Aminocoatings for improving implants’ tissue integration: understanding underlying biological mechanisms

发达国家的人口老龄化将进一步增加由于骨质疏松症等病理因素造成的骨缺乏症。因此，对能够整合到骨质疏松骨中的生物活性植入物的需求将显著增加。表面化学和表面修饰已被证明可以改善骨植入物的组织整合。在最近一项使用微细加工表面的常见工作中，我们展示了在影响骨细胞反应方面，化学因素相对于地形因素的优势。用聚烯丙基胺等离子体聚合物对具有几何凹槽的钛衬底进行胺官能化，能够消除沿微凹槽的细胞接触导向。这是第一次展示了通过改变表面化学来克服强烈地形信号的可能性。已经提出了几个假设来解释这种效应：(A)带负电荷的细胞膜和带正电荷的氨基残基之间必须发生的高度静电相互作用；(B)从血清中以更有效的构象与整合素受体相互作用的细胞黏附蛋白的吸附增加；(C)在介质中释放的多胺残基促进细胞突起形成的能力。然而，我们两个小组使用聚烯丙基胺等离子体聚合物涂层所获得的原始结果现在需要更深入的分析，以确定物理化学表面属性对这种细胞行为的作用和涉及的生物学机制。为了确定氨基的表面和/或体积密度在细胞响应中的作用，我们建议使用三种不同的技术来开发受控的富氨基纳米层，从而提高对化学成分的控制水平：(A)烯丙胺的等离子体聚合，(B)不同氨基含量的聚合物基富氨基纳米涂层的共价接枝，以及(C)带有氨基末端基团的自组装单分子层。在这些完美表征的富含氨基的有机表面上，我们将深入探索血清中的哪些蛋白质被吸附在表面，吸附的数量以及它们是如何构象的。为了验证这些不同表面的黏附和去除潜力与氨基的密度和组织的关系，我们将在活细胞和固定细胞上对人骨细胞的形态进行评估。细胞骨架和焦点粘连的组织和动力学将被量化，以实现硅细胞模型，并确定细胞在富含氨基的纳米层上的粘附力和机械性能。为了更深入地分析细胞反应中涉及的细胞机制，我们将分析细胞的信号和基因表达。对这些富含氨基的纳米层的作用机制的了解将为改进生物活性植入物治疗缺乏的老化骨带来必要的基础知识。

Population aging in developed countries will further increase bone deficiencies due to pathologies such as osteoporosis. Thus, the need of bioactive implants with the capacity to integrate inside osteoporotic bone will raise significantly. Surface chemistry and topography modifications have been shown to improve bone implants tissue integration. In a recent common work using microfabricated surfaces, we demonstrated the predominance of chemistry versus topography in influencing bone cell response. Amine functionalization of geometrically grooved titanium substrates with poly(allylamine) plasma polymer was able to abrogate cell contact guidance along the microgrooves. This was the first demonstration of the possibility to overcome a strong topographical signal by changing the surface chemistry. Several hypotheses have been proposed to explain this effect: (a) the high electrostatic interactions that must occur between a negatively-charged cell membrane and the positively-charged amino residues; (b) the increased adsorption of cell-adhesive proteins from the serum with more efficient conformation for interaction with integrin receptors; (c) the capacity of polyamines residues released in medium to promote cell protrusion formation. However, this original result obtained by our two groups with poly(allylamine) plasma polymer coatings needs now to be analyzed more deeply to determine the role of physico-chemical surface properties on this cell behavior and the biological mechanisms involved. With the objective of determining the role of surface and/or volume density of amino groups in this cell response, we propose to develop controlled amino-rich nano-layers using three different techniques allowing increasing levels of control of chemical composition: (a) plasma polymerization of allylamine, (b) covalent grafting of polymer-based amino-rich nano-coatings with varying content in amino groups, and (c) self-assembled monolayers with amino terminal groups. On these perfectly characterized amino-rich organic surfaces, we will explore in depth which proteins from the serum are adsorbed on the surface, in which quantity and how they are conformed.To verify the adhesion and abrogation potential of these different surfaces in relationship with the density and organization of amino groups, the morphology of human bone cells will be evaluated in living and fixed cells on coated grooved substrates. The organization and dynamics of the cytoskeleton and focal adhesions will be quantified to implement an in silico cell model and determine the adhesion force and mechanical properties of cells adhering on amino-rich nano-layers. To go deeper into the analysis of the cellular mechanisms involved in cell response, both the signalling and the gene expression of the cells will be analyzed. The understanding of the mechanism of action of these amino-rich nano-layers shall bring basic knowledge essential for improving bioactive implants for deficient aged bone.