低强度脉冲超声促进牵张成骨新骨成熟及种植体骨结合的研究
|
摘要
肿瘤、创伤等原因导致颌骨缺损的矫治不仅要求恢复外形,更重要的是恢复咀嚼功能,牵张成骨是新兴的、成熟的、有效的矫治颅颌面畸形及颌骨缺损的外科技术,而牙种植技术则可以有效的恢复咀嚼功能。牵张成骨技术要求骨段牵张结束后藉牵张器固定6～8周,以待延长段的新骨骨化,再拆除牵张器。长时间的固定期给患者的生活、学习、工作带来极大不便,且牵张成骨术后并发症有钉道感染、骨折、骨化不良及骨不连等。因此,众多学者均在寻求可以有效的促进牵张成骨新生骨痂生成、骨化的方法,以早日拆除牵张器,缩短治疗时间,增强新骨机械性能。而种植体植入颌骨后,必须形成牢固的骨结合后才能负载咀嚼力,一般至少2～3月。低强度脉冲超声(low-intensitypulsedultrasound,LIPUS)是近年来公认的能促进骨折愈合的一种物理治疗方法,大量的动物和临床试验均证明超声能加快并加强新鲜骨折的愈合。超声是一种较理想的、非创伤性的促进新生骨痂生成的物理刺激因素。本课题通过建立犬双侧下颌牵张成骨和兔股骨和胫骨骨骺端螺旋状种植体植入动物模型,观察低强度脉冲超声能否促进牵张成骨新骨成熟以及种植体的骨结合。采用99mTc-MDP放射性核素骨扫描观察两侧牵张新骨区血供和骨组织代谢变化,X线平片观察新骨成熟度、双能X线骨密度监测、三维CT扫描重建测量新骨体积和表面积,组织学观察新骨组织形成特点,材料力学检测观察新骨强度的变化;通过组织学观察种植体金属-骨界面愈合情况,种植体拔出实验检测种植体固位强度。
实验一:低强度脉冲超声促进牵张成骨新骨成熟动物模型建立。成年杂种犬7只,在双侧下颌骨第1、2前磨牙间行骨切开术,植入内置式牵张器,间歇期为7天,牵引速率为每天2次,0.5mm/次,共20天。随机选一侧为实验侧,另一侧为对照,在牵张过程中实验侧每天定时给予低强度(40mW/cm~2)脉冲超声波刺激,每次10分钟,每天两次,共20天。牵张结束后的0、1、2、4、6、8、12周各处死动物一只,切取双侧下颌骨标本。
实验二:99mTc-MDP核素颌骨扫描。动物处死前静脉注射99mTc-MDP740毫居,注射药物4小时后SPECT扫描采集断层显像,重建三维立体影像。用计算机感兴趣区(ROI)技术分别在实验侧和对照侧对应部位选择兴趣区,并求出实验侧骨/对照侧骨的放射性计数比值,作为半定量分析指标。ROI检查结果:牵张完成早期实验侧较对照侧放射性计数有显著性差异(P<0.05),表明实验侧与对照侧相比骨代谢水平在相同时间内有差别。实验侧早期有较强的成骨现象和再血管化迹象,实验侧成骨活跃,与对照侧相比骨的代谢水平要高,显示了在骨生成方面优于对照侧。牵张完成中后期实验侧新骨处于骨改建期,而对照侧处于骨加速形成期,此时对照侧骨代谢程度高,因而核素浓聚要高于实验侧。实验侧与对照侧在牵张完成4周内骨代谢活跃,随着时间推移,牵张完成6～8周骨代谢趋于稳定,至12周时骨代谢已降低至正常水平。实验证实:超声机械刺激可影响成骨细胞的代谢和增加局部血流量来促进早期牵张新骨的成熟。
实验三:X线平片、三维CT、骨密度和强度检测:拍摄常规X线平片观察双侧牵张新骨的成熟情况。双侧下颌骨标本前部作三维CT扫描并重建,将采集的数据录入图像分析软件计算出牵张新骨区的体积和表面积,进行配对t检验;双能X线骨密度仪测量双侧牵张新骨区对称感兴趣区(RIO)骨密度值,进行配对t检验。一半标本取材后-20℃生理盐水保存,解冻后,室温晾干48小时。用金刚砂磨片切割厚度为3mm的骨片,使用电脑控制的材料力学分析仪进行压缩实验,骨片置于载物台上,连接传感器的压缩杆以1mm/min的速度压迫骨片直至变形;计算机自动记录压力—挠度曲线,得出骨的生物材料力学参数:最大负荷和压缩距离,进行配对t检验。
X线平片显示相同时间内超声照射侧新生骨的生长和成熟早于对照侧。骨密度测量结果显示新骨骨密度随着固定时间延长而增强,相同时间点实验侧与对照侧相比骨密度值有显著性差异(P<0.05)。新骨体积和表面积测量结果显示实验侧与对照侧相比体积和表面积无显著性差异(P>0.05)。新骨强度材料力学检测结果显示实验侧骨最大负荷和压缩距离均高于对照侧(P<0.05)。实验结果表明相同时间内超声照射侧新生骨的生长和成熟快于对照侧,验证了LIPUS可促进牵张新骨的成熟。实验组的骨密度和强度高于对照侧,但三维CT重建显示超声刺激并不能增加骨量。
实验四:牵张新骨组织学观察:一半标本甲醛固定48h,20%EDTA脱钙四周,脱水,石蜡包埋,制作石蜡切片,行常规HE及Masson’s三色法染色,光镜观察。结果显示早期实验侧牵张间隙骨小梁数目和直径大于对照侧,实验侧可见散在的软骨内成骨,后期新骨成熟早于对照侧。表明低强度超声可能通过刺激软骨生成而促进新骨生成。
实验五:低强度脉冲超声促进种植体骨结合的作用观察。成年新西兰大白兔10只,双侧膝关节股骨和胫骨骨骺端植入直径2mm螺旋状纯钛种植体各一枚,共40枚。随机选一侧为实验侧,另一侧为对照,实验侧每天给予低强度(40mW/cm2)脉冲超声波刺激,每次10分钟,每天两次,共20天。结束后的0、2、4、6、8周各处死动物两只,切取含种植体骨标本,拍摄X线牙片,一半标本进行种植体拔出实验。标本取材后-20℃生理盐水保存,解冻后,擦干。使用电脑控制的材料力学分析仪进行种植体拔出实验,得出力学参数:最大负荷和拔出位移,进行配对t检验。X线显示实验组和对照组种植体周围没有放射透视影,形成良好的骨结合。种植体拔出实验结果实验组最大负荷和拔出位移负荷均大于对照侧(P<0.05)。表明低强度脉冲超声可以促进种植体骨愈合,增强骨结合。
实验六:组织学观察低强度脉冲超声促进种植体骨结合。一半标本酒精脱水,树脂包埋,制作不脱钙组织切片,Goldner’s三色染色。结果显示实验组骨结合早于对照侧,骨成熟早于对照侧。表明低强度脉冲超声可以促进种植体界面骨结合。
综上所述,本实验结果显示低强度脉冲超声可以促进牵张新骨区早期血供和骨代谢水平,实验侧牵张新骨成熟早于对照侧,新骨密度和强度增加,但骨量无显著变化。超声可能通过刺激软骨内成骨而促进新骨成熟。同时,显示低强度脉冲超声可以促进种植体的早期骨结合,增强种植体的固位。
The treatment of maxillofacial bone defect due to tumor and trauma is notonly to reconstruct the facial contour but also to resume mastication function.Distraction osteogenesis (DO) has had an enormous impact and played a majorrole in the reconstruction of congenital and acquired deformities of thecraniofacial skeleton. Dental implants can resume mastication functionefficiently. However, the main disadvantage of DO is the long treatment timewhich requires the distracted bone segment be fixed by the distraction device fora certain period of consolidation to facilitate the new bone mature andremodeled. This imposes a psychological effect on the patient and familybecause several months are needed before the woven bone becomes compactbone to allow for distractor removal. Ilizarov suggested that the consolidationtime should be no less than two or three times as long as（6～8weeks usually）the distraction time. Long-time retention can result in inconvenience andexpensive inpatient cost and there were also some complications such as pseudarthrosis and relapse were reported. After dental implants inserted intobone, it costs at least 2～3 months to achieve osseointegration before acceptingdenture loading, which means dental implant is almost completely covered by acompact, mature, newly formed bone. An early good biological fixation mayallow the shortening of time before loading the implant, favoring the clinicalprocedure of early or immediate implant loading. Now, how to shorten theconsolidation time and enhance the mechanical property of the new distractedbone is the focus research field of DO. Also how to intensify osseointegration ofdental implants is the front study of dentistry.
Low-intensity pulsed ultrasound (LIPUS) is a form of mechanical energywhich is transmitted through and into biological tissues as an acoustic pressurewave and has been widely used in medicine as a diagnostic and therapeutic tool.Application of LIPUS (30～50mW/cm2) was considered to have little thermaleffect and to produce stable cavitation and streaming. There are many papers anddocuments published to establish LIPUS can accelerate the healing of fresh bonefracture and nonunion. LIPUS is an ideal physical stimulus to enhance boneformation. If ultrasound stimulation could accelerate the rate of the distractedcallus formation and remodification, the treatment period could be shortened,complications would decrease. Similarly, If ultrasound stimulation couldimprove the osseointegration of dental implants, patients would eat what theywant to as soon as possible. In this study, we observed the effect of LIPUS onthe enhancement of bone formation during mandible distraction osteogenesis indogs and improvement of osseointegration of dental implant in rabbits.
