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: 8234157
• 负责人: ANTONIOS G. MIKOS
• 金额: $ 31.69万
• 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/8234157
• 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质干细胞（MSC）的流动性生物反应器培养到生物活性，可生物降解的可生物降解聚合物/细胞外基质（ECM）杂交构建体的培养。本提案的重点是这种创新方法在制造双层构造以修复骨软骨缺损的情况下的开发和应用。据推测，在电纺多（5-辅助酮）（PCL）（PCL）培养基中的流动灌注生物反应器培养，培养基增强的纳米纤维支架，并用成骨或软骨的补充剂增强生物活性聚合物/ECM聚合物/ECM Hybrid构建体，含有ECM成分，以表格和稳定性因素与稳定性的构成，并具有稳态的因素，并且具有反应性的稳定性。培养条件和支架的特性。进一步假设，在脱细胞和植入后，这些细胞成骨和软骨的聚合物/ECM杂交构建体将分别指导宿主祖细胞的分化降低骨或骨骼组织的产生。假设由纳米纤维组成的支架将导致ECM与沉积在微纤维支架上的ECM相比，含有更多的成骨或软骨发育因素，因为纳米纤维支架更接近近似于本机ECM分子的尺度，并且由于较小的孔径而产生的孔径较小。施加剪切应力，脚手架的结构（微纤维与纳米纤维）的影响以及培养条件对所产生的成骨和软骨造型杂交结构的影响，将通过监测相应组织类型的分子特征（例如，II型胶原型I型II型II型II型I II类型的分子特征）来研究。此外，将对ECM生成的培养持续时间进行调节，以检查脱细胞杂交构建体的ECM成分的成熟度对体外种子MSC的成骨细胞和软骨细胞分化的成熟成分的影响（如随后通过差异标志物（如碱性磷酸化酶）和糖磷酸化的类型，酸性和乳糖素元素的类型，以差异标记来测量。兔模型中骨软骨缺损中的体内组织形成（通过组织学和组织形态计量法测量）。最后，细胞双层聚合物/ECM杂交构建体将用成骨层和软骨层层制造，然后植入兔骨软骨缺陷模型中，以评估构建体的潜力，以影响骨骼和骨骼中骨骼和软骨的骨骼和软骨的空间分化的潜力。这种新型的方法是制造含有富含ECM的ECM的细胞生物活性降解的组织工程结构，该结构富含由细胞在体外的细胞产生的生长因子产生的生长因子，这在体外具有巨大的潜力，可在广泛的组织的指导再生中应用。对于能够促进受伤或损害组织的修复和再生（例如关节软骨受损的关节软骨）的新型植入物材料，存在着重要的临床需求。确实，由于软骨的自然能力有限，关节软骨和潜在的骨骼的损害通常会导致临床问题，这些临床问题困扰着全球数百万人，包括疼痛，流动性有限和骨关节炎。本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物材料，这些植入物可以促进受体中的细胞以再生或修复特定受损的组织，在这种情况下，在这种情况下是关节软骨和潜在的骨骼。