种植体表面改性—光诱导超亲水性表面骨结合机制研究
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摘要
【背景】
骨结合界面的破坏是目前临床上导致种植体发生失败的最主要原因，特别是即刻种植、即刻负重等新技术的广泛应用，对骨结合的效果提出了更高的要求。虽然已经有很多表面粗化处理的方法都能增强骨结合效果，但是骨结合率仍未达到非常理想的地步（仅介于30%-70%之间），而且部分学者认为这种粗糙的表面也成为种植体周围炎不易控制的重要影响因素之一。
最近，相关研究报道了在骨结合早期，经过放置4周的种植体骨结合率为60%，而刚刚加工完成的―新鲜‖表面则能达到90%，这应该归功于刚刚处理过的钛表面具有的生物活性。目前，有生产厂家尝试将种植体封装在PH等于5的生理盐水中来―保存‖生物活性，但是从加工完成到临床应用往往经历数月，会失去保存的意义；更重要的是，刚刚加工处理后的钛金属表面被证实带有正电荷，而疏水性种植体表面由于存在惰性氧化膜的保护不带电荷，而当种植体植入之前，制备后窝洞内的血浆蛋白带有负电荷，这就意味着在理论上极可能存在与以往不同的骨-种植体连接方式，即―直接‖骨连接。
有研究显示经过短时间内的光催化诱导，氧化钛表现出超强的亲水性能和生物活性；同时，紫外光（UV）照射能量超过3.2eV时，钛表面会释放电子，产生氧空穴，这种经过电荷改变的表面被证实更能吸引成骨细胞的粘附和同时促进细胞分化与增殖。所以，我们认为手术前即刻恢复植入材料表面生物活性要比长时间―保存‖的方式更加有效率；且这种钛表面电化学改变引起的、尚需被进一步证实的―直接‖骨结合的机理尚不清楚。
【目的】
1、进一步寻找具有生物活性的超亲水性表面―直接‖骨结合存在的证据，并验证超亲水性种植体―直接‖骨连接的效果。
2、探明光催化诱导钛种植体表面恢复超亲水性（接触角接近于0°）后增强骨结合能力的机制，为进一步指导临床应用提供理论依据。
【方法】
1.48枚实验用钛种植体及52个钛片（商业纯钛四级）采用微弧氧化法（MAO）表面粗化处理后随机分为实验组与对照组，并在黑暗的室内放置8周以强化其生物老化效果，然后对实验组的样本进行UVC光催化诱导48小时后，立即将实验组与对照组样本同时进行体内、体外实验。
2.分别对UVC光催化前后的钛片样本表面进行x射线光电子谱检测，分析光催化前后钛表面元素含量的变化。
3.分别对进行UVC光催化诱导后的钛片样本在特定连续时间段内进行接触角测量来观察生物老化现象的变化情况。
4.对实验组与对照组钛片样本进行成骨细胞与材料粘附共培养，观察成骨细胞对超亲水性表面与疏水性表面粘附、分化和增殖能力的差别。
5.48枚实验种植体随机分为实验组与对照组后进行体内动物实验，借助显微CT、组织形态学染色、生物力学检测及种植体-骨复合物的表面生物组织能谱分析等手段，观察超亲水性表面与疏水性表面的骨结合能力的差别。
【结果】
1.通过XPS检测，可以发现经过UVC光催化诱导后的钛表面的几种元素均发生了变化：其中在453.8eV的Ti4+和在458.3eV的Ti3+都升高说明光催化诱导方法可以暴露出更多的钛元素；此外，光催化诱导后的超亲水性表面Ti4+和Ti3+之间的―电势差值‖也增加了，原因可能因为光催化诱导的能量导致钛表面发生电子跃迁，进而产生了电荷加载，表面电化学结构发生改变。除了钛元素发生改变，氧元素的含量有所升高，这一现象可以间接表明光催化诱导后钛金属表面的羟基自由基的含量升高了。最后，碳元素的含量大大降低，说明光催化诱导有效地去除了杂质的附着。
2.表面接触角实验证实刚刚经过UVC光催化诱导后的超亲水性表面的接触角接近于0°，随着暴露时间的增加，接触角也增大，而经过7天的暴露后，接触角又恢复到70°以上，基本与对照组（疏水性表面）的接触角没有差别。
3.生物力学检测（最大抗拔出力实验）显示，在4周与12周的时候，实验组与对照组骨结合强度无明显差别；然而8周的时候，实验组最大抗拔出力值为70.4±9.3N，对照组为56.7±6.6N（P<0.01）,说明在骨结合早期，超亲水性表面较疏水性表面更能提高骨结合强度，原因可能是因为形成了化学性的连接，而在骨结合晚期未见明显优势。
4.通过EDS检测及扫描电镜观测可以发现，在骨-种植体结合界面内，对照组（疏水性表面）钛金属与骨之间有一个间隙；而经过UVC催化诱导后的超亲水性表面与骨之间则有着更为紧密的连接，即―直接‖骨-种植体连接。
5.显微CT观察的结果显示，在4周与12周时，超亲水性表面的骨矿化率（TMD）较疏水性表面只有较轻微的增加，而到了8周，则出现了明显的增加。
6.通过组织形态学分析可以得知，从4周到12周即骨结合早期与晚期，实验组（超亲水性表面）的骨结合效果均优于对照组（疏水性表面）。
【结论】
从上述实验研究中，得出以下五种推论：
1. UVC光催化诱导能够有效地将生物老化的钛表面重新恢复成为具有生物活性的超亲水性表面。
2.紫外线通过去除材料表面的碳等杂元素而纯化钛金属表面。
3.经过UVC光催化诱导后，XPS检测氧元素含量升高，表明具有生物活性的钛金属表面与空气中水接触后，会产生更多的羟基自由基。
4.经过UVC光催化诱导后，Ti4+与Ti3+之间的电势差增加了，使得生物惰性的钛金属由不带电荷转变成带有正电荷的表面，这样就有可能使钛金属表面直接吸引带有负点的血浆蛋白粘附，而加快骨结合的进度。
