Electrochemically Guided Collagen Synthesis for Functional Tissue Engineering

描述（由申请人提供）：胶原蛋白在再生组织替代策略中作为生物材料和支架起着核心作用。现有的胶原蛋白合成类似物与它们要替代的组织相比，具有极其差的生物力学性能，部分原因是在纤维水平之上缺乏层次顺序的定向。该研究的总体目标是证明一种新型的具有生物力学能力的胶原组织工程支架的可行性，该支架的制备方法：1)通过非常规的电化学过程获得前所未有的分子排列水平和分子堆积密度，并在多个结构层次上持续存在；2)通过使用仿生装饰素样连接分子来控制纤维间附着。拟议研究的第一阶段将阐明胶原蛋白溶液在直接应用于溶液的弱电作用下实现长程有序的机制。研究pH梯度、电流振幅和胶原浓度对胶原分层组织的影响，优化合成工艺。通过确定genipin和戊二醛之间交联的适当类型和浓度，可以提高定向胶原凝胶的强度。{通过调节磷酸盐缓冲盐水处理条件，可以改善d带模式和胶原纤维直径。定向结构的粘弹性特性将被由附着在肽基序上的皮肤硫酸酯组成的decorin模拟物修饰，这些肽基序选择性地结合到I型胶原分子上。由此产生的合成结构的力学性能将通过宏观力学测试进行评估，并与大鼠肌腱（一种参考天然组织）的力学性能进行比较。{第2阶段将评估骨髓间充质干细胞祖细胞在这种材料上向细胞分化，并通过机械提示促进这种分化途径。总的来说，该研究将：a)优化一种新型制造方法的制造过程变量，以获得高度定向的肌腱模拟结构；b)通过研究在由多个胶原束组成的三维结构中植入的骨髓来源的干细胞祖细胞的表型和基因型行为，评估材料作为组织工程支架的可行性。如果目前的研究证明其可行性，这种性质的生物材料可能在创造替代负重结缔组织（如肌腱、韧带、骨骼和血管壁）的策略方面发挥关键作用。

DESCRIPTION (provided by applicant): Collagen plays a central role as a biomaterial and as a scaffold in the regenerative tissue replacement strategies. Existing synthetic analogs of collagen have extremely poor biomechanical properties in comparison to the tissues they are targeted to replace due, in part, to the lack of orientation in hierarchical orders above the level of fibers. The overall goal of the proposed study is to demonstrate the feasibility of a novel biomechanically competent collagenous tissue engineering scaffold fabricated by: 1) an unconventional electrochemical process to attain an unprecedented level of molecular alignment and molecular packing density persistent across multiple levels of structural hierarchies, and, 2) the control of interfibrillar attachment by use of a biomimetic decorin-like linkage molecule. Phase 1 of proposed studies will elucidate the mechanisms by which collagen solutions achieve long-range order under the effect of weak currents applied to directly to the solutions. The effects of pH gradient electric current amplitude and collagen concentration on the hierarchical organization of collagen will be investigated to optimize the synthetic process. The strength of resulting oriented collagen gels will be improved by identifying the appropriate type and concentration of crosslinking amongst genipin and glutaraldehyde. {The D-banding pattern and collagen fibril diameter will be improved by modulating the phosphate buffered saline treatment conditions.} The viscoelastic properties of oriented constructs will be modified by decorin mimics consisting of dermatan sulfate attached to peptide motifs which selectively bind to type I collagen molecules. Mechanical properties of resulting synthetic constructs will be assessed by macroscale mechanical tests and compared to those of rat tendon, a reference natural tissue. {Phase 2 is going to assess the differentiation of bone marrow derived mesenchymal stem cell progenitors on this material towards tenocytic lineage and foster this differentiation pathway by the mechanical cue.} Overall, the proposed study will: a) optimize the fabrication process variables of a novel fabrication method to obtain highly oriented tendon-mimicking constructs, and, b) assess the feasibility of the material as a tissue engineering scaffold by investigating phenotypic and genotypic behavior of bone marrow derived stem-cell progenitors seeded within the three-dimensional structures consisting of multiple collagenous bundles. A biomaterial of this nature may play a key role in creating strategies towards replacement of load bearing connective tissues such as tendons, ligaments, bones and vascular walls provided that the current study proves its feasibility. PUBLIC HEALTH RELEVANCE: The overall goal of the proposed study is to demonstrate the feasibility of a novel collagenous tissue engineering scaffold with mechanical properties converging those of tendon or ligaments. The study will assess the promise of this novel material by investigating whether adult stem cells seeded on this material differentiate to act like tendon cells.