Experiment one was the animal model creation of enhancement of boneformation of mandible distraction osteogenesis by LIPUS. Bilateral surgical cutswere made in the mandible of seven dogs between the first and second premolar region. The anterior mandibles were lengthened by 20mm at the rate of 1mm/d,twice a day. During the distraction period one lateral distraction gap wasirradiated by LIPUS 10 minutes, twice a day and the other side was shamirradiated as control. After distraction was completed, the dogs were sacrificedon 0，1，2，4，6，8，12 week. The mandible samples were harvested.
Experiment two was 99mTc-methylene diphosphonate (MDP) bone imaging.Before the animals sacrifice, the animals were injected bolus 740 MBq 99mTc-MDP intravenously. Four hours later, delayed static bone scanning was obtainedby SPECT. For semiquantitative analysis, we set manually the region of interest(ROI) on the LIPUS stimulated distraction area and set a symmetric ROI on thecontralateral area . The result showed the uptake of 99mTc-MDP in theexperimental side was significant (P<0.05) higher than that of the control side inthe early period of consolidation (before the fourth week). But later the situationwas reversed, the uptake of 99mTc-MDP in the control side was significanthigher than that of the experimental side (P<0.05). There were no significantradioactivity differences between two sides at the 12th week. It was indicated theLIPUS stimulated area had more blood supply and metabolic activity. LIPUSradiation had positive effect on the healing and osteogenesis course of the newbone.
Experiment three was X-ray plain, three-dimension (3D) CT scan，bonemineral density (BMD) and biomechanical property measurement. The wholemandible samples were harvested and regular plain X-ray photographs weretaken. The front part of the mandibles were scanned with LightSpeed VCT 64-slice Scanner and 3D-images was obtained. Input 3D-CT scan data of themandible to the computer and used graphic information processing software tocalculate the volume and the surface area of the distracted bone. The BMD of the distracted bone was measured by dual-energy X-ray absorptiometry. Half of thebone samples were stored in saline at–20°C. Before testing, the bone wasthawed and dried for 48 hours at room temperature. The bone block was sectioninto 3mm thick slices by diamond blade. The biomechanical test was performedin a computed servohydraulic materials testing system. The slices were placed onthe on the smooth surface of a steel disk and axially compressed by the smoothsurface of a steel rod attached to load cell at a constant speed of 1 mm/min. Bothdisplacement and load were recorded for later analysis.
X-ray plain showed the new bone in the experimental side was matureearlier than that of the control side. The volume and the surface areameasurement of distracted area showed there was not significant differencebetween the LIPUS stimulated side and the control side ( paired t test, P > 0.05) .BMD measurement showed at the same time the BMD of LIPUS stimulated sidewas significantly higher than that of the control side (paired t test, P < 0.05). Theresult showed LIPUS could enhance the biomechanical property of new bone.
It was indicated the LIPUS radiation accelerate bone mature and increasethe BMD and mechanical property during mandible distraction osteogenesis buthad no effect on the volume of the new bone.
Experiment four was histological examination. One half of the bone sampleswere fixed in 4% paraformaldehyde for 48 hours and decalcified in 20% EDTAfor four weeks. The specimens were dehydrated in ethanol and embedded inparaffin, sectioned longitudinally at 4μm thick, stained for HE and Masson’sstain. The trabecula of the experimental side was more and thicker than that ofthe control side at the early period of consolidation. But in the late period, therewas not significant difference. We observed endochondral bone formation in theexperimental side at the 2w and 4w of consolidation. LIPUS may stimulate synthesis of extracellular matrix proteins altering chondrocyte maturation andendochondral bone formation. The modulation by ultrasound may occur byaccelerating endochondral ossification through action on chondrocytes, yetdistraction osteogenesis is mainly intramembranous.
Experiment five was the effect of LIPUS on the osseointegration of titaniumdental implants. 10 New Zealand rabbits were used in this study. Epiphyses ofboth femur and tibiae were inserted one screw titanium implants (? 2mm)separately in bilateral knee joints, total forty. One lateral knee joint includingimplants was irradiated by LIPUS（40mW/cm2）10 minutes, twice a day for 20days. The other side was dammed as control. The dogs were sacrificed on 0, 2, 4,6, 8 week after LIPUS irradiation. The bone samples including implants wereharvested. X-ray of dental implants apical image was taken. Half of the sampleswere stored in saline at–20°C. For pull-out test of dent implant, the bone wasthawed and dried. The bone block was fixed stable, the external part of dentimplant was held fast by steel pliers linked to load cell and distracted at aconstant displacement rate of 0.5mm/min. Both displacement and load wererecorded for later analysis. The result showed both the max load and extractiondisplacement were greater significantly in the experimental group than in thecontrol group (paired t test, P<0.05). The result indicated LIPUS can improve theretention of dental implant.
Experiment six was histological examination of the mental-bone surface ofdental implants. Half of the samples were dehydrated in ethanol and embeddedin methylmethacrylate, sectioned longitudinally at 100-200μm thick forGoldner’s stain. The result showed fibrillar calcified layer and trabecular bonearound the dental implant observable at the implant surface earlier in theexperimental group than in the control group. Later, trabecular bone was gradually substituted by lamellar bone and mature earlier in the experimentalgroup. The result indicated the LIPUS can accelerate osseointegration of dentalimplants.
In summary, based on our study showed LIPUS could enhance blood supplyand metabolic activity at the early period of consolidation and had positive effecton the healing and osteogenesis course of the new bone. LIPUS radiationaccelerated bone formation and mature, increased the BMD and biomechanicalproperty but had no effect on the volume of the new bone. LIPUS may stimulatesynthesis of extracellular matrix proteins altering chondrocyte maturation andendochondral bone formation. LIPUS also can accelerate osseointegration andenhance the retention of dental implants.
实验一:低强度脉冲超声促进牵张成骨新骨成熟动物模型建立。成年杂种犬7只,在双侧下颌骨第1、2前磨牙间行骨切开术,植入内置式牵张器,间歇期为7天,牵引速率为每天2次,0.5mm/次,共20天。随机选一侧为实验侧,另一侧为对照,在牵张过程中实验侧每天定时给予低强度(40mW/cm~2)脉冲超声波刺激,每次10分钟,每天两次,共20天。牵张结束后的0、1、2、4、6、8、12周各处死动物一只,切取双侧下颌骨标本。
实验二:99mTc-MDP核素颌骨扫描。动物处死前静脉注射99mTc-MDP740毫居,注射药物4小时后SPECT扫描采集断层显像,重建三维立体影像。用计算机感兴趣区(ROI)技术分别在实验侧和对照侧对应部位选择兴趣区,并求出实验侧骨/对照侧骨的放射性计数比值,作为半定量分析指标。ROI检查结果:牵张完成早期实验侧较对照侧放射性计数有显著性差异(P<0.05),表明实验侧与对照侧相比骨代谢水平在相同时间内有差别。实验侧早期有较强的成骨现象和再血管化迹象,实验侧成骨活跃,与对照侧相比骨的代谢水平要高,显示了在骨生成方面优于对照侧。牵张完成中后期实验侧新骨处于骨改建期,而对照侧处于骨加速形成期,此时对照侧骨代谢程度高,因而核素浓聚要高于实验侧。实验侧与对照侧在牵张完成4周内骨代谢活跃,随着时间推移,牵张完成6～8周骨代谢趋于稳定,至12周时骨代谢已降低至正常水平。实验证实:超声机械刺激可影响成骨细胞的代谢和增加局部血流量来促进早期牵张新骨的成熟。
实验三:X线平片、三维CT、骨密度和强度检测:拍摄常规X线平片观察双侧牵张新骨的成熟情况。双侧下颌骨标本前部作三维CT扫描并重建,将采集的数据录入图像分析软件计算出牵张新骨区的体积和表面积,进行配对t检验;双能X线骨密度仪测量双侧牵张新骨区对称感兴趣区(RIO)骨密度值,进行配对t检验。一半标本取材后-20℃生理盐水保存,解冻后,室温晾干48小时。用金刚砂磨片切割厚度为3mm的骨片,使用电脑控制的材料力学分析仪进行压缩实验,骨片置于载物台上,连接传感器的压缩杆以1mm/min的速度压迫骨片直至变形;计算机自动记录压力—挠度曲线,得出骨的生物材料力学参数:最大负荷和压缩距离,进行配对t检验。
X线平片显示相同时间内超声照射侧新生骨的生长和成熟早于对照侧。骨密度测量结果显示新骨骨密度随着固定时间延长而增强,相同时间点实验侧与对照侧相比骨密度值有显著性差异(P<0.05)。新骨体积和表面积测量结果显示实验侧与对照侧相比体积和表面积无显著性差异(P>0.05)。新骨强度材料力学检测结果显示实验侧骨最大负荷和压缩距离均高于对照侧(P<0.05)。实验结果表明相同时间内超声照射侧新生骨的生长和成熟快于对照侧,验证了LIPUS可促进牵张新骨的成熟。实验组的骨密度和强度高于对照侧,但三维CT重建显示超声刺激并不能增加骨量。
实验四:牵张新骨组织学观察:一半标本甲醛固定48h,20%EDTA脱钙四周,脱水,石蜡包埋,制作石蜡切片,行常规HE及Masson’s三色法染色,光镜观察。结果显示早期实验侧牵张间隙骨小梁数目和直径大于对照侧,实验侧可见散在的软骨内成骨,后期新骨成熟早于对照侧。表明低强度超声可能通过刺激软骨生成而促进新骨生成。
实验五:低强度脉冲超声促进种植体骨结合的作用观察。成年新西兰大白兔10只,双侧膝关节股骨和胫骨骨骺端植入直径2mm螺旋状纯钛种植体各一枚,共40枚。随机选一侧为实验侧,另一侧为对照,实验侧每天给予低强度(40mW/cm2)脉冲超声波刺激,每次10分钟,每天两次,共20天。结束后的0、2、4、6、8周各处死动物两只,切取含种植体骨标本,拍摄X线牙片,一半标本进行种植体拔出实验。标本取材后-20℃生理盐水保存,解冻后,擦干。使用电脑控制的材料力学分析仪进行种植体拔出实验,得出力学参数:最大负荷和拔出位移,进行配对t检验。X线显示实验组和对照组种植体周围没有放射透视影,形成良好的骨结合。种植体拔出实验结果实验组最大负荷和拔出位移负荷均大于对照侧(P<0.05)。表明低强度脉冲超声可以促进种植体骨愈合,增强骨结合。
实验六:组织学观察低强度脉冲超声促进种植体骨结合。一半标本酒精脱水,树脂包埋,制作不脱钙组织切片,Goldner’s三色染色。结果显示实验组骨结合早于对照侧,骨成熟早于对照侧。表明低强度脉冲超声可以促进种植体界面骨结合。
