Strategies for the Codelivery of Osteoclasts and Mesenchymal Stem Cells in 3D-Printable Osteochondral Scaffolds.

Osteochondral defects, characterized by structural compromises to articular cartilage and subchondral bone, can cause pain and lead to progressive cartilage damage and eventual osteoarthritis. Unfortunately, repairing these defects remains difficult because of the poor regenerative properties of cartilage and complex mechanical demands of the joint. As such, the field of tissue engineering aims to develop multiphasic implants that replace pathological cartilage and bone tissue and restore mechanical functionality to the joint. Recent bone physiology investigations have demonstrated that osteoclast (OC) lineage cells are inextricably involved in osteoblastic bone formation through an extensive network of anabolic signaling pathways, and so the codelivery OC and osteoblast (OB) lineage cells within scaffolds is being actively explored for bone tissue engineering purposes. However, it remains unclear how these cells can be incorporated into the design of multiphasic osteochondral scaffolds to potentially enhance subchondral bone formation and subsequent implant osseointegration. To explore this question, we examined direct surface seeding and hydrogel encapsulation as potential scaffold cellularization strategies. First, we examined how OC precursor cells and peripheral blood monocytes (PBMCs) influence early-stage bone matrix development and osteogenesis in 2D coculture. Then, we evaluated the osteogenic potential of mesenchymal stem cells (MSCs) and PBMCs cocultures encapsulated within a gelatin methacrylate (GelMA) hydrogel system. Our findings demonstrate that coculturing PBMCs with MSCs in 2D cultures significantly enhanced cell proliferation, early bone matrix deposition, and the formation of cell clusters by Day 28. However, we observed no significant difference in type I collagen deposition between GelMA hydrogel scaffolds cultured in basal and OC conditions during the same period. In addition, we found that the GelMA hydrogel system with MSC/PBMC cocultures in OC conditions exhibited decreased osteogenic activity by Day 28. Collectively, our findings support the osteogenic potential of OC-lineage cells in 2D culture conditions, and the potential benefits of surface-seeding for the codelivery of OC-lineage cells and MSCs in osteo-scaffolds for enhanced osteochondral regeneration and broader bone tissue engineering purposes.

Bioceramic-mediated chondrocyte hypertrophy promotes calcified cartilage formation for rabbit osteochondral defect repair.

Osteochondral tissue is a highly specialized and complex tissue composed of articular cartilage and subchondral bone that are separated by a calcified cartilage interface. Multilayered or gradient scaffolds, often in conjunction with stem cells and growth factors, have been developed to mimic the respective layers for osteochondral defect repair. In this study, we designed a hyaline cartilage-hypertrophic cartilage bilayer graft (RGD/RGDW) with chondrocytes. Previously, we demonstrated that RGD peptide-modified chondroitin sulfate cryogel (RGD group) is chondro-conductive and capable of hyaline cartilage formation. Here, we incorporated whitlockite (WH), a Mg2+-containing calcium phosphate, into RGD cryogel (RGDW group) to induce chondrocyte hypertrophy and form collagen X-rich hypertrophic cartilage. This is the first study to use WH to produce hypertrophic cartilage. Chondrocytes-laden RGDW cryogel exhibited significantly upregulated expression of hypertrophy markers in vitro and formed ectopic hypertrophic cartilage in vivo, which mineralized into calcified cartilage in bone microenvironment. Subsequently, RGD cryogel and RGDW cryogel were combined into bilayer (RGD/RGDW group) and implanted into rabbit osteochondral defect, where RGD layer supports hyaline cartilage regeneration and bioceramic-containing RGDW layer promotes calcified cartilage formation. While the RGD group (monolayer) formed hyaline-like neotissue that extends into the subchondral bone, the RGD/RGDW group (bilayer) regenerated hyaline cartilage tissue confined to its respective layer and promoted osseointegration for integrative defect repair.

Cytokine Receptor-like Factor 1 (CRLF1) and Its Role in Osteochondral Repair.

