An oriented-collagen scaffold including Wnt5a promotes osteochondral regeneration and cartilage interface integration in a rabbit model.

Articular cartilage regeneration remains a major challenge in orthopedics. Noncanonical Wnt5a is a particularly attractive growth factor in this context; Wnt5a inhibits chondrocyte hypertrophy but maintains chondrogenesis. We designed a novel, vertically oriented-collagen scaffold. The effect of Wnt5a on MSCs and chondrocytes and the therapeutic effects of the Wnt5a/oriented-collagen scaffold in terms of osteochondral repair and cartilage integration were evaluated. In vitro, the proliferation, migration, and differentiation of MSCs and chondrocytes treated with Wnt5a, and the mechanisms thereof, were assessed. mRNA microarray analysis was performed to compare the expression profiles of MSCs before and after Wnt5a treatment. In vivo, full-thickness cylindrical osteochondral defects (4 mm in diameter, 3 mm in depth) were created in the patellar grooves of 24 New Zealand white rabbits and implanted with oriented-collagen scaffolds (n = 8), Wnt5a/oriented-collagen scaffolds (n = 8), or nothing (n = 8). After 6 and 12 weeks, integration and tissue responses were evaluated. The proliferation, migration, chondrogenic differentiation, and extracellular matrix formation of/by MSCs and chondrocytes improved greatly after treatment with Wnt5a. Western blotting showed that the PI3K/AKT/JNK signaling pathway was activated. Microarray analysis revealed that the Wnt5a group exhibited a significant upregulation of the PI3K pathway. Reactome GSEA pathway interaction analysis revealed that such upregulation was associated with collagen and extracellular matrix formation. In vivo, the Wnt5a/oriented-collagen scaffold group exhibited optimal interface integration, cartilage regeneration, and collagenous fiber arrangement, accompanied by significantly increased glycosaminoglycan and collagen accumulations in the zones of regeneration and integration, compared to the other groups. Gene expression analysis showed that the levels of mRNAs encoding genes involved in cartilage formation were significantly increased in the Wnt5a/oriented, collagen scaffold group (all P < .05). Wnt5a promoted the proliferation, migration, and chondrogenic differentiation of MSCs and chondrocytes via the activation of the PI3K/AKT/JNK signaling pathway. The Wnt5a/oriented-collagen constructs enhanced the structure-specific regeneration of hyaline cartilage in a rabbit model and may be a promising treatment for the repair of human cartilage defects.

Type I collagen scaffold with WNT5A plasmid for in situ cartilage tissue engineering.

BACKGROUND: Cartilage tissue lacks the ability to heal. Cartilage tissue engineering using cell-free scaffolds has been increasingly used in recent years. OBJECTIVE: This study describes the use of a type I collagen scaffold combined with WNT5A plasmid to promote chondrocyte proliferation and differentiation in a rabbit osteochondral defect model. METHODS: Type I collagen was extracted and fabricated into a collagen scaffold. To improve gene transfection efficiency, a cationic chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) vector was used. A solution of TMC/WNT5A complexes was adsorbed to the collagen scaffold to prepare a WNT5A scaffold. Osteochondral defects were created in the femoral condyles of rabbits. The rabbits were divided into defect, scaffold, and scaffold with WNT5A groups. At 6 and 12 weeks after creation of the osteochondral defects, samples were collected from all groups for macroscopic observation and gene expression analysis. RESULTS: Samples from the defect group exhibited incomplete cartilage repair, while those from the scaffold and scaffold with WNT5A groups exhibited "preliminary cartilage" covering the defect. Cartilage regeneration was superior in the scaffold with WNT5A group compared to the scaffold group. Safranin O staining revealed more proteoglycans in the scaffold and scaffold with WNT5A groups compared to the defect group. The expression levels of aggrecan, collagen type II, and SOX9 genes were significantly higher in the scaffold with WNT5A group compared to the other two groups. CONCLUSIONS: Type I collagen scaffold showed effective adsorption and guided the three-dimensional arrangement of stem cells. WNT5A plasmid promoted cartilage repair by stimulating the expression of aggrecan, type II collagen, and SOX9 genes and proteins, as well as inhibiting cartilage hypertrophy.

Application of the cell sheet technique in tissue engineering.

