The Experimental Study On Repair Of Rat Dorsal Ischemic Flaps With VEGF/PELA/bFGF Mixed Microcapsules Combined With Bone Mesenchymal Stem Cells

The microvascular system of the bone includes the cortical vasculature and the bone marrow capillary system,which form a large vascular network and brings soluble factors and circulating stem/progenitor cells to the local bone tissue,maintains the normal metabolism and regeneration of the bone.Clinically,due to bone tumor resection,trauma,tuberculosis,etc.,it often causes large-area bone defects and the accompanying destruction of the vascular system.Traditional bone filler materials have been used in orthopedics for decades.They have great advantages in repairing small-sized bone defects.However,when repairing large bone defects,they often lack essential nutrients or oxygen in the inner of the material.The formation of necrotic areas in the site,which hinders the repair and regeneration process of bone tissue.Studies have shown that the maximum distance between capillaries in normal tissues does not exceed 200?m in order to adapt to the diffusion of matter,while in bone tissue,bone cell migration is limited to 100?m in the circumference of the tube.Therefore,in order to achieve effective repair of large bone defects and restart the self-repairing regeneration process of bone tissue,a functional vascular network must be formed inside the material as early as possible.The formation of blood vessels in the body depends on the vascular endothelial progenitor cells,which form a primitive capillary vascular network by recruitment and migration.Subsequently,pericyte cells and smooth muscle cells(SMCs)protrude from the pseudopods under the control of various cytokines,and migrate to cover the surface of the immature capillary network,the permeability of the immature vascular network is reduced,the mechanical strength increases,which in turn forms a mature vascular network,this process is aptly called "vascular tightening".Vascular endothelial growth factor(VEGF)plays a key role in regulating the targeted migration of cells into endothelial cells.The study have found that human VEGF has five kinds of monomers,namely VEGF121,VEGF145,VEGF165,VEGF189 and VEGF206,of which VEGF165 promotes the most angiogenic effect.Another factor that has a synergistic effect with VEGF in promoting angiogenesis is basic fibroblast growth factor(bFGF).It has been reported that VEGF can induce endothelial progenitor cells(EPCs)to migrate and proliferate in angiogenesis,forming a primitive tubular structure,while bFGF can recruit surrounding SMCs and pericytes to "package" the original lumen.Under the joint action of various factors,the original vascular structure reaches a mature and stable state.By studying the characteristics of pro-angiogenic factor release in the early stage of tissue damage,it has been found that the expression of bioactive molecules is strictly regulated in space and time.Liu Xiaotong et al.established a rat model of skin flap ischemia to detect the local release of VEGF and bFGF in the early stage of ischemic tissue and found that VEGF gradually increased after surgery and peaked at 3 days,while bFGF sustained release and peaked at 10 days.Some recent studies have also shown that the effect of multi-factor sequential controlled release promotes angiogenesis in the ischemic area is greater than single factor one-time release.Therefore,by coating the growth factor with special materials,it is possible to simulate and amplify the release process of pro-angiogenic cytokines in the process of injury repair under physiological conditions,which may got twice the result with half the effort.In the early stage,the PELA microcapsule scaffolds with sequential release of rhBMP-2 and rhBMP-7 were successfully prepared by our group.The parameters were optimized by the response surface method to achieve the highest encapsulation efficiency of the growth factors.The material has showed good biocompatibility,degradability,long sustained release period and the ability to induce osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs)in vitro experiments.The animal experiments confirmed that it has a good ability to repair large femoral bone defects in rats.In this study,PELA microcapsules encapsulating bFGF were prepared on the basis of this,and the angiogenesis-inducing ability was enhanced by the addition of VEGF to achieve the effect of two-factor sequential release and long-term sustained release of bFGF.The in vitro experiment will be carried out to investigate the effect of VEGF/PELA/bFGF mixed microcapsules on