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Meniscal Tissue Engineering Using Aligned Collagen Fibrous Scaffolds: Comparison of Different Human Cell Sources.
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April 2018

Third-generation autologous chondrocyte implantation versus mosaicplasty for knee cartilage injury: 2-year randomized trial
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Annulus Fibrosus Repair Using High-Density Collagen Gel: An In Vivo Ovine Model
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January 2018

Focal adhesion signaling affects regeneration by human nucleus pulposus cells in collagen- but not carbohydrate-based hydrogels
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December 2017

Engineering a highly elastic human protein-based sealant for surgical applications
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Mechanical confinement regulates cartilage matrix formation by chondrocytes
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Stress relaxing hyaluronic acid-collagen hydrogels promote cell spreading, fiber remodeling, and focal adhesion formation in 3D cell culture
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November 2017

Hyaluronic Acid Hydrogel Functionalized with Self-Assembled Micelles of Amphiphilic PEGylated Kartogenin for the Treatment of Osteoarthritis
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Sulfated hyaluronic acid hydrogels with retarded degradation and enhanced growth factor retention promote hMSC chondrogenesis and articular cartilage integrity with reduced hypertrophy
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October 2017

Timing of mesenchymal stem cell delivery impacts the fate and therapeutic potential in intervertebral disc repair
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In situ handheld three‐dimensional bioprinting for cartilage regeneration
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September 2017

Flexible shape-memory scaffold for minimally invasive delivery of functional tissues.
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Treatment of Articular Cartilage Defects With Microfracture and Autologous Matrix-Induced Chondrogenesis Leads to Extensive Subchondral Bone Cyst Formation in a Sheep Model
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July 2017

Allogeneic Mesenchymal Stem Cells Stimulate Cartilage Regeneration and Are Safe for Single-Stage Cartilage Repair in Humans upon Mixture with Recycled Autologous Chondrons.
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Bone Marrow-Derived Mesenchymal Stem Cells as Autologous Therapy in Dogs with Naturally Occurring Intervertebral Disc Disease: Feasibility, Safety, and Preliminary Results.
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May 2017

Cell-Based Meniscus Repair and Regeneration: At the Brink of Clinical Translation?: A Systematic Review of Preclinical Studies.
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Constrained Cage Culture Improves Engineered Cartilage Functional Properties by Enhancing Collagen Network Stability
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April 2017

Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel.
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February 2017

Local Administration of Simvastatin Stimulates Healing of an Avascular Meniscus in a Rabbit Model of a Meniscal Defect.
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January 2017

Kaempferol slows intervertebral disc degeneration by modifying LPS-induced osteogenesis/adipogenesis imbalance and inflammation response in BMSCs.
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November 2016

Regenerative Potential of Tissue-Engineered Nasal Chondrocytes in Goat Articular Cartilage Defects.
PubMed Article 
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Mumme M, Steinitz A, Nuss KM, Klein K, Feliciano S, Kronen P, Jakob M, von Rechenberg B, Martin I, Barbero A, Pelttari K.

Nasal chondrocytes (NC) were previously demonstrated to remain viable and to participate in the repair of articular cartilage defects in goats. Here, we investigated critical features of tissue-engineered grafts generated by NC in this large animal model, namely cell retention at the implantation site, architecture and integration with adjacent tissues, and effects on subchondral bone changes. In this study, isolated autologous goat NC (gNC) and goat articular chondrocytes (gAC, as control) were expanded, green fluorescent protein-labelled and seeded on a type I/III collagen membrane. After chondrogenic differentiation, tissue-engineered grafts were implanted into chondral defects (6 mm in diameter) in the stifle joint for 3 or 6 months. At the time of explantation, surrounding tissues showed no or very low (only in the infrapatellar fat pad <0.32%) migration of the grafted cells. In repair tissue, gNC formed typical structures of articular cartilage, such as flattened cells at the surface and column-like clusters in the middle layers. Semi-quantitative histological evaluation revealed efficient integration of the grafted tissues with the adjacent native cartilage and underlying subchondral bone. A significantly increased subchondral bone area, as a sign for the onset of osteoarthritis, was observed following treatment of cartilage defects with gAC-, but not with gNC-grafts. Our results reinforce the use of NC-based engineered tissue for articular cartilage repair and preliminarily indicate their potential for the treatment of early osteoarthritic defects.

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Lumbar intervertebral disc allograft transplantation:healing and remodelling of the bony structure
PubMed Article 
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Huang YC, Xiao J, Leung WY, Lu WW, Hu Y, Luk KD.

Previous human study suggested that fresh-frozen intervertebral disc allograft transplantation can relieve neurological symptoms and restore segmental kinematics. Before wide clinical application, research into the pathophysiology of the postoperative disc allograft is needed. One important question that remains to be answered in disc allografting is the healing process of the host-graft interface and the subsequent change of the endplates. With the goat model for lumbar disc allografting, histology, micro-computed tomography analysis, scanning electron microscopy and energy-dispersive X-ray spectroscopy mapping were applied to evaluate the healing of the host-graft interfaces, the remodelling of subchondral bone, and the changes of the bony and cartilaginous endplates after transplantation. It was found that healing of the host-graft interfaces started at 1.5 months and was completed at 6 months by natural remodelling. This bony remodelling was also noted in the subchondral bone area after 6 months. The bony endplate was well preserved initially, but was gradually replaced by trabecular bone afterwards; on the other hand, the cartilaginous endplate became atrophic at 6 months and nearly disappeared at the final follow-up. Collectively, after intervertebral disc allograft transplantation, bony healing and remodelling were seen which ensured the stability and mobility of the disc-transplanted segment, but the integrity of bony and cartilaginous endplates was gradually lost and nearly disappeared finally.

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October 2016

Transplantation of autologous synovial mesenchymal stem cells promotes meniscus regeneration in aged primates.
PubMed Article 
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Kondo S, Muneta T, Nakagawa Y, Koga H, Watanabe T, Tsuji K, Sotome S, Okawa A, Kiuchi S, Ono H, Mizuno M, Sekiya I.

