喷砂酸蚀钛种植体表面接触成骨现象及其影响因素的动物学实验研究
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摘要
研究背景：
在当今所有的种植体材料中,纯钛因其良好的生物相容性和机械加工性能,已经广泛应用于口腔种植领域。种植牙因为美观、舒适、并能很好的恢复咀嚼功能,作为人类的第三副牙齿,近年来被广泛接受。但是种植牙仍存在一些问题,如骨结合率较低,愈合时间较长,种植体植入体内到临床负重一般都需要6-12周的时间,这一定程度上限制了种植修复的临床应用。研究表明,大部分种植失败发生于种植体早期愈合的过程中,愈合速度越慢,种植体受到各种风险因素的影响越大,种植失败的风险越高。
影响种植体表面早期成骨有两个因素：1.受区的生物学特性；2.种植体的表面特性。
在种植早期,种植体植入机体后,与骨组织间存在两种不同的愈合模式：接触成骨和距离成骨。接触成骨,指成骨细胞与种植体表面接触后直接分泌骨基质,其骨生长方向是由种植体表面向种植窝周围的骨组织表面生长。而距离成骨与之相反,是新生骨基质沉积在种植体附近和种植窝周围的原有骨组织表面,其生长的方向为种植窝周围骨组织向种植表面生长。研究发现：接触成骨在种植体表面直接形成的编织骨有利于增加种植体初期和早期机械稳定性。尽快建立接触成骨所需的条件,有利于促进骨组织形成。骨组织愈合的速率和程度依赖于种植体表面接触成骨的程度。
影响种植早期表面接触成骨有两个因素：1.受区的生物学特性；2.种植体的表面特性。
受区的生物学特性是指受区骨的解剖生理学,包括骨代谢特点、骨密度、血供、生长因子、成骨细胞来源等。不同的骨质结构导致了不同的骨生物力学和不同的骨愈合骨改建能力。研究证明：不同的动物在相同部位植入相同的材料,实验结果有差异,在同一动物,将生物材料植入不同的部位,结果也有差异。Guehennec LL将骨代用品分别植入兔的股骨、颅骨和胫骨,观察4周,发现骨愈合在兔股骨是48.5%,颅骨是22.9%,胫骨是12.6%。这可能是由于不同部位的骨结构不同,包括皮质骨和松质骨分布特点不同。不同的骨质结构导致了不同的骨愈合骨改建能力。同一植入部位不同区域的骨愈合能力也有所差异。本课题组曾在兔胫骨距离干骺端三个不同距离处分别植入三枚种植体,结果显示,距离干骺端最近的骨-种植体接触率最高,接触成骨最明显,骨愈合最快。然而,在颌骨的不同部位,种植体表面的成骨是否有差异呢?在颌骨的哪一区域,接触成骨效果最好呢?目前尚无较肯定的研究结果。
种植体的表面特性是指种植体的表面形貌、元素组成、分子结构、电荷状态、表面自由能、亲疏水性特性等。种植体表面经过处理,可改变其表面特性,促进接触成骨,缩短骨整合所需的时间。为了有效地缩短种植体-骨结合所需的时间,在过去的二十年中,一系列钛表面处理技术不断涌现：如钛浆涂层、羟基磷灰石涂层、喷砂酸蚀、激光处理,微弧氧化等。其中,喷砂酸蚀是被研究得最多的一种表面处理方法,无论从其表面形貌、表面粗糙度还是生物相容性和骨引导性等方面,都有许多的研究报道,它已成为国际上使用最为广泛的种植体表面之一,其表面良好的机械生物相容性已经得到了公认。大量的研究已经证实：喷砂酸蚀的种植体表面具有良好的早期骨结合效果,是一种比较成熟的表面处理方法。
然而,传统的喷砂加酸蚀表面处理制备完成后被置于空气中干燥会由相对亲水性变成疏水性。研究显示,疏水性表面将减少蛋白吸附,同时润湿性不足也会影响材料表面接触血液成分的初始状态以及后续的细胞反应。种植体表面润湿性在很大程度上取决于表面自由能。表面自由能增加能使材料表面润湿性增加。种植体润湿性和表面自由能的增加可以增强种植体表面与生物环境的相互作用,从而影响到种植体生物活性的发挥,甚至骨结合的长期效果。
不同的表面处理方法会影响表面自由能的大小。自1997年由Wang等领导的研究小组,首次使用紫外线对二氧化钛膜进行表面处理,发现经紫外线处理过的二氧化钛膜层由疏水性能向超亲水性能转变,并将其成果发表在Nature上之后,研究人员开始关注紫外线改性钛表面。到目前为止,较多的研究证明：紫外光催化反应是改性钛表面,提高表面自由能并获得超亲水特性的一种简单、方便和高效的方法。
本课题组前期对紫外线辐照喷砂酸蚀纯钛表面进行了理化分析和细胞学实验,结果显示：UV+SLA表面处理降低了碳氢化合物的含量低,同时,与SLA表面相比, UV-SLA表面具有亲水性和高表面能,促进了MG63成骨细胞的粘附、增殖、分化和矿化；体外矿化诱导能力检测显示,UV-SLA表面诱导羟基磷灰石矿化沉积的速度是SLA表面的8倍。这些结果与国外有关紫外线研究是一致的。
大量的研究表明：那些由于长时间存放而老化的种植体在受到紫外线辐照后,其表面活性得以复活,提高表面自由能并同时获得亲水特性,促进了蛋白吸附、成骨细胞粘附、增殖、分化和矿化,甚至骨结合,显示出良好的生物相容性。
然而,就目前而言,国内外有关紫外线辐照的研究大部分集中在理化性质和细胞学实验,体内实验方面的研究相对较缺乏。那么,在体内实验中,通过紫外线辐照纯钛表面,改变喷砂酸蚀纯钛表面这一经典表面的自由能,钛种植体表面的成骨现象又是又是怎样的呢?
