Targeted Cellular Repair of Articular Cartilage

描述（由申请人提供）：已知成人软骨几乎没有修复能力，主要是由于缺乏活性软骨形成祖细胞。因此，治疗骨关节炎的替代方案有限，其中大多数针对疾病的症状而不是治愈。从患者分离的间充质干细胞和培养的前软骨细胞具有应用于软骨修复的潜力，如果它们可以被递送到需要的部位。假设用软骨基质特异性抗体包被的前软骨形成细胞可以用作再生或修复受损软骨的方法。该提案的重点是开发针对前软骨细胞的靶向策略，以便这些细胞可用于修复体内关节软骨缺损。为了实现这一目标，MSC和前软骨细胞将用棕榈酸化蛋白G（或A [PPG/A]）包被，然后与II型胶原蛋白或聚集蛋白聚糖分子上的表位的抗体一起孵育。这些抗体将与PPNG结合，并且它们的抗原结合位点将向外定向。将在兔关节软骨的冷冻切片和外植体上测试用不同抗体单独或组合包被的这些细胞结合软骨基质的能力。将前软骨细胞用活体荧光染料染色，用靶向抗体包被，添加到切片或外植体中，洗涤，然后观察是否存在荧光细胞。将对包被细胞进行体外研究，以确认包被程序不会显著阻碍细胞增殖或分化为软骨细胞的能力。一旦证明了软骨基质的靶向和合成，将测试这些靶向的前软骨细胞在体内修复兔中标准关节软骨缺损的能力。修复程度将通过关节软骨缺损的组织学检查和形态学检查进行评估。待测试的实验组是：（1）对照未处理的缺损，（2）靶向细胞注射，（3）PPA/g包被的，和（4）未处理的细胞注射。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引导到软骨基质，以及这些靶向细胞是否有助于修复受损的软骨。这些研究的长期目标是开发骨关节炎的治疗方法。

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.