Transplantation of aggregates of synovial mesenchymal stem cells (MSCs) enhanced meniscus regeneration in rats. Anatomy and biological properties of the meniscus depend on animal species. To apply this technique clinically, it is valuable to investigate the use of animals genetically close to humans. We investigated whether transplantation of aggregates of autologous synovial MSCs promoted meniscal regeneration in aged primates. Chynomolgus primates between 12-13 years old were used. After the anterior halves of the medial menisci in both knees were removed, an average of 14 aggregates consisting of 250,000 synovial MSCs were transplanted onto the meniscus defect. No aggregates were transplanted to the opposite knee for the control. Meniscus and articular cartilage were analyzed macroscopically, histologically, and by MRI T1rho mapping at 8 (n = 3) and 16 weeks (n = 4). The medial meniscus was larger and the modified Pauli's histological score for the regenerated meniscus was better in the MSC group than in the control group in each primate at 8 and 16 weeks. Mankin's score for the medial femoral condyle cartilage was better in the MSC group than in the control group in all primates at 16 weeks. T1rho value for both the regenerated meniscus and adjacent articular cartilage in the MSC group was closer to the normal meniscus than in the control group in all primates at 16 weeks. Transplantation of aggregates of autologous synovial MSCs promoted meniscus regeneration and delayed progression of degeneration of articular cartilage in aged primates. This is the first report dealing with meniscus regeneration in primates.

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Timing of mesenchymal stem cell delivery impacts the fate and therapeutic potential in intervertebral disc repair
PubMed Article 
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Robert Maidhof, Asfi Rafiuddin, Farzana Chowdhury, Timothy Jacobsen, Nadeen O. Chahine.

Cell-based therapies offer a promising approach to treat intervertebral disc (IVD) degeneration. The impact of the injury microenvironment on treatment efficacy has not been established. This study used a rat disc stab injury model with administration of mesenchymal stromal cells (MSCs) at 3, 14, or 30 days post injury (dpi) to evaluate the impact of interventional timing on IVD biochemistry and biomechanics. We also evaluated cellular localization within the disc with near infrared imaging to track the time and spatial profile of cellular migration after in vivo delivery. Results showed that upon injection into a healthy disc, MSCs began to gradually migrate outwards over the course of 14 days, as indicated by decreased signal intensity from the disc space and increased signal within the adjacent vertebrae. Cells administered 14 or 30 dpi also tended to migrate out 14 days after injection but cells injected 3dpi were retained at a significantly higher amount versus the other groups (p < 0.05). Correspondingly the 3dpi group, but not 14 or 30dpi groups, had a higher GAG content in the MSC group (p = 0.06). Enrichment of MSCs and increased GAG content in 3dpi group did not lead to increased compressive biomechanical properties. Findings suggest that cell therapies administered at an early stage of injury/disease progression may have greater chances of mitigating matrix loss, possibly through promotion of MSC activity by the inflammatory microenvironment associated with injury. Future studies will evaluate the mode and driving factors that regulate cellular migration out of the disc. 

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September 2016

Treatment of Articular Cartilage Defects With Microfracture and Autologous Matrix-Induced Chondrogenesis Leads to Extensive Subchondral Bone Cyst Formation in a Sheep Model.
PubMed Article 
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Beck A, Murphy DJ, Carey-Smith R, Wood DJ, Zheng MH.

BACKGROUND:

Microfracture and the autologous matrix-induced chondrogenesis (AMIC) technique are popular for the treatment of articular cartilage defects. However, breaching of the subchondral bone plate could compromise the subchondral bone structure.HYPOTHESIS:

Microfracture and AMIC will cause deleterious effects on the subchondral bone structure.

STUDY DESIGN:

Controlled laboratory study.

METHODS:

A total of 36 sheep received an 8-mm-diameter cartilage defect in the left medial femoral condyle. Control animals (n = 12) received no further treatment, and the rest received 5 microfracture holes either with a type I/III collagen scaffold implanted (n = 12; AMIC group) or without the collagen scaffold (n = 12; microfracture group). Macroscopic infill of defects, histology, and histomorphometry of the subchondral bone were performed at 13 and 26 weeks postoperatively, and micro-computed tomography (CT) was also performed at 26 weeks postoperatively.

RESULTS:

Microfracture and AMIC resulted in subchondral bone cyst formation in 5 of 12 (42%) and 11 of 12 (92%) specimens at 13 and 26 weeks, respectively. Subchondral bone changes induced by microfracture and AMIC were characterized by an increased percentage of bone volume, increased trabecular thickness, and a decreased trabecular separation, and extended beyond the area below the defect. High numbers of osteoclasts were observed at the cyst periphery, and all cysts communicated with the microfracture holes. Cartilage repair tissue was of poor quality and quantity at both time points and rarely reached the tidemark at 13 weeks.

CONCLUSION:

Microfracture technique caused bone cyst formation and induced severe pathology of the subchondral bone in a sheep model.

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August 2016

BioCartilage Improves Cartilage Repair Compared With Microfracture Alone in an Equine Model of Full-Thickness Cartilage Loss.
PubMed Article 
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Fortier LA, Chapman HS, Pownder SL, Roller BL, Cross JA, Cook JL, Cole BJ.

BACKGROUND:

Microfracture (MFx) remains a dominant treatment strategy for symptomatic articular cartilage defects. Biologic scaffold adjuncts, such as particulated allograft articular cartilage (BioCartilage) combined with platelet-rich plasma (PRP), offer promise in improving clinical outcomes as an adjunct to MFx.

PURPOSE:

To evaluate the safety, biocompatibility, and efficacy of BioCartilage and PRP for cartilage repair in a preclinical equine model of full-thickness articular cartilage loss.

STUDY DESIGN:

Controlled laboratory study.

METHODS:

Two 10-mm-diameter full-thickness cartilage defects were created in 5 horses in the trochlear ridge of both knees: one proximal (high load) and another distal (low load). Complete blood counts were performed on each peripheral blood and resultant PRP sample. In each horse, one knee received MFx with BioCartilage + PRP, and the other knee received MFx alone. Horses were euthanized at 13 months. Outcomes were assessed with serial arthroscopy, magnetic resonance imaging (MRI), micro-computed tomography (micro-CT), and histology. Statistics were performed using a mixed-effects model with response variable contrasts.

