Injectable, In Situ Forming Hydrogels for Craniofacial Tissue Engineering

描述(由申请人提供)：建议研究的基本原理是基于微创策略的需要，以提供由创伤和疾病引起的复杂颅面部缺陷的功能和美学重建。本研究的目的是开发一种可注射的、原位形成的水凝胶，它利用独特的双凝胶机制在生理温度下快速形成凝胶，通过化学交联来保持水凝胶的稳定性，并通过生物降解来实现组织再生。这项拟议的研究将检验以下基本假设：可注射水凝胶将为间充质干细胞(MSC)的增殖提供支持和矿化的基质，在体外实现成骨分化，并在体内促进骨再生。这项研究将通过三个具体目标完成：1)开发和表征可注射的、原位形成的、可生物降解的、可填充空间的水凝胶作为脱细胞组织工程疗法；2)评估可注射的双凝胶水凝胶在体外促进包裹的MSCs的增殖和成骨分化以及体内骨再生的有效性；以及3)评估细胞种植密度和预分化阶段对可注射的双凝胶水凝胶在体内再生能力的影响。合成了具有可交联性和可降解性官能团的新型大分子聚合物，并用核磁共振氢谱、差示扫描量热法和流变仪对其进行了表征。通过实时定量聚合酶链式反应检测成骨标志物基因的表达，以及细胞增殖、矿化细胞外基质产生和碱性磷酸酶活性的必要生化检测，将检测细胞相容性、包裹的MSC活性和体外成骨分化。为了评估水凝胶促进骨再生的能力，组织学和组织形态计量学评分，将使用微型计算机断层扫描分析和力学测试来量化组织反应和表征功能性骨形成。在这些研究完成后，预期的结果是成功地制造出能够生物降解、生物矿化和干细胞输送的原位形成、可注射的水凝胶，并对该系统的骨再生效果进行广泛的评估。这项拟议的工作不仅将提高对以生物材料为基础的微创组织再生疗法作为可行的临床替代方案的理解和测试，而且还将为 热敏材料的合理设计和水凝胶的生物矿化。此外，该系统为复合组织再生和受控输送提供了一种新的平台，可应用于各种不同的组织。

DESCRIPTION (provided by applicant): The rationale for the proposed research is based on the need for minimally invasive strategies to provide functional and aesthetic reconstruction in complex craniofacial defects caused by trauma and disease. The objective of this research is to develop an injectable, in situ forming hydrogel that utilizes a unique dual gelation mechanism to rapidly form gels at physiological temperature, chemically crosslink to maintain hydrogel stability, and biodegrade for full tissue regeneration. The proposed research will test the fundamental hypothesis that the injectable hydrogel will provide a supportive and mineralized substrate for mesenchymal stem cell (MSC) proliferation, enable osteogenic differentiation in vitro, and promote bone regeneration in vivo. The proposed research will be accomplished through three specific aims: 1) To develop and characterize injectable, in situ forming, biodegradable and space-filling hydrogels as an acellular tissue engineering therapeutic; 2) To assess the effectiveness of the injectable, dual-gelling hydrogels to promote the proliferation and osteogenic differentiation of encapsulated MSCs in vitro and bone regeneration in vivo; and 3) To evaluate the effects of cell seeding density and predifferentiation stage on the regenerative capacity of injectable, dual-gelling hydrogels in vivo. The novel macromers with crosslinkable and degradable functional groups will be synthesized and characterized using 1HNMR spectroscopy, differential scanning calorimetry, and rheometry. Cytocompatibility, encapsulated MSC viability and in vitro osteogenic differentiation will be measured via real-time PCR for osteogenic marker gene expression and essential biochemical assays for cell proliferation, mineralized extracellular matrix production and alkaline phosphatase activity. In order to evaluate the capacity of the hydrogels to promote bone regeneration, histological and histomorphometric scoring, microcomputed tomography analysis and mechanical testing will be used to quantify the tissue response and characterize functional bone formation. At the completion of these studies, the expected outcomes are successful fabrication of an in situ forming, injectable hydrogel capable of biodegradation, biomineralization, and stem cell delivery and the extensive evaluation of the system's efficacy for bone regeneration. The proposed work will not only improve the understanding and testing of biomaterials-based therapies for minimally invasive tissue regeneration as viable clinical alternatives, but provide new insights in the rational design of thermosensitive materials and hydrogel biomineralization. Moreover, the proposed system provides a novel platform for composite tissue regeneration and controlled delivery for application in a variety of different tissues.