纳米二氧化钛薄膜材料生物相容性的分析研究
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摘要
硬骨组织修复材料是生物医用材料的一种,主要应用于诊断、治疗、修复和替换病损骨组织,增进老损骨组织的功能,是一类需求量大,市场前景广泛的材料。目前临床使用的硬骨组织修复材料组要为金属、生物陶瓷、生物玻璃和高分子聚合物支架,其寿命通常为10-15年。这样短的寿命为患者带来不便,也易引起二次痛苦。
骨植入体在植入生物体后需要发生与周围组织的整合,其过程通常需要成骨细胞的大量粘附。成骨细胞早期大量粘附于植入体,细胞间迅速进行通信,完成细胞增殖、分化和其它生理功能,从而与生物体整合,发挥正常的生理功能。影响成骨细胞在材料表面早期粘附行为的因素包括材料表面的化学构成、拓扑形貌、粗糙度及亲水性等。研究表明,现有硬骨替代材料通常因为成纤维细胞包裹而导致发炎、坏死,使之无法发挥正常功能。而成纤维细胞和成骨细胞在材料表面的粘附比例显示与材料表面粗糙度有关,即材料表面粗糙度大时,成骨细胞易粘附,反之则造成成纤维细胞的大量附着。通过对生物材料表面改性,尤其是对表面粗糙度的改变可以提高成骨细胞在材料表面的粘附,提高植入体的成活率。
对天然骨组织的研究发现,天然骨表面覆盖一层直径为1nm的胶原颗粒,这使得纳米材料作为骨植入材料的研究得到了强有力的理论支持。钛金属以及其合金材料是生物材料学的研究热点,纳米尺度钛材料显示出相对于其他金属材料的低毒性,受到全世界科学家的瞩目。目前对纳米二氧化钛材料的研究普遍集中于颗粒物质的体内、体外毒性研究,以及合金材料作为骨植入体的相关研究,对于表面覆盖纳米二氧化钛薄膜的钛基底材料的相关研究还较为少见。
本实验分为两个部分,在第一部分中讨论了表面形貌分别为管状、网状、颗粒状的三种纳米二氧化钛薄膜材料在体外实验中的毒性测试,使用的实验手段包括扫描电子显微镜下观察材料表面粘附细胞的形态和数量,粘附细胞的乳酸脱氢酶泄露和细胞增殖。实验发现三种形貌的纳米二氧化钛薄膜材料中,颗粒状纳米二氧化钛覆盖的薄膜材料相对其他两种材料具有更高的细胞亲和力和较低的乳酸脱氢酶泄露,是相对理想的材料。
第二部分实验承接第一部分实验的结果,着重讨论了表面覆盖1层、2层、3层、4层纳米二氧化钛颗粒的薄膜材料的生物相容性。实验采用国际流行的Layer-by-Layer自组装技术在纯钛基底表面组装不同层数的纳米二氧化钛颗粒,并使用扫面电子显微镜对材料进行表征,通过接触角分析实验对材料的亲水性进行检测。生物实验部分,采用鼠成骨瘤细胞与四种材料共培养,分别研究了材料表面粘附细胞的形貌和数量,使用荧光染料对粘附细胞内的ROS和DNA进行染色观察,测量了细胞乳酸脱氢酶泄露量和细胞增殖情况。结果发现材料表面粗糙度随着薄膜层数的增加而增大,亲水性也随之增大。而薄膜层数多的材料在生物学研究指标上也表现出优势,即薄膜层数越多,粗糙度越大的材料,其表面粘附细胞数量越多,细胞生长状况约好,受氧化损伤程度低,细胞凋亡和坏死现象少。
Hard bone tissue repair material is a kind of biomedical materials, mainly used in diagnosis, treatment, repair and replacement of osteoporosis, osteoarthritis and other hard tissue diseases and defects, enhance the function of old bone tissue, which is has potential broad market[1,2] Synthetic bioactive materials such as Bioglass, glassceramic A-W, sintered P-tricalcium phosphate (TCP) and sintered hydroxyapatite (HA), due to a variety of problems, could only endure10-15years[3]. Changing for new implants will not only bring indescribable suffering to the patients, but also deteriorate the patients' state of illness.
Bone implants, after implantation, needs to integrate with the surrounding tissue, and the process usually starts from a large number of osteoblast adhesion[4-6]. After the osteoblast's initial adhesion, cells begin to rapidly send cell to cell communicate, which will help the forward cell proliferation and differentiation, and other physiological function. Many factors could affect the osteoblasts initial behavior, include the chemical composition of the material, surface topology morphology, roughness and hydrophilic, etc. Study shows that the hard bone substitute turn to failure usually because of the fibroblasts package, which will afterwards cause inflammation and necrosis, and finally lead to implant disability[7].
