下牙槽神经调节下颌骨骨折愈合的实验研究
摘要
随着人们对骨折愈合机制研究的不断深入,神经对骨折愈合过程的调节作用逐渐成为创伤修复学研究的热点。下牙槽神经走行于下颌骨体内,在下颌骨骨折的过程中常伴有下牙槽神经损伤,不仅会导致支配区感觉异常,也可能影响骨折愈合。本实验通过设计一种下颌骨骨折伴或不伴下牙槽神经损伤的模型,通过HE-染色、变色酸2R-亮绿染色、P物质及VEGF免疫组织化学染色及Ⅰ型胶原原位杂交等观察发现,伴神经损伤的骨折愈合延迟,长时间停留在纤维骨痂阶段,外骨痂明显。同保留下牙槽神经的骨折相比,在相同时相,VEGF免疫组织化学染色及Ⅰ型胶原原位杂交结果不同,差异有统计学意义。本实验成功的建立了下牙槽神经游离及保护下牙槽神经骨折模型,并进一步证明下颌骨在愈合过程中既有膜性成骨又有软骨内成骨;下牙槽神经对下颌骨骨折愈合有调节作用;在对骨折骨痂的调节过程中能增加VEGF及Ⅰ型胶原mRNA的表达。
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.
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.
引文
[1] Jose M. Marchena DMD, Bonnie L. Padwa DMD, MD and Leonard B. Kaban DMD, MD, et al. Sensory abnormalities associated with mandibular fractures: Incidence and natural history. Journal of Oral and Maxillofacial Surgery 1998;56(7):822-825
[2] Stefan Schultze-Mosgau, Martin Erbe, Dirk Rudolph, et al. Prospective study on post-traumatic and postoperative sensory disturbances of the inferior alveolar nerve and infraorbital nerve in mandibular and midfacial fractures. Journal of Cranio-Maxillofacial Surgery 1999; 27 (2):86-93
[3] Leslie R. Halpern DDS, MD, PhD, MPH, Leonard B. Kaban DMD, MD and Thomas B. Dodson DMD, MPH. Perioperative neurosensory changes associated with treatment of mandibular fractures. Journal of Oral and Maxillofacial Surgery 2004;62(5):576-581
[4] Lai Xinan, Li Suguang, Zhang Liangchao, Change in substance P in a firearm wound and its significance. Peptides 1998;19(7):1209-1212
[5] Hill and R. Elde, Distribution of CGRP-, VIP-, D beta H-, SP-, and NPY-immunoreactive nerves in the periosteum of the rat. Cell. Tissue Res. 1991;264:469–480.
[6] D.B. Mach, S.D. Rogers, M.C. Sabino, et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience 2002;113:155-166.
[7] Lundberg and U.H. Lerner, Expression and regulatory role of receptors for vasoactive intestinal peptide in bone cells. Microsc. Res. Tech. 2002; 58:98-103.
[8] S.C. Offley, T.Z. Guo, T. Wei, et al. Capsaicin-sensitive sensory neurons contribute to the maintenance of trabecular bone integrity. J. Bone Miner. Res. 2005;20:257-267.
[9] B. Burt-Pichat, M.H. Lafage-Proust, F. Duboeuf, et al. Dramatic decrease of innervation density in bone after ovariectomy. Endocrinology 2005;146:503-510.
[10] Barde Y, Atrophic factors and neuronal suvival. Neuron 1989;2:1525
[11] Grills BL, Schuijers JA, Immunohistochemical localization of nerve growth Factor infractured and unfractured rat bone. Acta Orthop Scand 1998; 69: 415
[12] Frenkel SR, Guerral A, Mitchello G, et al. Nerve growth factor in skeletal Tissues of tembryonic chick. Cell Tissue Res 1990;260:507
[13] J. Li, T. Ahmad, M. Spetea, et al. Bone reinnervation after fracture: a study in the rat. J Bone Miner Res 2001;16:1505–1510.
[14] A. Bjurholm, Neuroendocrine peptides in bone. Int Orthop 1991; 15: 325 –329.
[15] T. Kawase, G.A. Howard, B.A. Roos, et al. Calcitonin gene-related peptide rapidly inhibits calcium uptake in osteoblastic cell lines via activation of adenosine triphosphate-sensitive potassium channels. Endocrinology 1996; 137:984–990.
