摘要
【目的】通过大鼠拔牙后即刻种植体洞型的骨损伤模型,利用组织学和免疫组织化学的方法,结合统计学分析,观察即刻种植窝预备区域早期骨愈合过程中降钙素基因相关肽(calcitonin gene-related peptide, CGRP)的表达特点。探讨降钙素基因相关肽在牙槽骨愈合和改建时的表达规律,为把降钙素基因相关肽引入到牙槽外科和口腔种植基础及临床医学研究中进行初步探索。
【方法】16只Wistar成年雄性大鼠随机分到4个组中,每组4只,所有动物在同一天制备即刻种植窝预备牙槽骨损伤模型,1至4组依次分别在模型制备后4、7、14、21天取标本。即刻种植窝预备牙槽骨损伤模型制备:大鼠称重后以0.4%戊巴比妥钠(1ml/100g)通过腹腔注射麻醉大鼠,麻醉成功后用橡皮套将大鼠四肢及上颌下颌固定在实验手术台上,用普通镊子由助手协助显露上颌第一磨牙区域,手术区常规消毒、铺巾,在无菌的条件下用小尖头探针分离牙龈,用牙镊拔出左上颌第一磨牙然后用小螺纹钻预备深度和直径合适的种植体窝,术后用消毒小棉球压迫止血。种植窝制备后满4、7、14、21天后在模型制备区域取一小段牙槽骨组织块,所有标本经10%甲醛溶液固定24小时后取出,中性EDTA脱钙液脱钙,然后脱蜡、包埋,切片,免疫组织化学染色、HE染色(观察骨愈合区组织形态),最后光镜下免疫阳性细胞计数,采集图像,应用Image-Pro plus 6.0图像分析系统测量累积光密度值,统计学分析。
【结果】1.模型制备成功:16只动物在试验期间种植洞形制备顺利,术区周围组织未见明显肿胀感染现象。
2.HE观察结果显示,4天、7天时,骨损伤修复区纤维性组织较多;整个骨损伤修复区域细胞的增殖在4天,7天时最为活跃,细胞核染色较深,14天,21天时,细胞核小、染色相对较浅;四个时期在骨修复区边缘均可见到前成骨样细胞及破骨细胞,中心区域未曾发现。
3.骨组织免疫组织化学染色显示,CGRP免疫组织化学染色阳性的细胞,胞浆着色;CGRP在成熟的骨细胞中未见着色,在骨修复区边缘的类成骨细样细胞着色;在模型建立后第7天取标本组CGRP主要在骨修复改建中心区的成纤维细胞中着色,与其它不同时间段种植区骨愈合组织相比,此期CGRP在骨修复改建中心区域染色阳性细胞数最多,染色强度最强,4组经秩和检验CGRP阳性细胞数和IOD值均是在第七天时最高,具有统计学意义(P<0.01)。
【结论】1. CGRP在即刻种植术骨愈合区域存在高表达现象,该现象发生在成骨样细胞高增殖状态(骨细胞形成的关键时期)。CGRP在正常的牙槽骨及颌骨未见明显表达,从相反角度证明了,只有在骨修复改建期细胞功能活跃时CGRP才呈现阳性表达。推测CGRP可能是促进骨愈合和抑制骨吸收的一个重要因子。
2. CGRP主要在纤维性骨痂向骨性骨痂过度时呈高峰态表达,而CGRP的表达高峰期也是成纤维细胞被诱导表达其成骨作用的关键时期,提示CGRP有可能是诱导成纤维细胞表达其成骨作用的一个重要因子。
3.CGRP主要在前成骨细胞和被诱导表达成骨作用的成纤维细胞胞质中染色呈强阳性,考虑CGRP的表达可能是前成骨细胞形成和增殖活动活跃的一个标志,也可能是成纤维细胞被诱导开始表达成骨作用的一个标志。
[Objective] Established the rat alveolar bone injured models of the oral immediately implant bed preparation , which aim was to observe the characteristics of calcitonin gene related peptide expression in the alveolar bone healing process by HE and immunohistochemistry assay. We discussed the experession of calcitonin gene-related peptidein alveolar bone healings and remodeling , and investigated the peptide action mechanism on alveolar surgery and oral implant.
