骨修复用形状记忆聚氨酯的细胞相容性研究
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摘要
形状记忆聚氨酯(Shape memory polyurethane, SMPU)是一种能感知外界温度变化并发生形状改变的新型智能材料。因其良好的形状记忆功能而成为功能材料研究的热点之一,在生物医学领域尤其是微创手术中具有重要的应用价值。本文以丙交酯、对二氧环己酮、乙二醇、1,6-六亚甲基二异氰酸酯(HDI)、丁二胺为主要原料采用两步法合成了具有不同玻璃化转变温度的新型形状记忆聚氨酯。采用静态水接触角和吸水率测定综合评价了SMPU的亲/疏水性。采用体外细胞培养法,以SD大鼠颅骨成骨细胞为模型细胞,以聚(DL-乳酸)(PDLLA)为对照,将细胞与各SMPU膜复合培养,从细胞形态、细胞附着与铺展、细胞增殖与迁移、细胞分化与矿化能力等方面考查SMPU与成骨细胞的细胞相容性;同时,考查了SMPU拉伸-复形形变对细胞行为的影响,从而更综合地评价了SMPU与成骨细胞的细胞相容性,为动物体内实验和临床应用提供理论依据。主要工作和结论如下:
(1)细胞培养及鉴定:采用组织块法培养了大鼠乳鼠颅骨成骨细胞,并利用差时贴壁法对其进行有效纯化。通过形态观察、ALP染色鉴定表明具有成骨细胞的典型生物学行为。细胞经过传代培养至第三代用于后续实验。
(2)SMPU的合成及亲/疏水性测定:以丙交酯、对二氧环己酮和乙二醇为主要原料先合成大分子二醇(HO-P(LA-PDO)-OH),并以合成的大分子二醇作为软段,1,6-六亚甲基二异氰酸酯(HDI)和丁二胺作为硬段,通过调节软硬段比例,合成了“NCO/OH”比例分别为1.1、1.15和1.2的三种聚氨酯,即SMPU1、SMPU2和SMPU3。采用静态水接触角和吸水率综合评价了材料的亲/疏水性能。结果表明,随SMPU中硬段含量的增加,材料的亲水性增强;
(3)SMPU膜对成骨细胞生长行为的影响:以SD大鼠颅骨成骨细胞为模型细胞,以PDLLA为对照,将成骨细胞与SMPU膜复合培养,考查了成骨细胞在不同SMPU膜上的形态、附着、铺展、增殖、迁移速率和净位移。结果表明,细胞在SMPU膜上的适应期长于PDLLA膜,其早期的细胞形态、黏附、铺展和增殖都略次于PDLLA膜。但随接种时间延长,细胞在SMPU上的增殖速率明显加快,且随硬段含量的增大,细胞的增殖速率和迁移速率也相应加快。
(4)SMPU膜对成骨细胞分化、矿化的影响:采用BCA法、酶动力法、比色法分别测定了细胞与SMPU复合培养第7、10、13d的总蛋白质含量、碱性磷酸酶(ALP)活性和无机钙含量,RT-PCR法检测了成骨细胞I型胶原mRNA的表达情况。结果表明,细胞接种后10d内,SMPU膜上的细胞的总蛋白含量、ALP活性和无机钙的分泌量等功能指标均低于PDLLA组,但在接种后10-13d,SMPU膜上细胞的各指标增长速率均明显优于PDLLA组,且SMPU3>SMPU2>SMPU1。这表明SMPU材料比PDLLA具有更好的促进细胞分化和矿化的能力,但并未缩短细胞的生长分化周期。SMPU3膜上细胞的I型胶原表达量高于PDLLA和SMPU2、SMPU1,这进一步说明SMPU3具有优良的细胞相容性。
(5)拉伸-复形形变对SMPU的细胞相容性的影响:以玻璃化转变温度在体温附近的SMPU1为研究对象,将SMPU1膜分别在Tg+15℃、Tg-15℃和Tg+15℃下拉伸,固定,复形,考察形状记忆形变对膜表面结构,继而对成骨细胞行为的影响。结果表明,SMPU1在形状记忆形变前后,其表面都存在明显的规则的相分离结构;形状记忆形变后的SMPU1表面形成了微沟槽结构,这种微沟槽结构促进了细胞的黏附与增殖,并使细胞形态表现出一定的取向性。
综上所述,尽管SMPU上成骨细胞的早期黏附、铺展和增殖不及PDLLA,但长期接种后,SMPU具有更好的促进成骨细胞增殖、分化和矿化的能力。且随SMPU中硬段含量的增大,成骨细胞的增殖、分化和矿化的能力都逐渐增强。拉伸-复形过程可改变SMPU膜的表面形态,并在SMPU表面形成微沟槽结构。这种微沟槽结构有利于细胞的黏附和增殖,并使细胞表现出一定的取向性。这些结果都提示本研究所制备的形状记忆聚氨酯具有良好的成骨细胞相容性,为进一步的体内动物实验和SMPU在骨修复和再生中的应用提供了有用的信息。
Shape memory polyurethane (SMPU) is a novel intelligent material that is able to response to external temperature change and exhibit shape memory. It has received wide attentions in biomedical fields, especially in minimally invasive surgery because of its shape memory property. In this study, a series of novel SMPUs with different glass transition temperature (Tg) was synthesized through a two-step method with DL-lactide (LA), p-dioxanone (PDO), ethylene glycol (EG),hexamethylene diisocyanate (HDI) and ethylenediamine (BDA) as the main monomers. The hydrophilicity of SMPUs was characterized by water contact angle and water absorption. In addition, the cytocompatibility of SMPUs was studied by employing primary SD rat osteoblasts as the model cells and poly(DL-lactic acid) (PDLLA) as the control. The osteoblasts morphology, cell attachment and spreading, cell migration and proliferation, cell differentiation and mineralization ability were detected to indicate the cytocompatibility of SMPUs. Furthermore, the effects of stretching-shape recovering process of SMPU on ostoblasts behaviors were also examined. Thereby, a comprehensive investigation and understanding of the SMPUs cytocompatibility with osteoblasts were achieved. The main works and conclusions are included as follows:
(1) Cells culture and identify: The primary SD rats osteoblasts were cultured from the calvaria of new-born rats by the tissue piece culture method. The osteoblasts were purified by the difference of the adhibition time with other type of cells and identified by the morphology, ALP staining. The third-generation osteoblasts were used in the subsequent experiments.
(2) Synthesis and hydrophilicity determination of SMPUs: Two-step method was used to synthesize the SMPUs. In brief, LA, PDO and EG was firstly copolymerized to form a macrodiol HO-P(LA-PDO)-OH, then HO-P(LA-PDO)-OH as soft segments was extended by HDI and BDA as hard segments, resulting in the final SMPU copolymers. By adjusting the ratio of -NCO to–OH (NCO/OH=1.1, 1.15, 1.20), three SMPUs labeled as SMPU1, SMPU2 and SMPU3, respectively, were obtained. Water contact angle and water absorption showed the hydrophilicity of SMPUs increased with the increase of NCO/OH.
(3) Effects of SMPUs on osteoblasts growth behaviors: The morphology, attachment, spreading, migration and proliferation of SD osteoblasts were examined on various SMPUs films with PDLLA as the control. The results indicated that the initial morphology, adhesion, spreading and proliferation of osteoblasts on all SMPUs films is no better than those on PDLLA film. Despite this, the cell doubling rate and migration rate on SMPUs elevated with the increasing culture time. Furthermore, with the increased amount of hard segments in SMPUs, both cell doubling rate and migration rate correspondingly increased, which is mostly possibly attributed to the improved hydrophilicity.
(4) Effects of SMPUs on osteoblast differentiation and mineralization ability: Total protein content, alkaline phosphatase (ALP) activity, Ca secretion and collagen type I amount were measured by BCA method, enzyme dynamic method, chromometry and RT-PCR, repectively. The results showed that osteoblast on different SMPUs films all exhibited lower physiological functions compared to those on PDLLA films within the first 10 days after seeding. Thereafter, however, the osteoblasts on SMPUs demonstrated better differentiation and mineralization than those on PDLLA films, and SMPU3 > SMPU2 > SMPU1.
(5) Effects of stretching-shape recovering process on cell adhesion and proliferation: SMPU1 exibited a Tg close to body temperature, thus it was suitable for bone repair biomaterials in term of its shape memory temperature and was chose as a model SMPU for studying the effects of stretching-shape recovering process on cell adhesion and proliferation. For this purpose, SMPU1 films were stretched by 200%, then fixed, and finally recovered to its original shape at Tg+15℃, Tg-15℃and Tg+15℃, respectively. The surface morphology and phase separation were probed by means of tapping mode AFM, and then the osteoblast adhesion and proliberation were examined before and after the stretching-shape recovering process. The AFM results showed that there were obvious and regular phase separation resulted from soft segments and hard segments in SMPU, and some groove-ridge architectures within a scale of micrometers were produced by the stretching-shape recovering process. These special micropatterned structures promoted osteoblasts adhesion and proliferation, and also resulted in partially oriented cell growth along the grooves.
