Fabrication and In Vitro Study of Tissue-Engineered Cartilage Scaffold Derived from Wharton's Jelly Extracellular Matrix.
ABSTRACT: The scaffold is a key element in cartilage tissue engineering. The components of Wharton's jelly are similar to those of articular cartilage and it also contains some chondrogenic growth factors, such as insulin-like growth factor I and transforming growth factor-?. We fabricated a tissue-engineered cartilage scaffold derived from Wharton's jelly extracellular matrix (WJECM) and compared it with a scaffold derived from articular cartilage ECM (ACECM) using freeze-drying. The results demonstrated that both WJECM and ACECM scaffolds possessed favorable pore sizes and porosities; moreover, they showed good water uptake ratios and compressive moduli. Histological staining confirmed that the WJECM and ACECM scaffolds contained similar ECM. Moreover, both scaffolds showed good cellular adherence, bioactivity, and biocompatibility. MTT and DNA content assessments confirmed that the ACECM scaffold tended to be more beneficial for improving cell proliferation than the WJECM scaffold. However, RT-qPCR results demonstrated that the WJECM scaffold was more favorable to enhance cellular chondrogenesis than the ACECM scaffold, showing more collagen II and aggrecan mRNA expression. These results were confirmed indirectly by glycosaminoglycan and collagen content assessments and partially confirmed by histology and immunofluorescent staining. In conclusion, these results suggest that a WJECM scaffold may be favorable for future cartilage tissue engineering.
Project description:Articular cartilage defect repair is a problem that has long plagued clinicians. Although mesenchymal stem cells (MSCs) have the potential to regenerate articular cartilage, they also have many limitations. Recent studies have found that MSC-derived exosomes (MSC-Exos) play an important role in tissue regeneration. The purpose of this study was to verify whether MSC-Exos can enhance the reparative effect of the acellular cartilage extracellular matrix (ACECM) scaffold and to explore the underlying mechanism. The results of in vitro experiments show that human umbilical cord Wharton's jelly MSC-Exos (hWJMSC-Exos) can promote the migration and proliferation of bone marrow-derived MSCs (BMSCs) and the proliferation of chondrocytes. We also found that hWJMSC-Exos can promote the polarization of macrophages toward the M2 phenotype. The results of a rabbit knee osteochondral defect repair model confirmed that hWJMSC-Exos can enhance the effect of the ACECM scaffold and promote osteochondral regeneration. We demonstrated that hWJMSC-Exos can regulate the microenvironment of the articular cavity using a rat knee joint osteochondral defect model. This effect was mainly manifested in promoting the polarization of macrophages toward the M2 phenotype and inhibiting the inflammatory response, which may be a promoting factor for osteochondral regeneration. In addition, microRNA (miRNA) sequencing confirmed that hWJMSC-Exos contain many miRNAs that can promote the regeneration of hyaline cartilage. We further clarified the role of hWJMSC-Exos in osteochondral regeneration through target gene prediction and pathway enrichment analysis. In summary, this study confirms that hWJMSC-Exos can enhance the effect of the ACECM scaffold and promote osteochondral regeneration.
Project description:Articular cartilage lesions are a particular challenge for regenerative medicine due to cartilage low self-ability repair in case of damage. Hence, a significant goal of musculoskeletal tissue engineering is the development of suitable structures in virtue of their matrix composition and biomechanical properties. The objective of our study was to design in vitro a supporting structure for autologous chondrocyte growth. We realized a biohybrid composite scaffold combining a novel and nonspecific extracellular matrix (ECM), which is decellularized Wharton's jelly ECM, with the biomechanical properties of the synthetic hydrogel polyvinyl alcohol (PVA). Wharton's jelly ECM was tested for its ability in promoting scaffold colonization by chondrocytes and compared with polyvinyl alcohol itself and the more specific decellularized cartilage matrix. Our preliminary evidences highlighted the chance of using Wharton's jelly ECM in combination with PVA hydrogels as an innovative and easily available scaffold for cartilage restoration.
Project description:Seed cells of articular cartilage tissue engineering face many obstacles in their application because of the dedifferentiation of chondrocytes or unstable chondrogenic differentiation status of pluripotent stem cells. To overcome mentioned dilemmas, a simulation of the articular cartilage microenvironment was constructed by primary articular cartilage cells (pACs) and acellular cartilage extracellular matrix- (ACECM-) oriented scaffold cocultured with human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hWJMSCs) in vitro. The coculture groups showed more affluent cartilage special matrix ingredients including collagen II and aggrecan based on the results of histological staining and western blotting and cut down as many pACs as possible. The RT-PCR and cell viability experiments also demonstrated that hWJMSCs were successfully induced to differentiate into chondrocytes when cultured in the simulated cartilage microenvironment, as confirmed by the significant upregulation of collagen II and aggrecan, while the cell proliferation activity of pACs was significantly improved by cell-cell interactions. Therefore, compared with monoculture and chondrogenic induction of inducers, coculture providing a simulated native articular microenvironment was a potential and temperate way to regulate the biological behaviors of pACs and hWJMSCs to regenerate the hyaline articular cartilage.
