Polycaprolactone/nano-hydroxyapatite Scaffold Conjugated With E7 Peptide For The Construction Of Tissue Engineered Bone

BackgroundThe technique of tissue engineering can repair the bone defect,better than the traditional treatment.The research of tissue engineering scaffold is an important part of the research on tissue engineering and regenerative medicine.PCL and nHA are the biomaterials with excellent biocompatibility and mechanical properties.Because of its own nature,PCL is less adhesive to seed cells.In the current study,the scientists screened an affinity peptide of hBMSCs.The amino acid sequence is EPLQLKM.This peptide will be conjugated on the surface of PCL/nHA for the construction of tissue engineered bone.Objective· To create a porous tissue engineering scaffold named PCL/nHA with the help of 3D printing technology,and do the research on mechanical properties.To modify the scaffold PCL/nHA with MSCs-affinity peptide named E7,and assess the degradation performance.· To identify the biological effects of E7-PCL/nHA scaffold on hBMSCs.· To identify the osteogenesis ability of E7-PCL/nHA scaffold combined with hBMSCs.Methods· The nHA were synthesized via the hydrothermal synthesis,and the porous tissue engineering scaffolds PCL/nHA were created with the help of 3D printing technology.The scaffolds were observed using scanning electron microscope(SEM).PCL/nHA scaffolds with different HA contents(10%,20%,30%)were evaluated on its mechanical properties with compression test and bending test.The scaffolds conjugated with FITC-E7 were observed using fluorescence microscope.Implanting the E7-PCL/nHA scaffolds under the skin of the rabbits,to evaluate the degradation rate.· The hBMSCs were isolated by density gradient centrifugation.The hBMSCs were induced into adipocyte and osteoblast.Using alizarin red staining,oil red O staining for identification.The hBMSCs surface markers were detected by flow cytometry.After being induced into osteoblast,we detected osteogenesis gene expression level.· Both of the E7-PCL/nHA scaffolds and PCL/nHA scaffolds were combined with hBMSCs.Cell growth situation was observed using scanning electron microscope(SEM).Spatial distribution of cells was observed using laser scanning confocal microscope(LSCM),while 3-hours cell adhesion condition was also observed using LSCM.The hBMSCs combined with different scaffolds(E7-PCL/nHA PCL/nHA?-TCP CHA)were compared on adhension,proliferation and osteogenesis gene expression level.· E7-PCL/nHA+ hBMSCs and PCL/nHA+ hBMSCs were immersed in osteogenesis induction culture medium.After 2 weeks,we implanted them under the skin of nude mice.3 months later,we got the tissue engineering bone(TEB),the TEB were compared by histologic analysis.Results· The nHA were obtained via the hydrothermal synthesis.The tissue engineering scaffolds PCL/nHA were created,and the pores are uniform.Biomechanical testing results showed that,with the increasing of nHA content,the indices such as modulus of elasticity also increase.The scaffolds conjugated with FITC-E7 had a green fluorescent.The scaffolds can be degraded in vivo,degradation is very slow.· The hBMSCs isolated by density gradient centrifugation could be induced into adipocyte and osteoblast.· The hBMSCs on the E7-PCL/nHA scaffolds and PCL/nHA scaffolds growed in good condition.A great amount of cell matrix can be found around the scaffolds.We found that the cell density of E7-PCL/nHA scaffolds is a little bit bigger than the cell density of PCL/nHA scaffolds by LSCM observation.The cell adhesion rate,proliferation of hBMSCs seeded on PCL/nHA were significantly lower than E7-PCL/nHA,?-TCP and CHA groups.The OCN and OPN gene expression level of hBMSCs seeded on P-TCP were higher than E7-PCL/nHA,PCL/nHA,CHA groups.The gene expression level of ALP and RUNX2 had no differences in these 4 groups.· After being implanted 3 months,the E7-PCL/nHA and PCL/nHA group converted into TEB.There were no differences in bone square between the two groups.Conclusions· The porous E7-PCL/nHA tissue engineering scaffolds can be made successfully.· The hBMSCs can be isolated by density gradient centrifugation.· The E7-PCL/nHA scaffolds have good biocompatibility and have shown an apparent MSC-homing property.· The E7-PCL/nHA scaffolds were demonstrated to be successful for construction of TEB.