不同比例PLGA/β-TCP电纺纤维支架的制备与性能研究

目的：通过静电纺丝法制备不同比例PLGA/β-TCP纳米纤维支架,筛选出最合适比例,以便为进一步的体内植入提供依据。方法：利用静电纺丝法制备比例为10：0、9：1、8：2、7：3、6：4、5：5的PLGA/β-TCP纳米纤维支架,扫描电镜观察纤维支架的多孔结构,液体置换法测量支架的孔隙率;分别于1%胰酶PBS溶液中进行体外降解,测定材料的降解性;接触角仪测量材料的接触角,评价其亲水性能。结果：电镜观察显示制备的不同比例的PLGA/β-TCP纤维中（除6：4、5：5组）,直径均一,呈相互联通的三维多孔结构,各组支架材料的孔隙率均〉80%,其中6：4、5：5组的孔隙率〉85%,电镜下观察纤维成分有限,大部分为颗粒状,不具有多孔支架的三维结构;体外降解实验中7：3、6：4、5：5组均在第7周时完全降解。结论：通过静电纺丝法制备不同比例的PLGA/TCP支架材料,9：1、8：2、7：3组均符合骨组织工程要求,体外降解实验中,7：3组的降解率较另两组材料为优,具有用作体内骨修复材料的潜力。     

丝素蛋白-软骨脱细胞外基质复合取向支架的制备及评价

目的 制备丝素蛋白（SF）-软骨脱细胞外基质（CECM）仿生支架材料,检测其理化性能特性。方法 采用改良温度梯度热诱导相分离（TIPS）技术结合冷冻干燥法制备出CECM取向支架,然后将CECM浆料与制备好的SF溶液按照质量比1∶1混合后配制成质量分数6%的浆料,通过TIPS技术制备出SF-CECM复合取向支架。对SF-CECM复合取向支架进行扫描电子显微镜（SEM）观察和组织学染色,同时测定支架孔隙率、吸水性以及力学性能。结果 SEM观察可见,支架横断面呈多孔网状结构,纵剖面呈垂直的管状结构。组织学染色显示复合支架脱细胞彻底,有蛋白多糖、胶原成分,与天然软骨成分相似。支架孔隙率、吸水率和纵向压缩弹性模量分别为95.733%±1.010%、94.309%±1.302%和（65.40±4.09）kPa。结论 SF-CECM复合取向支架具有良好的理化特性和生物力学性能,有望成为较理想的组织工程软骨支架。     

3D打印白硅钙石支架材料在大鼠Onlay骨移植中的应用

目的 评价3D打印白硅钙石（bredigite,BRT）骨支架材料在大鼠Onlay骨移植中的效果。方法 通过3D打印技术制备骨BRT支架材料作为实验组,β-磷酸三钙（β-tricalcium phosphate,β-TCP）骨支架材料作为对照组,通过扫描电镜（scanning electron microscopy,SEM）、X射线衍射（X-ray diffraction,XRD）、力学测试和体外降解测试实验对其进行表征观察。建立SD大鼠颅骨Onlay骨移植的动物模型,根据移植物的不同,分为自体骨移植组（Auto组）、β-TCP组及BRT组。术后12周获取标本行大体观察、micro-CT扫描检测分析、组织学HE染色。结果 SEM观察显示,BRT有规则的多孔结构,XRD衍射峰较为锐利。与β-TCP组（11.29±1.30）MPa相比,BRT支架材料（46.80±3.44）MPa具有更好的力学强度（P<0.001）;在体外第35天时,BRT降解率为27.18%±1.41%,并且在降解过程中释放钙、镁、硅离子。micro-CT及组织学染色实验结果提示,Auto组有一定程度的骨吸收,β-...     

