软骨细胞三维培养扩增与液态凝胶复合构建软骨的实验研究

目的通过在微载体上进行三维培养扩增软骨细胞,并结合液态胶原构建组织工程软骨。方法比较兔软骨细胞在单层培养与微载体上进行三维培养扩增软骨细胞的保持表型能力。幼兔软骨细胞分别进行单层和微载体三维培养扩增,并进行体外球型培养评价软骨细胞保持表型能力和糖胺多糖的定量生化分析。三维培养扩增软骨细胞与液态鼠尾胶原复合构建组织工程软骨,分别以低细胞密度（2×10^6个／mL）和高细胞密度（1．2×10^7个／mL）两种细胞密度接种,培养14d后通过组织学特种染色鉴定构建组织特性。结果微载体培养的软骨细胞可以保持良好活力和保持表型能力,与单层培养体系相比较,细胞糖胺多糖的定量生化分析的差异具有统计学意义（P〈0．05）。三维培养扩增软骨细胞复合液态鼠尾胶原构建组织工程软骨,体外14d后发现高细胞密度接种时可形成形态稳定的软骨组织。组织学染色显示为透明软骨样组织。结论在微载体上进行三维培养扩增软骨细胞可以加强细胞保持表型能力。软骨细胞与液态胶原合成后,以高细胞密度（1．2×10^7个／mL）可以在体外形成形态稳定的组织工程软骨。     

改良猪耳郭软骨细胞体外培养的实验研究

目的：为组织工程化软骨修复机体软骨缺损提供实验基础。方法：用消化组织块培养法体外培养猪耳郭软骨细胞,并观察其在Polyglycolicacid（简称PGA）支架上的生长情况。结果：软骨细胞在pH值为7.0,含10%胎牛血清的DMEM培养液中生长良好,可传代10代,细胞数目扩增为原来的400～500倍;软骨细胞在PGA支架上生长良好,分泌基质旺盛。结论：改良消化组织块方法培养软骨细胞是一种可以大量扩增软骨细胞数目的确实可行的方法,PGA是软骨细胞良好的生长支架。     

胶原-透明质酸支架的制备及其与软骨细胞复合培养的实验研究

目的制备胶原-透明质酸支架,评价其与兔髁状突软骨细胞的生物相容性,探讨其应用于关节软骨组织工程的可行性。方法冷冻干燥法制备胶原-透明质酸复合多孔海绵支架材料,将其与碳化二亚胺[1-ethyl-3-（3-dimethyl aminopropyl）carbodiimide,EDC]进行化学交联。分别采用体积法和体外酶解实验测定支架材料孔隙率和降解率,扫描电镜观察交联前后支架材料的形态变化。取3周龄新西兰兔髁状突软骨细胞,体外培养5d后,甲苯胺蓝染色,倒置相差显微镜下观察行细胞鉴定。取消化后第2代软骨细胞接种至支架材料复合培养,倒置相差显微镜下观察细胞生长情况。复合培养1、3、5、7和10d后,PBS液清洗3次,置入24孔板并以0.25%胰酶和0.1%EDTA消化细胞,采集细胞进行细胞计数并绘制细胞生长曲线。另取部分样本继续培养5d,行组织学和扫描电镜观察。结果胶原-透明质酸支架材料经化学交联后,具有合适的三维多孔结构,孔隙率为83.7%,孔径100-120μm;交联后抗酶解能力显著增加。髁状突软骨细胞在其表面和内部贴附良好,形成细胞-材料复合体,细胞增殖实验显示,复合培养1d,材料中细胞数为3.7×104/支架,10d后增至8.2×104/支架。电镜观察见细胞周围有基质分泌。结论EDC交联后的胶原-透明质酸支架具有良好空间结构和生物相容性,可作为支架材料用于髁状突软骨组织工程的研究。     

几丁糖作为软骨细胞培养支架的实验研究

目的：探讨几丁糖作为组织工程技术中软骨细胞培养支架的可行性。方法：采用几丁糖无纺网作为细胞培养支架,使用前首先用10%多聚赖氨酸包埋以增加其对软骨细胞的吸附性,然后把几丁糖支架与软骨细胞一起进行体外培养,在倒置显微镜下观察几丁糖对软骨细胞生长的影响。结果：包埋后的几丁糖支架与软骨细胞一起进行体外培养,在倒置显微镜下观察几丁糖对软骨细胞生长的影响。结果：包埋后的几丁糖支架与软骨细胞一起体外培养10d,软骨细胞数目增加约6倍,并且与几丁糖支架牢固结合不易丢失。结论：几个糖无纺网经10%多聚赖氨酸包埋后,对软骨细胞吸附性增强,并且几丁糖具有良好的生物学特性,因此几丁糖符合组织工程对细胞培养支架的要求。有可能成为组织工程中一种良好的细胞培养支架。     