综上所述,本实验结果显示低强度脉冲超声可以促进牵张新骨区早期血供和骨代谢水平,实验侧牵张新骨成熟早于对照侧,新骨密度和强度增加,但骨量无显著变化。超声可能通过刺激软骨内成骨而促进新骨成熟。同时,显示低强度脉冲超声可以促进种植体的早期骨结合,增强种植体的固位。
The treatment of maxillofacial bone defect due to tumor and trauma is notonly to reconstruct the facial contour but also to resume mastication function.Distraction osteogenesis (DO) has had an enormous impact and played a majorrole in the reconstruction of congenital and acquired deformities of thecraniofacial skeleton. Dental implants can resume mastication functionefficiently. However, the main disadvantage of DO is the long treatment timewhich requires the distracted bone segment be fixed by the distraction device fora certain period of consolidation to facilitate the new bone mature andremodeled. This imposes a psychological effect on the patient and familybecause several months are needed before the woven bone becomes compactbone to allow for distractor removal. Ilizarov suggested that the consolidationtime should be no less than two or three times as long as（6～8weeks usually）the distraction time. Long-time retention can result in inconvenience andexpensive inpatient cost and there were also some complications such as pseudarthrosis and relapse were reported. After dental implants inserted intobone, it costs at least 2～3 months to achieve osseointegration before acceptingdenture loading, which means dental implant is almost completely covered by acompact, mature, newly formed bone. An early good biological fixation mayallow the shortening of time before loading the implant, favoring the clinicalprocedure of early or immediate implant loading. Now, how to shorten theconsolidation time and enhance the mechanical property of the new distractedbone is the focus research field of DO. Also how to intensify osseointegration ofdental implants is the front study of dentistry.
Low-intensity pulsed ultrasound (LIPUS) is a form of mechanical energywhich is transmitted through and into biological tissues as an acoustic pressurewave and has been widely used in medicine as a diagnostic and therapeutic tool.Application of LIPUS (30～50mW/cm2) was considered to have little thermaleffect and to produce stable cavitation and streaming. There are many papers anddocuments published to establish LIPUS can accelerate the healing of fresh bonefracture and nonunion. LIPUS is an ideal physical stimulus to enhance boneformation. If ultrasound stimulation could accelerate the rate of the distractedcallus formation and remodification, the treatment period could be shortened,complications would decrease. Similarly, If ultrasound stimulation couldimprove the osseointegration of dental implants, patients would eat what theywant to as soon as possible. In this study, we observed the effect of LIPUS onthe enhancement of bone formation during mandible distraction osteogenesis indogs and improvement of osseointegration of dental implant in rabbits.
Experiment one was the animal model creation of enhancement of boneformation of mandible distraction osteogenesis by LIPUS. Bilateral surgical cutswere made in the mandible of seven dogs between the first and second premolar region. The anterior mandibles were lengthened by 20mm at the rate of 1mm/d,twice a day. During the distraction period one lateral distraction gap wasirradiated by LIPUS 10 minutes, twice a day and the other side was shamirradiated as control. After distraction was completed, the dogs were sacrificedon 0，1，2，4，6，8，12 week. The mandible samples were harvested.
Experiment two was 99mTc-methylene diphosphonate (MDP) bone imaging.Before the animals sacrifice, the animals were injected bolus 740 MBq 99mTc-MDP intravenously. Four hours later, delayed static bone scanning was obtainedby SPECT. For semiquantitative analysis, we set manually the region of interest(ROI) on the LIPUS stimulated distraction area and set a symmetric ROI on thecontralateral area . The result showed the uptake of 99mTc-MDP in theexperimental side was significant (P<0.05) higher than that of the control side inthe early period of consolidation (before the fourth week). But later the situationwas reversed, the uptake of 99mTc-MDP in the control side was significanthigher than that of the experimental side (P<0.05). There were no significantradioactivity differences between two sides at the 12th week. It was indicated theLIPUS stimulated area had more blood supply and metabolic activity. LIPUSradiation had positive effect on the healing and osteogenesis course of the newbone.
Experiment three was X-ray plain, three-dimension (3D) CT scan，bonemineral density (BMD) and biomechanical property measurement. The wholemandible samples were harvested and regular plain X-ray photographs weretaken. The front part of the mandibles were scanned with LightSpeed VCT 64-slice Scanner and 3D-images was obtained. Input 3D-CT scan data of themandible to the computer and used graphic information processing software tocalculate the volume and the surface area of the distracted bone. The BMD of the distracted bone was measured by dual-energy X-ray absorptiometry. Half of thebone samples were stored in saline at–20°C. Before testing, the bone wasthawed and dried for 48 hours at room temperature. The bone block was sectioninto 3mm thick slices by diamond blade. The biomechanical test was performedin a computed servohydraulic materials testing system. The slices were placed onthe on the smooth surface of a steel disk and axially compressed by the smoothsurface of a steel rod attached to load cell at a constant speed of 1 mm/min. Bothdisplacement and load were recorded for later analysis.
X-ray plain showed the new bone in the experimental side was matureearlier than that of the control side. The volume and the surface areameasurement of distracted area showed there was not significant differencebetween the LIPUS stimulated side and the control side ( paired t test, P > 0.05) .BMD measurement showed at the same time the BMD of LIPUS stimulated sidewas significantly higher than that of the control side (paired t test, P < 0.05). Theresult showed LIPUS could enhance the biomechanical property of new bone.
It was indicated the LIPUS radiation accelerate bone mature and increasethe BMD and mechanical property during mandible distraction osteogenesis buthad no effect on the volume of the new bone.
Experiment four was histological examination. One half of the bone sampleswere fixed in 4% paraformaldehyde for 48 hours and decalcified in 20% EDTAfor four weeks. The specimens were dehydrated in ethanol and embedded inparaffin, sectioned longitudinally at 4μm thick, stained for HE and Masson’sstain. The trabecula of the experimental side was more and thicker than that ofthe control side at the early period of consolidation. But in the late period, therewas not significant difference. We observed endochondral bone formation in theexperimental side at the 2w and 4w of consolidation. LIPUS may stimulate synthesis of extracellular matrix proteins altering chondrocyte maturation andendochondral bone formation. The modulation by ultrasound may occur byaccelerating endochondral ossification through action on chondrocytes, yetdistraction osteogenesis is mainly intramembranous.
Experiment five was the effect of LIPUS on the osseointegration of titaniumdental implants. 10 New Zealand rabbits were used in this study. Epiphyses ofboth femur and tibiae were inserted one screw titanium implants (? 2mm)separately in bilateral knee joints, total forty. One lateral knee joint includingimplants was irradiated by LIPUS（40mW/cm2）10 minutes, twice a day for 20days. The other side was dammed as control. The dogs were sacrificed on 0, 2, 4,6, 8 week after LIPUS irradiation. The bone samples including implants wereharvested. X-ray of dental implants apical image was taken. Half of the sampleswere stored in saline at–20°C. For pull-out test of dent implant, the bone wasthawed and dried. The bone block was fixed stable, the external part of dentimplant was held fast by steel pliers linked to load cell and distracted at aconstant displacement rate of 0.5mm/min. Both displacement and load wererecorded for later analysis. The result showed both the max load and extractiondisplacement were greater significantly in the experimental group than in thecontrol group (paired t test, P<0.05). The result indicated LIPUS can improve theretention of dental implant.
