Cartilage storage solution for chondrocyte viability and biomaterial preservation

描述(申请人提供)：冷藏同种异体骨软骨软骨表面置换在临床上用于修复创伤和骨关节炎引起的关节软骨表面损伤。同种异体骨软骨移植后关节软骨细胞活性被认为是影响移植结果的决定因素之一。在临床使用软骨之前，需要仔细评估用于软骨存储的冷藏方法，因为在存储过程中，组织可能会发生显著的临床恶化。我们最近研究了软骨细胞在培养液和储存液中储存期间的细胞活性和基质渗透性，发现两者在用于临床操作的时间范围内都会恶化。在冷藏条件下保存软骨细胞活性最好的培养液不能保持基质通透性，反之亦然，营养不良的保持基质通透性的溶液细胞活力显著降低。这一目标将在三个具体目标中发展。在这些目标中，将研究两种溶液配方，一种基于细胞内设计，另一种基于细胞外环境设计。在猪软骨保存期间，软骨细胞的活力、化学成分、生物材料特性和基因表达将随着时间的推移而进行比较。基因表达研究将确定哪种配方能维持未经处理的软骨的正常表达，包括Sox9、aggrecan、II型胶原(与去分化标记物I型胶原相比)、软骨低聚基质、基质吸收标记物加上蛋白质和肥大标记物基因。该溶液可提供保存时间最长的软骨细胞，具有正常的未经处理的软骨细胞表型，且软骨生物材料变化最小，将被选择用于进一步的体内研究，并在随后的第二阶段SBIR应用中转化为人类软骨。

DESCRIPTION (provided by applicant): Resurfacing of articular cartilage with cold stored osteochondral allografts is employed clinically for repair of trauma and osteoarthritis-induced articular cartilage surface damage. Chondrocyte viability of transplanted articular cartilage is accepted as one of the determinants of outcome following osteochondral allograft transplantation. Refrigerated storage methods used for cartilage storage prior to clinical cartilage utilization need to be carefully evaluated because the tissue may be experiencing clinically significant deterioration during storage. We have recently investigated cartilage cell viability and matrix permeability during storage in culture medium, as well as storage solutions, and found that both deteriorate within the time frames that they are utilized for clinical procedures. Culture medium that preserves chondrocyte viability best under cold refrigerated storage conditions does not preserve matrix permeability and, vice versa, nutritionally deficient solutions that preserve matrix permeability have significantly less cell viability. This objective ill be developed in three specific aims. In these aims two solution formulations, one based on intracellular and the other on extracellular milieu designs will be investigated. Chondrocyte viability, chemistry, biomaterial properties and gene expression will be compared over time during porcine cartilage storage. The gene expression studies will determine which formulation maintains normal untreated cartilage expression of Sox9, aggrecan, collagen type II (versus dedifferentiation marker collagen type I), cartilage oligomeric matrix, a matrix resorption marker plus protein and hypertrophic marker genes. The solution that provides the longest preservation of chondrocytes with a normal untreated chondrocyte phenotype with minimal if any cartilage biomaterial changes will be selected for further investigation in vivo and translation to human cartilage in a subsequent Phase II SBIR application.