不同培养条件下脂肪干细胞与软骨细胞共培养的研究

探讨病理状态下软骨细胞能否诱导脂肪干细胞(ADSC)向软骨细胞分化以及可能的最佳条件,以便为临床修复关节软骨损伤提供可能的新途径.[方法]分离共培养新西兰大白兔骨关节炎模型的ADSC和软骨细胞,根据不同血清浓度(10% FBS和2% FBS)和不同培养空间(纤维蛋白凝胶支架和无支架单层培养)分组,共培养14 d后,胰酶消化终止.倒置显微镜观察和透射电镜观察共培养后ADSC的形态变化.甲苯胺蓝染色法和免疫细胞化学检测共培养后ADSC的蛋白多糖和Ⅱ型胶原的表达水平.RT-PCR检测蛋白多糖和Ⅱ型胶原的基因转录水平.[结果]共培养7 d后部分ADSC变圆,14d时ADSC形态高度分化与成熟软骨细胞相似,其蛋白多糖和Ⅱ型胶原的基因转录和蛋白表达均增高,尤以10%FBS支架共培养组最为明显.[结论]与病理状态下软骨细胞共培养后,ADSC可以被诱导成软骨样细胞.高浓度血清三维培养可以增强这种诱导作用.     

模拟微重力培养肝细胞的形态特点

目的　模拟微重力方法培养大鼠原代肝细胞 ,初步分析其形态学特点及其意义。方法　改良Seglen原位胶原酶灌注法获得大鼠肝脏单细胞悬液 ,2 .2× 10 5个 /ml加微载体Cytodex 3(4 g/L)接种 ,采用旋转细胞培养系统 (RCCS)进行模拟微重力培养。第 0、6、2 4、72、12 0、168小时取样 ,相差、体视显微镜观察活细胞形态 ,第 2 4小时标本苏木素 伊红 (HE)染色观察组织学形态 ,电镜观察超微结构。结果　模拟微重力培养中肝细胞 2 4h内贴附微载体并出现三维结构 ,2 4～ 72h发展为独特的肝细胞 微载体聚球体。电镜下可见细胞膜的 3种不同形态 ,其分布与功能相一致。结论　模拟微重力培养方法能使肝细胞形成分化的三维类组织结构 ,在组织工程领域存在良好的应用前景     

体外培养软骨细胞凋亡与caspase-3表达的实验研究

目的探讨软骨细胞体外培养过程中凋亡发生以及caspase-3的表达. 方法采用Annexin Ⅴ和TUNEL法检测第1～4代软骨细胞在体外正常培养体系中第3天和第7天的凋亡率;RT-PCR和Western blot分析caspase-3表达水平;ELISA检测caspase-3蛋白酶活性. 结果第1～4代软骨细胞在体外培养过程中均存在不同程度的凋亡现象,各代次软骨细胞第7天的凋亡率15.7%±0.3%,明显高于第3天的8.9%±0.6%,有统计学意义(P＜0.01).caspase-3 mRNA和蛋白质在整个培养过程中都有表达,随着时间延长表达上调,第7天的表达水平高于第3天.体外培养7天后caspase-3被激活,可检测到分子量为20 ku的裂解片段.caspase-3蛋白酶活性随着培养时间延长也逐渐增高,与细胞凋亡程度基本一致. 结论软骨细胞在体外培养过程中存在凋亡,caspase-3参与了凋亡事件并发挥着重要作用.     

