Self-assembling Biomimetic Hydrogels with Bioadhesive Properties for Intervertebr

组织工程是一个多学科领域，旨在修复或再生体内丢失或受损的组织和器官。组织工程的基础包括三个基本策略，特别是细胞，生物化学和基于支架的方法。对于身体的某些承重部分的修复，组织再生策略的成功可能取决于支架粘附或与周围宿主组织的整合以防止脱位。一个这样的领域是椎间盘（IVD）的髓核（NP）的再生。下背痛是世界上最常见的医学问题之一，神经鞘的组织工程被认为是治疗下背痛的潜在策略。一些研究人员已经专注于在三维基质中接种细胞以实现新的NP基质的形成。研究还表明，脂肪来源的干细胞（ASC）可以在体外和体内分化为NP样细胞。虽然这些发现是有希望的，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，并有助于在植入物和组织之间的界面上提供足够的力传递。尽管支架与组织的整合可以使用生物粘附聚合物来实现，但是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。在再生医学领域中存在对生物粘附性聚合物的需求。设计一种与周围细胞外基质成分共价结合并提供允许包封细胞存活和分化的环境的材料，不仅是IVD工程的一个重大进步，而且是骨科组织工程的一个重大进步。 在这个建议中，我们详细介绍了一种新型的“智能”水凝胶ASC封装，部分组成的热敏聚合物聚（N-异丙基丙烯酰胺）（PNIPAAm）的发展。在低于其32 ℃的低临界溶解温度（LCST）时，聚合物与水形成可混溶的溶液。在LCST以上，它变得疏水，因此聚合物和水分离，形成致密的凝胶。因此，PNIPAAm的水溶液可以通过小规格针非侵入性地植入并原位固化。将生物聚合物硫酸软骨素（CS）（天然IVD组织的ECM组分）掺入PNIPAAm基质中以形成具有PNIPAAm的有利机械特性和CS的酶降解性、抗炎活性、水和营养吸收的半合成可注射水凝胶。此外，CS可以用醛基（CS醛）修饰，使其通过席夫碱反应与胺反应，从而使水凝胶在与细胞外基质蛋白的胺接触时具有生物粘附性。然而，反应性醛基的存在可能损害包封细胞的活力。该提议中的新策略是通过包含脂质体来规避该问题，所述脂质体被设计为在聚合物粘附到组织并达到生理温度后递送ECM组分。ECM组分的放电将通过标记仿生基质的组装以及与凝胶内的醛官能团共价反应或“封端”来增强材料的生物相容性，所述醛官能团在接触时不参与与组织的结合。该工作基于以下假设：三组分生物粘附剂（PNIPAAm、CS醛和装载ECM的脂质体）将支持ASC的长期存活力和向NP表型的分化，使其成为用于IVD组织工程的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.