A Study On Physicochemical Properties,Biocompatibility And Osteogenesis Of Borosilicate Bioactive Glass Scaffold Releasing CGRP In Vitro

OBJECTIVE: To research The preparation of the borosilicate bioactive glass(BBG)scaffold releasing CGRP,while studying its physicochemical properties,,and biocompatibility and osteogenesis in vitro.METHODS: Materials science experiments:(1)13-93B1.5 scaffolds were prepared by three-dimensional(3D)printing technology with a bioactive glass composition of 6Na2O--8K2O--8Mg O--22 Ca O--27B2O3--27 Si O2--2P2O5(mol%),and then the scaffolds were placed in sodium alginate(SA)solution with a mass fraction of 0.5% and cross-linked with 5% Ca Cl2 solution for 20 min to obtain 13-93B1.5-SA composite scaffolds.(2)To characterize the physical properties of 13-93B1.5 scaffold and 13-93B1.5-SA composite scaffold,we used the Micro-computed tomography(Micro-CT)to calculate the pore size distribution and porosity of 13-93B1.5 scaffold,and the electronic static and dynamic universal material testing machine was used to test the compressive strength of 13-93B1.5-SA composite scaffold.To characterize the degradation of13-93B1.5-SA composite scaffold in vitro,we used the Inductively coupled plasma atomic emission spectra(ICP-AES)to detect the ions release,while measuring the weight loss and the p H value of the immersion solution in different time after soaking in SBF.To characterize the biological activity of13-93B1.5-SA composite scaffold in vitro,Scanning electron microscope(SEM)was used to observe the changes in the morphology of the composite scaffolds,Energy disperse spectroscopy(EDS)was used to detect the surface components,the phase constitution and functional groups changes were examined by X-ray diffractometer(XRD)and Fourier transform infrared spectroscopy(FTIR).(3)CGRP was loaded into the composite scaffolds and the release was detected by ELISA.Cells science experiments:(1)We used The materials to co-culture with HBMSCs in vitro.Then the proliferation of cells was quantified by cck-8,and the adhesion and growth morphology of cells on the composite scaffolds surface were observed by SEM.(2)Alkaline phosphatase(ALP)activity was determined by ALP assay kit.Ability of osteogenic differentiation and extracellular matrix mineralization were tested by ALP staining and alizarin red staining.RESULTS: Materials science experiments:(1)The 13-93B1.5 scaffolds were successfully prepared by 3D printing with a porosity of 59.85%±6.04%,and the pore size distributed between 350-400 μ m.The 13-93B1.5-SA composite scaffolds were successfully prepared with the SA hydrogel fully distributed into the pores of the 13-93B1.5 scaffolds.The 13-93B1.5-SA composite scaffolds have a vertical compressive strength of 23.75± 2.61 MPa,after immersing in SBF for 120 d,the compressive strength could still maintain around 8MPa.(2)The SA hydrogel degraded in 14 days in vitro,which did not significantly affect the degradation of 13-93B1.5 scaffolds,and the degradation rate of composite scaffolds is suitable.A hydroxyapatite(HA)layer formed on the scaffolds surface after soaking in SBF for a certain time,which represents a good biological activity.And the p H value of soaking liquid increased.(3)CGRP was successfully loaded into the 13-93B1.5-SA composite scaffolds,and its release time was up to 7 days in vitro,which could prevent the CGRP from rapid degradation and losing its activity to prolong the service time.Cells science experiments:(1)The cck-8 experiment showed that,the 13-93B1.5-SA composite scaffolds worked no significant cytotoxicity to HBMSCs,and CGRP-loaded composite scaffolds significantly promoted the proliferation of HBMSCs with a CGRP concentration dependence.The difference was statistically significant(p < 0.05).The adhesion and growth morphology of cells on the composite scaffolds surface were observed by SEM.(2)ALP activity testing,ALP staining and alizarin red staining experiments showed that,both the13-93B1.5-SA composite scaffolds and the CGRP-loaded composite scaffolds could improve the ALP activity and promote the osteogenic differentiation of HBMSCs,but the effect of CGRP-loaded scaffolds was stronger than the former with a CGRP concentration dependence.It means that CGRP can enhance the effect of 13-93B1.5-SA composite scaffolds on the proliferation and osteogenic differentiation of HBMSCs.CONCLUSION: The 13-93B1.5 scaffolds prepared in the present study meet the requirements of bone tissue engineering with an appropriate porosity and pore size.The 13-93B1.5-SA composite scaffold has an excellent mechanical strength,a suitable degradation rate,and good biological activity due to a HA layer formed with prolonging the immersion time.After CGRP was loaded into the 13-93B1.5-SA composite scaffolds,the SA hydrogel can prevent the CGRP from rapid degradation to prolong the service time.The cytological experiments have confirmed that,13-93B1.5-SA composite scaffold exhibits excellent cellular compatibility due to it works no cytotoxicity to HBMSCs and cells can grow on the scaffolds surface.CGRP has a synergistic effect in significantly enhancing the effect on prolification and osteogenic differentiation of HBMSCs from 13-93B1.5-SA composite scaffolds,and this effect is CGRP concentration dependent.The CGRP-loaded composite scaffold can be researched to serve as materials to repair limb large-size bone defects,and providing a little theoretical basis for the research on materials to repair large-size bone defects.