RESULTS:

No complications occurred. PRP generated in all subjects yielded an increase in platelet fold of 3.8 ± 4.7. Leukocyte concentration decreased in PRP samples by an average fold change of 5 ± 0.1. The overall International Cartilage Repair Society repair score in both the proximal and distal defects was significantly higher (better) in the BioCartilage group compared with MFx (proximal BioCartilage: 7.4 ± 0.51, MFx 4.8 ± 0.1, P = .041; distal BioCartilage: 5.6 ± 0.98, MFx 2.6 ± 1.5, P = .022). BioCartilage-treated proximal defects demonstrated improved histologic scores for repair-host integration (BioCartilage, 96 ± 9; MFx, 68 ± 18; P = .02), base integration (BioCartilage, 100 ± 0; MFx, 70 ± 37; P = .04), and formation of collagen type II (BioCartilage, 82 ± 8; MFx, 58 ± 11; P = .05) compared with the positive control. On MRI, T2 relaxation time was significantly shorter (better) in the superficial region of BioCartilage-treated distal defects compared with MFx (P = .05). There were no significant differences between BioCartilage and MFx on micro-CT analysis.

CONCLUSION:

BioCartilage with PRP safely improved cartilage repair compared with MFx alone in an equine model of articular cartilage defects up to 13 months after implantation.

CLINICAL RELEVANCE:

The 1-year results of BioCartilage + PRP suggest that homologous allograft tissue provides a safe and effective augmentation of traditional MFx.

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June 2016

Implantation of a polycaprolactone scaffold with subchondral bone anchoring ameliorates nodules formation and other tissue alterations.
PubMed Article 
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Vikingsson L, Sancho-Tello M, Ruiz-Saurí A, Martínez Díaz S, Gómez-Tejedor J, Gallego Ferrer G, Carda C, Monllau J, Gómez Ribelles J.

PURPOSE:

Articular cartilage has limited repair capacity. Two different implant devices for articular cartilage regeneration were tested in vivo in a sheep model to evaluate the effect of subchondral bone anchoring for tissue repair.

METHODS:

The implants were placed with press-fit technique in a cartilage defect after microfracture surgery in the femoral condyle of the knee joint of the sheep and histologic and mechanical evaluation was done 4.5 months later. The first group consisted of a biodegradable polycaprolactone (PCL) scaffold with double porosity. The second test group consisted of a PCL scaffold attached to a poly(L-lactic acid) (PLLA) pin anchored to the subchondral bone.

RESULTS:

For both groups most of the defects (75%) showed an articular surface that was completely or almost completely repaired with a neotissue. Nevertheless, the surface had a rougher appearance than controls and the repair tissue was immature. In the trials with solely scaffold implantation, severe subchondral bone alterations were seen with many large nodular formations. These alterations were ameliorated when implanting the scaffold with a subchondral bone anchoring pin.

DISCUSSIONS:

The results show that tissue repair is improved by implanting a PCL scaffold compared to solely microfracture surgery, and most importantly, that subchondral bone alterations, normally seen after microfracture surgery, were partially prevented when implanting the PCL scaffold with a fixation system to the subchondral bone.

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One-Year Outcomes of Total Meniscus Reconstruction Using a Novel Fiber-Reinforced Scaffold in an Ovine Model
PubMed Article 
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Jay M. Patel, ME, Aaron R. Merriam, PhD, Brian M. Culp, MD, Charles J. Gatt Jr, MD, and Michael G. Dunn, PhD.

Background: Meniscus injuries and resulting meniscectomies lead to joint deterioration, causing pain, discomfort, and instability. Tissue-engineered devices to replace the meniscus have not shown consistent success with regard to function, mechanical integrity, or protection of cartilage.

Purpose: To evaluate a novel resorbable polymer fiber–reinforced meniscus reconstruction scaffold in an ovine model for 52 weeks and assess its integrity, tensile and compressive mechanics, cell phenotypes, matrix organization and content, and protection of the articular cartilage surfaces.

Study Design: Controlled laboratory study.

Methods: Eight skeletally mature ewes were implanted with the fiber-reinforced scaffold after total meniscectomy, and 2 additional animals had untreated total meniscectomies. Animals were sacrificed at 52 weeks, and the explants and articular surfaces were analyzed macroscopically. Explants were characterized by ultimate tensile testing, confined compression creep testing, and biochemical, histological, and immunohistochemical analyses. Cartilage damage was characterized using the Mankin score on histologic slides from both the femur and tibia.

Results: One sheep was removed from the study because of a torn extensor tendon; the remaining 7 explants remained fully intact and incorporated into the bone tunnels. All explants exhibited functional tensile loads, tensile stiffnesses, and compressive moduli. Fibrocartilagenous repair with both types 1 and 2 collagen were observed, with areas of matrix organization and biochemical content similar to native tissue. Narrowing in the body region was observed in 5 of 7 explants. Mankin scores showed less cartilage damage in the explant group (femoral condyle: 3.43 ± 0.79, tibial plateau: 3.50 ± 1.63) than in the meniscectomy group (femoral condyle: 8.50 ± 3.54, tibial plateau: 6.75 ± 2.47) and were comparable with Mankin scores at the previously reported 16- and 32-week time points.

Conclusion: A resorbable fiber-reinforced meniscus scaffold supports formation of functional neomeniscus tissue, with the potential to prevent joint degeneration that typically occurs after total meniscectomy. Further studies with improvements to the initial mechanics of the scaffold and testing for longer time periods are warranted.

Clinical Relevance: Meniscectomy is an extremely common orthopaedic procedure, and few options currently exist for the treatment of significant loss of meniscus tissue. Successful development of a tissue-engineered meniscus scaffold could substantially reduce the incidence of postmeniscectomy joint degeneration and the subsequent procedures used for its treatment.

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May 2016

Developing a pro-regenerative biomaterial scaffold microenvironment requires T helper 2 cells
PubMed Article 
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Kaitlyn Sadtler, Kenneth Estrellas, Brian W. Allen, Matthew T. Wolf, Hongni Fan, Ada J. Tam, Chirag H. Patel, Brandon S. Luber, Hao Wang, Kathryn R. Wagner, Jonathan D. Powell, Franck Housseau, Drew M. Pardoll, Jennifer H. Elisseeff.

Immune-mediated tissue regeneration driven by a biomaterial scaffold is emerging as an innovative regenerative strategy to repair damaged tissues. We investigated how biomaterial scaffolds shape the immune microenvironment in traumatic muscle wounds to improve tissue regeneration. The scaffolds induced a pro-regenerative response, characterized by an mTOR/Rictor-dependent T helper 2 pathway that guides interleukin-4–dependent macrophage polarization, which is critical for functional muscle recovery. Manipulating the adaptive immune system using biomaterials engineering may support the development of therapies that promote both systemic and local pro-regenerative immune responses, ultimately stimulating tissue repair.