BACKGROUND: Since cytokine receptor-like factor 1 (CRLF1) has been implicated in tissue regeneration, we hypothesized that CRLF1 released by mesenchymal stem cells can promote the repair of osteochondral defects. METHODS: The degree of a femoral osteochondral defect repair in rabbits after intra-articular injections of bone marrow-derived mesenchymal stem cells (BMSCs) that were transduced with empty adeno-associated virus (AAV) or AAV containing CRLF1 was determined by morphological, histological, and micro computer tomography (CT) analyses. The effects of CRLF1 on chondrogenic differentiation of BMSCs or catabolic events of interleukin-1beta-treated chondrocyte cell line TC28a2 were determined by alcian blue staining, gene expression levels of cartilage and catabolic marker genes using real-time PCR analysis, and immunoblot analysis of Smad2/3 and STAT3 signaling. RESULTS: Intra-articular injections of BMSCs overexpressing CRLF1 markedly improved repair of a rabbit femoral osteochondral defect. Overexpression of CRLF1 in BMSCs resulted in the release of a homodimeric CRLF1 complex that stimulated chondrogenic differentiation of BMSCs via enhancing Smad2/3 signaling, whereas the suppression of CRLF1 expression inhibited chondrogenic differentiation. In addition, CRLF1 inhibited catabolic events in TC28a2 cells cultured in an inflammatory environment, while a heterodimeric complex of CRLF1 and cardiotrophin-like Cytokine (CLC) stimulated catabolic events via STAT3 activation. CONCLUSION: A homodimeric CRLF1 complex released by BMSCs enhanced the repair of osteochondral defects via the inhibition of catabolic events in chondrocytes and the stimulation of chondrogenic differentiation of precursor cells.

High quality repair of osteochondral defects in rats using the extracellular matrix of antler stem cells.

BACKGROUND: Cartilage defects are some of the most common causes of arthritis. Cartilage lesions caused by inflammation, trauma or degenerative disease normally result in osteochondral defects. Previous studies have shown that decellularized extracellular matrix (ECM) derived from autologous, allogenic, or xenogeneic mesenchymal stromal cells (MSCs) can effectively restore osteochondral integrity. AIM: To determine whether the decellularized ECM of antler reserve mesenchymal cells (RMCs), a xenogeneic material from antler stem cells, is superior to the currently available treatments for osteochondral defects. METHODS: We isolated the RMCs from a 60-d-old sika deer antler and cultured them in vitro to 70% confluence; 50 mg/mL L-ascorbic acid was then added to the medium to stimulate ECM deposition. Decellularized sheets of adipocyte-derived MSCs (aMSCs) and antlerogenic periosteal cells (another type of antler stem cells) were used as the controls. Three weeks after ascorbic acid stimulation, the ECM sheets were harvested and applied to the osteochondral defects in rat knee joints. RESULTS: The defects were successfully repaired by applying the ECM-sheets. The highest quality of repair was achieved in the RMC-ECM group both in vitro (including cell attachment and proliferation), and in vivo (including the simultaneous regeneration of well-vascularized subchondral bone and avascular articular hyaline cartilage integrated with surrounding native tissues). Notably, the antler-stem-cell-derived ECM (xenogeneic) performed better than the aMSC-ECM (allogenic), while the ECM of the active antler stem cells was superior to that of the quiescent antler stem cells. CONCLUSION: Decellularized xenogeneic ECM derived from the antler stem cell, particularly the active form (RMC-ECM), can achieve high quality repair/reconstruction of osteochondral defects, suggesting that selection of decellularized ECM for such repair should be focused more on bioactivity rather than kinship.

In vitro and in vivo investigation of a zonal microstructured scaffold for osteochondral defect repair.

Articular cartilage is comprised of zones that vary in architecture, extracellular matrix composition, and mechanical properties. Here, we designed and engineered a porous zonal microstructured scaffold from a single biocompatible polymer (poly [ϵ-caprolactone]) using multiple fabrication strategies: electrospinning, spherical porogen leaching, directional freezing, and melt electrowriting. With this approach we mimicked the zonal structure of articular cartilage and produced a stiffness gradient through the scaffold which aligns with the mechanics of the native tissue. Chondrocyte-seeded scaffolds accumulated extracellular matrix including glycosaminoglycans and collagen II over four weeks in vitro. This prompted us to further study the repair efficacy in a skeletally mature porcine model. Two osteochondral lesions were produced in the trochlear groove of 12 animals and repaired using four treatment conditions: (1) microstructured scaffold, (2) chondrocyte seeded microstructured scaffold, (3) MaioRegen™, and (4) empty defect. After 6 months the defect sites were harvested and analyzed using histology, micro computed tomography, and Raman microspectroscopy mapping. Overall, the scaffolds were retained in the defect space, repair quality was repeatable, and there was clear evidence of osteointegration. The repair quality of the microstructured scaffolds was not superior to the control based on histological scoring; however, the lower score was biased by the lack of histological staining due to the limited degradation of the implant at 6 months. Longer follow up studies (e.g., 1 yr) will be required to fully evaluate the efficacy of the microstructured scaffold. In conclusion, we found consistent scaffold retention, osteointegration, and prolonged degradation of the microstructured scaffold, which we propose may have beneficial effects for the long-term repair of osteochondral defects.

