Enhancing cartilage regeneration and repair through bioactive and biomechanical modification of 3D acellular dermal matrix.

Acellular dermal matrix (ADM) shows promise for cartilage regeneration and repair. However, an effective decellularization technique that removes cellular components while preserving the extracellular matrix, the transformation of 2D-ADM into a suitable 3D scaffold with porosity and the enhancement of bioactive and biomechanical properties in the 3D-ADM scaffold are yet to be fully addressed. In this study, we present an innovative decellularization method involving 0.125% trypsin and 0.5% SDS and a 1% Triton X-100 solution for preparing ADM and converting 2D-ADM into 3D-ADM scaffolds. These scaffolds exhibit favorable physicochemical properties, exceptional biocompatibility and significant potential for driving cartilage regeneration in vitro and in vivo. To further enhance the cartilage regeneration potential of 3D-ADM scaffolds, we incorporated porcine-derived small intestinal submucosa (SIS) for bioactivity and calcium sulfate hemihydrate (CSH) for biomechanical reinforcement. The resulting 3D-ADM+SIS scaffolds displayed heightened biological activity, while the 3D-ADM+CSH scaffolds notably bolstered biomechanical strength. Both scaffold types showed promise for cartilage regeneration and repair in vitro and in vivo, with considerable improvements observed in repairing cartilage defects within a rabbit articular cartilage model. In summary, this research introduces a versatile 3D-ADM scaffold with customizable bioactive and biomechanical properties, poised to revolutionize the field of cartilage regeneration.

Integrating coaxial electrospinning and 3D printing technologies for the development of biphasic porous scaffolds enabling spatiotemporal control in tumor ablation and osteochondral regeneration.

The osteochondral defects (OCDs) resulting from the treatment of giant cell tumors of bone (GCTB) often present two challenges for clinicians: tumor residue leading to local recurrence and non-healing of OCDs. Therefore, this study focuses on developing a double-layer PGPC-PGPH scaffold using shell-core structure nanofibers to achieve "spatiotemporal control" for treating OCDs caused by GCTB. It addresses two key challenges: eliminating tumor residue after local excision and stimulating osteochondral regeneration in non-healing OCD cases. With a shell layer of protoporphyrin IX (PpIX)/gelatin (GT) and inner cores containing chondroitin sulfate (CS)/poly(lactic-co-glycolic acid) (PLGA) or hydroxyapatite (HA)/PLGA, coaxial electrospinning technology was used to create shell-core structured PpIX/GT-CS/PLGA and PpIX/GT-HA/PLGA nanofibers. These nanofibers were shattered into nano-scaled short fibers, and then combined with polyethylene oxide and hyaluronan to formulate distinct 3D printing inks. The upper layer consists of PpIX/GT-CS/PLGA ink, and the lower layer is made from PpIX/GT-HA/PLGA ink, allowing for the creation of a double-layer PGPC-PGPH scaffold using 3D printing technique. After GCTB lesion removal, the PGPC-PGPH scaffold is surgically implanted into the OCDs. The sonosensitizer PpIX in the shell layer undergoes sonodynamic therapy to selectively damage GCTB tissue, effectively eradicating residual tumors. Subsequently, the thermal effect of sonodynamic therapy accelerates the shell degradation and release of CS and HA within the core layer, promoting stem cell differentiation into cartilage and bone tissues at the OCD site in the correct anatomical position. This innovative scaffold provides temporal control for anti-tumor treatment followed by tissue repair and spatial control for precise osteochondral regeneration.

Development of Tri-Layered Biomimetic Atelocollagen Scaffolds with Interfaces for Osteochondral Tissue Engineering.

The development of biomimetic scaffolds containing cartilage, calcified cartilage, and bone regeneration for precise osteochondral repair remains a challenge. Herein, a novel tri-layered scaffold-with a top layer containing type II atelocollagen and chondroitin sulphate for cartilage regeneration, an intermediate layer with type II atelocollagen and hydroxyapatite for calcified cartilage formation, and a bottom layer with type I atelocollagen and hydroxyapatite for bone growth-that can be built using liquid-phase cosynthesis, is described. The tri-layered scaffolds are mechanically demonstrably superior and have a lower risk of delamination than monolayer scaffolds. This is due to higher cohesion arising from the interfaces between each layer. In vitro results show that although monolayer scaffolds can stimulate bone marrow stem cells to differentiate and form cartilage, calcified cartilage, and bone separately (detected using quantitative polymerase chain reaction analysis and staining with safranin-O and Alizarin Red S), the tri-layered scaffolds support the regeneration of cartilage, calcified cartilage, and bone simultaneously after 2 and 4 months of implantation (detected using gross and micro-computed tomography images, histological staining, and Avizo, a software used to detect microlevel defects in metals). This work presents data on a promising approach in devising strategies for the precise repair of osteochondral defects.

