骨多肽生长素、珊瑚骨、地榆诱导小白鼠额面骨再生的实验研究
摘要
目的 评价骨多肽生长素(Bone Active Polypeptide，BAP)应用于小白鼠额面骨缺损区诱导骨再生修复的效果，探讨骨多肽生长素诱导成骨的机制。方法 健康昆明小白鼠72只，随机分3组，其中1组为实验组，2组为对照组，每组有24只小白鼠，在每只小白鼠额面骨建立骨缺损动物模型，用1％利多卡因额面浸润麻醉，切开皮肤、骨膜，显露额面骨，并制成3mm×2mm×1mm骨缺损区，止血完善。实验组植入骨多肽生长素复合珊瑚骨；对照A组植入单纯珊瑚骨；对照B组植入珊瑚骨复合地榆，分别用于修复额面骨缺损。严密缝合关闭手术创口，回笼饲养。术后7d、28d、90d分别处死24只小白鼠，每组8只，取材作切片。切取实验区额面骨4mm×3mm×1mm送检。在光镜下观察骨组织形态变化和新生骨结构，计量每视野新生成骨细胞数；在透射电镜下观察再生骨的超微结构变化。结果 组织病理学观察显示，术后7d，实验组与各对照组镜下表现差别不大，骨缺损区周围有破骨细胞、间充质细胞和大量炎性细胞浸润，骨变性坏死。术后28d，实验组可见炎性渗出明显吸收，成纤维细胞、毛细血管生长活跃，可见骨样组织和大量骨组织；对照A组可见炎性渗出吸收，骨缺损区周围有间充质细胞和成骨细胞；对照B组可见炎性渗出吸收，成纤维细胞、毛细血管生长活跃，骨缺损区周围有成骨细胞。术后90d，实验组可见新生骨组织范围扩大，钙化程度加强，几乎接近正常骨质结构；对照A组、B组皆可见骨缺损边缘有较多新骨沉积。于光镜下观察，计量3组新生成骨细胞数，运用单因素方差分析及两两比较，P＜0.01，实验组与各对照组有差异。再通过透射电镜观察得到进一步证明，术后90d，实验组骨缺损边缘有较多新骨沉积；对照A组、B组皆可见新骨样结构与骨创边缘的交界处仍有骨损线，分布有毛细血管及成骨细胞。结论 骨多肽生长素能诱导小白鼠额面骨再生，应用骨多肽生长素复合载体材料珊瑚骨可明显促进骨缺损的修复。骨多肽生长素(BAP)的诱导骨再生机制是含有多种与骨代谢有关的生长因子，从而诱导成骨细胞新生。
Objective: To evaluate the effect on the frontal bone regeneration of the mice induced by Bone Active Polypeptide (BAP), to study the mechanism of bone regeneration induced by BAP. Methods: 72 Kun Ming mice were divided into 3 groups randomly, one was experimental group , the two were control groups, each was respectively 24. The mice were operated . Bone defects of 3 mm . 2 mm . 1mm in site were created on frontal bone with the use of local anesthesia, and then the defects had been filled with BAP composite Coralline in the experimental group; only Coralline in the control group A; Coralline composite Sanguisorba Officinalis L (SO) in the control group B. Postoperatively, the mice were killed at intervals of 7, 28 and 90 days, the specimens were examined histopathologically by light microscope and transmission electron microscope. Osteoblasts were counted and studied quantitatively by analysis of variance(ANOVA) to compare each group. Results: 7 days of postoperation, it was revealled that the bone defects
were infiltrated by the white blood cells, degeneration and necrosis in all groups; after 28 days, the inflammation was absorbed, fibroblasts and new fine capillaries were developing actively, new bone formation were also found in the experimental group; the inflammation was absorbed in the control group A, and in the control group B, fibroblasts and many new capillaries were found. After 90 days, ossification was obvious in the experimental group, but not in the control groups. Osteoblasts were counted, P < 0.01, statistical analysis showed difference between the experimental and control groups. It was also proved by TEM. Conclusions: The frontal bone regeneration in the mice can be induced by BAP, and BAP composite Coralline was more remarkable. The mechanism of bone regeneration induced by BAP is that BAP is a variety of growth factors.