Natural bone tissue is a highly organized hierarchical structure which is composed of nano-, micro-, and macro-scale building blocks[8]. Non-collageneous organic proteins, fibrillar collagen and hydroxyapatite crystals which on the surface of bone are all nanoscale structures. From the biomimetic point of view, Ti with its surface coated with nanoscale TiO2may provide a more suitable surface topography for cell functions as it can better mimic the sructure of the natural extracellular matrix. Although all these results implies that nano-sized TiO2may has superior bioactivity and be more suitable for application as a bone substitute than the traditional biomaterials, different types of TiO2were fully researched.
This research is divided into2parts. In the first part, three types of titanium (Ti) based nanoscale titania (TiO2) were produced by controlling the reaction conditions, including TiO2nanotube, TiO2nanonetwork and TiO2nanoparticle. Human Osteosarcoma cell (MG63) was used in this study to evaluate the bioactivity and cytocompatibility of the new materials. Compared to pure Ti, cell adhesion of the materials coated with nanoscale TiO2was significantly enhanced after24h,48h, and72h of co-incubation, according to SEM photographs. More importantly, compare to the control group, the lactate dehydrogenase (LDH) released into the culture media and the absorbance of MTT showed no obvious difference after72h of co-incubation. Therefore, it is suggested that nanoscale TiO2is a bioactive and cytocompatible biomedical material.
The second part of the experiment to undertake the first part the result of the experiment, emphasized on the cytocompatibility of4different kind of material which have1larer,2layers,3layers and4layers of TiO2nanofilm. The materials were assembled by the Layer-by-Layer (LBL) self-assembly technique, which has been wildly used to fabricate multilayer thin films of controlled composition, thickness, and architecture. Surface roughness w measured by field emission scanning electron microscopy (SEM). Hydrophilicity was measured using the contact angle meter. Cell attachment to the materials was observed using SEM. Fluorescent dyes were used to observe the generation of reactive oxygen species and DNA damage. Apoptosis was measured through lactate dehydrogenase leakage assay. The2-(4,5-dimethyl-2-thiazol-2-yl)-3,5-diphenyl-2H-tetrazolium (MTT) assay was used to determine the viability of the cells attached to the materials. This new film was found to produce better cell proliferation and function; it is comparatively cytocompatible, and therefore may be a better material for orthopedic surgery.
骨植入体在植入生物体后需要发生与周围组织的整合,其过程通常需要成骨细胞的大量粘附。成骨细胞早期大量粘附于植入体,细胞间迅速进行通信,完成细胞增殖、分化和其它生理功能,从而与生物体整合,发挥正常的生理功能。影响成骨细胞在材料表面早期粘附行为的因素包括材料表面的化学构成、拓扑形貌、粗糙度及亲水性等。研究表明,现有硬骨替代材料通常因为成纤维细胞包裹而导致发炎、坏死,使之无法发挥正常功能。而成纤维细胞和成骨细胞在材料表面的粘附比例显示与材料表面粗糙度有关,即材料表面粗糙度大时,成骨细胞易粘附,反之则造成成纤维细胞的大量附着。通过对生物材料表面改性,尤其是对表面粗糙度的改变可以提高成骨细胞在材料表面的粘附,提高植入体的成活率。
对天然骨组织的研究发现,天然骨表面覆盖一层直径为1nm的胶原颗粒,这使得纳米材料作为骨植入材料的研究得到了强有力的理论支持。钛金属以及其合金材料是生物材料学的研究热点,纳米尺度钛材料显示出相对于其他金属材料的低毒性,受到全世界科学家的瞩目。目前对纳米二氧化钛材料的研究普遍集中于颗粒物质的体内、体外毒性研究,以及合金材料作为骨植入体的相关研究,对于表面覆盖纳米二氧化钛薄膜的钛基底材料的相关研究还较为少见。
本实验分为两个部分,在第一部分中讨论了表面形貌分别为管状、网状、颗粒状的三种纳米二氧化钛薄膜材料在体外实验中的毒性测试,使用的实验手段包括扫描电子显微镜下观察材料表面粘附细胞的形态和数量,粘附细胞的乳酸脱氢酶泄露和细胞增殖。实验发现三种形貌的纳米二氧化钛薄膜材料中,颗粒状纳米二氧化钛覆盖的薄膜材料相对其他两种材料具有更高的细胞亲和力和较低的乳酸脱氢酶泄露,是相对理想的材料。
第二部分实验承接第一部分实验的结果,着重讨论了表面覆盖1层、2层、3层、4层纳米二氧化钛颗粒的薄膜材料的生物相容性。实验采用国际流行的Layer-by-Layer自组装技术在纯钛基底表面组装不同层数的纳米二氧化钛颗粒,并使用扫面电子显微镜对材料进行表征,通过接触角分析实验对材料的亲水性进行检测。生物实验部分,采用鼠成骨瘤细胞与四种材料共培养,分别研究了材料表面粘附细胞的形貌和数量,使用荧光染料对粘附细胞内的ROS和DNA进行染色观察,测量了细胞乳酸脱氢酶泄露量和细胞增殖情况。结果发现材料表面粗糙度随着薄膜层数的增加而增大,亲水性也随之增大。而薄膜层数多的材料在生物学研究指标上也表现出优势,即薄膜层数越多,粗糙度越大的材料,其表面粘附细胞数量越多,细胞生长状况约好,受氧化损伤程度低,细胞凋亡和坏死现象少。