Experiment six was histological examination of the mental-bone surface ofdental implants. Half of the samples were dehydrated in ethanol and embeddedin methylmethacrylate, sectioned longitudinally at 100-200μm thick forGoldner’s stain. The result showed fibrillar calcified layer and trabecular bonearound the dental implant observable at the implant surface earlier in theexperimental group than in the control group. Later, trabecular bone was gradually substituted by lamellar bone and mature earlier in the experimentalgroup. The result indicated the LIPUS can accelerate osseointegration of dentalimplants.
In summary, based on our study showed LIPUS could enhance blood supplyand metabolic activity at the early period of consolidation and had positive effecton the healing and osteogenesis course of the new bone. LIPUS radiationaccelerated bone formation and mature, increased the BMD and biomechanicalproperty but had no effect on the volume of the new bone. LIPUS may stimulatesynthesis of extracellular matrix proteins altering chondrocyte maturation andendochondral bone formation. LIPUS also can accelerate osseointegration andenhance the retention of dental implants.
引文
1. Ilizarov GA. Basic principle of transosseus compression and distractionosteogenesis. Ortop Travmatol protez. 1971;32:7
2. Swennen G, Schliephake H, Dempf R, Schierle H, Malevez C.Craniofacial distraction osteogenesis: a review of the literature: Part 1:clinical studies. Int J Oral Maxillofac Surg 2001 Apr;30(2):89-103
3.刘印文，汤荣光，蔡体栋.低强度超声促进骨折愈合研究进展.国外医学骨科学分册，2001;22(1):28-31
4. US Food and Drug Administration (FDA) : Sonic Accelerated FractureHealing System (SAFHS) , Model 2A: Summary of Safety andEffectiveness. Premarket Approval P900009. Exogen , Inc , Rockville ,MD , US Food and Drug Administration , October 5 ,1994.
5. US Food and Drug Administration ( FDA) : Exogen 2000 , 3000 , or SonicAccelerated Fracture Healing System (SAFHS) : Summary of Safety andEffectiveness. Premarket Appreval P900009/ Supplement 6. Exogen , Inc ,Rockville , MD. US Food and Drug Administration , February 22 ,2000.
6. Heybeli N, Ye?ilda? A, Oyar O, Gülsoy UK, Tekinsoy MA, Mumcu EF.Diagnostic ultrasound treatment increases the bone fracture healing rate inan internally fixed rat femoral osteotomy model. J Ultrasound Med 2002Dec;21(12):1357-63
7. Busse JW, Bhandari M, Kulkarni AV, Tunks E. The effect of low-intensitypulsed ultrasound therapy on time to fracture healing: a meta-analysis.CMAJ 2002 Feb 19;166(4):437-41
8. Nolte PA, van der Krans A, Patka P, Janssen IM, Ryaby JP, Albers GH.Low-intensity pulsed ultrasound in the treatment of nonunions. J Trauma2001 Oct;51(4):693-702
9. Ilizarov GA. Clinical application of the tension stress effect for limblengthening [ J ]. Clin Orthop, 1990, 250: 8.
10. Corcoran J, Hubli EH, Salyer KE. Distraction osteogenesis ofcostochondral neomandibles: a clinical experience [ J ]. Plast ReconstrSurg, 1997, 100: 311- 5.
11. Wiltfang J, Kessler P,Merten HA, et al. Continuous and intermittent bonedistraction using a microhydraulic cylinder: an experimental study inminipigs [ J ]. Br J OralMaxillofac Surg, 2001, 39: 2-7.
12. Kessler PA, Merten HA, Neukam FW, et al. The effects of magnitude andfrequency of distraction factors on tissue regeneration in distractionosteogenesis of the mandible [ J ]. Plast Reconstr Surg, 2002, 109: 171- 80.
13. Smith SW, Sachdeva RC, Cope JB. Evaluation of the consolidation periodduring osteodistraction using computed tomography. Am J OrthodDentofacial Orthop. 1999;116(3):254-63.
14.刘彦普，邵祯，刘宝林，陈富林下颌骨牵张成骨和牵张器拆除时机的评价中国临床康复2003；7（2）：42～3
15.牛学刚，赵铱民，吴仲寅，韩小宪犬颧骨牵张成骨后种植体植入时机的实验研究中国口腔种植学杂志2006；11（2）：59
16.龙洁，樊瑜波，田卫东，吴玲，郑晓辉下颌骨牵张成骨中牵张器拆除时机的实验研究口腔颌面外科杂志2006;16（2）：102
17.刘瑞峰，周树夏，王翔，邵祯，刘彦普下颌骨延长术后牵张器拆除时机的实验研究临床口腔医学杂志2005;21（2）：67
18.周诺，麦华明，梁飞新，韦山良，范颖峰，赵亮狗下颌骨牵张成骨稳定性的头影测量分析广西医科大学学报2004;21（5）：655
19. Cho BC , Moon J H , Chung HY, et al. The bone regenerative effect ofgrowth hormone on consolidation in mandibular distraction osteogenesis ofa dog model. [J ] . J Craniofac Surg , 2003 ,14 (3) : 417-425.
20. Bail HJ , Raschke MJ , Kolbeck S ,et al. Recombinant species specificgrowth hormone increases hard callus formation in distraction osteogenesis.[J ] .Bone ,2002 ,30 (1) :117-124.
21. Seebach C , Skripitz R , Andreassen TT , et al. Intermittent parathyroidhormone enhances mechanical strength and density of new bone afterdistraction osteogenesis in rats. [J ] . Orthop Res ,2004 , 22 (3) :472-478.
22. Kitakoji T , Takashi S , Ono Y, et al . Effect of hyperbaric oxygenationtreatment on lengthened callus. Undersea Hyper Med , 1999 ,26 :165-168.
23. Tsubota S, Tsuchiya H, Shinokawa Y, et al. Transplantation of osteoblastlikecells to the distracted callus in rabbits [J]. J Bone Joint Surg1999;81B(1):125-129.
24. Richards M , Huibregtse BA , Caplan AI , et al. Marrow-derived progenitorcell injections enhance new bone formation during distraction [J ] . JOrthop Res ,1999 ,17 (6) :900-908.
25. Takamine Y, Tsuchiya H , Kitakoji T , et al. Distraction osteogenesisenhanced by osteoblastlike cells and collagen gel [ J ] . Clin Orthop , 2002,64 (399) :240-246.
26. Hagino T, Hamada Y. Accelerating bone formation and earlier healing afterusing demineralized bone matrix for limb lengthening in rabbits [J]. JOrthop Res 1999;17(2):232-237.
27. Little DG, Cornell MS, Briody J, et al. Intravenous pamidronate reducesosteoporosis and improves formation of the regenerate during distractionosteogenesis.A study in immature rabbits [J]. J Bone Joint Surg,2001;83B(7):1069-1074.
28. Willams PR, Smith NC, Cooke Yarborough, et al. Bisphosphonates andnephrocalcinosis in a rabbit leg lengthening model: a histological andtherapeutic comparison[J].Pharmacol Toxicol, 2001;89(3):149-152.
29. Al Ruhaimi KA. Effect of calcium sulphate on the rate of osteogenesis indistracted bone. Int J Oral Maxillofac Surg , 2001 ,30 : 228-233.
30.王志国，胡静，邹淑娟等重组人骨形成蛋白22促进兔下颌牵张成骨的研究[J ]华西口腔医学杂志,2004 ,22 (3) :286-288
31. Terheyden H , Wang H , Warnke PH , et al. Acceleration of callusmaturation using rhOP21 (bmp27) in mandibular distraction osteogenesisin a rat model. [J ] J Oral Maxillofac Surg , 2003;32 (5) :528-533.
32. Hasse A , Porksen M , Schultze S , et al. Effect of bFGF on regeneration ofdistracted mandibles after radiation [ J ] . Mund Kiefer Gesichtschir 2000,4 (Suppl2) :423～427.
33. Okazaki H , Kurokawa T , Nakamura K, et al. Stimulation of boneformation by recombinant fibroblast growth factor22 in callotasis bonelengthening of rabbits [ J ] . Calcif Tissue Int , 1999 , 64 ( 6) : 542 -546.
34. Stewart KJ , Weyand B , Van’t Hof RJ , et al . A quantitative analysis of theeffect of insulin-like growth factor-1 infusion during mandibular distractionosteogenesis in rabbit s. Br J Plast Surg , 1999 ,52 : 343-350.
35. Sciadini MF , Dawson JM , Banit D , et al . Growth factor modulation ofdistraction osteogenesis in a segmental defect model . Clin Ort hop , 2000 ,381 :266-277.
36.骆泉丰,王兴,王晓霞等富含血小板血浆对牵引成骨过程中新骨生成的影响[J ]中华整形外科杂志2004；20 (5) :367-379
37. Kitoh H , Kitakoji T , Tsuchiya H , et al. Transplantation of marrow-derivedmesenchymal stem cells and platelet2rich plasma during distractionosteogenesis-a preliminary result of three cases [ J ] . Bone , 2004；35 (4):892-898.
38.胡静，戚孟春，韩立赤等. BMP27基因促进大鼠下颌牵张成骨的研究[ J ].实用口腔医学杂志, 2006, 22 (5) : 635-638.
39. ZhaoM, Zhao Z, Koh JT, et al. Combinatorial gene therapy for boneregeneration: cooperative interactions between adenovirus vectorexpressing bone morphogenetic proteins 2, 4, and 7 [ J ]. Cell Biochem,2005, 95 (1) : 1-16.