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Evaluation of an injectable thermoresponsive hyaluronan hydrogel in a rabbit osteochondral defect model
PubMed Article 
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D'Este M, Sprecher CM, Milz S, Nehrbass D, Dresing I, Zeiter S, Alini M, Eglin D.

Articular cartilage displays very little self-healing capabilities, generating a major clinical need. Here, we introduce a thermoresponsive hyaluronan hydrogel for cartilage repair obtained by covalently grafting poly(N-isopropylacrylamide) to hyaluronan, to give a brush co-polymer HpN. The gel is fluid at room temperature and becomes gel at body temperature. In this pilot study HpN safety and repair response were evaluated in an osteochondral defect model in rabbit. Follow-up was of 1 week and 12 weeks and the empty defect served as a control, for a total of four experimental groups. At 12 weeks the defect sites were evaluated macroscopically and histologically. Local lymph nodes, spleen, liver, and kidneys were analyzed for histopathological evaluation. HpN could be easily injected and remained into the defect throughout the study. The macroscopic score was statistically superior for HpN versus empty. Histological score gave opposite trend but not statistically significant. A slight tissue reaction was observed around HpN, however, vascularization and subchondral bone formation were not impeded. An upper proteoglycans rich fibro-cartilaginous tissue with fairly good continuity and lateral integration into the existing articular cartilage was observed in all cases. No signs of local or systemic acute or subacute toxicity were observed. In conclusion, HpN is easily injectable, remains into an osteochondral defect within a moving synovial joint, is biocompatible and does not interfere with the intrinsic healing response of osteochondral defects in a rabbit model. 

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April 2016

Cell-free multi-layered collagen-based scaffolds demonstrate layer specific regeneration of functional osteochondral tissue in caprine joints
PubMed Article 
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Tanya J. Levingstonea, Ashwanth Ramesha, Robert T. Bradya, Pieter A.J. Bramad, Clodagh Kearneyd, John P. Gleesona, Fergal J. O'Briena.

Developing repair strategies for osteochondral tissue presents complex challenges due to its interfacial nature and complex zonal structure, consisting of subchondral bone, intermediate calcified cartilage and the superficial cartilage regions. In this study, the long term ability of a multi-layered biomimetic collagen-based scaffold to repair osteochondral defects is investigated in a large animal model: namely critical sized lateral trochlear ridge (TR) and medial femoral condyle (MC) defects in the caprine stifle joint. The study thus presents the first data in a clinically applicable large animal model. Scaffold fixation and early integration was demonstrated at 2 weeks post implantation. Macroscopic analysis demonstrated improved healing in the multi-layered scaffold group compared to empty defects and a market approved synthetic polymer osteochondral scaffold groups at 6 and 12 months post implantation. Radiological analysis demonstrated superior subchondral bone formation in both defect sites in the multi-layered scaffold group as early as 3 months, with complete regeneration of subchondral bone by 12 months. Histological analysis confirmed the formation of well-structured subchondral trabecular bone and hyaline-like cartilage tissue in the multi-layered scaffold group by 12 months with restoration of the anatomical tidemark. Demonstration of improved healing following treatment with this natural polymer scaffold, through the recruitment of host cells with no requirement for pre-culture, shows the potential of this device for the treatment of patients presenting with osteochondal lesions.

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February 2016

Addition of Mesenchymal Stem Cells to Autologous Platelet-Enhanced Fibrin Scaffolds in Chondral Defects: Does It Enhance Repair?
PubMed Article 
View Abstract

Goodrich LR, Chen AC, Werpy NM, Williams AA, Kisiday JD, Su AW, Cory E, Morley PS, McIlwraith CW, Sah RL, Chu CR.

BACKGROUND:

The chondrogenic potential of culture-expanded bone-marrow-derived mesenchymal stem cells (BMDMSCs) is well described. Numerous studies have also shown enhanced repair when BMDMSCs, scaffolds, and growth factors are placed into chondral defects. Platelets provide a rich milieu of growth factors and, along with fibrin, are readily available for clinical use. The objective of this study was to determine if the addition of BMDMSCs to an autologous platelet-enriched fibrin (APEF) scaffold enhances chondral repair compared with APEF alone.

METHODS:

A 15-mm-diameter full-thickness chondral defect was created on the lateral trochlear ridge of both stifle joints of twelve adult horses. In each animal, one defect was randomly assigned to receive APEF+BMDMSCs and the contralateral defect received APEF alone. Repair tissues were evaluated one year later with arthroscopy, histological examination, magnetic resonance imaging (MRI), micro-computed tomography (micro-CT), and biomechanical testing.

RESULTS:

The arthroscopic findings, MRI T2 map, histological scores, structural stiffness, and material stiffness were similar (p > 0.05) between the APEF and APEF+BMDMSC-treated repairs at one year. Ectopic bone was observed within the repair tissue in four of twelve APEF+BMDMSC-treated defects. Defects repaired with APEF alone had less trabecular bone edema (as seen on MRI) compared with defects repaired with APEF+BMDMSCs. Micro-CT analysis showed thinner repair tissue in defects repaired with APEF+BMDMSCs than in those treated with APEF alone (p < 0.05).

CONCLUSIONS:

APEF alone resulted in thicker repair tissue than was seen with APEF+BMDMSCs. The addition of BMDMSCs to APEF did not enhance cartilage repair and stimulated bone formation in some cartilage defects.

CLINICAL RELEVANCE:

APEF supported repair of critical-size full-thickness chondral defects in horses, which was not improved by the addition of BMDMSCs. This work supports further investigation to determine whether APEF enhances cartilage repair in humans.

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January 2016

A chondrocyte infiltrated collagen type I/III membrane (MACI® implant) improves cartilage healing in the equine patellofemoral joint model.
PubMed Article 
View Abstract

Nixon AJ, Rickey E, Butler TJ, Scimeca MS, Moran N, Matthews GL.

Autologous chondrocyte implantation (ACI) has improved outcome in long-term studies of joint repair in man. However, ACI requires sutured periosteal flaps to secure the cells, which precludes minimally-invasive implantation, and introduces complications with arthrofibrosis and graft hypertrophy. This study evaluated ACI on a collagen type I/III scaffold (matrix-induced autologous chondrocyte implantation; MACI(®)) in critical sized defects in the equine model.

