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

骨髓间充质干细胞原位再生透明软骨

• 批准号: 9206892
• 负责人: CONG-QIU CHU
• 金额: --
• 依托单位: PORTLAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9206892
• 关键词: Adipose tissue; Adult; Affect; Alpha Cell; Animal Model; Animals; Antibodies; Autologous; Biological Assay; Biomechanics; Bone Marrow; Cartilage; Cartilage injury; Cell Differentiation process; Cell Proliferation; Cells; Characteristics; Charge; Chimeric Proteins; Chondrocytes; Chondrogenesis; Chronic; Clinical Trials; Collagen; Collagen Type I; Collagen Type II; Contralateral; Data; Defect; Degenerative polyarthritis; Delayed Hypersensitivity; Development; Ensure; Failure; Family; Fibrocartilages; Fluorescence; Future; Genes; HMG Domain; Histologic; Histology; Human; Hyaline Cartilage; Hybrids; Immune response; Immunohistochemistry; Impairment; In Situ; In Vitro; Knee; Knowledge; Lead; Lesion; Life; Measures; Mediating; Membrane; Mesenchymal Differentiation; Mesenchymal Stem Cells; Methods; Modeling; Molecular; Natural regeneration; Oryctolagus cuniculus; Outcome; Pathology; Pathway interactions; Phenotype; Prevention; Procedures; Process; Production; Property; Proteins; Reaction; Recruitment Activity; Regulation; Repression; Research; SOX9 protein; Serological; Serum; Signaling Protein; Site; Source; Synovial Membrane; Synovial joint; T cell response; T-Lymphocyte; Techniques; Tertiary Protein Structure; Testing; Therapeutic Effect; Time; Tissues; Transplantation; Treatment Efficacy; United States; Variant; aggrecan; articular cartilage; cartilage development; cartilage regeneration; cartilage repair; clinical development; cost; design; experimental study; functional improvement; immunogenicity; improved; improved outcome; in vivo; in vivo regeneration; lymphocyte proliferation; migration; minimally invasive; novel therapeutics; prevent; public health relevance; repaired; self-renewal; subchondral bone; success; therapy design; tissue repair; transcription factor

 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.

 描述（由申请人提供）： 腕关节损伤是常见的，其可导致以关节软骨的进行性破坏为特征的骨关节炎（OA）的进展，并最终导致滑膜关节的功能衰竭。软骨再生已成为软骨修复和治疗OA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，能够自我更新并分化为软骨细胞，是软骨原位再生的潜在来源。微骨折诱导骨髓MSC迁移到软骨缺损部位并促进纤维软骨的产生。然而，纤维软骨是不持久的，长期功能不足。SOX 9是属于SOX（Sry型HMG盒）家族的转录因子，并且已被鉴定为软骨细胞表型的“主调节因子”。我们已经产生了一种细胞穿透，超正电荷的SOX 9融合蛋白（scSOX 9）。体外研究表明，scSOX 9蛋白诱导骨髓来源的MSC增殖并分化为软骨细胞。这通过减少I型胶原和X型胶原的产生而增加II型胶原和聚集蛋白聚糖（其是关节软骨细胞的特征）来证明。在21天的培养物中，最初一次性添加scSOX 9足以使MSC分化为软骨细胞并维持软骨细胞表型。初步的体内数据表明，scSOX 9在短期内显著改善了软骨修复中微骨折的结果。 在本提案中，我们的目标是通过刺激兔软骨缺损模型中的骨髓MSC来促进软骨原位再生。设计实验以在有缺陷的软骨的微骨折部位施用scSOX 9蛋白。scSOX 9的施用将通过在微骨折部位用生物支架、胶原蛋白膜直接递送来进行。在12周和24周时，通过检查聚集蛋白聚糖和I型、II型和X型胶原蛋白以及骨钙素的含量，使用组织学和免疫组织化学评估软骨再生的透明软骨特征。还将评估修复软骨的生物力学特性。将通过测量血清抗体和对scS0X9的T细胞反应来评估对scS0X9的免疫应答。将在先前在对侧膝关节中接受scS0X9的动物中评估具有微骨折的scS0X9的再施用的功效。将研究scSOX 9介导的透明软骨修复的分子机制。 该研究计划利用微创、简单、低成本的微骨折吸引骨髓MSC和scSOX 9的促软骨形成特性原位再生关节软骨用于治疗软骨缺损。这个项目的成功 将为设计使用相同技术治疗软骨病变和预防进展为OA的临床试验提供宝贵的信息。