改良型富血小板纤维蛋白在兔颅骨诱导成骨中的组织学观察
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摘要
背景:前期实验验证了富血小板纤维蛋白具有成骨的效果,并且选定了人与兔改良型富血小板纤维蛋白膜结构最相似时的转速和离心时间。目的:观察改良型富血小板纤维蛋白在诱导成骨中的组织学变化。方法:随机将30只日本大耳兔分为2组各15只,在兔颅顶骨制造圆形全层骨缺损来构建动物模型,改良型富血小板纤维蛋白组填入改良型富血小板纤维蛋白膜,空白组不填任何材料,于建模后当天、2周、4周、8周、12周处死动物,大体观察造模区成骨情况,苏木精-伊红染色、masson三色染色、免疫组织化学观察造模区组织学变化。结果与结论:(1)空白组骨缺损在实验结束时不能自行愈合;(2)建模后当天、4周、8周、12周2组标本的大体观察、组织学检查,均显示改良型富血小板纤维蛋白组的成骨速度、量及成熟度,皆明显优于空白组,差异具有统计学意义;(3)结果说明,制备的骨缺损实验期内不能自行愈合,符合临界性骨缺损的标准;改良型富血小板纤维蛋白能促进新骨形成,随着时间的延长,成骨数量更多,质量更佳;改良型富血小板纤维蛋白促使破骨细胞与成骨细胞协同作用,促进新骨生成。
BACKGROUND:Previous studies have proved platelet-rich fibrin(PRF) with osteoinduction ability,and the centrifugal speed and time to prepare rabbit advanced PRF(A-PRF) with the most similar structure to that of human PRF have been determined.OBJECTIVE:To observe the histological changes during A-PRF-induced osteogenesis.METHODS:Thirty Japanese white rabbits were randomly divided into A-PRF and blank control groups(n=15 per group).The full-thickness defect models were established on the rabbit parietal bone,followed by implanted with A-PRF or nothing,respectively.The model rabbits were killed immediately,at 2,4,8 and 12 weeks after modeling,to grossly observe the bone formation,and the histological changes in the defect region were observed through hematoxylin-eosin staining,Masson staining and immunohistochemistry.RESULTS AND CONCLUSION:Unhealed defects were observed in the blank control group.Gross and histological observations showed that the speed,amount and maturity of bone formation in the A-PRF group were significantly better than those in the blank control group immediately,4,8 and 12 weeks after modeling(P < 0.05).Our findings suggest that the rabbit skull bone defect is successfully established.A-PRF can induce osteogenesis,and more mature newly born bones appear with time.Additionally,osteoclasts can act with osteoblasts synergically under the A-PRF induction to promote the bone formation.
BACKGROUND:Previous studies have proved platelet-rich fibrin(PRF) with osteoinduction ability,and the centrifugal speed and time to prepare rabbit advanced PRF(A-PRF) with the most similar structure to that of human PRF have been determined.OBJECTIVE:To observe the histological changes during A-PRF-induced osteogenesis.METHODS:Thirty Japanese white rabbits were randomly divided into A-PRF and blank control groups(n=15 per group).The full-thickness defect models were established on the rabbit parietal bone,followed by implanted with A-PRF or nothing,respectively.The model rabbits were killed immediately,at 2,4,8 and 12 weeks after modeling,to grossly observe the bone formation,and the histological changes in the defect region were observed through hematoxylin-eosin staining,Masson staining and immunohistochemistry.RESULTS AND CONCLUSION:Unhealed defects were observed in the blank control group.Gross and histological observations showed that the speed,amount and maturity of bone formation in the A-PRF group were significantly better than those in the blank control group immediately,4,8 and 12 weeks after modeling(P < 0.05).Our findings suggest that the rabbit skull bone defect is successfully established.A-PRF can induce osteogenesis,and more mature newly born bones appear with time.Additionally,osteoclasts can act with osteoblasts synergically under the A-PRF induction to promote the bone formation.
引文
[1]张辉秋,孙连碧,蒋剑晖.骨劈开术在口腔种植骨增量中的应用[J].临床军医杂志,2012,40(5):1178-1180.
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[3]周磊.即刻种植术中引导骨增量技术的应用[J].中国实用口腔科杂志,2012,5(4):197-202.
[4]王晓娜,赵静辉,储顺礼,等.骨替代材料在口腔种植领域中的成骨效果[J].国际口腔医学杂志,2016,43(1):113-117.