5.超亲水性表面的这种―直接‖骨结合方式具有较疏水性表面更加优越的骨结合效果，表现为结合强度更高，结合面积更大。虽然从宏观上看，对骨结合早期（8周）影响明显，但是这种带有正负电荷的化学性连接在骨结合的后期作用则不明显，而且这种新的连接方式可能将改变以往人们普遍接受的骨-种植体需要依赖Ca/P半桥粒过渡的理念。
总之，在表面粗化处理和基因、蛋白质分子自组装技术遇到瓶颈后，采用紫外线特别是UVC光催化诱导钛种植体表面后，使其发生改性，从而促进骨结合的研究是非常有价值和意义的。
Most implants fail because the bone-implant interface is destroyed, the osseointegrationis showing its important practical significance following the immediate implant techniquedevelop. Although lots of methods of roughness the surface have adopted for enhancingthe bone-implant integration, however, bone-implant contact area (BIC) still not ideal (justbetween30%and70%). Meanwhile, this roughness surface which could be one factorhaving an influence on the peri-implantitis controlling.
Recently, similar results were reported in studies conducted by, which revealed that inthe early stage of healing, the bone-implant contact (BIC) remained less than60%for4-week-old implants, whereas this metric reached over90%for new implants. Nowadays,some researches―remaining‖the bioactivity and hydrophilic by immerging the implant inthe solution (PH=5). But this method is still oppugning.
More important, the plasma protein shows negative charge in the surgical site beforeinserting the implant and the titanium surface show positive charge after processing. Thismeans in theory, there may be two different bone-implant connected style exist.
Scientists have found that TiO2is also known to be an effective photocatalyzingmaterial that can be used to improve the hydrophobicity of the titanium surface. Largelevels of UV energy (greater than3.2eV) are required to induce photocatalytic activity,i.e., to excite an electron from the valence band to the conduction band, which allows for the transmission of electric current. Many studies have reported that this photocatalyticsemiconductor surface substantially strengthens osteoblast retention, an effect that wasassociated with the enhancement of intracellular structural development during celladhesion. However, the biological mechanisms by which electric charge modifications onsuper-hydrophilic surfaces enhance bone-implant integration are unclear.The objective of this study was to investigate the evidence of the―direct‖bone implantconnected and further test the effect on the photo-induced, super-hydrophilic bioactivitytitanium surface.