Innovative Tissue-Engineered Strategies for Osteochondral Defect Repair and Regeneration: Current Progress and Challenges.

Clinical treatments for the repair of osteochondral defects (OCD) are merely palliative, not completely curative, and thus enormously unfulfilled challenges. With the in-depth studies of biology, medicine, materials, and engineering technology, the conception of OCD repair and regeneration should be renewed. During the past decades, many innovative tissue-engineered approaches for repairing and regenerating damaged osteochondral units have been widely explored. Various scaffold-free and scaffold-based strategies, such as monophasic, biphasic, and currently fabricated multiphasic and gradient architectures have been proposed and evaluated. Meanwhile, progenitor cells and tissue-specific cells have also been intensively investigated in vivo as well as ex vivo. Concerning bioactive factors and drugs, they have been combined with scaffolds and/or living cells, and even released in a spatiotemporally controlled manner. Although tremendous progress has been achieved, further research and development (R&D) is needed to convert preclinical outcomes into clinical applications. Here, the osteochondral unit structure, its defect classifications, and diagnosis are summarized. Commonly used clinical reparative techniques, tissue-engineered strategies, emerging 3D-bioprinting technologies, and the status of their clinical applications are discussed. Existing challenges to translation are also discussed and potential solutions for future R&D directions are proposed.

Mussel-Inspired Tough Hydrogel with In Situ Nanohydroxyapatite Mineralization for Osteochondral Defect Repair.

Repairing osteochondral defects is a considerable challenge because it involves the breakdown of articular cartilage and underlying bone. Traditional hydrogels with a homogenized single-layer structure cannot fully restore the function of osteochondral cartilage tissue. In this study, a mussel-inspired hydrogel with a bilayer structure is developed to repair osteochondral defects. The hydrogel is synthesized by simultaneously polymerizing two layers using a one-pot method. The resulting upper and lower gelatin methacryloyl-polydopamine hydrogel layers are used as cartilage and subchondral bone repair layers, respectively. Polydopamine-induced hydroxyapatite in situ mineralization takes place in the lower layer to mimic the structure of subchondral bone. The bilayer hydrogel exhibits good mechanical properties for the synergistic effect of covalent and noncovalent bonds, as well as nanoreinforcement of mineralized hydroxyapatite. To improve the tissue-inducibility of hydrogels, transforming growth factor ß3 is immobilized in the upper layer to induce cartilage regeneration, while bone morphogenetic protein 2 is immobilized in the lower layer to induce bone regeneration. Bone and cartilage repair performance of the hydrogel is examined by implantation into a full-thickness cartilage defect of a rabbit knee joint. The bilayer-structure hydrogel promotes regeneration of osteochondral tissue, thus providing a new option for repair of osteochondral defects.

Remodeling of Human Osteochondral Defects via rAAV-Mediated Co-Overexpression of TGF-ß and IGF-I from Implanted Human Bone Marrow-Derived Mesenchymal Stromal Cells.

The application of chondrogenic gene sequences to human bone marrow-derived mesenchymal stromal cells (hMSCs) is an attractive strategy to activate the reparative activities of these cells as a means to enhance the processes of cartilage repair using indirect cell transplantation procedures that may improve the repopulation of cartilage lesions. In the present study, we examined the feasibility of co-delivering the highly competent transforming growth factor beta (TGF-ß) with the insulin-like growth factor I (IGF-I) in hMSCs via recombinant adeno-associated virus (rAAV) vector-mediated gene transfer prior to implantation in a human model of osteochondral defect (OCD) ex vivo that provides a microenvironment similar to that of focal cartilage lesions. The successful co-overexpression of rAAV TGF-ß/IGF-I in implanted hMSCs promoted the durable remodeling of tissue injury in human OCDs over a prolonged period of time (21 days) relative to individual gene transfer and the control (reporter lacZ gene) treatment, with enhanced levels of cell proliferation and matrix deposition (proteoglycans, type-II collagen) both in the lesions and at a distance, while hypertrophic, osteogenic, and catabolic processes could be advantageously delayed. These findings demonstrate the value of indirect, progenitor cell-based combined rAAV gene therapy to treat human focal cartilage defects in a natural environment as a basis for future clinical applications.