Study On Pro-Osteogenic,Pro-Angiogenic And Antibacterial Activities Of Polydatin/CuO/PCL Bionic Periosteum

PurposeThe present study aims to produce a PCL-based bionic periosteum loaded with polydatin（PD）and Cu O nanoparticles（Cu ONPs）by employing the electrospinning technology,and to explore its material characterization and osteogenic,angiogenic and antibacterial activities.MethodsPCL/PD and PCL/Cu O nanofiber membranes with different PD and Cu ONPs content were produced by the electrospinning technology.As per the results of cell proliferation,migration,cell viability assays,osteogenic-related indicators（ALP,Alizarin Red staining）,and NO secretion assays,suitable PD and Cu ONPs load quantities were chosen so as to produce PCL/PD/Cu O nanofiber membranes loaded with both PD and Cu ONPs,which served as a bionic periosteum.The surface morphology,element and composition analysis,hydrophilicity,elastic modulus,and release effects of PD and Cu²⁺were evaluated using scanning electron microscopy（SEM）,spectroscopy,infrared spectroscopy,near-infrared spectroscopy,and UV spectrophotometry.Cell proliferation,adhesion,migration,osteogenesis,angiogenesis,and antibacterial effects were tested;the effect of the bionic periosteum on osteogenesis,angiogenesis,and antibacterial effects was verified in vitro.A rat skull defect model was constructed;the bone healing effect was observed via Micro-CT.The new bone volume（BV）and bone volume fraction（BV/TV）of each group were analyzed using the CTan software;the three-dimensional structure of the rat skull was reconstructed using Mimics Research software.The effects of the bionic periosteum on osteogenesis and angiogenesis were validated in vivo.Results（1）When the PD content was 0.04wt‰and 0.08wt‰,the PCL/PD nanofiber membrane had a good promoting effect on the proliferation and migration of osteoblast precursor cells MC3T3-E1,and few dead cells were observed in cell viability assays.The results of osteogenic-related experiments also showed that PCL/PD nanofiber membranes at these two PD content levels had a desirable osteogenic effect.When the Cu ONPs content was 0.3wt%,the PCL/Cu O nanofiber membrane had no significant difference in promoting the proliferation of MC3T3-E1 and HUVEC cells compared to the no-treatment control group,and the membrane at this content level could promote the secretion of NO by HUVEC cells.（2）Material characterization of the PCL/PD/Cu O bionic periosteum:SEM results showed that the surface of the bionic periosteum was smooth,with relatively uniform fiber thickness and basically no bead formation,which indicated that the fiber morphology of the bionic periosteum could provide a bionic surface for cell growth and proliferation.UV spectrophotometry and spectroscopy analysis confirmed respectively the successful loading of PD and Cu ONPs in the bionic periosteum.The measurement of water contact angle suggested that the bionic periosteum was a hydrophilic scaffold,which could provide convenience for cell adhesion.Furthermore,the elastic modulus of the bionic periosteum was superior to that of pure PCL nanofiber membrane,and the characteristics of PD and Cu²⁺release curves also achieved the goal of continuously promoting bone and angiogenesis and antibacterial effect.（3）In vitro experiments of PCL/PD/Cu O bionic periosteum:ALP activity,Alizarin Red staining,osteogenic-related gene real-time PCR quantification analysis and osteogenic-related protein WB results all showed that the bionic periosteum had good osteogenic induction activity.Meanwhile,experiments on HUVEC cells NO secretion,angiogenic ability,angiogenic-related gene real-time PCR quantification analysis,angiogenic-related protein WB,angiogenic-related antigen immunofluorescence and four antibacterial experiments all indicated that Cu ONPs was capable of promoting angiogenesis and boasted certain antibacterial activity.（4）In vivo experiments of PCL/PD/Cu O bionic periosteum:The results showed that new bone tissue was visible in all groups,and there were differences in bone volume and bone volume fraction between the groups,with the PCL/PD/Cu O bionic periosteum having a significantly better osteogenic effect than the other groups.The HE and Masson’s trichrome staining of the bone defect area showed that the PCL/PD/Cu O bionic periosteum covered the defect and had a regular lamellar bone structure at the eighth week,while the no-treatment control group showed only fibrous tissue and no new bone formation.Similarly,Masson’s trichrome staining also showed that the PCL/PD/Cu O bionic periosteum group had more new bone formation.To explore the effect of Cu ONPs on promoting angiogenesis in vivo,CD31 immunohistochemical staining was performed on the defect area,which was consistent with the in vitro tube formation assay.Positive CD31 cells were identified in all groups of defect areas.Compared with the no-treatment control group,the PCL/PD/Cu O bionic periosteum group had significantly more newly formed blood vessels,which indicated that the bionic periosteum played a pivotal role in promoting blood vessel formation.Conclusion（1）A PCL-based bionic periosteum loaded with 0.08wt‰PD and0.3wt%Cu ONPs was produced using the electrospinning technology.Highly compatible with bionic periosteum in terms of material characterization,it had a high bioavailability as well as the potential to be used as an artificial bone substitute.（2）The PCL/PD/Cu O bionic periosteum exhibited good osteogenic,angiogenic,and antibacterial abilities in vitro compared with the no-treatment control group.It did not affect cell proliferation and adhesion,and could promote cell migration to a certain extent,which was conducive to the repair of bone defects.（3）The PCL/PD/Cu O bionic periosteum had a positive effect on promoting osteogenesis and angiogenesis compared with the no-treatment control group in vivo.