METHODS:

Chondrocytes were isolated from horses, expanded and seeded onto a collagen I/III membrane (ACI-Mai™) and implanted into one of two 15-mm defects in the femoral trochlear ridge of six horses. Control defects remained empty as ungrafted debrided defects. The animals were examined daily, scored by second look arthroscopy at 12 weeks, and necropsy examination 6 months after implantation. Reaction to the implant was determined by lameness, and synovial fluid constituents and synovial membrane histology. Cartilage healing was assessed by arthroscopic scores, gross assessment, repair tissue histology and immunohistochemistry, cartilage glycosaminoglycan (GAG) and DNA assay, and mechanical testing.

RESULTS:

MACI(®) implanted defects had improved arthroscopic second-look, gross healing, and composite histologic scores, compared to spontaneously healing empty defects. Cartilage GAG and DNA content in the defects repaired by MACI implant were significantly improved compared to controls. Mechanical properties were improved but remained inferior to normal cartilage. There was minimal evidence of reaction to the implant in the synovial fluid, synovial membrane, subchondral bone, or cartilage.

CONCLUSIONS:

The MACI(®) implant appeared to improve cartilage healing in a critical sized defect in the equine model evaluated over 6 months.

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Annular puncture with tumor necrosis factor-alpha injection enhances painful behavior with disc degeneration in vivo
PubMed Article 
View Abstract

Lai A, Moon A, Purmessur D, Skovrlj B, Laudier DM, Winkelstein BA, Cho SK, Hecht AC, Iatridis JC.

BACKGROUND CONTEXT:

Painfulintervertebral disc degeneration is extremely common and costly. Effective treatments are lacking because the nature of discogenic pain is complex with limited capacity to distinguish painful conditions from age-related changes in the spine. Hypothesized sources of discogenic pain include chronic inflammation, neurovascular ingrowth, and structural disruption.

PURPOSE:

This study aimed to investigate inflammation, pro-neurovascular growth factors, and structural disruption as sources of painful disc degeneration

STUDY DESIGN/SETTING:

This study used an in vivo study to address these hypothesized mechanisms with anterior intradiscal injections of tumor necrosis factor-alpha (TNFα), pro-neurovascular growth factors: nerve growth factor and vascular endothelial growth factor (NGF and VEGF), and saline with additional sham surgery and naïve controls. Depth of annular puncture was also evaluated for its effects on structural and painful degeneration.

METHODS:

Rat lumbar discs were punctured (shallow or deeper puncture) and intradiscally injected with saline, TNFα, or NGF and VEGF. Structural disc degeneration was assessed using X-ray, magnetic resonance imaging (MRI), and histology. The rat painful condition was evaluated using Von Frey hyperalgesia measurements, and substance P immunostaining in dorsal root ganglion (DRG) was performed to determine the source of pain.

RESULTS:

Saline injection increased painful responses with degenerative changes in disc height, MRI intensity, and morphologies of disc structure and cell. TNFα and NGF/VEGF accelerated painful behavior, and TNFα-injected animals had increased substance P in DRGs. Deeper punctures led to more severe disc degeneration. Multiple regression analysis showed that the painful behavior was correlated with disc height loss.

CONCLUSIONS:

We concluded that rate and severity of structural disc degeneration was associated with the amount of annular disruption and puncture depth. The painful behavior was associated with disc height loss and discal inflammatory state, whereas pro-inflammatory cytokines might play a more important role in the level of pain, which might have resulted from enhanced DRG sensitization. These in vivo painful disc degeneration models with different severities of structural changes may be useful for investigating discogenic pain mechanisms and for screening therapies, although interpretations must note the differences between all surgically induced animal models and the human condition.

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December 2015

Chitosan microspheres with an extracellular matrix-mimicking nanofibrous structure as cell-carrier building blocks for bottom-up cartilage tissue engineering.
PubMed Article 
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Zhou Y, Gao HL, Shen LL, Pan Z, Mao LB, Wu T, He JC, Zou DH, Zhang ZY, Yu SH.

Scaffolds for tissue engineering (TE) which closely mimic the physicochemical properties of the natural extracellular matrix (ECM) have been proven to advantageously favor cell attachment, proliferation, migration and new tissue formation. Recently, as a valuable alternative, a bottom-up TE approach utilizing cell-loaded micrometer-scale modular components as building blocks to reconstruct a new tissue in vitro or in vivo has been proved to demonstrate a number of desirable advantages compared with the traditional bulk scaffold based top-down TE approach. Nevertheless, micro-components with an ECM-mimicking nanofibrous structure are still very scarce and highly desirable. Chitosan (CS), an accessible natural polymer, has demonstrated appealing intrinsic properties and promising application potential for TE, especially the cartilage tissue regeneration. According to this background, we report here the fabrication of chitosan microspheres with an ECM-mimicking nanofibrous structure for the first time based on a physical gelation process. By combining this physical fabrication procedure with microfluidic technology, uniform CS microspheres (CMS) with controlled nanofibrous microstructure and tunable sizes can be facilely obtained. Especially, no potentially toxic or denaturizing chemical crosslinking agent was introduced into the products. Notably, in vitro chondrocyte culture tests revealed that enhanced cell attachment and proliferation were realized, and a macroscopic 3D geometrically shaped cartilage-like composite can be easily constructed with the nanofibrous CMS (NCMS) and chondrocytes, which demonstrate significant application potential of NCMS as the bottom-up cell-carrier components for cartilage tissue engineering.

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November, 2015

Positive effects of cell-free porous PLGA implants and early loading exercise on hyaline cartilage regeneration in rabbits.
PubMed Article 
View Abstract

Chang NJ, Lin CC, Shie MY, Yeh ML, Li CF, Liang PI, Lee KW, Shen PH, Chu CJ.