Project description:In this paper, we aim to explore the application value of tissue engineering for the construction of artificial cartilage in vitro. Chondrocytes from healthy porcine articular cartilage tissue were seeded on articular cartilage extracellular matrix (ACECM) scaffolds and cultivated. Type II collagen immunofluorescent staining was used to assess secretion from the extracellular matrix. Chondrocytes, which were mainly polygonal and cobblestone-shaped, were inoculated on ACECM-oriented scaffolding for 7 days, and the neo-tissue showed translucent shape and toughness. Using inverted and fluorescence microscopy, we found that chondrocytes on the scaffolds performed well in terms of adhesion and growth, and they secreted collagen type II. Moreover, the porcine ACECM scaffolds had good biocompatibility. The inflammatory cell detection, cellular immune response assay and humoral immune response assay showed porcine ACECM scaffolds were used for xenotransplantation without significant immune inflammatory response. All these findings reveal that ACECM-oriented scaffold is an ideal natural biomaterial for cartilage tissue engineering.
Project description:Diseases in articular cartilages have affected millions of people globally. Although the biochemical and cellular composition of articular cartilages is relatively simple, there is a limitation in the self-repair ability of the cartilage. Therefore, developing strategies for cartilage repair is very important. Here, we report on a new liquid resin preparation process of water-based polyurethane based photosensitive materials with hyaluronic acid with application of the materials for 3D printed customized cartilage scaffolds. The scaffold has high cytocompatibility and is one that closely mimics the mechanical properties of articular cartilages. It is suitable for culturing human Wharton's jelly mesenchymal stem cells (hWJMSCs) and the cells in this case showed an excellent chondrogenic differentiation capacity. We consider that the 3D printing hybrid scaffolds may have potential in customized tissue engineering and also facilitate the development of cartilage tissue engineering.
Project description:<h4>Background</h4>Osteochondral interface regeneration is challenging for functional and integrated cartilage repair. Various layered scaffolds have been used to reconstruct the complex interface, yet the influence of the permeability of the layered structure on cartilage defect healing remains largely unknown.<h4>Methods</h4>We designed and fabricated a novel bilayered scaffold using articular cartilage extracellular matrix (ACECM) and hydroxyapatite (HAp), involving a porous, oriented upper layer and a dense, mineralised lower layer. By optimising the HAp/ACECM ratio, differing pore sizes and porosities were obtained simultaneously in the two layers. To evaluate the effects of permeability on cell behaviour, rabbit chondrocytes were seeded.<h4>Results</h4>Morphological observations demonstrated that a gradual interfacial region was formed with pore sizes varying from 128.2?±?20.3 to 21.2?±?3.1 ?m. The permeability of the bilayered scaffold decreased with increasing compressive strain and HAp content. Mechanical tests indicated that the interface was stable to bearing compressive and shear loads. Accordingly, the optimum HAp/ACECM ratio (7 w/v%) in the layer to mimic native calcified cartilage was found. Chondrocytes could not penetrate the interface and resided only in the upper layer, where they showed high cellularity and abundant matrix deposition.<h4>Conclusions</h4>Our findings suggest that a bilayered scaffold with low permeability, rather than complete isolation, represents a promising candidate for osteochondral interface tissue engineering.
Project description:It remains scientifically challenging to regenerate injured cartilage in orthopedics. Recently, an endogenous cell recruitment strategy based on a combination of acellular scaffolds and chemoattractants to specifically and effectively recruit host cells and promote chondrogenic differentiation has brought new hope for <i>in situ</i> articular cartilage regeneration. In this study, a transforming growth factor-β3 (TGF-β3)-loaded biomimetic natural scaffold based on demineralized cancellous bone (DCB) and acellular cartilage extracellular matrix (ECM) was developed and found to improve chondral repair by enhancing cell migration and chondrogenesis. The DCB/ECM scaffold has porous microstructures (pore size: 67.76 ± 8.95 μm; porosity: 71.04 ± 1.62%), allowing the prolonged release of TGF-β3 (up to 50% after 42 days <i>in vitro</i>) and infrapatellar fat pad adipose-derived stem cells (IPFSCs) that maintain high cell viability (>96%) and favorable cell distribution and phenotype after seeding onto the DCB/ECM scaffold. The DCB/ECM scaffold itself can also provide a sustained release system to effectively promote IPFSC migration (nearly twofold <i>in vitro</i>). Moreover, TGF-β3 loaded on scaffolds showed enhanced chondrogenic differentiation (such as collagen II, ACAN, and SOX9) of IPFSCs after 3 weeks of culture. After implanting the composite scaffold into the knee joints of rabbits, enhanced chondrogenic differentiation was discovered at 1, 2, and 4 weeks post-surgery, and improved repair of cartilage defects in terms of biochemical, biomechanical, radiological, and histological results was identified at 3 and 6 months post-implantation. To conclude, our study demonstrates that the growth factor (GF)-loaded scaffold can facilitate cell homing, migration, and chondrogenic differentiation and promote the reconstructive effects of <i>in vivo</i> cartilage formation, revealing that this staged regeneration strategy combined with endogenous cell recruitment and pro-chondrogenesis is promising for <i>in situ</i> articular cartilage regeneration.