[Methods] 16 healthy adult male Wistar rats were randomly divided into 4 groups , there were 4 each group. Established the rat alveolar bone injured models of the oral Immediately implant bed preparation , and collected specimens group after 4、7、14、21days. The bone injured models of immediate implantation fossa preparation : Rats were weighed and anesthetized by 0.4% sodium pentobarbital intraperitoneal injection at a dose of 1 ml per gramme . Under sterile conditions, pulled out the first molar by dental tweezers , and then prepared implants nest in proper depth and diameter with a small impact drilling . Taken a short alveolar bone (4 mm×4 mm×4 mm) in the model preparation area as specimens respectively after 4、7、14、21days. all specimens were fixed 24 hours by 10% formaldehyde solution , And decalcified by neutral EDTA , dewaxed , embedded, sliced,SP stained, HE stained . Finally ,counted immunoreactive cells under the light microscope , captured images, measured cumulative optical density by Image-Pro plus 6.0 system, done statistical analysis .
[Results] 1. Established Models successfully: 16 rats were well during the procedure of implanting hole shape, and the surgical operation areas had no significant swelling and infection .
2. HE staining showed that more fibrous tissuse in bone repair areas at the 4th day and the 7th day; The repair bone cell were most activtive in bone repair areas at the 4th day and the 7th day, which nuclei were deeply stained; There had osteoblasts and osteoclasts apppeared at the edge of bone repair areas. But no in the central areas .
3. Osseous tissue immunohistochemistry staining show : masculine cells’endochylema were stained ; the ripe osseous cells weren’t stained , but the osteoblast were stained in the margin of osseous repair area; 7 days after established models , the fibroblast were stained in the center of osseous repair area , the positive cell amount was the most and stain intensity was the the most strong in this stage ,which compared to the bone healing tissue of implantation area in other different time . Rank sum test of 4 groups , the amount of CGRP positive cells and IOD came to the peak in the seventh day , and it was have the covariance learns meaning .
[Conclusions] 1.There was higher CGRP expression in bone healing area , meanwhile osteoblast-like cell higher proliferation (the critical period of bone cell formation). CGRP alveolar bone were significantly downregulated in the normal, proved the opposite point of view , which CGRP showed positive active only when the bone remodeling cells’function was active. It may be an important factor to promote bone formation and to inhibit bone resorption.
2.The peak expression of CGRP appeared at the phase ,which the fibrous callus change to the bony callus, and the phase was also important at the fibroblasts were induced to express bone formation .It suggested that CGRP may be an important factor for inducing fibroblasts to express the bone formation function .
3. CGRP showed strong positive experssion in the cytoplasm of former osteoblasts and fibroblasts of expressing bone formation function . we consideried the positive expression of CGRP not only a sign of the former osteoblasts formation and proliferation , but also inducing fibroblast expressing bone formation function .
引文
1.李文荟,郭莉,王晓洁.外伤前牙即刻种植的临床观察.实用临床医学, 2007,8(10):90-91.
2.张云飞,段银钟.正畸牙齿移动中降钙素基因相关肽的研究进展.国外医学口腔医学分册,1999,26(2) :9-92.
3.Frost HM. The biology of fracture healing-an overview for clinicians, partⅠ,Ⅱ. Clin Orthop, 1989, 9; (248) :283-309.
4.Frost HM.Skeletal structural adaptations to mechanical usage (SATMU):
1. Redefining Wolff's law: the bone modeling problem.Anat Rec. 1990 Apr;226(4):403-13. Review.
5.郭春,阎玉仙,张西正.力学拉伸强度对小鼠单核细胞RAW264.7诱导分化为破骨细胞的影响[J].中国组织工程研究与临床康复,2009,13(37):7211~7216.
6. Wang XY, Guo X, Qu SX, et al.Temporal and spatial CGRP innervation in recombinant human bone morphogenetic protein induced spinal fusion in rabbits.Spine (Phila Pa 1976). 2009 Oct 15;34(22):2363-8.
7. Ozono K,Clin Calcium.Hormones and osteoporosis update. Calcitonin, CGRP and bone. 2009 Jul;19(7):929-34.
8. Naot .D, Cornish J. The role of peptides and receptors of the calcitonin family in the regulation of bone metabolism. Bone. 2008 Nov;43(5):813-818.
9. Huebner AK, Keller J, Catala-Lehnen P, et al.The role of calcitonin and alpha-calcitonin gene-related peptide in bone formation. Arch Biochem Biophys. 2008 May 15;473(2):210-7. Epub 2008 Feb 16. Review.
