Self Assembly of Cartilage by Mesenchymal Stem Cells on Porous Chitosan/CaP

描述（由申请人提供）：本研究的目的是开始开发一种使用工程软骨进行全关节表面置换的方法。该方法基于产生多孔壳聚糖/磷酸钙和透明软骨的双相构建体，软骨由附着并堆积在支架上的间充质干细胞（MSC）产生。本研究的主要目的是：1）研究细胞接种密度和生长因子对人骨髓间充质干细胞自组装软骨生物化学和力学性能的影响; 2）优化工程化软骨与多孔壳聚糖-纳米晶磷酸钙的整合和附着3）工程化连接到蘑菇形壳聚糖/CaP支架的股骨头大小的软骨半球壳; 4）利用来源于同种异体骨髓MSC的双相软骨/CaP-壳聚糖构建体修复新西兰白色兔股骨转子沟中手术产生的临界尺寸缺损。具体目标1仅涉及在无支架模型中通过MSC增强软骨形成。由于MSC是一种稀缺商品，因此重要的是要了解最佳软骨形成需要多少MSC。我们还将评估TGF-21、TGF-23和BMP-13单独和联合使用对加速软骨诱导和细胞外基质快速积累的作用。具体目标2集中于将工程化软骨结合到刚性、多孔、骨传导性支架上，天然骨可以整合到该支架上以锚工程化软骨。我们将确定支架微结构和纤维连接蛋白涂层如何影响软骨形成和软骨-支架界面强度。作为特定目标1和2的一部分，将根据组织学、生物化学和生物力学特性评价工程化软骨。特定目标3增加了将工程软骨和支撑支架成型为股骨头表面置换所需的半球形的复杂性。我们将尝试在附着于多孔壳聚糖/CaP支架的半球形壳中产生厚度均匀的软骨层。将测量该软骨的厚度、球度和摩擦系数，以评估其是否适合关节表面置换。如果成功，Specific Aim 4将通过在活体动物中将透明软骨恢复到全层缺损来提供概念验证。该项目将通过展示一种用工程软骨进行关节表面重建的可行方法来影响公众健康。该提案针对一个特定的挑战，即股骨头表面置换，并代表了克服这一挑战所需的第一步。 公共卫生相关性：该项目解决了骨关节炎治疗替代方案开发中的重要问题，骨关节炎是一种影响数百万美国人的退行性疾病，每年造成数十亿美元的工资和生产力损失。软骨组织工程有可能用健康的功能性组织重建骨关节炎关节的表面，因此不需要用金属、塑料或陶瓷部件进行关节置换。该项目开发了一种关节表面重建策略，该策略基于将自组装软骨连接到多孔磷酸钙-壳聚糖复合支架的双相结构。

DESCRIPTION (provided by applicant): The objective of this research is to begin developing a method for total joint resurfacing with engineered cartilage. The approach is based on generating a biphasic construct of porous chitosan/calcium phosphate and hyaline cartilage, the cartilage having been produced by mesenchymal stem cells (MSCs) attached to and piled atop the scaffold. This proposal encompasses four aims: 1) Investigate the effects of cell seeding density and growth factor on the biochemical and mechanical properties of cartilage produced from human marrow-derived MSCs by the self-assembling process; 2) Optimize integration and attachment of engineered cartilage to a porous chitosan-nanocrystalline calcium phosphate (chitosan/CaP) scaffold; 3) Engineer a femoral head-sized hemispherical shell of cartilage attached to a mushroom-shaped chitosan/CaP scaffold; 4) Use biphasic cartilage/CaP-chitosan constructs derived from allogenic bone marrow MSCs to repair critical size defects surgically created in the trochlear groove of New Zealand White rabbit femora. Specific Aim 1 deals just with enhancing chondrogenesis by MSCs in a scaffold-free model. Since MSCs are a scarce commodity it is important to understand how many will be needed for optimal chondrogenesis. We will also assess the effects of TGF-21, TGF-23, and BMP-13, alone and in combination, for accelerated chondroinduction and rapid accumulation of extracellular matrix. Specific Aim 2 is focused on bonding of the engineered cartilage to a rigid, porous, osteoconductive scaffold to which native bone could integrate to anchor the engineered cartilage. We will determine how scaffold microstructure and coating with fibronectin affect chondrogenesis and the cartilage-scaffold interfacial strength. Engineered cartilage produced as part of Specific Aims 1 and 2 will be evaluated based on histological, biochemical, and biomechanical properties. Specific Aim 3 adds the complexity of molding the engineered cartilage and supporting scaffold into a hemispherical shape as would be required for femoral head resurfacing. We will attempt to produce a uniform thickness layer of cartilage in a hemispherical shell attached to the porous chitosan/CaP scaffold. The thickness, sphericity, and coefficient of friction of this cartilage will be measured to assess its suitability for joint resurfacing. If successful, Specific Aim 4 will provide a proof-of-concept by restoring hyaline cartilage to a full-thickness defect in a living animal. This project will impact public health by demonstrating a feasible approach to joint resurfacing with engineered cartilage. This proposal targets a specific challenge, femoral head resurfacing, and represents the first steps necessary to overcome this challenge. PUBLIC HEALTH RELEVANCE: This project addresses important issues in the development of treatment alternatives for osteoarthritis, a degenerative disease that affects millions of Americans and is responsible for billions of dollars annually in lost wages and productivity. Cartilage tissue engineering has the potential for resurfacing an osteoarthritic joint with healthy, functional tissue so that joint replacement with metal, plastic, or ceramic components is unnecessary. This project develops a joint resurfacing strategy based on a biphasic construct of self-assembled cartilage attached to a porous calcium phosphate-chitosan composite scaffold.