应力状态下软骨细胞研究进展

在应力状态下,软骨细胞的基本力学性能、代谢、病理、组织构建将发生一系列变化。该文介绍了近年来应力状态下软骨细胞的研究进展。     

微重力环境下体外构建组织工程化椎间盘

[目的]利用微重力培养环境,构建高质量组织工程化椎间盘。[方法]选用成年新西兰大白兔椎间盘细胞作为种子细胞,复合聚乳酸-羟基乙酸(PLGA)支架,分别在微重力培养环境和普通培养板环境下培养,利用倒置显微镜、MTT比色法、扫描电镜、组织学观察椎间盘细胞、支架和复合结构质量。[结果]体外单层培养的椎间盘细胞呈多角形符合传代要求,微重力环境比普通培养环境能更好地促进椎间盘细胞的增殖,同时椎间盘细胞在支架内分布更加均匀。[结论]微重力培养环境更适于椎间盘细胞在三维培养结构中均匀增殖,有利于构建高质量组织工程化椎间盘用于深入研究。     

脱细胞软骨细胞外基质取向支架复合软骨细胞构建组织工程软骨的实验研究北大核心CSCD

目的探讨脱细胞软骨细胞外基质(acellular cartilage extracellular matrix,ACECM)取向支架复合软骨细胞构建组织工程软骨的可行性。方法取市售猪关节软骨组织,分离培养关节软骨细胞并传代。取第3代软骨细胞行PKH26荧光标记,MTT检测标记对细胞增殖无影响后,分别取标记及未标记的软骨细胞复合ACECM取向支架并体外培养后,大体观察支架形态,倒置显微镜、荧光显微镜观察软骨细胞在支架中的黏附、生长和分布情况,扫描电镜观察支架中细胞形态,Ⅱ型胶原免疫荧光染色观察软骨细胞外基质分泌情况。将PKH26标记的软骨细胞-支架复合物植入裸鼠背部皮下腔隙,术后观察裸鼠一般情况,4周后分子荧光活体成像系统无创伤性评估细胞-支架复合物生长情况,取材行大体观察以及番红O、甲苯胺蓝、Ⅱ型胶原免疫组织化学染色观察,评价形成软骨组织的能力。结果细胞-支架复合物体外培养7 d,大体观察呈半透明并具有一定硬度;倒置显微镜和荧光显微镜观察软骨细胞在支架上能良好黏附生长,并沿支架管道方向生长,分泌Ⅱ型胶原。细胞-支架复合物植入裸鼠皮下后,分子荧光活体成像系统观察示细胞均存活;术后4周,大体观察见复合物呈类软骨样组织,组织学染色及Ⅱ型胶原免疫组织化学染色示细胞周围软骨细胞外基质分泌,可见"陷窝"样结构形成。结论 ACECM取向支架有利于软骨细胞的黏附、增殖及取向性分布类似于正常软骨结构,并在裸鼠皮下成功异位构建组织工程软骨。     

Effects of a chitosan scaffold containing TGF-beta1 encapsulated chitosan microspheres on in vitro chondrocyte culture

The objectives of this study were (1) to develop a three-dimensional chitosan scaffold in combination with transforming growth factor-beta1 (TGF-beta1)-loaded chitosan microspheres and (2) to evaluate the effect of the TGF-beta1 release on the chondrogenic potential of rabbit chondrocytes in the scaffolds. TGF-beta1 was loaded into chitosan microspheres using an emulsion-crosslinking method, resulting in spherical shapes with a size ranging from 0.3 to 1.5 microm. Controlled release of TGF-beta1, as measured by enzyme-linked immunosorbent assay (ELISA), was observed with chitosan microspheres over 7 days. Chitosan solutions (2% and 3%) were fabricated into two types of scaffolds with different pore morphologies and mechanical properties using a freeze-drying technique, with the result that scaffold with higher concentrations showed smaller pores and lower porosity, leading to a much stronger scaffold. The TGF-beta1 microspheres were incorporated into the scaffolds at a concentration of 10 ng TGF-beta1/scaffold and then chondrocytes seeded into each scaffold and incubated in vitro for 2 weeks. The 2% chitosan scaffolds showed higher cell attachment levels than the 3% chitosan scaffolds (P < 0.01), regardless of the TGF-beta1 microspheres. Both the proliferation rate and glycosaminoglycan (GAG) production were significantly higher for scaffolds incorporating TGF-beta1 microspheres than for the control scaffolds without microspheres 10 days after incubation. Extracellular matrix staining by Safranin O and immunohistochemistry for type II collagen both significantly increased in scaffolds containing TGF-beta1 microspheres. These results suggest that the TGF-beta1 microsphere incorporated in scaffolds have the potential to enhance cartilage formation.     

