Function Of BMP-2-Loaded SF/CNC Composite Scaffold On Rat BMSCs Osteogenesis

Objective:This study is aimed to develop a composite hierarchical porous scaffold composed of silk fibroin(SF)and CNC(SF/CNC)for the controlled delivery of BMP-2,and evaluate its In vitro releasing behavior,biocompatibility and osteogenic performance,as well as its In vivo bone repair of critical-sized bone defects.Methods:1.CNC was well fabricated,and characterized by field emission scanning electron microscope(FESEM),transmission electron microscope(TEM),X-ray diffraction(XRD)and so on.The effect of CNC on rat BMSCs proliferation was evaluated by CCK-8 assay.In addition,the effect of single treatment or synergic treatment of CNC and BMP-2 on osteogenic differentiation was assessed by ALP staining,ALP activity analysis and RT-PCR.2.A SF/CNC composite scaffold was well fabricated,and characterized.The effect of CNC on rat BMSCs proliferation and adhesion was evaluated by CCK-8 assay and confocal laser scanning.Furthermore,the effect of single treatment or synergic treatment of CNC and BMP-2 on osteogenic differentiation was assessed in detail.3.A BMP-2 loaded SF/CNC composite hierarchical scaffold was well fabricated,and its releasing behavior was tested in vitro.In addition,the effect of BMP-2 loaded SF/CNC composite hierarchical scaffold on osteogenic gene expression was evaluated by RT-PCR.4.A rat model of calvarial defect was established to evaluate the effect of BMP-2 loaded SF/CNC composite hierarchical scaffold on bone repair in vivo.Results:1.FESEM showed that CNC is composed of spherical particles with relatively uniform size,with a graphitized structure characterized by XRD.The CCK-8 assay indicated that CNC has no toxicity to BMSCs in the concentration range of 0-80 μg/mL.The results of ALP staining,ALP activity analysis and Alizarin red staining staining suggested the synergy between CNC and BMP-2 in osteogenic differentiation.2.FESEM showed that the SF/CNC composite scaffold had desirable three-dimensional macroporous structures,with the large pores communicating with each other,the diameter of which was about 100-200 nm.The confocal laser scanning illustrated that the SF/CNC composite scaffold promoted the adhesion and proliferation of BMSCs.CCK-8 assay indicated that both SF/CNC-0.1 group and SF/CNC-1 group had no toxicity to BMSCs.ALP staining suggested that SF/CNC composite scaffold exhibited more evident osteogenic differentiation of BMSCs in vitro,compared with that in SF and control groups.The same result was observed in ALP activity.The results of RT-PCR demonstrated that,the SF/CNC-5 scaffold had the most significant up-regulated effect on ALP,BSP,RUNX2,and OCN,compared with other groups at day 3.Meanwhile,the expression was also significantly promoted at day 7.The SF/CNC-1 scaffold had the most significant up-regulated effect on ALP,BSP,and OCN,compared with other groups.The SF/CNC-0.1 scaffold also significantly promoted the expression of ALP and OCN,compared with SF scaffold.Alizarin red staining and semiquantitative results showed that SF/CNC-0.1 and SF/CNC-1 groups could significantly improve the osteogenic differentiation and mineralization performance of BMSCs,and up-regulate the expressions of key osteogenic proteins ALP and OCN.Among them,SF/CNC-1 group had the most ideal osteogenic effect.3.The results of BMPs releasing profile indicated that the SF/CNC composite exhibited a satisfactory releasing behavior,with 23.9%after 24 h and 47.7%after 7 d.The results of RTqPCR demonstrated that BMP-2/CNC and BMP-2/SF/CNC scaffolds significantly promoted the ALP gene expression at day 3,compared with BMP-2/SF scaffolds.Also,the expression level of ALP.RUNX2 and OCN were significantly up-regulated at day 7.Compared with BMP2/CNC scaffold,the BMP-2/SF/CNC scaffold showed up-regulated expression of ALP and RUNX2 at day 7.The results of ALP staining,ALP activity analysis,alizarin red staining and semi-quantitative analysis showed that BMP-2/SF/CNC scaffold had the most obvious promoting effect on osteogenic differentiation and mineralization.Western blot results showed that the BMP-2/SF/CNC scaffold could promote osteogenic differentiation by activating the Wnt/β-catenin pathway and up-regulating the protein expression level of RUNX2,a key transcription factor for osteogenic differentiation.The results of RNA sequencing further suggested that the composite scaffold of BMP-2/SF/CNC might activate the PI3K/Akt/GSK3β/Wnt/β-catenin signaling pathway in BMSCs,thereby promoting the expression of RUNX2 transcription factors related to osteogenic differentiation and upregulating osteogenic proteins such as ALP and OCN.4.The results of micro-CT analysis and Masson staining demonstrated that the BMP2/SF/CNC group exhibited the most prominent bone repair performance.The quantitative analysis of micro-CT showed that the new bone volume percentage(BV/TV)in the BLANK group.SF group,SF/CNC group,BMP-2/SF group and BMP-2/SF/CNC group was 15.1 ± 0.3%.24.9 ± 2.6%,40.6 ± 3.2%,44.5 ± 4.7%and 70.6 ± 4.1%,respectively.The percentage of new bone volume was statistically significant between the Control group and the SF group,the SF/CNC group and the BMP-2/SF/CNC group.SF/CNC,BMP-2/SF and BMP-2/SF/CNC increased in BV/TV/Tb.Th and BMD,but BS/BV.The results of immunohistochemistry and fluorescent double-labeled staining showed that the BMP-2/SF/CNC group had a significant effect on promoting new bone formation,and the bone mineralization deposition rate(μm/day)increased most significantly compared with the control group.Conclusion:In the present study,a composite hierarchical porous delivery system was developed by combining CNC with inter connected porous SF scaffolds,which could well control the delivery of BMP-2 and promote osteogenesis synergistically.The results demonstrated that the CNCs with pro-osteogenic activity could optimize the efficacy of BMP-2 loaded in the composite scaffolds by prolonging its local retention and promoting its biological activity.The developed composite scaffold exhibited an appropriate physical structure,good bioactivity,and biocompatibility.Moreover,osteogenic differentiation in vitro was activated,which has the potential to promote bone regeneration in cases of bone defects.