The Experimental And Clinical Study Of Large Free Infectious Bone Revascularization By Ectopic Transplantation

Research background:There are more opportunities for leg impacted and extruded by directly violence. Those open comminuted fractures caused by traffic injury, machine injury, war trauma and so on, had higher rates of bone-infection, even evolved into chronic bone infection, which Accompanied by a large area of skin and soft tissue defects with bone exposure or more of the larger segment of bone fracture fragments. Only when the infected bone and the whole segment inflammatory tissue were completely removed, it will be possible to cure. Thorough debridement is one of the effective measures for treating bone-infection, but it can lead to bone defect which bring new problems to the treatment.At present, bone graft is a common method for treating bone defect, related research involve in autogenous bone, allograft bone, artificial bone, tissue engineering, transgenic engineering, guided bone regeneration technique, these methods have their respective indications and advantages and disadvantages. Some are still in the animal or in vitro experimental stage, and has not been widely used in clinical. Vascularized autogenous bone graft to repair bone defects known as the "gold standard", has been widely used in clinical practice, what be known for repairing large segmental bone defect. But the source of autogenous bone is limited and the bone infection which complicated with skin and soft tissue and vascular injury, or the vascular degeneration which caused by chronic inflammation, is also increases the risk of vascular crisis during the course of vascularized autogenous bone graft, and the risk of operation failure increased along with; or can not transplant vascularized bone due to no vessels for anastomosis. Allogeneic bone is an ideal substitute of bone graft, and can be apply for the treatment of smaller bone defect and nonunion. But it have the existence potential risks of rejection and cross infection, Bone transport technology has been widely used in clinic, but it should not be applied for large segmental bone defects, especially near the knee flat or poor blood supply to the lower part of the tibia bone defects. As a matter of fact, for the large segment of infected bone or necrosis bone after severe trauma, we always abandon most of them in clinic, and can not take advantage of them. There are still lack of ideal methods to deal with bulk (segment) of free infected bone, and the related orthopedics experimental and clinical studies are few.Research objective:In order to explore the feasibility of making the large segmental infected bone into activated bone with blood supply then be reused of, the large infectious bone, from animal model of rabbit tibia infection, was sterilized in vitro and embedded in the site with rich muscle and beside renowned vascular to make them revascularized at the same time. After the infection were controlled, it is time to replant autogenous bone to repair bone defect, so as to achieve free bone infection re-use purposes.Part one:Preparate the model of rabbit tibia infectionMethods:20 healthy Chinese white rabbits were randomly divided into experimental group and control group with 10 rats in each group, fixed the rabbits onthe animal test bench with supine position after anesthesia, then disinfects surgical field and drape aseptic towel conventionally. Drill a hole at 2.5cm under the left tibial tuberosity (1.5mm diameter)until to the medullary cavity, then packing gauzes into the medullary cavity. Using a micropipettor extract equal amount of prepared suspension of Staphylococcus aureus and sterile physiological saline to injected into the gauze respectively, then closed the hole with bone wax.Evaluating the preparation effect of bone infection model of rabbit tibia though different time nodes, anatomical observation, histological section observation, microbiological detection of the two groups.Results:Anatomic observation:Purulent secretion overflowed from the left leg incision of experimental rabbit and skin incision of control group rabbits gradually healed, with no redness and leakage. Histopathological observation:As a continuation of the time, experimental bone tissue slice by a large number of neutrophil infiltration gradually transformed into a large number of lymphocyte infiltration and the control bone tissue slice gradually became normal bone tissue. Microbial detection:The-results of microbial cultures of the purulent secretion, the contents of the medullary cavity and bone tissue which were took out of the experimental group at 2 weeks,4 weeks postoperatively were suggesting the growth of staphylococcus aureus, with the control group no bacterial growth.Conclusion:The effectively animal models of tibia bone infection can be prepared by injection injection 0.3ml medium concentration (3×108cfu/ml) suspension of Staphylococcus aureus into rabbit tibia bone marrow cavity.Part two:The experimental study of