白细胞介素1β对组织工程化椎间盘细胞基质金属蛋白酶13表达的作用

[目的]观察不同浓度白细胞介素1β对组织工程化椎间盘细胞基质金属蛋白酶13表达的影响.[方法]将体外培养的兔组织工程化椎间盘细胞随机分为1、10、100 μg/L浓度白细胞介素1β作用组及空白对照组,免疫组织化学检测基质金属蛋白酶13表达.[结果]1、10、100 μg/L浓度白细胞介素1β组基质金属蛋白酶13阳性表达率均较对照组增高(P＜0.05).随着白细胞介素1 β浓度的增高,基质金属蛋白酶13阳性表达率显著升高,100 μg/L浓度高于1、10 μg/L浓度组(P＜0.05),10 μg/L浓度组高于1.μg/L浓度组(P＜0.05).[结论]白细胞介素1β对兔组织工程化椎间盘细胞基质金属蛋白酶13表达的调控作用随其浓度的升高而呈正相关.     

微重力培养提高冻存大鼠胰岛移植的效果

目的观察大鼠胰岛冻存后经三维微重力细胞旋转培养系统(RCCS)培养后移植给受者能否明显提高胰岛移植的效果。方法将分离纯化的大鼠胰岛分为3组。实验1组:即将胰岛在RCCS中培养3d后,悬浮于4℃低温保存液(HTS)中,放置30min,然后进行冻存步骤;冻存1个月后进行复苏,继续在RCCS中培养30d;实验2组:新鲜大鼠胰岛在RCCS中培养30d;对照组:大鼠胰岛在冻存前后均在普通培养基中培养,培养步骤和时间同实验1组。上述3组胰岛复苏后再按各组应用的培养体系培养7d,然后分别以2000IEQ冻存或新鲜胰岛移植入糖尿病大鼠体内,并观察10周。结果每个胰腺纯化后最多收获胰岛1058.47IEQ,最少411.88IEQ,平均(826.95±93.42)IEQ。实验1组、实验2组的胰岛收获率、细胞内胰岛素和DNA含量以及胰岛素刺激指数均高于对照组(P<0.05);实验2组和实验1组的2000IEQ新鲜胰岛或冻存胰岛在移植后1周内可100%纠正糖尿病。结论大鼠胰岛冻存前后经微重力培养后移植可以明显提高1型糖尿病大鼠的治疗效果。     

A novel bioartificial liver with culture of porcine hepatocyte aggregates under simulated microgravity

An extracorporeal bioartificial liver device could provide vital support to patients suffering from acute liver failure. We designed a novel, customized bioreactor for use as a bioartificial liver (patent pending). The Innsbruck Bioartificial Liver (IBAL) contains aggregates of porcine hepatocytes grown under simulated microgravity. The culture vessel rotates around its longitudinal axis and is perfused by two independent circuits. The circuit responsible for exchange of plasma components with the patient consists of a dialysis tube winding spirally around the internal wall of the culture vessel. IBAL was evaluated in vitro. Viability tests showed sufficient viability of hepatocytes for up to 10 days. Cytologic examination of samples from the bioreactor showed liver cell aggregates. These were also examined by electron microscopy. A number of biochemical parameters were analyzed. In conclusion, cell culture is possible for at least 10 days in the IBAL system, organoid hepatocyte aggregates are formed and synthetic activity of the hepatocytes was demonstrated.     

软骨细胞体外培养负性相关因素研究进展

软骨细胞体外培养受到众多复杂因素的影响.白介素-1(IL-1)和肿瘤坏死因子-α(TNF-α),可通过核因子-κB等信号途径干扰软骨细胞的正常代谢,两者具有协同作用;IL-6、IL-17、IL-18经相应途径上调炎症和软骨破坏相关基因,基质金属蛋白酶及其抑制剂的平衡失调是引起软骨退变的重要原因;含钙结晶体能通过两个独立的途径引起关节组织退变;过度的应力刺激、低氧、高渗溶液可不同程度地影响软骨细胞生长;各种因素引起的内质网过度紧张可导致软骨细胞调亡,软骨外植和切碎过程能引起IL-1表达升高.     

Arthroscopic implantation of a three dimensional scaffold for autologous chondrocyte transplantation

The arthroscopic M-ACT technique is applicable for defects at the femoral condyle up to 5 cm2. The size of the defect has to be assessed with a specific scaled, percutaneously inserted needle. Then an 8 mm water-stop-cannula is positioned in a suprameniscal portal. The chondrocyte seeded matrix is trimmed to size the defect. The scaffold is introduced in the joint through the cannula and placed into the defect with a blunt arthroscopic grasp instrument to prevent damage of the scaffold. Then a specific drill guide is inserted through an additional anteromedial portal to place it on the scaffold in a perpendicular angle. The position of the drill guide should not be changed during the next two steps. It may be helpful to hold the matrix in place with a probe inserted through the cannula. A 1.5 mm K-wire is drilled at least 16 mm into the subchondral bone. Then the biodegradable pin (length 16 mm) is placed in the drill guide and carefully hammered into the subchondral bone. The joint is flexed so that the drill guide can be placed on the posterior end of the scaffold. Another hole is drilled with the K-wire and a second pin is inserted. Finally the stability of the matrix is tested with a probe and the joint is mobilized.     

