Flow Perfusion Bioreactor Fabrication of Bioactive Polymer/ECM Hybrid Constructs

描述(申请人提供)：拟议研究的总体目标是将间充质干细胞(MSCs)的流动灌流生物反应器培养应用于构建用于组织工程的生物活性、可生物降解的聚合物/细胞外基质(ECM)杂化结构。本方案的重点是开发和应用这种创新的方法来制造用于修复骨软骨缺损的双层结构。假设MSCs在电纺聚(5-己内酯)(PCL)纳米纤维支架上流动灌流培养，在添加成骨或软骨补充的介质中将产生具有生物活性的聚合物/ECM杂化结构，其中ECM成分分别含有成骨或软骨形成因子，ECM的性质受培养条件和支架的性质的影响。进一步的假设是，在脱细胞和植入后，这些去细胞成骨和成软骨聚合物/ECM杂化结构将分别引导宿主祖细胞向骨或软骨组织的分化。假设由纳米纤维组成的支架将导致含有更多成骨或软骨因子的ECM沉积在微纤维支架上，因为纳米纤维支架更接近天然ECM分子的规模，并且由于孔径较小，在给定的流速下产生更大的剪应力。所施加的剪应力、支架的结构(微纤维和纳米纤维)以及培养条件对所产生的成骨和软骨混合构建物的影响将通过监测不同组织类型(例如，用于骨的I型胶原和用于软骨的II型胶原)的分子的存在来研究。此外，ECM生成的培养时间将被调整，以检测脱细胞混合构建物的ECM成分的成熟度对随后种植的MSCs在体外的成骨细胞和软骨细胞分化的影响(通过分化标记物，如碱性磷酸酶活性、钙和糖胺聚糖含量，以及I型和II型胶原蛋白的存在来衡量)，以及对兔模型骨软骨缺损体内组织形成的影响(根据组织学和组织形态计量学的衡量)。最后，将无细胞双层聚合物/ECM杂交物构建成成骨层和软骨化层，然后将其植入兔骨软骨缺损模型中，以评估构建物影响宿主祖细胞在各自层中形成骨和软骨的空间分化的潜力。这种由细胞在体外工程条件下产生的富含生长因子的细胞外基质，构建了一种新型的无细胞生物活性可降解组织工程结构，在引导组织再生方面具有巨大的应用潜力。临床上需要能够促进损伤或受损组织修复和再生的新型植入材料，例如受损的关节软骨。事实上，由于软骨自我修复的自然能力有限，关节软骨和底层骨骼的损伤往往会导致相当大的临床问题，这些问题困扰着全球数百万人，包括疼痛、活动受限和骨关节炎。这项提案中提出的研究项目旨在应用先进的细胞培养技术来制造具有生物活性的植入材料，这种材料可以促进受者体内的细胞再生或修复特定的受损组织，在这种情况下是关节软骨和底层骨。

DESCRIPTION (provided by applicant): The overall goal of the proposed research is to apply flow perfusion bioreactor culture of mesenchymal stem cells (MSCs) toward the fabrication of bioactive, biodegradable polymer/extracellular matrix (ECM) hybrid constructs for tissue engineering. The present proposal focuses upon the development and application of this innovative approach to fabricate bi-layered constructs for the repair of osteochondral defects. It is hypothesized that flow perfusion bioreactor culture of MSCs upon electrospun poly(5-caprolactone) (PCL) nanofiber scaffolds in medium augmented with osteogenic or chondrogenic supplements will produce bioactive polymer/ECM hybrid constructs with an ECM component containing osteogenic or chondrogenic factors, respectively, and that the character of the ECM is influenced by the culture conditions and the properties of the scaffold. It is further hypothesized that, following decellularization and implantation, these acellular osteogenic and chondrogenic polymer/ECM hybrid constructs will direct the differentiation of host progenitor cells toward the generation of bone or cartilage tissue, respectively. It is hypothesized that scaffolds composed of nanofibers will result in the deposition of ECM containing more osteogenic or chondrogenic factors than ECM deposited on microfiber scaffolds, as nanofiber scaffolds more closely approximate the scale of native ECM molecules and, due to the smaller pore size, produce increased shear stress at a given flow rate. The effects of the applied shear stress, the architecture of the scaffold (microfibers vs. nanofibers), and the culture conditions on the generated osteogenic and chondrogenic hybrid constructs will be investigated by monitoring the presence of molecules characteristic of the respective tissue types (e.g., collagen type I for bone and collagen type II for cartilage). Further, the culture duration for ECM generation will be modulated to examine the effect of the maturity of the ECM component of the decellularized hybrid constructs upon the osteoblastic and chondrocytic differentiation of subsequently seeded MSCs in vitro (as measured by differentiation markers such as alkaline phosphatase activity, calcium and glycosaminoglycan content, and the presence of collage types I and II) and upon tissue formation in vivo in an osteochondral defect in a rabbit model (as measured by histology and histomorphometry). Finally, acellular bi-layered polymer/ECM hybrid constructs will be fabricated with an osteogenic layer and a chondrogenic layer and then implanted in a rabbit osteochondral defect model to assess the potential of the constructs to influence the spatial differentiation of progenitor cells of the host to form bone and cartilage in the respective layers. This novel approach to fabricate acellular bioactive degradable tissue engineering constructs containing ECM rich in growth factors produced by cells under engineered conditions in vitro presents tremendous potential for application in the guided regeneration of a wide range of tissues. A significant clinical need exists for novel implant materials capable of promoting the repair and regeneration of injured or compromised tissues, such as damaged articular cartilage. Indeed, as cartilage has a limited natural capacity to repair itself, damage to articular cartilage and underlying bone often leads to considerable clinical problems that afflict million of people worldwide, including pain, limited mobility and osteoarthritis. The research project presented in this proposal seeks to apply advanced cell culture technologies to fabricate biologically active implant materials that can promote cells within the recipient to regenerate or repair specific damaged tissues, in this case articular cartilage and underlying bone.