Flow Perfusion Bioreactor Fabrication of Bioactive Polymer/ECM Hybrid Constructs

生物活性聚合物/ECM 混合结构的流动灌注生物反应器制造

• 批准号: 7799085
• 负责人: ANTONIOS G. MIKOS
• 金额: $ 33.01万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7799085
• 关键词: Alkaline Phosphatase; Architecture; Biomimetics; Bioreactors; Bone Regeneration; Calcium; Caliber; Cartilage; Cell Culture Techniques; Cells; Characteristics; Clinical; Collagen Type I; Collagen Type II; Defect; Degenerative polyarthritis; Deposition; Development; Differentiation Antigens; Engineering; Extracellular Matrix; Fiber; Generations; Glycosaminoglycans; Goals; Growth Factor; Histocompatibility Testing; Histology; Hybrids; Implant; In Situ; In Vitro; Measures; Mesenchymal Stem Cells; Modeling; Monitor; Natural regeneration; Oryctolagus cuniculus; Pain; Perfusion; Poly-5; Polymers; Property; Research; Research Project Grants; Signal Transduction; Stem cells; Technology; Tissue Engineering; Tissues; articular cartilage; biodegradable polymer; bone; caprolactone; cartilage regeneration; density; implant material; implantation; in vivo; injured; innovation; nanofiber; novel; novel strategies; osteoblast differentiation; osteochondral repair; osteochondral tissue; osteogenic; repaired; scaffold; shear stress

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.

描述（由申请人提供）：拟议研究的总体目标是将间充质干细胞（MSC）的流动灌注生物反应器培养应用于制造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）混合构建体。本提案的重点是开发和应用这种创新的方法来制造双层结构修复骨软骨缺损。假设MSC在电纺聚（5-己内酯）（PCL）支架上在用成骨或软骨形成补充剂增强的培养基中的流动灌注生物反应器培养将产生具有ECM组分的生物活性聚合物/ECM混合构建体，所述ECM组分分别含有成骨或软骨形成因子，并且ECM的特征受培养条件和支架性质的影响。进一步假设，在脱细胞化和植入后，这些脱细胞成骨和软骨形成聚合物/ECM混合构建体将分别引导宿主祖细胞分化成骨或软骨组织。假设由纳米纤维组成的支架将导致ECM的沉积，所述ECM比沉积在微纤维支架上的ECM含有更多的成骨或软骨形成因子，因为纳米纤维支架更接近天然ECM分子的规模，并且由于较小的孔径，在给定的流速下产生增加的剪切应力。将通过监测相应组织类型的分子特征（例如，I型胶原用于骨，II型胶原用于软骨）。此外，将调节ECM产生的培养持续时间，以检查脱细胞杂交构建体的ECM组分的成熟度对随后接种的MSC体外成骨细胞和软骨细胞分化的影响（如通过分化标志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）和在兔模型中骨软骨缺损的体内组织形成时（如通过组织学和组织形态计量学测量的）。最后，将用成骨层和成软骨层制造无细胞双层聚合物/ECM混合构建体，然后植入兔骨软骨缺损模型中，以评估构建体影响宿主祖细胞的空间分化以在相应层中形成骨和软骨的潜力。这种新的方法来制造脱细胞生物活性可降解的组织工程构建体含有ECM丰富的生长因子产生的细胞在体外工程条件下，在广泛的组织的引导再生的应用呈现出巨大的潜力。对于能够促进损伤或受损组织（例如受损的关节软骨）的修复和再生的新型植入物材料存在显著的临床需求。事实上，由于软骨具有有限的天然自我修复能力，关节软骨和底层骨的损伤通常导致相当大的临床问题，这些问题困扰着全世界数百万人，包括疼痛、活动受限和骨关节炎。该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，这些材料可以促进受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。