40. Ashinoff RL, Cetrulo CL, Galiano RD, et al. Bone morphogenic protein22gene therapy for mandibular distraction osteogenesis. [ J ] Ann Plast Surg,2004, 52 (6) : 585-590.
41.周诺，黄旋平，廖妮等.人骨形态发生蛋白22基因克隆及真核表达载体的构建[ J ].华西口腔医学杂志, 2007, 25 ( 5 ) :487-489.
42. Hagiwara T, Bell WH. Effect of electrical stimulation on mandibulardistraction osteogenesis[J]. J Craniomaxillofac Surg, 2000,28(1):12-19.
43.王战鑫,彭铁男,孙宏晨.弱激光对牵张成骨作用的实验研究[ J ].口腔医学研究2004;20 (3) : 256-258.
44. Shimazaki A, Inui k, Azuma Y, et al. Low～intensity pulsed ultrasoundaccelerates bone maturation in distraction osteogenesis in rabbits. J BoneJoint Surg Br 2000 Sep;82(7):1077～82
45. Sakurakichi K, Tsuchiya H, Uehara K, et al. Effects of timing of lowintensitypulsed ultrasound on distraction osteogenesis. J Orthop Res.2004 Mar;22(2):395-403.
46. Tsumaki N, Kakiuchi M, Saaski J, et al. Low-intensity pulsed ultrasoundaccelerates maturation of callus in patients treated with opening-wedgehigh tibial osteotomy by hemicallotasis. J Bone Joint Surg Am. 2004Nov;86-A(11):2399-405.
47. Esenwein SA, Dudda M, Pommer A, et al. Efficiency of low intensitypulsed ultrasound on distraction osteogenesis in case of delayed callotasisclinical results. [J] J Zentralbl Chir, 2004,129(5):413-20
48. Machen MS, Tis JE, Inoue N, Meffert RH, et al. The effect of lowintensity pulsed ultrasound on regenerate bone in a less than rigidbiomechanical environment. Biomed Mater Eng 2002;12(3):239-47
49. Eberson CP, Hogan KA, Moore DC, et al. Effect of low-intensityultrasound stimulation on consolidation of the regenerate zone in a ratmodel of distraction osteogenesis. J Pediatr Orthop 2003 Jan～Feb;23(1):46-51
50. Tis JE, Meffert CR, Inoue N. The effect of low intensity pulsedultrasound applied to rabbit tibiae during the consolidation phase ofdistraction osteogenesis. J Orthop Res 2002 Jul;20(4):793-800
51. Yasuda I. Electrical callus and callus formation by electret. Clin Orthop ,1977；124 :5-8.
52. Parvizi J, Parpura J, Greenleaf JF, et al. Calcium signaling is required forultrasound-stimulated aggrecan synthesis by rat chondrocytes. J OrthopRes, 2002, 20（1）：51- 57.
53. Rawool NM, Goldberg BB, Forsberg F, et al. Power Doppler assessmentof vascular changes during fracture treatment with low-intensity ultrasound.J Ultrasound Med, 2003, 22（2）：145- 153.
54. Ryaby JT, Bacher EJ ,Bendo JA , et al. Low-intensity pulsed ultrasoundincreased calcium incorporation in both differentiating cartilage and bonecell cultures. Trans Ortho Res Soc ,1996 ,21 :622.
55. Hadjiargyrou M, McLeod K, Ryaby JP, Rubin C. Enhancement offracture healing by low-intensity ultrasound. J Clin Orthop1998,355(suppl):216-218
56. Parvizi J, Wu CC, Lewallen DG, Greenleaf JF, Bolander ME. Lowintensityultrasound stimulates proteoglycan synthesis in rat chondrocytesby increasing aggrecan gene expression. J Orthop Res 1999Jul;17(4):488-94
57. Warden SJ, Favaloro JM, Bennell KL, McMeeken JM, Ng KW, Zajac JD,Wark JD. Low-intensity pulsed ultrasound stimulates a bone formingresponse in UMR-106 cells. Biochem Biophys Res Commun 2001 Aug24;286(3):443-50
58. Sun JS, Hong RC, Chang WH, Chen LT, Lin FH, Liu HC. In vitro effects oflow-intensity ultrasound stimulation on the bone cells. J Biomed MaterRes 2001 Dec 5;57(3):449-56
59.韩雪，宋九余；口腔种植体骨界面骨整合的思考.医学与哲学(临床决策论坛版) 2007;28(11):40-41
60. Ishikawa K, Miyamoto Y, Nagayama M, Asaoka K. Blast coatingmethod: new method of coating titanium surface with hydroxyapatite atroom temperature. J Biomed Mater Res. 1997 Summer;38(2):129-34.
61. Chang YL, Stanford CM, Wefel JS, et al. Osteoblastic cell attachment tohydroxyapatite coated implant surfaces in vitro[ J ]. Int J OralMaxillofacImplants, 1999, 14 (2)∶239– 247
62. Soskolne WA, Cohen S, Sennerby L, et al. The effect of titanium surfaceroughness on the adhesion of monocytes and their secretion of TNF - alphaand PGE2 [ J ]. Clin Oral Imp lants Res, 2002, 13 (1)∶86– 93
63. Zhang H, Ahmad M, Gronowicz G. Effects of transforming growth factorbeta 1 (TGF-beta1) on in vitro mineralization of human osteoblasts onimplant materials. Biomaterials. 2003 May;24(12):2013-20.
64. Pilliar RM, Deporter DA, Watson PA, Todescan R. The Endopore implantenhanced osseointegration with a sintered porous-surfaced design. OralHealth. 1998 Jul;88(7):61-4.
65. Eric J, Jenneke K, Elisabeth H, et al. Transforming growth factor -β1incorporated in calcium phosphate cement stimulates osteotransductivity inrat bone defects [ J ]. Clin Oral Implants Res, 2001, 12 (6)∶609– 616
66. Fini M, Cadossi R, Cane V, et all. The effect of pulsed electromagneticfields on the osteointegration of hydroxyapatite implants in cancellousbone: a morphologic and microstructural in vivo study. J Orthop Res2002,20:756-763
67. Fini M, Giavaresi G, Giardino R, et all. Histomorphometric andmechanical analysis of the hydroxyapatite-bone interface afterelectromagnetic stimulation: an experimental study in rabbits. J Bone JointSurg Br 2006,88:123-128
68. Ottani V, Raspanti M, Martini D, et all. Electromagnetic stimulation on thebone growth using backscattered electron imaging. Micron 2002,33:121-125
69. Matsumoto H, Ochi M, Abiko Y, et all. Pulsed electromagnetic fieldspromote bone formation around dental implants inserted into the femur ofrabbits. Clin Oral Implants Res.2000 Aug, 11 (4) :354-360
70.张树标,朱健,刘江峰,潘广嗣He～Ne激光影响纯钛种植体骨性愈合的实验研究中国口腔种植学杂志2001;6(1):4
71.李炎洋氦氖激光对促进人工种植牙骨整合的临床研究应用激光2001 ;21(5):354
72.徐淑兰,关虹,孙晓东,毕绍臣微直流电对非埋植型即刻钛牙种植体骨整合影响的实验研究广东牙病防治2001;9(3):166
73. Tanzer M, Kantor S, Bobyn JD. Enhancement of bone growth into porousintramedullary implants using noninvasive low intensity ultrasound. JOrthop Res,2001,19:195-199
74. Iwai T, Harada Y, Imura K, et all. Low-intensity pulsed ultrasoundincreases bone ingrowth into porous hydroxyapatite ceramic. J Bone MinerMetab, 2007,25 (6) :392-399
75. Dyson M, Brookes M. Non-invasive low-intersity pulsed ultrasoundaccelerates bone healing in the rabbit. Ultrasond Med Biol ,1983,(suppl)2:61-66.
76. Duarte LR. The stimulation of bone growth by ultrasound. ArchOrthopTrauma Surg 1983 ,101 :153～159.
77. Warden SJ, Bennell KL, McMeeken JM, Wark JD. Acceleration of freshfracture repair using the sonic accelerated fracture healing system (SAFHS):a review. Calcif Tissue Int. 2000 Feb;66(2):157-63.
78. Tsai CL, Chang WH, Liu TK, Song GM. Ultrasound can affect bonehealing both locally and systemically. Chin J Physiol. 1991;34(2):213-22.
79. Reher P, Elbeshir NI, Harvey W, Meghji S, Harris M. The stimulation ofbone formation in vitro by therapeutic ultrasound. Ultrasound Med Biol.1997;23(8):1251－8.
80. Wang SJ ,Lewallen DG,Bolander ME , et al. Low intensity ultrasoundtreatment increases strength in a rat femoral fracture model. OrthopRes ,1994 ,12 :40-47.
81. Tsai CL, Chang WH, Liu TK, Song GM. Ultrasonic effect on fracture repairand prostaglandin E2 production. Chin J Physiol. 1992;35(1):27-34.
82. Jingushi S , Azuma V , lto M, et al. Effects of non invasive pulsed lowintensityultrasound on rat femoral fracture. In Proceedings of the ThirdWorld Congress of Biomechanics ,1998 ,175.
83. Schumann D, Kujat R, Zellner J, et al. Treatment of human mesenchymalstem cells with pulsed low intensity ultrasound enhances the chondrogenicphenotype in vitro. Biorheology, 2006, 43（3/4）：431- 443.