10.王军海,夏仁云.神经肽与骨折愈合.中国骨伤, 2005,18( 3):183-184.
11.Valentijn K, Gutow AP, Troiano N, et al. Effects of calcitonin gene-related peptide on bone turnover in ovariectomized rats.Bone. 1997 Sep;21(3):269-74.
12.赵昱辉,段银钟,孙应明. CGRP1受体在大鼠牙齿及牙周组织中的表达[J].临床口腔医学杂志. 2004,20( 11):3-645.
13.廉凯,杜靖远.降钙素基因相关肽对鼠成骨细胞IGF-I及IGF-IR mRNA表达的影响[J].中国矫形外科,2001, 8(11):1084-1087.
14.Michitaka N, Rumiko A,Takeshi K, et al. A novel member of the calcitonin gene-related peptide family, calcitonin receptor-stimulating peptide, inhibits the formation and activity of osteoclasts[J]. European Journal of Pharmacology , 2007 (560): 234 -239.
15.Florent E. Regulation of bone remodeling by the central and peripheral nervous system[J].Arch Biochem Biophys,2008,473(2):231-236.
16.Michitaka N, Rumiko A, Takeshi K, et al. A novel member of the calcitonin gene-related peptide family, calcitonin receptor-stimulating peptide, inhibits the formation and activity of osteoclasts. European Journal of Pharmacology, 2007 (560): 234 -239.
17.李焰,谭颖徽,张纲.兔下颌骨骨折愈合中降钙素基因相关肽对一氧化氮合成酶调控的实验研究实用口腔医学杂志,2008 Jan 24(1):78-81.
18.周彬,蒋滔.牙种植体—骨组织界面的细胞学研究.国外医学.口腔医学分册,2002,02:102-102.
19.王国平,陈安玉.非埋植型和埋植型骨内牙种植体—骨组织界面形态学观察和定量组织学研究.华西口腔医学杂志1993,03:223-225.
20.李义召,孙琳,刘爱芬等.脑血管病患者CGRP的临床研究.脑与神经疾病杂志, 2000,1(8):5-8.
21. Boronat López A, Balaguer Martínez J, Lamas Pelayo J,et al. Resonance frequency analysis of dental implant stability during the healing period. Med Oral Patol Oral Cir Bucal. 2008 Apr 1; 13(4): E244-7.
22. Kieswetter K, Schwartz Z, Dean D,et al.The role of implant surface characteristics in the healing of bone. Crit Rev Oral Biol Med.1996; 7(4):329-45.
23. Tomasi C, Sanz M, Cecchinato D,et al. Bone dimensional variations at implants placed in fresh extraction sockets: a multilevel multivariate analysis. Clin Oral Implants Res. 2010 Jan; 21(1):30-6.
24. O'Mahony A, Spencer P. Osseointegrated implant failures. J Ir Dent Assoc. 1999; 45(2):44-51.
25. Brunski JB. Biomechanical factors affecting the bone-dental implant interface. Clin Mater. 1992; 10(3): 153-201.
26.种植体的表面改性与促进成骨.国际口腔医学杂志.2008,35(增): 225-257
27.Pietrokovski J,Massler M.Ridge Remodeling after Tooth Extraction in Rats. J Dent Res. 1967, 46: 222-231.
28.吴婉芳,徐敏生(等).建立新生儿缺氧缺血性脑病动物模型[J].新生儿科杂志,1992,7(6):265.
29.王晓云,郭霞,钱忠明.骨组织中降钙素基因相关肽阳性神经的分布及生理作用.中华骨科杂志.2005,25(3),185-188.
30.H. DrissiM. Lieberherr,M. Hott,M. P. J. Marie and F. Lasmoles. Calcitonin Gene-Related Peptide (CGRP) increases intracellular free Ca2 + concentrations but not cyclic AMP formation in CGRP receptor2+ positive osteosarcoma cells(OHS 4) [J].Cytokine,1999,11 (3): 200(8237)
31卢政好,詹瑞森,王卫国,等.正常SANFH大鼠骨组织中CGRP的分布特点及意义[J].医学临床研究. 2007, 24(5): 750 - 753.
32. Imai S, Matsusue Y. Neuronal regulation of bonemetabolism and anab2olism: calcitonin gene2 related peptide2, substance P2, and tyrosine hydroxylase2 containing nerves and the bone. Microsc2 Res2 Tech. 2002, 58 (2): 61-69.
