Preparation Of VEGF Mimetic Peptide Loaded On Gelatin/Octacalcium Phosphate Composite Hydrogels And Osteogenesis Performance Study

Common bone grafts include autologous bone,allograft bone and artificial bone.Autologous bone and allograft have corresponding defects,which limit their application.In recent years,researchers had conducted extensive and in-depth research on artificial bone replacement materials,in the hope of eventually replacing autologous bone and allograft bone.At present,there are mainly two kinds of artificial bone substitute materials:first,artificial inorganic materials,such as calcium phosphate,calcium sulfate ceramics.Calcium phosphate ceramics including hydroxyapatite(HA)and beta tricalcium phosphate(beta-TCP),has been widely used clinically;Organic polymer materials and calcium phosphate composite materials can be loaded with active factors.Organic polymer materials include natural biological derivative materials and synthetic polymer materials.Gelatin(Gel)made from animal collagen and similar to amino acids in normal bone matrix,is widely used in bone tissue regeneration engineering.However,common gelatin hydrogels are difficult to be applied in clinical practice alone due to high swelling rate,poor mechanical properties and fast degradation in vivo.Octacalcium phosphate(OCP),is an important precursor of HA in the body,in recent years,research has shown that it has high conductivity,good biocompatibility and bone induced,and matching with bone formation velocity of degradation rate.However,the mechanical properties of OCP powder were poor,and it was unstable in aqueous solution and easy to be converted to HA.Mixing Gel and OCP to prepare composite material can solve this problem well.The vascularization of bone substitute material in vivo is a necessary process for the graft to be finally degraded and replaced by new bone.How to accelerate the blood vessel growth of bone substitute material in vivo is the key to the success of bone transplantation.Vascular endothelial growth factor(VEGF)has a good effect in inducing endothelial cell growth and angiogenesis.KLTWQELYQLKYKGI(QK)is a mimetic peptide of VEGF,which has been proved to have similar biological activity.However,the compound prepared by simple mixing is prone to sudden release of polypeptide molecules for a short time and may have side effects.Beta-cyclodextrin(?-CD)is widely used as a carrier of controlled release and sustained release of a variety of drugs,and acrylic modified beta-cyclodextrin(Ac-?-CD)can significantly improve the drug loading and controlled release performance.This paper aims to build Gel/OCP composite hydrogel materials that can slowly and continuously release QK,evaluate the structure,mechanical properties,biocompatibility of the materials and the efficiency of loading QK polypeptides in vitro and in vivo experiments,and investigate the performance of osteogenesis and repairing.This study was divided into three parts:the first part,the preparation and characterization of Gel/OCP composite hydrogel loaded with VEGF mimetic peptide;In the second part,the biocompatibility characterization of Gel/OCP hydrogel loaded with VEGF mimetic peptide in vitro;In the third part,the in vivo performance of Gel/OCP combined with VEGF mimetic peptide was studied.Part ?:The Preparation and Characterization of Gel/OCP Composite Hydrogel Loaded with VEGF Mimetic PeptideObjective:HA precursor octaphosphate(OCP)and gelatin(Gel)were selected as the base material,and beta-cyclodextrin modified by acrylic acid(Ac-?-CD)was added to improve the performance of the composite material loading and releasing QK.The morphological structure characterization,mechanical properties,invitro mineralization properties,loading and releasing QK properties of composites were studiedMethods:Na2HPO4 and Ca(CH3COO)2 solutions were prepared,OCP powder was prepared by homogeneous precipitation,and its characterization was observed and measured by FTIR,XRD,SEM and TEM.The hydrogels of Gel(G)were prepared by Ac-[3-CD and Gel.For example,in 1 ml of hydrogel solution,according to take 100 mg Ac-?-CD and 80 mg of Gel,add 0.9 ml PBS and 0.1 mL 12959(0.5%)solution,vortex blending in 37 ?,ultrasound to bubbles,add to the mold,under ultraviolet light(365 nm)for 30 min.When Gel/OCP(GP)complex hydrogel was prepared,10mg OCP powder was first dissolved in 0.1ml 12959(0.5%)solution,and then added to the above solution to prepare it.The internal structure of the material was observed by optical microscope and SEM.The dimensional stability of the material was characterized by immersion experiment.The mechanical properties of the material were tested by universal mechanical testing machine.The prepared material was immersed in the simulated body fluid(SBF)for 2 weeks to observe its invitro mineralization performance.The material was soaked in 2mg/ml QK solution,loaded with QK and taken out after 12 hours,and then the material was placed in PBS solution to test its QK release performance.Micro BCA kit and enzyme marker were used to determine the concentration of QK in the solution,so as to calculate the drug loading rate of the material with QK and the cumulative release curve.Results:The characteristic absorption band and the characteristic peak of the crystal surface of OCP were detected by FTIR and XRD,which proved that the OCP was successfully prepared.The OCP presented typical thin sheet crystalline morphology under SEM and TEM.The G and GP hydrogels were three-dimensional network structures under microscope and SEM,with high porosity.The variation rate of the diameter of GP was significantly smaller than that of G hydrogel,the compression strength in wet state was significantly improved,and more hydroxyapatite(HA)crystals were formed on the surface during invitro mineralization.The drug loading rate of GP hydrogel on QK was 49.6+15.8%,which was better than G.Conclusion:The OCP crystal was successfully prepared in this experiment.Gel/OCP composite hydrogel has good internal pore structure,size stability,mechanical properties and invitro mineralization properties.The ?