实验目的：
鉴于以上问题和争议,本研究的第一个目的是研究受区生物学特性与喷砂酸蚀钛种植体表面接触成骨的关系；第二个目的是研究紫外光处理对喷砂酸蚀钛种植体接触成骨的影响。
研究方法：
1.实验动物：成年雄性健康的Beagle犬共4只,12月龄,体重为14-15kg。牙齿牙列完整,无慢性牙周病及龋齿,口腔粘膜色泽正常,咬合关系无明显异常。
2.Beagle犬拔牙：4只beagle犬适应性饲养1周后,术前12小时禁饮食,术前半小时肌注青霉素钠40万u,速眠新和戊巴比妥钠复合麻醉下微创拔除双侧第二、三、四前磨牙和第一磨牙,术后连续三天给予青霉素80万单位肌注,以预防感染。术后进软食一周。拔牙创愈合三个月。
3.种植体选择：种植体为WEGO种植体,长度为9mm,直径为3.8mm,表面处理是SLA,作为对照组。
4.种植体UV辐照：选用Philips的UVC灭菌灯,使用UVC灭菌灯前先用75%酒精棉球擦拭灯管,再在无菌环境下距离材料表面10cm,对SLA种植体表面照射48h。SLA+UV处理,作为实验组。
5.分组：使用随机数字表按随机分配的原则,将4只成年雄性健康Beagle犬分为2周组和4周组,每组各有2只。
采用左右半口对照设计,每只beagle犬每边下颌骨植入5颗种植体,一边为实验组(SLA+UV),一边为对照组(SLA),每只beagle犬植入10颗种植体,4只beagle犬共植入40颗种植体。其中SLA组和SLA+UV组各有20个样本。SLA组的20个样本先用于分析受区生物学特性与接触成骨的关系。而后SLA组的20个样本和SLA+UV组的20个样本用于分析对比SLA表面处理和SLA+UV表面处理钛种植体表面接触成骨现象。
6.种植手术：拔牙创愈合三个月后。术前12小时禁饮食,术前半小时肌注青霉素钠40万u,速眠新和戊巴比妥钠肌肉注射麻醉后,常规消毒铺巾,下颌骨牙槽脊顶切开,翻瓣,球钻定位,逐级预备种植窝,成型钻成形种植窝,植入种植体,种植窝大小与种植体是一致的,使种植体外螺纹贴着种植窝骨壁。对位缝合创口。术后常规抗炎处理。
7.荧光双标记法：所有成年雄性健康Beagle犬于处死前的第13、14天在皮下注射盐酸四环素25mg/kg;处死前的第3、4天皮下注射钙黄绿素5mg/kg,每种试剂使用时连续注射两天,每日注射一次。
8.标本处理：分别在2周和4周各处死两只Beagle犬,获得带种植体的Beagle犬下颌骨,以0.9%生理盐水反复冲洗,将标本切分成单独种植体,用10%中性福尔马林固定液浸泡,置于4℃恒温冰箱保存48-72小时,然后换成70%乙醇浸泡。
9.显微CT检查：取4个含种植体标本,进行显微CT数据扫描,并取种植体粗螺纹区周围45μm-300vm及300-600u m的空间进行三维重建。
10.不脱钙的种植体骨切片的制备：EXAKT510系统自动脱水,包埋后,用EXAKT300CP切片机切片,然后用EXAKT400CS系统自动磨片至30um,荧光观察,再用亚甲基蓝酸性品红染色。
11.对比显微CT的结果和硬组织切片,选择更能反映种植体表面成骨的实验方法。
12.取喷砂酸蚀组切片,观察种植体在颌骨不同部位的成骨差异,第一部分是种植体螺纹底部到皮质骨大约300μ m距离的部分,第二部分是种植体螺纹底部到皮质骨大约300μ m-600μ m距离的部分,第三部分是种植体螺纹底部到皮质骨大于600μ m距离的部分。
12.对喷砂酸蚀组及喷砂酸蚀加紫外光辐照组按实验目的行荧光双标记及染色观察分析,评估种植体周围的骨愈合情况和计算BIC。
13.统计学处理：测定数据结果均采用x±s表示,使用SPSS13.0统计分析软件,正态分布数据先用析因设计的方差分析分析处理组与时间段之间是否有交互效应,如有再用单因素方差分析(one-way ANOVA),预先用Levene's test检验方差是否齐性,如方差齐则采用样品均数见的两两比较的LSD,方差不齐则采取校正的方法Welch方法,多重比较用Dunnett's T3法。当P<0.05时,认为差异具有统计学意义.
实验结果：
1.实验动物情况：4只beagle犬术后6-9小时清醒进食,无死亡动物。种植手术伤口未见异常,均可达到一期愈合,无明显出血。无种植体松动。
2.样本大体观察：种植体顶部被周围骨组织和纤维组织等包埋,未见明显骨吸收的现象,种植体无松动。
3.微型CT观察：可通过调整切面,观察到种植体周围每个面的成骨情况。可进行目标整体分割测量,在任意方向得到切片,得到较完整数据。对种植体粗螺纹周围45μm-300μm范围,300μm-600μm范围进行三维重建,两周时和四周时均可见较多新生骨小梁长入螺纹内部,但0-45u m由于金属伪影的影响显示不清,无法进行三维重建。硬组织切片只能取一个方向的截面,但是对种植体表面0-45u m区域显示清晰,可以很好地观察到种植体表面成骨的方向,可以清晰地观察到接触成骨现象。
4.喷砂酸蚀种植体与皮质骨的不同距离对接触成骨的影响：
亚甲基蓝酸性品红染色：可见种植体螺纹与皮质骨距离不同,螺纹内新骨的量不同。2周：种植体螺纹凹槽底部与皮质骨大概300u m时,即皮质骨是贴着种植体外螺纹的,此时有部分新骨长入螺纹内部,种植体表面包括螺纹凹槽底部可见新生骨小梁,有接触成骨；种植体螺纹凹槽底部与皮质骨大概300-600μm时,可见少量新生骨小梁顺着螺纹壁攀爬,种植体螺纹斜壁有少量新生骨小梁,螺纹凹槽底部未见明显新生骨小梁；种植体螺纹凹槽底部与皮质骨距离大于600μm时,未见明显新骨生成。4周：种植体螺纹凹槽底部与皮质骨大概300μm时,此时很多新骨长入螺纹内部,分布于螺纹腔及螺纹底壁,斜壁,可见接触成骨；种植体螺纹凹槽底部与皮质骨大概300-600μ m时,可见少量新生骨小梁顺着螺纹壁攀爬,螺纹底壁新生骨小梁不明显。种植体螺纹凹槽底部与皮质骨距离大于600μ m时,极少量新骨生成。
骨组织计量学：两周及四周时,对种植体螺纹凹槽底部到皮质骨的距离300μm,300-600μm,≥600μm这三个组种植体周围的BIC,新生骨面积进行测量,发现：新生种植体螺纹凹槽底部到皮质骨的距离300μ m时BIC及新生骨面积>种植体螺纹凹槽底部到皮质骨的距离300-600μm时BIC及新生骨面积>种植体螺纹凹槽底部到皮质骨的距离大于或等于600μ m的BIC及新生骨面积,差异均有统计学意义,螺纹凹槽底部与皮质骨越近,种植体—骨接触效果越好。5.紫外光处理喷砂酸蚀纯钛种植体表面早期成骨效应组织学观察。
(1)荧光分析：2周组：可见种植体周围及种植体螺纹内都有散在荧光,荧光主要集中在种植体螺纹处,皮质骨区域比骨髓腔区域荧光分布多。SLA荧光主要集中在螺纹斜壁上和种植体周围,SLA+UV组,除了种植体周围和螺纹壁上外,螺纹底部也有更多荧光分布。4周组：可见种植体周围及种植体螺纹内都有散在荧光,荧光主要集中在种植体螺纹处,与2周比,可见骨髓腔区域(即离皮质骨远的地方)也有新骨生成,且新生骨小梁较2周时粗大。SLA+UV组较SLA组更多荧光分布。且SLA+UV组较SLA组荧光条带粗。
(2)骨磨片亚甲基蓝-酸性品红染色：2周,SLA组可见螺纹腔底部无明显新骨生成,新生骨小梁多来源于周围基骨,沿着种植体螺纹腔斜壁爬行而来,新生骨小梁细长,颜色稍深；SLA+UV组：见新生骨小梁不仅沿着种植体螺纹腔斜壁爬行,在离基骨远的螺纹腔底壁,也有较多新骨生成。4周,SLA组可见新骨由周围基骨往螺纹腔内延伸,螺纹腔中间有新生骨小梁,也有新生骨小梁沿着螺纹腔斜壁往内生长,螺纹腔底壁散在分布着少量新生骨小梁,种植窝骨壁见大量新形成的哈弗氏系统,螺纹内部未见明显的哈弗氏系统。SLA+UV组：见新生骨小梁不仅不仅分布于离周围成熟骨近的螺纹腔斜壁,在离基骨远的螺纹腔底壁也见大量新生骨小梁,接触成骨非常明显,同时,螺纹腔侧壁和底壁新生骨小梁都较2周时增粗明显,除了种植窝骨壁见大量新形成的哈弗氏系统,螺纹内部也有新生成的哈弗氏系统。
(3)染色片的骨组织计量学分析：两周和四周时,SLA+UV组种植体—骨结合率>SLA组种植体—骨结合率,差异有统计学意义。两周和四周时：SLA组与SLA+UV组新生骨面积百分数差异无统计学意义。
结论
1.本实验采用beagle犬下颌骨延期种植模型,拔牙后3个月植入种植体,beagle犬下颌骨与人接近,可比性强,较符合种植临床实际情况。新的备洞和种植体植入模式,能更清晰地观察到不同表面处理方法对成骨的影响。
2.显微CT可以测定骨三维结构,具有连续性和完整性的优势。可以宏观地观察到种植体周围骨质情况,但是由于金属伪影的存在,显微CT对种植体表面接触成骨现象显示不够清晰。硬组织是一种单层组织切片,制作过程较复杂,而且会破坏标本的完整性,从而使得一个标本不能同时用于结构参数分析以外的其他检测。但是它对于种植体表面成骨的细微变化反映较清晰。
3.SLA钛种植体表面可观察到接触成骨,接触成骨与受区的生物学特性相关,与皮质骨距离近的区域,接触成骨效果好,种植体BIC高,BA也高；与皮质骨距离大于600u m的区域,接触成骨不明显。
4.SLA+UV组接触成骨效果好,在两周和四周时,SLA+UV组种植体—骨结合率均好于SLA组种植体一骨结合率。
Backgrond
The pure titanium is widely used in the field of dental implantology due to its good physical and mechanical properties and highly biocompatibility. Implant denture as human the third vice teeth is widely accepted in recent years for its beauty, comfort and can be a very good recovery on chewing function.
There are two different modes of bone healing:contact osteogenesis and distance osteogenesis. Contact osteogenesis is that osteoblasts secrete bone matrix to implant surface directly and then form the bone. The growth direction is from the surface of the implant to the base bone surround the implant. On the contrary, distance osteogenesis is that new bone form in the surface of base bone.The growth direction is from the surface of the base bone surrounding to the surface of implant. The woven bone directly form by contact osteogenesis in the surface of the implant is beneficial to increase the mechanical stability of the implant in the early stage. Establish the conditions of contact osteogenesis as soon as possible would promote bone formation.The rate and degree of bone healing depends on the extent of the implant surface contact osteogenesis.
