牛心包衍生膜材料的制备及理化性能检测

目的：筛选脱牛心包衍生膜材料的制备方法,观察其组织结构和力学性能,探讨作为支架材料的可能性。方法：分别采用0．1mol／L NaCl 0．5％ Triton X-100、0．1mol／L NaCl 0．25％Trypsin、0．25％Trypsin 0．5%Triton X-100、0．1mol／L NaCl 0．25％Trypsin 0．5% Triton X-100对新鲜牛心包进行脱细胞处理,脱细胞牛心包经过碳化二亚胺(EDAC)交联。观察脱细胞牛心包基质材料的组织结构、测定其理化性质。结果：经0．25% Trypsin 0．5%Triton X-100酶联脱细胞处理获得的脱细胞牛心包内无细胞成分,其双层结构及理化性能保存良好。结论：0．25％Trypsin 0．5％Triton X-100酶联脱细胞法能有效地脱去牛心包内的细胞成分,获得的脱细胞牛心包的组织结构和理化性能符合生物支架材料的要求。     

牛心包衍生材料引导骨再生的实验研究

目的:评价碳化二亚胺(EDAC)交联处理的脱细胞牛心包的引导成骨作用。方法:在新西兰白兔双侧下颌体各制备7 mm×7 mm大小的骨缺损,一侧骨缺损覆盖EDAC交联脱细胞牛心包,另一侧不覆膜作为对照。术后4周、8周、12周、16周行X线、DXA测量骨密度及组织学观察骨缺损修复情况。结果:术后各组新西兰白兔伤口愈合良好;各期EDAC交联脱细胞牛心包组骨缺损区的BMD均高于同期对照组,有统计学意义(P<0.05);DAC交联脱细胞牛心包组骨缺损修复速度快于对照组。结论:EDAC交联脱细胞牛心包有良好的引导骨组织再生作用。     

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。     

脱细胞腮腺基质的制备及组织学特征初步观察

目的:探讨脱细胞腮腺支架材料的制备方法,制备涎腺组织工程的理想支架材料。方法:对新西兰大白兔腮腺进行冻融联合酶消化,最后冷冻干燥,观察其组织学特征。结果:制备的脱细胞腮腺基质的细胞成分能被有效去除,材料保留了利于腮腺细胞生长的三维网状基质胶原结构,有一定的强度和韧性。结论:采用反复冻融联合酶消化、冷冻干燥法能获得一种无细胞的理想组织工程腮腺支架材料。     

天然衍生脱细胞牛心包复合BMP-2转染的BMSCs引导骨组织再生的体外实验研究

目的: 研究天然衍生脱细胞牛心包复合BMP-2转染BMSCs引导骨组织再生的可行性和有效性。方法: 天然衍生脱细胞牛心包体外复合BMP-2转染BMSCs,SEM评估细胞生长与增殖,MTT法检测牛心包对转染细胞的毒性,ELISA 检测BMP-2含量,结晶紫染色法测定BMSCs细胞粘附能力,ALP活性实验检测转染BMSCs成骨性能。结果: SEM发现天然衍生脱细胞牛心包复合BMP-2基因转染的BMSCs后,细胞生长并增殖良好;各组细胞的存活率没有显著性差异;天然衍生脱细胞牛心包增强了BMP-2转染BMSCs的细胞粘附能力和成骨细胞转化能力。结论: 天然衍生脱细胞牛心包复合BMP-2基因转染BMSCs后,具有良好的生物和细胞相容性,有望成为一种优良的天然GBR膜材料。     

载乳铁蛋白壳聚糖微球/nHA/Co复合材料的制备

目的:制备载乳铁蛋白壳聚糖微球/nHA/Co复合材料作为组织工程材料拟修复骨缺损。方法:通过乳化交联法法制备载乳铁蛋白壳聚糖微球,化学沉淀法制备纳米羟基磷灰石,改良酸酶法提取Ⅰ型胶原,应用京尼平交联制备复合材料,SEM检测材料的微观结构,测定壳聚糖微球对乳铁蛋白的载药率和包封率。结果:复合材料及各组成成份扫描电镜观察均具有较好的微观形态,壳聚糖微球对乳铁蛋白的载药率为(1.06±0.06)%,包封率为(88.2±2.9)%。结论:载乳铁蛋白壳聚糖微球/nHA/Co复合材料具有较好的微观形态。     

