Development of an Ostoechondral Xenograft for Articular Cartilage Repair

 描述(申请人提供)：膝关节的关节软骨很容易受到损伤，这种损伤通常是疼痛的，并可能发展为骨关节炎。自体骨软骨移植和同种异体软骨移植是目前唯一能立即恢复受损关节透明软骨的治疗方案。这些方法的缺点，如供区发病率和组织可获得性有限，激发了人们对异种骨软骨移植的兴趣，因为它具有成本低、供应充足的潜在优势。这项研究的长期目标是开发一种脱细胞猪骨软骨异种移植(OCXG)，一旦发现软骨损伤即可植入。开发包括优化脱细胞和交联方法，以降低免疫原性，提高移植物的耐用性和与宿主组织的整合。具体目的如下：1.比较含糖胺多糖和不含糖胺多糖的猪骨软骨钉的力学性能和体外支持干细胞侵袭和软骨诱导的能力。2.评价四种不同植物来源的无毒交联剂对GAG(+)和GAG(-)OCXGs物理力学性能的影响，以及对原代培养人膝关节软骨细胞行为的影响。3.在兔关节内模型中，评价GAG(+)和GAG(-)脱细胞的猪OCXGs的急性炎症反应，无论是使用SA2中最有前途的试剂进行交联还是非交联。4.测定去细胞交联型OCXGs修复成年犬滑车沟全层缺损的能力。第一个目标将提供有关脱细胞的根本不同方法的重要信息，一种寻求保存所有细胞外基质，另一种是故意提取非胶原性成分(特别是糖胺聚糖)以创造更多的孔洞。评估包括DNA和GAG含量的测量，移植物软骨的双相特性，以及在移植物上培养的人软骨细胞的附着、增殖和基质产生。第一个目标还包括开发一种新的脱细胞方案。第二个目标将揭示天然、无毒交联剂提高异种移植物机械性能和胶原酶抗性的潜力。它还将决定软骨细胞是否对交联度敏感。第三个目的是将OCXGs短期移植到成年兔股骨内侧髁内，主要是为了研究OCXG引起的滑膜炎。通过滑液分析和组织学检查脱细胞方法和交联法的效果。最终目标将是 评价猪骨软骨异种移植物用于填补犬膝关节手术造成的缺陷的功能。除了压痕测试外，大体外观和组织学的半定量评分将证明在这个临床前模型中，异种骨软骨移植是否能够恢复健康的关节表面。

 DESCRIPTION (provided by applicant): Articular cartilage of the knee is quite susceptible to injuries which are often painful and which may progress to osteoarthritis. Osteochondral autografting and allografting are currently the only treatment options which immediately restore hyaline cartilage to the injured joint. Drawbacks to these procedures such as donor site morbidity and limited tissue availability have stimulated interest in osteochondral xenografts, which have the potential advantages of low cost and abundant supply. The long-term objective of the proposed research is to develop a decellularized porcine osteochondral xenograft (OCXG) which can be available for implantation as soon as a cartilage lesion is discovered (i.e., "off-the-shelf"). Development includes optimization of decellularization and crosslinking methods to reduce immunogenicity and improve graft durability and integration with host tissue. The specific Aims are as follows: 1. Compare glycosaminoglycan-containing and glycosaminoglycan-free porcine osteochondral dowels in terms of their mechanical properties and ability to support stem cell infiltration and chondroinduction in vitro. 2. Evaluate the effect of four different plant- derived, nontoxic crosslinking agents on the physical and mechanical properties of GAG(+) and GAG(-) OCXGs, as well as their effects on primary human knee chondrocyte behavior in vitro. 3. Evaluate the acute inflammatory response to GAG(+) and GAG(-) decellularized porcine OCXGs, both crosslinked and non- crosslinked using the most promising agent from SA2, in an intraarticular rabbit model. 4. Determine the capacity for crosslinked, decellularized OCXGs to repair full-thickness defects in the trochlear groove of adult dogs. The first aim will provide important information about fundamentally different approaches to decellularization, one which seeks to preserve all extracellular matrix and one which intentionally extracts non- collagenous components (especially glycosaminoglycan) to create more porosity. Evaluations include measurement of the DNA and GAG contents, the graft cartilage's biphasic properties, and the attachment, proliferation, and matrix production of human chondrocytes cultured on the grafts. The first aim also includes development of a novel decellularization protocol. The second aim will reveal the potential for natural, nontoxic crosslinking agents to enhance xenograft mechanical properties and collagenase resistance. It will also determine whether chondrocytes are sensitive to the degree of crosslinking. The third aim involves short-term transplantation of OCXGs into the medial femoral condyle of mature rabbits, mainly to investigate OCXG- induced synovitis. The effects of decellularization method and crosslinking will be examined through synovial fluid analysis and histology. The final aim will evaluate the functionality of porcine osteochondral xenografts used to fill surgically created defects in the canine knee joint. Semiquantitative scoring of macroscopic appearance and histology, in addition to indentation testing, will demonstrate whether osteochondral xengrafts are capable of restoring a healthy joint surface in this preclinical model.