| BackgroundWith the development of social productivity,various open injuries caused by high energy are on the increase,especially in orthopedics,where the incidence of open fractures is high and postoperative bone infection is easy to occur[1].Although the infection can be controlled by aggressive debridement,large bone defects are left with skin and soft tissue defects.For infectious bone defects accompanied by skin and soft tissue injuries,local injuries are complex and there is no skin and soft tissue coverage,which has always been a difficult problem for orthopedics[2].In the past,the infected bone was not fully utilized,the treatment time was long,the operation frequency was many,and the postoperative function was poor[3-4].Autologous bone flap transplantation does not require a slow"crawling replacement"process,and can cover the wound surface and fill the tissue defect.However,the sampling location of autologous bone flap is limited,and it is difficult to meet various types of injuries,which limits its application and development[5-6].Different types of tissue flaps can be preformed by modifying the flap appropriately,so that the preformed flap can repair various types of skin tissue defects[7-9].In clinical practice,the bone flap can be preformed by heterotopic foster of the large segment?block?autologous bone flap.After6-12 months,the large segment?block?autologous bone flap can be vascularized[10].Because human bone cortices are thick and hard,it takes a long time to complete vascularization.How to shorten the process of vascularization has always been the focus of our research.Skeletal muscle can induce large bone angiogenesis and increase bone activity without the addition of any exogenous seed cells and factors[11].Muscle is rich in a large population of bone-induced cells,which secrete a variety of cytokines and growth factors related to fracture healing.In the process of prefabricated bone flap,the angiogenesis and osteogenesis of skeletal muscle are rarely reported.The in-depth study of skeletal muscle can provide theoretical basis for ensuring the survival of prefabricated bone flap and shortening the process of prefabricated bone flap.ObjectiveTo investigate the mechanism of vascular endothelial growth factor?VEGF?and bone morphogenetic protein-2?BMP-2?secreted by skeletal muscle in rabbits and the effect on the large segment autogenous bone ectopic preconstruction of vascularized bone flap.MethodsA total of 70 adult Chinese white rabbits?male or female?were randomly selected as the experimental group,and 60 of them were randomly selected as the experimental group.The bone segment of the middle tibia of the left hind limb?1.5cm?was cut off to remove the intramedullary cavity and the attached soft tissue on the surface of the bone segment.The bone flap was prefabricated in the muscle pocket of the right rectus femoris after being sterilized by high temperature water bath.The remaining 10 were used as blank control group.Experimental group at 2,4,6,8,10,12 weeks each executed only and remove the large section of the autologous bone and the whole piece of rectus muscle,large autologous bone gross observation,histological,HE staining of CD34 and type I collagen immunofluorescence staining detection and bone vascularization degree activity,Western blotting detection rectus muscle tissue VEGF and BMP-2 expression.In the blank control group,only the bone segment of 1.5cm in the middle tibia of the left hind limb and the whole rectus femoris muscle on the right side were intercepted,and the detection indexes were the same as those in the experimental group.Results1 General observation of large segments of autogenous boneIn the experimental group,the incision healed well and there was no inflammatory reaction around the prefabricated bone flap.The fibrous connective tissue adhered to the bone cortical surface at both ends of the large segment of autologous bone marrow cavity gradually thickened and became more and more dense,and adhered to the bone segment gradually.After the removal of fibrous connective tissue,bone resorption pits can be seen on the bone surface,and the depressions become deeper and deeper with the extension of time,and the bone cortex gradually becomes thinner.At week 8,the fibrous connective tissue wrapped bone segment was similar to the normal thickness of autologous bone periosteum,and was difficult to separate.2 Results of HE staining of large segment autogenous boneFibrous granulation tissue was rarely seen on the bone surface and lumen of the experimental group at the