In Vitro Study Of BMP-9-Loaded SerMA/GO Composite Bionic Scaffold In Disc Tissue Engineering

Background: Intervertebral disc degeneration(IVDD)is known as significant causation of Intervertebral disc(IVD)-derived low back pain,which is one of the common diseases in today’s society and impacting the quality of lives of several millions of people.Spinal fusion,the gold standard of clinical treatment for IVDD,is a widely used classical procedure,but the procedure is often accompanied by complications such as pseudarthrosis,fusion displacement,vertebral collapse and bone nonunion.Currently,a composite bionic scaffold based on Disc Tissue Engineering(DTE)is expected to eliminate these phenomena and be used for the clinical treatment of IVDD.Objective: To prepare a graphene oxide(GO)methacrylated sericin Methacrloyl(SerMA)bionic scaffold with photocrosslinking ability,and preliminarily investigate the ability of Bone morphogenetic protein-9(BMP-9)-loaded SerMA/GO composite bionic scaffold loaded with BMP-9 was used to elicit in vitro the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods: Part I: Preparation and property characterization of SerMA/GO bionic scaffolds.(1)Construction of SerMA/GO bionic scaffolds with photocrosslinking properties;(2)Scanning electron microcopy(SEM)to observe the microstructure of the bionic scaffold;(3)Fourier infrared spectroscopy to analyze the secondary structure of bionic scaffolds;(4)Electronic universal testing machine to test the mechanical properties of bionic scaffolds;(5)Weighing method to determine the swelling rate of bionic scaffolds;(6)Weighing method to test the degradation rate of bionic scaffolds in Phosphate buffer solution(PBS);(7)Resuscitation,passaging and freezing of rat BMSCs;(8)Cell Counting Kit-8(CCK-8)and fluorescent staining of live-dead cells to detect cytotoxicity of bionic scaffolds.Part II: Construction of BMP-9-loaded SerMA/GO composite bionic scaffolds and in vitro osteogenic performance study.(1)Construction of BMP-9-loaded SerMA/GO bionic scaffolds;(2)Enzyme-linked immunosorbent assay for slow release of BMP-9in BMP-9-loaded bionic scaffolds;(3)Transwell cell migration assay to test the effect of BMP-9-loaded composite bionic scaffolds on the migration of BMSCs;(4)ALP and ARS staining to compare the migration of BMSCs loaded with different levels of BMP-9.(4)Alkaline phosphatase(ALP)and Alizarin red S(ARS)staining to compare the in vitro osteogenic induction performance of composite bionic scaffolds loaded with different levels of BMP-9;(5)Cell counting to calculate the early cell adhesion rate of BMSCs after inoculation with composite bionic scaffolds;(6)Effect of CCK-8 and livedead cell fluorescence staining to detect the proliferation and activity of composite bionic scaffolds on BMSCs;(7)ALP staining and ARS staining to detect ALP and mineralized matrix production after inoculation of BMSCs with composite bionic scaffolds,quantitative real time-PCR(q RT-PCR)was performed to detect the expression of osteogenic-related genes in BMSCs after inoculation with composite bionic scaffolds,and immunofluorescence was performed to detect the expression of osteogenic-related proteins in BMSCs after inoculation with composite bionic scaffolds.Results:(1)The SerMA/GO bionic scaffold had a pore-like structure inside,the addition of GO did not affect the secondary structure of SerMA,and no chemical structure was formed between them.the combination of GO enhanced the mechanical properties of the bionic scaffold,and the mechanical properties of the bionic scaffold were gradually enhanced with the increase of GO content.On the contrary,the swelling rate and degradation rate of the bionic scaffold showed a gradual decrease with the increase of GO content.The results of in vitro biocompatibility study showed that the high concentration of GO inhibited the proliferation and affected the activity of BMSCs,while there was no significant difference between the low concentration group and the control group,showing a better cellular biocompatibility in vitro.(2)In the BMP-9release assay,the addition of GO slowed down the sudden release of BMP-9 and prolonged the release period.The cell migration results showed that the highest number of cells migrated in the bionic scaffold group containing 400 ng/m L BMP-9.ALP and ARS staining results showed that the expression of ALP and mineralized matrix was significantly more in the 400 ng/m L and 800 ng/m L bionic scaffolds than in the remaining two groups,but there was no significant difference between these two groups.(3)The in vitro results of the composite bionic scaffold were as follows: the early adhesion rate of BMSCs after inoculation showed a gradual increase with the extension of culture time in both the control group and the composite bionic scaffold group,and the composite bionic scaffold group was higher than the control group.The results of CCK-8 and live-dead cell fluorescence staining showed that the proliferation of BMSCs in the composite bionic scaffold group was significantly stronger than that in the Control group,and there was no significant difference in cell activity between the two groups,suggesting that the composite bionic scaffold has better biological descriptiveness in vitro.ALP and ARS results showed that the expression of ALP and mineralized matrix was significantly higher in the composite bionic scaffold group than in the SerMA/GO1 group and the control group.q RT-PCR results showed that at the early osteogenesis stage on the 7th day,the expression of ALP,Runt-related transcription factor-2(RUNX2),osteopontin(OPN),and osteocalcin(OCN)expressions were higher in the composite bionic scaffold group than in the Control group at the early osteogenesis stage on day 7,especially for ALP and RUNX2;At the middle osteogenesis stage on the 14 th day,the expression of these four osteogenesisrelated genes in the composite bionic scaffold group was significantly higher than that in the Control group,and there were also significant differences compared with the SerMA/GO1 group,with the expression of RUNX2 and OCN being the most significant at this time.Immunofluorescence staining results showed that the fluorescence of the red area characterizing RUNX2 and OCN in the composite bionic scaffold group was significantly higher than that in the Control and SerMA/GO1 groups,suggesting that the composite bionic scaffold could promote the secretion of osteogenic-related proteins in vitro.Conclusions:(1)SerMA/GO bionic scaffolds have relatively tunable physicochemical properties,and their mechanical properties,pore size,and swelling properties can be mediated by GO content,and the degradation properties meet the DTE requirements.(2)BMSCs exhibit good biocompatibility in SerMA/GO bionic scaffolds.Meanwhile,the SerMA/GO composite bionic scaffold has the property of slow-release BMP-9.The composite bionic scaffold showed the best material properties and biological characteristics when the GO content was 0.05%.(3)The SerMA/GO composite bionic scaffold loaded with BMP-9 has the potentiation to support and strengthen osteogenic differentiation of in vitro BMSCs.