Driving tissue formation by inductive stem cell sheet technology

描述(申请人提供)：骨关节炎(OA)是一种关节软骨退行性疾病，影响全球数百万人，包括超过70%的65岁以上的人。当关节软骨受损时，由于其细胞密度低、无血管等特点，自身修复能力不足。目前的技术通常不能恢复全部功能，因此需要一种替代疗法。软骨组织工程作为一种可行的技术来满足这一需求具有很大的潜力。来自骨髓的人骨髓间充质干细胞(HMSCs)是这些软骨组织工程策略的一种有前途的、临床相关的细胞来源。高的初始细胞密度和转化生长因子β的作用是体外诱导hMSCs向软骨分化的两个重要因素。我们设计了一种含有生长因子释放水凝胶微球的hMSC自组装片材系统。明胶被用作微球的基础生物材料，因为它形成了生物相容的、可生物降解的水凝胶，可以在细胞分泌的蛋白酶存在的情况下，随着时间的推移促进转化生长因子-1的受控输送，同时保持生长因子的生物活性。这种自组装、微球包埋的hMSC片材系统能够在外源转化生长因子-1存在的情况下形成软骨，或者通过微球中释放的转化生长因子-1形成软骨。携带转化生长因子1的微球可以消除外源性生长因子补充的需要，克服外源性补充的运输限制，缩短新骨构建物植入前所需的培养时间，并通过延长hMSCs对软骨生长因子的局部暴露而避免体内软骨形成表型缺失的问题。此外，组织工程化软骨片的动态压缩和灌流已被证明可以改善力学性能，并增加所产生的组织的细胞外基质合成。我们的中心假设是，这些hMSC-微球片的软骨形成潜力可以通过在存在或不存在机械加载和灌流的情况下通过控制生长因子递送到细胞来调节。具体目的是：(1)确定软骨形成因子和细胞数量的时空表达在hMSC片状软骨形成中的作用；(2)单独和联合动态压缩机械载荷和灌流，确定hMSC片状系统对软骨基质组成和组织以及由此产生的结构力学性能的影响；(3)检测该hMSC片状系统修复兔全层临界大小软骨缺损区关节软骨的能力。这项拟议工作的成功完成将为修复受损关节软骨的临床相关组织工程策略提供基础。 公共卫生相关性：受损的关节软骨是一种细胞很少的无血管组织，本身就没有自我修复的能力。我们建议将含有生长因子微球的软骨源性人骨髓间充质干细胞(HMSC)构建成软骨，并展示其在体内修复全层关节软骨缺损的潜力。拟议工作的成功实施将提供 这将为修复受损关节软骨的临床相关组织工程策略奠定基础，这将是推进骨关节炎患者软骨丢失或损伤治疗的一项强大技术。

DESCRIPTION (provided by applicant): Osteoarthritis (OA) is a degenerative disease of articular cartilage affecting millions of people worldwide, including over 70% of people over 65. When articular cartilage is damaged, it has inadequate intrinsic ability to repair itself due to it low cellularity and avascular nature. Current techniques do not typically restore total function, and thus an alternative therapy is needed. Tissue engineering of cartilage tissue has great potential as a viable technology to meet this need. Human mesenchymal stem cells (hMSCs) from bone marrow are a promising, clinically relevant cell source for these cartilage tissue engineering strategies. Two important factors for the in vitro chondrogenic induction of hMSCs are high initial cell density and exposure to transforming growth factor-¿ (TGF-¿). We have engineered a system of self-assembling hMSC sheets incorporated with growth factor releasing hydrogel microspheres. Gelatin is used as the base biomaterial for the microspheres, as it forms biocompatible, biodegradable hydrogels that can facilitate the controlled delivery of TGF-¿1 over time in the presence of cell-secreted proteases while preserving bioactivity of the growth factor. This system of self-assembled, microsphere-incorporated hMSC sheets is capable of forming cartilage in the presence of exogenous TGF-¿1 or with TGF-¿1 released from the incorporated microspheres. The incorporation of TGF-¿1 loaded microspheres could eliminate the need for exogenous growth factor supplementation, overcome transport limitations of exogenous supplementation, decrease culture time necessary prior to implantation of neocartilage constructs, and circumvent the problem of los of the chondrogenic phenotype in vivo by providing prolonged local exposure of hMSCs to chondrogenic growth factor. In addition, dynamic compression and perfusion of tissue engineered cartilage sheets have been shown to improve the mechanical properties and increase extracellular matrix synthesis of resulting tissue. Our central hypothesis is that the chondrogenic potential of these hMSC-microsphere sheets can be regulated by controlling growth factor presentation to the cells in the presence or absence of mechanical loading and perfusion. The Specific Aims are: (1) Determine the role of spatial and temporal presentation of chondrogenic factors and cell number on hMSC sheet chondrogenesis; (2) Determine the effects of dynamic compressive mechanical loading and perfusion, separately and in combination, of the hMSC sheet system on cartilage matrix composition and organization and resulting construct mechanical properties; and (3) Examine the capacity of this hMSC sheet system to repair articular cartilage in a full-thickness critical-sized cartilage defect in a rabbit model. The successful completion of the proposed work will provide the basis for a clinically relevant tissue engineering strategy for the repair of damaged articular cartilage. PUBLIC HEALTH RELEVANCE: Damaged articular cartilage, which is an avascular tissue with few cells, does not have an intrinsic capacity to repair itself. We propose to engineer chondrogenic human mesenchymal stem cell (hMSC) sheets containing growth factor-laden microspheres to form cartilage and to demonstrate their potential in healing full thickness articular cartilage defects in vivo. The successful execution of the proposed work will provide the basis for a clinically relevant tissue engineering strategy for the regeneration of damaged articular cartilage, which would be a powerful technology for advancing the treatment of patients suffering from cartilage loss or damage as is present in osteoarthritis.