large free infectious bone revascularization by ectopic transplantationMethods:180 healthy Chinese white rabbits were divided into the boiled bone group, antibiotic soaked bone group, normal autogenous bone group with 60 rats in each group. Cut out 2.0cm infected tibial, then boiled and antibiotic solution soaked respectively, transplant the sterilized tibia into the contralateral thigh rectus femoris and vastus medialis muscle gap where near by saphenous artery, and used 1.0 Kirschner wire fixed it on the femoral. The tibial segment only soaked with sterilized physiological saline in normal autologous bone group, the remaining steps ibid.10 rabbits were killed respectively after surgery 0,4,6,8,10,12 weeks in the three groups, though CD34 and VEGF immunohistochemical staining method, detection of ectopic vascularized bone microvascular density and the expression of VEGF protein relative gray values,what indirectly reflect the degree of ectopic bone revasculariza-tion.Results:Postoperative infection situation analysis of three groups:the boiling bone group and autologous normal bone group had no statistical difference in terms of postoperative infection(P>0.05), and they compared with the antibiotics soak bone group, with statistical difference(P<0.05); postoperative vascularization analysis of three groups:CD34 positive blood vessel number and grey value of VEGF protein expression of the antibiotics soak bone group and autologous normal bone group peaked at 8 weeks postoperatively, with no statistical difference(P>0.05), and the ones of boiling bone peaked at 10 weeks postoperatively, but which compared with the ones of autologous normal bone at 8 weeks postoperatively, with no statistical difference(P>0.05).Conclusion:Infection Bone by boiled, soaked by antibiotic could achieve sterilization effect. Both the infection bone soaked by antibiotic and the autologous bone completed the vascularization in 8 weeks after surgery. The boiled bone also completed the vascularization in 10 weeks after surgery.Part three:The experimental study of large free infectious bone ectopic revascularization and bone activityMethods:On the basis of the second experiment part, the specimens at different times nodes of the boiled bone and normal autologous bone. By BMP-2 and type I collagen immunohistochemical staining, the gray value of BMP-2 and type I collagen in ectopic vasculariealth bone was detect. Choose 40 healthy Chinese white rabbits, that were randomly divided into the boiled bone group and normal autologous bone group with 20 rats in each group. On 14,16 week, the gray value of BMP-2 and type I collagen in bone was detect, which could indirectly reflect the relationship between bone activity and vascularization of ectopic vascularized bone.Results:The grey value of BMP-2 and type I collagen protein expression from autologous normal bone group peaked at 10 weeks postoperatively, which compared with the ones of boiling bone at 16 weeks postoperatively. There was no statistical difference(P> 0.05).Conclusion:Autologous normal bone was changed into activation bone at 10 weeks after surgery, and the boiled bone was done at 16 weeks.Part four:The clinical study of large free infectious bone ectopic revascularization by second stage replantationMethods:In clinic, we select the appropriate cases and get the large segment of infected bone out, then sterilized in vitro. The sterilized infected bone was transplanted into rectus femoris muscle and vastus lateralis muscle gap near by the lateral femoral circumflex artery on the anterior thigh until the revascularization is completed, the pedicled bone flap which carrying the lateral circumflex femoral artery and vein as blood vessel of ectopic vascularized bone to in situ was free translated to repair large segmental bone defect. We observe the vascularization and bone healing condition after the ectopic bone planting back by angiography, X-ray, nuclide bone scan.Results:After operation of soaked antibiotics bone ectopic revascularization at 4 months and boiled bone at 11 months, DSA shows that there is a lot of blood vessels around the back bone. Postoperation of retransplantation at 6 months and 8 months respectively, X-ray showed that the fracture line were unclear or disappeared. Postoperation of retransplantation at 1 month and 2 months radionuclide bone scan and PET-CT showed that graft bone metabolism was active.Conclusion:After sterilized in vitro, Large Free Infectious Bone could completed the revascularization by ectopic transplantation and could be prefabricated the complex tissue flap with vascular pedicle. While the infected wounds was healed, the composited bone flap with two well-known vascular pedicle bone could be replantated situ to repair bone defects. This method reduced the risk of reinfection from bone graft second replanted to repair bone defect and achieved the purpose of re-use of abandoned infected bone segments. It provides a new surgical procedures for the treatment of large bone defects and infection.