模拟微重力培养大鼠胰岛的形态学观察

目的应用电子显微镜观察模拟微重力培养对大鼠胰岛形态的影响。方法将新鲜分离和消化的大鼠胰岛分别进行普通培养和模拟微重力条件培养 ,应用扫描电镜和透射电镜观察各组大鼠胰岛的大体结构及超微结构的变化 ,并与新鲜分离的胰岛对照。结果培养 7d时透射电镜下观察微重力组的胰岛形态类似新鲜组的胰岛 ,胞浆内有发育良好的分泌颗粒和丰富的线粒体 ;扫描电镜显示只有微重力组胰岛与胰岛之间形成了许多小洞。结论模拟微重力培养时 ,胰岛的形态保持良好。     

Influences of buffer systems on chondrocyte growth during long-term culture in alginate

OBJECTIVE: Chondrocyte behavior is very sensitive to culture environment such as physical and biochemical conditions. As extracellular pH (pHo) and the existence of bicarbonate could affect the chondrocyte fate, hence, the purpose of this study is to investigate the buffer system effect on chondrocyte fate during relatively long-term culture. METHODS: In order to examine whether effects seen were due to bicarbonate or to pHo, we had to devise a system which could differentiate between the two effects. Culture media buffered by N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) only and the combination of HEPES and bicarbonate were used. Bovine articular chondrocytes were cultured in alginate beads for up to 12 days. pHo was kept constant by culture of 3 beads in 2 ml culture medium. Cell density, intracellular pH (pHi) and glycosaminoglycan (GAG) were measured at day 5 and day 12. Cell morphology, distribution and viability in alginate beads were monitored over 12 days of culture. RESULTS: Compared to culture in the absence of bicarbonate, a higher proliferation rate of chondrocytes was observed in the presence of bicarbonate. pHi was more alkaline, about 0.2 pH unit, in the presence of bicarbonate than that in the absence of bicarbonate. About 50% more GAG was deposited in alginate beads when chondrocytes were cultured in the combination of HEPES and bicarbonate, compared to chondrocytes cultured in the absence of NaHCO3 at the end of 12 days of culture. CONCLUSION: The presence of bicarbonate results in more alkaline in the pHi of bovine chondrocytes after long-term culture. The combination of bicarbonate and HEPES in culture medium improves cell growth, matrix production in three-dimensional alginate beads.     

Rapid and large-scale formation of chondrocyte aggregates by rotational culture

Chondrocytes in articular cartilage synthesize collagen type II and large sulfated proteoglycans, whereas the same cells cultured in monolayer (2D) dedifferentiate into fibroblastic cells and express collagen type I and small proteoglycans. On the other hand, a pellet culture system was developed as a method for preventing the phenotypic modulation of chondrocytes and promoting the redifferentiation of dedifferentiated ones. Because the pellet culture system forms only one cell aggregate each tube by a centrifugator, the pellet could not be applied to produce a tissue-engineered cartilage. Therefore, we tried to form chondrocyte aggregates by a rotational culture, expecting to form a large number of aggregates at once. In order to increase cell-cell interactions and decrease chondrocyte-material interaction, dishes with low retention of protein adsorption and cell adhesiveness were used. In addition, rotational shaking of the dish including cells was attempted to increase the cell-cell interaction. The shaking speed was set at 80 rpm, so the cells would be distributed in the center of the dish to augment the frequency of cell-cell contact. Under these conditions, bovine articular chondrocytes started aggregating in a few hours. At 24-36 h of rotational culture, aggregates with smooth surfaces were observed. Parameters such as increase of culture time and addition of TGF-beta controlled diameters of the aggregates. There were many fusiform cells at the periphery of the aggregates, where the cells tended to form a multilayered zone in cross sections. In addition, lacune-like structure, which was almost the same as pellet culture, was observed. It was found that the internal structure of the aggregates was similar to that of pellets reported previously. Therefore, the aggregates formed by a rotational culture could become an essential component to make tissue-engineered artificial cartilage.