3D打印聚乙烯醇/纳米羟基磷灰石支架与丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架的性能比较

目的　采用3D打印技术制备3D打印聚乙烯醇 /纳米羟基磷灰石支架与丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架,并对其进行表征。方法　采用3D打印技术制作聚乙烯醇/纳米羟基磷灰石支架以及丝素蛋白/聚乙烯醇/纳米羟基磷灰石复合支架。进行孔隙率、扫描电镜、压缩力学性能及细胞毒性检测。 结果　①扫描电镜观察：丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架结构规则,网状结构清晰,交通支连续,层层之间搭接良好,支架空隙均一。相同倍数下,聚乙烯醇/纳米羟基磷灰石支架网状结构连续性较差。②压缩力学性能：相同应力情况下（10 MPa）,3D打印丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架的应变大于3D打印聚乙烯醇/纳米羟基磷灰石支架。③孔隙率：3D打印丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架的孔隙率大于3D打印聚乙烯醇/纳米羟基磷灰石支架。④细胞毒性检测：不同时间点两组支架的细胞增殖率无明显差别。结论　结果表明：3D打印聚乙烯醇 /纳米羟基磷灰石支架与丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架具有良好的理化性能和细胞相容性。     

鹿角粉复合支架与纳米级羟基磷灰石复合支架的性能比较

目的探索不同胶体材料结合鹿角粉和纳米级羟基磷灰石(n-HA)支架的理化、生物性能。方法取鹿角粉和纳米级羟基磷灰石,分别与15%聚乙烯醇(PVA)、5%丝素蛋白液(SF)、15%PVA和5%丝素蛋白液(4∶1)混合,制备鹿角粉/PVA支架、鹿角粉/SF支架、鹿角粉/PVA/SF支架、n-HA/PVA支架、n-HA/SF支架、n-HA/PVA/SF支架,检测其抗溶解性能、抗压缩力、抗剪切力及支架细胞毒性。结果鹿角粉/SF支架、n-HA/SF支架溶解变形程度为Ⅲ级,其余4组为Ⅱ级。鹿角粉/SF支架、n-HA/SF支架抗剪切力、抗压缩力均较差,鹿角粉/PVA支架、鹿角粉/PVA/SF支架分别与n-HA/PVA支架、nHA/PVA/SF支架抗剪切性能相似,鹿角粉/PVA支架、鹿角粉/PVA/SF支架抗压缩性能分别优于n-HA/PVA支架、n-HA/PVA/SF支架。鹿角粉/PVA支架、鹿角粉/SF支架、鹿角粉/PVA/SF支架细胞相容性优于n-HA/PVA支架、n-HA/SF支架、n HA/PVA/SF支架。结论鹿角粉复合支架具有良好的机械性能及细胞相容性,可以作为异种骨支架材料研究的新方向。单纯丝素作为水凝胶复合支架抗溶解性不佳,机械性能差,无法满足骨组织工程支架要求。     

京尼平交联改性可溶性蛋壳膜蛋白冻干支架的制备及检测

目的：评价京尼平交联改性前后可溶性蛋壳膜蛋白（soluble eggshell membrane protein,SEP）冻干组织工程支架的理化性能和生物相容性。方法：冷冻干燥法制备SEP的冻干支架,浸泡于京尼平溶液中交联改性。通过扫描电镜观察其表面形貌。测量其孔隙率、抗拉强度以及降解率,采用溶血实验、亚急性全身毒性实验和细胞毒性实验初步评价其生物相容性。结果：京尼平交联改性前后的SEP冻干支架孔径分别为（280±71）μm和（263±89）μm,孔隙率分别为（90.4±7.6）%和（87.9±9.7）%;交联改性显著提高了SEP冻干支架的拉伸强度和弹性模量,降低了支架的失重率（P〈0.01）;交联前后的材料均无溶血现象;亚急性短期全身毒性实验中组织切片均未见病理变化;细胞毒性检测均为0级。结论：京尼平交联改性在提高SEP冻干支架的力学强度和抗降解能力的同时,仍可保持支架材料良好的生物相容性。     