【Methods】
1.For this study,48titanium implant rods (diameter2mm, length6mm) and52disks(diameter20mm, thickness1mm) were fabricated from pure titanium grade4(Northwest Metallurgy Institute, Shann’Xi, China). The surfaces of the24testimplants were MAO treated and were randomly divided into two groups. Theimplants from both group A and group B were stored under dark conditions for8weeks following processing. Following this phase, the group B specimens werefurther UVC-treated for48h.
2.We used an X-ray photoelectron spectra (XPS) system (Model PHI-5072, PhysicalElectronics Inc., China) using Mg K R radiation (250W, pass energy of29.35eV)to analyze the changes to the titanium surface following UVC irradiation.
3.The52super-hydrophilicity of the disk specimens was measured by assessing thestatic contact angles using a contact angle analyzer (Face, Kyowa Interface ScienceCo. Ltd., Japan).
4.The52super-hydrophilicity of the disk specimens were cultured with osteoblast (OB)to observe the different of differentiation and proliferation on the surper-hydrophilicsurface and dydrophobic surface.
5.The specimens with inserted implants were examined by micro-CT scanner,histological observation, push-out test and EDS test.
【Results】
1. The notable differences in the intensities of the457and453.8eV Ti2p peaks between the UV-treated and un-treated groups indicated that more Ti element exposed.Furthermore, XPS analyses revealed that the intensity of carbon was decreased bynearly half on the UV-photolyzed surfaces means the contaminations are removed andthat the intensity of the O1s peak was also increased which indicate that the hydroxylradical increased.
2. The initially super-hydrophilic (contact angle <10) UV-treated surfaces becamehydrophobic (contact angle>70) following7days of exposure to the atmosphere.
3. No significant difference was observed in the week4group and12week group;however, the bone anchorage strength was detected in the test group (70.4±9.3N)compared to the control group (56.7±6.6N) at8weeks.
4. In the bone-implant interface area, a layer of new bone was―directly‖contacted withthe implant on the UV-treated surface, on the contrast, a significant―gap‖structurewas found on the un-treated surface. The Ca, P, O and Ti element spectra line alsoindicated the bone structure was―directly‖integrated with the titanium on theUV-treated surface, however, there was an oxygen layer generally exhibited betweenthe bone and implant on the un-treated surface.
5. At4weeks and12weeks, the bone volume (BV) and tissue mineral density (TMD)were slightly increased in the test group than that in control group. However, at8weeks, the UV-treated group had visually greater bone formation than that in theun-treated group in the ROI, and the BV and TMD were significantly increased in thetest group compared to the control group.
6. The histological evaluation showed that the bone-implant integration in the uv-treatedsurper-hydrophilic surface group is better than the un-treated hydrophobic surfacegroup both from4week to12week.
【Conclusion】
1. UVC irradiation is sufficient to overcome the influence of biologicaltime-dependent degradation and to recover the hydrophobic surface tosurper-hydrophilic surface.
2. UVC irradiation can purify the titanium surface by removing the carboncontamination layer.
3. XPS test showed―O‖element increased may indicate that the dydroxyl radicalincreased on the UV-treated titanium surface.
4. UVC irradiation can lead potential difference increased. This notable differentelectric exchanged on super-hydrophilic titanium surface effectively increases theadhesion of the osteopontin and the osteo-conductivity.
5. The surper-hydrophilic surface effect on the osseointegration in the early healingstage (8w) and this notable―direct‖bone-implant integration connective structuremay change the concept of the traditional type of osseointegration depend on theCa/P hemidesmosome connective structure.
All in all, there are significant to adopt UVC induced the titanium bioactivity toenhance the osseointegration when the roughness technique, gene and biomoleculelayer by layer molecular self-assembly technique surfer the bottlenecks.