33.韩庆林,苟三怀,王琪.降钙素基因相关肽(CGRP)调控成骨细胞功能研究进展.中国矫形外科杂志.2005, 7 (13),544-549.
34. Wimalawansa SJ.CGRP and its receptors:molecular genetics,physiology,pathophysio1ogy,and the rapeutic potentials.Endocr Rev,1996,17(5):533.
35. Barros I, Muramoto T, Soma K. Effects of occlusal loading on alveolar bone remodeling and changes in the distribution of neuropeptides after tooth replantation in rats. J Med Dent Sci. 2007, 54(1):49-56.
36.Inaba K, Matsunaga S, Ishidou Y, Imamura T, Yoshida H.Effect of transforming growth factor-beta on fibroblasts in ossification of the posterior longitudinal ligament. In Vivo. 1996 Jul-Aug;10(4):445-9.
37.裘世静,柴本甫.不同接骨板固定后骨折修复的超微结构研究.中华外科杂志,1990, 28(2):88
38.柴本甫,汤雪明.实验性骨折愈合的超微结构研究-成纤维细胞成骨作用的电子显微镜观察.中华实验外科杂志,1985, (4) :57
39.Tang xM, Chai BF. Ultrastructural investition of experimental fracture healing-Ⅳ:electron microscopic observation on transformation and fate of fibroblast and chondrocytes. Clin Med J,1981,94(5):291
40.柴本甫,汤雪明,李慧.骨折二期愈合过程中的成纤维细胞成骨作用.中华骨科杂志,1996,16(4) :5
41.徐荣辉,饶寒敏,朱雅萍,等.皮肤成纤维细胞在体外培养中的成骨作用.中华外科杂志,1994,32(3) :190
42.Dodda rA, Merry K, Littlewood A, et al. Expression of mRNA or IL-1 beta, IL-6and IGF beta in developing human bone and cartilage. J Histochem Cytochem, 1994,42:73
43.邓廉夫,柴本甫,齐进。损伤性和骨关节炎性滑膜细胞成骨作用的体外研究.中华骨科杂志,1997,17(11) :693
44. Laflamme C, Curt S, Rouabhia M. Epidermal growth factor and bone morphogenetic proteins upregulate osteoblast proliferation and osteoblastic markers and inhibit bone nodule formation. Arch Oral Biol. 2010; 55 (9): 689-701.
45.Mrak E, Guidobono F, Moro G, et al.Calcitonin gene-related peptide (CGRP) inhibits apoptosis in human osteoblasts byβ-catenin stabilization.Cell Physiol. 2010 Nov;225(3):701-708.
46. Elefteriou F.Neuronal signaling and the regulation of bone remodeling. Cell Mol Life Sci. 2005 Oct;62(19-20):2339-49.
47. Irie K, Hara-Irie F, Ozawa H, Yajima T.Calcitonin gene-related peptide (CGRP)-containing nerve fibers in bone tissue and their involvement in bone remodeling. Microsc Res Tech. 2002 Jul 15;58(2):85-90.
1.Ramiro ET,Catherine W,Gustavo WL,et al.Molecular cloning and expression of equine calcitonin, calcitonin gene-related peptide-I,and calcitonin gene-related peptide-II [J].Olecular and Cellular Endocrinology ,2003,199(1-2):119 -128.
2.Toshio S,Nobuo S,Nobuyuki F,et al.Calcitoninin a protochordate,ciona intestinalis-the prototype of the vertebrate calcitonin/calcitonin gene-related peptide,superfamily[J].FEBS,2009,276(16):4437-4447.
3.Susan DB,Helen MC. Neuropeptides and their receptors: innovative science providing novel the rapeutic targets[J].British Journal of Pharmacology,2006,147:202-211.
4.Florent Elefteriou. Regulation of bone remodeling by the central and peripheral nervous system[J].Arch Biochem Biophys,2008,473(2):231-236.
5.赵昱辉,段银钟,孙应明. CGRP1受体在大鼠牙齿及牙周组织中的表达[J].临床口腔医学杂志. 2004,20(11):643-645.
6.Tomoyuki K,Kazuhiro O,Douglas M.Immature human osteoblastic MG63 cells predominantly express a subtype 1-like CGRP receptor that inactivates extracellular signal response kinase by a cAMP-dependent mechanism[J].European Journal of Pharmacology,2003,470(3): 125-130.