Hard bone tissue repair material is a kind of biomedical materials, mainly used in diagnosis, treatment, repair and replacement of osteoporosis, osteoarthritis and other hard tissue diseases and defects, enhance the function of old bone tissue, which is has potential broad market[1,2] Synthetic bioactive materials such as Bioglass, glassceramic A-W, sintered P-tricalcium phosphate (TCP) and sintered hydroxyapatite (HA), due to a variety of problems, could only endure10-15years[3]. Changing for new implants will not only bring indescribable suffering to the patients, but also deteriorate the patients' state of illness.
Bone implants, after implantation, needs to integrate with the surrounding tissue, and the process usually starts from a large number of osteoblast adhesion[4-6]. After the osteoblast's initial adhesion, cells begin to rapidly send cell to cell communicate, which will help the forward cell proliferation and differentiation, and other physiological function. Many factors could affect the osteoblasts initial behavior, include the chemical composition of the material, surface topology morphology, roughness and hydrophilic, etc. Study shows that the hard bone substitute turn to failure usually because of the fibroblasts package, which will afterwards cause inflammation and necrosis, and finally lead to implant disability[7].
Natural bone tissue is a highly organized hierarchical structure which is composed of nano-, micro-, and macro-scale building blocks[8]. Non-collageneous organic proteins, fibrillar collagen and hydroxyapatite crystals which on the surface of bone are all nanoscale structures. From the biomimetic point of view, Ti with its surface coated with nanoscale TiO2may provide a more suitable surface topography for cell functions as it can better mimic the sructure of the natural extracellular matrix. Although all these results implies that nano-sized TiO2may has superior bioactivity and be more suitable for application as a bone substitute than the traditional biomaterials, different types of TiO2were fully researched.
This research is divided into2parts. In the first part, three types of titanium (Ti) based nanoscale titania (TiO2) were produced by controlling the reaction conditions, including TiO2nanotube, TiO2nanonetwork and TiO2nanoparticle. Human Osteosarcoma cell (MG63) was used in this study to evaluate the bioactivity and cytocompatibility of the new materials. Compared to pure Ti, cell adhesion of the materials coated with nanoscale TiO2was significantly enhanced after24h,48h, and72h of co-incubation, according to SEM photographs. More importantly, compare to the control group, the lactate dehydrogenase (LDH) released into the culture media and the absorbance of MTT showed no obvious difference after72h of co-incubation. Therefore, it is suggested that nanoscale TiO2is a bioactive and cytocompatible biomedical material.
The second part of the experiment to undertake the first part the result of the experiment, emphasized on the cytocompatibility of4different kind of material which have1larer,2layers,3layers and4layers of TiO2nanofilm. The materials were assembled by the Layer-by-Layer (LBL) self-assembly technique, which has been wildly used to fabricate multilayer thin films of controlled composition, thickness, and architecture. Surface roughness w measured by field emission scanning electron microscopy (SEM). Hydrophilicity was measured using the contact angle meter. Cell attachment to the materials was observed using SEM. Fluorescent dyes were used to observe the generation of reactive oxygen species and DNA damage. Apoptosis was measured through lactate dehydrogenase leakage assay. The2-(4,5-dimethyl-2-thiazol-2-yl)-3,5-diphenyl-2H-tetrazolium (MTT) assay was used to determine the viability of the cells attached to the materials. This new film was found to produce better cell proliferation and function; it is comparatively cytocompatible, and therefore may be a better material for orthopedic surgery.
引文
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