second week.At week 4,fibrous granulation tissue appeared on the bone surface,with small lacunae and a small amount of neovascularization and cellular components surrounding.At week 6,more blood vessels and cells were observed.The bone vortex on the surface of the bone adjacent to the blood vessels showed mature osteocytes.The haval lumen became larger and the trabecular bone began to grow slowly.Bone HE staining in the experimental group from week 8 was similar to that in the fresh tibia,with more vascular endothelial cells and osteocytes,as well as dense and mature bone trabeculae.3 Results of immunofluorescence detection of large segment autogenous boneImmunofluorescence results of CD34 showed that:the number of positive blood vessels of CD34 in the experimental group increased first and then decreased slowly,and the number of positive blood vessels of CD34 reached the maximum at week 8.The number of CD34 positive blood vessels in the experimental group at week 4 was higher than that at week 2,and the difference was significant?P<0.05?.The number of CD34positive blood vessels in the experimental group at week 6 was significantly higher than that at week 4?P<0.05?.The number of CD34 positive blood vessels in the experimental group at week 8 was higher than that at week 6,with a significant difference?P<0.05?.The number of positive blood vessels of CD34 in the control group was significantly higher than that in the experimental group at week 2,4,6 and 12?P<0.05?.?type collagen immune fluorescence intensity shows that:the experimental group?type collagen immune fluorescence intensity enhancement before they are waning,peaked at 10 weeks,the?type collagen fluorescence intensity is higher than 2,4,6,8,12weeks.The experimental group 4 weeks?type collagen immune fluorescence intensity is higher than its second week,obvious difference?P<0.05?;Experimental group 6 weeks?type collagen immune fluorescence intensity is higher than the 4th week,obvious difference?P<0.05?;Experimental group 8 weeks?type collagen immune fluorescence intensity is higher than the 6 weeks,obvious difference?P<0.05?;Experimental group 10weeks?type collagen immune fluorescence intensity is higher than the 8 weeks,significant difference?P<0.05?.Control group?type collagen immune fluorescence intensity is higher than experimental group 2,4,6,8,12 weeks,the differences?P<0.05?.4 Expression of VEGF and BMP-2 secretion in rectus femorisThe secretion of VEGF in muscle tissue samples of the experimental group increased first,maintained a certain amount and then decreased.It peaked at week 4 and remained at week 6,higher than week 2,8,10,and 12.In the experimental group,the secretion of VEGF in week 4 was higher than that in week 2,and the difference was significant?P<0.05?.The secretion of VEGF in the experimental group at week 8 was lower than that in week 6,with a significant difference?P<0.05?.Compared with the control group,the amount of secretion in the experimental group at week 2,4,6 and 8 was higher than that in the control group,with significant difference?P<0.05?.In the experimental group,the secretion of BMP-2 increased first and then decreased.It peaked at week 6,higher than week 2,8,10 and 12.The secretion of BMP-2 in the experimental group at week 4 was higher than that at week 2,with a significant difference?P<0.05?.The secretion of BMP-2 at week 6 was higher than that at week 4,and the difference was significant?P<0.05?.The secretion of BMP-2 at week 8 was lower than that at week 6,with a significant difference?P<0.05?.The secretion of BMP-2 at week 10 was lower than that at week 8,and the difference was significant?P<0.05?.The secretion of BMP-2 at week 12 was significantly lower than that at week 10?P<0.05?.Compared with the control group,theBMP-2 secretion of the experimental group at week 2,4,6 and 8 was higher than that of the control group?P<0.05?.The amount of secretion in the experimental group at week 10 and 12 was less than that in the control group,and the difference was statistically significant?P<0.05?.Conclusion1.The rabbit skeletal muscle can secrete VEGF and BMP-2 in the process of bone flap preformed by heterotopic vascularization of large autogenous bone.2.VEGF and BMP-2 can play a role in the early stage of the prefabricated bone flap,increasing gradually and then decreasing slowly.3.Skeletal muscle can promote both angiogenesis and bone activity during bone flap preconditioning.The completion of vascularization helps to continue to enhance bone activity. |
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