藻酸盐凝胶三维培养体系在TMJ骨关节病髁突软骨细胞体外培养中的应用

目的 将藻酸盐凝胶三维培养体系应用于颞下颌关节骨关节病（osteoarthritis of temporomandibular joint,TMJOA)髁突软骨细胞的体外培养。方法 利用机械与酶消化的方法从TMJOA患者手术切除患疾髁突软骨中获得髁突软骨细胞，部分细胞在二维贴壁培养条件下体外培养1周；部分细胞在高密度条件下转入藻酸盐凝胶培养介质，进行三维培养4周；分别对贴壁条件下培养细胞和藻酸盐细胞凝胶微球右蜡包埋切片进行Ⅱ型胶原和软骨特异性蛋白多糖的免疫组化鉴定。结果 从骨关节病髁突软骨组织中收获并行体外培养成活的细菌，鉴定为软骨细胞；体外培养的藻酸盐凝胶三维培养体系中的髁突软骨细胞生长状态良好；培养4周后细胞保持了良好的分化表型。结论 成功地体外培养成活了人TMJOA髁突软骨细胞；成功地将藻酸盐凝胶三维培养体系应用于人TMJOA髁突软骨细胞体外培养，在该体系中，软骨细胞生长状态与功能蛋白分泌能力良好。     

人髁突软骨细胞培养及细胞库建立

目的　建立生物学性状稳定的髁突软骨细胞库 ,为颞下颌关节髁突软骨缺损及关节盘的生物性修复奠定基础。方法　髁突软骨取自因母体健康原因需终止妊娠的人胚胎 ,经机械分离及胰蛋白酶、胶原酶联合消化获得 ,应用微载体培养技术进行髁突软骨细胞的体外扩增 ,采用液氮深低温保存细胞 ,并定期 (2周、1个月 )对复苏软骨细胞生物学特性鉴定。结果　微载体培养技术可在短期内获得大量的、高成活率的髁突软骨细胞。经液氮冻存的软骨细胞在细胞增殖动力学、表型特征及细胞代谢方面无显著变化 ,基本上保持了原代培养软骨细胞的生物学特性。结论　成功的建立了髁突软骨细胞库 ,可为应用组织工程技术修复关节软骨缺损及重建关节盘的研究提供良好的供体细胞     

HGF基因转染软骨细胞治疗兔颞下颌关节骨关节病的研究

目的 建立兔颞下颌骨关节病动物模型,观察携带有GFP-HGF cDNA基因体外转染兔髁突软骨细胞后对兔颞下颌骨关节病的治疗效果。方法 以Ⅱ型胶原酶进行颞下颌关节腔内注射,建立颞下颌骨关节病动物模型。取兔髁状突软骨细胞体外进行培养与增殖,第2代软骨细胞进行GFP-HGFcDNA转染。将收集的转染后的软细胞进行动物模型关节腔内注射,并与对照组(空白质粒组)比较,通过组织切片观察治疗效果。结果 兔颞下颌关节经两次法注射胶原酶可在2周时出现偏侧咀嚼症状;动物模型显示转染细胞可促进关节损伤的修复,促进愈合。结论 转染HGF的软骨细胞可促进颞下颌骨关节病的愈合。     

肉毒杆菌毒素对兔颞下颌关节骨关节病动物模型的关节组织病理学影响

目的:观察颞下颌关节骨关节病动物模型应用肉毒杆菌毒素后关节组织学改变,证明它对该病的治疗作用.方法:应用胶原酶注射法制备30例兔TMJOA的动物模型,分成对照组与实验组,实验组双侧颞肌、嚼肌内注射肉毒杆菌毒素,将实验组与对照组在4周末、8周末、12周末各处死5只.取出关节盘及髁状突,分别观察其在光镜及扫描电镜下的病理变化.结果:4周末时,对照组动物出现了明显的骨关节病变化,关节盘血管增生明显和软骨细胞增生活跃,实验组病变较轻,第8周末时,对照组病变更典型,出现软骨裂隙和凝胶样物质剥脱,实验组出现病变修复;12周末时,对照组出现严重的病理改变,软骨剥脱,骨赘形成,而实验组病变修复接近正常.结论:肉毒杆菌毒素可以抑制TMJOA的发展,促进关节的自身修复,对TMJOA有显著的治疗作用.     

颞下颌关节髁突软骨细胞体外培养中去分化现象研究

目的　研究去分化髁状突软骨细胞的细胞生物学特性。方法　连续在体外贴壁条件下培养传代正常乳兔髁突软骨细胞至第 2 0代 ,观察培养细胞的形态变化 ,免疫组化法和RT -PCR法对传代过程中的数代细胞对II型胶原与蛋白多糖聚糖体的表达情况进行连续检测 ,观察细胞软骨基质蛋白表达分泌的变化以及发生明显去分化软骨细胞的超微结构特点。结果　在贴壁培养条件下 ,髁状突软骨细胞去分化现象于细胞传代第 3代后逐渐显著 ,第 6代后培养的软骨细胞几乎完全去分化。去分化细胞胞体拉长 ,呈“成纤维细胞”样外观。去分化过程中 ,细胞对软骨基质蛋白的表达水平逐渐降低 ,细胞增殖速度增强。透射电镜观察显示 ,去分化软骨细胞细胞核明显呈分叶状 ,表现出异型核。结论　髁突软骨细胞在体外贴壁培养传代过程中会发生去分化现象 ,丧失软骨细胞特征性细胞表型。     

