Self-assembling Biomimetic Hydrogels with Bioadhesive Properties for Intervertebr

描述（申请人提供）：组织工程是一门旨在修复或再生体内丢失或受损的组织和器官的多学科领域。组织工程的基础包括三个基本策略，特别是细胞，生化和基于支架的方法。对于身体某些承重部位的修复，组织再生策略的成功可能取决于支架与周围宿主组织的粘附或整合，以防止脱位。其中一个区域是椎间盘髓核（NP）的再生。NP的组织工程被认为是治疗腰痛的潜在策略，腰痛是世界上最常见的医学问题之一。一些研究人员专注于在三维矩阵中播种细胞以实现新的NP矩阵的形成。研究还表明，脂肪源性干细胞（adipose derived stem cells, ASCs）在体内和体外均可分化为np样细胞。虽然这些发现很有希望，但下一代NP工程支架必须具有与周围椎间盘组织形成实质性界面的能力。这将减少或消除椎间盘脱位的风险，并有助于通过植入物和组织之间的界面提供足够的力传递。尽管使用生物粘附聚合物可以实现支架与组织的整合，但目前提出的具有高粘附性能的材料具有有限的生物相容性。再生医学领域对生物胶粘剂聚合物的需求很大。设计一种与周围细胞外基质成分共价结合的材料，为被封装细胞的生存和分化提供一个允许的环境，这不仅是IVD工程的重要一步，也是骨科组织工程的重要一步。在本提案中，我们详细介绍了用于ASC封装的新型“智能”水凝胶的开发，该水凝胶部分由热敏聚合物聚（n -异丙基丙烯酰胺）（PNIPAAm）组成。低于32℃的较低临界溶液温度（LCST），聚合物与水形成可混溶溶液。在LCST之上，它变得疏水，因此聚合物和水分离，形成致密的凝胶。因此，PNIPAAm水溶液可以通过小尺寸针头无创植入并原位固化。将天然IVD组织的ECM组分生物聚合物硫酸软骨素（CS）加入到PNIPAAm基质中，形成半合成的可注射水凝胶，该水凝胶具有PNIPAAm良好的力学特性和CS的酶解性、抗炎活性、水分和营养吸收。此外，CS可以用醛基（CS醛）修饰，使其通过希夫碱反应与胺发生反应，从而使水凝胶与细胞外基质蛋白的胺接触后具有生物粘附性。然而，活性醛的存在会损害被包膜细胞的生存能力。本提案中的新策略是通过包含脂质体来绕过这个问题，脂质体设计用于在聚合物粘附到组织并达到生理温度后递送ECM成分。ECM组分的释放将通过标记仿生基质的组装来增强材料的生物相容性，并且还与凝胶中不参与与组织结合的醛官能发生共价反应或“端盖”。这项工作是基于三组分生物粘合剂（PNIPAAm， CS醛和ecm负载脂质体）将支持ASCs向NP表型的长期生存和分化的假设，使其成为IVD组织工程中可行的三维培养系统。

DESCRIPTION (provided by applicant): Tissue engineering is a multidisciplinary field that aims to repair or regenerate lost or damaged tissues and organs in the body. The foundation of tissue engineering encompasses three fundamental strategies, specifically cellular, biochemical, and scaffold-based approaches. For the repair of certain load-bearing parts of the body, success of a tissue regeneration strategy can be dependent on scaffold adhesion or integration with the surrounding host tissue to prevent dislocation. One such area is the regeneration of the nucleus pulposus (NP) of the intervertebral disc (IVD). Tissue engineering of the NP is regarded as a potential strategy for the treatment of lower back pain, one of the most common medical problems in the world. Several researchers have focused on seeding cells in three-dimensional matrices to achieve formation of a new NP matrix. Studies have also shown that adipose derived stem cells (ASCs) can be differentiated into NP-like cells in vitro and in vivo. While these findings are promising, next generation NP engineering scaffolds must have the ability to form a substantial interface with surrounding disc tissue. This will reduce or eliminate the risk o dislocation in the disc and help to provide adequate transmission of force across the interface between the implant and the tissue. Although scaffold integration with tissue can be achieved using a bioadhesive polymer, the currently proposed materials with high adhesive properties have limited biocompatibility. A need for bioadhesive polymers exists in the area of regenerative medicine. The design of a material that covalently bonds with surrounding extracellular matrix components and provides an environment permissive to the survival and differentiation of encapsulated cells would be a major step forward not just in IVD engineering, but in orthopedic tissue engineering, in general. In this proposal, we detail the development of a novel "smart" hydrogel for ASC encapsulation, partially composed of the thermally sensitive polymer poly(N-isopropylacrylamide) (PNIPAAm). Below its lower critical solution temperature (LCST) at 32C, the polymer forms a miscible solution with water. Above the LCST, it becomes hydrophobic, so the polymer and water separate, forming a compact gel. Therefore, aqueous solutions of PNIPAAm can be implanted non-invasively through a small gauge needle and solidify in situ. The biopolymer chondroitin sulfate (CS), an ECM component of the native IVD tissue, is incorporated into the PNIPAAm matrix to form a semi-synthetic injectable hydrogel with the favorable mechanical characteristics of PNIPAAm and the enzymatic degradability, anti-inflammatory activity, water and nutrient absorption of CS. In addition, CS can be modified with aldehyde groups (CS aldehyde), allowing it to react with amines via Schiff's base reaction, thus rendering the hydrogel bioadhesive upon contact with amines of the extracellular matrix proteins. However, the presence of the reactive aldehyde groups can compromise the viability of encapsulated cells. The novel strategy in this proposal is to circumvent this problem with the inclusion of liposomes designed to deliver ECM components after the polymer has adhered to tissue and reached physiological temperature. The discharge of ECM components will enhance the biocompatibility of the material by marking the assembly of a biomimetic matrix, and also covalently reacting with, or "end-capping", the aldehyde functionalities within the gel that did no participate in bonding with tissue upon contact. This work is based on the hypothesis that the three-component bioadhesive (PNIPAAm, CS aldehyde, and ECM-loaded liposomes) will support long term viability and differentiation of ASCs toward a NP phenotype, making it a feasible three- dimensional culture system for use in IVD tissue engineering.