不同低功率He：Ne激光对种植体骨结合影响的实验研究
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摘要
目的：以低功率激光照射种植体植入部位对应体表,通过种植体-骨大体标本骨组织计量学及组织学观察,分析种植体骨结合过程及程度,探讨不同低功率激光照射对种植体-骨结合界面骨形成的影响,为低功率激光在口腔种植的临床应用提供一定的实验学依据。
方法：选用国际标准级雄性Beagle犬6只,按照BLB种植系统外科植入操作步骤于Beagle犬每侧后肢胫骨距近膝关节约3-9cm范围内制备4个种植窝,植入4枚BLB(?)3.3×8mm种植体,并将植体随机分为A、B、C、D组,D组设为空白对照组,共48枚,每组12枚,种植体间隔2cm,A、B、C组术后立即以波长632.8nm,频率50Hz±1Hz,光斑直径1.2cm的He：Ne激光垂直照射,A组15mW、B组25mW、C组35mW,20min/次,1次／天,持续2周。分别于术后1、2、3个月处死动物,处死前2周行四环素荧光双标记,每次处死2只,制取16个带种植体的骨标本,进行大体标本观察及制作带种植体的骨磨片,荧光正置显微镜下,采用骨形态分析系统(BIOQUANTOSTEO2009)测量两次荧光标记线长度及骨小梁周长,计算双荧光标记率(percent labeled perimeter %L.Pm),测量双荧光标记线间的垂直距离,计算矿化沉积率(mineral apposition rate MAR)。经Goldner's三色法染色后光学显微镜下进行组织学观察,在半自动数字化图像分析仪下分析骨磨片,测量记录新生骨与种植体接触面的总长度及埋入骨组织中种植体的总长度,计算种植体骨结合率(bone-implant contact, BIC),应用SPSS13.0软件对数据进统计分析,得出实验结论。
结果：1.种植体植入手术：种植体植入手术顺利,激光照射局部未见异常反应。术后犬创口愈合良好,无炎症反应,均健康成活。2.标本大体观察：实验组尤其A、B组较空白对照组种植体周围有较多新骨形成,覆盖种植体光滑面。3.标本X线显示：种植体无脱落,植入方向一致。4.荧光显微镜下观察及骨组织形态计量学分析：术后1个月A、B组荧光标记率、矿化沉积率显著高于空白对照组及C组p<0.05)。而A组与B组间无显著差异p>0.05),空白对照组与C组间亦无显著差异p>0.05)。术后2、3个月荧光标记率、矿化沉积率各组间均无显著差异(p>0.05)。同一剂量组在不同检测期,A组、B组荧光标记率及矿化沉积率术后1个月均显著高于术后2、3个月(p<0.05),C组及空白对照组荧光标记率、矿化沉积率术后1个月显著高于术后3个月p<0.05)。5.光学显微镜下观察及组织形态学分析：种植体骨接触率术后1、2个月A、B、C、D组间无显著差异(p>0.05),术后3个月A、B组明显高于C、D组p<0.05),A、B组间及C、D组间无显著差异p>0.05)。
结论：1.低功率激光对种植体周围早期新骨形成及增强种植体初期稳定性有明显促进作用。2.低功率激光能够促进种植体骨界面骨形成,并在一定程度上促进骨组织成熟,缩短骨结合时间。3.种植术后持续照射低功率He:Ne激光2周,15mW、25 mW对术后早期(1个月)促进种植体周围成骨细胞增殖提高成骨细胞活性的效果更佳。4.种植术后持续照射低功率He:Ne激光2周15 mW、25 mW低功率对最终(术后3个月)提高种植体骨结合率效果更佳。
Objective:This research aims to explore the best power of low-power laser irradiation on implant-osseointegration by observation,analysis of histology and bone histomorphometry, and provide a further scientific basis for the clinical application of a low-power laser in Oral Implantology.
Methods:6 female Beagle dogs were selected according to international standards. In accordance with the surgical implanting method of BLB implant system, surgeries were operated on each dog from both sides of the tibia, four implant sockets were implanted in 4 BLB (?) 3.3 x 8mm implant, every side 4 planting interval 2cm,which were divided into A, B, C,D group randomly. D group which as control group was not irradiated. A(15mW)、B(25Mw)、C(35Mw) groups for the experimental group were immediately irradiated after surgery.The period of irradiation is two weeks,20min/once, per day. Specimens were obtained 1、2 and 3 months after implantation, from two animals at each time point.Before killing the animals tetracycline double labeling were exposed. Making the 16 tibial implant specimens, for the general observation and production of bone implants with grinding plate, the semi-automatic digital image analysis system under the analysis of bone grinding, measuring and recording percent labeled perimeter(%L.Pm) and mineral apposition rate (MAR), after Goldner's trichrome the bone-implant contact(BIC) was measured.Analyzing the variance of data and then obtain the experimental results.