There are two influence factor of contact osteogenesis:1.The biological characteristics of the implantation sites;2. The surface character of the implant.
The biological characteristics of the implantation sites is the bone metabolism and bone physiology, including bone density, blood, etc. Different bone structure leads to the different bone biomechanics and different ability of bone healing and bone rebuilding.Studies have proven that when different animal implant the same materal in the same part of body, the results were different. The bone colonization appeared statistically higher in the femur of rabbits (48.5%) than in the tibia (12.6%) and calvaria (22.9%) sites. This slightly higher degree of bone healing was related to differences in the bone architecture of the implantation sites. These different types of bone architecture may lead to different types of biomechanical stress and thus different rates of bone healing and remodeling. Rong implant three implant in three part of rabbit tibial metaphysis. the different implantation sites demonstrated different early osseointegration. However, in the different parts of the jaw, is there any different on the surface of the implant? In which areas of the jaw, contact osteogenesis effect best? There is no more positive results.
The surface character of the implant are surface topography, elementary composition, molecular structure, electrical charge, surface free energy, wettability, etc. After treatment, implant can change its surface properties, thus promotes contact osteogenesis, shorten the time needed for bone integration, improve the success rate of planting. In order to shorten the time of implant-tissue intergration effectively, a series of surface treatment technologies have emerged in the past two decades. such as titanium plasma sprayed, hydroxyapatite coating, sandblasting with large grit and acid-etching, double etching,electron beamheat treatmentlaser treatment, anodic oxidation, micro-arc oxidation. Changes insurface topography of the implant surface, elemental composition, molecular structure,charge state,surface free energy, hydrophobic and hydrophilic characteristics, thus changing the biological characteristics of the implant surface. Sandblasting with large grit and acid-etching (SLA) treatment is one of the most common methods to improve implant surfaces in clinic.
The holes with diameter10-30μm in the implant surface can be obtained by the large particles blasting, these holes were good for the bone formation. Meanwhile, a large number of pores (size at about1-3μm) were formed by the acid-etched method.It could be said that, the primary roughness was helpful to the osteoblasts adhesion, and proliferation and differentiation of osteoblasts could be stimulate by secondly roughness. There are many research reports about its surface morphology, surface roughness, biocompatibility and osteoconductive properties. SLA suface is most widely used in the world currently.The SLA surface with multi-level pores has been demonstrated enhancement of bone deposition in histomorphometric studies and higher removal torque values in biomechanical testing.
However, titanium surface is time-dependent degradation. When the titanium placed in the dark for4weeks would due to aging and reduces protein adsorption compared to a fresh surface.The experimental prove that when the titanium stored in the dark at different times due to the Hydrocarbons increases, protein adhesion reduction, cell adhesion decrease.
Ultraviolet (UV) irradiation would prevent titanium surface from time-dependent degradation. Fuminori had found that UV has been applied in surface treatment of implant to obtain super-hydrophilic property, as well as the attachment of osteoblast. Aita found that titanium specimen treated additionally with UV show ssuper-hydrophilic property, and better promote attachment, proliferation, differentiation and mineralization of osteoblast. Studies have shown that the implant which used in clinical exist hydrocarbons contamination. There are statistically significant correlation between the decrease in biological activity and the increase in carbon atoms. Uetsuka and Henderson show that UV radiation can remove hydrocarbons in the titanium surface, and then Ti4+sites exposed. This site promotes the protein and cell-surface interactions, promotes protein adsorption and osteoblast attachment. There are also studies indicate that the static electricity stimulate by UV promote protein adsorption which exceed the effect of surface hydrophilicity in protein adsorption.
However, the study about UV focus on physicochemical properties and cytological experiments in the world at present. There are lack of animal experiments.
The first purpose of the present study was to establish an animal model of beagle dogs and implant the titanium implant with SLA surface, Observe the contact osteogenesis in different parts of the jaw bone. The second purpose of the present study was to establish an animal model of beagle dogs and implant the titanium implant with SLA surface and SLA+UV surface. Then compare the contact osteogenenisis between the two surface.
