功能化壳聚糖组成及表面形态与干细胞的相互作用
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摘要
目的:通过自身材料学因素控制和优化设计,赋予材料诱导组织再生的功能,已成为生物材料的研究热点之一。本研究首先通过壳聚糖(CS)磷酸化改性和在CS基质中添加钙磷盐,调控磷灰石晶体的形成与组装,探讨材料对骨髓间充质干细胞(MSCs)增殖和分化的影响;其次改变CS表面微观物理形貌,探讨材料拓扑结构对MSCs黏附生长的影响。为制备具有良好生物相容性及骨诱导性的生物可降解膜材奠定理论基础。
方法:(1)采用差速贴壁法分离、培养MSCs,观察其生物学特性;流式细胞技术和定向诱导分化法鉴定MSCs。(2)激光扫描共聚焦显微镜(LSCM)和原子力显微镜(AFM)观察MSCs在成骨诱导前后的变化。(3)制备亚甲基磷酸化壳聚糖(NMPC),与CS共混成膜并进行表征;与MSCs体外复合培养评价其生物相容性,实时荧光定量PCR(FQ-PCR)评价材料成骨诱导功能。(4)共沉淀法制备磷酸四钙(TTCP)、高温脱水法制备磷酸氢钙(DCPA),与CS共混成膜并进行表征;与MSCs体外复合培养检测其生物相容性,通过检测碱性磷酸酶(ALPase)活性和成骨相关基因表达评价其成骨诱导性。(5)利用相变法制备高分子聚苯乙烯(PS)多孔膜,以此为模板制备不同表面微观结构的CS膜,评价表面微观结构对MSCs黏附、生长的影响。
结果:(1)体外培养MSCs成纤维样、漩涡状生长,纯度随传代次数增加而提高;P3代MSCs表面抗原CD29、CD44表达阳性,CD45表达阴性;MSCs经定向诱导可分化为成骨细胞和脂肪细胞。(2)经成骨诱导后,MSCs胞内Ca~(2+)由均匀分布转变为点状、块状的高Ca~(2+)分布;胞质周围的微管荧光染色强度明显高于胞质中央,且排列稀疏而紊乱;细胞由长梭形转变为不规则状,细胞边缘网丝状伪足样突起减少,胞核增大,内有3-5个核仁,细胞膜表面粗糙,呈矿化物沉积特征。(3)CS与NMPC共混成膜制得CS/NMPC,与CS相比,表面接触角由77±2°降至的20±10°,吸水率由128±14%升至248±24.6%,拉伸强度和断裂伸长降低,弹性模量升高,材料能吸附培养基中的Ca~(2+);CS/NMPC更有利于细胞增殖,第5、7、9天差异显著(P＜0.05);骨钙素(OC)、骨桥蛋白(OPN)为CS膜上的275.1%和736.2%,表明NMPC能诱导MSCs向成骨分化。(4)TTCP和DCPA与CS溶液共混后成膜,获得CS基羟基磷灰石(CS/HA)膜,其表面接触角为57±5°,吸水率185±15%,拉伸强度、断裂伸长、弹性模量较纯CS膜降低,对培养基中Ca~(2+)吸附加速;与CS膜相比,CS/HA膜细胞生物相容性大幅提高,细胞增殖在第5、7天有显著差异(P＜0.05),第9天有极显著差异(P＜0.01);在成骨诱导培养12d后CS/HA膜上ALPase活性是CS膜的6.3倍,Ⅰ-型胶原(ColⅠ)、ALPase和OPN基因表达水平为CS膜的479.0%、377.1%和597.9%,表明CS/HA中的HA能显著诱导MSCs向成骨分化。(5)利用相分离技术制备不同孔洞PS膜,以此为模板制备微观结构完全匹配的CS膜,并在表面培养MSCs,结果表明CS膜上不同凸起对细胞黏附无显著差异;但凸起10-50μm不规则微球表面增殖率最高,其次为10μm规则紧密排列的半球状凸起,0.8-1μm短柱状凸起和光滑CS膜增值率最低。
结论:材料化学组成及微观结构对MSCs的黏附、生长、分化均有影响。在以CS为基质的材料中添加含磷酸根的有机物NMPC和无机物HA,均增加细胞的生物相容性,并能诱导MSCs向成骨分化,是一种良好的具有骨诱导性能的组织工程材料;CS表面10-50μm微球结构CS膜能促进MSCs在材料上增殖,为下一步研究CS膜的不同拓扑结构对MSCs分化的影响奠定基础。
Objective:Biomaterials endowed with the function of inducing tissue regeneration have been a hot topic.In this study,chitosan(CS) was first modified by phosphorylation and by addition of calcium phosphate cement to modulate nucleation and crystallization.The products were used to study effects of biomaterials on the proliferation and differentiation of mesenchymal stem cells(MSCs).Secondly CS membranes with different physical morphology were fabricated to study effects of biomaterials on attachment and proliferation of MSCs.They provide experimental basis for developing novel biodegradable materials which have good biocompatibility and osteo-inductivity in guiding bone tissue regeneration.
