骨髓来源成骨细胞复合纳米晶羟基磷灰石胶原材料培养的研究

目的 探讨纳米材料作为组织工程骨基质材料的可行性。方法 分离培养兔骨髓间充质干细胞，诱导为成骨细胞后作为种子细胞，与纳米晶羟基磷灰石胶原材料于体外联合培养，通过对复合物光镜、免疫组织化学染色及扫描电镜观察，了解细胞在材料中生长情况。结果 兔骨髓间充质干细胞可以诱导为成骨细胞，体外复合培养8天，分布于支架材料上的细胞大量分化增殖、分泌细胞外基质。结论 纳米晶羟基磷灰石胶原材料是一种构建组织工程骨的较好的支架材料。     

纳米陶瓷支架的研制及其生物相容性评价

目的制备纳米多孔羟基磷灰石(HA)和磷酸三钙(TCP)支架；研究新型陶瓷支架的生物相容性。方法用Sel-gel法和直接沉淀法分别制备纳米级的HA及TCP粉体，采用有机泡沫浸渍法制备多孔陶瓷支架；采用人骨膜成骨细胞(periosteal—derived osteoblast cells，PPB)与两种陶瓷支架的体外复合培养，观察细胞在材料上的生长及功能表达。结果制备出具有良好孔隙结构和一定强度的纳米多孔陶瓷支架材料；人POB在HA和TCP表面良好的生长，并发挥成骨功能。结论以HA、TCP的纳米级粉体为原料，采用有机泡沫浸渍法可制备出具有良好孔隙结构和较好力学强度的多孔陶瓷支架；新型多孔陶瓷支架有良好的生物相容性。     

仿生矿化PLGA/MWNTs/HA引导组织再生膜的细胞相容性研究

目的 通过仿生矿化的方法制备PLGA/MWNTs/HA牙周引导组织再生膜并对其细胞相容性进行表征.方法 先将聚乳酸-羟基乙酸共聚物[Poly(lactic-co-glycolic acid),PLGA]与多壁碳纳米管(Multi-walled carbon nanotubes,MWNTs)复合,采用溶液浇铸法制备PLGA/MWNTs复合膜;再将其仿生矿化,制得PLGA/MWNTs/羟基磷灰石(Hydroxyapatite,HA)复合膜,再将人牙周膜干细胞接种于PLGA/MWNTs/HA复合膜上培养,采用扫描电子显微镜(Scanning electron microscope,SEM)观察细胞在复合膜上的形貌,四甲基偶氮唑蓝(MTT)法分析细胞的增殖情况.结果 PLGA/MWNTs复合膜表面比较均匀、致密;矿化7天后,PLGA/MWNTs复合膜表面有磷灰石晶体形成;人牙周膜干细胞在PLGA/MWNTs/HA复合膜表面生长、增殖良好,HA的加入有利于细胞的粘附和生长.结论 通过仿生矿化法制得的PLGA/MWNTs/HA复合膜具有良好的细胞相容性,有望在牙周引导组织再生领域发挥作用.     

聚羟基丁酸酯-羟基戊酸酯作为软骨细胞支架的实验研究

目的探讨聚羟基丁酸酯-羟基戊酸酯（PHBV）作为软骨细胞支架的可行性。方法采用“压片-热处理-粒子析出”技术制备PHBV多孔支架并观测其结构。体外分离培养软骨细胞后接种到PHBV支架进行培养，第3、7、14天后取材行扫描电镜观察；培养至2、6、10周后行组织学观察。结果PHBV支架孔径在200-300μm之间孔隙率80％；软骨细胞-PHBV复合物在体外培养期间支架无塌陷及形变，软骨细胞在支架上黏附、增殖良好，并能分泌细胞外基质。随着培养时间延长，新生软骨的组织学特征日益明显。结论PHBV支架具有适宜的孔结构和生物相容性，可以作为软骨组织工程支架材料。     

纳米羟基磷灰石种植体的实验研究

采用冷冻干燥技术制备三种纳米羟基磷灰石支架材料:纳米羟基磷灰石(n-HA)、纳米羟基磷灰石/丝素蛋白(n-HA/SF)、纳米羟基磷灰石/丝素蛋白-壳聚糖(n-HA/SF-CS),并植入实验兔股骨下端缺损处,通过种植体在不同时期的骨结合界面情况的研究,探讨复合体的生物学行为,为建立更为理想的种植体提供理论依据。     