the differentiation of BMSCs.Further,the rat back flap ischemia model was used to verify the repairing effect of the mixed microcapsules combined with BMSCs on the ischemic site.It is expected to provide a research basis for further study of internal vascularization in the inner of bone repair materials finally.Object:To fabricate the VEGF/PELA/bFGF mixed microcapsule material,and the biocompatibility,cytotoxicity and drug release characteristics of the material will be studied.The inducing ability of the mixed microcapsules to induce angiogenesis of BMSCs will be investigated in vitro.Animal experiments will be carried out to evaluate the repair ability of rat ischemic flaps,in order to provide methods and ideas for internal vascularization of large bone repair materials.Method:1 Preparation and parameter detection of VEGF/PELA/bFGF mixed microcapsulesVEGF/PELA/bFGF(Group A),PELA/bFGF(Group B),VEGF/PELA(Group C)and PELA(Group D)were prepared using PLA-PEG-PLA triblock copolymer(PELA)as the microcapsule material by using the solvent-vulcanization method.The microcapsule morphology was observed by inverted microscope.The lyophilized microcapsules were dissolved in 37? PBS(pH 7.4),freeze-dried at a fixed time point and weighed,their in vitro degradation ability will be studied;The releasing ability of VEGF/PELA/bFGF mixed microcapsules were detected in the same condition,50mg of lyophilized mixed microcapsules were dissolved in an EP tube containing 1 mL of PBS,and placed in an incubator at 37?,and taken out at fixed time points(1,2,4,8,12,16,20 d),and collected.The supernatant is stored at-20?for final measurement.After the supernatant was collected for 20 days,the samples at each time point were taken out,and the concentrations of VEGF and bFGF in the supernatant were detected by reference to the rhVEGF165 and rhbFGF ELISA kits.Six replicate samples were set for each group to reduce errors.2 VEGF/PELA/bFGF mixed microcapsules in promoting angiogenic differentiation of rat bone marrow mesenchymal stem cells in vitroThe BMSCs were isolated by the method of whole bone marrow adherent,and sub_cultured.The passage 3 BMSCs were identified by Wright-Gimsa staining and flow cytometry,and used for subsequent experiments.The cytotoxicity of PELA microcapsules were determined by CCK-8 kit.The passage 3 BMSCs were co-cultured with the extracts of groups A,B,C,and D,separately.At 1,3,7,14,and 20 days after culturing,the morphological changes of induced BMSCs were recorded.At 21 days,the induced BMSCs were tested for DiI-labeled acetylated low density lipoprotein(Dil-ac-LDL)and FITC-labeled ulex europaeus agglutinin I(FITC-UEA-I)uptake ability.The tube-forming ability of the induced cells on Matrigel was also verified.The differences of the vascularize indexes in nodes,master junctions,master segments,and tot.master segments length in 4 groups were summarized and analyzed.Six replicate samples were set for each group to reduce errors.P<0.05 was considered to be statistically significant.3 The experimental study on repair of rat ischemic flap with VEGF/PELA/bFGF mixed microcapsules combined with bone mesenchymal stem cells30 male SD rat,8W age,were randomly divided into 5 groups,6 every group.The model of ischemic skin flap in the back of rats was established,4 groups of microcapsules described above were prepared simultaneously.Multipoint subcutaneous injection was performed using materials combined with BMSCs which were diluted by PBS.The suspensions of VEGF/PELA/bFGF+BMSCs,PELA/bFGF+BMSCs,VEGF/PELA+BMSCs,PELA+BMSCs and PELA alone were divided into groups A,B,C,D and E.Animals were killed at 14 days,and the area of ischemic necrosis was quantitatively evaluated by Image J software.HE staining and CD31 immunohistochemical staining were performed on the central flap of the distal end of the back,and the number of microvessels in each group was counted and analyzed.Using the LSD test if the variance was equal,in contrast using the Dunnett T3 test for two comparison,P<0.05 was considered to be statistically significant.Results:1 Preparation and parameter detection of VEGF/PELA/bFGF mixed microcapsulesAccording to the optimization parameters in the previous synthesis process(PELA=280mg,PVA concentration=0.8%,bFGF=3?g),the VEGF/PELA/bFGF,PELA/bFGF,VEGF/PELA and PELA microcapsule materials were successfully prepared.Under the inverted microscope,the PELA microcapsules were completely spherical,regular in shape,intact in the cyst wall,and occasionally partially fused.Most of the microcapsules had a diameter distribution between 30 and 60 ?m,and some of