钛表面构建生物活性分子与类骨磷灰石复合功能涂层
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摘要
钛及其合金是目前临床上广泛使用的一类植入物材料,具有良好的机械性能和生物相容性。然而,长期临床实践和相关研究表明,现有钛植入体材料尚面临许多问题,主要包括：骨再生能力差或再生缓慢,与周围骨组织整合不佳,平均使用寿命不能满足患者的需求。由于钛植入体在体内是通过其表面与周围人体组织相接触的,因此通过表面改性对获得综合性能优良的植入体具有现实意义。通过对材料进行表面改性,能够获得综合性能优良的钛植入体,这对于克服钛植入体的上述不足具有重要的现实意义。近年来,在医用钛植入体表面构建类骨磷灰石涂层,并进一步装载药物、生长因子等生物活性分子,已经成为医用钛领域的研究热点。这些表面改性技术赋予医用钛植入体特定的功能,例如骨传导性和骨诱导性,从而改善钛植入体的骨整合性能和使用寿命。
本论文选用具有促细胞粘附的纤连蛋白(Fibronectin, FN)和促细胞向成骨分化的成骨生长肽(Osteogenic growth peptide, OGP),在钛表面构建了一系列的生物活性分子与类骨磷灰石复合功能涂层。我们首先研究了钛表面类骨磷灰石涂层的制备,并对仿生矿化液中类骨磷灰石涂层的生成机理进行了探讨；其次,在钛表面构建了纤连蛋白-类骨磷灰石和成骨生长肽-类骨磷灰石两种复合涂层,并对复合涂层的物相组成、微观形貌、精细结构等进行了检测和分析,研究了生物活性分子对类骨磷灰石生长的影响及作用机理；最后,我们通过不同的生物活性分子固定方法(共沉积法和／或表面吸附法)以及不同的固定顺序,将纤连蛋白和成骨生长肽共固定到具有类骨磷灰石涂层的钛表面,并对改性表面进行了相关的物化表征和体外生物学评价。本文的研究结果包括以下三个方面：
(1)通过离子束辅助沉积和仿生矿化沉积相结合的方法,可以在钛表面构建出均匀且均一的类骨磷灰石涂层。实验结果表明,在类骨磷灰石涂层沉积的过程中,随着浸泡时间的延长,类骨磷灰石的XRD特征衍射峰增强,且逐渐窄化,DPBS溶液中钙离子浓度随着磷灰石薄膜的分解逐渐升高,到达峰值后,随着类骨磷灰石的沉积,溶液中钙离子的浓度逐渐降低。随着涂覆在钛表面的磷灰石薄膜结晶度的增加,仿生矿化液中钛表面类骨磷灰石成核的速率逐渐减慢,但随后晶核生长的速度有所增加。
(2)通过生物仿生矿化沉积法,可在磷灰石薄膜活化的钛表面沉积上纤连蛋白-类骨磷灰石或成骨生长肽-类骨磷灰石两种复合涂层。FESEM、confocal结果显示,在构建的复合涂层中,生物活性分子均匀地分布在类骨磷灰石涂层内。慢扫XRD数据的Rietveld精修结果表明,单纯的类骨磷灰石涂层无机化学组成为apatite和OCP,随着生物活性分子的共沉积,类骨磷灰石中OCP所占的百分比逐渐降低,apatite晶胞的c轴长度增加,a轴的长度发生改变。同时,生物活性分子的加入,会降低类骨磷灰石晶体的生长速率,并影响类骨磷灰石晶体的微观形貌。初步的物化表征和精细结构分析表明,生物活性分子的共沉积主要是由生物活性分子、材料表面及溶液中的离子三者之间的静电吸附介导的,在沉积过程中,生物活性分子参与了类骨磷灰石晶胞构建的过程。
(3)通过共沉积法、表面吸附法或两者相结合的方法能够将纤连蛋白和成骨生长肽共固定到具有类骨磷灰石涂层的钛表面。FESEM及XRD的的结果显示,表面吸附的生物活性分子对预沉积的类骨磷灰石的结构和物相没有影响,而共沉积的生物活性分子使沉积的类骨磷灰石的片层结构晶体增大,并且使沉积的类骨磷灰石的主相由apatite转变为OCP。体外释放结果证明,固定的生物活性分子在体外可以进行持续可控的释放。通过体外细胞实验研究表明,共固定的纤连蛋白／成骨生长肽能够显著的提高骨髓间充质干细胞的贴附和铺展能力,并能够有效的促进骨髓间充质干细胞的增殖和向成骨分化。研究证明,通过表面共吸附的方法能够在钛植入体表面同时固定多种生物活性分子,对生物医用植入体发挥长期疗效具有积极作用,在牙科和整形外科等领域具有良好的临床实用价值。
Titanium and its alloys are extensively used for clinical implants, because of their excellent mechanical properties, and good biocompatibility. The long-term clinical researches show that titanium implant is confronted with the following problems:poor bone regeneration or slow regeneration, poor integration with surrounding bone tissues, and limited service life. The events after implantation include interactions between tissues and implant surfaces. Therefore, it is extremely important to achieve the implant with good comprehensive performance through surface modification. In recent years, further immobilizing the bioactive molecules, such as therapeutic agents or growth factors, with bone-like mineral layer on titanium implants is becoming the research focus in the biomaterials fields. Such surface modification technology could incorporate osteoinductivity and osteoconductivity into the design of the supporting biomaterial, thus improving the osseointegration and extending the service life.