Ultrafast, tough, and adhesive hydrogel based on hybrid photocrosslinking for articular cartilage repair in water-filled arthroscopy.

A hydrogel scaffold for direct tissue-engineering application in water-irrigated, arthroscopic cartilage repair, is badly needed. However, such hydrogels must cure quickly under water, bind strongly and permanently to the surrounding tissue, and maintain sufficient mechanical strength to withstand the hydraulic pressure of arthroscopic irrigation (~10 kilopascal). To address these challenges, we report a versatile hybrid photocrosslinkable (HPC) hydrogel fabricated though a combination of photoinitiated radical polymerization and photoinduced imine cross-linking. The ultrafast gelation, high mechanical strength, and strong adhesion to native tissue enable the direct use of these hydrogels in irrigated arthroscopic treatments. We demonstrate, through in vivo articular cartilage defect repair in the weight-bearing regions of swine models, that the HPC hydrogel can serve as an arthroscopic autologous chondrocyte implantation scaffold for long-term cartilage regeneration, integration, and reconstruction of articular function.

A Biomimetic Biphasic Scaffold Consisting of Decellularized Cartilage and Decalcified Bone Matrixes for Osteochondral Defect Repair.

It is challenging to develop a biphasic scaffold with biomimetic compositional, structural, and functional properties to achieve concomitant repair of both superficial cartilage and subchondral bone in osteochondral defects (OCDs). This study developed a biomimsubchondraletic biphasic scaffold for OCD repair via an iterative layered lyophilization technique that controlled the composition, substrate stiffness, and pore size in each phase of the scaffold. The biphasic scaffold consisted of a superficial decellularized cartilage matrix (DCM) and underlying decalcified bone matrix (DBM) with distinct but seamlessly integrated phases that mimicked the composition and structure of osteochondral tissue, in which the DCM phase had relative low stiffness and small pores (approximately 134 µm) and the DBM phase had relative higher stiffness and larger pores (approximately 336 µm). In vitro results indicated that the biphasic scaffold was biocompatible for bone morrow stem cells (BMSCs) adhesion and proliferation, and the superficial DCM phase promoted chondrogenic differentiation of BMSCs, as indicated by the up-regulation of cartilage-specific gene expression (ACAN, Collagen II, and SOX9) and sGAG secretion; whereas the DBM phase was inducive for osteogenic differentiation of BMSCs, as indicated by the up-regulation of bone-specific gene expression (Collagen I, OCN, and RUNX2) and ALP deposition. Furthermore, compared with the untreated control group, the biphasic scaffold significantly enhanced concomitant repair of superficial cartilage and underlying subchondral bone in a rabbit OCD model, as evidenced by the ICRS macroscopic and O'Driscoll histological assessments. Our results demonstrate that the biomimetic biphasic scaffold has a good osteochondral repair effect.

Photocrosslinked natural hydrogel composed of hyaluronic acid and gelatin enhances cartilage regeneration of decellularized trachea matrix.

