山羊颞下颌关节盘细胞透射电镜观察

目的 用透射电镜的方法对两只1月龄健康山羊四只颞下颌关节盘的细胞及胶原结构进行了观察.方法 切取山羊双侧颞下颌关节盘,经组织学处理后行透射电镜观察细胞和胶原超微结构特征.结果 颞下颌关节盘由不均分布的细胞和胶原纤维组成,细胞有成纤维细胞样细胞和软骨细胞样细胞两种,其中成纤维细胞样细胞较软骨细胞样细胞占优势.成纤维细胞样细胞表现有细长的突起,核大,梭形或不规则形,细胞器较少.软骨细胞样细胞胞核呈圆形或椭圆形,周围少有电子透射区,细胞膜不明显,胞突少见.胶原纤维由平行排列的有周期性横纹特征的胶原原纤维组成.结论 颞下颌关节盘细胞有成纤维细胞样和软骨细胞样细胞两种,前者占优势,胶原原纤维表现有周期性横纹特征.这为颞下颌关节盘组织工程研究中细胞来源及表型分析奠定了一定基础.     

滑液干细胞对关节盘软骨细胞的作用研究

目的:初步探讨人颞下颌关节滑液来源的间充质干细胞条件培养基对大鼠关节盘软骨细胞的作用。方法:临床收取颞下颌关节紊乱病患者的滑液干细胞进行细胞培养,三向分化验证其细胞干性。激光共聚焦显微镜下观察获取的大鼠关节盘软骨细胞Ⅰ型胶原、Ⅱ型胶原免疫荧光表达,确认细胞属性。收集滑液干细胞培养24h的上清液制作条件培养基,设置空白对照组分别培养大鼠关节盘软骨细胞,CCK-8检测大鼠关节盘软骨细胞增殖情况,实时荧光定量PCR(RT-PCR)检测培养7天、14天大鼠关节盘软骨细胞相关基因Ⅰ、Ⅱ、Ⅹ型胶原表达变化。结果:获取的滑液干细胞具有成脂、成骨、成软骨三向分化潜能;大鼠关节盘软骨细胞表达Ⅰ型胶原、Ⅱ型胶原;CCK-8结果显示条件培养基组48h后细胞增殖速度较对照组明显加快(P<0.05);第7天、14天实时荧光定量PCR显示条件培养基组Ⅹ型胶原表达明显低于对照组。结论:人颞下颌关节滑液来源的间充质干细胞条件培养基促进大鼠关节盘软骨细胞增殖,抑制关节盘软骨细胞Ⅹ型胶原表达。     

自组装山羊颞下颌关节盘组织工程纤维软骨模型的构建

目的 构建山羊颞下颌关节盘纤维软骨自组装模型,观察自组装组织工程纤维软骨的生物学特征,为颞下颌关节盘及其他组织工程纤维软骨的进一步研究创造条件.方法 分离、培养山羊颞下颌关节盘细胞,按每井5.5×106个接种到预制的直径5 mm×深10 mm琼脂糖井内,每天换液,培养2周,观察和检测颞下颌关节盘形态和成分的变化.结果 ...     

经颧弓后上牵引下颌后关节盘后区适应性改建的实验研究

目的:观察经颧弓后上牵引兔下颌后颞下颌关节盘后区是否发生适应性改建及其意义.方法:在兔下颌角和颧弓处打孔后放置牵引装置,向后上方牵引下颌,于术后2、4、6 周分别处死动物,采用组织学及免疫组化方法观察负荷下关节盘后区组织的改变.结果:术后2 周兔牵引侧关节盘移位或变形,盘后区胶原密度增加;随着负荷时间的延长,关节后区成纤维细胞密度明显下降,关节盘后区结缔组织变得致密,组织内出现少量软骨样细胞出现,而对照组标本未见明显组织学改变.与对照组相比,术后2 周移位的关节盘功能区软骨基质、蛋白聚糖及Ⅱ型胶原呈弱表达,阿辛蓝染色变淡;移位至负重区的关节盘后区软骨基质、蛋白聚糖表达比对照组有所增加.随着关节负荷时间的延长,移位至负重区的关节盘后区软骨基质、蛋白聚糖及Ⅱ型胶原逐渐增强.结论:经颧弓后上牵引下颌后,关节盘后区组织内软骨基质合成增加,并有少量软骨细胞出现,发生改建以适应关节功能.     

