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

我们的研究小组旨在开发一种骨软骨复合材料，使用II型胶原凝胶与羟基磷灰石（HAp）沉积在一侧。将凝胶浸泡在Ca^2+和磷酸盐溶液中对于HAp沉积是必不可少的，因此在二维和三维培养中检测了细胞行为与Ca^2+浓度之间的关系。目前的结果表明，2-4 mM Ca^<2+>适合于成骨细胞的增殖和存活，而略高的浓度（6-8 mM）有利于成骨细胞的分化和基质矿化，在二维和三维培养中均如此。较高浓度（>10 mM）具有细胞毒性，在含磷酸盐的凝胶中培养细胞，在含Ca^2+的培养基中也会导致凝胶中HAp的时间依赖性形成。考虑到包埋细胞的活力，在Ca^2+浓度约为5 mM的培养基中培养支架有利于HAp沉积和成骨细胞的行为。一个 ...更多信息 原位凝胶系统的开发和软骨细胞包封和均匀分散在碱处理的胶原蛋白（AlCol）凝胶。MTT染色结果显示细胞在AlCol凝胶包裹后存活。生化分析表明，DNA含量的AlCol凝胶是恒定的3周后。随着培养时间的延长，糖胺聚糖含量和II型胶原和聚集蛋白聚糖的mRNA表达增加。这些结果表明，这种原位凝胶系统可用于体外软骨再生和软骨缺损的微创治疗。体内实验中，将包埋在II型胶原凝胶中的软骨细胞直接注射到兔全层软骨缺损中，并在几分钟内与邻近的软骨和骨形成凝胶并结合。根据O 'Drivel组织学评分，移植组和对照组在12周和24周时明显差异。免疫组化染色显示移植后8周再生软骨中有足够的II型胶原合成。在24周时仍观察到II型胶原的强染色。这些结果表明，II型胶原凝胶可以实现充分的软骨再生，无论是在外观上还是在组织学上，并且这种支架对于软骨修复非常有用。少

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