84.王成综述，曾融生低强度脉冲超声在颌骨牵张成骨中的应用国际口腔医学杂志2007；34（5）：374-377
85. Chan CW, Qin L, Lee KM, Zhang M, Cheng JC, Leung KS. Low intensitypulsed ultrasound accelerated bone remodeling during consolidation stageof distraction osteogenesis. J Orthop Res. 2006 Feb;24(2):263-70.
86.潘中允.临床核医学[M].北京:原子能出版社. 1994，271-291 .
87.杨东伟，温广武，高晓辉，王邦康核素骨显像在局部应用rh-BMP/bFGF加速山羊下颌骨牵张成骨实验研究中的应用价值临床口腔医学杂志2005；21（9）：518-21
88.李继华，胡静，王大章，祝颂松，应彬彬，韩立赤牵张成骨延长下颌骨过程中血管生成数量的定量检测口腔医学2005；2 5（6）：332-35
89. Kanishi D. 99mTc-MDP accumulation mechanisms in bone. Oral Surg OralMed Oral Pathol, 1993;75(2):239
90. Yasui N, Sato M, Ochi T, Kimura T, Kawahata H, Kitamura Y, Nomura S.Three modes of ossification during distraction osteogenesis in the rat. JBone Joint Surg Br. 1997 Sep;79(5):824-30.
91. Sun JS, Tsuang YH, Lin FH, Liu HC, Tsai CZ, Chang WH. Bone defecthealing enhanced by ultrasound stimulation: an in vitro tissue culturemodel. J Biomed Mater Res. 1999 Aug;46(2):253-61.
92. Leung KS, Cheung WH, Zhang C, Lee KM, Lo HK. Low intensity pulsedultrasound stimulates osteogenic activity of human periosteal cells. ClinOrthop Relat Res. 2004 Jan;(418):253-9.
93. Naruse K, Miyauchi A, Itoman M, Mikuni-Takagaki Y. Distinct anabolicresponse of osteoblast to low-intensity pulsed ultrasound. J Bone MinerRes. 2003 Feb;18(2):360-9.
94. Kudo O, Sabokbar A, Pocock A, Itonaga I, Fujikawa Y, Athanasou NA.Interleukin-6 and interleukin-11 support human osteoclast formation by aRANKL-independent mechanism. Bone. 2003 Jan;32(1):1-7.
95. Lee SE, Chung WJ, Kwak HB, Chung CH, Kwack KB, Lee ZH, Kim HH.Tumor necrosis factor-alpha supports the survival of osteoclasts throughthe activation of Akt and ERK. J Biol Chem. 2001 Dec 28;276(52):49343-9.
96. Wu CC，Lewallen DG，Bolander ME，et al. Exposure to low intensityultrasound stimulate aggrecan gene expression by cultured chondrocyte.Trans Ortho Res Soc ,1996 ,21 :622.
97. Mukai S, Ito H, Nakagawa Y, Akiyama H, Miyamoto M, Nakamura T.Transforming growth factor-beta1 mediates the effects of low-intensitypulsed ultrasound in chondrocytes. Ultrasound Med Biol. 2005Dec;31(12):1713-21.
98.黄静文综述，谢培豪。生物物理学刺激对种植体周围骨重建的影响中国口腔种植学杂志2009；14（1）：33-36
99. JoosU, BuchterA, Wiesmann HP, et al. Strain driven fast osseointegrationof implants. Head Face Med, 2005,1 (1) : 1～6
100. Romanos GE, Toh CG, Siar CH, et al. Histologic and histomophometricevaluation of peri -implant bone subjected to immediate loading: anexperimental study with macaca fascicularis. Int J Oral MaxillofacImplants,2002,17 (1) : 44～45
101. Periklis P, Kan J, Lozada J, et al. Effects of immediate loading withthreaded hydroxyapatite -coated root form implants on single premoalrreplacements: a preliminary report. Int J OralMaxillofac Implants, 2002,17(4) : 567～572
102. MarcoD, Giovanna P, Giovanna Z, et al. Histological evaluation of humanimmediately loaded implants inserted in the posterior mandible andretrieved after 6 months. J Oral Implantol, 2003,5 (2) : 223～230
103. MeyerU, JoosU, Mythili J, et al. Ultrastructural characterization of theimplant-bone interface of immediately loaded dental implants.Biomaterials, 2004,25(10) : 1959～1967
104. Vandamme K, Naert I, Geris L, et all. The effect of micro-motion on thetissue response around immediately loaded roughened titanium implants inthe rabbit. Eur J Oral Sci,2007 Feb,115 (1) :21～29
105. Barone A, Covani U, Cornelini R, et al. Radiographic bone density aroundimmediately loaded oral implants. Clin Oral Implants Res,2003 Oct,14 (5) :610～615
106. Turner CH, Robling AG. Exercise as an anabolic stimulus for bone. CurrPharmDes,2004,10 ( 21) :2629～2641
107. Cruber Helen. E. Adaptation of Goldner’s Masson Trichrome stain for thestudy of undecalcified plastic embedded bone [J].BiotechnicHistochemistry 1992,67(1)30-34
2. Swennen G, Schliephake H, Dempf R, Schierle H, Malevez C.Craniofacial distraction osteogenesis: a review of the literature: Part 1:clinical studies. Int J Oral Maxillofac Surg 2001 Apr;30(2):89-103
3.刘印文，汤荣光，蔡体栋.低强度超声促进骨折愈合研究进展.国外医学骨科学分册，2001;22(1):28-31
4. US Food and Drug Administration (FDA) : Sonic Accelerated FractureHealing System (SAFHS) , Model 2A: Summary of Safety andEffectiveness. Premarket Approval P900009. Exogen , Inc , Rockville ,MD , US Food and Drug Administration , October 5 ,1994.
5. US Food and Drug Administration ( FDA) : Exogen 2000 , 3000 , or SonicAccelerated Fracture Healing System (SAFHS) : Summary of Safety andEffectiveness. Premarket Appreval P900009/ Supplement 6. Exogen , Inc ,Rockville , MD. US Food and Drug Administration , February 22 ,2000.
6. Heybeli N, Ye?ilda? A, Oyar O, Gülsoy UK, Tekinsoy MA, Mumcu EF.Diagnostic ultrasound treatment increases the bone fracture healing rate inan internally fixed rat femoral osteotomy model. J Ultrasound Med 2002Dec;21(12):1357-63
7. Busse JW, Bhandari M, Kulkarni AV, Tunks E. The effect of low-intensitypulsed ultrasound therapy on time to fracture healing: a meta-analysis.CMAJ 2002 Feb 19;166(4):437-41
8. Nolte PA, van der Krans A, Patka P, Janssen IM, Ryaby JP, Albers GH.Low-intensity pulsed ultrasound in the treatment of nonunions. J Trauma2001 Oct;51(4):693-702
9. Ilizarov GA. Clinical application of the tension stress effect for limblengthening [ J ]. Clin Orthop, 1990, 250: 8.
10. Corcoran J, Hubli EH, Salyer KE. Distraction osteogenesis ofcostochondral neomandibles: a clinical experience [ J ]. Plast ReconstrSurg, 1997, 100: 311- 5.
11. Wiltfang J, Kessler P,Merten HA, et al. Continuous and intermittent bonedistraction using a microhydraulic cylinder: an experimental study inminipigs [ J ]. Br J OralMaxillofac Surg, 2001, 39: 2-7.
12. Kessler PA, Merten HA, Neukam FW, et al. The effects of magnitude andfrequency of distraction factors on tissue regeneration in distractionosteogenesis of the mandible [ J ]. Plast Reconstr Surg, 2002, 109: 171- 80.
13. Smith SW, Sachdeva RC, Cope JB. Evaluation of the consolidation periodduring osteodistraction using computed tomography. Am J OrthodDentofacial Orthop. 1999;116(3):254-63.
14.刘彦普，邵祯，刘宝林，陈富林下颌骨牵张成骨和牵张器拆除时机的评价中国临床康复2003；7（2）：42～3
15.牛学刚，赵铱民，吴仲寅，韩小宪犬颧骨牵张成骨后种植体植入时机的实验研究中国口腔种植学杂志2006；11（2）：59
16.龙洁，樊瑜波，田卫东，吴玲，郑晓辉下颌骨牵张成骨中牵张器拆除时机的实验研究口腔颌面外科杂志2006;16（2）：102
17.刘瑞峰，周树夏，王翔，邵祯，刘彦普下颌骨延长术后牵张器拆除时机的实验研究临床口腔医学杂志2005;21（2）：67
18.周诺，麦华明，梁飞新，韦山良，范颖峰，赵亮狗下颌骨牵张成骨稳定性的头影测量分析广西医科大学学报2004;21（5）：655
19. Cho BC , Moon J H , Chung HY, et al. The bone regenerative effect ofgrowth hormone on consolidation in mandibular distraction osteogenesis ofa dog model. [J ] . J Craniofac Surg , 2003 ,14 (3) : 417-425.
20. Bail HJ , Raschke MJ , Kolbeck S ,et al. Recombinant species specificgrowth hormone increases hard callus formation in distraction osteogenesis.[J ] .Bone ,2002 ,30 (1) :117-124.