Results:1. Surgical implants:The surgery of implanting implants in Beagle dog tibia went smoothly without any abnormal reaction for laser irradiation. All the dogs survived the surgery and behaved normally, with their wounds healing well and no inflammatory response.2. General observation of specimens. More new bones grew, with smooth surface, around the implants in the A、B groups than those of in the C、D groups.3. Roentgenologic visualtation of specimens.any of the implants which were implanted in the same direction were without devulvium.4. Fluorescence microscope observation and bone histomorphometry analysis:One month after operation,there was a statistically significant difference between A、B and C、D groups in mineral apposition rate (MAR) and percent labeled perimeter(%L.Pm) (p<0.05).The MAR and %L.Pm of A、B groups was significantly higher than C、D groups, there was no statistically significant difference between A and B group (p>0.05). and there was no statistically significant difference between C and D group either. Two and three months after operation there was no statistically significant difference between among four groups (p>0.05). InA、B groups the percent labeled perimeter(%L.Pm) and mineral apposition rate (MAR) of 1 month after operation was significantly higher than it of 2、3 months after operation (p<0.05).Between C、D groups the significant difference was found only between 1 and 3 months(p<0.05). 5. Optical microscope observation and histomorphology analysis:On bone-implant contact(BIC) one and two months after operation there was no statistically significant difference between among four groups (p>0.05). Three months after operation there was a statistically significant difference between between A、B and C、D groups (p<0.05). The bone-implant contact (BIC) of A、B groups was significantly higher than C、D groups. But there was no statistically significant difference between A and B group (p>0.05) and there was no statistically significant difference between C and D group either. (p>0.05).
Conclusions:1. Low-power laser can promote early peri-implant bone formation,enhance the initial implant stability, so make the implant more stable.2. Low-power laser can promote the formation of bone-implant interface and, to some extent promote the mature of bone tissue and shorten the osseo-integration time.3. Two weeks'irradiation of low-power laser play a significant promotion role in implant-osseointegration and the effect of 15mW and 25mW low-power on increasing the activity and the proliferation of osteoblasts aroud implant at early period is better.4. Two weeks'irradiation of low-power laser play a significant promotion role in implant-osseointegration and the effect of 15mW and 25mW low-power on increasing final bone-implant contact (BIC) is better.
方法：选用国际标准级雄性Beagle犬6只,按照BLB种植系统外科植入操作步骤于Beagle犬每侧后肢胫骨距近膝关节约3-9cm范围内制备4个种植窝,植入4枚BLB(?)3.3×8mm种植体,并将植体随机分为A、B、C、D组,D组设为空白对照组,共48枚,每组12枚,种植体间隔2cm,A、B、C组术后立即以波长632.8nm,频率50Hz±1Hz,光斑直径1.2cm的He：Ne激光垂直照射,A组15mW、B组25mW、C组35mW,20min/次,1次／天,持续2周。分别于术后1、2、3个月处死动物,处死前2周行四环素荧光双标记,每次处死2只,制取16个带种植体的骨标本,进行大体标本观察及制作带种植体的骨磨片,荧光正置显微镜下,采用骨形态分析系统(BIOQUANTOSTEO2009)测量两次荧光标记线长度及骨小梁周长,计算双荧光标记率(percent labeled perimeter %L.Pm),测量双荧光标记线间的垂直距离,计算矿化沉积率(mineral apposition rate MAR)。经Goldner's三色法染色后光学显微镜下进行组织学观察,在半自动数字化图像分析仪下分析骨磨片,测量记录新生骨与种植体接触面的总长度及埋入骨组织中种植体的总长度,计算种植体骨结合率(bone-implant contact, BIC),应用SPSS13.0软件对数据进统计分析,得出实验结论。
结果：1.种植体植入手术：种植体植入手术顺利,激光照射局部未见异常反应。术后犬创口愈合良好,无炎症反应,均健康成活。2.标本大体观察：实验组尤其A、B组较空白对照组种植体周围有较多新骨形成,覆盖种植体光滑面。3.标本X线显示：种植体无脱落,植入方向一致。4.荧光显微镜下观察及骨组织形态计量学分析：术后1个月A、B组荧光标记率、矿化沉积率显著高于空白对照组及C组p<0.05)。而A组与B组间无显著差异p>0.05),空白对照组与C组间亦无显著差异p>0.05)。术后2、3个月荧光标记率、矿化沉积率各组间均无显著差异(p>0.05)。同一剂量组在不同检测期,A组、B组荧光标记率及矿化沉积率术后1个月均显著高于术后2、3个月(p<0.05),C组及空白对照组荧光标记率、矿化沉积率术后1个月显著高于术后3个月p<0.05)。5.光学显微镜下观察及组织形态学分析：种植体骨接触率术后1、2个月A、B、C、D组间无显著差异(p>0.05),术后3个月A、B组明显高于C、D组p<0.05),A、B组间及C、D组间无显著差异p>0.05)。
结论：1.低功率激光对种植体周围早期新骨形成及增强种植体初期稳定性有明显促进作用。2.低功率激光能够促进种植体骨界面骨形成,并在一定程度上促进骨组织成熟,缩短骨结合时间。3.种植术后持续照射低功率He:Ne激光2周,15mW、25 mW对术后早期(1个月)促进种植体周围成骨细胞增殖提高成骨细胞活性的效果更佳。4.种植术后持续照射低功率He:Ne激光2周15 mW、25 mW低功率对最终(术后3个月)提高种植体骨结合率效果更佳。
Objective:This research aims to explore the best power of low-power laser irradiation on implant-osseointegration by observation,analysis of histology and bone histomorphometry, and provide a further scientific basis for the clinical application of a low-power laser in Oral Implantology.
