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Inducing in vivo cartilage formation using extracellular matrix based hydrogel

초록/요약

Introduction: Hydrogel is one of the most commonly used scaffolds for cell delivery and tissue regeneration because of its highly hydrated three-dimensional polymeric networks which are structurally similar to the extracellular matrix, providing sites for adhesion, proliferation and even differentiation of cells. But there are some shortcomings, which include mechanical ability and rapid degradation, resulting in volume losses of implanted material. In order to support anatomical structure following implantation we need to develop the new cell delivery material. For this, we have focused on the use of porcine articular cartilage extracellular matrix in suspension form as a candidate material for cartilage tissue engineering. We designed material in concept that rapid differentiation of cell into cartilage would prevent the reduction of mass. The suspension made from porcine cartilage extracellular matrix powder (PCP) is mainly composed of glycosaminoglycan and collagen type II, similar to normal cartilage. In previous study, we confirmed that they provided favorable environment for cartilage formation of chondrocytes and in vivo gel formation property. Fibrin is the most commonly used by providing favorable environment to cells. Therefore, PCP suspension/fibrin mixtures is expected to induce the cartilage formation and more likely to maintain volume. This study aimed to develop a cell delivery system inducing cartilage formation and maintaining volume of mound to be used to cartilage tissue engineering. Materials and Methods: We prepared PCP separated from the pig's knee cartilage slices. Viscous liquid was made by the process of protein dissolving using pepsin and hydrochloric acid then freeze-dried. Powder was mixed with phosphate-buffered saline (PBS). PCP-suspension was dispersed in PBS to yield the desired concentrations PCP-10, PCP-20, PCP-30 and PCP-40 (100,200,300,400 mg/ml). Cellular toxicity test was carried out using water soluble tetrazolium salt (WST) assay on chondrocyte. In -vivo gel formation was observed. However volume reduction of injected PCP suspension was occurred. We used PCP suspension/Fibrin mixture to maintain volume. We prepared PCP-20, PCP-30, 2% alginate and PCP/Fibrin mixture groups for in vivo experiment. These groups were injected in subcutaneous tissue of nude mice. Cartilage formation was evaluated by safranin-O staining. And analysis of injected PCP suspension volume change using micro-CT was performed. Results: In vitro study, viability of Chondrocyte was decreased with PCP-40 (400mg/ml) group. So, in vivo experiments were conducted in the PCP-20 (200mg/ml) and 30 (300mg/ml) group. In analysis of injected PCP suspension volume change, all most of group gels are degraded time-dependently. We confirmed maintaining injected volume about 70% in 2% alginate groups. The volume of PCP groups was decreased fewer than 10% of initial volume, the PCP/Fibrin mixture groups were confirmed maintaining 40% and 2% alginate group maintained well, but mound shift occurred terribly and was not positively stained in glycosaminoglycan. We found lacunae and chondrocytes around matrix in PCP/Fibrin mixture groups overall. PCP suspension/Fibrin mixture groups were positively stained for safranin-O staining. Conclusions: It is demonstrated that the porcine cartilage was successfully decellularized and made to a soluble form for delivering chondrocytes. PCP suspension/Fibrin mixture is a good cell delivery system for inducing cartilage formation and maintaining injected volume.

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ABSTRACT ⅰ
TABLE OF CONTENTS iv
LIST OF FIGURES v
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 4
III. RESULTS 11
IV. DISCUSSION 21
Ⅴ. CONCLUSION 25
REFERENCES 26
국문요약 29

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