Repair of long segmental trachea defects is always a great challenge in the clinic. The key to solving this problem is to develop an ideal trachea substitute with biological function. Using of a decellularized trachea matrix based on laser micropore technique (LDTM) demonstrated the possibility of preparing ideal trachea substitutes with tubular shape and satisfactory cartilage regeneration for tissue-engineered trachea regeneration. However, as a result of the very low cell adhesion of LDTM, an overly high concentration of seeding cell is required, which greatly restricts its clinical translation. To address this issue, the current study proposed a novel strategy using a photocrosslinked natural hydrogel (PNH) carrier to enhance cell retention efficiency and improve tracheal cartilage regeneration. Our results demonstrated that PNH underwent a rapid liquid-solid phase conversion under ultraviolet light. Moreover, the photo-generated aldehyde groups in PNH could rapidly react with inherent amino groups on LDTM surfaces to form imine bonds, which efficiently immobilized the cell-PNH composite to the surfaces of LDTM and/or maintained the composite in the LDTM micropores. Therefore, PNH significantly enhanced cell-seeding efficiency and achieved both stable cell retention and homogenous cell distribution throughout the LDTM. Moreover, PNH exhibited excellent biocompatibility and low cytotoxicity, and provided a natural three-dimensional biomimetic microenvironment to efficiently promote chondrocyte survival and proliferation, extracellular matrix production, and cartilage regeneration. Most importantly, at a relatively low cell-seeding concentration, homogeneous tubular cartilage was successfully regenerated with an accurate tracheal shape, sufficient mechanical strength, good elasticity, typical lacuna structure, and cartilage-specific extracellular matrix deposition. Our findings establish a versatile and efficient cell-seeding strategy for regeneration of various tissue and provide a satisfactory trachea substitute for repair and functional reconstruction of long segmental tracheal defects.

Covalent grafting of PEG and heparin improves biological performance of electrospun vascular grafts for carotid artery replacement.

Rapid endothelialization of small-diameter vascular grafts remains a significant challenge in clinical practice. In addition, compliance mismatch causes intimal hyperplasia and finally leads to graft failure. To achieve compliance match and rapid endothelialization, we synthesized low-initial-modulus poly(ester-urethane)urea (PEUU) elastomer and prepared it into electrospun tubular grafts and then functionalized the grafts with poly(ethylene glycol) (PEG) and heparin via covalent grafting. The PEG- and heparin-functionalized PEUU (PEUU@PEG-Hep) graft had comparable mechanical properties with the native blood vessel. In vitro data demonstrated that the grafts are of good cytocompatibility and blood compatibility. Covalent grafting of PEG and heparin significantly promoted the adhesion, spreading, and proliferation of human umbilical vein endothelial cells (HUVECs) and upregulated the expression of vascular endothelial cell-related genes, as well as increased the capability of grafts in preventing platelet deposition. In vivo assessments indicated good biocompatibility of the PEUU@PEG-Hep graft as it did not induce severe immune responses. Replacement of resected carotid artery with the PEUU@PEG-Hep graft in a rabbit model showed that the graft was capable of rapid endothelialization, initiated vascular remodeling, and maintained patency. This study demonstrates the PEUU@PEG-Hep vascular graft with compliance match and efficacious antithrombosis might find opportunities for bioactive blood vessel substitutes.

Porous fish collagen for cartilage tissue engineering.

Cartilage defects repair is still a challenge in clinical practice until now. Although many breakthroughs have been achieved in cartilage repair using tissue engineering technology, there are still no scaffolds available for large-scale clinical applications. Currently, fish collagen (FC) is a natural source that is considered as an alternative to mammal-derived collagen in engineering cartilage tissue due to its excellent biocompatibility, suitable biodegradability, lack of immunogenicity, rich sources, low cost and minimal risk of transmitting zoonoses, which implies great potential for use in cartilage regeneration. Herein, we successfully prepared three-dimensional porous FC scaffolds from three different concentrations of FC (0.5%, 1% and 2%) by freeze-drying technology. Our results indicated that increasing the FC concentration resulted in comparable levels of suitable biodegradability and good biocompatibility but lead to a concurrent decrease in pore size and porosity and a significant increase in water absorption capacity and mechanical properties; further, initial scaffold dimension was only sustained in the 2% FC concentration. Moreover, the in vivo immunological evaluation suggested that the FC scaffold evoke low immunogenicity. In addition, our results confirmed that the porous FC scaffold facilitated cartilage formation both in vitro and when placed subcutaneously in rabbits. The gross and autopsy outcomes at 12 weeks postoperation suggested that the porous FC scaffold achieved superior cartilage repair effect than what was observed in the empty group with no scaffold. Overall, our results demonstrated that porous FC scaffolds represent a promising prospective natural material for use in engineering cartilage for clinical applications.

Tanshinone IIA Delivery Silk Fibroin Scaffolds Significantly Enhance Articular Cartilage Defect Repairing via Promoting Cartilage Regeneration.