Methods:
(1) MSCs were isolated and purified by differential adherence and their biological characteristics were observed.Cells were identified by surface molecules and the ability to differentiate into osteoblasts or adipoctyes.
(2) Laser scanning confocal microscope(LSCM) and atomic force microscopy(AFM) were employed to observe changes of MSCs due to osteogenic differentiation.
(3) CS was mixed with home-made N-methylene phosphonic chitosan(NMPC) to form CS/NMPC membrane and its performance was then characterized.MSCs were cultured on the CS/NMPC membrane to evaluate its biocompatibility.The osteogenic induction of materials were detected by fluorescence quantitative real-time PCR (FQ-PCR).
(4) Tetracalcium phosphate(TTCP) made by co-precipitation and dicalicium phosphate anhydrous(DCPA) made by dehydration were mixed with CS to form CS/hydroxyapatite(CS/HA) membrane and its performance was then detected.To evaluate the biocompatibility and osteogenic induction of CS/HA,MSCs was cultured on the materials for detection of the activity of alkaline phosphatase(ALPase) and osteogenic mRNA.
(5) Polystyrene(PS) membranes with pits of different sizes were presented by liquid phase separation and then convex CS membranes were prepared with the PS membranes as templates.The attachment and proliferation of the MSCs on the surface of CS with different microstructures were evaluated.
Results:
(1) MSCs cultured in vitro demonstrated uniform appearance under the inverted microscope.The morphous of cells was fibroblast shape and swirled when they assembled together.By flow cytometer,surface antigens was positive for CD29, CD44 and negative for CD45.MSCs isolated could be successfully induced into osteoblasts and adipocytes.
(2) After osteo-induction,the distribution of Ca~(2+) was changed from uniform to punctiform and massive.The microtubules showed rarefaction and derangement.The morphology of MSCs changed from fusiform to irregular form.The filipodium of cells decreased and nucleus increased after induction.Membrane of cells became rough just like deposited mineralizer.
(3) When NMPC was mixed with chitsan,CS/NMPC membrane was formed.The phenotype of membrane showed that the water contact angle decreased from CS's 77±2°to CS/NMPC's 20±10°and the water absorption increased from CS's 128±14%to CS/NMPC's 248±24.6%.The tensile strength and breaking elongation decreased but elastic modulus increased slightly.When immersed into culture medium, the materials could absorb Ca~(2+).Cell culture assay showed that proliferation of MSCs on CS/NMPC was higher than that on pure CS 5,7 and 9 days after inoculation.There were significant differences between the two groups(P＜0.05).After 14 days' growth in basic culture medium on materials,the expression of osteocalcin(OC) and osteopontin(OPN) of MSCs on CS/NMPC was determined as 275.1%and 736.2% respectively of that on CS by FQ-PCR.This indicated that NMPC played an important role in MSCs osteogenic differentiation.
(4) Home-made TTCP and DCPA were added into CS solution to form CS/HA membrane.Water contact angle decreased from CS's 77±2°to CS/HA's 57±5°and water absorption increased from CS's 128±14%to CS/HA's 185±15%.The tensile strength,breaking elongation and elastic modulus decreased compared with CS.CS/HA absorbed Ca~(2+) quickly and low crystalline hydroxyapatite deposited on the surface of the membranes.MTT showed that proliferation of stem cells on CS/HA was higher than that on CS 3,5 and 9 days after inoculation.There were significant differences between the two groups after 3 and 5 days of culture(P＜0.05) and extremely significant differences after 9days(P＜0.01).ALPase activity value of MSCs on CS/HA was 6.3 times of that on CS detected by ALPase kit.The expression mRNA levels ofⅠ-collagen(ColⅠ),ALPase and OPN of MSCs on CS/HA were determined as 479.0%,377.1%and 597.9%respectively of that on CS by FQ-PCR. These results indicated that hydroxyapatite on CS could induce MSCs to differentiate into osteoblasts,
(5) The PS membranes with different morphologies and microstructures were obtained and the correspondingly scaled convex CS films were then prepared based on the template technology.MSCs cultured on the surface of CS with different microstructures showed no significant difference in cell's attachment on the first day. But the proliferation of MSCs was highest on CS 10-50 followed by CS10 and lowest on the surface with smooth CS and CS0.8-1 by MTT.