脱细胞气管基质制备及其生物相容性研究

目的:比较研究家兔、SD大鼠脱细胞气管基质材料生物相容性,为组织工程化人工涎腺样组织的体外构建实验准备材料。方法:用改良去污剂-酶联合多步法对SD大鼠、家兔气管组织进行脱细胞处理,并分别对2种材料行组织学、内壁拓扑结构及生物相容性研究。结果:处理后的2种气管组织中细胞均脱落完全,生物相容性好。结论:自制的脱细胞气管基质材料可用于组织工程化涎腺样组织体外构建的实验研究。     

牛心包引导骨组织再生的实验研究

目的评价戊二醛(GA)处理的牛心包作为引导骨组织再生材料的可行性。方法在II只狗的双侧下领骨颊侧骨板各制备一个1.0 cm x1.Ocmx0.5cm大小的骨缺损,随机在一侧骨缺损覆盖GA牛心包,另一侧不覆盖膜作为对照侧。术后2周,4周,8周,16周采用Dental-CT及组织学切片观察骨缺损修复情况。结果①10只狗伤口愈合良好,1只狗(16周组)牛心包处理区伤口愈合欠佳;316周内牛心包未见明显吸收,周围仅少量炎性细胞浸润;③牛心包覆盖侧骨缺损修复速度快于对照侧。结论①GA牛心包有良好的引导骨组织再生作用,牛心包有可能成为一种新的引导骨组织再生材料;②GA处理的牛心包有良好的生物相容性。     

脱细胞气管基质制备及组织相容性初步研究

目的:制作脱细胞气管基质,为组织工程化人工涎腺样组织的体外构建研究提供天然衍生生物导管支架材料。方法:用改良的去污剂-酶联合多步法脱除家兔、SD大鼠气管组织中的细胞,并将处理后的气管行HE染色,观察脱细胞及组织结构情况;扫描电镜下观察气管壁表面结构。然后将脱细胞气管植入SD大鼠面颊部,并于1、4、12周后取出进行组织学观察。结果:处理后的气管组织中细胞脱落完全,管腔内壁纤维组织完整、能保持气管原有的中空状管形。扫描电镜见气管内壁为规则的粗糙沟槽状结构。植入体内12周后与周围组织相容较好,未见明显管腔塌陷及炎性反应。结论:制备的气管材料,细胞脱落完全,是进行组织工程化人工涎腺样组织体外构建研究较理想的细胞外基质材料。     

增龄因素对人牙髓干细胞生物学行为影响的研究

目的 对不同年龄患者的牙髓干细胞(human dental pulp stem cells,hDPSCs)进行分离培养,并比较其生物学特性.方法 应用组织块联合胰酶消化法培养不同年龄患者的hDPSCs,记录细胞爬出时间,显微镜下观察比较细胞形态.流式细胞术检测细胞表面抗原,并对细胞行体外成骨、成脂诱导,观察矿化结节和脂...     

Effects of different irrigation solutions on root dentine microhardness,smear layer removal and erosion

This study aimed to compare the effects of different irrigants on root dentine microhardness, erosion and smear layer removal. A total of 72 root dentine slices were divided into six groups, according to the final irrigants used: Group 1: 17% ethylenediamine tetra‐acetic acid (EDTA) + 2.5% NaOCl, Group 2: 7% maleic acid (MA) + 2.5% sodium hypochloride (NaOCl), Group 3: 1.3% NaOCl + mixture of tetracycline, acid and detergent (MTAD), Group 4: Smear Clear + 2.5% NaOCl, Group 5: 5% NaOCl, Group 6: saline. Vickers microhardness values were measured before and after treatment. In total, 42 root‐halves were prepared for scanning electron microscope to evaluate the amount of smear and erosion in the coronal, middle and apical thirds. Data were analysed using two‐way anova , Duncan and two‐proportion z‐tests. Maleic acid showed the greatest reduction in dentine microhardness (P < 0.05), followed by EDTA and MTAD. EDTA, maleic acid, MTAD and Smear Clear removed smear layer efficiently in the coronal and middle thirds of root canal. However, in the apical region, maleic acid showed more efficient removal of the smear layer than the other irrigants (P < 0.05).     