The regeneration of hyaline cartilage remains clinically challenging. Here, we evaluated the therapeutic effects of using cell-free porous poly(lactic-co-glycolic acid) (PLGA) graft implants (PGIs) along with early loading exercise to repair a full-thickness osteochondral defect. Rabbits were randomly allocated to a treadmill exercise (TRE) group or a sedentary (SED) group and were prepared as either a PGI model or an empty defect (ED) model. TRE was performed as a short-term loading exercise; SED was physical inactivity in a free cage. The knees were evaluated at 6 and 12weeks after surgery. At the end of testing, none of the knees developed synovitis, formed osteophytes, or became infected. Macroscopically, the PGI-TRE group regenerated a smooth articular surface, with transparent new hyaline-like tissue soundly integrated with the neighboring cartilage, but the other groups remained distinct at the margins with fibrous or opaque tissues. In a micro-CT analysis, the synthesized bone volume/tissue volume (BV/TV) was significantly higher in the PGI-TRE group, which also had integrating architecture in the regeneration site. The thickness of the trabecular (subchondral) bone was improved in all groups from 6 to 12weeks. Histologically, remarkable differences in the cartilage regeneration were visible. At week 6, compared with SED groups, the TRE groups manifested modest inflammatory cells with pro-inflammatory cytokines (i.e., TNF-α and IL-6), improved collagen alignment and higher glycosaminoglycan (GAG) content, particularly in the PGI-TRE group. At week 12, the PGI-TRE group had the best regeneration outcomes, showing the formation of hyaline-like cartilage, the development of columnar rounded chondrocytes that expressed enriched levels of collagen type II and GAG, and functionalized trabecular bone with osteocytes. In summary, the combination of implanting cell-free PLGA and performing an early loading exercise can significantly promote the full-thickness osteochondral regeneration in rabbit knee joint models.

STATEMENT OF SIGNIFICANCE:

Promoting effective hyaline cartilage regeneration rather than fibrocartilage scar tissue remains clinically challenging. To address the obstacle, we fabricated a spongy cell-free PLGA scaffold, and designed a reasonable exercise program to generate combined therapeutic effects. First, the implanting scaffold generates an affordable mechanical structure to bear the loading forces and bridge with the host to offer a space in the full-thickness osteochondral regeneration in rabbit knee joint. After implantation, rabbits were performed by an early treadmill exercise 15min/day, 5days/week for 2weeks that directly exerts in situ endogenous growth factor and anti-inflammatory effects in the reparative site. The advanced therapeutic strategy showed that neo-hyaline cartilage formation with enriched collagen type II, higher glycosaminoglycan, integrating subchondral bone formation and modest inflammation.

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Successful Total Meniscus Reconstruction Using a Novel Fiber-Reinforced Scaffold: A 16- and 32-Week Study in an Ovine Model.
PubMed Article 
View Abstract

Merriam AR, Patel JM, Culp BM, Gatt CJ Jr, Dunn MG.

BACKGROUND:

Meniscus injuries in the United States result in an estimated 850,000 surgical procedures each year. Although meniscectomies are the most commonly performed orthopaedic surgery, little advancement has been made in meniscus replacement and regeneration, and there is currently no total meniscus replacement device approved by the Food and Drug Administration.

HYPOTHESIS:

A novel fiber-reinforced meniscus scaffold can be used as a functional total meniscus replacement.

STUDY DESIGN:

Controlled laboratory study.

METHODS:

A tyrosine-derived, polymer fiber-reinforced collagen sponge meniscus scaffold was evaluated mechanically (tensile and compressive testing) and histologically after 16 and 32 weeks of implantation in an ovine total meniscectomy model (N = 20; 16 implants plus 4 meniscectomies, divided equally over the 2 time periods). The extent of cartilage damage was also measured on tibial plateaus by use of toluidine blue surface staining and on femoral condyles by use of Mankin scores on histological slides.

RESULTS:

Scaffolds induced formation of neomeniscus tissue that remained intact and functional, with breaking loads approximating 250 N at both 16 and 32 weeks compared with 552 N for native menisci. Tensile stiffness values (99 and 74 N/mm at 16 and 32 weeks, respectively) were also comparable with those of the native meniscus (147 N/mm). The compressive modulus of the neomeniscus tissue (0.33 MPa at both 16 and 32 weeks) was significantly increased compared with unimplanted (time 0) scaffolds (0.15 MPa). There was histological evidence of extensive tissue ingrowth and extracellular matrix deposition, with immunohistochemical evidence of types I and II collagen. Based on significantly decreased surface damage scores as well as Mankin scores, the scaffold implants provided greater protection of articular cartilage compared with the untreated total meniscectomy.

CONCLUSION:

This novel fiber-reinforced meniscus scaffold can act as a functional meniscus replacement, with mechanical properties similar to those of the native meniscus, while protecting the articular cartilage of the knee from the extensive damage after a total meniscectomy.

CLINICAL RELEVANCE:

This meniscus replacement scaffold has the potential to improve surgical treatment and provide better long-term outcomes for those suffering from severe meniscus damage.

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October 2015

Intervention of rAAV-hTERT-Transducted Nucleus Pulposus Cells in Early Stage of Intervertebral Disc Degeneration: A Study in Canine Model.
PubMed Article 
View Abstract

Shi Z, Gu T, Xin H, Wu J, Xu C, Zhang C, He Q, Ruan D.

OBJECTIVES:

To investigate the efficacy of recombinant adeno-associated virus (rAAV)-human telomerase reverse transcriptase (hTERT)-transducted nucleus pulposus cells (NPCs) in disc degeneration process in a canine disc degeneration model.

METHODS:

The intervertebral disc degeneration of lumbar (L) 1-2, L3-4, and L5-6, from 12 female mongrels was prepared with the 20-gauge biopsy gun. Four weeks after animal model preparation, intervention experiment with rAAV-hTERT-transducted NPCs was conducted: group A, L1-2, serum-free medium with rAAV-hTERT modified NPCs; group B, L3-4, serum-free medium with NPCs; group C, L5-6, serum-free medium alone. Canines underwent digital radiography and magnetic resonance imaging 1 day before intervention, and 4, 8, and 12 weeks after intervention to evaluate the change of disc height and hydration status of interventional intervertebral discs. Twelve weeks after intervention, histological, biomechanical, and biochemical studies were carried out.

RESULTS:

The rAAV-hTERT-transducted NPCs were constructed successfully. The mRNA level of hTERT from rAAV-hTERT-transfected NPCs increased obviously. There was no significant change of disc height index observed between groups and within groups. The relative grayscale index (RGI) was maintained 8 weeks after the intervention in group A, whereas in group B and group C, the RGI decreased significantly (p<0.05). No significant differences of the angle of lateral bending and extension-flexion bending were observed in group A compared with other groups (p>0.05). The morphology of disc structure was preserved in group A. In group B, the structure of inner annulus was broken down and the jelly-like nucleus pulposus (NP) tissue transmitted into the fibrocartilaginous tissue. In group C, the jelly-like NP tissue was completely replaced by fibrocartilaginous tissue. In the NP, the content of proteoglycan (PG) and collagen II was higher in group A than in group C (p<0.05). The content of PG was 13, 8.9, and 15.6 times higher than the content of collagen II in group A, group B, and group C, respectively.