[5]宁江海,刘洪臣.人工植骨材料的研究进展[J].口腔颌面修复学杂志,2008,9(4):299-302
[6]Lindhe J,Cecchinato D,Donati M,et al.Ridge preservation with the use of deproteinized bovine bone mineral.Clin Oral Implants Res.2014;25(7):786-790.
[7]王兴,刘洪臣.自体骨移植修复种植位点骨缺损的研究进展[J].口腔颌面修复学杂志,2016,17(1):49-52.
[8]Ortel TL,Mercer MC,Thames E H,et al.Immunologic impact and clinical outcomes after surgical exposure to bovine thrombin.Ann Surg.2001;233(1):88-96.
[9]Choukroun J,Diss A,Simonpieri A,et al.Platelet-rich fibrin(PRF):a second-generation platelet concentrate.Part V:histologic evaluations of PRF effects on bone allograft maturation in sinus lift.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2006;101(3):299-303.
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[11]李永斌.浓缩生长因子纤维蛋白与富血小板纤维蛋白体外降解的对比研究[D].天津医科大学,2014.
[12]Sam G,Vadakkekuttical RJ,Amol NV.In vitro evaluation of mechanical properties of platelet-rich fibrin membrane and scanning electron microscopic examination of its surface characteristics.J Indian Soc Periodontol.2015;19(1):32-36.
[13]Dohan ED,Del CM,Inchingolo F,et al.Selecting a relevant in vitro cell model for testing and comparing the effects of a Choukroun's platelet-rich fibrin(PRF)membrane and a platelet-rich plasma(PRP)gel:tricks and traps.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2010;110(4):409-411.
[14]Ghanaati S,Booms P,Orlowska A,et al.Advanced platelet-rich fibrin:a new concept for cell-based tissue engineering by means of inflammatory cells.J Oral Implantol.2014;40(6):679-689.
[15]Kim TH,Kim SH,Sandor GK,et al.Comparison of platelet-rich plasma(PRP),platelet-rich fibrin(PRF),and concentrated growth factor(CGF)in rabbit-skull defect healing.Arch Oral Biol.2014;59(5):550-558.
[16]万永.富血小板纤维蛋白在牙槽嵴位点保存应用中的临床研究[D].泸州医学院,2014.
[17]何通文,徐庚池,韩耀辉,等.构建兔颅顶骨临界骨缺损模型:确立颅顶临界骨缺损的参考值[J].中国组织工程研究,2014,18(18):2789-2794.
[18]李雅巍,孙晓梅,滕利,等.牙齿种植骨量不足的相关研究进展[J].组织工程与重建外科杂志,2015,11(1):50-54.
[19]付冬梅,肖琼,杨琴秋,等.富血小板纤维蛋白新生诱导骨的组织学观察[J].中国组织工程研究,2016,20(7):933-939.
[20]万永.富血小板纤维蛋白在牙槽嵴位点保存应用中的临床研究[D].泸州医学院,2014.
[21]Schmitz J P,Hollinger J O.The critical size defect as an experimental model for craniomandibulofacial nonunions.Clin Orthop Relat Res.1986;(205):299-308.
[22]Hollinger JO,Kleinschmidt JC.The critical size defect as anexperimental model to test bone repair materials.JCraniofac Surg.1990;1(1):60-68.
[23]李英.SPF级新西兰实验兔生物学特性的研究[D].第一军医大学,2004.
[24]毛俊丽,赵峰,陈红亮,等.兔PRF、A-PRF制备方法的筛选[J].西南国防医药,2016,26(6):593-596.
[25]周春梅,温齐古丽·乃库力,于莉,等.骨诱导活性材料复合富血小板纤维蛋白在拔牙位点引导新骨形成的骨计量学研究[J].临床口腔医学杂志,2016,32(2):67-69.
[26]徐翔.富血小板纤维蛋白(PRF)作为支架材料修复颌骨骨量不足的实验研究[D].辽宁医学院,2012.
[27]李京旭,李龙和,玄云泽.Bio-Oss/PRF复合支架结合骨髓基质细胞构建组织工程骨实验研究[J].延边大学医学学报,2014,37(4):235-238.
[28]何璇.富血小板纤维蛋白凝胶作为牙髓再生支架的体外实验研究[D].广西医科大学,2015.
[29]Shubhashini N,Kumar RV,Shija AS,et al.Platelet-rich fibrin in treatment of periapical lesions:a novel therapeutic option.Chin J Dent Res.2013;16(1):79-82.
[30]Schmitz JP,Hollinger JO.The bio lo gy of platelet-rich plasma.Oral Maxillofac Surg.2003;59(9):1119-1121.