3D打印丝素蛋白/聚乙烯醇/纳米羟基磷灰石支架体内降解的研究

目的 使用3D打印技术打印出丝素蛋白/聚乙烯醇/纳米羟基磷灰石(SF/PVA/n?HA)支架与聚乙烯醇/纳米羟基磷灰石(PVA/n?HA)支架并用细胞膜片包裹后回植到动物体内,研究并探讨SF/PVA/n?HA与PVA/n?HA支架的降解性能.方法 将SF/PVA/n?HA支架与PVA/n?HA支架分别植入羊下颌骨内,在术后1、2、3个月处死实验动物,取出支架所在部位下颌骨,行影像学观察、HE染色观察、实时定量PCR(RT?PCR),探讨两种支架在体内降解情况的区别以及降解相关因子之间的关系.结果 ①影像学结果:SF/PVA/n?HA支架组与PVA/n?HA支架组可见骨缺损处低密度影均减小,而二者相比,SF/PVA/n?HA支架组低密度影范围较小,高密度影部位也较PVA/n?HA支架明显.②HE染色结果:SF/PVA/n?HA支架组在前两个月仍可见部分支架残余,支架周围前两个月可见炎性细胞,3个月末炎性逐渐消退,可见明显骨陷窝.③RT?PCR:同一时间不同材料比较,SF/PVA/n?HA支架组与PVA/n?HA支架组相比,IL?1、IL?6、M?CSF、NFATc1四种因子表达趋势相近.在术后1、2个月,SF/PVA/n?HA支架组四种因子的mRNA表达量均高于PVA/n?HA支架组;术后3个月,四种因子mRNA表达量SF/PVA/n?HA支架组均低于PVA/n?HA支架组(P<0.05).结论 SF/PVA/n?HA支架具有一定的降解性能,且在降解过程中,能促进破骨相关因子IL?1、IL?6、M?CSF、NFATc1的表达.     

快速成型技术构建聚己内酯支架作为骨支架材料的研究

目的：运用快速成型技术（RP）构建聚己内酯（PCL）支架,检测其生物相容性,探讨成为组织工程骨支架的潜力。方法：以聚己内酯为原料利用熔融沉积成型法（FDM）制备无孔隙及孔径300μm、500μm 3种PCL支架,比重瓶法测孔隙率。利用MTT法检测支架材料对细胞增殖的影响。倒置荧光显微镜、扫描电子显微镜观察聚己内酯支架上的细胞粘附情况及细胞形态。结果：肉眼观察可见300μm及500μm孔径支架材料孔隙大小均匀,排列规整,层次分明,两种孔径支架都具有良好的孔隙连通率。MTT检测结果显示1d、2d、3d各时间点均无明显细胞毒性,细胞毒性为1级。荧光倒置显微镜下观察发现细胞粘附数量由无孔隙组、500μm孔径组、300μm孔径组依次增加。扫描电子显微镜下观察细胞紧密粘附于支架。结论：用快速成型技术制备的聚己内酯支架具有较高的孔隙率,孔隙之间连通率良好,生物相容性良好,细胞粘附良好,有望成为骨组织工程支架。     

丝素蛋白/左旋聚乳酸支架材料与软骨细胞体外细胞相容性研究

目的:观察静电纺丝支架材料丝素蛋白/左旋聚乳酸(SF/PLLA)的体外细胞相容性,探索其作为软骨组织工程支架材料的可行性,为进一步的体内及动物实验奠定基础。方法:将兔膝关节软骨细胞与丝素蛋白/左旋聚乳酸(SF/PLLA)支架材料复合培养,在第3、7、14天分别作HE染色和阿利新蓝+核固红染色,扫描电镜检验细胞黏着情况,MTT试验检测细胞在支架上的增殖情况。结果:细胞在支架上可以获得良好的粘附,细胞增殖良好,无细胞表型的变化。结论:丝素蛋白/左旋聚乳酸(SF/PLLA)具有良好的细胞相容性,可作为软骨组织工程支架材料。     

