Microbubble Infused Hydrogels for Cartilage Tissue Engineering

描述(由申请人提供)：在这项潜在的高回报R21提案中，我们探索了脂壳充气微泡(临床用作超声造影剂)作为一种用于软骨组织工程的细胞负载微孔水凝胶的新应用。与传统的致孔剂浸出技术(通常是有毒的)不同，微泡的溶解可以通过施加静水压力或超声波来“按需”触发。后者在水凝胶中提供了独特的微孔形成的空间控制，我们预计这将促进机械功能的大型(解剖形状)工程软骨结构的培养发展，最终用作大型同种异体移植或关节植入物的临床替代品。微泡溶解过程产生均匀分布在凝胶中的微孔，同时使得凝胶中的细胞能够直接从头开始固定。重要的是，初步数据显示，含有微泡衍生微孔的软骨细胞种子水凝胶的机械性能比对照凝胶提高了2倍。这种影响比我们使用软骨性介质施加变形载荷时观察到的更大。我们也有证据表明，微气泡促进了更均匀的轴向特性。本研究的目的是在以下假设(H)和特定目标(SA)的指导下进行研究：H1：微泡注入的水凝胶支架以微泡剂量依赖的方式增加溶质渗透性。SA1.制备具有微泡浓度的软骨细胞种子水凝胶结构，与不含微泡的水凝胶(0%)相比，转化生长因子β3(转化生长因子-23)的渗透性最初可高出25%、50%、100%。转化生长因子β3是功能软骨工程中的关键化学因素。测量溶质渗透性(P)和材料特性，包括杨氏弹性系数(EY)和动态弹性系数(G*)。H2：与没有微泡的相同支架相比，含有微泡的软骨细胞种植的水凝胶支架将产生性能更接近天然组织的工程化组织。H_2A。具有微泡的构造物的性质取决于微泡溶解的时间。H_2B。应用外加动态变形载荷增强了微泡注入水凝胶的有益效果。SA2a。利用SA1的微泡条件(增加25%、50%、100%的转化生长因子-23通透性)，培养56天，在第0天或第14天触发充气微泡的溶解。在第0、14、28和56天测量材料和生化性质、溶质通透性并进行组织学检查。SA2B。对第0天和第14天触发的微泡，使用最佳响应组重复SA2a，但施加每日动态变形载荷(10%变形，1赫兹，3小时/天)。

DESCRIPTION (provided by applicant): In this potentially high-reward R21 proposal, we explore the novel application of lipid-shelled, gas-filled microbubbles (used clinically as ultrasound contrast agents) as a method for creating cell laden microporous hydogels for cartilage tissue engineering. Rather than classical techniques of porogen leaching (which are often toxic), microbubble dissolution can be triggered "on-demand" by applied hydrostatic pressure or ultrasound. The latter affords unique spatial control of micropore formation in the hydrogel, which we anticipate will promote culture development of mechanically functional, large (anatomically-shaped) engineered cartilage constructs to serve ultimately as clinical alternatives to large allografts or joint implants. The microbubble dissolution process generates micropores that are homogeneously distributed within the gels, while enabling the direct ab initio immobilization of cels within the gels. Importantly, preliminary data demonstrates a 2-fold increase in mechanical properties of chondrocyte-seeded hydrogels with microbubble- derived microporosity versus control gels. This effect is greater than we have observed with applied deformational loading using chondrogenic media. We also have evidence that microbubbles promote more homogeneous axial properties. The proposed research to fabricate patella constructs is guided by these Hypotheses (H) & Specific Aims (SA): H1: Microbubble-infused hydrogel scaffolds exhibit increasing solute permeability in a microbubble dose- dependent manner. SA1. Fabricate chondrocyte-seeded hydrogel constructs with microbubble concentrations yielding initially 25%, 50%, 100% greater permeability of transforming growth factor beta 3 (TGF-23), a critical chemical factor in engineering of functional cartilage, than the hydrogel without microbubbles (0%). Measure solute permeability (P) and material properties including Young's modulus (EY) and dynamic modulus (G*). H2: Chondrocyte-seeded, hydrogel scaffolds incorporated with microbubbles will yield engineered tissues with properties closer to the native tissue compared to the same scaffolds without microbubbles. H2a. The properties of constructs with microbubbles are dependent on timing of microbubble dissolution. H2b. Application of applied dynamic deformational loading enhances the beneficial effects of microbubble-infused hydrogels. SA2a. Using microbubble conditions of SA1 (25%, 50%, 100% increase in TGF-23 permeability), culture constructs for 56 days with triggered dissolution of gas-filled microbubbles on day 0 or day 14. Measure material and biochemical properties, solute permeability, and perform histology on day 0, 14, 28 and 56. SA2b. Repeat SA2a using the best responding groups for microbubbles triggered on day 0 and day 14, but with application of daily dynamic deformational loading (10% deformation at 1 Hz, 3 hours/day).