Optimizing Methods for Bovine Dental Pulp Decellularization

IntroductionThis study aimed to characterize the decellularization effects of different treatment protocols on the bovine dental pulp extracellular matrix (ECM) for tissue regeneration.MethodsSeven different decellularization protocols consisting of trypsin/EDTA (for 1 hour, 24 hours, or 48 hours), sodium dodecyl sulfate (SDS, for 24 hours or 48 hours), Triton X-100 (for 1 hour), and deoxyribonuclease treatments were tested on bovine dental pulp tissue. The posttreatment samples were evaluated for remaining DNA and cellular contents, structural durability, immunofluorescence analysis, and in vivo immune responses.ResultsA complete decellularization process in all of the experimental groups was observed. The protocol that included 1 hour of Triton X-100 treatment and 12 hours of trypsin/EDTA treatment with no SDS treatment (P7 [12E-0S-1T]) showed the highest retention of glycosaminoglycan and the absence of nuclei in 4,6-diamidino-2-phenylindole. All groups showed significantly lower DNA content compared with native pulp tissue (P < .05), whereas compared with other protocols, protocols 1 (1 hour of EDTA/trypsin, 24 hours of SDS, and 1 hour of Triton X-100) and 4 (1 hour of EDTA/Trypsin, 48 hours of SDS, and no Triton X-100) resulted in the highest DNA contents (P < .05). Based on these results, P7 was further evaluated by immunofluorescence and in vivo immunogenicity. P7 specimens preserved collagen type I, whereas mononuclear cell infiltration along with neovascularization was observed in vivo.ConclusionsAll tested treatments displayed the potential ability to decellularize pulp tissue and are viable options for a xenogeneic dental pulp ECM scaffold. The P7 (12E-0S-1T) protocol resulted in decellularized ECM with minimal organic matrix/ultrastructural detriments and an acceptable host immune response.     

Photodynamically crosslinked and chitosan-incorporated dentin collagen

A lingering concern with restored root-filled teeth is the loss of structural integrity of the dentin and dentin-sealer interface over time. We hypothesized that crosslinking of dentin collagen with simultaneous incorporation of a biopolymer into collagen matrix would improve its structural stability. This study aimed to investigate the effects of combining chemical/photodynamic crosslinking of dentin collagen with the incorporation of carboxymethyl-chitosan (CMCS) on the resistance to enzymatic degradation and mechanical properties of dentin collagen. Ninety-six demineralized dentin collagen specimens (human, n = 72; and bovine, n = 24) were prepared and crosslinked chemically/ photodynamically, with/without CMCS. Glutaraldehyde and carbodiimides were used for chemical crosslinking, while rose Bengal activated with a non-coherent light (540 nm) at 20 J/cm(2) was applied for photodynamic crosslinking. The crosslinked human dentin collagen was subjected to chemical characterization, 7 days enzymatic degradation, and transmission electron microscopy (TEM), while the bovine dentin collagen was used for tensile-testing. Crosslinked collagen showed significantly higher resistance to enzymatic degradation (p < 0.01), stable ultrastructure, and increased tensile strength (p < 0.05). Crosslinking CMCS with collagen matrix as observed in the TEM further improved the mechanical properties of dentin collagen (p < 0.01). This study highlighted the possibility of improving the resistance and toughness of dentin collagen by chemically/photodynamically crosslinking collagen matrix with CMCS.     

人体口腔粘膜上皮细胞体外培养和鉴定

目的 建立稳定的人口腔颊粘膜上皮细胞体外培养方法。方法 取正常人口腔颊粘膜，中性蛋白酶和胰蛋白酶消化后，获取的粘膜上皮细胞接种于10％含胎牛血清培养基中连续培养，探索最适培养条件，进行细胞定性及形态学观察。结果 体外培养细胞中无成纤维细胞混杂，长满后呈上皮细胞特有的铺路石样外观。体外可连续传9～10代，生长期50～60天。细胞角蛋白免疫组化染色阳性，透射电镜下可见上皮细胞间特有的桥粒结构。结论 采用含血清培养基体外连续培养口腔粘膜上皮细胞获得成功，4代以内培养上皮细胞形态结构与原代无差别，可用于组织工程化口腔粘膜构建。     