丝素蛋白抗菌性修饰在皮肤组织工程中的应用

丝素蛋白（silk fibroin，SF）是一种天然高分子纤维蛋白，常被作为理想敷料应用于口腔颌面部及全身皮肤创面的修复中。SF具备良好的生物相容性、可降解性、机械性能，可促进创面愈合，但其本身不具备抗菌性。通过不同抗菌性修饰赋予SF材料抗菌性能已得到广泛的研究和关注。本文对SF抗菌性修饰材料在皮肤组织工程中的应用进行系统性介绍，为今后开展SF抗菌性支架材料研究提供思路和指导。     

重组人骨保护素抑制破骨细胞及促进羟磷灰石修复去势大鼠下颌骨缺损的研究

目的 探讨转染人骨保护素（hOPG）基因的大鼠骨髓间充质干细胞（rBMSCs）复合羟磷灰石（HA）支架对去势大鼠下颌骨缺损的修复作用。方法 将重组腺病毒pDC316-hOPG-EGFP转染rBMSCs,蛋白质印迹法和骨磨片试验分别检测hOPG的表达水平和抑制破骨细胞功能;构建骨质疏松大鼠模型,分别将HA支架、未转染rBMSCs复合HA支架、转染rBMSCs复合HA支架植入大鼠下颌骨骨缺损,6周后通过抗酒石酸酸性磷酸酶、苏木精-伊红染色检测骨缺损区破骨细胞及骨修复情况。结果 体外携载有hOPG基因的腺病毒成功转染rBMSCs,转染后的rBMSCs表达具有抑制破骨细胞活性功能的hOPG;表达hOPG的rBMSCs复合HA支架后骨缺损处破骨细胞明显减少,成骨增多。结论 转染hOPG基因的rBMSCs在体内外均具有抑制破骨细胞功能的作用,且转染rBMSCs复合HA支架可促进骨质疏松大鼠的下颌骨缺损修复。     

The acquisition and loss of fluoride by topically fluoridated human tooth enamel

Intact tooth surfaces were treated in vitro with acidulated-phosphate-fluoride and stannous fluoride solutions. In addition a number of teeth were treated in vivo with acidulated-phosphate-fluoride. Results indicate an initially high level of fluoride acquisition followed by a loss of as much as two-thirds of the fluoride within 1 day, in both in vitro and in vivo experiments. When the treated enamel was washed in water for 1 week all acquired fluoride was lost. This secondary loss of fluoride did not occur in vivo or when in vitro treated enamel was washed with solutions saturated with calcium and phosphate.

It is suggested that the initial rapid loss of fluoride is due to removal of unreacted fluoride while that part lost in water upon longer washing results from a slow dissolution of a slightly soluble compound.     

Collagen synthesis is a major component of protein synthesis in the periodontal ligament in various species

The incorporation of radioactive proline into collagen and non-collagen proteins of periodontal ligament has been studied in vivo and in three in vitro systems. Approximately 90 per cent of the protein-bound proline label was recovered in collagen from rat first molar ligaments in vivo; more than 50 per cent in proteins synthesized and secreted by fibroblasts isolated from monkey periodontal ligament and maintained in cell culture in vitro; almost 30 per cent in ligaments of mouse first molar periodontium maintained in ascorbic acid-deficient organ culture in vitro; and less than 2 per cent in ligaments of pig, rat and rabbit in tissue culture in vitro. These results provide strong evidence that collagen synthesis is a major component of protein synthesis in the periodontal ligament. The lack of collagen synthesis found in isolated periodontal ligament in tissue culture, as well as a published report that collagen synthesis by periodontal ligament expiants in culture is marginal, can be explained by our observation that only a limited number of cells remain viable under these culture conditions.     

白皮杉醇抗恶性黑色素瘤的体内外实验研究

目的 研究白皮杉醇(PIC)的体内外抗恶性黑色素瘤作用.方法 体外培养来源于小鼠皮肤的恶性黑色素瘤细胞系B16F10,以梯度浓度PIC处理细胞后甲基噻唑基四唑(MTT)法检测细胞活力;蛋白印迹法检测金属基质蛋白酶(MMP)-2及MMP-9、血管内皮生长因子(VEGF)、脾酪氨酸激酶(Syk)及p-Syk的表达;划痕实验...     