7.Tomoyuki K, Kazuhiro O, Douglas MB.Effects of calcitonin gene-related peptide and amylin on human osteoblast -like cells proliferation Isabella Villa a, Raffaella Melzi[J].European Journal of Pharmacology , 2000, 409:273 -278.
8. Vause CV, Durham PL .CGRP stimulation of iNOS and NO release from trigeminal ganglion glial cells involves mitogen-activated protein kinase pathways[J].Neurochem,2009,110(3):811–821.
9.Yan L, Yinghui T,Gang Z, et al. Effects of Calcitonin gene-related peptide on the expression and activity of nitric oxide synthase during mandibular bone healing in rabbits: an experimental study[J].American Association of Oral and Maxillofacial Surgeons,2009,67(2):273-279.
10.廉凯,杜靖远.降钙素基因相关肽对鼠成骨细胞IGF-I及IGF-IR mRNA表达的影响[J].中国矫形外科,2001, 8 (11):1084-1087
11.Michitaka N, Rumiko A,Takeshi K, et al. A novel member of the calcitonin gene-related peptide family, calcitonin receptor-stimulating peptide, inhibits the formation and activity of osteoclasts[J]. European Journal of Pharmacology , 2007 ,560(2-3): 234 -239.
12.廉凯,杜靖远,饶振玉,等.降钙素基因相关肽抑制白细胞介素一1介导骨的吸收作用[J].第四军医大学学报,2002,23(9):776-779.
13秦晗,杨富生,吴礼安,等.降钙素对小鼠牙齿萌出途中破骨细胞的影响[J].牙体牙髓牙周病学杂志,2007,17(12):683.
14.Kyoko I,Koji N, Hiroshi N,et al.Inhibitory effect of CGRP on osteoclast formation by mouse bone marrow cells treated with isoprtereno[J].Neuroscience Letters ,2005 ,379(1):47–51.
15. Lerner UH. Deletions of genes encoding calcitonin/α-CGRP, amylin and calcitonin receptor have given new and unexpected insights into the function of calcitonin receptors and calcitonin receptor-like receptors in bone[J].Musculoskelet Neuronal Interact,2006, 6(1):87-95.
16.卢政好,詹瑞森,王卫国,等.正常SANFH大鼠骨组织中CGRP的分布特点及意义[J].医学临床研究, 2007,24(5):750-753.
17.徐格,吴雪晖,许建中,等.CGRP对MSCs细胞问缝隙连接影响的研究[J] .华南国防医学杂志,2008,22(2):18-20.
18.杨琴,陈红雷,陈德基,等.降钙素基因相关肽诱导脂肪干细胞向成骨细胞的分化[J].中国组织工程研究与临床康复.2008,12(43):8539-8542.
19.王新,宋跃明,裴福兴.神经肽对大鼠成骨细胞胰岛素样生因子-1及其受体基因表达的影响[J].中华实验外科杂志,2006,12(23) :1515 -1518.
20. Adeghate E, Ponery AS.Diabetes mellitus influences the degree of colocalization of calcitonin gen-related peptide with insulin and somatostatin in the rat pancreas[J].Pancreas,2004,29(4):311-318.
21.周磊.牙种植学新进展[J].华西口腔医学杂志,2009,27(1):10.
22.Russell C, Susmita B,Amit B. Bone cell–materials interaction on Simicrochannels with bioiner coatings[J].Acta Biomaterialia, 2007,3(4): 523.
23.Guehennec.LL, Soueidan A, Layrolle P,et al. Surface treatments of titanium dental implants for rapid osseointegration[J].Dental Materials , 2007,23(7): 844–854.
24.Paulo SV, Marcia SS,Ivan NB,et al.Improvement of in vitro titanium bioactivity by three different surface treatments[J].Dental Materials, 2006,22(3): 275–282.
25.Davies JE.Bone bonding at natural and biomaterial surfaces. [J].Biomaterials,2007,28 (34) :5058-5067.
26.周磊.牙种植学新进展.华西口腔医学杂志[J] .2009,27(1):8-12
27.Mombelli A,Schimid B,Rutar A,et al.Local antibiotic therapy guided by microbiological diagnosis[J].Clin Perodontol,2002,29(8):734-749.