Regeneration of hyaline cartilage in situ with bone marrow mesenchymal stem cells

 描述(由申请人提供)： 软骨损伤是一种常见的疾病，可导致以关节软骨进行性破坏为特征的骨性关节炎的进展，最终导致滑膜关节的功能衰竭。软骨再生已成为骨性关节炎软骨修复和治疗的一种有吸引力的方法。然而，自体软骨细胞移植在体外扩增过程中存在细胞供应不足、供体部位新的损伤和软骨细胞去分化等问题。间充质干细胞(MSC)存在于骨髓和许多成人组织中，具有自我更新和分化为软骨细胞的能力，是软骨原位再生的潜在来源。微骨折诱导骨髓间充质干细胞向软骨缺损处迁移，促进纤维软骨的生成。然而，从长远来看，纤维软骨是不可持续的，功能不够充分。Sox9是一种属于Sox(Sry-type HMG box)家族的转录因子，被认为是软骨细胞表型的主要调节因子。我们已经产生了一种穿透细胞、带超正电的SOX9融合蛋白(ScSOX9)。体外研究表明，scSOX9蛋白可诱导骨髓间充质干细胞增殖和分化为软骨细胞。这明显表现为I型和X型胶原的产生减少，而II型胶原和聚集素的增加，这是关节软骨细胞的特征。在第21天的培养中，最初一次性加入scSOX9足以将MSC分化为软骨细胞，并保持软骨细胞的表型。初步的体内数据表明，scSOX9在短期内显著改善了软骨修复中微骨折的结果。在这项建议中，我们的目标是通过刺激骨髓间充质干细胞来促进软骨的原位再生。实验的目的是在受损软骨的微骨折部位应用scSOX9蛋白。ScSOX9的给药将通过直接使用生物支架和微骨折处的胶原膜进行。术后12周和24周用组织学和免疫组织化学方法观察软骨再生情况，并检测软骨组织中聚集素、I、II、X型胶原和骨钙素的含量，以观察透明软骨的特性。修复后的软骨的生物力学性能也将被评估。对scSOX9的免疫反应将通过检测血清抗体和T细胞对scSOX9的反应来评估。在以前在对侧膝关节接受scSOX9治疗的动物身上，将评估再次注射scSOX9合并微骨折的疗效。ScSOX9介导的透明软骨修复的分子机制将被研究。该研究计划利用微创、简单、低成本的微骨折来吸引骨髓间充质干细胞和scSOX9的促软骨形成特性来原位再生关节软骨，用于软骨缺损的治疗。这个项目的成功 将为设计一项临床试验提供宝贵的信息，该试验使用相同的技术治疗软骨损伤并防止进展为骨性关节炎。

 DESCRIPTION (provided by applicant): Cartilage injury is common which can lead to progression of osteoarthritis (OA) which is characterized by progressive breakdown of articular cartilage, and ultimately leads to functional failure of synovial joints. Regeneration of cartilage has been an attractive approach to cartilage repair and treatment of OA. However, autologous chondrocyte transplantation suffers from insufficient cell supply, new damage to the donor sites and chondrocytes dedifferentiation during in vitro expansion. Mesenchymal stem cells (MSC) which reside in the bone marrow and many adult tissues are capable of self-renewal and differentiation into chondrocytes and are a potential source for cartilage in situ regeneration. Microfracture induces migration of bone marrow MSC to the site of cartilage defect and promotes fibrocartilage production. However, fibrocartilage is non-durable and functionally inadequate in the long-term. SOX9 is a transcription factor belonging to the SOX (Sry-type HMG box) family and has been identified as a "master regulator" of the chondrocyte phenotype. We have generated a cell penetrating, super positively charged SOX9 fusion protein (scSOX9). In vitro studies have demonstrated that scSOX9 protein induced bone marrow derived MSC proliferation and differentiation into chondrocytes. This was evident by decreased production of collagen type I and type X but increased collagen type II and aggrecan, which are characteristics of articular chondrocytes. In a 21 day culture, the initial one time addition of scSOX9 was sufficient to differentiate MSC into chondrocytes and maintain the chondrocyte phenotype. Preliminary in vivo data demonstrated scSOX9 significantly improved the outcome of microfracture in cartilage repair in short-term. In this proposal, we aim to promote cartilage in situ regeneration by stimulating bone marrow MSC in a rabbit model of cartilage defect. Experiments are designed to administer scSOX9 protein at the site of microfracture of the defective cartilage. Administration of scSOX9 will be carried out by either direct delivery with a bioscaffold, collagen membrane at the site of microfracture. Regeneration of cartilage will be assessed at 12 and 24 weeks using histology and immunohistochemistry for characters of hyaline cartilage by examination of content of aggrecan and collagen type I, II and X and osteocalcine. The biomechanical properties of the repaired cartilage will also be evaluated. Immune response to scSOX9 will be assessed by measuring serum antibodies and T cells reaction to scSOX9. Efficacy of re-administration of scSOX9 with microfracture will be assessed in animals who received scSOX9 previously in contralateral knee. Molecular mechanism for which scSOX9 mediated hyaline cartilage repair will be investigated. The research plan takes advantage of the minimal invasive, simple and low cost microfracture to attract bone marrow MSC and pro-chondrogenic property of scSOX9 to regenerate articular cartilage in situ for therapy of cartilage defect. The success of this project will provide invaluable information for design of a clinical trial using the same techniques for therapy of cartilage lesions and prevention of progression to OA.