In sum, SMPUs stimulated osteoblast migration, proliferation, differentiation and mineralization after a longer period of seeding, although they exhibited reduced initial osteoblasts attachment, spreading and proliferation compared to PDLLA film. Furthermore, the increased amount of hard segments in SMPUs is beneficial to osteoblast growth, differentiation and mineralization. In addition, stretching-shape recovering process could change the surface morphology of SMPU films and contribute to enhanced cell adhesion and proliferation, and even produce oriented arrangement of osteoblasts. All above suggest the sound cytocompatibility of SMPUs with osteoblasts, providing valuable information for further in vivo experiments and applications of SMPUs in bone repair and regeneration.
(1)细胞培养及鉴定:采用组织块法培养了大鼠乳鼠颅骨成骨细胞,并利用差时贴壁法对其进行有效纯化。通过形态观察、ALP染色鉴定表明具有成骨细胞的典型生物学行为。细胞经过传代培养至第三代用于后续实验。
(2)SMPU的合成及亲/疏水性测定:以丙交酯、对二氧环己酮和乙二醇为主要原料先合成大分子二醇(HO-P(LA-PDO)-OH),并以合成的大分子二醇作为软段,1,6-六亚甲基二异氰酸酯(HDI)和丁二胺作为硬段,通过调节软硬段比例,合成了“NCO/OH”比例分别为1.1、1.15和1.2的三种聚氨酯,即SMPU1、SMPU2和SMPU3。采用静态水接触角和吸水率综合评价了材料的亲/疏水性能。结果表明,随SMPU中硬段含量的增加,材料的亲水性增强;
(3)SMPU膜对成骨细胞生长行为的影响:以SD大鼠颅骨成骨细胞为模型细胞,以PDLLA为对照,将成骨细胞与SMPU膜复合培养,考查了成骨细胞在不同SMPU膜上的形态、附着、铺展、增殖、迁移速率和净位移。结果表明,细胞在SMPU膜上的适应期长于PDLLA膜,其早期的细胞形态、黏附、铺展和增殖都略次于PDLLA膜。但随接种时间延长,细胞在SMPU上的增殖速率明显加快,且随硬段含量的增大,细胞的增殖速率和迁移速率也相应加快。
(4)SMPU膜对成骨细胞分化、矿化的影响:采用BCA法、酶动力法、比色法分别测定了细胞与SMPU复合培养第7、10、13d的总蛋白质含量、碱性磷酸酶(ALP)活性和无机钙含量,RT-PCR法检测了成骨细胞I型胶原mRNA的表达情况。结果表明,细胞接种后10d内,SMPU膜上的细胞的总蛋白含量、ALP活性和无机钙的分泌量等功能指标均低于PDLLA组,但在接种后10-13d,SMPU膜上细胞的各指标增长速率均明显优于PDLLA组,且SMPU3>SMPU2>SMPU1。这表明SMPU材料比PDLLA具有更好的促进细胞分化和矿化的能力,但并未缩短细胞的生长分化周期。SMPU3膜上细胞的I型胶原表达量高于PDLLA和SMPU2、SMPU1,这进一步说明SMPU3具有优良的细胞相容性。
(5)拉伸-复形形变对SMPU的细胞相容性的影响:以玻璃化转变温度在体温附近的SMPU1为研究对象,将SMPU1膜分别在Tg+15℃、Tg-15℃和Tg+15℃下拉伸,固定,复形,考察形状记忆形变对膜表面结构,继而对成骨细胞行为的影响。结果表明,SMPU1在形状记忆形变前后,其表面都存在明显的规则的相分离结构;形状记忆形变后的SMPU1表面形成了微沟槽结构,这种微沟槽结构促进了细胞的黏附与增殖,并使细胞形态表现出一定的取向性。
综上所述,尽管SMPU上成骨细胞的早期黏附、铺展和增殖不及PDLLA,但长期接种后,SMPU具有更好的促进成骨细胞增殖、分化和矿化的能力。且随SMPU中硬段含量的增大,成骨细胞的增殖、分化和矿化的能力都逐渐增强。拉伸-复形过程可改变SMPU膜的表面形态,并在SMPU表面形成微沟槽结构。这种微沟槽结构有利于细胞的黏附和增殖,并使细胞表现出一定的取向性。这些结果都提示本研究所制备的形状记忆聚氨酯具有良好的成骨细胞相容性,为进一步的体内动物实验和SMPU在骨修复和再生中的应用提供了有用的信息。
Shape memory polyurethane (SMPU) is a novel intelligent material that is able to response to external temperature change and exhibit shape memory. It has received wide attentions in biomedical fields, especially in minimally invasive surgery because of its shape memory property. In this study, a series of novel SMPUs with different glass transition temperature (Tg) was synthesized through a two-step method with DL-lactide (LA), p-dioxanone (PDO), ethylene glycol (EG),hexamethylene diisocyanate (HDI) and ethylenediamine (BDA) as the main monomers. The hydrophilicity of SMPUs was characterized by water contact angle and water absorption. In addition, the cytocompatibility of SMPUs was studied by employing primary SD rat osteoblasts as the model cells and poly(DL-lactic acid) (PDLLA) as the control. The osteoblasts morphology, cell attachment and spreading, cell migration and proliferation, cell differentiation and mineralization ability were detected to indicate the cytocompatibility of SMPUs. Furthermore, the effects of stretching-shape recovering process of SMPU on ostoblasts behaviors were also examined. Thereby, a comprehensive investigation and understanding of the SMPUs cytocompatibility with osteoblasts were achieved. The main works and conclusions are included as follows:
(1) Cells culture and identify: The primary SD rats osteoblasts were cultured from the calvaria of new-born rats by the tissue piece culture method. The osteoblasts were purified by the difference of the adhibition time with other type of cells and identified by the morphology, ALP staining. The third-generation osteoblasts were used in the subsequent experiments.
