成骨细胞与血管内皮细胞复合PDLLA的体外实验研究

目的：探讨兔成骨细胞与血管内皮细胞体外复合外消旋聚乳酸（PDLLA）的可能性,为血管化组织工程骨构建打下实验基础.方法：制备PDLLA浸提液培养血管内皮细胞,MTT法检测细胞活性.将制备好的PDLLA预湿、Ⅰ型胶原包埋;体外培养兔成骨细胞及血管内皮细胞后,与PDLLA复合10 d,扫描电子显微镜观察其在支架上的生长情况.结果：PDLLA浸提液对血管内皮细胞无毒性,成骨细胞与血管内皮细胞在支架上成片状分布,可见细胞外基质分泌.结论：两种细胞在经Ⅰ型胶原包埋的PDLLA上生长状态良好,可用于血管化组织工程骨的构建.     

无机诱导因子骨组织工程支架材料体外细胞相容性的实验研究

目的：细胞毒性和细胞相容性是生物材料应用于临床首先必须面对的问题.通过对无机诱导因子骨组织工程支架材料的体外实验,来研究其细胞毒性和细胞相容性,为其临床应用作前期准备.方法：将生长状态良好的人骨髓基质细胞与无机诱导因子支架材料浸提液共同培养,通过镜下观察细胞形态、MTT比色法、流式细胞仪检测法评价生物材料浸提液对人骨髓基质细胞生长和增殖的影响;并将人骨髓基质细胞接种于无机诱导因子支架材料上,扫描电镜观察材料细胞复合物生长状况.结果：人骨髓基质细胞能够在无机诱导因子支架材料上生长,支架材料对体外培养的人骨髓基质细胞的形态不构成损害,对细胞的生长和增殖无明显抑制作用.结论：无机诱导因子支架材料具有良好的细胞生物学相容性,是一良好的组织工程骨构建支架材料.     

一种新型纳米羟基磷灰石材料应用于牙周组织工程的可行性研究

目的:对一种新型纳米羟基磷灰石材料 -纳米羟晶 -胶原仿生骨材料 (nHAC)进行细胞相容性和细胞毒性的研究,以初步探讨其应用于牙周组织工程的可行性。方法:将体外培养的人牙周膜细胞(PDLCs)接种于nHAC三维支架上复合培养,采用细胞计数和扫描电镜观察PDLCs在nHAC支架上的附着、生长情况,并通过MTT测试法和碱性磷酸酶(ALP)活性检测法研究nHAC浸提液对PDLCs增殖和功能表达的影响。结果:人PDLCs在nHAC支架上形成良好贴附并增殖,扫描电镜可见nHAC具有良好的多孔网状结构,细胞在nHAC上生长旺盛,伸展充分,而PDLCs在不同浓度的材料浸提液中的生长、增殖及ALP活性与空白对照组相比无显著性差异。结论:nHAC具有良好的三维空间结构和细胞相容性,且无细胞毒性,有望成为牙周组织工程的支架材料。     

壳核型n-HA／ZrO2支架材料对L929细胞增殖活性的影响

目的研究核壳型纳米羟基磷灰石包覆二氧化锆（壳核型n-HA／ZrO2）复合生物陶瓷支架材料对L929细胞增殖活性的影响及细胞毒性反应。方法将L929细胞接种于96孔板,随机分为3组进行培养,实验组用壳核型n-HA／ZrO2支架材料浸提液处理,阳性对照组用苯酚溶液,阴性对照组用聚乙烯浸提液,于24、48、72h后观察细胞生长形态,通过MTT比色法检测各组细胞的相对增殖率（relative growth rate,RGR）,按GB／T16175-1996标准评价材料的细胞毒性。结果实验组L929细胞24、48、72h后的RGR随时间延长而升高,分别为95.86％、98.29％和102.73％,与阴性对照组L929细胞在增殖活性方面的差异无统计学意义,两组受试材料细胞毒性评级为0-I级。结论核壳型n-HA／ZrO2支架材料不影响L929细胞的增殖活性,无细胞毒性。     

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目的探讨丝素胶原支架用作牙周组织工程支架材料的可行性。方法本研究于2008年9月在辽宁医学院实验中心进行。通过冻干法制备丝素胶原支架,用扫描电镜(SEM)观察、噻唑蓝(MTT)法及碱性磷酸酶(ALP)活性检测,了解人牙周膜成纤维细胞(PDLF)附着于丝素胶原支架的生长情况,评价其生物相容性。结果制备的复合膜孔隙率为80.7%;PDLF在材料浸提液中的生长、增殖状况与对照组相比,差异无统计学意义(P>0.05);ALP活性与对照组相比差异亦无统计学意义(P>0.05)。SEM观察可见复合材料具有良好的多孔网状结构,细胞在各复合膜上生长旺盛,伸展充分,形成良好。结论通过冻干法自制的丝素胶原复合物的孔隙率符合牙周组织工程的要求。其良好的三维空间结构和细胞相容性促进了PDLF的生长、黏附及增殖,且无细胞毒性,有望成为牙周组织工程的支架材料。     