In vivo and in vitro study of the effects of chlorpromazine on tooth mineralization in rats and mice

The effects of chlorpromazine (CPZ) on tooth mineralization were examined using incisor dentine in adult rats and cultured tooth germs of mandibular first molars dissected from mouse embryos. CPZ (10, 50 and 250 mg/kg, s.c.) substantially inhibited dentine mineralization as evaluated by contact microradiographs. Plasma calcium and phosphorus concentrations were not decreased by CPZ (10 and 50 mg/kg). Physicochemical effects were not involved in the action of CPZ on the mineralization. In vitro experiments showed that CPZ (1 and 10 μM) inhibited mineralization and alkaline phosphatase (ALP) activity in the tooth germs. As CPZ has the properties of a calmodulin antagonist, the calmodulin antagonists W-7 and W-5 were also examined. Both inhibited mineralization and ALP activity in tooth germs; W-5 had less effect than W-7. These in vivo and in vitro findings suggest that CPZ inhibited cell-mediated mineralization in dentine without affecting the calciumdashregulating system and physicochemical mineral deposition. In addition, calmodulin could be involved in cell-mediated mineralization.     

可载药的磁性聚己内酯/明胶微球支架的制备及其体外成骨性能的研究

目的 制备出可载药的磁性颗粒支架,并研究其体外成骨性能.方法 使用复乳法制备聚己内酯(polycaprolactone,PCL)/明胶微球(PCL/gel microsphere,PGM)支架,通过相差显微镜、场发射电子显微镜(field emission scanning electron micro?scope,F...     

A comparison between osteoclasts in vitro and in vivo in the periodontal ligament of the adult mouse

Twelve expiants of the first mandibular molar and surrounding periodontium of young adult mice were cultured for 7 days in vitro. The number and size of osteoclasts, the number of nuclei per osteoclast and the distribution of the osteoclasts in the periodontal ligament were compared with osteoclasts in periodontal ligament in vivo in mice of the same age as the mice from which the expiants were taken. Light microscope comparison of the two groups indicated that resorption of bone had occurred in vitro. There was a significant increase in the number and size of osteoclasts in the cultured expiants compared with the in vivo material. The numbers of nuclei per osteoclast and the distribution of osteoclasts in the periodontal ligament was similar in the two groups, but the greatest increase in osteoclast numbers occurred on the distal aspect of the distal root where there were most osteoclasts in vivo. It was concluded that there was active bone resorption and that osteoclasts formed de novo during culture of these expiants.     