Cartilage tissue engineering is a promising approach for repairing articular cartilage defects and requires proper scaffolds and necessary growth factors. Herein, tanshinone IIA (TAN) delivery silk fibroin scaffolds were prepared for efficient cartilage defect repair by bioactivities of TAN. By incubating with the TAN delivery silk fibroin scaffold, the transcription of the chondrocytic activity-related genes was enhanced in chondrocytes, and it also can inhibit cell apoptosis and reduce the oxidative stress by regulating the transcription of related genes, indicating that these scaffolds may promote cartilage regeneration. TAN10 delivery silk fibroin scaffolds, in which the concentration of TAN is 10 µg/mL, significantly promotes chondrocytes to generate the cartilage-specific extracellular matrix and tissue both in vitro and in vivo, compared with silk fibroin scaffolds. By treating rabbit articular cartilage defects with TAN10 delivery silk fibroin scaffolds, cartilage defects were filled with hyaline-cartilage-like tissue that integrated with the surrounding cartilage perfectly and displayed strong mechanical properties and higher extracellular matrix content. Hence, TAN facilitates cartilage regeneration, and TAN delivery silk fibroin scaffolds can be potentially applied in the clinics treating cartilage defects in the future.

Melanocyte Chitosan/Gelatin Composite Fabrication with Human Outer Root Sheath-Derived Cells to Produce Pigment.

The hair follicle serves as a melanocyte reservoir for both hair and skin pigmentation. Melanocyte stem cells (MelSCs) and melanocyte progenitors reside in the bulge/sub-bulge region of the lower permanent portion of the hair follicle and play a vital role for repigmentation in vitiligo. It would be beneficial to isolate MelSCs in order to further study their function in pigmentary disorders; however, due to the lack of specific molecular surface markers, this has not yet been successfully accomplished in human hair follicles (HuHF). One potential method for MelSCs isolation is the "side population" technique, which is frequently used to isolate hematopoietic and tumor stem cells. In the present study, we decided to isolate HuHF MelSCs using "side population" to investigate their melanotic function. By analyzing mRNA expression of TYR, SOX10, and MITF, melanosome structure, and immunofluorescence with melanocyte-specific markers, we revealed that the SP-fraction contained MelSCs with an admixture of differentiated melanocytes. Furthermore, our in vivo studies indicated that differentiated SP-fraction cells, when fabricated into a cell-chitosan/gelatin composite, could transiently repopulate immunologically compromised mice skin to regain pigmentation. In summary, the SP technique is capable of isolating HuHF MelSCs that can potentially be used to repopulate skin for pigmentation.

Lyophilized Scaffolds Fabricated from 3D-Printed Photocurable Natural Hydrogel for Cartilage Regeneration.

Repair of cartilage defects is highly challenging in clinical treatment. Tissue engineering provides a promising approach for cartilage regeneration and repair. As a core component of tissue engineering, scaffolds have a crucial influence on cartilage regeneration, especially in immunocompetent large animal and human. Native polymers, such as gelatin and hyaluronic acid, have known as ideal biomimetic scaffold sources for cartilage regeneration. However, how to precisely control their structure, degradation rate, and mechanical properties suitable for cartilage regeneration remains a great challenge. To address these issues, a series of strategies were introduced in the current study to optimize the scaffold fabrication. First, gelatin and hyaluronic acid were prepared into a hydrogel and 3D printing was adopted to ensure precise control in both the outer 3D shape and internal pore structure. Second, methacrylic anhydride and a photoinitiator were introduced into the hydrogel system to make the material photocurable during 3D printing. Finally, lyophilization was used to further enhance mechanical properties and prolong degradation time. According to the current results, by integrating photocuring 3D printing and lyophilization techniques, gelatin and hyaluronic acid were successfully fabricated into human ear- and nose-shaped scaffolds, and both scaffolds achieved shape similarity levels over 90% compared with the original digital models. The scaffolds with 50% infill density achieved proper internal pore structure suitable for cell distribution, adhesion, and proliferation. Besides, lyophilization further enhanced mechanical strength of the 3D-printed hydrogel and slowed its degradation rate matching to cartilage regeneration. Most importantly, the scaffolds combined with chondrocytes successfully regenerated mature cartilage with typical lacunae structure and cartilage-specific extracellular matrixes both in vitro and in the autologous goat model. The current study established novel scaffold-fabricated strategies for native polymers and provided a novel natural 3D scaffold with satisfactory outer shape, pore structure, mechanical strength, degradation rate, and weak immunogenicity for cartilage regeneration.