骨结合界面的破坏是目前临床上导致种植体发生失败的最主要原因，特别是即刻种植、即刻负重等新技术的广泛应用，对骨结合的效果提出了更高的要求。虽然已经有很多表面粗化处理的方法都能增强骨结合效果，但是骨结合率仍未达到非常理想的地步（仅介于30%-70%之间），而且部分学者认为这种粗糙的表面也成为种植体周围炎不易控制的重要影响因素之一。
最近，相关研究报道了在骨结合早期，经过放置4周的种植体骨结合率为60%，而刚刚加工完成的―新鲜‖表面则能达到90%，这应该归功于刚刚处理过的钛表面具有的生物活性。目前，有生产厂家尝试将种植体封装在PH等于5的生理盐水中来―保存‖生物活性，但是从加工完成到临床应用往往经历数月，会失去保存的意义；更重要的是，刚刚加工处理后的钛金属表面被证实带有正电荷，而疏水性种植体表面由于存在惰性氧化膜的保护不带电荷，而当种植体植入之前，制备后窝洞内的血浆蛋白带有负电荷，这就意味着在理论上极可能存在与以往不同的骨-种植体连接方式，即―直接‖骨连接。
有研究显示经过短时间内的光催化诱导，氧化钛表现出超强的亲水性能和生物活性；同时，紫外光（UV）照射能量超过3.2eV时，钛表面会释放电子，产生氧空穴，这种经过电荷改变的表面被证实更能吸引成骨细胞的粘附和同时促进细胞分化与增殖。所以，我们认为手术前即刻恢复植入材料表面生物活性要比长时间―保存‖的方式更加有效率；且这种钛表面电化学改变引起的、尚需被进一步证实的―直接‖骨结合的机理尚不清楚。
【目的】
1、进一步寻找具有生物活性的超亲水性表面―直接‖骨结合存在的证据，并验证超亲水性种植体―直接‖骨连接的效果。
2、探明光催化诱导钛种植体表面恢复超亲水性（接触角接近于0°）后增强骨结合能力的机制，为进一步指导临床应用提供理论依据。
【方法】
1.48枚实验用钛种植体及52个钛片（商业纯钛四级）采用微弧氧化法（MAO）表面粗化处理后随机分为实验组与对照组，并在黑暗的室内放置8周以强化其生物老化效果，然后对实验组的样本进行UVC光催化诱导48小时后，立即将实验组与对照组样本同时进行体内、体外实验。
2.分别对UVC光催化前后的钛片样本表面进行x射线光电子谱检测，分析光催化前后钛表面元素含量的变化。
3.分别对进行UVC光催化诱导后的钛片样本在特定连续时间段内进行接触角测量来观察生物老化现象的变化情况。
4.对实验组与对照组钛片样本进行成骨细胞与材料粘附共培养，观察成骨细胞对超亲水性表面与疏水性表面粘附、分化和增殖能力的差别。
5.48枚实验种植体随机分为实验组与对照组后进行体内动物实验，借助显微CT、组织形态学染色、生物力学检测及种植体-骨复合物的表面生物组织能谱分析等手段，观察超亲水性表面与疏水性表面的骨结合能力的差别。
【结果】
1.通过XPS检测，可以发现经过UVC光催化诱导后的钛表面的几种元素均发生了变化：其中在453.8eV的Ti4+和在458.3eV的Ti3+都升高说明光催化诱导方法可以暴露出更多的钛元素；此外，光催化诱导后的超亲水性表面Ti4+和Ti3+之间的―电势差值‖也增加了，原因可能因为光催化诱导的能量导致钛表面发生电子跃迁，进而产生了电荷加载，表面电化学结构发生改变。除了钛元素发生改变，氧元素的含量有所升高，这一现象可以间接表明光催化诱导后钛金属表面的羟基自由基的含量升高了。最后，碳元素的含量大大降低，说明光催化诱导有效地去除了杂质的附着。
2.表面接触角实验证实刚刚经过UVC光催化诱导后的超亲水性表面的接触角接近于0°，随着暴露时间的增加，接触角也增大，而经过7天的暴露后，接触角又恢复到70°以上，基本与对照组（疏水性表面）的接触角没有差别。
3.生物力学检测（最大抗拔出力实验）显示，在4周与12周的时候，实验组与对照组骨结合强度无明显差别；然而8周的时候，实验组最大抗拔出力值为70.4±9.3N，对照组为56.7±6.6N（P<0.01）,说明在骨结合早期，超亲水性表面较疏水性表面更能提高骨结合强度，原因可能是因为形成了化学性的连接，而在骨结合晚期未见明显优势。
4.通过EDS检测及扫描电镜观测可以发现，在骨-种植体结合界面内，对照组（疏水性表面）钛金属与骨之间有一个间隙；而经过UVC催化诱导后的超亲水性表面与骨之间则有着更为紧密的连接，即―直接‖骨-种植体连接。
5.显微CT观察的结果显示，在4周与12周时，超亲水性表面的骨矿化率（TMD）较疏水性表面只有较轻微的增加，而到了8周，则出现了明显的增加。
6.通过组织形态学分析可以得知，从4周到12周即骨结合早期与晚期，实验组（超亲水性表面）的骨结合效果均优于对照组（疏水性表面）。
【结论】
从上述实验研究中，得出以下五种推论：
1. UVC光催化诱导能够有效地将生物老化的钛表面重新恢复成为具有生物活性的超亲水性表面。
2.紫外线通过去除材料表面的碳等杂元素而纯化钛金属表面。
3.经过UVC光催化诱导后，XPS检测氧元素含量升高，表明具有生物活性的钛金属表面与空气中水接触后，会产生更多的羟基自由基。
4.经过UVC光催化诱导后，Ti4+与Ti3+之间的电势差增加了，使得生物惰性的钛金属由不带电荷转变成带有正电荷的表面，这样就有可能使钛金属表面直接吸引带有负点的血浆蛋白粘附，而加快骨结合的进度。
