Study Of Pathway Of Absorbed Proteins Mediating Cells Adhesion And Growth On Biomaterials

Biocompatibility is the key issue in the research of biomaterials, and the cells adhesion and growth are the important contents of the biocompatibility evaluation. Universality, protein adsorption is the first biological event occurred on biomaterial surface, followed by cell adhesion and growth on biomaterial surface, when biomaterials are implanted in vivo or tested in vitro. Gene expression microarray and PCR array are used to evaluate genes expression and further study the pathway associated with cells adhesion and growth, then explain the interaction between the biomaterials and cells from the molecular level.The aim of this paper is:(1) Investigate genes expression in PC12 cells effected by chitosan and collagen/chitosan composite films based on gene expression microarray, and find change of genome with our previously absorbed proteomics results; (2) Investigate genes expression of endothelial cells effected by bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys based on PCR array to test the specific pathways, and find influence of cell adhesion with our previously important pathways. The molecular mechanism of absorbed proteins mediating cells adhesion and growth on biomaterials are explained by above results.The main contents of this paper are as follows:1. Chitosan and collagen/chitosan composite films are prepared by spin-coating method. Acridine orange stains the PC 12 cells to observe cells morphology. CCK-8 is used to evaluate the rate of cell proliferation. The results showed that after cultured for 4h and 24h, collagen/chitosan composite films are benefit to PC 12 cells adhesion and spreading. After cultured for 4h, cells proliferation is similar, and after cultured for 24h, cells proliferation on collagen/chitosan composite films is greater than chitosan films. Therefor collagen/chitosan composite can promote PC 12 cells adhension and growth compared with chitosan film.2. Gene expression microarray is used to evaluate genes expression of PC 12 cells on chitosan and collagen/chitosan composite films after cultured for 4h and 24h. The results showed that differentially expressed genes in the cells on chitosan films are greater, Moreover, the RT-PCR is used to evaluate the expression of Pak1, Actn1, Bcar1, Akt1 and these are consistent with results of gene expression microarray. Additionally, PCR array is used to evaluate the genes expression in "TGFβ/BMP signaling pathway" and "cytoskeleton gegulators" of human umbilical vein endothelial cells (HUVECs) on bare and TiN coated NiTi alloys after cultured for 4h and 24h. The results showed that differentially expressed genes in HUVECs on TiN coated NiTi alloys are greater.3. DAVID Functional Annotation is used to analyse Gene Ontology (GO) of differentially expressed genes in PC12 cells on chitosan and collagen/chitosan composite films after cultured for 4h and 24h. The results showed that the genes mainly participate in biological process, molecular function and cell component. Biological process consists of cellular process, processes relevant metabolic, cell differentiation and development. Molecular function consists of molecular function relevant nucleotide, DNA and protein. Cell component consists of intracellular cell compoment function, plasma membrane and extracellular relevant function. The analysis of pathway showed that biomatrials effects on focal adhesion, regulation of actin cytoskeleton, insulin signaling and EGFR1 pathway. By comprehensive analysis of genomics results and our previously obtaind proteomics results, it is found that absorbed proteins binding receptors on cell membrance, transmit extracellular signals into cells through the cytoskeleton transmembrane connection, then active focal adhesion, MAPK, PI3K. pathway, and participate in the rearrangement of focal adhesion and actin cytoskeleton. Through the interaction between pathways, collagen/chitosan composite films are benefit to PC 12 cells adhesion and growth.4. The bioinformatics analysis of differentially expressed genes in HUVECs on bare and TiN coated NiTi alloys after cultured for 4h and 24h showed the activation of "TGFβ/BMP signaling pathway" and "cytoskeleton regulators". By comprehensive analysis of PCR array, our previously obtaind proteomics and genomics results, it is found that in phase of cells adhesion, cells promote expression of integin α/β subunits through activation of TGFβ pathway, and proteins containing RGD sequence bind to actived integin and active focal adhesion and regulation of actin cytoskeleton to promote cells ashesion. In phase of cells growth and proliferation, TiN coated NiTi alloys can effectively prevent the release of nickel ions from NiTi alloy, and active regulation of actin cytoskeleton, focal adhesion, energy metabolism, inflammatory response pathway to inhibit cell apoptosis.By comprehensive analysis of these results of interactions between biomaterials and cells, it is found that focal adhesion, regulation of actin cytoskeleton are important pathways in cells adhesion and growth on biomaterials surface.