The Experimental Study Of Inferior Alveolar Nerve Regulating Mandibular Fracture Healing

At present, An increasing number of evidence indicate that nervous system (NS) play an important role in the regulation of bone metabolism. With bone being richly innervated by both sympathetic and sensory neurons,while these nerves serve sensory and vascular functions, they may also influence bone cell activities. Bone cells are equipped with receptors for these peptides and activation of these receptors alters bone cell activity. Selectively damaging amyelinate sensory neurons can make neuropeptide exhaustion, will influence bone metabolism. Inversely, bone metabolism also will affect innervation intensity. It is indicated that there is functional conjunction between nervous system and bone tissue, any one changes will affect the other one. Likewise, the intimate relationship also exists between the nervous system and bone tissue during fracture. Nerve growth factor (NGF) emerged at local fracture site post-fracture, the induction of NGF made the sensory nerve fiber grow into fracture callus, and then the healing process was regulated by peptides released from nerve fibers. Now it is confirmed that peptides released from nerves that influence bone remodeling and fracture repair include calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), substance P (SP), and neuropeptide Y (NPY) et al.Facial injury is most commonly accompanied with mandibular fracture, the incidence is 23%-57%。The inferior alveolar nerve (IAN), part of the mandibular division of the trigeminal nerve, runs within a bony canal in the mandible and is in close proximity to the root tips of the mandibular molar teeth. These features make it vulnerable to damage during mandibular fracture. While many patients who sustain such an injury will regain normal sensation, a significant proportion are left with some form of permanent sensory disturbance. This can include the development of severe dysaesthesia involving the lower teeth, parts of the oral mucosa and the lower lip and chin, making it difficult for patients to talk and masticate effectively. Meanwhile, the IAN injury may also impact fracture healing.The aim of this experiment is to study the regulating effect of IAN for mandiblar fracture healing through dissecting inferior alveolar nerve canal and establishing mandible defect model accompanied with or without IAN injury after IAN was liberated. Twenty four big ear rabbits were selected, After revealing the lingual surface of mandible ramus and mandibular body, the lingual cortical bone of inferior alveolar nerve canal from mandibular foramen to close to meso-symphysis was removed, till to reveal inferior alveolar nerve blood vessel bundle membrane, then liberated the right inferior alveolar nerve and made 2×5mm mandible defect 1 cm before antegonial notch, at last, the IAN was put back into the inferior alveolar nerve canal. Then the left IAN was cut off at mandibular foramen with the proximal part was ligated and the distal end was avulsed, likewise the same mandible defect was made like the right one. Close suture was made eventually after small amount of penicillin (20pan-u) was put in. Pin-prick test was undertook at 3d,5d,7d,14d,21d,28d after surgery. Then the rabbits were perfused and sacrificed respectively at 1,2,3 and 4 weeks. Step by step chromotropic acid 2R-bright green staining of IAN was made at day 28 and then fracture calluses samples were observed,stained and analyzed for further study.Pin-prick test and chromotropic acid 2R-bright green staining of liberated IAN confirmed that the anatomic structure and physical function is conserved well. Further study through naked eye observation and HE,chromotropic acid 2R-bright green staining indicated that mandibular fracture healing was delayed cause of mutilated injury of IAN, and the fibrous callus stage was at a standstill with obvious external callus for a long time. While well innervated mandibular fracture can accomplish the healing process successfully. Meanwhile, intramembranous ossification and endochondral ossification were observed within both bilateral fracture callus during fracture healing.It is well known the sensory neuropeptide substance P (SP) produced in sensory neurons and stored at terminal ends of sensory nerves not only conveys information about noci-influence by injury to the central nervous system (afferent function), but also is rapidly released from the axons into the injured tissue to be bound the specific cell receptors (efferent function), which induces a potent vasodilatation and neurogenic inflammation related to wound healing. In vitro studies have further proved that the SP induces neutrophil chemotaxis and mitogenesis of T cells, macrophages, smooth muscle cells, endothelial cells, and fibroblasts. This experiment discovered that in IAN conserved group the irregular morphous bone producing cell is more considerable than IAN mutilated group. In cell-intensive zone, the immunoreactivity of SP is also intensive. Author believe that this is the cytology fundament of the fracture healing differences of the two groups.Nascent capillaries are imperative during mandibular fracture healing, either in IAN conserved group, nor in the IAN mutilated group. Capillaries invade into cartilage zone is the prerequisite of cartilage being replaced by mineralized bone during the process of endochondral ossification. The most important biological activity of VEGF is to act in endothelial cell to promote its proliferation and angiogenesis. In both of the IAN conserved group and IAN mutilated group of our experiment, intramembranous ossification,endochondral ossification,systema Haversii formation and bony callus rebuilding et al. appeared one after another, and simultaneously being accompanied with bony matrix mineralization and blood vessel invasion events. It is thus evident that bone cell,cartilage cell and vascular endothelial cell(VEC) have intimate relationship with each other. VEGF may have important regulating effect during osseous-derived and cartilaginous-derived ossification. In the same phase, the expression of VEGF is obviously statistics different between the IAN conserved group and the IAN mutilated group. It is known that SP can enhance fracture healing through acting at osteocytes and osteoblasts. Histological observation have confirmed that the osteocytes and osteoblasts amounts in fracture callus of IAN conserved group is higher than IAN mutilated group, with more intensive VEGF expression meanwhile. On the other hand, there is VEGF receptor on osteoblasts, VEGF can produce chemotaxis to osteoblasts through acting with VEGF receptor. This positive feedback process made VEGF increasing to submit geometry multiple, thus made VEGF expression obviously different between the two groups.CollagenⅠis the main collagen of osseous tissue, so determining collagen synthesis is the main biological index of determining bone formation, stimulating collagen synthesis in fracture callus can also stimulating fracture healing. This experiment determined collagenⅠmRNA expression of the two groups to estimate the influence of IAN to fracture healing. The results is that collagenⅠmRNA expression is observed in both groups, and mainly expressed in osteoblasts and fibroblasts, while the intensive expression appears earlier in IAN conserved groups, and to incline to disappear at week 4; in mutilated group, collagenⅠmRNA expression appears even later, expressed midrange or weaker persistently till to week 4. Thus indicate that fracture healing is slow-moving in mutilated groups, and also coincide with the histological observation. CollagenⅠis the marker of bone formation and chondrogenesis, in this study we guess that conserving IAN can change the earlier expression of collagenⅠmRNA which was confirmed through observation, we found that the expression crest-time appears at week 2 in conserved groups, however, in mutilated groups there is no obvious peak amplitude, they all expressed gently tendency at 2,3,4 weeks.From above-mentioned investigation, we can draw such conclusions:1,The fracture model liberating and conserving IAN was successfully established;2,IAN have regulating effect for mandibular fracture healing; 3,IAN can enhance the expression of VEGF and collagenⅠmRNA during regulating the mandibular fracture healing;4,Mandibular fracture healing have both intramembranous ossification and endochondral ossification process.