人胚颞颌关节软骨细胞培养及生物学特性研究

采用机械分离及胰蛋白酶和Ⅱ型胶原酶联合消化的方法，简便、快速地获得了大量成活率高的人ＴＭＪ软骨细胞。在ＤＭＥＭ培养基中进行原代和传代培养，并对原代培养软骨细胞进行了组化及免疫组化鉴定，相差显微镜、光镜及超微结构的观察。结果显示，光镜观察，原代培养细胞呈多角形，单层排列，电镜可见细胞内有丰富的粗面内质网及线粒体，细胞呈多极性，表面有突起。ＴＭＪ软骨细胞免疫组化Ⅱ型胶原染色阳性。甲苯胺蓝染色细胞质内有     

不同方法诱导的骨髓基质细胞用于修复山羊髁突软骨缺失的实验研究

目的:比较用诱导因子诱导及用部分成体软骨细胞诱导的骨髓基质细胞(BMSCs)复合生物材料修复山羊颞下颌关节髁突软骨全层缺失的效果.方法:取山羊6只,分为2个实验组及1个对照组,每组2只.实验Ⅰ组植入经诱导因子(TGF-131、IGF-1、地塞米松)诱导的BMSCs与支架(PIuronic F-127溶胶)复合物,实验Ⅱ组植入软骨细胞-BMSCs(按3:7比例混合)的细胞-支架复合物,对照组仅植入支架材料.于术后8周取材,进行颞下颌关节软骨面缺失修复的大体观察、修复组织的HE染色、Ⅱ型胶原分泌的免疫组化染色.结果:术后8周实验Ⅱ组髁突软骨缺失区由软骨样组织修复.HE染色及免疫组化染色结果均提示修复组织为成熟的软骨组织.而实验Ⅰ组及材料对照组缺损区仅由少量的纤维性组织修复,不能形成成熟的软骨组织.结论:骨髓基质细胞在自体软骨细胞基质的诱导下,可以修复山羊髁突软骨面全层缺失,二维培养诱导后的BMSCs在山羊髁突软骨缺失区难以达到修复作用.     

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。     

颞下颌关节盘前移位后双板区组织软骨化生机制的实验研究

目的:探讨颞下颌关节盘前移位后双板区内化生软骨细胞的来源。方法:将12只日本成年大耳白兔随机分为A、B两组,每组包括4只实验兔和2只对照兔,实验兔的右侧关节盘被手术前移并固定在前方的颧弓上,A组用于HE染色和抗增殖细胞核抗原(PCNA)、抗成纤维细胞生长因子受体3(FGFR3)免疫组化检测,B组用于透射电镜观察。结果:所有实验组动物的关节盘双板区内均出现了软骨细胞化生,部分化生软骨细胞内PCNA和FGFR3均为阳性。超微结构观察可见:部分细胞具有成纤维细胞和软骨细胞的双重特征,部分软骨细胞具有幼稚细胞的特征。结论:颞下颌关节盘前移位后,双板区内化生的软骨细胞可能部分由间充质干细胞增殖分化而来。     

壳聚糖聚电解质复合物再生纤维软骨的实验研究

目的:探讨壳聚糖电介质复合物与髁突软骨细胞体内形成纤维软骨情况,以期为工程化软骨再生修复颞下颌关节软骨缺损奠定基础。方法:合成磷酸化壳聚糖电解质复合物(chitosan-polyelectrolyte complex,CS-PEC)制备三维凝胶支架,并与髁突软骨细胞复合培养后植入裸鼠体内,分别于4、8周取出新生物,通过组织学和免疫组化观察新生物的生物学特性。结果:软骨细胞在CS-PEC支架中形态铺展良好。植入体内4周后,支架保持原有结构,可见肥大样软骨细胞出现,免疫组化染色显示中央新软骨形成区Ⅱ胶原为阳性。8周后,支架降解,见新生软骨形成,阿新兰染色显示软骨基质形成,免疫组化染色显示软骨形成区Ⅱ胶原为阳性,周围纤维样细胞中Ⅰ胶原阳性;对照组无新生软骨形成,软骨特异性标志Ⅱ胶原及软骨基质特染均为阴性。结论:CS-PEC复合材料有望成为软骨再生的支架载体。     

