| Due to diseases or traumas,bone defects have become a serious health problem for humans now.As a promising strategy,artificial scaffolds in various types have been sythesized and invesited for reparing bone defects.The repair strategy based on porous scaffolds,which composed of inorganic-organic composites,such as HAP/ gelatin,has been widely applicated in bone tissue engineering.Its promoting effects on bone formation was largely influenced by revascularization,and without proper nutrition supplying from vascular channels,regenerated bone tissue would necrose and the scaffolds’ osteogenesis effects in vivo was limited.In order to enhance the long-term efficacy of bone repair,it is necessary to design a porous scaffold with both osteogenesis and angiogenesis capacity.Copper(Cu)is considered to play an important role in osteogenesis and angiogenesis.Therefore,we synthesized a copper-doped micronbelt hydroxylapatite(Cu HARB).Based on Cu HARB,we further prepared a bone tissue scaffold with gelatin to repair critical-size rat calvarial defect and its promoting effects on bone formation were well evaluated in vitro and in vivo,trying to provide a new selection for the bone defects treatment.Inspired by the structure and morphology of hydroxyapatite in natural bone,by taking the advantage of solvothermal method,where calcium nonanoate and copper nonanoate used as precursors to avoid the usages of double bond,we innovatively synthesized Cu HARB with different amounts of Cu amout.After detecting by XRD,XRF,FTIR,FSEM and FTEM,it was found that when the Cu /(Ca + Cu)molar ratio was no more than 8%,the products showed the pure HA phase,and Cu was successfully doped into the HA lattice.The synthesized products were micronbelts,which orderly arranged by monocrystal hydroxyapatite nanorods,whose length was about 500 nm,and diameter was nearly 30 nm,by a self-assembly way.In the in vitro co-cultured experiment of Cu HARB extract with rat bone mesenchymal stem cells(r BMSCs).When using CCK-8 kits to test the r BMSCs activity,it was found that the doping amount of Cu will affect the cytocompatibility of HA.When the extract concentration of the Cu doped HA,whose Cu doped content was 8%,was less than 40 mg/ml,Cu HARB showed good cytocompatibility on r BMSCs.Meanwhile,the extract had a positive effect on the alkaline phosphatase(ALP)activity expression of r BMSCs,showing that Cu HARB has potential to promote osteogenic differentiation of r BMSCs.We quantitatively analyzed the ALP relative expression of r BMSCs by PCR technology,and furthermoere confirmed Cu HARB’s excellent osteogenesis induction activity.Above all,the 40 mg/ml extract concentration of Cu HARB with 8% Cu doping amount not only has good cytocompatibility but also havs promoting effects on osteogenic differentiation of r BMSCs.We constructed the Cu HARB/gelatin porous scaffold by freeze-drying method.The observation results of the micron morphology of the scaffold and the cell adhesion showed that the inside of scaffold had microcellular structure and its surface was suitable for r BMSCs to adhere and grow.Subsequently,a critical size(8mm)SD rat cranial defect model was established to investigate the promoting effect on bone regeneration of Cu HARB/gelatin scaffold in vivo.By integrated utilization of Micro-CT,histological staining and immune staining teachnology,we evaluated the recovery progress of bone defects,confirmed that the Cu HARB/gelatin scaffold had osteogenesis and angiogenesis capacity,which could stimulate bone repair in vivo.In general,this study innovatively synthesized a Cu-doped micronbelt HA,which showed good cytocompatibility and promoting effects on osteogenesis differentiationan of r BMSCs.The porous scaffold,which composed of Cu HARB and geletin,presented both good osteogenesis capacity and proangiogenic potential.Our study not only provided a new potential material for bone defect repair,but also a new method for ion-doped HA synthesis. |
PDF Full Text Request