Materials and Methods:
1. Experimental animal:Four one-year-old male beagle dogs (approximate weight14-15kg) were Included in the experiment
2. Tooth extraction:After adaptive feeding for one week, the mandibular second, third, and fourth premolar and first molar was extract bilaterally.
3. Dental implant:WEGO implant,3.8x9mm, surface treatment is SLA.
4. UV radiation:Some of the SLA implant were irradiated by15W UVC germicidal lamp in a sterile environment,10cm close to the material surface, and irradiated48h.
5. Grouping:4healthy adult male Beagle dogs were randomly divided into two and four weeks group. A split-mouth study was designed. One side of the mandibular was experiment group, the other side of the mandibular was control group. Every Beagle dog had10implant,5were experiment group and5were control group.
6. Implant surgery:After three months of healing, surgical implantation of implant was performed. After general anesthesia which was induced by the injection of Sumian Xin and pentobarbital sodium intramuscularly, the skin and oral mucosa was disinfected. And then a flap was elevated to expose the mandibular bone. With the drill, the planting holes were prepared and10implants were place for every beagle dogs. Each implant received a cover-screw and the mucoperiosteal flaps were sutured to submerge the implants.
7. Fluorochrome bone labels:Fluorochrome bone labels were given as single injections subcutaneously oxytetracycline for25mg/kg body weight13days before sacrifice and calcein for5mg/kg body weight3days before sacrifice.
8.The animals were sacrificed after2and4weeks. The jaws were dissected, and blocks containing the implant specimens were obtained. All specimens were fixed in10%neutral buffered formalin solution for48h.
9.Micro CT examination:The mandibular specimens were fixed in10%formalin,and then move to70%alcohol. X-ray tube voltage:90KV, Electricity:45μA, Projection:720perspectives, Integral time:0.5s, Image resolution:15μm. Take the area around implant coarse thread45μm to300μm and300μm to600μm for3D reconstruction and then calculate the bone volume fraction.
10. The preparation of bone biopsy:The specimens were dehydrated,infiltrated and embedded for nondecalcified sectioning. Blocks were serial sectioned parallel with the long axis of the implant in a buccolingual direction and ground to a final thickness of approximately30μm. Upon completion of the fluorescent light examination, all sections were stained with methylene blue-fuchsin acid for histopathologic and histomorphometric analysis using light microscopy.
11.Observe osteogenesis differences from different parts of the implant in the jaw. The first part is300μm distance from the bottom of the implant to Cortical bone, the second part is300-600μm distance from the bottom of the implant to Cortical bone, the third part is exceed600μm distance from the bottom of the implant to Cortical bone.
12.Evaluation of new bone area around implant and calculate the BIC in group SLA and SLA+UV.
13.Statistical analyses:The experimental data are expressed as mean±standard deviation (SD). Statistical analysis was carried out by SPSS(?) v13.0software (SPSS Inc., Chicago, USA). For the single factor data, when meet the normal distribution and homogeneity of variance, using factorial classification ANOVA, then one way ANOVA was using. The LSD multiple comparison test was used for post-hoc analysis to determine which groups were significantly different, and the Levene statistic was used to test homogeneity of variances. If did not pass the Levene test, The Dunnett's T3multiple comparison test was used. A p<0.05was considered statistically significant.
Results:
1.During the experiment, all animals showed uneventful healing of the surgical area. Finally, postoperative infections were not observed.
2.The sample general observation:the histological sections revealed that none of the titanium implants were dislocated from their original position.
3.Micro CT observation:From adjusted by section, every facial of the implant can be observed. Target can be the overall division measurement, slice in any direction, get more complete data. Three-dimensional reconstructed45-300μm,300-600μm around implant coarse thread. Many slender bone trabeculae grew into the thread. Because of the influence of the metal artifacts,0-45μm is not clear. Hard tissue section can only take one direction of cross section, but of the implant surface0-45microns region shows clear. It is a good way to observe the direction of the implant surface osteogenesis, contact osteogenesis phenomenon can be seen clearly.
4.Magenta acidic methylene blue staining:Different distances between implant and cortical bone, different amounts of new bone in the thread. In two weeks, when the distance between the bottom of implant and cortical bone was about300μm, many new bone grew into the thread; when the distance between the bottom of implant and cortical bone was about300-600μm, some new bone grew into the thread; when the distance between the bottom of implant and cortical bone was exceed600μm, few new bone grew into the thread. In four weeks, when the distance between the bottom of implant and cortical bone was about300μm, many new bone grew into the thread; when the distance between the bottom of implant and cortical bone was about300-600μm, some new bone grew into the thread; when the distance between the bottom of implant and cortical bone was exceed600μm, few new bone grew into the thread.
Bone histomorphometry:Test BIC and new bone area of the different distance between implant and cortical bone. The BIC and new bone area of the area under300μm between implant and cortical bone>the area300-600μm between implant and cortical bone> the area more than600μm between implant and cortical bone in two weeks and four weeks. The closer between the bottom of the thread and cortical bone, the better results of implant-bone contact.
5.The early osteogenenisis of Ultraviolet Irradiation on Sandblasted and Acid-etched Titanium implant Surfaces.(1) Fluorescence analysis。In two weeks, fluorescence are scattered around the implant and thread, mainly focus on implant thread. More fluorescence distribute at cortical bone than bone marrow cavity. In groups SLA, fluorescence are mainly focus on the skew wall of the thread and the arounding of implant. In groups SLA+UV, except the skew wall of the thread and the arounding of the implant, many fluorescence focus on the bottom of implant thread. In four weeks, except the inside of the thread and the arounding of the implant, new bone was found in the area of bone marrow cavity. There are more fluorescence scattered in group SLA than group SLA+UV.
(2)Methylene blue-acid fuchsin staining:At two weeks, New bone trabecular number comes from the surrounding bone,crawling along the implant thread in group SLA. In gorup SLA+UV, new bone not only grew along the inclined wall of the thread, but also in the bottom of the thread. At four weeks, new bone grew among the thread, haversian system was not seen obvious among the thread in group SLA, in group SLA+UV, many new bone was not only found in the inclined wall of the thread but also at the bottom of the thread. Contact osteogenesis is very obvious. Haversian system was seen among the thread.
(3)Bone tissue metrology analysis:In two weeks and four weeks, the BIC of group SLA+UV> the BIC ofgroup SLA. The difference was statistically significant.But the difference of the new bone area with two groups were not statistically significant.
Conclusion:
l.This beagle dog mandible model simulated the clinical situation and can be used to investigate bone formation around implant. The new planting model can be more clearly observed the effects of different surface treatments on osteogenesis.
2.Micro CT can be used to measure the bone with three-dimensional structure,and it has the advantage of continuity and integrity. But because of the existence of the metal artifacts, the implant-bone interface of osteogenesis reflect poor condition. Hard tissue sliciing is a single-layer tissue section and the production process is complicated, and will destroy the integrity of the specimen. But the subtle changes of implant surface osteogenesis reflect clearly.
3.The closer from bottom of thread to cortical bone, the better implant-bone contact.