[31]郭建刚,赵然,侯桂英,等.骨组织成分与Masson三色染色反应的关系分析[J].中医正骨,2001,13(11):5-6+63.
[32]郭蕊欣,李金源.基质金属蛋白酶在牙周病发展中的作用[J].军医进修学院学报,2010,31(5):519-520.
[33]沈敏华,束蓉.基质金属蛋白酶与牙周病关系的研究进展[J].牙体牙髓牙周病学杂志,2007,17(1):44-47.
[34]王昱翔,张宏其,郭超峰,等.雌激素受体β基因沉默对成骨样MG63细胞骨保护素和RANKL表达的影响[J].中国组织工程研究,2013,17(41):7188-7198.
[35]鄢林霞.生物活性玻璃对家兔股骨髁骨缺损的修复实验研究[D].四川农业大学,2013.
[36]张嫒儒.Beagle犬下颌骨前磨牙拔牙后位点保存的实验研究[D].泸州医学院,2011.
[37]Blair JM,Zheng Y,Dunstan CR.RANK ligand.Int J Biochem Cell Biol.2007;39(6):1077-1081.
[38]Simonet W S,Lacey DL,Dunstan CR,et al.Osteoprotegerin:a novel secreted protein involved in the regu lation og bo ne density.Cell.1997;89(2):309-319.
[39]田虹,樊瑜波.OPG、RANK、RANKL的结构、作用机制和在骨疾病中的作用[J].现代生物医学进展,2010,10(20):3963-3966.
[40]陈雁南.OPG对实验大鼠正畸牙移动的影响[D].重庆医科大学,2012.
[41]Parfitt AM.Targeted and nontargeted bone remodeling:relationship to asic multicellular unit origination and progression.Bone.2002;30(1):5-7.
[42]Kearns AE,Khosla S,Kostenuik PJ.Receptor activator of nuclear factor B ligand and osteoprotegerin regulation of bone remodeling in health and disease.Endocr Rev.2008;29(2):155-192.
[2]文勇,徐欣.骨增量技术在口腔种植中的应用[J].中国口腔种植学杂志,2008,13(4):204-207.
[3]周磊.即刻种植术中引导骨增量技术的应用[J].中国实用口腔科杂志,2012,5(4):197-202.
[4]王晓娜,赵静辉,储顺礼,等.骨替代材料在口腔种植领域中的成骨效果[J].国际口腔医学杂志,2016,43(1):113-117.
[5]宁江海,刘洪臣.人工植骨材料的研究进展[J].口腔颌面修复学杂志,2008,9(4):299-302
[6]Lindhe J,Cecchinato D,Donati M,et al.Ridge preservation with the use of deproteinized bovine bone mineral.Clin Oral Implants Res.2014;25(7):786-790.
[7]王兴,刘洪臣.自体骨移植修复种植位点骨缺损的研究进展[J].口腔颌面修复学杂志,2016,17(1):49-52.
[8]Ortel TL,Mercer MC,Thames E H,et al.Immunologic impact and clinical outcomes after surgical exposure to bovine thrombin.Ann Surg.2001;233(1):88-96.
[9]Choukroun J,Diss A,Simonpieri A,et al.Platelet-rich fibrin(PRF):a second-generation platelet concentrate.Part V:histologic evaluations of PRF effects on bone allograft maturation in sinus lift.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2006;101(3):299-303.
[10]Sacco L.Lecture,International academy of implant prosthesis and osteoconnection.2006;12:4.
[11]李永斌.浓缩生长因子纤维蛋白与富血小板纤维蛋白体外降解的对比研究[D].天津医科大学,2014.
[12]Sam G,Vadakkekuttical RJ,Amol NV.In vitro evaluation of mechanical properties of platelet-rich fibrin membrane and scanning electron microscopic examination of its surface characteristics.J Indian Soc Periodontol.2015;19(1):32-36.
[13]Dohan ED,Del CM,Inchingolo F,et al.Selecting a relevant in vitro cell model for testing and comparing the effects of a Choukroun's platelet-rich fibrin(PRF)membrane and a platelet-rich plasma(PRP)gel:tricks and traps.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2010;110(4):409-411.
[14]Ghanaati S,Booms P,Orlowska A,et al.Advanced platelet-rich fibrin:a new concept for cell-based tissue engineering by means of inflammatory cells.J Oral Implantol.2014;40(6):679-689.
[15]Kim TH,Kim SH,Sandor GK,et al.Comparison of platelet-rich plasma(PRP),platelet-rich fibrin(PRF),and concentrated growth factor(CGF)in rabbit-skull defect healing.Arch Oral Biol.2014;59(5):550-558.