-CD in it makes the material have good loading and controlled release function to QK.Part ?:The Characterization of Biocompatibility of Gel/OCP Composite Hydrogel Loaded with VEGF Mimetic Peptide in VitroObjective:To observe the adhesion,growth and proliferation of rat bone marrow mesenchymal stem cells(BMSC)and human vascular endothelial cells(HUVEC)on Gel/OCP composite hydrogels,to study the biocompatibility and toxicity of the materials in vitro.To study the bone differentiation ability of hydrogel materials in vitro by osteoblast induced differentiation culture.Methods:The Gel(G),Gel/OCP(GP),Gel-QK(GQ)and Gel/OCP-QK(GPQ)were prepared on the 48-well culture plate according to the addition of OCP and the loading of QK.BMSC and HUVEC were prepared into 1×105?/ml suspension and inoculated on the culture plate for 3 hours,and the cell morphology was observed by ordinary microscopy and SEM.Dil fluorescent stain was added to the cell suspension and inoculated to the culture plate.BMSC was cultured for 3 days to observe the growth of cells on hydrogel through laser confocal microscopy,and HUVEC was cultured for 2 days to observe by inverted fluorescence microscopy.The extracts of G,GQ,GP and GPQ were prepared by adding osteogenic induction medium into the culture plate,and the non-osteogenic induction medium was used as the control group.The BMSC was inoculated to the culture plate and replaced with fresh extract solution every day.Alkaline phosphatase(ALP)staining was performed after 7 days and alizarin red staining was performed after 21 days to characterize the invitro osteogenic properties of the material.Results:All four groups of hydrogel materials could well support the growth of BMSC and HUVEC.The good adhesion and growth of BMSCS on the surface of materials were observed by SEM.Laser confocal 3D imaging showed that BMSCS could not only grow on the surface of materials,but also infiltrate and grow into the materials.Under the optical microscope,HUVEC presented a typical oval shape,rapidly proliferating and covering the entire hydrogel.Inverted fluorescence microscopy also confirmed that HUVEC can grow well on the material surface.The osteogenic induction experiment showed that the expression of ALP in 7 days and the number of calcium nodules in 21 days were significantly increased in the hydrogel materials loaded QK or doped OCP.Conclusion:The Gel/OCP hydrogels loaded with QK have good biocompatibility in vitro,which can support the adhesion and growth of BMSC and HUVEC on the surface of materials,and have good invitro osteogenesis.Part?:The Performance of Gel/OCP Composite Hydrogel Loaded with VEGF Mimetic Peptide in VivoObjective:To investigate the angiogenic capacity of Gel/OCP composite hydrogel materials loaded with VEGF Mimetic Peptide.To study the ability to repair skull defects in rats.Methods:The Gel(G),Gel/OCP(GP),Gel-QK(GO)and Gel/OCP-QK(GPQ)were prepared.The material was made into a round sheet with a diameter of 5mm and a thickness of 1mm.The chicken embryo villus allantoic membrane(CAM)was used as the experimental model of invivo angiogenesis.Incubated the fertilized egg for 7 days,opened a window in the air chamber,and uncovered the egg membrane to expose the chick's CAM.Chicken embryos were randomly divided into four groups(G,GQ,GP,GPQ).Four groups of hydrogel discs were placed on the allantoic membrane in the experimental group while no materials were used in the control group.After sealing,incubated for 48h.To observe the changes of blood vessels on the allantoic membrane,to take photos and record,and to make statistics on the development of blood vessels and the relative length of vessels to evaluate the effect of promoting blood vessel growth in each group.Thirty healthy SD rats were selected and divided into 6 groups.No treatment was performed in the normal group,while the remaining rats were treated with a bone defect of 5mm each in the right and left parietal bone,and no material was implanted in the blank group.The remaining four groups were implanted with G,GQ,GP and GPQ.Collected 8 weeks and 16 weeks after surgery for Micro-CT detection and HE staining to observe the bone formation in the defect area.At 16 weeks after the operation,ink perfusion was prepared before the skull was taken out to mark the blood vessels with new bone formation.Results:The GQ and GPQ group loaded with QK showed obvious promoting CAM angiogenesis,with relatively large relative vessel length.Eight weeks after the skull defect repair,Micro-CT found that there were a lot of new bone formation in the four groups of hydrogels,not only at the junction of material and natural bone,but also in the center of the defect.HE staining also confirmed the above findings,but undegraded materials were observed in all four groups.There were more new bone formation in GP and GPQ group.At 16 weeks after operation,no good effect of bone repair was found in the blank group.The experimental group had better bone repair effect and obvious material degradation.In the GPQ group in particular,the hydrogel material was completely replaced by new bone,but the thickness of new bone was different from that of natural bone.Through ink-infusion,it was found that there were a lot of neovascularization in the center of GQ group materials.GPQ group showed a large area of new bone with a small number of blood vessels,which was close to the natural bone.Conclusion:The hydrogel material loaded with QK has a good effect of promoting invivo angiogenesis.The hydrogel materials prepared in this experiment can degrade gradually with the extension of transplantation time.Meanwhile,new bone can be formed at the interface between the material and bone and inside the material.Gel/OCP hydrogel loaded with QK can be used as an excellent substitute material for bone repair.