The development and application of the tissue engineering technique has shown a significant potential in regenerative medicine. However, the limitations of conventional tissue engineering methods (cell suspensions, scaffolds and/or growth factors) restrict its application in certain fields. The novel cell sheet technique can overcome such disadvantages. Cultured cells can be harvested as intact sheets without the use of proteolytic enzymes, such as trypsin or dispase, which can result in cell damage and loss of differentiated phenotypes. The cell sheet is a complete layer, which contains extracellular matrix, ion channel, growth factor receptors, nexin and other important cell surface proteins. Mesenchymal stem cells (MSCs), which have the potential for multiple differentiation, are promising candidate seed cells for tissue engineering. The MSC sheet technique may have potential in the fields of regenerative medicine and tissue engineering in general. Additionally, induced pluripotent stem cell and embryonic stem cell-derived cell sheets have been proposed for tissue regeneration. Currently, the application of cell sheet for tissue reconstruction includes: Direct recipient sites implantation, superposition of cell sheets to construct three-dimensional structure for implantation, or cell sheet combined with scaffolds. The present review discusses the progress in cell sheet techniques, particularly stem cell sheet techniques, in tissue engineering.

Combining mesenchymal stem cell sheets with platelet-rich plasma gel/calcium phosphate particles: a novel strategy to promote bone regeneration.

BACKGROUND: Promotion of bone regeneration is important for successful repair of bony defects. This study aimed to investigate whether combining bone marrow-derived mesenchymal stem cell (BMSC) sheets with platelet-rich plasma (PRP) gel/calcium phosphate particles could promote bone formation in the femoral bone defects of rats. METHODS: The proliferation and differentiation of BMSCs or BMSC sheets cultured with calcium phosphate particles and/or PRP were investigated in in vitro. In vivo, 36 2.5 × 5 mm bone defects were randomly divided into groups and treated with either BMSCs/PRP gel, calcium phosphate particles, PRP gel/calcium phosphate particles, a BMSC sheet/calcium phosphate particles, a BMSC sheet/PRP gel/calcium phosphate particles, or were left untreated (n = 6/group). A further 15 bone defects were treated with chloromethyl-benzamidodialkylcarbocyanine (CM-Dil)-labelled BMSC sheet/PRP gel/calcium phosphate particles and observed using a small animal in vivo fluorescence imaging system to trace the implanted BMSCs at 1 day, 3 days, 7 days, 2 weeks, and 4 weeks after surgery. RESULTS: The expression of collagen type I and osteocalcin genes of BMSCs or BMSC sheets treated with PRP and calcium phosphate particles was significantly higher than that of BMSCs or BMSC sheets treated with calcium phosphate particles or the controls (P <0.05). PRP can promote gene expression of collagen III and tenomodulin by BMSCs and in BMSC sheets. The VEGF, collagen I and osteocalcin gene expression levels were higher in the BMSC sheet than in cultured BMSCs (P <0.05). Moreover, alizarin red staining quantification, ALP quantification and calcein blue fluorescence showed the osteogenic potential of BMSCs treated with PRP and calcium phosphate particles The implanted BMSCs were detectable at 1 day, 3 days, 7 days, 2 weeks and 4 weeks after surgery by a small animal in vivo fluorescence imaging system and were visualized in the defect zones by confocal microscopy. At 4 weeks after implantation, the defects treated with the BMSC sheet/PRP gel/calcium phosphate particles showed significantly more bone formation than the other five groups. CONCLUSIONS: Incorporation of an BMSC sheet into the PRP gel/calcium phosphate particles greatly promoted bone regeneration. These BMSC sheet and tissue engineering strategies offer therapeutic opportunities for promoting bone defect repair clinically.

Reconstruction of segmental bone defects in the rabbit ulna using periosteum encapsulated mesenchymal stem cells-loaded poly (lactic-co-glycolic acid) scaffolds.

BACKGROUND: Repair of large bone defects remains a challenge for clinicians. The present study investigated the ability of mesenchymal stem cells (MSCs) and/or periosteum-loaded poly (lactic-co-glycolic acid) (PLGA) to promote new bone formation within rabbit ulnar segmental bone defects. METHODS: Rabbit bone marrow-derived MSCs (passage 3) were seeded onto porous PLGA scaffolds. Forty segmental bone defects, each 15 mm in length, were created in the rabbit ulna, from which periosteum was obtained. Bone defects were treated with either PLGA alone (group A), PLGA + MSCs (group B), periosteum-wrapped PLGA (group C) or periosteum-wrapped PLGA/MSCs (group D). At 6 and 12 weeks post-surgery, samples were detected by gross observation, radiological examination (X-ray and micro-CT) and histological analyses. RESULTS: Group D, comprising both periosteum and MSCs, showed better bone quality, higher X-ray scores and a greater amount of bone volume compared with the other three groups at each time point (P < 0.05). No significant differences in radiological scores and amount of bone volume were found between groups B and C (P > 0.05), both of which were significantly higher than group A (P < 0.05). CONCLUSIONS: Implanted MSCs combined with periosteum have a synergistic effect on segmental bone regeneration and that periosteum plays a critical role in the process. Fabrication of angiogenic and osteogenic cellular constructs or tissue-engineered periosteum will have broad applications in bone tissue engineering.