Objective: To evaluate the effect on the frontal bone regeneration of the mice induced by Bone Active Polypeptide (BAP), to study the mechanism of bone regeneration induced by BAP. Methods: 72 Kun Ming mice were divided into 3 groups randomly, one was experimental group , the two were control groups, each was respectively 24. The mice were operated . Bone defects of 3 mm . 2 mm . 1mm in site were created on frontal bone with the use of local anesthesia, and then the defects had been filled with BAP composite Coralline in the experimental group; only Coralline in the control group A; Coralline composite Sanguisorba Officinalis L (SO) in the control group B. Postoperatively, the mice were killed at intervals of 7, 28 and 90 days, the specimens were examined histopathologically by light microscope and transmission electron microscope. Osteoblasts were counted and studied quantitatively by analysis of variance(ANOVA) to compare each group. Results: 7 days of postoperation, it was revealled that the bone defects
were infiltrated by the white blood cells, degeneration and necrosis in all groups; after 28 days, the inflammation was absorbed, fibroblasts and new fine capillaries were developing actively, new bone formation were also found in the experimental group; the inflammation was absorbed in the control group A, and in the control group B, fibroblasts and many new capillaries were found. After 90 days, ossification was obvious in the experimental group, but not in the control groups. Osteoblasts were counted, P < 0.01, statistical analysis showed difference between the experimental and control groups. It was also proved by TEM. Conclusions: The frontal bone regeneration in the mice can be induced by BAP, and BAP composite Coralline was more remarkable. The mechanism of bone regeneration induced by BAP is that BAP is a variety of growth factors.
引文
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[21] Arnaud-E,De-Pollak-C,Meunier-A, et al.Ostergenesis with coral is increased by BMP and BMC in a rat cranioplasty. Ann-Chir-Gynaecol-Suppl, 1999,20(20):1908-1918
[22] 程东亮．中药地榆的化学成分研究．甘肃药学，1995．05：10(1)：1-3
[23] 周印锁．中药地榆粉末显微鉴定研究．中国药学杂志，1995．01．08：30(1)：3-7
[24] 戴毅敏，陈新梅，毛天球．基因重组人骨形成蛋白-2与珊瑚／聚乳酸复合异位诱导成骨的实验研究．口腔医学纵横，2002，18(1)：14-15
[25] 云蔓，邓芳成．骨吸收机理与破骨细胞分子生物学研究的进展．中国热带医学，2002，2(1)：60-63
[26] Kong, YY, Yoshida, H., Sarosi, L., et al. OPGL is a key regulator of osteo clastogenesis, lymphocyte development and lymph-node organogenesis. Nature, 1999,397:315-323
[27] Felix, R., Hofstetter, W. & Cecchini, M.G Recent developments in the understanding of the pathophysiology of osteopetrosis. Eur. J. Endocrinol, 1996, 134: 143-156
[28] Teitelbaum S.I. The osteoclast and osteoporosis. Mt Sinai J. Med, 1996, 63: 399-402
[29] Roodman, G.D. Advances in bone biology: the osteoclast. Endocr, 1996,17:308-332
[30] Yasuda H, Shima N, Nakagawa N, et al. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegeria (OPG): A Mechanism by with OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology, 1998, 139(3):1329-1337
[2] 杨阳，曾融生，陈松龄，等．珊瑚骨人工骨复合骨形成蛋白修复狗下颌骨缺损．中华口腔医学杂志，1997，18(3)：195-197