(2) Synthesis and hydrophilicity determination of SMPUs: Two-step method was used to synthesize the SMPUs. In brief, LA, PDO and EG was firstly copolymerized to form a macrodiol HO-P(LA-PDO)-OH, then HO-P(LA-PDO)-OH as soft segments was extended by HDI and BDA as hard segments, resulting in the final SMPU copolymers. By adjusting the ratio of -NCO to–OH (NCO/OH=1.1, 1.15, 1.20), three SMPUs labeled as SMPU1, SMPU2 and SMPU3, respectively, were obtained. Water contact angle and water absorption showed the hydrophilicity of SMPUs increased with the increase of NCO/OH.
(3) Effects of SMPUs on osteoblasts growth behaviors: The morphology, attachment, spreading, migration and proliferation of SD osteoblasts were examined on various SMPUs films with PDLLA as the control. The results indicated that the initial morphology, adhesion, spreading and proliferation of osteoblasts on all SMPUs films is no better than those on PDLLA film. Despite this, the cell doubling rate and migration rate on SMPUs elevated with the increasing culture time. Furthermore, with the increased amount of hard segments in SMPUs, both cell doubling rate and migration rate correspondingly increased, which is mostly possibly attributed to the improved hydrophilicity.
(4) Effects of SMPUs on osteoblast differentiation and mineralization ability: Total protein content, alkaline phosphatase (ALP) activity, Ca secretion and collagen type I amount were measured by BCA method, enzyme dynamic method, chromometry and RT-PCR, repectively. The results showed that osteoblast on different SMPUs films all exhibited lower physiological functions compared to those on PDLLA films within the first 10 days after seeding. Thereafter, however, the osteoblasts on SMPUs demonstrated better differentiation and mineralization than those on PDLLA films, and SMPU3 > SMPU2 > SMPU1.
(5) Effects of stretching-shape recovering process on cell adhesion and proliferation: SMPU1 exibited a Tg close to body temperature, thus it was suitable for bone repair biomaterials in term of its shape memory temperature and was chose as a model SMPU for studying the effects of stretching-shape recovering process on cell adhesion and proliferation. For this purpose, SMPU1 films were stretched by 200%, then fixed, and finally recovered to its original shape at Tg+15℃, Tg-15℃and Tg+15℃, respectively. The surface morphology and phase separation were probed by means of tapping mode AFM, and then the osteoblast adhesion and proliberation were examined before and after the stretching-shape recovering process. The AFM results showed that there were obvious and regular phase separation resulted from soft segments and hard segments in SMPU, and some groove-ridge architectures within a scale of micrometers were produced by the stretching-shape recovering process. These special micropatterned structures promoted osteoblasts adhesion and proliferation, and also resulted in partially oriented cell growth along the grooves.
In sum, SMPUs stimulated osteoblast migration, proliferation, differentiation and mineralization after a longer period of seeding, although they exhibited reduced initial osteoblasts attachment, spreading and proliferation compared to PDLLA film. Furthermore, the increased amount of hard segments in SMPUs is beneficial to osteoblast growth, differentiation and mineralization. In addition, stretching-shape recovering process could change the surface morphology of SMPU films and contribute to enhanced cell adhesion and proliferation, and even produce oriented arrangement of osteoblasts. All above suggest the sound cytocompatibility of SMPUs with osteoblasts, providing valuable information for further in vivo experiments and applications of SMPUs in bone repair and regeneration.
引文
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