耻骨联合软骨细胞的体外培养及形态学观察

目的:分离获取大鼠耻骨联合软骨细胞,体外培养并进行形态学观察。方法;采用机械及胰蛋白酶、胶原酶序贯消化法,从大鼠耻骨联合软骨组织中分离出软骨细胞,用含15?S的DMEM/F12培养液原代和传代培养,倒置显微镜下动态观察细胞形态及生长情况,阿尔辛蓝组织化学、Ⅱ型胶原免疫组化染色鉴定,通过扫描及透射电镜观察培养细胞的超微结构。结果:体外培养的大鼠耻骨联合软骨细胞呈短梭形、三角形,有短细胞突起,细胞浆内质网发达,线粒体丰富,富含分泌颗粒。细胞胞浆糖胺聚糖、Ⅱ型胶原染色均阳性。结论:耻骨联合细胞体外培养过程中,较好地保持了生长期软骨细胞的形态特征。     

Substance P and mast cells: preliminary histologic analysis of the human temporomandibular joint

PURPOSE: Neuropeptide-containing nerves can serve as a mechanism for nervous system regulation of host defense responses. Because bacteria associated with reactive arthritis have been identified in the temporomandibular joint (TMJ), this study investigates whether the presence of substance P (SP) neuropeptide-containing nerves and mast cells can be identified in the TMJ. MATERIAL AND METHODS: Posterior bilaminar tissue removed during TMJ surgery from 9 women was evaluated for the presence of neuropeptide-containing nerves by staining with a monoclonal antibody to SP. Staining of the TMJ tissue sections with 0.5% toluidine blue was performed to identify the presence of mast cells. RESULTS: SP-containing nerves and mast cells were identified within the posterior bilaminar tissue associated with the vasculature. CONCLUSIONS: The presence of neuropeptide nerves and mast cells within the TMJ has been shown. Mast cell degranulation products and SP release can contribute to TMJ inflammation.     

Cell type and distribution in the porcine temporomandibular joint disc.

PURPOSE: Surprisingly little is known about the cellular composition of the temporomandibular joint (TMJ) disc, which is a crucial piece of the puzzle in tissue engineering efforts. Toward this end, cell types were identified and quantified regionally in the TMJ disc. MATERIALS AND METHODS: Porcine TMJ discs were examined by histology, electron microscopy, and immunohistochemistry. Histology consisted of hematoxylin and eosin staining to identify regional variation of cell type and cell numbers. Transmission electron microscopy was used to elucidate differences in organelle content and pericellular matrix between TMJ disc cells and chondrocytes from hyaline cartilage. Immunohistochemistry was used to assess the presence of smooth and skeletal muscle character in the TMJ disc. RESULTS: The overall ratio of fibroblasts to chondrocyte-like cells in the TMJ disc was approximately 2.35 to 1, with the highest relative number of chondrocyte-like cells in the intermediate zone. Electron microscopy revealed distinct differences between TMJ disc chondrocyte-like cells and chondrocytes from hyaline cartilage with respect to organelles and the pericellular region. Immunostaining identified smooth muscle in the form of vessels, which were most prominent in the anterior band. Skeletal muscle was not observed. CONCLUSION: The cells of the TMJ disc are distinctly different from cells of hyaline cartilage, and consequently should not be referred to as chondrocytes. TMJ disc cells are comprised of heterogeneously distributed subpopulations, with fibroblasts predominating over fibrochondrocytes.     

不同体积分数氧气对山羊颞下颌关节盘细胞骨架改建的影响

目的 探讨不同体积分数的氧气对山羊颞下颌关节盘细胞3种细胞骨架蛋白改建的影响。方法 体外分离、培养山羊颞下颌关节盘细胞,传代至第2代,分别于常氧21%O₂及低氧8%O₂、4%O₂、2%O₂下培养。甲苯胺蓝、天狼星红及Ⅰ型胶原免疫细胞化学染色观察不同氧体积分数下细胞显型的变化。细胞免疫荧光染色和实时定量逆转录聚合酶链反应检测细胞骨架蛋白,包括肌动蛋白、微管蛋白和波形蛋白的表达情况。结果 不同氧体积分数下关节盘细胞仍然具有成纤维特性,3种细胞骨架蛋白有规律地排列。4%O₂时,肌动蛋白和波形蛋白荧光强度最低（P<0.05）;2%O₂时,微管蛋白荧光强度最高（P<0.05）,其他各组间差异无统计学意义（P>0.05）。检测肌动蛋白mRNA表达,21%O₂组最高,而2%O₂组和4%O₂组最低（P<0.05）;微管蛋白mRNA表达在2%O₂组最高,8%O₂组最低（P<0.05）;波形蛋白mRNA表达,在4%O₂组最低,21%O₂组最高,差异有统计学意义（P<0.05）。结论 在不同氧体积分数条件下,细胞骨架蛋白有不同程度的改建,2%O₂可能是适合颞下颌关节盘细胞扩增的最佳氧体积分数。     