Comparative Efficacy of Autologous Stromal Vascular Fraction and Autologous Adipose-Derived Mesenchymal Stem Cells Combined With Hyaluronic Acid for the Treatment of Sheep Osteoarthritis.

The current study explored whether intra-articular (IA) injection of autologous adipose mesenchymal stem cells (ASCs) combined with hyaluronic acid (HA) achieved better therapeutic efficacy than autologous stromal vascular fraction (SVF) combined with HA to prevent osteoarthritis (OA) progression and determined how long autologous ASCs combined with HA must remain in the joint to observe efficacy. OA models were established by performing anterior cruciate ligament transection (ACLT) and medial meniscectomy (MM). Autologous SVF (1×107 mononuclear cells), autologous low-dose ASCs (1×107), and autologous high-dose ASCs (5×107) combined with HA, and HA alone, or saline alone were injected into the OA model animals at 12 and 15 weeks after surgery, respectively. Compared with SVF+HA treatment, low-dose ASC+HA treatment yielded better magnetic resonance imaging (MRI) scores and macroscopic results, while the cartilage thickness of the tibial plateau did not differ between low, high ASC+HA and SVF+HA treatments detected by micro-computed tomography (µCT). Immunohistochemistry revealed that high-dose ASC+HA treatment rescued hypertrophic chondrocytes expressing collagen X in the deep area of articular cartilage. Western blotting analysis indicated the high- and low-dose ASC+HA groups expressed more collagen X than did the SVF+HA group. Enzyme-linked immunosorbent assay showed treatment with both ASC+HA and SVF+HA resulted in differing anti-inflammatory and trophic effects. Moreover, superparamagnetic iron oxide particle (SPIO)-labeled autologous ASC signals were detected by MRI at 2 and 18 weeks post-injection and were found in the lateral meniscus at 2 weeks and in the marrow cavity of the femoral condyle at 18 weeks post-injection. Thus, IA injection of autologous ASC+HA may demonstrate better efficacy than autologous SVF+HA in blocking OA progression and promoting cartilage regeneration, and autologous ASCs (5×107 cells) combined with HA potentially survive for at least 18 weeks after IA injection.

Efficacy and Persistence of Allogeneic Adipose-Derived Mesenchymal Stem Cells Combined with Hyaluronic Acid in Osteoarthritis After Intra-articular Injection in a Sheep Model.

Although a number of studies have reported efficacy of autologous adipose-derived mesenchymal stem cells (AD-MSCs) in treating osteoarthritis (OA) no reliable evidences demonstrate whether allogeneic AD-MSCs can efficiently block OA progression in a large animal model. This study explored the efficacy and survival of allogeneic AD-MSCs combined with hyaluronic acid (HA) after intra-articular (IA) injection in a sheep OA model, which were conventionally established by anterior cruciate ligament resection and medial meniscectomy. Allogeneic AD-MSCs from donor sheep at high (5 × 107 cells) and low (1 × 107 cells) doses combined with HA, HA alone, or saline alone were injected into the OA sheep at 3 and 6 weeks after surgery, respectively. Evaluations by magnetic resonance imaging (MRI), macroscopy, micro-computed tomography, and cartilage-specific staining demonstrated that AD-MSCs+HA treated groups preserved typical articular cartilage feature. Inflammatory factors from synovial fluid of AD-MSCs+HA treated groups were significantly lower than those in the HA alone group. Notably, transforming growth factor beta 1 and insulin-like growth factor 1 were detected in the supernatant of cultured AD-MSCs. In addition, labeling signals of allogeneic AD-MSCs could be detected by MRI after 14 weeks of injection and be found in synovium by histology. These results indicated that IA injection of allogeneic AD-MSCs combined with HA could efficiently block OA progression and promote cartilage regeneration and allogeneic AD-MSCs might survive at least 14 weeks after IA injection.

Cell yield, chondrogenic potential, and regenerated cartilage type of chondrocytes derived from ear, nasoseptal, and costal cartilage.

