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Bone tissue engineering using human fetal cartilage-derived progenitor cells and porcine cartilage acellular matrix scaffold

초록/요약

PURPOSE. In this study, engineered artificial bone tissue was prepared by using human fetal cartilage-derived progenitor cells (hFCPCs) and scaffold made of extracellular matrix derived from porcine cartilage. In many studies, it has been known that markers related to osteogenic differentiation are up-regulated when inoculating mesenchymal stem cells into the extracellular matrix scaffold and inducing osteogenic differentiation in vitro. It also reported that mesenchymal stem cells having multi-lineage differentiative potency were inoculated into cartilage-derived extracellular matrix scaffolds to fabricate artificial bone tissue in vivo. With this regard, this study aimed to confirm the possibility that artificial bone tissue can be produced by inoculating hFCPCs with osteogenic differentiation ability into extracellular matrix scaffolds derived from porcine cartilage. METHODS. Porous cartilage extracellular matrix scaffolds were prepared through a salt-extraction method. In vitro environment, hFCPCs and hBMSCs were inoculated in a porcine cartilage extracellular matrix scaffold and in vitro cultured in a bone differentiation medium to induce bone differentiation. In addition, in order to see the bone formation in vivo of the artificial bone tissue thus produced, a defect was observed in the tibia of a rabbit after transplantation. RESULTS. As a result of in vitro experiments, in the same environment, the hFCPCs group showed no significant difference in the ALP activity and bone formation gene expression of the hBMSCs group at 0, 7, 14 days after differentiation. On day 21, the expression of the Osterix and Osteocalcin genes was significantly higher in hFCPCs than in hBMSCs. In vivo experiments, hFCPCs were used to confirm that the porcine cartilage-derived extracellular matrix scaffolds inoculated with hFCPCs helped bone regeneration when transplanted into a tibial defect in a rabbit and were compared with the group treated with no treatment as a control group. As a result of analysis through micro-computed tomography and histological staining, the hFCPCs group showed a tendency to increase rapidly regenerated bone volume when going from 8 to 12 weeks. And at 12 weeks, it was shown that the hFCPCs group induced significantly more bone regeneration than rBMSCs. And also, the group transplanted with hFCPCs after 12 weeks showed an effect on bone regeneration similar to the negative control without any treatment. Through histological staining, newly formed bone structures were identified at artificially created defect sites, and the fact that the transplanted hFCPCs differentiated into osteoblasts and angiogenesis in tissues was also confirmed by the method of immunostaining. CONCLUSIONS. The hFCPCs-seeded CAM scaffold group showed relatively low regenerated bone volume at 4 and 8 weeks when compared to the negative control with no treatment. At 12 weeks, the hFCPCs group did not show any significant difference from the negative control group. In conclusion, when the bone tissue was prepared by inoculating hFCPCs in a cartilage-derived extracellular matrix scaffold of pigs, the transplantation into a tibial defect of a rabbit did not prove significantly better bone tissue regeneration than a negative control group, the conclusion was not reached. Based on these results, it is judged that positive results will be obtained for the evaluation of bone regeneration ability of the experimental group by supplementing the experimental design in the future.

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목차

1. Introduction 1
2. Materials and Methods 3
2.1 Cell isolation and culture 3
2.2 Fabrication and characterization of porcine cartilage-derived acellular matrix (CAM) powder scaffolds 4
2.3 Seeding hFCPCs on a CAM scaffold 4
2.4 Osteogenic differentiation of hFCPCs on a CAM scaffold 5
2.5 Alkaline Phosphatase (ALP) Activity 5
2.6 Quantitative Real Time Polymer (qRT-PCR) analysis 6
2.7 Histological evaluation 6
2.8 Immunohistochemistry analysis 7
2.9 The effect on bone regeneration by hFCPCs-seeded CAM scaffold in rabbit tibia; gross view and micro-CT analysis 7
2.10 Histological evaluation 9
2.11 Immunohistochemistry analysis 9
2.12 Statistical analysis 10
3. Results 14
3.1 Fabrication of porous CAM scaffolds 14
3.2 preparation of cell-seeded CAM scaffolds 17
3.3 In vitro osteogenesis of hFCPCs in CAM scaffolds 17
3.4 In vivo bone repair using construct fabricated with hFCPCs-seeded CAM scaffolds on a rabbit tibial defect 21
3.5 Evaluation of new bone formation for hFCPCs-seeded CAM scaffolds on tibia defect in rabbit model using micro-CT 23
3.6 hFCPCs-seeded CAM scaffold promoted new bone formation in rabbits 25
4. Discussion 29
References 32

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