细胞增殖与程序性细胞死亡在颞下颌关节发育中的意义

目的：探讨细胞增殖与程序性细胞死亡在颞下颌关节发育中的作用。方法：利用免疫组织化学技术及原位末端标记法,对胚胎及生后一周ＳＤ大鼠颞下颌关节不同发育时期髁状突软骨中增殖细胞核抗原（ＰＣＮＡ）的表达及程序性细胞死亡（ＰＣＤ）进行了观察。结果：胎鼠颞下颌关节髁状突软骨增殖层ＰＣＮＡ阳性细胞率最高,进入浅层肥厚层ＰＣＮＡ阳性细胞数减少,深层肥厚层中则未见ＰＣＮＡ阳性细胞。出生后髁状突软骨ＰＣＮＡ阳性细胞率均较出生前低。ＰＣＤ阳性细胞主要分布于软骨增殖层及邻近深层肥厚层的浅层肥厚层中,在关节腔开始形成时髁状突软骨表面ＰＣＤ明显。结论：细胞增殖与程序性细胞死亡密切相关,协同参与调控颞下颌关节的生长发育与塑形。     

Lubricin immunohistochemical expression in human temporomandibular joint disc with internal derangement

J Oral Pathol Med (2011) 40 : 587–592 Lubricin is a chondroprotective, mucinous glycoprotein which contribute to joint lubrication, especially to boundary lubrication and maintains joint integrity. The present investigation aimed to study the immunolocalization of lubricin in TMJ discs from patients affected by anterior disc displacement with reduction (ADDwR) ADDwoR. Eighteen TMJ displaced disc affected by ADDwoR were processed immunohistochemically, with a polyclonal anti‐lubricin antibody, used at 1:50 working dilution. The percentage of lubricin immunopositive cells (extent score = ES) and the extent of lubricin staining of the disc extracellular matrix (ECM), were evaluated. Each sample was scored for histopathological changes. Percentage of immunostained surface disc cells was the same (ES = 4) in both control and ADDwOR cells, being this data not statistically significant (P < 0.05). In pathological specimens the percentages of lubricin‐stained cells was very high with an ES of 4 respect to control specimen, and this difference was statistically significant different (P > 0.05). The extracellular matrix (ECM) of discs at the disc surfaces of both pathological and normal specimens was very heavily stained (++++). Both the ES and ECM staining were not statistically correlated to the TMJ degeneration score according to the Spearman’s rank correlation coefficient. According to our findings, a longstanding TMJ disc injury, affects lubricin expression in the TMJ disc tissue and not its surfaces, moreover, lubricin immunostaining is not correlated to TMJ disc histopathological changes.     

The effects of protein-coated surfaces on passaged porcine TMJ disc cells

OBJECTIVE: Implantation of synthetic temporomandibular joint (TMJ) disc replacements aimed to alleviate pain and restore functional losses caused by TMJ disorders. Unfortunately, these synthetic replacements have been largely unsuccessful and in some instances have incited severe immune responses. Tissue engineering, however, may provide viable TMJ disc replacements. Towards this end, we have studied TMJ disc gene expression as a measure of protein production potential. With passage, collagen type I and aggrecan gene expression decrease in TMJ disc cell cultures. We hypothesize that surfaces coated with TMJ disc proteins may rapidly recover the lost gene expression in passaged TMJ disc cells. DESIGN: To study these effects, passages 0, 1, and 2 TMJ disc cells were plated in wells coated with aggrecan, collagen type I, collagen type II, or decorin. Safranin O staining was conducted to visualize cell aggregation. RESULTS: At passage 0, cultures appeared similar on each surface; however, by passages 1 and 2, aggrecan-coated and decorin-coated surfaces appeared to have more cell aggregates. Gene expression data did not correspond to these visual changes. No treated surface offered a significant change in aggrecan, collagen type I, or decorin expression relative to untreated controls. Furthermore, aggrecan and collagen type I gene expression dropped relative to samples taken prior to plating. CONCLUSIONS: These results indicate that, despite visual changes described by cell aggregates, protein coatings have limited effects for recovering TMJ disc gene expression in monolayer cultures.