胶原基纳米骨的遗传毒性及对体外培养细胞影响的研究

目的：体外研究胶原基纳米骨的遗传毒性,及对原代培养的人牙周膜成纤维样细胞、兔成骨细胞的影响。方法：采用Ames致突变试验、MTT法及碱性磷酸酶（ALP）检测法,测定不同浓度胶原基纳米骨（nHAC）的浸提液对鼠伤寒沙门菌的致突变比值的影响和对人牙周膜成纤维样细胞及兔成骨细胞的影响。结果：nHAC的浸提液各剂量组对鼠伤寒沙门菌的致突变比值均小于2,nHAC不会引起鼠伤寒沙门菌的回复突变数增加。不同时间点用不同浓度浸提液培养的人牙周膜成纤维样细胞正常增殖,浸提液不影响兔成骨细胞的功能表达。结论：胶原基纳米骨无遗传毒性,不影响人牙周膜成纤维样细胞的增殖活性和兔成骨细胞的成骨活性,是一种组织工程骨支架的良好材料。     

含铜不锈钢对血管内皮细胞黏附、增殖及凋亡的影响

目的 观察含铜不锈钢对血管内皮细胞黏附、增殖及凋亡的影响,并与316L不锈钢进行比较。方法 将血管内皮细胞接种于两种材料表面,在培养1、2、3 d后,应用吖啶橙染色及甲基噻唑基四唑（MTT）法检测细胞在 两种材料表面黏附和增殖活性。并制备两种材料的浸提液,用浸提液培养血管内皮细胞,采用流式细胞术检测细 胞凋亡率。结果 荧光显微镜下,细胞在两种材料表面均伸展良好,呈铺路石状生长。在培养1、2 d时,含铜不锈 钢组黏附的细胞数多于316L不锈钢组（P<0.05）;3 d时两组间差异无统计学意义（P>0.05）。MTT结果显示,含铜不锈 钢组在1、2 d的吸光度值高于316L不锈钢组,差异有统计学意义（P<0.05）;而在培养3 d后,两组间差异无统计学意 义（P>0.05）。316L不锈钢组的早期凋亡率高于含铜不锈钢组,差异有统计学意义（P<0.05）。结论 含铜不锈钢较 316L不锈钢更利于血管内皮细胞的黏附及增殖,并可以降低血管内皮细胞的早期凋亡率。     

PGS/PLLA共纺膜片的降解性能及生物相容性研究

目的:观察PGS/PLLA共纺膜片体外降解性及细胞相容性,探究其作为山羊颞下颌关节盘组织工程支架的可行性,为进一步动物实验奠定基础。方法:将PGS/PLLA共纺膜片浸泡在磷酸盐缓冲液中(pH=7.4),不同时间点取样,对材料微观形貌、pH、力学性能等进行检测;将关节盘细胞-PGS/PLLA支架复合培养,MTT法检测支架的细胞毒性及对关节盘细胞增殖影响;在14 d时,取出关节盘细胞-PGS/PLLA复合物,行HE、番红O、免疫组化染色。结果:PGS/PLLA支架降解性能适宜;MTT结果示,支架无毒性,细胞增殖良好。结论:颞下颌关节盘细胞在支架上粘附良好、增殖明显、代谢活跃,PGS/PLLA支架有望作为颞下颌关节盘组织工程支架。     