4. Group SLA+UV have better contact osteogenesis, and the BIC%is higher in group SLA+UV than in group SLA.
在当今所有的种植体材料中,纯钛因其良好的生物相容性和机械加工性能,已经广泛应用于口腔种植领域。种植牙因为美观、舒适、并能很好的恢复咀嚼功能,作为人类的第三副牙齿,近年来被广泛接受。但是种植牙仍存在一些问题,如骨结合率较低,愈合时间较长,种植体植入体内到临床负重一般都需要6-12周的时间,这一定程度上限制了种植修复的临床应用。研究表明,大部分种植失败发生于种植体早期愈合的过程中,愈合速度越慢,种植体受到各种风险因素的影响越大,种植失败的风险越高。
影响种植体表面早期成骨有两个因素：1.受区的生物学特性；2.种植体的表面特性。
在种植早期,种植体植入机体后,与骨组织间存在两种不同的愈合模式：接触成骨和距离成骨。接触成骨,指成骨细胞与种植体表面接触后直接分泌骨基质,其骨生长方向是由种植体表面向种植窝周围的骨组织表面生长。而距离成骨与之相反,是新生骨基质沉积在种植体附近和种植窝周围的原有骨组织表面,其生长的方向为种植窝周围骨组织向种植表面生长。研究发现：接触成骨在种植体表面直接形成的编织骨有利于增加种植体初期和早期机械稳定性。尽快建立接触成骨所需的条件,有利于促进骨组织形成。骨组织愈合的速率和程度依赖于种植体表面接触成骨的程度。
影响种植早期表面接触成骨有两个因素：1.受区的生物学特性；2.种植体的表面特性。
受区的生物学特性是指受区骨的解剖生理学,包括骨代谢特点、骨密度、血供、生长因子、成骨细胞来源等。不同的骨质结构导致了不同的骨生物力学和不同的骨愈合骨改建能力。研究证明：不同的动物在相同部位植入相同的材料,实验结果有差异,在同一动物,将生物材料植入不同的部位,结果也有差异。Guehennec LL将骨代用品分别植入兔的股骨、颅骨和胫骨,观察4周,发现骨愈合在兔股骨是48.5%,颅骨是22.9%,胫骨是12.6%。这可能是由于不同部位的骨结构不同,包括皮质骨和松质骨分布特点不同。不同的骨质结构导致了不同的骨愈合骨改建能力。同一植入部位不同区域的骨愈合能力也有所差异。本课题组曾在兔胫骨距离干骺端三个不同距离处分别植入三枚种植体,结果显示,距离干骺端最近的骨-种植体接触率最高,接触成骨最明显,骨愈合最快。然而,在颌骨的不同部位,种植体表面的成骨是否有差异呢?在颌骨的哪一区域,接触成骨效果最好呢?目前尚无较肯定的研究结果。
种植体的表面特性是指种植体的表面形貌、元素组成、分子结构、电荷状态、表面自由能、亲疏水性特性等。种植体表面经过处理,可改变其表面特性,促进接触成骨,缩短骨整合所需的时间。为了有效地缩短种植体-骨结合所需的时间,在过去的二十年中,一系列钛表面处理技术不断涌现：如钛浆涂层、羟基磷灰石涂层、喷砂酸蚀、激光处理,微弧氧化等。其中,喷砂酸蚀是被研究得最多的一种表面处理方法,无论从其表面形貌、表面粗糙度还是生物相容性和骨引导性等方面,都有许多的研究报道,它已成为国际上使用最为广泛的种植体表面之一,其表面良好的机械生物相容性已经得到了公认。大量的研究已经证实：喷砂酸蚀的种植体表面具有良好的早期骨结合效果,是一种比较成熟的表面处理方法。
然而,传统的喷砂加酸蚀表面处理制备完成后被置于空气中干燥会由相对亲水性变成疏水性。研究显示,疏水性表面将减少蛋白吸附,同时润湿性不足也会影响材料表面接触血液成分的初始状态以及后续的细胞反应。种植体表面润湿性在很大程度上取决于表面自由能。表面自由能增加能使材料表面润湿性增加。种植体润湿性和表面自由能的增加可以增强种植体表面与生物环境的相互作用,从而影响到种植体生物活性的发挥,甚至骨结合的长期效果。
不同的表面处理方法会影响表面自由能的大小。自1997年由Wang等领导的研究小组,首次使用紫外线对二氧化钛膜进行表面处理,发现经紫外线处理过的二氧化钛膜层由疏水性能向超亲水性能转变,并将其成果发表在Nature上之后,研究人员开始关注紫外线改性钛表面。到目前为止,较多的研究证明：紫外光催化反应是改性钛表面,提高表面自由能并获得超亲水特性的一种简单、方便和高效的方法。
本课题组前期对紫外线辐照喷砂酸蚀纯钛表面进行了理化分析和细胞学实验,结果显示：UV+SLA表面处理降低了碳氢化合物的含量低,同时,与SLA表面相比, UV-SLA表面具有亲水性和高表面能,促进了MG63成骨细胞的粘附、增殖、分化和矿化；体外矿化诱导能力检测显示,UV-SLA表面诱导羟基磷灰石矿化沉积的速度是SLA表面的8倍。这些结果与国外有关紫外线研究是一致的。
大量的研究表明：那些由于长时间存放而老化的种植体在受到紫外线辐照后,其表面活性得以复活,提高表面自由能并同时获得亲水特性,促进了蛋白吸附、成骨细胞粘附、增殖、分化和矿化,甚至骨结合,显示出良好的生物相容性。
然而,就目前而言,国内外有关紫外线辐照的研究大部分集中在理化性质和细胞学实验,体内实验方面的研究相对较缺乏。那么,在体内实验中,通过紫外线辐照纯钛表面,改变喷砂酸蚀纯钛表面这一经典表面的自由能,钛种植体表面的成骨现象又是又是怎样的呢?