In this study, we chose fibronectin (FN) and osteogenic growth peptide (OGP) to prepare a series of bioactive molecules-mineral functional composite layers on titanium surfaces. Firstly, the precipitation of bone-like mineral layer on titanium surfaces was studied, and a detailed insight into bone-like mineral nucleation and growth was discussed. Secondly, a coprecipitation method to build FN-mineral or OGP-mineral composite layers on titanium was developed. The phase composition, micro-morphology and fine structure of composite layers were investigated. The mechanisms underlying the coprecipitation of biomolecules into the mineral layer were also discussed. Finally, the fibronectin and osteogenic growth peptide were immobilized with bone-like mineral layer on titanium with varying immobilizing sequences through the methods of coprecipitation and/or physical adsorption. There are three parts in this dissertation, including:
1. A uniform, homogeneous apatite layer was biomimetically precipitated on hydroxyapatite thin film coated titanium through combing IBAD with biomimetic precipitation. XRD patterns indicate that, during the bone-like apatite precipitation, with the increase of immersion time, the density of apatite's characteristic peak increased and the peak became narrower. The calcium concentration in DPBS solution increased with the dissolution of hydroxyapatite thin film. And then the calcium concentration decreased with the precipitation of apatite. Bone-like apatite was nucleated earliest on the hydroxyapatite thin film with the lowest degree of crystallinity. The rate of nucleation growth increased with increasing crystallinity of the coated hydroxyapatite thin film.
2. FN-mineral and OGP-mineral composite layers were biomimetically induced on an active hydroxyapatite thin film through coprecipitation method. FESEM and confocal observations show that, the biomimetic coprecipitation process allowed biomolecules to be incorporated uniformly throughout the mineral layer. Rietveld refinement results suggest that, the mineral layer was indexed of a mixture of apatite and OCP. The phases of FN-mineral and OGP-mineral were partially transformed from OCP to apatite. The lattice parameter c of apatite was higher in the bioactive molecules-mineral composite layers than in the mineral layers, and the parameter a of apatite was changed when the bioactive molecules were coprecipitated on the surfaces. The biomimetic coprecipitated bioactive molecules slowed down the rate of mineral growth and influenced the ultimate structure of mineral. Primary physicochemical characterization and fine structure analysis indicate that the coprecipitation process was mainly mediated by electrostatic attraction among bioactive molecules, material surfaces and the ions in the solutions, and the bioactive molecules participated in the formation of the crystal latticework.