多孔细菌纤维素/聚乳酸-羟基乙酸聚合物/羟基磷灰石复合纤维支架生物相容性研究

目的探讨多孔细菌纤维素/聚乳酸-羟基乙酸聚合物/羟基磷灰石（BC-PLGA-HA）复合支架的生物相容性。方法采用溶液浇铸/粒子沥滤法进行BC-PLGA-HA多孔复合支架的制备,测定支架抗张强度和孔隙率,作细胞MG-63培养为对照组,对支架对MG-63细胞增殖、黏附活性以及对碱性磷酸酶（ALP）活性的影响进行评价。结果多孔BC-PLGA-HAP支架孔隙率及抗拉强度分别为（64.3±5.6）%、（8.925±0.913）N/mm2,BC-PLGA-HAP支架对成骨样细胞具有较高的增殖及ALP活性。结论多孔BC-PLGA-HAP复合支架作为一种新型支架材料,具良好的生物相容性及孔隙率。     

细胞与组织工程血管支架的黏附方法选择

目的 选择观察细胞和组织工程血管支架黏附的最佳方法.方法 用生物可降解材料聚β羟基丁酯(PHB)作为支架材料,运用盐析方法制成实心和不同孔径的膜片;将培养的第3代血管平滑肌细胞种植于膜片上,于培养的第1、3、7、11、14天,采用倒置显微镜下观察、苏木素-伊红(HE)染色、甲苯胺蓝染色、扫描电镜检查及MTT法检测材料上细胞附着和生长情况.结果 倒置显微镜下无法观察附着于材料上的细胞;HE和扫描电镜标本制备过程中细胞丢失多,不能为材料提供准确的信息;甲苯胺蓝染色可快速观察材料上细胞附着情况;MTT法可定量测定材料上的细胞数.经比较发现,150～200μm孔径的PHB膜片上血管平滑肌细胞附着和生长最佳.结论 甲苯胺蓝染色和MTT比色法是观察细胞在材料上黏附和生长的最佳方法.     

三种不同载体材料体外生物相容性的比较

目的探讨纳米晶羟基磷灰石胶原复合材料(NHAC)、非纳米晶羟基磷灰石胶原复合材料(HAC)和珊瑚羟基磷灰石(CHA)的体外生物相容性,为组织工程提供理想的细胞基质载体材料。方法将人骨髓基质干细胞分别与上述三种载体材料体外复合培养,应用倒置显微镜、扫描电镜、四唑盐比色试验(MTT法)及碱性磷酸酶(ALP)活性测定,对材料上复合培养的细胞进行形态学和功能测定。结果骨髓基质干细胞能在NHAC和CHA两种材料上良好地黏附、增殖和生长。细胞的活性和碱性磷酸酶活性未受到材料的影响;而HAC不利于人骨髓基质干细胞的黏附、增殖,并使碱性磷酸酶活性降低。结论NHAC和CHA两种材料具有良好的生物相容性,可作为骨组织工程理想的骨替代材料;HAC不适合做细胞外基质。     

以外消旋聚乳酸为支架体外培养软骨细胞

目的：观察国产外消旋聚乳酸（PDLLA）材料为细胞载体对软骨细胞吸附生长增殖的影响。方法：将生长状态良好的兔软骨细胞接种于PDLLA海绵后行体外培养，通过细胞计数检测材料细胞吸附能力，相关显微镜观察附着情况及细胞形态，共聚焦显微镜观察细胞生长增殖活性，结果：软骨细胞接种后吸附好。继续生长增殖，并具良好活性。结论：国产PDLLA材料可以作为软骨细胞生长的支架载体。     

新型三维复合仿生网络的构建及其组织相容性评价

目的构建并评价新型三维复合仿生网络的组织相容性。方法采用仿生学方法,将壳聚糖、羟基磷灰石、明胶、果胶按一定比例制成新型三维复合仿生网络,将小鼠胚胎成骨细胞MC3T3-E1与材料进行复合培养,通过倒置相差显微镜、石蜡切片常规染色、扫描电镜、F-DA荧光染色法评价细胞相容性;将制备好的生物支架材料植入SD大鼠的背部皮下,术后2、4、8、12周评价组织相容性、血管化能力及体内降解情况。结果新型三维复合仿生网络呈三维多孔状,细胞在材料上贴附生长良好,呈多角形或梭形,形态饱满;皮下包埋实验发现:早期有轻微的炎症反应,随时间延长而消退,后期有血管化发生,材料降解吸收比较缓慢。结论新型三维复合仿生网络组织相容性好,易于血管化,是一种很好的骨组织工程支架材料。     