5.超亲水性表面的这种―直接‖骨结合方式具有较疏水性表面更加优越的骨结合效果，表现为结合强度更高，结合面积更大。虽然从宏观上看，对骨结合早期（8周）影响明显，但是这种带有正负电荷的化学性连接在骨结合的后期作用则不明显，而且这种新的连接方式可能将改变以往人们普遍接受的骨-种植体需要依赖Ca/P半桥粒过渡的理念。
总之，在表面粗化处理和基因、蛋白质分子自组装技术遇到瓶颈后，采用紫外线特别是UVC光催化诱导钛种植体表面后，使其发生改性，从而促进骨结合的研究是非常有价值和意义的。
Most implants fail because the bone-implant interface is destroyed, the osseointegrationis showing its important practical significance following the immediate implant techniquedevelop. Although lots of methods of roughness the surface have adopted for enhancingthe bone-implant integration, however, bone-implant contact area (BIC) still not ideal (justbetween30%and70%). Meanwhile, this roughness surface which could be one factorhaving an influence on the peri-implantitis controlling.
Recently, similar results were reported in studies conducted by, which revealed that inthe early stage of healing, the bone-implant contact (BIC) remained less than60%for4-week-old implants, whereas this metric reached over90%for new implants. Nowadays,some researches―remaining‖the bioactivity and hydrophilic by immerging the implant inthe solution (PH=5). But this method is still oppugning.
More important, the plasma protein shows negative charge in the surgical site beforeinserting the implant and the titanium surface show positive charge after processing. Thismeans in theory, there may be two different bone-implant connected style exist.
Scientists have found that TiO2is also known to be an effective photocatalyzingmaterial that can be used to improve the hydrophobicity of the titanium surface. Largelevels of UV energy (greater than3.2eV) are required to induce photocatalytic activity,i.e., to excite an electron from the valence band to the conduction band, which allows for the transmission of electric current. Many studies have reported that this photocatalyticsemiconductor surface substantially strengthens osteoblast retention, an effect that wasassociated with the enhancement of intracellular structural development during celladhesion. However, the biological mechanisms by which electric charge modifications onsuper-hydrophilic surfaces enhance bone-implant integration are unclear.The objective of this study was to investigate the evidence of the―direct‖bone implantconnected and further test the effect on the photo-induced, super-hydrophilic bioactivitytitanium surface.