Conclusion:Chemical composition and micro-structure of biomaterials can affect the attachment,growth and differentiation of MSCs.When materials with phosphate radical just as organic NMPC and inorganic HA are added to CS,it not only increases the biocompatibility but also accelerates the differentiation of MSCs into osteoblast.It is a good tissue engineering materials that have favorable osteo-inductivity.The microstructure of CS membrane surface affects the distribution of MSCs and results in increased proliferation of MSCs.
方法:(1)采用差速贴壁法分离、培养MSCs,观察其生物学特性;流式细胞技术和定向诱导分化法鉴定MSCs。(2)激光扫描共聚焦显微镜(LSCM)和原子力显微镜(AFM)观察MSCs在成骨诱导前后的变化。(3)制备亚甲基磷酸化壳聚糖(NMPC),与CS共混成膜并进行表征;与MSCs体外复合培养评价其生物相容性,实时荧光定量PCR(FQ-PCR)评价材料成骨诱导功能。(4)共沉淀法制备磷酸四钙(TTCP)、高温脱水法制备磷酸氢钙(DCPA),与CS共混成膜并进行表征;与MSCs体外复合培养检测其生物相容性,通过检测碱性磷酸酶(ALPase)活性和成骨相关基因表达评价其成骨诱导性。(5)利用相变法制备高分子聚苯乙烯(PS)多孔膜,以此为模板制备不同表面微观结构的CS膜,评价表面微观结构对MSCs黏附、生长的影响。
结果:(1)体外培养MSCs成纤维样、漩涡状生长,纯度随传代次数增加而提高;P3代MSCs表面抗原CD29、CD44表达阳性,CD45表达阴性;MSCs经定向诱导可分化为成骨细胞和脂肪细胞。(2)经成骨诱导后,MSCs胞内Ca~(2+)由均匀分布转变为点状、块状的高Ca~(2+)分布;胞质周围的微管荧光染色强度明显高于胞质中央,且排列稀疏而紊乱;细胞由长梭形转变为不规则状,细胞边缘网丝状伪足样突起减少,胞核增大,内有3-5个核仁,细胞膜表面粗糙,呈矿化物沉积特征。(3)CS与NMPC共混成膜制得CS/NMPC,与CS相比,表面接触角由77±2°降至的20±10°,吸水率由128±14%升至248±24.6%,拉伸强度和断裂伸长降低,弹性模量升高,材料能吸附培养基中的Ca~(2+);CS/NMPC更有利于细胞增殖,第5、7、9天差异显著(P＜0.05);骨钙素(OC)、骨桥蛋白(OPN)为CS膜上的275.1%和736.2%,表明NMPC能诱导MSCs向成骨分化。(4)TTCP和DCPA与CS溶液共混后成膜,获得CS基羟基磷灰石(CS/HA)膜,其表面接触角为57±5°,吸水率185±15%,拉伸强度、断裂伸长、弹性模量较纯CS膜降低,对培养基中Ca~(2+)吸附加速;与CS膜相比,CS/HA膜细胞生物相容性大幅提高,细胞增殖在第5、7天有显著差异(P＜0.05),第9天有极显著差异(P＜0.01);在成骨诱导培养12d后CS/HA膜上ALPase活性是CS膜的6.3倍,Ⅰ-型胶原(ColⅠ)、ALPase和OPN基因表达水平为CS膜的479.0%、377.1%和597.9%,表明CS/HA中的HA能显著诱导MSCs向成骨分化。(5)利用相分离技术制备不同孔洞PS膜,以此为模板制备微观结构完全匹配的CS膜,并在表面培养MSCs,结果表明CS膜上不同凸起对细胞黏附无显著差异;但凸起10-50μm不规则微球表面增殖率最高,其次为10μm规则紧密排列的半球状凸起,0.8-1μm短柱状凸起和光滑CS膜增值率最低。
结论:材料化学组成及微观结构对MSCs的黏附、生长、分化均有影响。在以CS为基质的材料中添加含磷酸根的有机物NMPC和无机物HA,均增加细胞的生物相容性,并能诱导MSCs向成骨分化,是一种良好的具有骨诱导性能的组织工程材料;CS表面10-50μm微球结构CS膜能促进MSCs在材料上增殖,为下一步研究CS膜的不同拓扑结构对MSCs分化的影响奠定基础。
Objective:Biomaterials endowed with the function of inducing tissue regeneration have been a hot topic.In this study,chitosan(CS) was first modified by phosphorylation and by addition of calcium phosphate cement to modulate nucleation and crystallization.The products were used to study effects of biomaterials on the proliferation and differentiation of mesenchymal stem cells(MSCs).Secondly CS membranes with different physical morphology were fabricated to study effects of biomaterials on attachment and proliferation of MSCs.They provide experimental basis for developing novel biodegradable materials which have good biocompatibility and osteo-inductivity in guiding bone tissue regeneration.