人牙周韧带细胞在SGBG/PHBV三维支架材料上分泌功能的研究

目的研究人牙周韧带细胞（periodontal ligament cells PDLCs）在溶胶-凝胶生物活性玻璃（sol-gel bioglass,SGBG）/聚羟基烷酸酯（poly-3-hydroxy butyrate-co-3-hydroxy valerate,PHBV）三维支架上的分泌功能,为牙周组织工程支架的选择提供依据。方法通过体外细胞培养实验,人牙周韧带细胞与SGBG/PHBV体外三维培养的直接接触法,测定上清液羟脯氨酸含量,检测材料对细胞分泌功能的影响。结果实验组上清液羟脯氨酸含量测定值与对照组有显著性差异,材料不影响细胞的分泌功能,同时支架材料的三维结构更能促进细胞的分泌功能。结论 SGBG/PHBV作为支架材料,有良好的生物相容性,有望应用于牙周组织缺损的组织工程修复。     

电纺PLGA/HA复合支架的细胞相容性研究

目的通过静电纺丝的方法制备PLGA/HA复合支架并对其细胞相容性进行表征。方法先通过高压静电纺丝的方法制备PLGA/HA复合生物支架,再将大鼠的骨髓间充质干细胞接种于复合支架上培养,采用SEM观察细胞在支架上的形貌、MTT法分析细胞的增殖情况。结果 PLGA/HA复合支架是由超细纤维构成的三维多孔结构,与天然的细胞外基质结构相似,骨髓间充质干细胞在其表面附着、铺展良好,HA纳米颗粒的加入明显提高了细胞的粘附率。结论静电纺丝法制得的PLGA/HA复合支架有可能作为骨组织再生的支架在组织工程领域发挥作用。     

Scaffold: a novel carrier for cell and drug delivery

Scaffolds are implants or injects, which are used to deliver cells, drugs, and genes into the body. Different forms of polymeric scaffolds for cell/drug delivery are available: (1) a typical three-dimensional porous matrix, (2) a nanofibrous matrix, (3) a thermosensitive sol-gel transition hydrogel, and (4) a porous microsphere. A scaffold provides a suitable substrate for cell attachment, cell proliferation, differentiated function, and cell migration. Scaffold matrices can be used to achieve drug delivery with high loading and efficiency to specific sites. Biomaterials used for fabrication of scaffold may be natural polymers such as alginate, proteins, collagens, gelatin, fibrins, and albumin, or synthetic polymers such as polyvinyl alcohol and polyglycolide. Bioceramics such as hydroxyapatites and tricalcium phosphates also are used. Techniques used for fabrication of a scaffold include particulate leaching, freeze-drying, supercritical fluid technology, thermally induced phase separation, rapid prototyping, powder compaction, sol-gel, and melt moulding. These techniques allow the preparation of porous structures with regular porosity. Scaffold are used successfully in various fields of tissue engineering such as bone formation, periodontal regeneration, repair of nasal and auricular malformations, cartilage development, as artificial corneas, as heart valves, in tendon repair ,in ligament replacement, and in tumors. They also are used in joint pain inflammation, diabetes, heart disease, osteochondrogenesis, and wound dressings. Their application of late has extended to delivery of drugs and genetic materials, including plasmid DNA, at a controlled rate over a long period of time. In addition, the incorporation of drugs (i.e., inflammatory inhibitors and/or antibiotics) into scaffolds may be used to prevent infection after surgery and other disease for longer duration. Scaffold also can be used to provide adequate signals (e.g., through the use of adhesion peptides and growth factors) to the cells, to induce and maintain them in their desired differentiation stage, and to maintain their survival and growth. The present review gives a detailed account of the need for the development of scaffolds along with the materials used and techniques adopted to manufacture scaffolds for tissue engineering and for prolonged drug delivery.     

壳聚糖-丝素蛋白-磷酸三钙复合体用于牙周组织工程支架初探

目的构建壳聚糖-丝素蛋白-磷酸三钙多孔复合体,观察其微观结构与体内外降解情况,探讨其作为牙周组织工程软骨支架的可行性。方法采用二次冻干技术制备壳聚糖-丝素蛋白-磷酸三钙复合体,冰冻切片和扫描电镜观察微观形态;将壳聚糖-丝素蛋白-磷酸三钙复合体支架置于人工降解液及植入大鼠背部肌肉,观察体内外降解情况及生物相容性。结果壳聚糖-丝素蛋白-磷酸三钙复合体呈海绵状,孔隙较均匀,孔形态不规则,孔隙之间彼此联通,孔隙率约80%,孔径200μm左右。4周体外降解率60%,在植入早期可见一过性炎性反应,随后逐渐降解,12周时基本降解吸收。结论壳聚糖-丝素蛋白-磷酸三钙多孔复合体具有良好的三维立体结构及可降解性,是一种可行的牙周组织工程支架。     

