Biocompatibility Of Pure Titanium Modified By Human Endothelial Cell-Derived Extracellular Matrix

It has some limitation of immobilizing single or mixed ECM components on biomaterial surface for improving cell adhesion and proliferation ability. However, depositing extracellular matrix (ECM) derived from endothelial cells on biomaterial surface has great potential and value in biomedical modification and application areas.In this work, human umbilical vein endothelial cells (HUVECs) were cultured on polished titanium surface (P) and acid treated-titanium surface (AT), and then stripped from the underlying matrix after 10 days. Then native basement membrane like ECM on the two different titanium surfaces was obtained. To determine component of the ECM proteins, diffuse reflectance fourier infrared spectroscopy (FTIR) was carried out before and after depositing ECM on the titanium surfaces. The chemical composition and binding types of the ECM proteins were determined by X-ray photoelectron spectroscopy (XPS) The ECM morphology on different samples was analyzed using light microscopy, SEM and atomic force microscopy (AFM). The water contact angles on the sample surfaces were measured using a JY-82 contact angle goniometer. To study the distribution pattern and relative amounts of ECM proteins, indirect immunofluorescence staining for fibronectin (FN), laminin (LN) and type IV collagen (IV-COL) were performed. The biological behavior of cultured HUVECs, distribution pattern of endothelial cell actin cytoskeleton and adherent platelets on on the titanium surfaces were investigated by in vitro HUVECs culture and platelet adhesion before and after depositing ECM.FTIR test proved the existence of amide I and amide II band in the ECM modified P and AT titanium surfaces. XPS further confirmed the chemical composition and binding types of the ECM proteins on the different titanium substrates. The results of light microscopy, SEM and AFM exhibit the morphology of HUVEC derived-ECM on the two kinds of titanium surfaces. There were higher water contact angles on the ECM modified samples. Furthermore, some ECM components, including FN, LN and IV-COL were presented on the ECM-covered titanium surfaces by immunofluorescence staining. These results suggested that ECM is successfully deposited on P and AT surfaces. Light microscopy, SEM and Alamar Blue test results of endothelial cell adhesion and proliferation on the two kinds of titanium surfaces proved that ECs cultured on ECM-covered P and AT surfaces proliferated faster than those on unmodified titamium surfaces at 1 day,3 day and 5 day. Actin cytoskeleton proteins were clearly visible with close cell arrangement, little burr-like protrusions and more endothelial cells on ECM-covered titanium surfaces compared with ECs grown on unmodified titamium surfaces. The SEM results indicated the ECM-covered P and AT surfaces inhibited adhesion and activation of platelets while unmodified P and AT surfaces promoted stronger platelet activation. Thus, this kind of processing may provide a basis for preparing anticoagulant titanium surfaces for application in cardiovascular implants.