微小RNA-29a-3p调节卷曲蛋白4表达影响高脂血症大鼠种植体骨整合的实验研究

目的 研究microRNA-29a-3p（miR-29a-3p）对高脂环境下大鼠骨髓间充质干细胞（BMSCs）成骨分化和高脂血症大鼠种植体骨整合的影响及其作用位点。方法 1）体外实验：对BMSCs分别进行普通和高脂成骨诱导,通过逆转录实时定量聚合酶链反应和Western blot检测miR-29a-3p及成骨相关因子碱性磷酸酶（ALP）、Runt相关基因2（Runx2）的基因和蛋白质表达;高脂培养的BMSCs分别转染miR-29a-3p模拟物、抑制物及阴性对照（NC）质粒,RT-qPCR检测miR-29a-3p、ALP及Runx2基因表达情况,Western blot检测ALP、Runx2蛋白表达情况。通过靶基因预测软件（Target Scan、MiRNA.org等）预测miR-29a-3p与成骨相关的靶基因为卷曲蛋白4（Fzd4）,双荧光素酶报告检测miR-29a-3p与Fzd4的相互作用关系。2）体内实验：高脂血症大鼠为实验组,普通大鼠为对照组,两组分别植入种植体,检测种植体周围骨组织中miR-29a-3p、ALP、Runx2的表达差异;行种植体-骨组织的硬组织切片,亚甲基蓝-酸性品红染色,进行组织学观察。分别将miR-29a-3p过表达慢病毒载体及空白对照慢病毒载体注射入高脂血症大鼠,种植体植入后3、10 d检测种植体周围骨组织中ALP、Runx2的表达变化以研究miR-29a-3p对高脂血症大鼠成骨的作用。结果 高脂组与普通组相比,ALP、Runx2和miR-29a-3p表达下调,BMSCs成骨分化能力下降。miR-29a-3p模拟物组与抑制物组相比,ALP、Runx2表达升高,BMSCs成骨分化能力增强。体内实验也得到了相似结果。双荧光素酶报告基因分析证实miR29a-3p通过直接结合3’-UTR抑制Fzd4表达。结论 miR-29a-3p对高脂环境下大鼠BMSCs成骨分化起正向调节作用,可直接与Fzd4结合调节成骨分化;miR-29a-3p能够促进高脂血症大鼠种植体周围成骨标志基因的表达,有利于骨整合。     

选择性激光烧结多孔钛种植体及其性能研究

目的 分析选择性激光烧结（SLS）多孔钛种植体的机械性能及生物相容性,探讨其与壳聚糖（CS）/羟磷灰石（HA）复合涂层结合后的促骨结合作用。方法 制备Ti6Al4V试件,部分试件表面进行CS/HA涂层处理;对试件进行扫描电子显微镜观察和机械性能检测;体外培养MC3T3-E1细胞,进行活/死细胞染色、甲基噻唑基四唑（MTT）及碱性磷酸酶（ALP）水平检测;将柱状螺纹种植体植入兔股骨髁部,分析其体内生物学性能。结果 试件准弹性梯度随孔隙率增大而减小,孔隙率为30%时与皮质骨接近,70%时与松质骨接近;试件具有良好的生物相容性。复合CS/HA涂层后可促进MC3T3-E1细胞增殖、分化,有利于骨组织长入孔隙,形成良好的骨结合。结论 多孔钛种植体具有良好的机械性能和生物相容性,与CS/HA涂层结合后具有骨诱导性,利于形成稳定的骨结合。     

Saliva mediated adherence, aggregation and prevalence in dental plaque of Streptococcus mutans, streptococcus sanguis and actinomyces spp. in young and elderly humans

Salivary components in the pellicle mediate bacterial adherence to the tooth. Such components may also aggregate bacteria in saliva and prevent them becoming established in dental plaque. In the present study, the adherence and aggregation of Streptococcus mutans strain Ingbritt, S. sanguis strain 10556 and Actinomyces viscosus strain 19246 mediated by parotid and whole saliva from groups of young and elderly people were examined. Significant differences were found between test strains, salivary secretions and age groups. S. sanguis 10556 and A. viscosus 19246 generally adhered more strongly than S. mutans Ingbritt, which adhered better to pellicles from parotid saliva than from whole saliva. Strain 19246 bound in higher numbers to parotid saliva pellicles from elderly compared to young individuals. Strain 10556 adhered better to whole saliva than parotid saliva pellicles, and the difference was significant among the young individuals, indicating reduced adherence ability in elderly whole saliva. The streptococci were aggregated by parotid and whole saliva, and S. sanguis aggregation was less with whole saliva from the elderly than from the young participants. Besides a correlation between whole saliva aggregation of S. mutans and proportions of bacteria in plaque, no correlations were found for the individual binding properties of saliva and prevalence of bacteria in vivo. However, the level of saliva-mediated adherence in vitro was in the following order: S. mutans < Actinomyces S. sanguis, which corresponded to their isolation frequency in plaque. These findings emphasize the importance of initial adherence to salivary receptors in bacterial colonization on teeth. Further studies are needed to reveal if individual patterns in the in vitro binding characteristics of saliva lead to variation of colonization in vivo.