21. Seebach C , Skripitz R , Andreassen TT , et al. Intermittent parathyroidhormone enhances mechanical strength and density of new bone afterdistraction osteogenesis in rats. [J ] . Orthop Res ,2004 , 22 (3) :472-478.
22. Kitakoji T , Takashi S , Ono Y, et al . Effect of hyperbaric oxygenationtreatment on lengthened callus. Undersea Hyper Med , 1999 ,26 :165-168.
23. Tsubota S, Tsuchiya H, Shinokawa Y, et al. Transplantation of osteoblastlikecells to the distracted callus in rabbits [J]. J Bone Joint Surg1999;81B(1):125-129.
24. Richards M , Huibregtse BA , Caplan AI , et al. Marrow-derived progenitorcell injections enhance new bone formation during distraction [J ] . JOrthop Res ,1999 ,17 (6) :900-908.
25. Takamine Y, Tsuchiya H , Kitakoji T , et al. Distraction osteogenesisenhanced by osteoblastlike cells and collagen gel [ J ] . Clin Orthop , 2002,64 (399) :240-246.
26. Hagino T, Hamada Y. Accelerating bone formation and earlier healing afterusing demineralized bone matrix for limb lengthening in rabbits [J]. JOrthop Res 1999;17(2):232-237.
27. Little DG, Cornell MS, Briody J, et al. Intravenous pamidronate reducesosteoporosis and improves formation of the regenerate during distractionosteogenesis.A study in immature rabbits [J]. J Bone Joint Surg,2001;83B(7):1069-1074.
28. Willams PR, Smith NC, Cooke Yarborough, et al. Bisphosphonates andnephrocalcinosis in a rabbit leg lengthening model: a histological andtherapeutic comparison[J].Pharmacol Toxicol, 2001;89(3):149-152.
29. Al Ruhaimi KA. Effect of calcium sulphate on the rate of osteogenesis indistracted bone. Int J Oral Maxillofac Surg , 2001 ,30 : 228-233.
30.王志国，胡静，邹淑娟等重组人骨形成蛋白22促进兔下颌牵张成骨的研究[J ]华西口腔医学杂志,2004 ,22 (3) :286-288
31. Terheyden H , Wang H , Warnke PH , et al. Acceleration of callusmaturation using rhOP21 (bmp27) in mandibular distraction osteogenesisin a rat model. [J ] J Oral Maxillofac Surg , 2003;32 (5) :528-533.
32. Hasse A , Porksen M , Schultze S , et al. Effect of bFGF on regeneration ofdistracted mandibles after radiation [ J ] . Mund Kiefer Gesichtschir 2000,4 (Suppl2) :423～427.
33. Okazaki H , Kurokawa T , Nakamura K, et al. Stimulation of boneformation by recombinant fibroblast growth factor22 in callotasis bonelengthening of rabbits [ J ] . Calcif Tissue Int , 1999 , 64 ( 6) : 542 -546.
34. Stewart KJ , Weyand B , Van’t Hof RJ , et al . A quantitative analysis of theeffect of insulin-like growth factor-1 infusion during mandibular distractionosteogenesis in rabbit s. Br J Plast Surg , 1999 ,52 : 343-350.
35. Sciadini MF , Dawson JM , Banit D , et al . Growth factor modulation ofdistraction osteogenesis in a segmental defect model . Clin Ort hop , 2000 ,381 :266-277.
36.骆泉丰,王兴,王晓霞等富含血小板血浆对牵引成骨过程中新骨生成的影响[J ]中华整形外科杂志2004；20 (5) :367-379
37. Kitoh H , Kitakoji T , Tsuchiya H , et al. Transplantation of marrow-derivedmesenchymal stem cells and platelet2rich plasma during distractionosteogenesis-a preliminary result of three cases [ J ] . Bone , 2004；35 (4):892-898.
38.胡静，戚孟春，韩立赤等. BMP27基因促进大鼠下颌牵张成骨的研究[ J ].实用口腔医学杂志, 2006, 22 (5) : 635-638.
39. ZhaoM, Zhao Z, Koh JT, et al. Combinatorial gene therapy for boneregeneration: cooperative interactions between adenovirus vectorexpressing bone morphogenetic proteins 2, 4, and 7 [ J ]. Cell Biochem,2005, 95 (1) : 1-16.
40. Ashinoff RL, Cetrulo CL, Galiano RD, et al. Bone morphogenic protein22gene therapy for mandibular distraction osteogenesis. [ J ] Ann Plast Surg,2004, 52 (6) : 585-590.
41.周诺，黄旋平，廖妮等.人骨形态发生蛋白22基因克隆及真核表达载体的构建[ J ].华西口腔医学杂志, 2007, 25 ( 5 ) :487-489.
42. Hagiwara T, Bell WH. Effect of electrical stimulation on mandibulardistraction osteogenesis[J]. J Craniomaxillofac Surg, 2000,28(1):12-19.
43.王战鑫,彭铁男,孙宏晨.弱激光对牵张成骨作用的实验研究[ J ].口腔医学研究2004;20 (3) : 256-258.
44. Shimazaki A, Inui k, Azuma Y, et al. Low～intensity pulsed ultrasoundaccelerates bone maturation in distraction osteogenesis in rabbits. J BoneJoint Surg Br 2000 Sep;82(7):1077～82
45. Sakurakichi K, Tsuchiya H, Uehara K, et al. Effects of timing of lowintensitypulsed ultrasound on distraction osteogenesis. J Orthop Res.2004 Mar;22(2):395-403.
46. Tsumaki N, Kakiuchi M, Saaski J, et al. Low-intensity pulsed ultrasoundaccelerates maturation of callus in patients treated with opening-wedgehigh tibial osteotomy by hemicallotasis. J Bone Joint Surg Am. 2004Nov;86-A(11):2399-405.
47. Esenwein SA, Dudda M, Pommer A, et al. Efficiency of low intensitypulsed ultrasound on distraction osteogenesis in case of delayed callotasisclinical results. [J] J Zentralbl Chir, 2004,129(5):413-20
48. Machen MS, Tis JE, Inoue N, Meffert RH, et al. The effect of lowintensity pulsed ultrasound on regenerate bone in a less than rigidbiomechanical environment. Biomed Mater Eng 2002;12(3):239-47
49. Eberson CP, Hogan KA, Moore DC, et al. Effect of low-intensityultrasound stimulation on consolidation of the regenerate zone in a ratmodel of distraction osteogenesis. J Pediatr Orthop 2003 Jan～Feb;23(1):46-51
50. Tis JE, Meffert CR, Inoue N. The effect of low intensity pulsedultrasound applied to rabbit tibiae during the consolidation phase ofdistraction osteogenesis. J Orthop Res 2002 Jul;20(4):793-800
51. Yasuda I. Electrical callus and callus formation by electret. Clin Orthop ,1977；124 :5-8.
52. Parvizi J, Parpura J, Greenleaf JF, et al. Calcium signaling is required forultrasound-stimulated aggrecan synthesis by rat chondrocytes. J OrthopRes, 2002, 20（1）：51- 57.
53. Rawool NM, Goldberg BB, Forsberg F, et al. Power Doppler assessmentof vascular changes during fracture treatment with low-intensity ultrasound.J Ultrasound Med, 2003, 22（2）：145- 153.
54. Ryaby JT, Bacher EJ ,Bendo JA , et al. Low-intensity pulsed ultrasoundincreased calcium incorporation in both differentiating cartilage and bonecell cultures. Trans Ortho Res Soc ,1996 ,21 :622.
55. Hadjiargyrou M, McLeod K, Ryaby JP, Rubin C. Enhancement offracture healing by low-intensity ultrasound. J Clin Orthop1998,355(suppl):216-218
56. Parvizi J, Wu CC, Lewallen DG, Greenleaf JF, Bolander ME. Lowintensityultrasound stimulates proteoglycan synthesis in rat chondrocytesby increasing aggrecan gene expression. J Orthop Res 1999Jul;17(4):488-94
57. Warden SJ, Favaloro JM, Bennell KL, McMeeken JM, Ng KW, Zajac JD,Wark JD. Low-intensity pulsed ultrasound stimulates a bone formingresponse in UMR-106 cells. Biochem Biophys Res Commun 2001 Aug24;286(3):443-50
58. Sun JS, Hong RC, Chang WH, Chen LT, Lin FH, Liu HC. In vitro effects oflow-intensity ultrasound stimulation on the bone cells. J Biomed MaterRes 2001 Dec 5;57(3):449-56
59.韩雪，宋九余；口腔种植体骨界面骨整合的思考.医学与哲学(临床决策论坛版) 2007;28(11):40-41
60. Ishikawa K, Miyamoto Y, Nagayama M, Asaoka K. Blast coatingmethod: new method of coating titanium surface with hydroxyapatite atroom temperature. J Biomed Mater Res. 1997 Summer;38(2):129-34.
61. Chang YL, Stanford CM, Wefel JS, et al. Osteoblastic cell attachment tohydroxyapatite coated implant surfaces in vitro[ J ]. Int J OralMaxillofacImplants, 1999, 14 (2)∶239– 247
62. Soskolne WA, Cohen S, Sennerby L, et al. The effect of titanium surfaceroughness on the adhesion of monocytes and their secretion of TNF - alphaand PGE2 [ J ]. Clin Oral Imp lants Res, 2002, 13 (1)∶86– 93
63. Zhang H, Ahmad M, Gronowicz G. Effects of transforming growth factorbeta 1 (TGF-beta1) on in vitro mineralization of human osteoblasts onimplant materials. Biomaterials. 2003 May;24(12):2013-20.
