Microbubble Infused Hydrogels for Cartilage Tissue Engineering

描述（由申请人提供）：在这个潜在的高回报R21提案中，我们探索了脂质外壳，充满气体的微泡（临床上用作超声造影剂）的新应用，作为软骨组织工程中制造充满细胞的微孔水凝胶的方法。与传统的孔隙浸出技术（通常是有毒的）不同，微泡溶解可以通过施加静水压力或超声波“按需”触发。后者为水凝胶中微孔形成提供了独特的空间控制，我们预计这将促进机械功能、大型（解剖形状）工程软骨结构的培养发展，最终作为大型同种异体移植物或关节植入物的临床替代品。微泡溶解过程产生了均匀分布在凝胶内的微孔，同时使凝胶内的细胞能够直接从头固定。重要的是，初步数据表明，与对照凝胶相比，具有微泡衍生微孔的软骨细胞种子水凝胶的机械性能增加了2倍。这种效果比我们观察到的使用软骨介质施加变形载荷的效果更大。我们也有证据表明，微气泡促进更均匀的轴向性质。所提出的制造髌骨构建体的研究是基于以下假设(H)和特定目标（SA）： 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率。SA1。制备含微泡浓度的软骨细胞种子水凝胶，其转化生长因子β 3 （TGF-23）的渗透性比不含微泡的水凝胶（0%）高25%、50%和100%，TGF-23是功能性软骨工程的关键化学因子。测量溶质渗透率(P)和材料性能，包括杨氏模量（EY）和动态模量（G*）。H2：与没有微泡的相同支架相比，含有软骨细胞种子的水凝胶支架将产生更接近天然组织特性的工程组织。H2a。微泡结构的性质取决于微泡溶解的时间。H2b。施加动态变形载荷增强了微泡注入水凝胶的有益效果。SA2a。采用SA1微泡条件（TGF-23通透性增加25%、50%、100%），培养56天，在第0天或第14天触发充气微泡溶解。在第0、14、28和56天测量材料和生化特性、溶质渗透率，并进行组织学检查。SA2b。使用在第0天和第14天触发的微气泡的最佳响应组重复SA2a，但应用每日动态变形加载（10%变形，1hz， 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).