两种多孔生物玻璃支架材料的体外细胞相容性研究

目的 对比研究采用同样原料但以不同方法制备的两种多孔生物玻璃支架材料的体外细胞相容性。方法 溶胶－凝胶法制备A/W生物活性微晶玻璃（4006材料）,熔融法制备多孔生物活性玻璃（45S5材料）。体外诱导分离培养及鉴定兔骨髓基质细胞（BMSCs）,通过材料浸提液细胞毒性实验（MTT法）、细胞黏附实验、倒置相差显微镜、扫描电镜和环境扫描电镜比较4006材料和45S5材料对BMSCs细胞黏附和生长的影响,并探索与材料复合的最佳BMSCs细胞悬液浓度。结果 4006材料浸提液培养1 d时,其细胞活力与纯培养液间无统计学差异（P>0.05）;但培养3 d后,其细胞活力低于纯培养液（P<0.01）。45S5材料浸提液培养的细胞活力明显低于纯培养液（P<0.01）。细胞与材料复合培养后,镜下见BMSCs在4006材料孔隙内贴壁生长良好,分泌基质活跃;而在45S5材料上细胞黏附生长较差。BMSCs与4006材料的黏附量随接种细胞浓度升高而升高,细胞悬液浓度为2×10⁷个•mL^-1时的细胞黏附量最高。结论 溶胶－凝胶法制备的A/W生物活性微晶玻璃具有良好的生物活性和细胞相容性,具有作为骨组织工程支架材料的潜能。与其复合的细胞悬液浓度需要2×107 个•mL-1或以上。     

新型多孔HA/PDLLA支架体外细胞相容性的实验研究

目的:研究新型多孔羟基磷灰石/聚乳酸(HA/PDLLA)支架材料的体外细胞相容性。方法:贴壁法培养兔骨髓基质细胞(bone marrow stromal cells,BMSCs),经体外矿化诱导培养、扩增后,与实验组 A(含2%HA 的 HA/PDLLA)、实验组 B(含4%HA 的 HA/PDLLA)及对照组(PDLLA)分别进行体外复合培养;并通过定性及定量检测细胞在材料表面的粘附能力、增殖活力,验证细胞材料复合体的成骨活性,比较分析各组支架材料之间的差异。结果:兔 BMSCs在三组支架材料的表面均能生长,经体外诱导后在支架材料的表面形成钙结节,实验组 A 与 B 细胞的粘附及增殖能力均强于对照组(P<0.05)。结论:兔 BMSCs 与新型多孔 HA/PDLLA 支架材料有良好的细胞相容性。     

复合血管内皮细胞的组织工程骨构建

目的:探讨将血管内皮细胞(vascular endothelial cell,VEC)与成骨细胞(osteoblast,OB)复合后用于血管化组织工程骨预构的可能性。方法:6只日本大耳白兔分为两组,分别在背阔肌下植入(OB+VEC)/外消旋聚乳酸(PDLLA)、OB/PDLLA。术后4、8周处死动物,大体标本、HE染色镜下观察体内成骨与血管化情况,以及血管化与成骨之间关系。结果:OB/PDLLA、(OB+VEC)/PDLLA两组植入物表面均有大量毛细血管肌纤维长入。OB/PDLLA组植入4周后可见骨组织形成,材料周围可见有小血管长入,随时间延长成骨量及血管化程度均有所增加。OB+VEC/PDLLA组可见在支架内部有大量毛细血管形成,支架周围成骨细胞活跃,成骨能力较强,在体内成骨能力及血管化程度均高于单纯成骨细胞复合支架组。结论:复合血管内皮细胞的组织工程骨成骨能力与血管化程度均高于单纯成骨细胞构建的组织工程骨。     

复合血管内皮细胞组织工程骨修复兔下颌骨缺损的研究

目的探讨血管内皮细胞构建的组织工程骨修复兔下颌骨缺损。方法实验分为三组,A组：兔成骨细胞（rabbit osteoblast,ROB）和血管内皮细胞（rabbit vascular endothelial cell,RVEC）复合外消旋聚乳酸（poly-DL-lacide,PDLLA）;B组：单纯成骨细胞复合PDLLA,C组：单纯PDLLA。分别修复兔下颌骨缺损,手术后4周、8周通过形态学、X线观察骨缺损修复及血管化情况。结果A组骨组织形成与血管化程度均高于B组,在材料中心区可见有血管形成,越靠近血管成骨越多。X线观察,A组：骨缺损区阴影面积明显减小,材料植入区可见骨组织影像;B组：缺损面积减小,骨组织影像增加,中心区影像低于周围区域。C组：缺损区未见骨组织影像,周围可见骨痂形成。结论复合血管内皮细胞和成骨细胞的细胞支架复合体无论在成骨还是血管化方面均好于单纯成骨细胞复合支架植入组。     