不同方法制备抗原致敏的树突状细胞介导T淋巴细胞对Tca8113细胞杀伤作用的影响

目的探讨树突状细胞（DCs）疫苗治疗舌癌的可行性,并选择较优的抗原负载方式。方法分别采用弱酸洗脱法、反复冻融法制备Tca8113细胞抗原,并分为3组：弱酸洗脱抗原组、反复冻融抗原组、对照组（不加肿瘤抗原）。从外周血单核细胞中诱导DCs,分离出T淋巴细胞。以不同效靶比将DCs和T淋巴细胞混合培养,MTT法测光密度值,计算刺激指数。DCs抗原负载后,与T淋巴细胞混合,以不同效靶比加入预先接种了Tca8113细胞的培养板。MTT法测光密度值,计算杀伤率。结果2种方法体外成功地构建了DCs疫苗,抗原致敏组与对照组比较差异显著,且弱酸洗脱组优于反复冻融组。DCs疫苗可诱导细胞毒性T淋巴细胞有效的杀伤Tca8113细胞,并表现明显的剂量效应。结论DCs疫苗致敏T淋巴细胞能够有效的杀伤Tca8113细胞,酸洗脱抗原冲击致敏的DCs疫苗在舌癌的免疫治疗中优于冻融抗原。     

Assessment of static and perfusion methods for decellularization of PCL membrane-supported periodontal ligament cell sheet constructs

ObjectivesDecellularization aims to harness the regenerative properties of native extracellular matrix. The objective of this study was to evaluate different methods of decellularization of periodontal ligament cell sheets whilst maintaining their structural and biological integrity.DesignHuman periodontal ligament cell sheets were placed onto melt electrospun polycaprolactone (PCL) membranes that reinforced the cell sheets during the various decellularization protocols. These cell sheet constructs (CSCs) were decellularized under static/perfusion conditions using a) 20 mM ammonium hydroxide (NH4OH)/Triton X-100, 0.5% v/v; and b) sodium dodecyl sulfate (SDS, 0.2% v/v), both +/− DNase besides Freeze–thaw (F/T) cycling method. CSCs were assessed using a collagen quantification assay, immunostaining and scanning electron microscopy. Residual fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were assessed with Bio-plex assays.ResultsDNA removal without DNase was higher under static conditions. However, after DNase treatment, there were no differences between the different decellularization methods with virtually 100% DNA removal. DNA elimination in F/T was less efficient even after DNase treatment. Collagen content was preserved with all techniques, except with SDS treatment. Structural integrity was preserved after NH4OH/Triton X-100 and F/T treatment, while SDS altered the extracellular matrix structure. Growth factor amounts were reduced after decellularization with all methods, with the greatest reduction (to virtually undetectable amounts) following SDS treatment, while NH4OH/Triton X-100 and DNase treatment resulted in approximately 10% retention.ConclusionsThis study showed that treatment with NH4OH/Triton X-100 and DNase solution was the most efficient method for DNA removal and the preservation of extracellular matrix integrity and growth factors retention.     

单纯疱疹病毒1型体外感染人口腔上皮细胞的初步探讨

目的 观察单纯疱疹病毒1型(herpes simplex virus type 1,HSV-1)体外感染人口腔上皮细胞的途径和方式,为研究HSV-1导致牙周病的机制提供依据.方法 在体外扩增获得HSV-1,将其感染人口腔上皮细胞,再转移感染Vero细胞.倒置显微镜及透射电镜下观察.应用聚合酶链反应(PCR)技术对病毒核酸进行检测.结果 倒置显微镜下人口腔上皮细胞未见细胞病变,转移感染后的Vero细胞发生典型细胞病变.透射电镜下在人口腔上皮细胞内未观察到病毒颗粒,在转移感染后的Vero细胞里可见典型的病毒颗粒.HSV-1感染后的人口腔上皮细胞内可检测到病毒核酸.结论 HSV-1可直接感染人口腔上皮细胞,为进一步研究HSV-1导致牙周病的机制提供了较好的体外模型.