64. Pilliar RM, Deporter DA, Watson PA, Todescan R. The Endopore implantenhanced osseointegration with a sintered porous-surfaced design. OralHealth. 1998 Jul;88(7):61-4.
65. Eric J, Jenneke K, Elisabeth H, et al. Transforming growth factor -β1incorporated in calcium phosphate cement stimulates osteotransductivity inrat bone defects [ J ]. Clin Oral Implants Res, 2001, 12 (6)∶609– 616
66. Fini M, Cadossi R, Cane V, et all. The effect of pulsed electromagneticfields on the osteointegration of hydroxyapatite implants in cancellousbone: a morphologic and microstructural in vivo study. J Orthop Res2002,20:756-763
67. Fini M, Giavaresi G, Giardino R, et all. Histomorphometric andmechanical analysis of the hydroxyapatite-bone interface afterelectromagnetic stimulation: an experimental study in rabbits. J Bone JointSurg Br 2006,88:123-128
68. Ottani V, Raspanti M, Martini D, et all. Electromagnetic stimulation on thebone growth using backscattered electron imaging. Micron 2002,33:121-125
69. Matsumoto H, Ochi M, Abiko Y, et all. Pulsed electromagnetic fieldspromote bone formation around dental implants inserted into the femur ofrabbits. Clin Oral Implants Res.2000 Aug, 11 (4) :354-360
70.张树标,朱健,刘江峰,潘广嗣He～Ne激光影响纯钛种植体骨性愈合的实验研究中国口腔种植学杂志2001;6(1):4
71.李炎洋氦氖激光对促进人工种植牙骨整合的临床研究应用激光2001 ;21(5):354
72.徐淑兰,关虹,孙晓东,毕绍臣微直流电对非埋植型即刻钛牙种植体骨整合影响的实验研究广东牙病防治2001;9(3):166
73. Tanzer M, Kantor S, Bobyn JD. Enhancement of bone growth into porousintramedullary implants using noninvasive low intensity ultrasound. JOrthop Res,2001,19:195-199
74. Iwai T, Harada Y, Imura K, et all. Low-intensity pulsed ultrasoundincreases bone ingrowth into porous hydroxyapatite ceramic. J Bone MinerMetab, 2007,25 (6) :392-399
75. Dyson M, Brookes M. Non-invasive low-intersity pulsed ultrasoundaccelerates bone healing in the rabbit. Ultrasond Med Biol ,1983,(suppl)2:61-66.
76. Duarte LR. The stimulation of bone growth by ultrasound. ArchOrthopTrauma Surg 1983 ,101 :153～159.
77. Warden SJ, Bennell KL, McMeeken JM, Wark JD. Acceleration of freshfracture repair using the sonic accelerated fracture healing system (SAFHS):a review. Calcif Tissue Int. 2000 Feb;66(2):157-63.
78. Tsai CL, Chang WH, Liu TK, Song GM. Ultrasound can affect bonehealing both locally and systemically. Chin J Physiol. 1991;34(2):213-22.
79. Reher P, Elbeshir NI, Harvey W, Meghji S, Harris M. The stimulation ofbone formation in vitro by therapeutic ultrasound. Ultrasound Med Biol.1997;23(8):1251－8.
80. Wang SJ ,Lewallen DG,Bolander ME , et al. Low intensity ultrasoundtreatment increases strength in a rat femoral fracture model. OrthopRes ,1994 ,12 :40-47.
81. Tsai CL, Chang WH, Liu TK, Song GM. Ultrasonic effect on fracture repairand prostaglandin E2 production. Chin J Physiol. 1992;35(1):27-34.
82. Jingushi S , Azuma V , lto M, et al. Effects of non invasive pulsed lowintensityultrasound on rat femoral fracture. In Proceedings of the ThirdWorld Congress of Biomechanics ,1998 ,175.
83. Schumann D, Kujat R, Zellner J, et al. Treatment of human mesenchymalstem cells with pulsed low intensity ultrasound enhances the chondrogenicphenotype in vitro. Biorheology, 2006, 43（3/4）：431- 443.
84.王成综述，曾融生低强度脉冲超声在颌骨牵张成骨中的应用国际口腔医学杂志2007；34（5）：374-377
85. Chan CW, Qin L, Lee KM, Zhang M, Cheng JC, Leung KS. Low intensitypulsed ultrasound accelerated bone remodeling during consolidation stageof distraction osteogenesis. J Orthop Res. 2006 Feb;24(2):263-70.
86.潘中允.临床核医学[M].北京:原子能出版社. 1994，271-291 .
87.杨东伟，温广武，高晓辉，王邦康核素骨显像在局部应用rh-BMP/bFGF加速山羊下颌骨牵张成骨实验研究中的应用价值临床口腔医学杂志2005；21（9）：518-21
88.李继华，胡静，王大章，祝颂松，应彬彬，韩立赤牵张成骨延长下颌骨过程中血管生成数量的定量检测口腔医学2005；2 5（6）：332-35
89. Kanishi D. 99mTc-MDP accumulation mechanisms in bone. Oral Surg OralMed Oral Pathol, 1993;75(2):239
90. Yasui N, Sato M, Ochi T, Kimura T, Kawahata H, Kitamura Y, Nomura S.Three modes of ossification during distraction osteogenesis in the rat. JBone Joint Surg Br. 1997 Sep;79(5):824-30.
91. Sun JS, Tsuang YH, Lin FH, Liu HC, Tsai CZ, Chang WH. Bone defecthealing enhanced by ultrasound stimulation: an in vitro tissue culturemodel. J Biomed Mater Res. 1999 Aug;46(2):253-61.
92. Leung KS, Cheung WH, Zhang C, Lee KM, Lo HK. Low intensity pulsedultrasound stimulates osteogenic activity of human periosteal cells. ClinOrthop Relat Res. 2004 Jan;(418):253-9.
93. Naruse K, Miyauchi A, Itoman M, Mikuni-Takagaki Y. Distinct anabolicresponse of osteoblast to low-intensity pulsed ultrasound. J Bone MinerRes. 2003 Feb;18(2):360-9.
94. Kudo O, Sabokbar A, Pocock A, Itonaga I, Fujikawa Y, Athanasou NA.Interleukin-6 and interleukin-11 support human osteoclast formation by aRANKL-independent mechanism. Bone. 2003 Jan;32(1):1-7.
95. Lee SE, Chung WJ, Kwak HB, Chung CH, Kwack KB, Lee ZH, Kim HH.Tumor necrosis factor-alpha supports the survival of osteoclasts throughthe activation of Akt and ERK. J Biol Chem. 2001 Dec 28;276(52):49343-9.
96. Wu CC，Lewallen DG，Bolander ME，et al. Exposure to low intensityultrasound stimulate aggrecan gene expression by cultured chondrocyte.Trans Ortho Res Soc ,1996 ,21 :622.
97. Mukai S, Ito H, Nakagawa Y, Akiyama H, Miyamoto M, Nakamura T.Transforming growth factor-beta1 mediates the effects of low-intensitypulsed ultrasound in chondrocytes. Ultrasound Med Biol. 2005Dec;31(12):1713-21.
98.黄静文综述，谢培豪。生物物理学刺激对种植体周围骨重建的影响中国口腔种植学杂志2009；14（1）：33-36
99. JoosU, BuchterA, Wiesmann HP, et al. Strain driven fast osseointegrationof implants. Head Face Med, 2005,1 (1) : 1～6
100. Romanos GE, Toh CG, Siar CH, et al. Histologic and histomophometricevaluation of peri -implant bone subjected to immediate loading: anexperimental study with macaca fascicularis. Int J Oral MaxillofacImplants,2002,17 (1) : 44～45
101. Periklis P, Kan J, Lozada J, et al. Effects of immediate loading withthreaded hydroxyapatite -coated root form implants on single premoalrreplacements: a preliminary report. Int J OralMaxillofac Implants, 2002,17(4) : 567～572
102. MarcoD, Giovanna P, Giovanna Z, et al. Histological evaluation of humanimmediately loaded implants inserted in the posterior mandible andretrieved after 6 months. J Oral Implantol, 2003,5 (2) : 223～230
103. MeyerU, JoosU, Mythili J, et al. Ultrastructural characterization of theimplant-bone interface of immediately loaded dental implants.Biomaterials, 2004,25(10) : 1959～1967
104. Vandamme K, Naert I, Geris L, et all. The effect of micro-motion on thetissue response around immediately loaded roughened titanium implants inthe rabbit. Eur J Oral Sci,2007 Feb,115 (1) :21～29
105. Barone A, Covani U, Cornelini R, et al. Radiographic bone density aroundimmediately loaded oral implants. Clin Oral Implants Res,2003 Oct,14 (5) :610～615
106. Turner CH, Robling AG. Exercise as an anabolic stimulus for bone. CurrPharmDes,2004,10 ( 21) :2629～2641
107. Cruber Helen. E. Adaptation of Goldner’s Masson Trichrome stain for thestudy of undecalcified plastic embedded bone [J].BiotechnicHistochemistry 1992,67(1)30-34