实验目的：
鉴于以上问题和争议,本研究的第一个目的是研究受区生物学特性与喷砂酸蚀钛种植体表面接触成骨的关系；第二个目的是研究紫外光处理对喷砂酸蚀钛种植体接触成骨的影响。
研究方法：
1.实验动物：成年雄性健康的Beagle犬共4只,12月龄,体重为14-15kg。牙齿牙列完整,无慢性牙周病及龋齿,口腔粘膜色泽正常,咬合关系无明显异常。
2.Beagle犬拔牙：4只beagle犬适应性饲养1周后,术前12小时禁饮食,术前半小时肌注青霉素钠40万u,速眠新和戊巴比妥钠复合麻醉下微创拔除双侧第二、三、四前磨牙和第一磨牙,术后连续三天给予青霉素80万单位肌注,以预防感染。术后进软食一周。拔牙创愈合三个月。
3.种植体选择：种植体为WEGO种植体,长度为9mm,直径为3.8mm,表面处理是SLA,作为对照组。
4.种植体UV辐照：选用Philips的UVC灭菌灯,使用UVC灭菌灯前先用75%酒精棉球擦拭灯管,再在无菌环境下距离材料表面10cm,对SLA种植体表面照射48h。SLA+UV处理,作为实验组。
5.分组：使用随机数字表按随机分配的原则,将4只成年雄性健康Beagle犬分为2周组和4周组,每组各有2只。
采用左右半口对照设计,每只beagle犬每边下颌骨植入5颗种植体,一边为实验组(SLA+UV),一边为对照组(SLA),每只beagle犬植入10颗种植体,4只beagle犬共植入40颗种植体。其中SLA组和SLA+UV组各有20个样本。SLA组的20个样本先用于分析受区生物学特性与接触成骨的关系。而后SLA组的20个样本和SLA+UV组的20个样本用于分析对比SLA表面处理和SLA+UV表面处理钛种植体表面接触成骨现象。
6.种植手术：拔牙创愈合三个月后。术前12小时禁饮食,术前半小时肌注青霉素钠40万u,速眠新和戊巴比妥钠肌肉注射麻醉后,常规消毒铺巾,下颌骨牙槽脊顶切开,翻瓣,球钻定位,逐级预备种植窝,成型钻成形种植窝,植入种植体,种植窝大小与种植体是一致的,使种植体外螺纹贴着种植窝骨壁。对位缝合创口。术后常规抗炎处理。
7.荧光双标记法：所有成年雄性健康Beagle犬于处死前的第13、14天在皮下注射盐酸四环素25mg/kg;处死前的第3、4天皮下注射钙黄绿素5mg/kg,每种试剂使用时连续注射两天,每日注射一次。
8.标本处理：分别在2周和4周各处死两只Beagle犬,获得带种植体的Beagle犬下颌骨,以0.9%生理盐水反复冲洗,将标本切分成单独种植体,用10%中性福尔马林固定液浸泡,置于4℃恒温冰箱保存48-72小时,然后换成70%乙醇浸泡。
9.显微CT检查：取4个含种植体标本,进行显微CT数据扫描,并取种植体粗螺纹区周围45μm-300vm及300-600u m的空间进行三维重建。
10.不脱钙的种植体骨切片的制备：EXAKT510系统自动脱水,包埋后,用EXAKT300CP切片机切片,然后用EXAKT400CS系统自动磨片至30um,荧光观察,再用亚甲基蓝酸性品红染色。
11.对比显微CT的结果和硬组织切片,选择更能反映种植体表面成骨的实验方法。
12.取喷砂酸蚀组切片,观察种植体在颌骨不同部位的成骨差异,第一部分是种植体螺纹底部到皮质骨大约300μ m距离的部分,第二部分是种植体螺纹底部到皮质骨大约300μ m-600μ m距离的部分,第三部分是种植体螺纹底部到皮质骨大于600μ m距离的部分。
12.对喷砂酸蚀组及喷砂酸蚀加紫外光辐照组按实验目的行荧光双标记及染色观察分析,评估种植体周围的骨愈合情况和计算BIC。
13.统计学处理：测定数据结果均采用x±s表示,使用SPSS13.0统计分析软件,正态分布数据先用析因设计的方差分析分析处理组与时间段之间是否有交互效应,如有再用单因素方差分析(one-way ANOVA),预先用Levene's test检验方差是否齐性,如方差齐则采用样品均数见的两两比较的LSD,方差不齐则采取校正的方法Welch方法,多重比较用Dunnett's T3法。当P<0.05时,认为差异具有统计学意义.
实验结果：
1.实验动物情况：4只beagle犬术后6-9小时清醒进食,无死亡动物。种植手术伤口未见异常,均可达到一期愈合,无明显出血。无种植体松动。
2.样本大体观察：种植体顶部被周围骨组织和纤维组织等包埋,未见明显骨吸收的现象,种植体无松动。
3.微型CT观察：可通过调整切面,观察到种植体周围每个面的成骨情况。可进行目标整体分割测量,在任意方向得到切片,得到较完整数据。对种植体粗螺纹周围45μm-300μm范围,300μm-600μm范围进行三维重建,两周时和四周时均可见较多新生骨小梁长入螺纹内部,但0-45u m由于金属伪影的影响显示不清,无法进行三维重建。硬组织切片只能取一个方向的截面,但是对种植体表面0-45u m区域显示清晰,可以很好地观察到种植体表面成骨的方向,可以清晰地观察到接触成骨现象。
4.喷砂酸蚀种植体与皮质骨的不同距离对接触成骨的影响：
亚甲基蓝酸性品红染色：可见种植体螺纹与皮质骨距离不同,螺纹内新骨的量不同。2周：种植体螺纹凹槽底部与皮质骨大概300u m时,即皮质骨是贴着种植体外螺纹的,此时有部分新骨长入螺纹内部,种植体表面包括螺纹凹槽底部可见新生骨小梁,有接触成骨；种植体螺纹凹槽底部与皮质骨大概300-600μm时,可见少量新生骨小梁顺着螺纹壁攀爬,种植体螺纹斜壁有少量新生骨小梁,螺纹凹槽底部未见明显新生骨小梁；种植体螺纹凹槽底部与皮质骨距离大于600μm时,未见明显新骨生成。4周：种植体螺纹凹槽底部与皮质骨大概300μm时,此时很多新骨长入螺纹内部,分布于螺纹腔及螺纹底壁,斜壁,可见接触成骨；种植体螺纹凹槽底部与皮质骨大概300-600μ m时,可见少量新生骨小梁顺着螺纹壁攀爬,螺纹底壁新生骨小梁不明显。种植体螺纹凹槽底部与皮质骨距离大于600μ m时,极少量新骨生成。
骨组织计量学：两周及四周时,对种植体螺纹凹槽底部到皮质骨的距离300μm,300-600μm,≥600μm这三个组种植体周围的BIC,新生骨面积进行测量,发现：新生种植体螺纹凹槽底部到皮质骨的距离300μ m时BIC及新生骨面积>种植体螺纹凹槽底部到皮质骨的距离300-600μm时BIC及新生骨面积>种植体螺纹凹槽底部到皮质骨的距离大于或等于600μ m的BIC及新生骨面积,差异均有统计学意义,螺纹凹槽底部与皮质骨越近,种植体—骨接触效果越好。5.紫外光处理喷砂酸蚀纯钛种植体表面早期成骨效应组织学观察。
(1)荧光分析：2周组：可见种植体周围及种植体螺纹内都有散在荧光,荧光主要集中在种植体螺纹处,皮质骨区域比骨髓腔区域荧光分布多。SLA荧光主要集中在螺纹斜壁上和种植体周围,SLA+UV组,除了种植体周围和螺纹壁上外,螺纹底部也有更多荧光分布。4周组：可见种植体周围及种植体螺纹内都有散在荧光,荧光主要集中在种植体螺纹处,与2周比,可见骨髓腔区域(即离皮质骨远的地方)也有新骨生成,且新生骨小梁较2周时粗大。SLA+UV组较SLA组更多荧光分布。且SLA+UV组较SLA组荧光条带粗。
(2)骨磨片亚甲基蓝-酸性品红染色：2周,SLA组可见螺纹腔底部无明显新骨生成,新生骨小梁多来源于周围基骨,沿着种植体螺纹腔斜壁爬行而来,新生骨小梁细长,颜色稍深；SLA+UV组：见新生骨小梁不仅沿着种植体螺纹腔斜壁爬行,在离基骨远的螺纹腔底壁,也有较多新骨生成。4周,SLA组可见新骨由周围基骨往螺纹腔内延伸,螺纹腔中间有新生骨小梁,也有新生骨小梁沿着螺纹腔斜壁往内生长,螺纹腔底壁散在分布着少量新生骨小梁,种植窝骨壁见大量新形成的哈弗氏系统,螺纹内部未见明显的哈弗氏系统。SLA+UV组：见新生骨小梁不仅不仅分布于离周围成熟骨近的螺纹腔斜壁,在离基骨远的螺纹腔底壁也见大量新生骨小梁,接触成骨非常明显,同时,螺纹腔侧壁和底壁新生骨小梁都较2周时增粗明显,除了种植窝骨壁见大量新形成的哈弗氏系统,螺纹内部也有新生成的哈弗氏系统。
(3)染色片的骨组织计量学分析：两周和四周时,SLA+UV组种植体—骨结合率>SLA组种植体—骨结合率,差异有统计学意义。两周和四周时：SLA组与SLA+UV组新生骨面积百分数差异无统计学意义。
结论
1.本实验采用beagle犬下颌骨延期种植模型,拔牙后3个月植入种植体,beagle犬下颌骨与人接近,可比性强,较符合种植临床实际情况。新的备洞和种植体植入模式,能更清晰地观察到不同表面处理方法对成骨的影响。
2.显微CT可以测定骨三维结构,具有连续性和完整性的优势。可以宏观地观察到种植体周围骨质情况,但是由于金属伪影的存在,显微CT对种植体表面接触成骨现象显示不够清晰。硬组织是一种单层组织切片,制作过程较复杂,而且会破坏标本的完整性,从而使得一个标本不能同时用于结构参数分析以外的其他检测。但是它对于种植体表面成骨的细微变化反映较清晰。
3.SLA钛种植体表面可观察到接触成骨,接触成骨与受区的生物学特性相关,与皮质骨距离近的区域,接触成骨效果好,种植体BIC高,BA也高；与皮质骨距离大于600u m的区域,接触成骨不明显。
4.SLA+UV组接触成骨效果好,在两周和四周时,SLA+UV组种植体—骨结合率均好于SLA组种植体一骨结合率。
Backgrond
The pure titanium is widely used in the field of dental implantology due to its good physical and mechanical properties and highly biocompatibility. Implant denture as human the third vice teeth is widely accepted in recent years for its beauty, comfort and can be a very good recovery on chewing function.
There are two different modes of bone healing:contact osteogenesis and distance osteogenesis. Contact osteogenesis is that osteoblasts secrete bone matrix to implant surface directly and then form the bone. The growth direction is from the surface of the implant to the base bone surround the implant. On the contrary, distance osteogenesis is that new bone form in the surface of base bone.The growth direction is from the surface of the base bone surrounding to the surface of implant. The woven bone directly form by contact osteogenesis in the surface of the implant is beneficial to increase the mechanical stability of the implant in the early stage. Establish the conditions of contact osteogenesis as soon as possible would promote bone formation.The rate and degree of bone healing depends on the extent of the implant surface contact osteogenesis.
