Driving tissue formation by inductive stem cell sheet technology

描述（由申请人提供）：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岁以上人群。当关节软骨受损时，由于其低细胞性和无血管性，其自身修复能力不足。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。软骨组织的组织工程作为一种可行的技术具有很大的潜力来满足这一需求。来自骨髓的人间充质干细胞（hMSCs）是一种有前途的、临床相关的软骨组织工程策略的细胞来源。体外诱导hMSCs成软骨的两个重要因素是高初始细胞密度和暴露于转化生长因子-¿（TGF-¿）。我们设计了一个自组装的hMSC片与生长因子释放水凝胶微球结合的系统。明胶被用作微球的基础生物材料，因为它形成生物相容性，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TGF-¿1的受控递送，同时保持生长因子的生物活性。这种自组装、微球掺入的hMSC薄片系统能够在外源性TGF-¿1存在或TGF-¿1从掺入的微球释放的情况下形成软骨。TGF-¿1负载微球的掺入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，减少植入新软骨构建体前所需的培养时间，并通过延长hMSCs局部暴露于软骨生长因子，避免体内软骨表型缺失的问题。此外，组织工程软骨片的动态压缩和灌注已被证明可以改善机械性能并增加所产生组织的细胞外基质合成。我们的中心假设是，这些hmsc微球片的成软骨潜能可以通过在机械负荷和灌注存在或不存在的情况下控制生长因子向细胞的呈递来调节。具体目的是：(1)确定成软骨因子和细胞数量的时空分布对hMSC片软骨形成的影响；(2)确定动态压缩机械加载和灌注（单独或联合）对hMSC片材系统软骨基质组成和组织以及由此产生的构建体力学性能的影响；(3)在兔模型中检测这种hMSC薄片系统修复全层临界尺寸软骨缺损的能力。这项工作的成功完成将为临床相关的修复受损关节软骨的组织工程策略提供基础。

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.