Methods:6 female Beagle dogs were selected according to international standards. In accordance with the surgical implanting method of BLB implant system, surgeries were operated on each dog from both sides of the tibia, four implant sockets were implanted in 4 BLB (?) 3.3 x 8mm implant, every side 4 planting interval 2cm,which were divided into A, B, C,D group randomly. D group which as control group was not irradiated. A(15mW)、B(25Mw)、C(35Mw) groups for the experimental group were immediately irradiated after surgery.The period of irradiation is two weeks,20min/once, per day. Specimens were obtained 1、2 and 3 months after implantation, from two animals at each time point.Before killing the animals tetracycline double labeling were exposed. Making the 16 tibial implant specimens, for the general observation and production of bone implants with grinding plate, the semi-automatic digital image analysis system under the analysis of bone grinding, measuring and recording percent labeled perimeter(%L.Pm) and mineral apposition rate (MAR), after Goldner's trichrome the bone-implant contact(BIC) was measured.Analyzing the variance of data and then obtain the experimental results.
Results:1. Surgical implants:The surgery of implanting implants in Beagle dog tibia went smoothly without any abnormal reaction for laser irradiation. All the dogs survived the surgery and behaved normally, with their wounds healing well and no inflammatory response.2. General observation of specimens. More new bones grew, with smooth surface, around the implants in the A、B groups than those of in the C、D groups.3. Roentgenologic visualtation of specimens.any of the implants which were implanted in the same direction were without devulvium.4. Fluorescence microscope observation and bone histomorphometry analysis:One month after operation,there was a statistically significant difference between A、B and C、D groups in mineral apposition rate (MAR) and percent labeled perimeter(%L.Pm) (p<0.05).The MAR and %L.Pm of A、B groups was significantly higher than C、D groups, there was no statistically significant difference between A and B group (p>0.05). and there was no statistically significant difference between C and D group either. Two and three months after operation there was no statistically significant difference between among four groups (p>0.05). InA、B groups the percent labeled perimeter(%L.Pm) and mineral apposition rate (MAR) of 1 month after operation was significantly higher than it of 2、3 months after operation (p<0.05).Between C、D groups the significant difference was found only between 1 and 3 months(p<0.05). 5. Optical microscope observation and histomorphology analysis:On bone-implant contact(BIC) one and two months after operation there was no statistically significant difference between among four groups (p>0.05). Three months after operation there was a statistically significant difference between between A、B and C、D groups (p<0.05). The bone-implant contact (BIC) of A、B groups was significantly higher than C、D groups. But there was no statistically significant difference between A and B group (p>0.05) and there was no statistically significant difference between C and D group either. (p>0.05).
Conclusions:1. Low-power laser can promote early peri-implant bone formation,enhance the initial implant stability, so make the implant more stable.2. Low-power laser can promote the formation of bone-implant interface and, to some extent promote the mature of bone tissue and shorten the osseo-integration time.3. Two weeks'irradiation of low-power laser play a significant promotion role in implant-osseointegration and the effect of 15mW and 25mW low-power on increasing the activity and the proliferation of osteoblasts aroud implant at early period is better.4. Two weeks'irradiation of low-power laser play a significant promotion role in implant-osseointegration and the effect of 15mW and 25mW low-power on increasing final bone-implant contact (BIC) is better.
