The Mechanism Study About Passive Lengthening Nerve In Distraction Osteogenesis

Limb lengthening is one of the important trauma and orthopedic surgery techniques. Its biological mechanism is based on organism stress-tension principle. That is to say throwing traction to broken limb can make new bone growing in the detatched segmental space. This course is also called Distraction Osteogenesis (DO). Clinically, this technique has become one of the major procedures for limb lengthening, bone defects and infected nonunion etc. The biological responses and regeneration mechanisms underlying the distracted connection tissue surrounding the new bone during DO, especially the peripheral nerves expanding and lengthening, have yet to be documented. The incidence of related complication caused by the distracted nerves in patient still occurs and remains at relatively high rate. Thus increases necessary and instancy on researching distracted peripheral nerves. The peripheral nerve surrounding the distracted site must be damaged during DO, but spontaneously recovery during consolidation. There have been few studies on the mechanism of peripheral nerve recovery after DO.Objective:Study and discuss the mechanism of the nerves regeneration during DO. (1) Establish the tibial lengthening animal model attaining all experimental purposes successfully; (2) Observe the morphological changes tendency of new bone and related soft tissue at the horizontal sectional slides during consolidation period using ordinary X ray and celloidin embedding technique; (3) Observe the ultra structure changes of distracted peripheral nerve tissue, analysis the natural repairing process of peripheral nerve damaged by slow limb lengthening; (4) Observe and explore the expression pattern and mechanism of NGF and p75NGFR in extended tibia nerve after limb distraction osteogenesis, detect autocrine growth activity in tibia nerve following DO. Analysis the adaption course of surrounding lengthening tissues in DO, especially the changes in surrounding passive lengthening nerve, the functional mechanism of main growth factors.(5) Attempt the microwave application in the immunofluorescent staining.Methods:50 healthy Japan rabbits were performed by upper one-third tibia osteotomy of right hind limb and conducted by unilateral external distracter (0.5mm each, twice per day for 10 days after a 7-day lag phase post-surgery). Applying the ordinary X-ray knows growth condition of callus at osteotomy site during distraction and consolidation periods. The animals were killed at 0,7,14,28,56 days after completing distraction and divided into 5 groups randomly. The celloidin embedding continuous slices of distraction area were made. Observe the position relationship and measure main parameters about new bone and surrounding structures at typical layers; The distracted tibia nerve and contralateral control nerve were harvested. Observe the histological morphology and ultrastructure changes at each consolidation time by means of light microscope (HE staining) and transmission electron microscopy (fresh tissues). The extended tibial nerves of different groups performed immunohistochemical staining and double immunofluorescent staining to determine NGF,p75NGFR expression and co-expression. Detect the expression pattern of NGF and p75NGFR during different time. All data received were statistic analysis. Applying the microwave performs the antigen recovery and immunofluorescent staining detection.Results:1. Establish the animal models successively:the articular movements were gradually recovery following the consolidation period. It can be seen by X-ray that callus images gradually changed clearly, the amount of callus increased comparing with the previous callus images, the cortical bone combined, the medullary cavity appeared, clearly boundary between the cortical bone and medullary cavity. At last, the callus union strong, good alignment of prolonged limb, no deformity union; 2. The observe results of transverse planes on prolonged limb:the position relationship of main structures, measure and contrast of the new growth bone area and the distance between the new bone and surrounding nerves and vessels, analyses the adaptation processes of the organism tissue.3. The distracted nerves appeared the damage and recovery course caused by distraction. In early stage, nerve fibers arranged disordered under the light microscope, some of Ranvier's node area widened, myelin sheath swelled, nerve fiber expanded demyelinate. Under the electron microscope showed myelin laminar loose, vacuolar degeneration of myelin and the ring-shape body formation, swelling pressed axon.7 days after completely distraction, there was mild restoration of nerve fiber under the light microscope, newly formed myelin sheath under the electron microscope. After that, the nerve damage is gradually repaired. Until 8 weeks after lengthening, the nerves return to normal structure. The newly myelin sheath still can be seen.4. The expression of all cellular factors is different in different time of consolidation period after DO. The common rule is that levels of NGF,p75 expression were found to be significantly elevated at 7 and 14 days in Schwann cells located in the outer layer of axon, but were low detectable or almost undetectable at 0,28 and 56 days. In double immunofluorescent images, the co-expression of NGF and p75 were also detected at 7 and 14 days.5. Applying the microwave in immunofluorescent staining can greatly saves experimental time and achieves good staining results.Conclusions:1. Rabbit is an optimal option for limb lengthening animal model. X-ray is a traditional examination way detecting the new bone formation in different lengthening stages. The rabbit limb lengthening model established successfully. This model accorded with the experimental conditions entirely and acquired the precise data.2. The technique of celloidin embedding sectional slice is one of the major sectional anatomy research methods, which can clearly display the morphological position relations and change tendency of the relative structures in distracted zone. It can provide plenty of morphological material for clinic; 3. During slow limb lengthening, the subclinical peripheral nerve injury is reversible. The natural repair of the damaged nerves has begun as soon as the nerve was damaged; 4. p75 NGFR plays an important role in remyelination and adaptive nerve lengthening due to its abundant expression in Schwann cells of damaged nerves. NGF and p75NGFR are all autocrine growth factor present in distracted nerve during the early consolidation period after limb DO. NGF combines p75 differential, thus can adjust nerve cells activities and promote damage repair and reconstruction of nerves.5. Applying the microwave in immunofluorescent staining is worthwhile to be explored and promoted.