Methods:
(1) MSCs were isolated and purified by differential adherence and their biological characteristics were observed.Cells were identified by surface molecules and the ability to differentiate into osteoblasts or adipoctyes.
(2) Laser scanning confocal microscope(LSCM) and atomic force microscopy(AFM) were employed to observe changes of MSCs due to osteogenic differentiation.
(3) CS was mixed with home-made N-methylene phosphonic chitosan(NMPC) to form CS/NMPC membrane and its performance was then characterized.MSCs were cultured on the CS/NMPC membrane to evaluate its biocompatibility.The osteogenic induction of materials were detected by fluorescence quantitative real-time PCR (FQ-PCR).
(4) Tetracalcium phosphate(TTCP) made by co-precipitation and dicalicium phosphate anhydrous(DCPA) made by dehydration were mixed with CS to form CS/hydroxyapatite(CS/HA) membrane and its performance was then detected.To evaluate the biocompatibility and osteogenic induction of CS/HA,MSCs was cultured on the materials for detection of the activity of alkaline phosphatase(ALPase) and osteogenic mRNA.
(5) Polystyrene(PS) membranes with pits of different sizes were presented by liquid phase separation and then convex CS membranes were prepared with the PS membranes as templates.The attachment and proliferation of the MSCs on the surface of CS with different microstructures were evaluated.
Results:
(1) MSCs cultured in vitro demonstrated uniform appearance under the inverted microscope.The morphous of cells was fibroblast shape and swirled when they assembled together.By flow cytometer,surface antigens was positive for CD29, CD44 and negative for CD45.MSCs isolated could be successfully induced into osteoblasts and adipocytes.
(2) After osteo-induction,the distribution of Ca~(2+) was changed from uniform to punctiform and massive.The microtubules showed rarefaction and derangement.The morphology of MSCs changed from fusiform to irregular form.The filipodium of cells decreased and nucleus increased after induction.Membrane of cells became rough just like deposited mineralizer.
(3) When NMPC was mixed with chitsan,CS/NMPC membrane was formed.The phenotype of membrane showed that the water contact angle decreased from CS's 77±2°to CS/NMPC's 20±10°and the water absorption increased from CS's 128±14%to CS/NMPC's 248±24.6%.The tensile strength and breaking elongation decreased but elastic modulus increased slightly.When immersed into culture medium, the materials could absorb Ca~(2+).Cell culture assay showed that proliferation of MSCs on CS/NMPC was higher than that on pure CS 5,7 and 9 days after inoculation.There were significant differences between the two groups(P＜0.05).After 14 days' growth in basic culture medium on materials,the expression of osteocalcin(OC) and osteopontin(OPN) of MSCs on CS/NMPC was determined as 275.1%and 736.2% respectively of that on CS by FQ-PCR.This indicated that NMPC played an important role in MSCs osteogenic differentiation.
(4) Home-made TTCP and DCPA were added into CS solution to form CS/HA membrane.Water contact angle decreased from CS's 77±2°to CS/HA's 57±5°and water absorption increased from CS's 128±14%to CS/HA's 185±15%.The tensile strength,breaking elongation and elastic modulus decreased compared with CS.CS/HA absorbed Ca~(2+) quickly and low crystalline hydroxyapatite deposited on the surface of the membranes.MTT showed that proliferation of stem cells on CS/HA was higher than that on CS 3,5 and 9 days after inoculation.There were significant differences between the two groups after 3 and 5 days of culture(P＜0.05) and extremely significant differences after 9days(P＜0.01).ALPase activity value of MSCs on CS/HA was 6.3 times of that on CS detected by ALPase kit.The expression mRNA levels ofⅠ-collagen(ColⅠ),ALPase and OPN of MSCs on CS/HA were determined as 479.0%,377.1%and 597.9%respectively of that on CS by FQ-PCR. These results indicated that hydroxyapatite on CS could induce MSCs to differentiate into osteoblasts,
(5) The PS membranes with different morphologies and microstructures were obtained and the correspondingly scaled convex CS films were then prepared based on the template technology.MSCs cultured on the surface of CS with different microstructures showed no significant difference in cell's attachment on the first day. But the proliferation of MSCs was highest on CS 10-50 followed by CS10 and lowest on the surface with smooth CS and CS0.8-1 by MTT.
Conclusion:Chemical composition and micro-structure of biomaterials can affect the attachment,growth and differentiation of MSCs.When materials with phosphate radical just as organic NMPC and inorganic HA are added to CS,it not only increases the biocompatibility but also accelerates the differentiation of MSCs into osteoblast.It is a good tissue engineering materials that have favorable osteo-inductivity.The microstructure of CS membrane surface affects the distribution of MSCs and results in increased proliferation of MSCs.
引文
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