Effect Of Self-assembling Peptide Hydrogel Scaffold On Adhesion,Proliferation And Osteogenesis Of MC3T3-E1 Preosteoblasts

Background Bone defects would be caused by resection of bone tumor,trauma or infection,therefore,it need a lot of bone filling material to be implanted into the bone destructive and bone fusion region in arthroscopic surgical procedure or spine fusion surgery.At present,autologous bone graft was still the preferred method in clinical procedure.Due to limited available bone,pains for obtaining bone and increasing the infection risk,clinical application of autologous bone graft was restricted.In order to work out solutions for shortage of available autologous bone,synthetic bone materials such as ceramic material and natural hydroxyapatite have been applied in clinical treatment,however,there are disadvantages of low cell adhesion rate and poor biocompatibility.Along with a rapid development of scaffold materials of bone tissue engineering research in recent years,scientists created new solutions for solving these problems through surface modification or replacement of synthetic materials.RADA16-?was a representative self-assembling peptide(SAP),which was composed of 16 amino acid residues.Since triggered by physiological saline,16 peptide amino acid formed fold ?-sheet structure and to form complementary ionic bonds by mutual reciprocating arrangement and eventually resulting in self-assembly nanofiber(10~20nm in diameter).Studies showed that this kind of self-assembling peptide hydrogel can form reticular fiber structure characterized by a moisture content of more than 99% and pore size of 5~200nm,and it can create advantage conditions for cells of adhesion,proliferation and differentiation on the surface of scaffold materials in vitro,through simulation of extracellular matrix(ECM)resembled in vivo and micro environment.In recent years,scientists have constructed a new type of self-assembling peptide hydrogel scaffold materials with more excellent biological properties by the method of traditional protein C-terminal linker active function sequence on the structure of RADA16-?.Osteostatin(Thr-Arg-Ser-Ala-Trp)was a related protein of parathyroid hormone(PTHr P)on the structure of protein C-terminal domain.Studies showed that pentapeptide fragment of PTHr P(107~111)can promote differentiation and growth of osteoblast in vitro through mediating non-related receptors of PTH/PTHr P and can promote repairing bone defects in rabbits.Functional modification of protein C-terminal domain on the structure of traditional RADA16-?via PTHr P(107~111)fragments was performed in this study,aiming at obtaining a kind of self-assembling peptide hydrogel scaffold materials with related biological performance combing adhesion function with promoting osteogenesis.Objective(1)To explore nano-structure of this new self-assembling peptide hydrogel and detect it's self-assembly properties;(2)To evaluate adhesion function of MC3T3-E1 cells on the new self-assembling peptide hydrogel;(3)To detect proliferation and growth of MC3T3-E1 cells on the new self-assembling peptide hydrogel;(4)To evaluate effect of MC3T3-E1 promoting differentiation of osteogenesis on the new self-assembling peptide hydrogel.Methods(1).Diluents of self-assembled peptide solution were dropped on the surface of clean mica.Nano-structure of self-assembling peptide on the mica surface was observed through atomic force microscopy(AFM).(2).According to be grouped by adding gumforming compound of different materials on slides(14mm),cell slides were made to put into 24-well plates and undergo cell suspension inoculation,for observing the number of adherent cells on the surface of material of each grouping at different time points and detection of cell adhesive ability.(3).Proliferation of growing MC3T3-E1 cells on the surface of different materials was evaluated by the method of CCK-8 at different time points.(4).With confoeal laser scanning microscopy,growth of MC3T3-E1 cells on the surface of different materials at each point and after 14 days of cultivation on different HA/?-TCP composite material were observed.(5).formation of calcium nodule after 14 days' cultivation of MC3T3-E1 cells on the surface of different materials was determined by alizarin red-S staining.(6).Activity of alkaline phosphatase(ALP)of MC3T3-E1 cells cultured on the surface of different materials at different time-points alkaline phosphatase,and gene expression of ALP,osteocalcin(OCN)and vascular endothelial growth factor(VEGF)of MC3T3 E1 cells after14 days' cultivation on the material surface.Results(1)Under atomic force microscope(AFM),four groups of oligopeptides including TRSAW,RADA16-?,RADA16/TRSAWRADA16-?and TRASWmx were observed respectively.Results showed that cloud-like dispersoids were found in the TRSAW group,which unable to form nano-structure of three-dimensional reticular fiber,While RADA16/TRSAW groups formed irregular patchy fibers varied in size,but unable to carry each other.Notably,after mixed RADA16/TRSAW with RADA16-?diluents according to volume ratio 1:1,TRSAWmx formed long nanofiber similar to that in the RADA16-?and reticular structure and both with self-assembly properties.According to detection results of AFM,follow-up experiments were conducted after divided into blank group,RADA16-? group and TRSAWmx group.(2)Statistical analysis of MC3T3-E1 cells counts adhering on the surface of three kinds of material slices at each phase points was performed to find there was a significant difference between the blank group and RADA16-?after 10 minutes(P<0.05)At the time points of 30 min,60min and 90 min,there was significant differences between the blank group with TRSAWmx group and RADA16-?group respectively(P<0.05),while there was no statistical difference between the RADA16-?and TRSAWmx group(P> 0.05).(3)Detection results of MC3T3-E1 cells proliferation on the surface of different material slices via CCK-8 at each time point showed that there were significant differences among the blank group and TRSAWmx and RADA16-?group after one day cultivation(P<0.05),but there was no significant difference between the TRSAWmx group and RADA16-?group(P>0.05);while at the 3rd,5th and 7th day blank group,there were statistically differences among the blank group and TRSAWmx and RADA16-?group(P<0.05).(4)Under laser scanning confoeal microscopy,growth situation of MC3T3-E1 cells inoculated on different material slices respectively was observed to find out that cytoskeleton was arranged continuously with fiber structure extending along the long axis of cell after 3 days,7 days and 14 days;The number of cells in the TRSAWmx group at different time points was higher than that in the blank group and the RADA16-? group(P<0.05).After 14 days' cultivation on HA/?-TCP porous scaffolds,cell density of the TRSAWmx group was obviously higher than that of other groups.(5)ALP activity of MC3T3-E1 cells on the surface of different material slices at different time points was detected,and results showed that there were statistically differences among the TRASWmx group,blank group and RADA16-?group at the 3rd,7th and 14 th day(P<0.05).(6)According to staining results by alizarin red-S,cell density of the blank group after 14 days induced cultivation was lower than that of other two groups,with fewer calcium nodule formed in the middle.However,cell density of the RADA16-? group was higher with multiple calcium nodule,while much more orange calcium nodules were seen in the TRSAWmx group.(7)Results of Real-time PCR showed that gene expression of ALP,OCN and VEGF of cells cultured in the TRSAWmx group were relatively higher than that of the blank and RADA16-?group(P<0.05).Conclusions High-activity fragments-PTHr P(107~111)of parathyroid hormone related protein(PTHr P)was connected with the traditional RADA16-?protein C-terminal domain by solid phase peptide synthesis,based on which to obtain the novel self-assembling hydrogel peptides through mixed with traditional self-assembling peptide RADA16-?.Experiment results showed that the adhesion property and related performance such as promoting proliferation and osteogenic induction of MC3T3-E1 cells of the self-assembling peptide hydrogel in the TRSAWmx group was proved to be promising,which can be applied as a new type of scaffold material to provide suitable environment for cells culture in vitro.Moreover,it can be used in composite with hydroxyapatite(HA)and demineralized bone matrix(DBM)and so on to further explore related biological properties of composite materials in vivo,and provides a solid experimental foundation and theoretical basis for clinical application in the future.