[3] 尉文华，毛天球，王贺忠，等．rhbmp-2胶原复合珊瑚骨人工骨植入智齿牙槽窝的临床研究．现代口腔医学杂志，2002,16(2)：141-142
[4] Arnaud E, Morieux C, Wybier M, et al. Osteogenesis induced by the combination of grow factor, fibrin glue and coral,towards a substitute of autologous bone graft. An experimental study on the rabbit, Ann Chit Plast Esthet, 1994, 39(4):491-498
[5] Arnaud e, Molina f, Mendoza m, et al.Bone substitute with growth factor. Preliminary clinical cases for cranio and maxillo facial indication. Ann Chir Plast Esther, 1998,43(1):40-50
[6] 苗林，刘宝林．珊瑚修复骨缺损中转化生长因子β的观察．第二军医大学学报，1999，20(6)：391-393
[7] 邓芳成．骨多肽生长素与珊瑚骨诱导鼠额面骨再生的研究．口腔医学研究，2002，18(4)：265-267
[8] 梅陵宣．骨吸收机理的分子生物学进展．国外医学口腔医学分册，2001，(1)：40-42
[9] 王伟．BMP-2诱导骨形成研究进展．口腔材料器械杂志，1999，8(2)：87-89
[10] 张森林，毛天球，王会信，等．重组人骨形成蛋白-2珊瑚人工骨修复骨质缺损的实验研究．中华口腔医学杂志，1998，33(1)：13-14
[11] 王常勇．人rhBMP-2与异种骨复合移植的实验研究．中华口腔医学杂志，1997，32(6)：360-362
[12] 王伟．应用国产消旋聚乳酸载体复合rhBMP-2和hTGF-β1修复兔下颌骨缺损．中国口腔颌面外科杂志，2003，1(3)：166-171
[13] 张泽兵，高文涛，欧阳喈．重组大鼠骨形成蛋白-2成熟肽编码区cDNA克隆．口腔医学纵横，2000，16(1)7-9
[14] Noda M, Camilliere JJ. In vivo stimulation of bone formation by transforming growth factor-beta. Endocrinology, 1989,124:2991-2994
[15] 赵淑贤，王强，程政，等．转化生长因子β1诱导鼠牙槽骨缺损修复的实验研究．中华口腔医学杂志，2000，35(40)：292-293
[16] Pfeilschifter J, Oechsner M, Naumann A, et al. Stimulation of bone matrix apposition in vitro by local growth factors: a comparison between insulin-like growth factor Ⅰ, platelet-derived growth factor, and transforming growth factor beta. Endocrinology, 1990, 127: 69-75.
[17] Pepoff, SN & Schneider, GB. Animal models of osteopetrosis: the impact of recent molecular developments on novel strategies for therapeutic intervention. Mol. Med. Today, 1996,2:349-358
[18] Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regutates osteoclast differention and activation. Cell, 1998, 93: 165-176
[19] 曾融生，任材年，于秦羲．珊瑚人工骨作为颌面骨修复材料的初步报告．中华口腔医学杂志，1991，26：345-346
[20] Holmes RE. Bone Regeneration Within a Coralline Hydroxyapatite Implant. Plast Reconstr Surg, 1979,63(5): 626-633
[21] Arnaud-E,De-Pollak-C,Meunier-A, et al.Ostergenesis with coral is increased by BMP and BMC in a rat cranioplasty. Ann-Chir-Gynaecol-Suppl, 1999,20(20):1908-1918
[22] 程东亮．中药地榆的化学成分研究．甘肃药学，1995．05：10(1)：1-3
[23] 周印锁．中药地榆粉末显微鉴定研究．中国药学杂志，1995．01．08：30(1)：3-7
[24] 戴毅敏，陈新梅，毛天球．基因重组人骨形成蛋白-2与珊瑚／聚乳酸复合异位诱导成骨的实验研究．口腔医学纵横，2002，18(1)：14-15
[25] 云蔓，邓芳成．骨吸收机理与破骨细胞分子生物学研究的进展．中国热带医学，2002，2(1)：60-63
[26] Kong, YY, Yoshida, H., Sarosi, L., et al. OPGL is a key regulator of osteo clastogenesis, lymphocyte development and lymph-node organogenesis. Nature, 1999,397:315-323
[27] Felix, R., Hofstetter, W. & Cecchini, M.G Recent developments in the understanding of the pathophysiology of osteopetrosis. Eur. J. Endocrinol, 1996, 134: 143-156
[28] Teitelbaum S.I. The osteoclast and osteoporosis. Mt Sinai J. Med, 1996, 63: 399-402
[29] Roodman, G.D. Advances in bone biology: the osteoclast. Endocr, 1996,17:308-332
[30] Yasuda H, Shima N, Nakagawa N, et al. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegeria (OPG): A Mechanism by with OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology, 1998, 139(3):1329-1337