新型骨组织工程支架材料生物相容性的体内研究

目的通过比较2种新型骨组织工程支架材料即聚消旋乳酸/聚乳酸－聚乙二醇－聚乳酸/磷酸三钙（A）、聚消旋乳酸/聚乳酸－聚乙二醇－聚乳酸（B）与对照组聚消旋乳酸（C）修复兔下颌骨缺损的效果,探讨新型可吸收性生物支架材料体内埋植的生物相容性。方法24只成年新西兰大白兔按取材时间随机分为4组。双侧下颌骨下缘形成15 mm×6 mm全层骨质缺损, 每一缺损作为一个实验单位。每组内按完全随机化设计植入实验材料和对照材料。术后2、4、8、12周取材行大体标本、X线、组织学观察及计算机图像分析。结果复合支架材料A、B与聚消旋乳酸相比,复合支架B生物相容性好,同期成骨量最大;复合支架A出现明显的异物肉芽肿反应。结论新型复合支架材料B生物相容性好,效果优于聚消旋乳酸,有可能成为一种较理想的支架材料。复合支架A不适宜作为骨组织工程生物支架材料。     

Effect of endothelial differentiated adipose-derived stem cells on vascularity and osteogenesis in poly(D,L-lactide) scaffolds in vivo

Prevascularization of engineered bony constructs can potentially improve in vivo viability. However, the effect of endothelial cells on osteogenesis is unknown when placed in poly(D,L-lactide) (PLA) scaffolds alone. Adipose-derived stem cells (ASCs) have the ability to differentiate into both osteoblasts and endothelial cells by culture in specific media. We hypothesized that ASC-derived endothelial cells would improve vascularity with minimal contribution to bone formation when placed in scaffold alone. ASCs were successfully differentiated into endothelial cells (ASC-Endo) and osteoblasts (ASC-Osteo) using media supplemented with vascular endothelial growth factor and bone morphogenic protein 2, respectively. Tissue-engineered constructs were created with PLA matrices containing no cells (control), undifferentiated ASCs (ASCs), osteogenic-differentiated ASCs (ASC-Osteo), or endothelial differentiated ASCs (ASC-Endo), and these constructs were evaluated in critical-size Lewis rat calvarial defect model (n = 34). Eight weeks after implantation, the bone volume and microvessel population of bony constructs were evaluated by micro-computed tomography analysis and histologic staining. Bone volumes for ASCs and ASC-Osteo constructs, 0.7 and 0.91 mm(3), respectively, were statistically greater than that for ASC-Endo, 0.28 mm(3) (P < 0.05). There was no statistical difference between the PLA control (0.5 mm(3)) and ASC-Endo (0.28 mm(3)) constructs in bone formation. The percent area of microvessels within constructs was highest in the ASC-Endo group, although it did not reach statistical significance (0.065). Prevascularization of PLA scaffold with ASC-Endo cells will not increase bone formation by itself but may be used as a cell source for improving vascularization and potentially improving existing osteoblast function.     

Effects of 3-dimensional Bioprinting Alginate/Gelatin Hydrogel Scaffold Extract on Proliferation and Differentiation of Human Dental Pulp Stem Cells

IntroductionAlginate/gelatin hydrogel (Alg-Gel) scaffold has been applied in tissue engineering, but the research on its application in dental tissues regeneration is still lacking. We investigated the effect of this scaffold on human dental pulp stem cells (hDPSCs).MethodshDPSCs were cultured in both Alg-Gel and 3D-printed Alg-Gel scaffolds. Cell growth and adhesion were compared using fluorescein isothiocyanate–phalloidin staining and scanning electron microscopic micrographs. Changes in the proliferation in hDPSCs cultured in the complete culture medium containing aqueous extracts of the Alg-Gel or 3D-printed Alg-Gel scaffolds were examined using Cell Counting Kit-8 assay and flow cytometry analysis. Cells were cultured in the mineralization medium containing aqueous extracts of the Alg-Gel or 3D-printed Alg-Gel scaffolds for 7 or 14 days, and the differentiation of cells was shown by alizarin red S staining and alkaline phosphatase staining. The messenger RNA and protein expression of mineralization-related genes were detected with real-time polymerase chain reaction and Western blotting. Elemental analysis was used to test the material extract composition.ResultsMore cells were grown and adhered to the 3D-printed Alg-Gel scaffolds than the Alg-Gel scaffolds. The aqueous extracts of 3D-printed scaffolds can promote cell proliferation, and compared with Alg-Gel scaffolds, the extracts of 3D-printed scaffolds were more effective. Compared with the negative control group, 3D-printed Alg-Gel scaffold and Alg-Gel scaffold aqueous extracts promoted osteogenic/odontoblastic differentiation of hDPSCs with the enhanced formation of bone-like nodules and the alkaline phosphatase staining. The expression of mineralization-related genes was also up-regulated. 3D-printed scaffold aqueous extract contained more calcium and phosphorus ions than the Alg-Gel scaffold.ConclusionsThese findings suggest that compared with the Alg-Gel scaffold, 3D-printed Alg-Gel is more suitable for the growth of hDPSCs, and the scaffold extracts can better promote cell proliferation and differentiation.     