There are two influence factor of contact osteogenesis:1.The biological characteristics of the implantation sites;2. The surface character of the implant.
The biological characteristics of the implantation sites is the bone metabolism and bone physiology, including bone density, blood, etc. Different bone structure leads to the different bone biomechanics and different ability of bone healing and bone rebuilding.Studies have proven that when different animal implant the same materal in the same part of body, the results were different. The bone colonization appeared statistically higher in the femur of rabbits (48.5%) than in the tibia (12.6%) and calvaria (22.9%) sites. This slightly higher degree of bone healing was related to differences in the bone architecture of the implantation sites. These different types of bone architecture may lead to different types of biomechanical stress and thus different rates of bone healing and remodeling. Rong implant three implant in three part of rabbit tibial metaphysis. the different implantation sites demonstrated different early osseointegration. However, in the different parts of the jaw, is there any different on the surface of the implant? In which areas of the jaw, contact osteogenesis effect best? There is no more positive results.
The surface character of the implant are surface topography, elementary composition, molecular structure, electrical charge, surface free energy, wettability, etc. After treatment, implant can change its surface properties, thus promotes contact osteogenesis, shorten the time needed for bone integration, improve the success rate of planting. In order to shorten the time of implant-tissue intergration effectively, a series of surface treatment technologies have emerged in the past two decades. such as titanium plasma sprayed, hydroxyapatite coating, sandblasting with large grit and acid-etching, double etching,electron beamheat treatmentlaser treatment, anodic oxidation, micro-arc oxidation. Changes insurface topography of the implant surface, elemental composition, molecular structure,charge state,surface free energy, hydrophobic and hydrophilic characteristics, thus changing the biological characteristics of the implant surface. Sandblasting with large grit and acid-etching (SLA) treatment is one of the most common methods to improve implant surfaces in clinic.
The holes with diameter10-30μm in the implant surface can be obtained by the large particles blasting, these holes were good for the bone formation. Meanwhile, a large number of pores (size at about1-3μm) were formed by the acid-etched method.It could be said that, the primary roughness was helpful to the osteoblasts adhesion, and proliferation and differentiation of osteoblasts could be stimulate by secondly roughness. There are many research reports about its surface morphology, surface roughness, biocompatibility and osteoconductive properties. SLA suface is most widely used in the world currently.The SLA surface with multi-level pores has been demonstrated enhancement of bone deposition in histomorphometric studies and higher removal torque values in biomechanical testing.
However, titanium surface is time-dependent degradation. When the titanium placed in the dark for4weeks would due to aging and reduces protein adsorption compared to a fresh surface.The experimental prove that when the titanium stored in the dark at different times due to the Hydrocarbons increases, protein adhesion reduction, cell adhesion decrease.
Ultraviolet (UV) irradiation would prevent titanium surface from time-dependent degradation. Fuminori had found that UV has been applied in surface treatment of implant to obtain super-hydrophilic property, as well as the attachment of osteoblast. Aita found that titanium specimen treated additionally with UV show ssuper-hydrophilic property, and better promote attachment, proliferation, differentiation and mineralization of osteoblast. Studies have shown that the implant which used in clinical exist hydrocarbons contamination. There are statistically significant correlation between the decrease in biological activity and the increase in carbon atoms. Uetsuka and Henderson show that UV radiation can remove hydrocarbons in the titanium surface, and then Ti4+sites exposed. This site promotes the protein and cell-surface interactions, promotes protein adsorption and osteoblast attachment. There are also studies indicate that the static electricity stimulate by UV promote protein adsorption which exceed the effect of surface hydrophilicity in protein adsorption.
However, the study about UV focus on physicochemical properties and cytological experiments in the world at present. There are lack of animal experiments.
The first purpose of the present study was to establish an animal model of beagle dogs and implant the titanium implant with SLA surface, Observe the contact osteogenesis in different parts of the jaw bone. The second purpose of the present study was to establish an animal model of beagle dogs and implant the titanium implant with SLA surface and SLA+UV surface. Then compare the contact osteogenenisis between the two surface.
