Construction And Osteogenic Properties Of Injectable Multivariate RADA Peptide Gel

Objective:To develop an injectable gel system,which can contain and control the release of multiple bioactive factors.Use the RADA peptide gel and nano hydroxyapatite as scaffold,PLGA microsphere in nanometer packaging rhBMP-2 as growth factor and hPDLSCs as seed cells.We hypothesize that rhBMP-2 can release into the composite scaffold slowly and induce hPDLSCs differentiate into osteoblasts effectively,and the plasticity of scaffold can be improved by the injectable materials.Methods:1)Cell culture and isolation: Periodontal ligament cells are harvested from periodontium of the first premolar extracted from adolescents for orthodontics.Periodontal ligament stem cells are isolated from expanded periodontal ligament cells,and then identified by OCT4 expression through immunofluorescence;The expressions of CD34,CD45,CD79 a,CD73,CD90,CD105 are also identified by flow cytometry.2)Assembly of delivering system: Firstly,PLGA nanoparticles loading BMP-2 are prepared using the rhBMP-2 as modeling drug and PLGA as the carrier materials.Transmission electron microscopy is used to examine the particle shape.Laser granularity instrument is used to measure the particle size and particle size distribution.The coating rate and the drug-loading rate of BMP-2 contained in nanoparticles are measured by chemical kit.Secondly,trigger condition for gelatinization of RADA polypeptide is optimized.Finally,the release rates of BMP-2,BMP-2/PLGA-NP,BMP-2/RADA and BMP-2/PLGA-NP/RADA are determined.3)hPDLSCs are cocultured with BMP-2,nHA,nHA/RADA,BMP-2/NP,rhBMP-2/RADA,BMP-2/PLGA-NP and BMP-2/PLGA-NP/RADA/nHA respectively.Furthermore,ALP activity is examined at 7d,alizarin red staining at 14 d,and the expression level of two osteogenic markers ALP and OCN,are compared respectively at 7d and 14 d.4)hPDLSCs co-cultured with RADA、nHA、n HA/RADA、BMP-2/PLGA-NP、BMP-2/PLGA-NP/RADA and BMP-2/PLGA-NP/RADA/nHA are collected respectively,then seeded into the nude mice.The osteogenesis is examined after 4 weeks of post-implantation.Micro-CT scans the differences among different groups.Harvest samples are analyzed by HE staining,mineralized nodules are observed by alizarin red staining,the expression level of two osteogenesis markers ALP and OCN genes are detected by RT-PCR.Result:1)Cells expanded by limited dilution method showed polygon and spindle.The OCT4 expression was positive by immune fluorescence detection,a bright red fluorescence was observed,indicating its stem cell characteristics;Flow cytometry detected positive expression of CD105(93.1%),CD73(95.7%)and CD90(94.8%),and negative expression of CD34,CD45 and CD79 a.2)The nanoparticles BMP-2/PLGA-NP were examined by transmission electron microscopy.Particle size distribution was uniform.The particles were spherical,and particle size distribution was uniform.Particle size was about 100 nm,pdi was about 0.231,detected by laser particle analyzer.835 ng BMP-2 was packed in nanomaterials,around 20.27 ng per mg freeze-dried powder,the envelopment rate was 17%.Triggering condition of RADA was detected by single factor.The best condition of the RADA gel is: RADA1%,triggering agent 0.5% Tris,and triggering time 0.25 hours.The BMP-2 release rate can be slowed down by PLGA and RADA,but the effect wss not obvious when acting alone.3)hPDLSCs showed no obvious osteogenesis differentiation phenomenon after 14 d co-cultured with RADA.nHA can promote the osteogenesis differentiation of PDLSCs.BMP-2/PLGA-NP,BMP-2/PLGA-NP/RADA and BMP-2/PLGA-NP/RADA/nHA can promote the osteogenesis differentiation of hPDLSCs.The phenomenon of osteogenesis can be observed in three groups after 14 d.ALP testing found that RADA gel cannot improve the activity of ALP.In groups of BMP-2/PLGA-NP,BMP-2/PLGA-NP/RADA and BMP-2/PLGA-NP/RADA/hHA,the ALP activity was significantly increased after 7d,the difference was statistically significant(p<0.01).The results of alizarin red staining showed that BMP-2/PLGA-NP/RADA/nHA was most significant,the RADA group cannot be observed,bone formation can be observed in nHA.The expression level of osteogenesis marker gene ALP and OCN : RADA gel can not improve the expression level of gene,and the difference was not significant;but the expression of OCN and ALP were significantly increased in BMP-2/PLGA-NP,BMP-2/PLGA-NP/RADA and BMP-2/PLGA-NP/RADA/nHA,the difference was statistically significant(p<0.01).4)Materials of different groups were implanted in nude mice for 4 weeks.Micro CT found that RADA+hPDLSCs gel group had sparse gel organization,other groups compact tissue.Typical osteogenesis organization and mineralized nodules can be observed in these groups by HE and alizarin red staining.No difference of ALP and OCN expression level can be found between hPDLSCs and hPDLSCs+RADA gel.The expression of OCN and ALP gene were impromoted in BMP-2/PLGA-NP,BMP-2/PLGA-NP/RADA and BMP-2/PLGA-NP/RADA/nHA,the difference was statistically significant(p<0.01).Conclusion:In this study,the multiple RADA peptide gel scaffold complex was successfully prepared.It can be injected in the form of solution,and then turn into gel scaffold through ionization gelling.This scaffold can provide the 3D growth environment for hPDLSCs.Meanwhile,the biodegradable PLGA nanoparticles can slowly release BMP-2,and promote the osteoblastic differentiation of hPDLSCs and bone formation.The nHA had synergistic effect during this process.