Targeted Cellular Repair of Articular Cartilage

描述(申请人提供)：成人软骨已知几乎没有修复能力，主要是由于缺乏活跃的软骨前体细胞。因此，治疗骨性关节炎的选择有限，大多数针对的是疾病的症状，而不是治愈方法。从患者体内分离的间充质干细胞和培养的前软骨细胞，如果能被输送到需要的部位，就有可能应用于软骨修复。推测软骨基质特异性抗体包被的前软骨形成细胞可以作为一种再生或修复受损软骨的方法。这项提议的重点是开发一种针对前软骨细胞的靶向策略，以便这些细胞可以用于修复体内的关节软骨缺损。为了实现这一目标，MSCs和前软骨细胞将被棕榈化蛋白G(或A[PPG/A])包裹，然后与II型胶原抗体或凝集素分子上的抗原表位孵育。这些抗体将与PPNG结合，其抗原结合部位将向外定位。这些细胞被不同的抗体包裹，单独或联合使用，将测试它们在冰冻切片和兔关节软骨移植块上结合软骨基质的能力。前软骨细胞将被一种重要的荧光染料染色，涂上靶向抗体，加入切片或外植体，清洗，然后观察荧光细胞的存在。体外研究将在涂层细胞上进行，以确认涂层程序不会显著阻碍细胞增殖或分化为软骨细胞的能力。一旦证实了软骨基质的靶向和合成，这些靶向前软骨细胞将在体内测试它们修复标准兔关节软骨缺损的能力。修复程度将通过组织学检查和关节软骨缺损的形态计量学检查来评估。试验组为：(1)空白对照，(2)靶向细胞注射，(3)PPA/g涂层，(4)未处理细胞注射。这些实验将确定这种新的细胞靶向方法是否有效地将前软骨细胞定向到软骨基质，以及这些靶向细胞是否有助于受损软骨的修复。这些研究的长期目标是开发一种治疗骨关节炎的方法。

DESCRIPTION (provided by applicant): Adult cartilage is known to have little ability for repair, due primarily to a lack of active chondrogenic progenitor cells. As a result, there are limited alternatives for the treatment of osteoarthritis, most of which are targeted at the symptoms of the disease and not at a cure. Mesenchymal stem cells and cultured pre-chondrocytes isolated from a patient have the potential to be applied to the repair of cartilage if they can be delivered to the sites where needed. It is hypothesized that pre-chondrogenic cells coated with antibodies specific for cartilage matrix could be used as a method to regenerate or repair damaged cartilage. The focus of this proposal is to develop a targeting strategy for pre-chondrocytes so that these cells can be used to repair an articular cartilage defect in vivo. To achieve this goal, MSCs and pre-chondrocytes will be coated with palmitated protein G (or A [PPG/A]) and then incubated with antibodies to type II collagen or to epitopes on aggrecan molecules. These antibodies will bind to the PPNG and their antigen binding sites will be oriented outward. These cells coated with different antibodies, alone or in combination, will be tested for their ability to bind cartilage matrix on frozen sections and on explants of rabbit articular cartilage. Pre-chondrocytes will be stained with a vital fluorescent dye, coated with targeting antibodies, added to sections or explants, washed and then observed for the presence of fluorescent cells. In vitro studies will be carried out on coated cells to confirm that the coating procedure does not significantly impede the cells ability to proliferate or to differentiate into chondrocytes. Once targeting and synthesis of cartilage matrix have been demonstrated, these targeted pre-chondrocytes will be tested for their ability to repair a standard articular cartilage defect in rabbits, in vivo. The degree of repair will be assessed by histologic examination and morphometric examination of the articular cartilage defects. Experimental groups to be tested are: (1) control untreated defects, (2) targeted cell-injected, (3) PPA/g coated, and (4) untreated cell-injected. These experiments will determine if this novel cell targeting methodology effectively directs pre-chondrocytes to cartilage matrix and whether these targeted cells can contribute to the repair of damaged cartilage. The long-term goal of these studies is to develop a treatment for osteoarthritis.