nHACP/CF支架材料对人脐静脉内皮细胞早期附着生长影响的研究

目的 探讨甲壳素纤维增强多孔胶原基骨组织工程支架材料(nHACP/CF)对人脐静脉内皮细胞(human umbilical vein endothelial cells,hUVEC)早期附着生长情况的影响.方法 本研究于2008年8月至2010年1月在中国医科大学实验病理教研室实验室进行.体外培养hUVEC,并与nHACP/CF支架材料联合培养,观察其在支架上的生长情况,通过倒置显微镜、扫描电子显微镜以及HE染色等方法观察细胞在nHACP/CF支架材料上的早期附着情况.结果 hUVEC在nHACP/CF支架材料上生长良好.结论 nHACP/CF支架材料有利于hUVEC的早期黏附、生长,具有良好的细胞相容性,是一种很有前景的骨组织工程支架材料.     

Exogenous recombinant human BMP-2 has little initial effects on human osteoblastic cells cultured on collagen type I coated/noncoated hydroxyapatite ceramic granules.

PURPOSE: Tissue engineering of bone entails the successful interplay between osteoinductive factors, osteogenic cells, their extracellular environment, and an osteoconductive biomaterial scaffold. Naturally produced ceramics, like hydroxyapatite (HA) calcified from red algae, are the most promising materials for use as scaffolds in this field. We hypothesized that extracellular matrix compartments and osteoinductive factors could further ameliorate the bioactivity of the scaffold. MATERIALS AND METHODS: Osteosarcoma cells with proven osteogenic phenotype (SaOS-2) were cultured onto type I collagen coated (Coll I/HA) and noncollagen coated HA granules (NC/HA) gained from red algae (C GRAFT/Algipore). Cells grown on tissue culture polystyrene dishes (TCPS) were used as controls. Second, SaOS-2 cells cultured on Coll I/HA, NC/HA, and TCPS were treated with recombinant human bone morphogenetic protein-2 (rhBMP-2) in different concentrations (10, 100, and 500 ng/mL). Non rhBMP-2-treated cultures were used as controls. Cultures of both experiments were grown under osteogenic differentiation conditions and after 24, 48, and 72 hours assays for cell viability, apoptosis, alkaline phosphatase activity (ALP), and osteocalcin (OC) secretion were done. RESULTS: Coating of HA granules with type I collagen showed higher cell viability in rhBMP-2-treated and nontreated cells. Supplementation of cultured cells with exogenous rhBMP-2 showed a dose-dependent effect only in the TCPS group. No alterations of the apoptotic rate within 1 investigation group were found. Addition of rhBMP-2 did not significantly alter the specific OC secretion of cells grown on Coll I/HA and TCPS. CONCLUSION: These in vitro findings show that in the initial period of cultivation and up to 72 hours, the coating of HA granules with collagen type I had positive effects on cell viability and osteoblastic characteristics of osteoblastic cells. In contrast, the supplementation with exogenous rhBMP-2 shows no dose-dependent effects. The combination of collagen type I and exogenous rhBMP-2 did not ameliorate the bioactivity of hydroxyapatite calcified from red algae in the initial period of cultivation.     

血管内皮细胞对牙周组织细胞增殖的影响

目的：通过血管内皮细胞与牙周组织细胞复合培养模拟牙周组织再生环境,观察血管内皮细胞对牙周组织细胞增殖的作用规律,探讨血管内皮细胞对牙周组织修复再生的影响。方法：应用原代培养的血管内皮细胞,在Transwell嵌套中实现与人牙周膜成纤维细胞、牙龈成纤维细胞间的复合培养,分别于复合培养第2、4、6、8和10天,以细胞计数手段检测血管内皮细胞共存条件下2种牙周组织细胞的增殖情况,并与单独培养的2种牙周组织细胞作为对照,采用SPSS13.0软件包对数据进行统计学分析。结果：在血管内皮细胞存在条件下,2种牙周组织细胞的增殖速率均显著高于单独培养条件。血管内皮细胞存在时,牙周膜成纤维细胞增殖速率逐渐超越牙龈成纤维细胞(第6天起),并在第8天细胞数量上超过牙龈成纤维细胞,差异具有显著性(P<0.01)。结论：血管内皮细胞的存在,对牙周组织细胞的增殖具有促进作用,对牙周膜成纤维细胞增殖的促进作用显著高于牙龈成纤维细胞。