Regulation Of Construction Of Biomimetic Vascular Intima By Hyaluronic Acid Micro-Pattern On Ti Surface

Surface endothelialization was considered to be the main way to resolve thrombosis and hyperplasia after the cardiovascular devices implanted. Therefore, improving the endothelial surface quality of such biomaterials is very important and meaningful. Currently, in vitro studies have shown that complete endothelium coverage of the biomaterial surface has a certain inhibition effect on thrombosis and smooth muscle cell proliferation. However, monolayer formed by this single cell on the surface was found lots of problems after implantation, such as anti-coagulation deficiencies and easily falling off in a large area. To solve these problems, more research turned to in vivo endothelialization, while the studies on construction and improvement of the endothelial cells pericytes environment and imitation of fluid shear stress environment on the materials surface were also important. Thus, in this work research on building biomimetic pericytes environment of endothelial cells and in vivo fluid shear stress environment to contribute to biomimetic intimal building on the materials surface was performed.In this paper, titanium (Ti) with good biocompatibility was chosen as the base materials. Tensile role on the endothelial cells by in vivo fluid shear stress was simulated by micro-strip graphic of hyaluronic acid (HA) on the Ti surface. Bionic smooth muscle cells pericytes environment was provided for the endothelial cells through the limitation of HA micro-strip on the smooth muscle cells. We built two kinds of vascular endothelial cells and smooth muscle cells co-culture model named "SMCs-HAa-ECs" and "SMCs-ColIV-ECs". respectively. On this basis, bionic vascular intimal on Ti metal surface was initial built, and the biological function of the endometrium was evaluated. At first, the effects of three kinds of micro-pattern size, P10/40, P25/25, P40/10, on endothelial cell morphology, proliferation, functional cytokine secretion and anticoagulant function were studied. The micro-pattern size which was the most suitable for physiological functions of human umbilical vein endothelial cells was filtered out. Secondly, two kinds of vascular endothelial cells and smooth muscle cells co-culture model named "SMCs-HAa-ECs" and "SMCs-ColIV-ECs" were built on this basis. By comparing the two models on endothelial cell morphology, the number of anti-coagulation factor secretion, anti-coagulation and anti-shear stress function, the model which was more suitable for culturing bionic vascular intima on Ti substrate was filtered out. Finally, on the basis of the previous step, the initial planting density of smooth muscle cells was optimized, and ultimately the construction of blood vessels bionic initial on Ti surface was realized, and the biological function of the endometrium was evaluated. Utilizing scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle analysis, Fourier transform infrared spectroscopy (FTIR), enzyme-linked immunosorbent assay (ELISA) and immunofluorescence analysis methods to characterize physical and chemical properties and stability of the hyaluronic acid micro-graphics on Ti surface. We use enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining methods to evaluate endothelial cell morphology, number and secretory function, in particular using double staining to observe endothelial cells and smooth muscle cell morphology and behavior in the co-culture system. We use cell morphology measurement and calculation software (ImageJ) statisticed endothelial cell morphology index. By platelet adhesion and activation experiments and clotting time (APTT, PT) test, endothelial cells anticoagulation property was evaluated. In the flow chamber device, the human aortic flow shear stress was simulated to evaluate anti-shear-strength function of endothelial cells on the surface.Another relevant work of this paper is a combination of regulation of HA micro-pattern on endothelial cells and decellularization technology, which can build an orderly distribution of endothelial extracellular matrix surface on Ti substrate surface. The initial blood compatibility evaluation, evaluation of rapid endothelialization, smooth muscle cell culture evaluation and evaluation of macrophage cultures for the extracellular matrix surface were performed.Full results are as follows:1. Hyaluronic acid micro-graph on Ti substrate surface has good stability. P25/25better suits to play a physiological function of endothelial cells among P10/40, P25/25and P40/10three micro-graphs, including imitation situ morphological, functional cytokine secretion and anticoagulant function.2. Hyaluronic micropattern surface itself does not have ideal blood compatibility. Micro-pattern can regulate endothelial cell morphology and extracellular matrix synthesis. Combining decellularization technology, a patterned distribution of endothelial extracellular matrix can be achieved. This extracellular matrix surface has good blood compatibility, rapid endothelialization function, certain inhibitions of smooth muscle over proliferation and inhibition of macrophage adhesion by preliminary biological evaluation.3. Because hyaluronidase can change the property of hyaluronic acid that inhibit cell adhesion, we take advantage of this to achieve a "SMCs-HAa-ECs" co-culture system construction; We take advantage of ColIV shield impedance function of hyaluronic acid on endothelial cell, to achieve the "SMCs-ColIV-ECs" co-culture system construction; Comparing two models on anti-clotting factor secretion, anti-clotting function, inhibition of smooth muscle function and anti fluid shear stress of endothelial cells,"SMCs-ColIV-ECs" co-culture model is considered to have a greater physiological advantage。4. On the basis of "SMCs-ColIV-ECs" co-culture model, initial construction of bionic vascular intima has been realized by the initial planting density difference of endothelial cells and smooth muscle cells (1×105cells/ml:2.5×104cells/ml). After initial of the biological and mechanical evaluation, the biomimetic membrane surface compared with endothelial cells cultured alone, has the advantages of growing fast, anti-coagulation factor secretion and better ability to resist shear stress and so on。 This work provides important experimental basis for subsequent optimization of biomimetic membrane on inorganic material surface.