【Methods】
1.For this study,48titanium implant rods (diameter2mm, length6mm) and52disks(diameter20mm, thickness1mm) were fabricated from pure titanium grade4(Northwest Metallurgy Institute, Shann’Xi, China). The surfaces of the24testimplants were MAO treated and were randomly divided into two groups. Theimplants from both group A and group B were stored under dark conditions for8weeks following processing. Following this phase, the group B specimens werefurther UVC-treated for48h.
2.We used an X-ray photoelectron spectra (XPS) system (Model PHI-5072, PhysicalElectronics Inc., China) using Mg K R radiation (250W, pass energy of29.35eV)to analyze the changes to the titanium surface following UVC irradiation.
3.The52super-hydrophilicity of the disk specimens was measured by assessing thestatic contact angles using a contact angle analyzer (Face, Kyowa Interface ScienceCo. Ltd., Japan).
4.The52super-hydrophilicity of the disk specimens were cultured with osteoblast (OB)to observe the different of differentiation and proliferation on the surper-hydrophilicsurface and dydrophobic surface.
5.The specimens with inserted implants were examined by micro-CT scanner,histological observation, push-out test and EDS test.
【Results】
1. The notable differences in the intensities of the457and453.8eV Ti2p peaks between the UV-treated and un-treated groups indicated that more Ti element exposed.Furthermore, XPS analyses revealed that the intensity of carbon was decreased bynearly half on the UV-photolyzed surfaces means the contaminations are removed andthat the intensity of the O1s peak was also increased which indicate that the hydroxylradical increased.
2. The initially super-hydrophilic (contact angle <10) UV-treated surfaces becamehydrophobic (contact angle>70) following7days of exposure to the atmosphere.
3. No significant difference was observed in the week4group and12week group;however, the bone anchorage strength was detected in the test group (70.4±9.3N)compared to the control group (56.7±6.6N) at8weeks.
4. In the bone-implant interface area, a layer of new bone was―directly‖contacted withthe implant on the UV-treated surface, on the contrast, a significant―gap‖structurewas found on the un-treated surface. The Ca, P, O and Ti element spectra line alsoindicated the bone structure was―directly‖integrated with the titanium on theUV-treated surface, however, there was an oxygen layer generally exhibited betweenthe bone and implant on the un-treated surface.
5. At4weeks and12weeks, the bone volume (BV) and tissue mineral density (TMD)were slightly increased in the test group than that in control group. However, at8weeks, the UV-treated group had visually greater bone formation than that in theun-treated group in the ROI, and the BV and TMD were significantly increased in thetest group compared to the control group.
6. The histological evaluation showed that the bone-implant integration in the uv-treatedsurper-hydrophilic surface group is better than the un-treated hydrophobic surfacegroup both from4week to12week.
【Conclusion】
1. UVC irradiation is sufficient to overcome the influence of biologicaltime-dependent degradation and to recover the hydrophobic surface tosurper-hydrophilic surface.
2. UVC irradiation can purify the titanium surface by removing the carboncontamination layer.
3. XPS test showed―O‖element increased may indicate that the dydroxyl radicalincreased on the UV-treated titanium surface.
4. UVC irradiation can lead potential difference increased. This notable differentelectric exchanged on super-hydrophilic titanium surface effectively increases theadhesion of the osteopontin and the osteo-conductivity.
5. The surper-hydrophilic surface effect on the osseointegration in the early healingstage (8w) and this notable―direct‖bone-implant integration connective structuremay change the concept of the traditional type of osseointegration depend on theCa/P hemidesmosome connective structure.
All in all, there are significant to adopt UVC induced the titanium bioactivity toenhance the osseointegration when the roughness technique, gene and biomoleculelayer by layer molecular self-assembly technique surfer the bottlenecks.
引文
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