Development of artificial osteochondral composite with gradient apatite deposition for transplantation onto chondral defects

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Our research group aims to develop an osteochondral composite using type II collagen gel with hydroxyapatite (HAp) deposited on one side. Soaking gels in Ca^<2+> and phosphate solution is indispensable to HAp deposition, so relationships between cell behavior and Ca^<2+> concentration were examined in 2- and 3-dimensional cultures. The present results indicate that 2-4 mM Ca^<2+> is suitable for proliferation and survival of osteoblasts, whereas slightly higher concentrations (6-8 mM) favor osteoblast differentiation and matrix mineralization in both 2- and 3-dimensional cultures. Higher concentrations (>10 mM) are cytotoxic.Culturing cells in phosphate-containing gel in media with Ca^<2+> also leads to time-dependent formation of HAp in the gel. Considering the viability of embedded cells, culturing scaffolds in media with Ca^<2+> concentrations around 5 mM is useful for both HAp deposition and osteoblast behavior.Then, behavior of chondrocytes encapsulated in the gel was examined. An … More in situ gel system was developed and chondrocytes were encapsulated and dispersed homogeneously in alkali-treated collagen (AlCol) gels. Results of MTT staining showed that cells survived after encapsulation in AlCol gels. Biochemical analysis demonstrated that DNA content in AlCol gels was constant after 3 weeks. Glycosaminoglycan content and mRNA expression of type II collagen and aggrecan increased with culture time. These results suggest that this in situ gel system is useful for regenerating cartilage in vitro and for minimally invasive therapy for cartilage defects.In vivo experiment, chondrocytes embedded in the type II collagen gel were directly injected into rabbit full-thickness cartilage defects, gelled and bonded to the adjacent cartilage and bone in several minutes. According to O'Driscoll histological scores, significant differences between transplanted and control groups were apparent at 12 and 24 weeks. Immunohistochemical staining showed sufficient type II collagen synthesis in regenerated cartilage 8 weeks after transplantation. Intense staining of type II collagen was still observed at 24 weeks. These results indicated that the type II collagen gel can achieve sufficient cartilage regeneration, both grossly and histologically, and this scaffold is extremely useful for cartilage repair. Less