Project description:Heterogeneity of mesenchymal stem cells (MSCs) influences the cell therapy outcome and the application in tissue engineering. Also, the application of subpopulations of MSCs in cartilage regeneration remains poorly characterized. CD146+ MSCs are identified as the natural ancestors of MSCs and the expression of CD146 are indicative of greater pluripotency and self-renewal potential. Here, we sorted a CD146<sup>+</sup> subpopulation from adipose-derived mesenchymal stem cells (ADSCs) for cartilage regeneration. <b>Methods</b>: CD146<sup>+</sup> ADSCs were sorted using magnetic activated cell sorting (MACS). Cell surface markers, viability, apoptosis and proliferation were evaluated <i>in vitro</i>. The molecular signatures were analyzed by mRNA and protein expression profiling. By intra-articular injections of cells in a rat osteochondral defect model, we assessed the role of the specific subpopulation in cartilage microenvironment. Finally, CD146<sup>+</sup> ADSCs were combined with articular cartilage extracellular matrix (ACECM) scaffold for long term (3, 6 months) cartilage repair. <b>Results</b>: The enriched CD146<sup>+</sup> ADSCs showed a high expression of stem cell and pericyte markers, good viability, and immune characteristics to avoid allogeneic rejection. Gene and protein expression profiles revealed that the CD146<sup>+</sup> ADSCs had different cellular functions especially in regulation inflammation. In a rat model, CD146<sup>+</sup> ADSCs showed a better inflammation-modulating property in the early stage of intra-articular injections. Importantly, CD146<sup>+</sup> ADSCs exhibited good biocompatibility with the ACECM scaffold and the CD146<sup>+</sup> cell-scaffold composites produced less subcutaneous inflammation. The combination of CD146<sup>+</sup> ADSCs with ACECM scaffold can promote better cartilage regeneration in the long term. <b>Conclusion</b>: Our data elucidated the function of the CD146<sup>+</sup> ADSC subpopulation, established their role in promoting cartilage repair, and highlighted the significance of cell subpopulations as a novel therapeutic for cartilage regeneration.
Project description:The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-? 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.
Project description:Articular cartilage has a very limited regeneration capacity. Therefore, injury or degeneration of articular cartilage results in an inferior mechanical stability, load-bearing capacity, and lubrication capability. Here, we developed a biomimetic scaffold consisting of macroporous polyvinyl alcohol (PVA) sponges as a platform material for the incorporation of cell-embedded photocrosslinkable poly(ethylene glycol) diacrylate (PEGDA), PEGDA-methacrylated chondroitin sulfate (PEGDA-MeCS; PCS), or PEGDA-methacrylated hyaluronic acid (PEGDA-MeHA; PHA) within its pores to improve in vitro chondrocyte functions and subsequent in vivo ectopic cartilage tissue formation. Our findings demonstrated that chondrocytes encapsulated in PCS or PHA and loaded into macroporous PVA hybrid scaffolds maintained their physiological phenotypes during in vitro culture, as shown by the upregulation of various chondrogenic genes. Further, the cell-secreted extracellular matrix (ECM) improved the mechanical properties of the PVA-PCS and PVA-PHA hybrid scaffolds by 83.30% and 73.76%, respectively, compared to their acellular counterparts. After subcutaneous transplantation in vivo, chondrocytes on both PVA-PCS and PVA-PHA hybrid scaffolds significantly promoted ectopic cartilage tissue formation, which was confirmed by detecting cells positively stained with Safranin-O and for type II collagen. Consequently, the mechanical properties of the hybrid scaffolds were biomimetically reinforced by 80.53% and 210.74%, respectively, compared to their acellular counterparts. By enabling the recapitulation of biomimetically relevant structural and functional properties of articular cartilage and the regulation of in vivo mechanical reinforcement mediated by cell⁻matrix interactions, this biomimetic material offers an opportunity to control the desired mechanical properties of cell-laden scaffolds for cartilage tissue regeneration.