CONCLUSIONS:

In 12 weeks of observation, rAAV-hTERT-transducted NPCs could delay the degeneration process in the canine model which was superior than the capacity of NPCs in preserving structure integrity, content of extracellular matrix, and mechanical stability.

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September 2015

Short Term Evaluation of an Anatomically Shaped Polycarbonate Urethane Total Meniscus Replacement in a Goat Model
PubMed Article 
View Abstract

Vrancken AC, Madej W, Hannink G, Verdonschot N, van Tienen TG, Buma P.

PURPOSE:

Since the treatment options for symptomatic total meniscectomy patients are still limited, an anatomically shaped, polycarbonate urethane (PCU), total meniscus replacement was developed. This study evaluates the in vivo performance of the implant in a goat model, with a specific focus on the implant location in the joint, geometrical integrity of the implant and the effect of the implant on synovial membrane and articular cartilage histopathological condition.

METHODS:

The right medial meniscus of seven Saanen goats was replaced by the implant. Sham surgery (transection of the MCL, arthrotomy and MCL suturing) was performed in six animals. The contralateral knee joints of both groups served as control groups. After three months follow-up the following aspects of implant performance were evaluated: implant position, implant deformation and the histopathological condition of the synovium and cartilage.

RESULTS:

Implant geometry was well maintained during the three month implantation period. No signs of PCU wear were found and the implant did not induce an inflammatory response in the knee joint. In all animals, implant fixation was compromised due to suture breakage, wear or elongation, likely causing the increase in extrusion observed in the implant group. Both the femoral cartilage and tibial cartilage in direct contact with the implant showed increased damage compared to the sham and sham-control groups.

CONCLUSION:

This study demonstrates that the novel, anatomically shaped PCU total meniscal replacement is biocompatible and resistant to three months of physiological loading. Failure of the fixation sutures may have increased implant mobility, which probably induced implant extrusion and potentially stimulated cartilage degeneration. Evidently, redesigning the fixation method is necessary. Future animal studies should evaluate the improved fixation method and compare implant performance to current treatment standards, such as allografts.

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August 2015

Intervertebral disc repair with activated nucleus pulposus cell transplantation: a three-year, prospective clinical study of its safety.
PubMed Article 
View Abstract

Mochida J, Sakai D, Nakamura Y, Watanabe T, Yamamoto Y, Kato S.

Degeneration of the lumbar intervertebral discs is irreversible, with no treatment currently available. Building upon experimental studies that demonstrated the importance of the nucleus pulposus (NP) in preserving disc structure, we demonstrated that reinsertion of NP cells slowed further disc degeneration and that direct cell-to-cell contact co-culture with mesenchymal stromal cells (MSCs) significantly upregulated the viability of NP cells in basic and pre-clinical studies in vitro and in vivo using animal models and human cells. Here, we report a 3-year result of a prospective clinical study, aimed to assess the safety and efficacy of activated NP cell transplantation in the degenerate lumbar intervertebral disc. Candidates were 9 patients aged 20-29 years who had Pfirrmann's grade III disc degeneration at the level adjacent to the level scheduled for posterior lumbar intervertebral fusion. Viable NP cells from the fused disc were co-cultured in direct contact with autologous bone marrow-derived MSCs. One million activated NP cells were transplanted into the degenerated disc adjacent to the fused level at 7 d after the first fusion surgery. No adverse effects were observed during the 3-year follow-up period. Magnetic resonance imaging did not show any detrimental effects to the transplanted discs and revealed a mild improvement in 1 case. No cases reported any low back pain. Our clinical study confirmed the safety of activated NP cell transplantation, and the findings suggest the minimal efficacy of this treatment to slow the further degeneration of human intervertebral discs.

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July 2015

Scaffold-guided subchondral bone repair: implication of neutrophils and alternatively activated arginase-1+ macrophages.
PubMed Article 
View Abstract

Hoemann CD, Chen G, Marchand C, Tran-Khanh N, Thibault M, Chevrier A, Sun J, Shive MS, Fernandes MJ, Poubelle PE, Centola M, El-Gabalawy H.

BACKGROUND:

Microfracture and drilling elicit a cartilage repair whose quality depends on subchondral bone repair. Alternatively activated (AA) macrophages express arginase-1, release angiogenic factors, and could be potential mediators of trabecular bone repair.

HYPOTHESIS:

Chitosan-glycerol phosphate (GP)/blood implants elicit arginase-1+ macrophages in vivo through neutrophil-dependent mechanisms and improve trabecular bone repair of drilled defects compared with drilling alone.

STUDY DESIGN:

Controlled laboratory study.

METHODS:

Bilateral trochlear cartilage defects were created in 15 rabbits, microdrilled, and treated or not with chitosan-GP/blood implant to analyze AA macrophages, CD-31+ blood vessels, bone, and cartilage repair after 1, 2, or 8 weeks. Neutrophil and macrophage chemotaxis to rabbit subcutaneous implants of autologous blood and chitosan-GP (+/-blood) was quantified at 1 or 7 days. In vitro, sera from human chitosan-GP/blood and whole blood clots cultured at 37 degrees C were analyzed by proteomics and neutrophil chemotaxis assays.

RESULTS:

Chitosan-GP/blood clots and whole blood clots released a similar profile of chemotactic factors (PDGF-BB, IL-8/CXCL8, MCP-1/CCL2, and no IL-1beta or IL-6), although chitosan clot sera attracted more neutrophils in vitro. Subcutaneous chitosan-GP (+/-blood) implants attracted more neutrophils (P < .001) and AA macrophages than whole blood clots in vivo. In repairing subchondral drill holes, chitosan-GP/blood implant attracted more AA macrophages at 1 and 2 weeks and more blood vessels at 2 weeks compared with drilled controls. Treatment elicited a more complete woven bone repair at 8 weeks than controls (P = .0011) with a more uniform, integrated collagen type II+ cartilage repair tissue.

CONCLUSION AND CLINICAL RELEVANCE:

AA macrophages may play a role in the regeneration of subchondral bone, and chitosan-GP can attract and transiently accumulate these cells in the repair tissue. The resulting improved subchondral repair could be advantageous toward enhancing integration of a restored chondral surface to the subchondral bone.