Materials and Methods:
1. Experimental animal:Four one-year-old male beagle dogs (approximate weight14-15kg) were Included in the experiment
2. Tooth extraction:After adaptive feeding for one week, the mandibular second, third, and fourth premolar and first molar was extract bilaterally.
3. Dental implant:WEGO implant,3.8x9mm, surface treatment is SLA.
4. UV radiation:Some of the SLA implant were irradiated by15W UVC germicidal lamp in a sterile environment,10cm close to the material surface, and irradiated48h.
5. Grouping:4healthy adult male Beagle dogs were randomly divided into two and four weeks group. A split-mouth study was designed. One side of the mandibular was experiment group, the other side of the mandibular was control group. Every Beagle dog had10implant,5were experiment group and5were control group.
6. Implant surgery:After three months of healing, surgical implantation of implant was performed. After general anesthesia which was induced by the injection of Sumian Xin and pentobarbital sodium intramuscularly, the skin and oral mucosa was disinfected. And then a flap was elevated to expose the mandibular bone. With the drill, the planting holes were prepared and10implants were place for every beagle dogs. Each implant received a cover-screw and the mucoperiosteal flaps were sutured to submerge the implants.
7. Fluorochrome bone labels:Fluorochrome bone labels were given as single injections subcutaneously oxytetracycline for25mg/kg body weight13days before sacrifice and calcein for5mg/kg body weight3days before sacrifice.
8.The animals were sacrificed after2and4weeks. The jaws were dissected, and blocks containing the implant specimens were obtained. All specimens were fixed in10%neutral buffered formalin solution for48h.
9.Micro CT examination:The mandibular specimens were fixed in10%formalin,and then move to70%alcohol. X-ray tube voltage:90KV, Electricity:45μA, Projection:720perspectives, Integral time:0.5s, Image resolution:15μm. Take the area around implant coarse thread45μm to300μm and300μm to600μm for3D reconstruction and then calculate the bone volume fraction.
10. The preparation of bone biopsy:The specimens were dehydrated,infiltrated and embedded for nondecalcified sectioning. Blocks were serial sectioned parallel with the long axis of the implant in a buccolingual direction and ground to a final thickness of approximately30μm. Upon completion of the fluorescent light examination, all sections were stained with methylene blue-fuchsin acid for histopathologic and histomorphometric analysis using light microscopy.
11.Observe osteogenesis differences from different parts of the implant in the jaw. The first part is300μm distance from the bottom of the implant to Cortical bone, the second part is300-600μm distance from the bottom of the implant to Cortical bone, the third part is exceed600μm distance from the bottom of the implant to Cortical bone.
12.Evaluation of new bone area around implant and calculate the BIC in group SLA and SLA+UV.
13.Statistical analyses:The experimental data are expressed as mean±standard deviation (SD). Statistical analysis was carried out by SPSS(?) v13.0software (SPSS Inc., Chicago, USA). For the single factor data, when meet the normal distribution and homogeneity of variance, using factorial classification ANOVA, then one way ANOVA was using. The LSD multiple comparison test was used for post-hoc analysis to determine which groups were significantly different, and the Levene statistic was used to test homogeneity of variances. If did not pass the Levene test, The Dunnett's T3multiple comparison test was used. A p<0.05was considered statistically significant.
Results:
1.During the experiment, all animals showed uneventful healing of the surgical area. Finally, postoperative infections were not observed.
2.The sample general observation:the histological sections revealed that none of the titanium implants were dislocated from their original position.
3.Micro CT observation:From adjusted by section, every facial of the implant can be observed. Target can be the overall division measurement, slice in any direction, get more complete data. Three-dimensional reconstructed45-300μm,300-600μm around implant coarse thread. Many slender bone trabeculae grew into the thread. Because of the influence of the metal artifacts,0-45μm is not clear. Hard tissue section can only take one direction of cross section, but of the implant surface0-45microns region shows clear. It is a good way to observe the direction of the implant surface osteogenesis, contact osteogenesis phenomenon can be seen clearly.
4.Magenta acidic methylene blue staining:Different distances between implant and cortical bone, different amounts of new bone in the thread. In two weeks, when the distance between the bottom of implant and cortical bone was about300μm, many new bone grew into the thread; when the distance between the bottom of implant and cortical bone was about300-600μm, some new bone grew into the thread; when the distance between the bottom of implant and cortical bone was exceed600μm, few new bone grew into the thread. In four weeks, when the distance between the bottom of implant and cortical bone was about300μm, many new bone grew into the thread; when the distance between the bottom of implant and cortical bone was about300-600μm, some new bone grew into the thread; when the distance between the bottom of implant and cortical bone was exceed600μm, few new bone grew into the thread.
Bone histomorphometry:Test BIC and new bone area of the different distance between implant and cortical bone. The BIC and new bone area of the area under300μm between implant and cortical bone>the area300-600μm between implant and cortical bone> the area more than600μm between implant and cortical bone in two weeks and four weeks. The closer between the bottom of the thread and cortical bone, the better results of implant-bone contact.
5.The early osteogenenisis of Ultraviolet Irradiation on Sandblasted and Acid-etched Titanium implant Surfaces.(1) Fluorescence analysis。In two weeks, fluorescence are scattered around the implant and thread, mainly focus on implant thread. More fluorescence distribute at cortical bone than bone marrow cavity. In groups SLA, fluorescence are mainly focus on the skew wall of the thread and the arounding of implant. In groups SLA+UV, except the skew wall of the thread and the arounding of the implant, many fluorescence focus on the bottom of implant thread. In four weeks, except the inside of the thread and the arounding of the implant, new bone was found in the area of bone marrow cavity. There are more fluorescence scattered in group SLA than group SLA+UV.
(2)Methylene blue-acid fuchsin staining:At two weeks, New bone trabecular number comes from the surrounding bone,crawling along the implant thread in group SLA. In gorup SLA+UV, new bone not only grew along the inclined wall of the thread, but also in the bottom of the thread. At four weeks, new bone grew among the thread, haversian system was not seen obvious among the thread in group SLA, in group SLA+UV, many new bone was not only found in the inclined wall of the thread but also at the bottom of the thread. Contact osteogenesis is very obvious. Haversian system was seen among the thread.
(3)Bone tissue metrology analysis:In two weeks and four weeks, the BIC of group SLA+UV> the BIC ofgroup SLA. The difference was statistically significant.But the difference of the new bone area with two groups were not statistically significant.
Conclusion:
l.This beagle dog mandible model simulated the clinical situation and can be used to investigate bone formation around implant. The new planting model can be more clearly observed the effects of different surface treatments on osteogenesis.
2.Micro CT can be used to measure the bone with three-dimensional structure,and it has the advantage of continuity and integrity. But because of the existence of the metal artifacts, the implant-bone interface of osteogenesis reflect poor condition. Hard tissue sliciing is a single-layer tissue section and the production process is complicated, and will destroy the integrity of the specimen. But the subtle changes of implant surface osteogenesis reflect clearly.
3.The closer from bottom of thread to cortical bone, the better implant-bone contact.
4. Group SLA+UV have better contact osteogenesis, and the BIC%is higher in group SLA+UV than in group SLA.
引文
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