3. The coprecipitation and/or adsorption approaches were used to immobilize FN and/or OGP with biomimetic mineral layer onto titanium substrates. FESEM and XRD results show that, the adsorption of BMs did not change the morphology of mineral crystal and the main phase of mineral layer. Whereas, the coprecipitation of BMs enlarge the mineral crystal and change the main phase of mineral layer from apatite to OCP. Release studies demonstrated that either coprecipitation or adsorption approach provided sustained delivery of bioactive molecules. The dual immobilizing of FN and OGP had a significant impact on bMSCs attachment, spreading, proliferation and osteogenic differentiation compared to immobilizing of FN or OGP alone. The multi-immobilizing mineral with various bioactive molecules through adsorption approach has the potential to be used for orthopedic and dental applications.
本论文选用具有促细胞粘附的纤连蛋白(Fibronectin, FN)和促细胞向成骨分化的成骨生长肽(Osteogenic growth peptide, OGP),在钛表面构建了一系列的生物活性分子与类骨磷灰石复合功能涂层。我们首先研究了钛表面类骨磷灰石涂层的制备,并对仿生矿化液中类骨磷灰石涂层的生成机理进行了探讨；其次,在钛表面构建了纤连蛋白-类骨磷灰石和成骨生长肽-类骨磷灰石两种复合涂层,并对复合涂层的物相组成、微观形貌、精细结构等进行了检测和分析,研究了生物活性分子对类骨磷灰石生长的影响及作用机理；最后,我们通过不同的生物活性分子固定方法(共沉积法和／或表面吸附法)以及不同的固定顺序,将纤连蛋白和成骨生长肽共固定到具有类骨磷灰石涂层的钛表面,并对改性表面进行了相关的物化表征和体外生物学评价。本文的研究结果包括以下三个方面：
(1)通过离子束辅助沉积和仿生矿化沉积相结合的方法,可以在钛表面构建出均匀且均一的类骨磷灰石涂层。实验结果表明,在类骨磷灰石涂层沉积的过程中,随着浸泡时间的延长,类骨磷灰石的XRD特征衍射峰增强,且逐渐窄化,DPBS溶液中钙离子浓度随着磷灰石薄膜的分解逐渐升高,到达峰值后,随着类骨磷灰石的沉积,溶液中钙离子的浓度逐渐降低。随着涂覆在钛表面的磷灰石薄膜结晶度的增加,仿生矿化液中钛表面类骨磷灰石成核的速率逐渐减慢,但随后晶核生长的速度有所增加。
(2)通过生物仿生矿化沉积法,可在磷灰石薄膜活化的钛表面沉积上纤连蛋白-类骨磷灰石或成骨生长肽-类骨磷灰石两种复合涂层。FESEM、confocal结果显示,在构建的复合涂层中,生物活性分子均匀地分布在类骨磷灰石涂层内。慢扫XRD数据的Rietveld精修结果表明,单纯的类骨磷灰石涂层无机化学组成为apatite和OCP,随着生物活性分子的共沉积,类骨磷灰石中OCP所占的百分比逐渐降低,apatite晶胞的c轴长度增加,a轴的长度发生改变。同时,生物活性分子的加入,会降低类骨磷灰石晶体的生长速率,并影响类骨磷灰石晶体的微观形貌。初步的物化表征和精细结构分析表明,生物活性分子的共沉积主要是由生物活性分子、材料表面及溶液中的离子三者之间的静电吸附介导的,在沉积过程中,生物活性分子参与了类骨磷灰石晶胞构建的过程。
(3)通过共沉积法、表面吸附法或两者相结合的方法能够将纤连蛋白和成骨生长肽共固定到具有类骨磷灰石涂层的钛表面。FESEM及XRD的的结果显示,表面吸附的生物活性分子对预沉积的类骨磷灰石的结构和物相没有影响,而共沉积的生物活性分子使沉积的类骨磷灰石的片层结构晶体增大,并且使沉积的类骨磷灰石的主相由apatite转变为OCP。体外释放结果证明,固定的生物活性分子在体外可以进行持续可控的释放。通过体外细胞实验研究表明,共固定的纤连蛋白／成骨生长肽能够显著的提高骨髓间充质干细胞的贴附和铺展能力,并能够有效的促进骨髓间充质干细胞的增殖和向成骨分化。研究证明,通过表面共吸附的方法能够在钛植入体表面同时固定多种生物活性分子,对生物医用植入体发挥长期疗效具有积极作用,在牙科和整形外科等领域具有良好的临床实用价值。
Titanium and its alloys are extensively used for clinical implants, because of their excellent mechanical properties, and good biocompatibility. The long-term clinical researches show that titanium implant is confronted with the following problems:poor bone regeneration or slow regeneration, poor integration with surrounding bone tissues, and limited service life. The events after implantation include interactions between tissues and implant surfaces. Therefore, it is extremely important to achieve the implant with good comprehensive performance through surface modification. In recent years, further immobilizing the bioactive molecules, such as therapeutic agents or growth factors, with bone-like mineral layer on titanium implants is becoming the research focus in the biomaterials fields. Such surface modification technology could incorporate osteoinductivity and osteoconductivity into the design of the supporting biomaterial, thus improving the osseointegration and extending the service life.