them exceeded 100?m,which was similar to the previous research results.In the first 4 days,the degradation rate of the material was stable and slow,and it experienced a rapid degradation period from 4 to 12 days.After 10 days,the material degraded to 50%of the original mass,and then experienced a slow continuous degradation process.At 20 days,about 30%of the material remained.Since VEGF was not fixed and directly lyophilized on the surface of the microcapsules,it was most rapidly dissolved and released after being dissolved in PBS,which showed a cumulative release of more than 90%at 4 days,and then showed a relatively smooth micro-release process.At 20 d,the total amount released was over 95%.In contrast,bFGF encapsulated inside the microcapsules showed a sustained release process after 2 days,and a cumulative release of more than 80%was detected by 20 days.This process roughly mimics the release characteristics of two important cytokines,VEGF and bFGF,in the early stage of angiogenesis.2 VEGF/PELA/bFGF mixed microcapsules in promoting angiogenic differentiation of rat bone marrow mesenchymal stem cells in vitroThe BMSCs were isolated by the method of whole bone marrow adherent,and sub-cultured.The passage 3 BMSCs were identified by Wright-Gimsa staining and flow cytometry and identified as BMSCs.The cell viability of BMSCs co-cultured with PELA was detected by CCK-8 kit.The results showed that on the third day of co-culture(F=0.020,P=0.890),day 7(F=1.149,P=0.309)and day 14(F=1.182,P=0.302),there was no significant difference in the A-value between the PELA culture group and the pure cell control group,suggesting that PELA as the capsule material had no effect on the viability of rat BMSCs in a short period of time.The morphology of cells in groups A,B,and C were changed.The cells in groups A and B showed"typical of cobble-stone morphology"and with a higher ability to uptake Dil-ac-LDL and FITCUEA-I than other groups.The cells in groups A and B formed a grid-like structure on Matrigel.Quantitative analysis showed that the difference in the number of nodes,master junctions,master segments,and tot.master segments length in groups A and B were significant compared with those in groups C and D(P<0.05).The number of nodes and the tot.master segments length of group A were more than those of group B(P<0.05).There was no significant difference in the number of master junctions and master segments between 2 groups(P>0.05).Those results suggested that VEGF/PELA/bFGF mixed microcapsules have significantly ability to promote the angiogenic differentiation of rat BMSCs in vitro.3 The experimental study on repair of rat dorsal ischemic flaps with VEGF/PELA/bFGF mixed microcapsules combined with bone mesenchymal stem cellsWhen treated with different groups of materials for 14 days,The area of ischemic necrosis of the dorsal skin flap was minimal in group A,which was significantly different from the other four groups(P<0.01).There was no significant difference between groups B and C(P>0.05),but significant difference compared with groups D and E(P<0.01).The difference between groups D and E were not statistically significant(P>0.05).HE staining showed that the capillaries in the group A were rich,and the skin tissues of groups B,C,and D had different degrees of necrosis,the tissue was disorganized in group E,and no vascular structure was seen.Immunohistochemical staining of CD31 showed that the number of microvessels per unit area was significantly higher in group A than in the other four groups(P<0.01).There was no significant difference between groups B and C(P=0.235),but significant difference compared with group D and E(P<0.01).The difference between groups C and D were not statistically significant(P=0.059),but significantly different compared with group E(P<0.01).There was a statistically significant difference between groups D and E(P=0.029).These results suggested that VEGF/PELA/bFGF mixed microcapsules combined with bone marrow mesenchymal stem cells have a significant role in promoting microangiogenesis in the ischemic site.Conclusion:This study demonstrates that VEGF/PELA/bFGF mixed microcapsules have the efficiently ability to induce BMSCs to differentiate into endothelial-like cells;further study in rat confirms that the materials combined with BMSCs can significantly promote microvascular formation in the ischemic region.This provides a new way to overcome the insufficient internal vascularization into tissue engineering bone filling materials.At the same time,it provides a new idea for the revascularization in ischemic tissue clinically.