Functional reconstruction of large cartilage defects in subcutaneous sites remains clinically challenging because of limited donor cartilage. Tissue engineering is a promising and widely accepted strategy for cartilage regeneration. To date, however, this strategy has not achieved a significant breakthrough in clinical translation owing to a lack of detailed preclinical data on cell yield and functionality of clinically applicable chondrocytes. To address this issue, the current study investigated the initial cell yield, proliferative potential, chondrogenic capacity, and regenerated cartilage type of human chondrocytes derived from auricular, nasoseptal, and costal cartilage using a scaffold-free cartilage regeneration model (cartilage sheet). Chondrocytes from all sources exhibited high sensitivity to basic fibroblast growth factor within 8 passages. Nasoseptal chondrocytes presented the strongest proliferation rate, whereas auricular chondrocytes obtained the highest total cell amount using comparable cartilage sample weights. Importantly, all chondrocytes at fifth passage showed strong chondrogenic capacity both in vitro and in the subcutaneous environment of nude mice. Although some significant differences in histological structure, cartilage matrix content and cartilage type specific proteins were observed between the in vitro engineered cartilage and original tissue; the in vivo regenerated cartilage showed mature cartilage features with high similarity to their original native tissue, except for minor matrix changes influenced by the in vivo environment. The current study provides detailed preclinical data for choice of chondrocyte source and thus promotes the clinical translation of cartilage regeneration approach.

Improvement of In Vitro Three-Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor.

In vitro three-dimensional (3D) cartilage regeneration is a promising strategy for repair of cartilage defects. However, inferior mechanical strength and tissue homogeneity greatly restricted its clinical translation. Simulation of mechanical stress through a bioreactor is an important approach for improving in vitro cartilage regeneration. The current study developed a hydrostatic pressure (HP) bioreactor based on a novel pressure-transmitting mode achieved by slight deformation of a flexible membrane in a completely sealed stainless steel device. The newly developed bioreactor efficiently avoided the potential risks of previously reported pressure-transmitting modes and simultaneously addressed a series of important issues, such as pressure scopes, culture chamber sizes, sealability, contamination control, and CO2 balance. The whole bioreactor system realized stable long-term (8 weeks) culture under high HP (5-10 MPa) without the problems of medium leakage and contamination. Furthermore, the results of in vitro 3D tissue culture based on a cartilage regeneration model revealed that HP provided by the newly developed bioreactor efficiently promoted in vitro 3D cartilage formation by improving its mechanical strength, thickness, and homogeneity. Detailed analysis in cell proliferation, cartilage matrix production, and cross-linking level of collagen macromolecules, as well as density and alignment of collagen fibers, further revealed the possible mechanisms that HP regulated in vitro cartilage regeneration. The current study provided a highly efficient and stable bioreactor system for improving in vitro 3D cartilage regeneration and thus will help to accelerate its clinical translation. Stem Cells Translational Medicine 2017;6:982-991.

The influence of Chm-I knockout on ectopic cartilage regeneration and homeostasis maintenance.

Ectopic ossification of mesenchymal stem cell (MSC) regenerated cartilage has greatly restricted its application in repairing subcutaneous cartilage defects (such as nasal or auricular). Different from MSCs, chondrocytes can maintain stable chondrogenic phenotype in ectopic microenvironment, which was speculated to be related with the existence of antiangiogenic factors such as Chondromodulin-I (Chm-I). Therefore, the purpose of this study was to illustrate whether Chm-I was indispensable for stable ectopic chondrogenesis by chondrocyte, which may help to solve the problem of MSC ectopic ossification in the future. The current study demonstrated that Chm-I knockout did not obviously influence articular cartilage development in situ. However, native articular cartilage from Chm-I knockout (Chm-I(-/-), KO), but not wild-type (WT) mice, showed obvious ossification after subcutaneously implanted into nude mice for 16 days. Interestingly, cell morphology, cartilage-specific matrix expression, and pellet culture demonstrated that Chm-I knockout had no obvious influence on the phenotype, function, and chondrogenic ability of chondrocytes in vitro, except that cells in the WT group proliferated a little faster than those in the KO group. Nevertheless, Chm-I knockout directly interfered with in vivo ectopic cartilage regeneration when chondrocytes were subcutaneously injected into nude mice with matrigel. Moreover, Chm-I knockout obviously compromised ectopic stability of in vitro regenerated cartilage after subcutaneous implantation. These findings indicated that Chm-I was an indispensable factor for ectopic cartilage regeneration and the maintenance of cartilage homeostasis, which may provide a clue for solving the stability problem of MSC regenerated cartilage in ectopic niche. In addition, this study also provides a novel model based on tissue engineering strategy to properly evaluate the function of other targeted genes.