In this study, we chose fibronectin (FN) and osteogenic growth peptide (OGP) to prepare a series of bioactive molecules-mineral functional composite layers on titanium surfaces. Firstly, the precipitation of bone-like mineral layer on titanium surfaces was studied, and a detailed insight into bone-like mineral nucleation and growth was discussed. Secondly, a coprecipitation method to build FN-mineral or OGP-mineral composite layers on titanium was developed. The phase composition, micro-morphology and fine structure of composite layers were investigated. The mechanisms underlying the coprecipitation of biomolecules into the mineral layer were also discussed. Finally, the fibronectin and osteogenic growth peptide were immobilized with bone-like mineral layer on titanium with varying immobilizing sequences through the methods of coprecipitation and/or physical adsorption. There are three parts in this dissertation, including:
1. A uniform, homogeneous apatite layer was biomimetically precipitated on hydroxyapatite thin film coated titanium through combing IBAD with biomimetic precipitation. XRD patterns indicate that, during the bone-like apatite precipitation, with the increase of immersion time, the density of apatite's characteristic peak increased and the peak became narrower. The calcium concentration in DPBS solution increased with the dissolution of hydroxyapatite thin film. And then the calcium concentration decreased with the precipitation of apatite. Bone-like apatite was nucleated earliest on the hydroxyapatite thin film with the lowest degree of crystallinity. The rate of nucleation growth increased with increasing crystallinity of the coated hydroxyapatite thin film.
2. FN-mineral and OGP-mineral composite layers were biomimetically induced on an active hydroxyapatite thin film through coprecipitation method. FESEM and confocal observations show that, the biomimetic coprecipitation process allowed biomolecules to be incorporated uniformly throughout the mineral layer. Rietveld refinement results suggest that, the mineral layer was indexed of a mixture of apatite and OCP. The phases of FN-mineral and OGP-mineral were partially transformed from OCP to apatite. The lattice parameter c of apatite was higher in the bioactive molecules-mineral composite layers than in the mineral layers, and the parameter a of apatite was changed when the bioactive molecules were coprecipitated on the surfaces. The biomimetic coprecipitated bioactive molecules slowed down the rate of mineral growth and influenced the ultimate structure of mineral. Primary physicochemical characterization and fine structure analysis indicate that the coprecipitation process was mainly mediated by electrostatic attraction among bioactive molecules, material surfaces and the ions in the solutions, and the bioactive molecules participated in the formation of the crystal latticework.
3. The coprecipitation and/or adsorption approaches were used to immobilize FN and/or OGP with biomimetic mineral layer onto titanium substrates. FESEM and XRD results show that, the adsorption of BMs did not change the morphology of mineral crystal and the main phase of mineral layer. Whereas, the coprecipitation of BMs enlarge the mineral crystal and change the main phase of mineral layer from apatite to OCP. Release studies demonstrated that either coprecipitation or adsorption approach provided sustained delivery of bioactive molecules. The dual immobilizing of FN and OGP had a significant impact on bMSCs attachment, spreading, proliferation and osteogenic differentiation compared to immobilizing of FN or OGP alone. The multi-immobilizing mineral with various bioactive molecules through adsorption approach has the potential to be used for orthopedic and dental applications.
引文
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