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June 2015

Injectable microcryogels reinforced alginate encapsulation of mesenchymal stromal cells for leak-proof delivery and alleviation of canine disc degeneration
PubMed Article 
View Abstract

Yang Zeng, Chun Chen, Wei Liu, Qinyouen Fu, Zhihua Han, Yaqian Li, Siyu Feng, Xiaokang Li, Chunxiao Qi, Jianhong Wu, Deli Wang, Christopher Corbett, Barbara P. Chan, Dike Ruan, Yanan Du

In situ crosslinked thermo-responsive hydrogel applied for minimally invasive treatment of intervertebraldisc degeneration (IVDD) may not prevent extrusion of cell suspension from injection site due tohigh internal pressure of intervertebral disc (IVD), causing treatment failure or osteophyte formation. Inthis study, mesenchymal stromal cells (MSCs) were encapsulated in alginate precursor and loaded intopreviously developed macroporous PGEDA-derived microcryogels (PMs) to form three-dimensional (3D)microscale cellular niches, enabling non-thermo-responsive alginate hydrogel to be injectable. The PMsreinforced alginate hydrogel showed superior elasticity compared to alginate hydrogel alone and couldwell protect encapsulated cells through injection. Chondrogenic committed MSCs in the injectablemicroniches expressed higher level of nucleus pulposus (NP) cell markers compared to 2D cultured cells.In an ex vivo organ culture model, injection of MSCs-laden PMs into NP tissue prevented cell leakage,improved cell retention and survival compared to free cell injection. In canine IVDD models, alleviateddegeneration was observed in MSCs-laden PMs treated group after six months which was superior toother treated groups. Our results provide in-depth demonstration of injectable alginate hydrogel reinforcedby PMs as a leak-proof cell delivery system for augmented regenerative therapy of IVDD in canine

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May 2015

Adult human neural crest–derived cells for articular cartilage repair
PubMed Article 
View Abstract

Karoliina Pelttari, Benjamin Pippenger, Marcus Mumme, Sandra Feliciano, Celeste Scotti, Pierre Mainil-Varlet, Alfredo Procino, Brigitte von Rechenberg, Thomas Schwamborn, Marcel Jakob, Clemente Cillo, Andrea Barbero, Ivan Martin

In embryonic models and stem cell systems, mesenchymal cells derived from the neuroectoderm can be distinguished from mesoderm-derived cells by their Hox-negative profile—a phenotype associated with enhanced capacity of tissue regeneration. We investigated whether developmental origin and Hox negativity correlated with self-renewal and environmental plasticity also in differentiated cells from adults. Using hyaline cartilage as a model, we showed that adult human neuroectoderm-derived nasal chondrocytes (NCs) can be constitutively distinguished from mesoderm-derived articular chondrocytes (ACs) by lack of expression of specific HOX genes, including HOXC4 and HOXD8. In contrast to ACs, serially cloned NCs could be continuously reverted from differentiated to dedifferentiated states, conserving the ability to form cartilage tissue in vitro and in vivo. NCs could also bereprogrammed to stably express Hox genes typical of ACs upon implantation into goat articular cartilage defects, directly contributing to cartilage repair. Our findings identify previously unrecognized regenerative properties ofHOX-negative differentiated neuroectoderm cells in adults, implying a role for NCs in the unmet clinical challenge of articular cartilage repair. An ongoing phase 1 clinical trial preliminarily indicated the safety and feasibility of autologous NC–based engineered tissues for the treatment of traumatic articular cartilage lesions.

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April 2015

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

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 
View Abstract

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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March 2015

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

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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February 2015

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

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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January 2015

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

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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December 2014

Developing functional musculoskeletal tissues through hypoxia and lysyl oxidase-induced collagen cross-linking.
PubMed Article 
View Abstract

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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November 2014

Establishment of a Herniation Model and Experiments With an Anular Closure Device.
PubMed Article 
View Abstract

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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October 2014

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

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 
View Abstract

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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September 2014

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

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 
View Abstract

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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August 2014

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

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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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 
View Abstract

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 
View Abstract

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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June 2014

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

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 
View Abstract

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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May 2014

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

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 
View Abstract

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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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 
View Abstract

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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March 2014

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

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 
View Abstract

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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February 2014

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

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 
View Abstract

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 
View Abstract

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 
View Abstract

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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January 2014

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

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 
View Abstract

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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December 2013

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

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 
View Abstract

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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April 22, 2013

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

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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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 
View Abstract

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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December 17, 2012

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

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 
View Abstract

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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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 
View Abstract

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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October 22, 2012

Osteochondritis Dissecans Knee Histology Studies Have Variable Findings and Theories of Etiology.
PubMed Article 
View Abstract

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 
View Abstract

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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October 8, 2012

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

Kon E, Filardo G, Tschon M, Fini M, Giavaresi G, Marchesini Reggiani L, Chiari C, Nehrer S, Martin I, Salter DM, Ambrosio L, Marcacci M.

The aim of the study was to investigate the use of a hyaluronic acid/polycaprolactone material for meniscal tissue engineering and to evaluate the tissue regeneration after the augmentation of the implant with expanded autologous chondrocytes. Eighteen skeletally mature sheep were treated. The animals were divided into three groups: cell-free scaffold, scaffold seeded with autologous chondrocytes, and meniscectomy alone. The implant was sutured to the capsule and to the meniscal ligament. At a 12-month gross assessment, histology and histomorphometry were used to assess the meniscus implant, knee joint, and osteoarthritis development. All implants showed excellent capsular ingrowth at the periphery. The implant gross assessment showed significant differences between cell-seeded and cell-free groups (p=0.011). The histological analysis indicated a cellular colonization throughout the implanted constructs. Avascular cartilaginous tissue formation was significantly more frequent in the cell-seeded constructs. Joint gross assessment showed that sheep treated with scaffold implantation achieved a significant higher score than those underwent meniscectomy (p<0.0005), and the Osteoarthritis Research Society International score showed that osteoarthritic changes were significantly less in the cell-seeded group than in the meniscectomy group (p=0.047), even though results were not significantly superior to those of the cell-free scaffold. Seeding of the scaffold with autologous chondrocytes increases its tissue regeneration capacity, providing a better fibrocartilaginous tissue formation. The study suggests the potential of the novel hyaluronic acid/polycaprolactone scaffold for total meniscal substitution, although this approach has to be further improved before being applied into clinical practice.

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Isolation, characterization, and differentiation of stem cells for cartilage regeneration.
PubMed Article 
View Abstract

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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