引文
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48.宿玉成(主编).现代口腔种植学.北京：人民卫生出版社,2004：53
49. Ozawa Y, Shimizu N, Kariya G, et al. Low-energy laser irradiation stimulates bone nodule formation at early stages of cell culture in rat calvarial cells. Bone 1998;22(4):347-354
1. Basford JR.Low-energy laser therapy:controversies and new research findings[J]. Lasers Surg Med,1989,9(1):1-5
2. Mester E,Mester AF,MesterA,et al.The biomedical effects of laser application[J]. Lasers Surg Med,1985,5(1):31-39
3. Mustafa K,Silva LB,Huhenby K,et al.Attachment and proliferation of human oral fibroblasts to titanium surfaces blasted with TiO2 particles. A scanning electron microscopic and histomorphometric analysis[J].Clin Oral Implants Rcs,1998,9 (3):195-207
4. Khadra M,Lyngstadas SP,Haanaes HR,et al.Determining optimal dose of laser therapy for attachment and proliferation of human oral fibroblasts cultured on titanium implant material Biomaterials[J],2005,26(6):3503-3509
5. Khadra M,Kasem N,Lyngstadaas SP,et al.Laser therapy accelerates initial attachment and subsequent behaviour of human oral fibroblasts cultured on titanium implant material. A scanning electron microscope and histomorphometric analysis[J]. Clin Oral Implants Res,2005,16(2):168-175
6. Karu TI,Pyatibrat LV,Afanasyeva NI.Cellular effects of low power laser therapy can be mediated by nitric oxide[J].Lasers Surg Med,2005,36(4):307-314
7. Ozawa Y,Shimizu N,Kariya G,et al.Low-energy laser irradiation stimulates bone nodule formation at early stages of cell culture in rat calvarial cells[J]. Bone,1998,4(22):347-355
8. 李梅,周磊,王春先,等.半导体激光(Ga Al As)对大鼠成骨细胞增殖和分化的影响[J].中国口腔种植学杂志,2004,3(9)：3-5
9. Oyster DK,Parker WB,Gher ME,et al.CO2 lasers and temperature changes of titanium implants[J],1995,66(12):1017-1024
10. Takeda Y Irradiation effect of low-energy laser on alveolar bone after tooth extraction :experimental study in rats. International Jounrnal of Oral and Msxilla facial Surgery.1988.17:388-391
11. Nagasawa A,et al.Bone regeneration effect of low level laser including argon laser.Laser Therapy.1991,3:59-62
12. Rochkid 8,et al.Molecular stracture of the Bone tissue after experimental trauma to the mandibular region followed by laser therapy.Photomedicine and Laser Surgery. 204.22(3):249-253
13. Da Silva RV,et al.Experimental Study:Repair of Bone defects treated with aurogenous bone graft and low-power laser.J of Craniofacial Surgery 2006.17(2):297-301
14. Kim YD,Kim SS,Hwang DS,et al.Effect of low-level laser treatment after installation of dental titanium implant-immunohistochemical study of RANKL, RANK,OPG:An experimental study in rats[J].Lasers in Surgery and Medicine,2007,39:441-450
15.安玉.口腔种植学[M],成都：四川科技出版社,1991
16. Garavello-Freitas I,Baranauskas V,Joazeiro PP Low-power laser irradiation improves histomorphometrical parameters and bone matrix organization during tibia wound healing in rats[J].J Photochem Photobiol B,2003,70(2):81-9
17. Durtbudak O,Haas R,Mailath PG.Efect of low-power laser irradiation on bony implant sites[J].Clin Oral Impl Res,2002,13:288-292
18. Guzzardella P,Torricelli N,Gaetano A.Osseointegration of endosseous ceramic implants after postoperative low-power laser stimulation:an in vivo compareative study.Clin Oral Impl Res[J],2003,14:226-232
19. Mester E, Mester AF, Mester A. The biomedical ef2fects of laser application. Lasers Surg Med,1985,5:31
20. Basford JR. Low-energy laser therapy:Controversies and desearch findings. Laser Surg Med,1989,9:1
21.宿玉成(主编).现代口腔种植学.北京：人民卫生出版社,2004：53
22.邱蔚六,张震康主编口腔颌面外科学.第5版.北京：人民卫生出版社,2003
23.耿建华,陈英茂,魏晓芬.不同剂量He-Ne激光照射对骨折愈合影响的验研究.中国激光医学杂志,1998,7：34-37
24. Da Silva RV,et al.Experimental Study:Repair of Bone defects treated with auroge-nous bone graft and low-power laser.J of Craniofacial Surgery 2006.17(2):297-301