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[17] Ballica R, Valentijn K, Khachatryan A, et al. Targeted expression of alcitonin gene-related peptide osteoblasts increase bone density in mice. J Bone Miner Res 1999;14:1067
[18] Kundu B, Khare SK, Singh G, Role of polypeptides in the treatment and Diagnosis of steoporosis. Peptides 1999;20:523
[19] Vignery A, McCarthy TL, The neuropeptide calcitonin gene-related peptide stimulates insulin growth factor I production by primary fetal rat osteoblast. Bone 1996;8:331
[20] Shihc.Bernard CW, Neurogenic substance P stimulates osteogenesis in vitro. Peptides 1997;18:323
[21] Goto T, Yamaza T, Kidoma, et al. Light and electron microscopic study-The distribution of axons containing substance P and the localization of neurokinin-1 receptor in bone. Cell Tissue Res 1998; 293
[22] Hphmann El, Levine L, Tashjian AH, Vasoactive intestinal peptides stimulates bone resorption via acyclicadenosine 3’,5’ monophosphate- dependant mechanism. Endocrinology 1983;12:1233
[23] Lindblad BE, Nielsen LB, Jespersen SM, et al. Vasoconstrictive action of neuropeptide Y in bone. The porcine tibia perfused in vivo, Acta Orthop Scand 1994;65:629
[24] G. Sisask, A. Bjurholm, M. Ahmed and A. Kreicbergs, The development of autonomic innervation in bone and joints of the rat. J Autonom Nerv Syst 1996;59:27–33.
[25] 赵雅度主编.神经系统外伤.北京:人民军医出版社,2001 第 1 版,252
[26] C. Elcock, F. M. Boissonade and P. P. Robinson, Changes in neuro- peptide expression in the trigeminal ganglion following inferior alveolar nerve section in the ferret. Neuroscience 2001; 102(3): 655-667
[27] Kenji Iijima, Fumiko Harada, Kooji Hanada, et al. Temporal expression of immunoreactivity for heat shock protein 25 (Hsp25) in the rat periodontal ligament following transection of the inferior alveolar nerve. Brain Research 2003; 979 (1-2):146-152
[28] R.H. Haug, J. Prather and A.T. Indresano, An epidemiologic survey of facial fractures and concomitant injuries. Journal of Oral and Maxillofacial Surgery 1990;48:926–932.
[29] G.M. Kushner and B. Alpert, Open reduction and internal fixation to acute mandibular fractures in adults. Facial Plastic Surgery 1998; 14: 11–21.
[30] José R. Fernández, M. Gallas, M. Burguera, et al. A three- dimensional numerical simulation of mandible fracture reduction with screwed miniplates. Journal of Biomechanics 2003; 36 (3): 329 -337
[31]Tsutomu Nomura, Evan Gold, Michael P. Powers, et al.Micromechanics/structure relationships in the human mandible. Dental Materials 2003;19(3):167-173
[32] MD, DDS Tateyuki Iizuka and Christian Lindqvist, Rigid internal fixation of mandibular fractures: An analysis of 270 fractures treated using the AO/ASIF method. International Journal of Oral and Maxillofacial Surgery 1992;21(2):65-69
[33] 张益、孙勇刚主编.颌骨坚固内固定. 北京:北京大学医学出社. 2003 第 1 版,29-34
[34] P. Holzer, Local effector of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988; 24: 739-768.
[35] Gracey N. Onuoha. Circulating sensory peptide levels within 24 h of human bone. Peptides 2001;22(7):1107-1110.
[36] S. Toyosawa, N. Kanatani, S. Shintani, et al. Expression of dentin matrix protein 1 (DMP1) during fracture healing. Bone 2004; 35 (2):553-561
[37] P. Holzer, Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol Rev. 1991;43:143-201.
[38] Marianowski Rémi, Martins Carvalho Christine, Potard Gael, et al. Mandibular fractures in children long term results. International Journal of Pediatric Otorhinolaryngology 2003;67(1):25-30
[39] A. Hulth, Current concepts of fracture healing. Clin Orthop Rel Res 1989; 249: 265-284.
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[2] Stefan Schultze-Mosgau, Martin Erbe, Dirk Rudolph, et al. Prospective study on post-traumatic and postoperative sensory disturbances of the inferior alveolar nerve and infraorbital nerve in mandibular and midfacial fractures. Journal of Cranio-Maxillofacial Surgery 1999; 27 (2):86-93
[3] Leslie R. Halpern DDS, MD, PhD, MPH, Leonard B. Kaban DMD, MD and Thomas B. Dodson DMD, MPH. Perioperative neurosensory changes associated with treatment of mandibular fractures. Journal of Oral and Maxillofacial Surgery 2004;62(5):576-581
[4] Lai Xinan, Li Suguang, Zhang Liangchao, Change in substance P in a firearm wound and its significance. Peptides 1998;19(7):1209-1212
[5] Hill and R. Elde, Distribution of CGRP-, VIP-, D beta H-, SP-, and NPY-immunoreactive nerves in the periosteum of the rat. Cell. Tissue Res. 1991;264:469–480.
[6] D.B. Mach, S.D. Rogers, M.C. Sabino, et al. Origins of skeletal pain: sensory and sympathetic innervation of the mouse femur. Neuroscience 2002;113:155-166.
[7] Lundberg and U.H. Lerner, Expression and regulatory role of receptors for vasoactive intestinal peptide in bone cells. Microsc. Res. Tech. 2002; 58:98-103.
[8] S.C. Offley, T.Z. Guo, T. Wei, et al. Capsaicin-sensitive sensory neurons contribute to the maintenance of trabecular bone integrity. J. Bone Miner. Res. 2005;20:257-267.
