Control Release Of CGRP From Chitosan-Coating Strontium-Incorporated Calcium Phosphate Bone Cement And Effect Of It On Human Umbilical Vein Endothelial Cells Proliferation In Vitro

Objective:To research synthetic method, morphological structure, mechanical property, release profile of control release of calcitonin gene-related peptide from chitosan-coating strontium-incorporated calcium phosphate cement (CGRP/Chitosan-Sr-CPC), and effect of it on Human umbilical vein endothelial cells (HUVECs) proliferation in vitro. Methods:(1)Preparation of strontium-incorporated calcium phosphate cement (Sr-CPC) (Molar ratio of strontium and calcium is 1:9) according to previous experimental methods。 First, CGRP was absorbed into the matrix of per-setting Sr-CPC by vacuuming penetration and freeze drying, then a layer of chitosan was coated on CGRP/Sr-CPC to fabricate CGRP/Chitosan-Sr-CPC and chitosan-strontium-calcium phosphate cement (Chitosan-Sr-CPC) (without CGRP). Sr-CPC, Chitosan-Sr-CPC as control. (2) Characterization of materials:The surface morphology before and after immersed in SBF, porosity and compressive strength of material were detected by SEM, micro-CT, electro-mechanical universal testing machines. The release of CGRP from material was evaluated by Human CGRP ELISA KIT respectively, after soaking in simulated body fluid(SBF). (3)Cell experiment:to detect CGRP receptors of primary human umbilical vein endothelial cells(HUVECs) by immunofluorescence (IF), HUVECs on material surface was observed by cytoskeleton staining after culturing to evaluate cytoarchitecture. The cytotoxicity and proliferation stimulation of material to HUVECs was tested by CCK-8 assay. The release of vascular endothelial growth factor (VEGF) from cell in the culture medium was evaluated by Human VEGF ELISA KIT. Results:Preparation of CGRP /Chitosan-Sr-CPC is successful. Bone cement powder of material surface was gradually transformed into hydroxyapatite in SBF by SEM. Layer of chitosan is degradation, thinning gradually. The materials had a porosity of material structure, with pore size of 45-82 μm for all the groups. The compressive strength was not impaired in the Composites groups, but rather, further increased over time. After soaked in SBF, test group showed a sustained release of CGRP in 5 days. IF results show that, CGRP perhaps play a role on CGRP receptors of HUVECs. The cytoskeleton staining and cell adhesion results also presented a good spread and cell function. CCK-8 assay showed no cytotoxicity for all the groups, and HUVECs proliferation rate of the experimental group is higher than control group’s, with a significant difference (P<0.05), the same to VEGF levels of in Medium at the second day. Conclusions:CGRP/Chitosan-Sr-CPC is a porous bioactive composites, with chitosan coating and substrate sustainable into hydroxyapatite, The compressive strength was not impaired, but rather, further increased over time, and sustained release of CGRP. It showed bioactivity to HUVECs in vitro, be effect on CGRP receptors, possibly up regulation of VEGF secretion, stimulate proliferation. It may be desirable through well-constructed micro-circulation to promote osteoinductive and osseointegration of Sr-CPC in pathological fracture (such as osteoporotic fractures), expanding the scope of application.