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骨质疏松症等疾病导致的骨质缺乏。因此，对具有生物活性的植入物的需求将显著增加，这些植入物具有在骨质疏松性骨内整合的能力。表面化学和形貌修饰已被证明可以改善骨植入物的组织整合。在最近使用微加工表面的共同工作中，我们证明了化学与地形在影响骨细胞反应方面的优势。用聚烯丙胺等离子体聚合物对几何沟槽钛基板进行胺功能化，可以消除细胞沿微沟槽的接触引导。这是首次证明了通过改变表面化学来克服强地形信号的可能性。已经提出了几个假设来解释这种效应：(a)在带负电的细胞膜和带正电的氨基残基之间必须发生高静电相互作用；(b)与整合素受体相互作用的更有效构象的细胞粘附蛋白从血清中吸附增加；(c)在培养基中释放的多胺残基促进细胞突起形成的能力。然而，我们两组用聚烯丙胺等离子体聚合物涂层获得的原始结果现在需要更深入地分析，以确定物理化学表面特性在这种细胞行为中的作用以及所涉及的生物学机制。为了确定氨基的表面和/或体积密度在细胞反应中的作用，我们建议使用三种不同的技术来开发可控制的富氨基纳米层，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)具有氨基末端基团的自组装单层。在这些完美表征的富含氨基的有机表面上，我们将深入探讨血清中的哪些蛋白质被吸附在表面上，其数量和它们是如何被整合的。为了验证这些不同表面与氨基密度和组织的关系的粘附和消除潜力，人类骨细胞的形态将在涂覆的凹槽基质上的活细胞和固定细胞中进行评估。细胞骨架和黏附的组织和动力学将被量化，以实现硅细胞模型，并确定细胞粘附在富氨基纳米层上的黏附力和力学性能。为了更深入地分析参与细胞反应的细胞机制，我们将分析细胞的信号传导和基因表达。了解这些富含氨基酸的纳米层的作用机制将为改善缺乏老化骨的生物活性植入物提供必要的基础知识。

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.