[9] B. Burt-Pichat, M.H. Lafage-Proust, F. Duboeuf, et al. Dramatic decrease of innervation density in bone after ovariectomy. Endocrinology 2005;146:503-510.
[10] Barde Y, Atrophic factors and neuronal suvival. Neuron 1989;2:1525
[11] Grills BL, Schuijers JA, Immunohistochemical localization of nerve growth Factor infractured and unfractured rat bone. Acta Orthop Scand 1998; 69: 415
[12] Frenkel SR, Guerral A, Mitchello G, et al. Nerve growth factor in skeletal Tissues of tembryonic chick. Cell Tissue Res 1990;260:507
[13] J. Li, T. Ahmad, M. Spetea, et al. Bone reinnervation after fracture: a study in the rat. J Bone Miner Res 2001;16:1505–1510.
[14] A. Bjurholm, Neuroendocrine peptides in bone. Int Orthop 1991; 15: 325 –329.
[15] T. Kawase, G.A. Howard, B.A. Roos, et al. Calcitonin gene-related peptide rapidly inhibits calcium uptake in osteoblastic cell lines via activation of adenosine triphosphate-sensitive potassium channels. Endocrinology 1996; 137:984–990.
[16] K. Edoff, J. Hellman, J. Persliden et al. The developmental skeletal growth in the rat foot is reduced after denervation. Anat Embryol 1997;195:531–538.
[17] Ballica R, Valentijn K, Khachatryan A, et al. Targeted expression of alcitonin gene-related peptide osteoblasts increase bone density in mice. J Bone Miner Res 1999;14:1067
[18] Kundu B, Khare SK, Singh G, Role of polypeptides in the treatment and Diagnosis of steoporosis. Peptides 1999;20:523
[19] Vignery A, McCarthy TL, The neuropeptide calcitonin gene-related peptide stimulates insulin growth factor I production by primary fetal rat osteoblast. Bone 1996;8:331
[20] Shihc.Bernard CW, Neurogenic substance P stimulates osteogenesis in vitro. Peptides 1997;18:323
[21] Goto T, Yamaza T, Kidoma, et al. Light and electron microscopic study-The distribution of axons containing substance P and the localization of neurokinin-1 receptor in bone. Cell Tissue Res 1998; 293
[22] Hphmann El, Levine L, Tashjian AH, Vasoactive intestinal peptides stimulates bone resorption via acyclicadenosine 3’,5’ monophosphate- dependant mechanism. Endocrinology 1983;12:1233
[23] Lindblad BE, Nielsen LB, Jespersen SM, et al. Vasoconstrictive action of neuropeptide Y in bone. The porcine tibia perfused in vivo, Acta Orthop Scand 1994;65:629
[24] G. Sisask, A. Bjurholm, M. Ahmed and A. Kreicbergs, The development of autonomic innervation in bone and joints of the rat. J Autonom Nerv Syst 1996;59:27–33.
[25] 赵雅度主编.神经系统外伤.北京:人民军医出版社,2001 第 1 版,252
[26] C. Elcock, F. M. Boissonade and P. P. Robinson, Changes in neuro- peptide expression in the trigeminal ganglion following inferior alveolar nerve section in the ferret. Neuroscience 2001; 102(3): 655-667
[27] Kenji Iijima, Fumiko Harada, Kooji Hanada, et al. Temporal expression of immunoreactivity for heat shock protein 25 (Hsp25) in the rat periodontal ligament following transection of the inferior alveolar nerve. Brain Research 2003; 979 (1-2):146-152
[28] R.H. Haug, J. Prather and A.T. Indresano, An epidemiologic survey of facial fractures and concomitant injuries. Journal of Oral and Maxillofacial Surgery 1990;48:926–932.
[29] G.M. Kushner and B. Alpert, Open reduction and internal fixation to acute mandibular fractures in adults. Facial Plastic Surgery 1998; 14: 11–21.
[30] José R. Fernández, M. Gallas, M. Burguera, et al. A three- dimensional numerical simulation of mandible fracture reduction with screwed miniplates. Journal of Biomechanics 2003; 36 (3): 329 -337
[31]Tsutomu Nomura, Evan Gold, Michael P. Powers, et al.Micromechanics/structure relationships in the human mandible. Dental Materials 2003;19(3):167-173
[32] MD, DDS Tateyuki Iizuka and Christian Lindqvist, Rigid internal fixation of mandibular fractures: An analysis of 270 fractures treated using the AO/ASIF method. International Journal of Oral and Maxillofacial Surgery 1992;21(2):65-69
[33] 张益、孙勇刚主编.颌骨坚固内固定. 北京:北京大学医学出社. 2003 第 1 版,29-34
[34] P. Holzer, Local effector of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 1988; 24: 739-768.
[35] Gracey N. Onuoha. Circulating sensory peptide levels within 24 h of human bone. Peptides 2001;22(7):1107-1110.
[36] S. Toyosawa, N. Kanatani, S. Shintani, et al. Expression of dentin matrix protein 1 (DMP1) during fracture healing. Bone 2004; 35 (2):553-561
[37] P. Holzer, Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol Rev. 1991;43:143-201.
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