[16]万永.富血小板纤维蛋白在牙槽嵴位点保存应用中的临床研究[D].泸州医学院,2014.
[17]何通文,徐庚池,韩耀辉,等.构建兔颅顶骨临界骨缺损模型:确立颅顶临界骨缺损的参考值[J].中国组织工程研究,2014,18(18):2789-2794.
[18]李雅巍,孙晓梅,滕利,等.牙齿种植骨量不足的相关研究进展[J].组织工程与重建外科杂志,2015,11(1):50-54.
[19]付冬梅,肖琼,杨琴秋,等.富血小板纤维蛋白新生诱导骨的组织学观察[J].中国组织工程研究,2016,20(7):933-939.
[20]万永.富血小板纤维蛋白在牙槽嵴位点保存应用中的临床研究[D].泸州医学院,2014.
[21]Schmitz J P,Hollinger J O.The critical size defect as an experimental model for craniomandibulofacial nonunions.Clin Orthop Relat Res.1986;(205):299-308.
[22]Hollinger JO,Kleinschmidt JC.The critical size defect as anexperimental model to test bone repair materials.JCraniofac Surg.1990;1(1):60-68.
[23]李英.SPF级新西兰实验兔生物学特性的研究[D].第一军医大学,2004.
[24]毛俊丽,赵峰,陈红亮,等.兔PRF、A-PRF制备方法的筛选[J].西南国防医药,2016,26(6):593-596.
[25]周春梅,温齐古丽·乃库力,于莉,等.骨诱导活性材料复合富血小板纤维蛋白在拔牙位点引导新骨形成的骨计量学研究[J].临床口腔医学杂志,2016,32(2):67-69.
[26]徐翔.富血小板纤维蛋白(PRF)作为支架材料修复颌骨骨量不足的实验研究[D].辽宁医学院,2012.
[27]李京旭,李龙和,玄云泽.Bio-Oss/PRF复合支架结合骨髓基质细胞构建组织工程骨实验研究[J].延边大学医学学报,2014,37(4):235-238.
[28]何璇.富血小板纤维蛋白凝胶作为牙髓再生支架的体外实验研究[D].广西医科大学,2015.
[29]Shubhashini N,Kumar RV,Shija AS,et al.Platelet-rich fibrin in treatment of periapical lesions:a novel therapeutic option.Chin J Dent Res.2013;16(1):79-82.
[30]Schmitz JP,Hollinger JO.The bio lo gy of platelet-rich plasma.Oral Maxillofac Surg.2003;59(9):1119-1121.
[31]郭建刚,赵然,侯桂英,等.骨组织成分与Masson三色染色反应的关系分析[J].中医正骨,2001,13(11):5-6+63.
[32]郭蕊欣,李金源.基质金属蛋白酶在牙周病发展中的作用[J].军医进修学院学报,2010,31(5):519-520.
[33]沈敏华,束蓉.基质金属蛋白酶与牙周病关系的研究进展[J].牙体牙髓牙周病学杂志,2007,17(1):44-47.
[34]王昱翔,张宏其,郭超峰,等.雌激素受体β基因沉默对成骨样MG63细胞骨保护素和RANKL表达的影响[J].中国组织工程研究,2013,17(41):7188-7198.
[35]鄢林霞.生物活性玻璃对家兔股骨髁骨缺损的修复实验研究[D].四川农业大学,2013.
[36]张嫒儒.Beagle犬下颌骨前磨牙拔牙后位点保存的实验研究[D].泸州医学院,2011.
[37]Blair JM,Zheng Y,Dunstan CR.RANK ligand.Int J Biochem Cell Biol.2007;39(6):1077-1081.
[38]Simonet W S,Lacey DL,Dunstan CR,et al.Osteoprotegerin:a novel secreted protein involved in the regu lation og bo ne density.Cell.1997;89(2):309-319.
[39]田虹,樊瑜波.OPG、RANK、RANKL的结构、作用机制和在骨疾病中的作用[J].现代生物医学进展,2010,10(20):3963-3966.
[40]陈雁南.OPG对实验大鼠正畸牙移动的影响[D].重庆医科大学,2012.
[41]Parfitt AM.Targeted and nontargeted bone remodeling:relationship to asic multicellular unit origination and progression.Bone.2002;30(1):5-7.
[42]Kearns AE,Khosla S,Kostenuik PJ.Receptor activator of nuclear factor B ligand and osteoprotegerin regulation of bone remodeling in health and disease.Endocr Rev.2008;29(2):155-192.