前脂肪细胞与蚕丝支架体外复合培养的实验研究

目的 观察蚕丝对前脂肪细胞吸附作用及蚕丝对前脂肪细胞形态和功能的影响,为脂肪组织工程支架选择提供依据.方法 从家蚕蚕茧中抽丝所得的原料蚕丝,经胰蛋白酶消化后与原料蚕丝分别制成三维支架,将前脂肪细胞制成细胞悬液接种在支架上,倒置显微镜和扫描电镜观察前脂肪细胞的吸附和生长.结果 在消化后的蚕丝三维支架可看见前脂肪细胞1～2 d后完全贴壁.培养3～7 d细胞生长增殖十分活跃,两周时,蚕丝支架网眼充满前脂肪细胞,扫描电镜见细胞与支架紧密贴附适度伸展并有基质分泌.结论 蚕丝对前脂肪细胞具有良好的吸附作用,并能维持前脂肪细胞正常形态和功能,蚕丝是前脂肪细胞立体培养时的天然支架.     

人牙周膜成纤维细胞的原代培养及鉴定

目的体外分离培养人牙周膜成纤维细胞,进一步研究细胞因子对牙周膜细胞功能的调控作用。方法利用组织块法原代培养牙周膜成纤维细胞并传代,通过形态学及免疫细胞化学方法对其进行鉴定。结果牙周膜成纤维细胞培养成功传代,5代后细胞生长旺盛,表现为长梭形细胞,免疫细胞化学鉴定细胞抗波形丝蛋白阳性,抗角蛋白阴性,为中胚层来源的成纤维细胞。结论组织块法培养出的细胞符合牙周膜成纤维细胞的形态学及免疫学特征,生长状态良好,可作为体外的细胞模型为研究细胞因子对其功能的调控作用奠定基础。     

TGF—β1对人牙周膜成纤维细胞合成胶原的影响

目的：观察不同浓度的转化生长因子β1对体外培养的人牙周膜成纤维细胞合成胶原的影响。方法：采用组织块培养技术进行人牙周膜成纤维细胞的体外培养,采用羟脯氨酸试剂盒来观察细胞合成胶原的情况。结果：0．1、1．0、10．0、50．0ng／ml的转化生成因子β1可以促进牙周膜成纤维细胞合成胶原,其中10ng／ml的TGF—β1在48h促胶原合成作用最明显,差异有统计学意义（P〈0．05）。结论：转化生长因子β1有促进人牙周膜成纤维细胞合成胶原的作用。     

In vitro stem cell cultures from human dental pulp and periodontal ligament: new prospects in dentistry

In spite of the vast knowledge of tooth development and of the various kinds of specialized bone/tooth-associated cells, the characteristics and properties of their precursor cell populations present in the postnatal organism are little known, as is their possible therapeutic use. Taken together dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) possess stem-cell-like qualities, including self-renewal capability and multi-lineage differentiation. Regenerative medicine is based on stem cells, signals and scaffolds. Transplantation of those cells, which can be obtained from an easily accessible tissue resource and expanded in vitro, holds promise as a therapeutic approach for reconstruction of tissues and bone in vivo.     

Morphological and cytofluorimetric analysis of adult mesenchymal stem cells expanded ex vivo from periodontal ligament

Many adult tissues contain a population of stem cells that have the ability of regeneration after trauma, disease or aging. Recently, there has been great interest in mesenchymal stem cells and their roles in maintaining the physiological structure of tissues, and their studies have been considered very important and intriguing, after having shown that this cell population can be expanded ex vivo to regenerate tissues not only of the mesenchymal lineage, such as intervertebral disc cartilage, bone, tooth-associated tissue, cardiomyocytes, but also to differentiate into cells derived from other embryonic layers, including neurons. Currently, different efforts have been focused on the identification of odontogenic progenitors from oral tissues. In this study we isolated and characterized a population of homogeneous human mesenchymal stem cells proliferating in culture with an attached well-spread morphology derived from periodontal ligament, a tissue of ectomesenchymal origin, with the ability to form a specialized joint between alveolar bone and tooth. The adherent cells were harvested and expanded ex vivo under specific conditions and analysed by FACScan flow cytometer and morphological analysis was carried out by light, scanning and transmission electron microscopy. Our results displayed highly evident cells with a fibroblast-like morphology and a secretory apparatus, probably indicating that the enhanced function of the secretory apparatus of the mesenchymal stem cells may be associated with the secretion of molecules that are required to survive and proliferate. Moreover, the presence in periodontal ligament of CD90, CD29, CD44,CD166, CD 105, CD13 positive cells, antigens that are also identified as stromal precursors of the bone marrow, indicate that the periodontal ligament may turn out to be a new efficient source of the cells with intrinsic capacity to self-renewal, high ability to proliferate and differentiate, that can be utilized for a new approach to regenerative medicine and tissue engineering.