Tissue Engineering Part A最新文献

In vitro engineering of human articular cartilage (AC) is a regenerative medicine challenge. The main objective of this study was the development of a repeatable scaffold-free in vitro model of chondrocyte spheroid-based treatments of cartilage defects, to allow for systematic study and further optimization of this type of treatment. Human articular chondrocytes (HC) and immortalized mesenchymal cells differentiated in chondrocytes (Y201-Cs) were cultured in round-bottom 96-well plates to produce multicellular spheroids and their growth kinetics, and viability was evaluated over 7 days of culture. Then, the spheroids were assembled and cultured for 21 days on a gelatin-coated poly(lactic-co-glycolic acid) electrospun membrane (10 spheroids/cm²), following a protocol in line with the clinically approved Chondrosphere^® (CO.DON AG) technique. Both HC and Y201-C cells formed compact and viable spheroids after 7 days of culture with a reduction of diameter over the 7 days from 1300 ± 150 μm to 600 ± 90 μm and from 1250 ± 60 μm to 800 ± 20 μm for HC and Y201-C, respectively. When the spheroids were transferred onto the support membrane, these adhered on the membrane itself and fused themselves, producing collagen type II (COL2A1) and aggrecan (ACAN), according to gene expression and glycosaminoglycans quantification analyses. We detected higher expression of COL2A1 in HC cells, while the Y201-C constructs were characterized by an increased ACAN expression. The approach we presented allows a standardizable production of spheroids with predictable geometry and the creation of a reproducible scaffold-free in vitro AC-like construct showing high expression of chondrogenic markers, using both HC and Y201-C. In addition, the bankable Y201-C cells provide an effective base model for experimentation with the spheroid approach to further enhance the process. Impact statement This is first work on the development of a repeatable scaffold-free in vitro model based on an optimized protocol in line with a recent clinically approved Chondrosphere^® (CO.DON AG) technique. In addition, we demonstrated that a bankable cell type (Y201-C) could produce an engineered cartilage-like construct, giving a repeatable model as a key tool for experimentation of therapeutic treatment ahead of studies with heterogeneous cell populations.

Tissue engineering and regenerative medicine has gradually evolved as a promising therapeutic strategy to the modern health care of aging and diseased population. In this study, we developed a novel nanofibrous scaffold and verified its application in the critical bone defect regeneration. The metformin-incorporated nano-gelatin/hydroxyapatite fibers (NGF) was produced by electrospinning, cross-linked, and then characterized by X-ray powder diffractometer and Fourier-transform infrared spectroscopy. Cytotoxicity, cell adhesion, cell differentiation, and quantitative osteogenic gene and protein expression were analyzed by bone marrow stem cells (BMSCs) from rat. Rat forearm critical bone defect model was performed for the in vivo study. The NGF were characterized by their porous structures with proper interconnectivity without significant cytotoxic effects; the adhesion of BMSCs on the NGF could be enhanced. The osteogenic gene and protein expression were upregulated. Postimplantation, the new regenerated bone in bone defect was well demonstrated in the NGF samples. We demonstrated that the metformin-incorporated NGF greatly improved healing potential on the critical-size bone defect. Although metformin-incorporated NGF had advantageous effectiveness during bone regeneration, further validation is required before it can be applied to clinical applications. Impact statement Bone is the structure that supports the rest of the human body. Critical-size bone defect hinders the regeneration of damaged bone tissues and compromises the mechanical strength of the skeletal system. Characterized by their porous structures with proper interconnectivity, the electrospinning nano-gelatin/hydroxyapatite fibrous scaffold developed in this study can greatly improve the healing potential on the critical-size bone defect. Further validation can validate its potential clinical applications.

Due to the poor capacity for articular cartilage to regenerate, its damage tends to result in progressively degenerating conditions such as osteoarthritis. To repair the damage, the transplantation of allogeneic human induced pluripotent stem cell (iPSC)-derived cartilage is being considered. However, although allogeneic cartilage transplantation is effective, immunological reactions can occur. One hypothetical solution is to delete the expression of major histocompatibility complex (MHC) class I molecules to reduce the immunological reactions. For this purpose, we deleted the β2 microglobulin (B2M) gene in a cynomolgus monkey (crab-eating monkey [Macaca fascicularis]) iPS cells (cyiPSCs) to obtain B2M^-/- cyiPSCs using the CRISPR/Cas9 system. Western blot analysis confirmed B2M^-/- cyiPSCs lacked B2M protein, which is necessary for MHC class I molecules to be transported to and expressed on the cell surface by forming multimers with B2M. Flow cytometry analysis revealed no B2M^-/- cyiPSCs expressed MHC class I molecules on their surface. The transplantation of B2M^-/- cyiPSCs in immunodeficient mice resulted in teratoma that contained cartilage, indicating that the lack of MHC class I molecules on the cell surface affects neither the pluripotency nor the chondrogenic differentiation capacity of cyiPSCs. By modifying the chondrogenic differentiation protocol for human iPSCs, we succeeded at differentiating B2M^+/+ and B2M^-/- cyiPSCs toward chondrocytes followed by cartilage formation in vitro, as indicated by histological analysis showing that B2M^+/+ and B2M^-/- cyiPSC-derived cartilage were positively stained with safranin O and expressed type II collagen. Flow cytometry analysis confirmed that MHC class I molecules were not expressed on the cell surface of B2M^-/- chondrocytes isolated from B2M^-/- cyiPSC-derived cartilage. An in vitro mixed lymphocyte reaction assay showed that neither B2M^+/+ nor B2M^-/- cyiPSC-derived cartilage cells stimulated the proliferation of allogeneic peripheral blood mononuclear cells. On the contrary, osteochondral defects in monkey knee joints that received allogeneic transplantations of cyiPSC-derived cartilage showed an accumulation of leukocytes with more natural killer cells around B2M^-/- cyiPSC-derived cartilage than B2M^+/+ cartilage, suggesting complex mechanisms in the immune reaction of allogeneic cartilage transplanted in osteochondral defects in vivo. Impact statement The transplantation of allogeneic induced pluripotent stem cell (iPSC)-derived cartilage is expected to treat articular cartilage damage, although the effects of major histocompatibility complex (MHC) in immunological reactions have not been well studied. We succeeded at creat