颞下颌关节骨关节病动物模型的建立及组织病理学观察

目的 建立稳定的颞下颌关节骨关节病动物模型。方法 通过外科手术切除部分关节盘的方法，在成年兔上建立较为稳定的颞下颌关节骨关节病动物模型；分别于术后第15天、1个月、2个月、3个月、4个月、5个月和6个月将实验兔处死，取实验侧髁突组织进行组织病理学观察；1％甲苯胺蓝染色，观察病变髁突组织中蛋白多糖的分布范围与水平变化。结果 本研究中的颞下颌关节骨关节病模型从组织病理学上先后观察到了关节盘损伤后早期关节软骨的应激性修复增殖，中期软骨基质渐进性损耗，后期软骨基质损耗丧失、纤维组织修复覆盖及骨赘形成等软骨退行性变过程。甲苯胺蓝染色显示，病变早期肥大增厚的软骨组织中蛋白多糖含量降低，且分布不均匀；病变晚期的髁突较深层软骨组织中仍可见有蛋白多糖的合成与分布。结论 本研究所建立的颞下颌关节骨关节病动物模型能较好地模拟骨关节病的组织病理学变化过程，且具有结果稳定、可重复性好的优点。     

骨髓基质细胞修复山羊髁突软骨缺失的绿色荧光蛋白示踪

目的:采用绿色荧光蛋白(green fluorescent protein,GFP)对骨髓基质细胞体内修复髁突软骨全层缺失进行示踪观察。方法:扩增PG13细胞株,获得大量含GFP基因的假病毒液,并直接转染骨髓基质细胞(bone marrow stromal cells,BMSCs)。将含有GFP基因转染标记的BMSCs和少量软骨细胞与生物可降解材料复合后,植入山羊髁突软骨全层缺失处,1个月后应用激光共聚焦显微镜检测修复组织中GFP标记的BMSCs的分布。结果:标记的BMSCs植入关节软骨缺损1个月后,修复组织仍能高效表达GFP。激光共聚焦显微镜下显示:多数新生软骨陷窝内有GFP标记的细胞。结论:标记的BMSCs可在髁突软骨全层缺失区分化为成熟的软骨细胞,并在髁突软骨缺失的修复中发挥重要作用。     

颞下颌关节骨关节病髁突组织细胞凋亡的研究

目的研究颞下颌关节骨关节病骸突组织中软骨细胞凋亡发生的特点,探讨细胞凋亡在颞下颌关节骨关节病发病机制中的作用。方法通过手术切除部分关节盘的方法建立颞下颌关节骨关节病动物模型,15 d-6个月内采用TUNEL法标记裸突各相应时段的凋亡细胞,结合相应组织学变化,观察、分析颞下颌关节骨关节病裸突组织中细胞凋亡发生的特点。结果关节盘损伤后软骨产生明显应激性修复增殖反应时,凋亡细胞主要集中于关节表面的纤维层中;随着关节软骨和骨组织不断改建,软骨组织中逐渐出现大量的细胞凋亡现象,主要发生于表面纤维层与增殖层,尤其集中在软骨细胞簇中;在软骨组织消耗严重时,浅表区域内的细胞凋亡现象逐渐减少,肥大钙化层中出现了较多的凋亡细胞。结论颞下颌关节骨关节病骸突软骨组织中存在过量凋亡的现象,软骨细胞异常增殖与过度凋亡导致软骨基质自身调节机制的破坏可能是骨关节病发生的重要原因之一。     

胰岛素样生长因子-Ⅰ及其与髁突软骨发育的调控

出生后的髁突软骨处于复杂的力学微环境中,其生长改建的生物学基础是髁突软骨细胞的增殖和分化。胰岛素样生长因子(IGF)-Ⅰ是髁突软骨生长发育过程中重要的生长因子之一。在细胞学水平,IGF-Ⅰ受胰岛素样生长因子连接蛋白(IGFBP)调控,通过与其受体的结合、激活来发挥生物学效应,其中包括调控细胞增殖、分化及程序性死亡,加快髁突软骨基质合成,参与髁突软骨细胞内力学信号的转导等。本文就IGF-Ⅰ及其受体,IGFBP及IGF-Ⅰ调控髁突软骨发育及其机制的研究作一综述。     

生长期兔髁突软骨细胞体外培养方法及其生物学特性研究

目的 建立髁突软骨细胞体外培养模型,研究其生物学特性。 方法 分离4周龄健康新西兰大白兔双侧髁突软骨,采用胰酶和胶原酶联合消化方法收集4 h和12 h 2个时间点的髁突软骨细胞,连续体外培养并传代至P10。使用倒置显微镜观察细胞形态;采用甲苯胺蓝、阿利新蓝和免疫细胞化学染色对髁突软骨细胞进行鉴定;利用细胞计数绘制细胞生长曲线;通过Western 免疫印迹分析细胞Ⅱ型胶原、Ⅹ型胶原和SOX9在蛋白水平的表达情况。采用SPSS13.0软件包对Western 印迹条带灰度值进行统计学分析。 结果 兔髁突软骨细胞体积较大,呈纺锤形或多角形,铺路石样排列,细胞爬片染色结果为阳性。随着细胞传代“成纤维样”细胞比例逐渐加重,P2代之前的细胞形态差异较小。细胞生长曲线显示,兔髁突软骨细胞的体外培养符合经典的S形生长曲线。Western 印迹结果表明,4h和12 h所收髁突软骨细胞Ⅱ型胶原、Ⅹ型胶原和SOX9表达无显著差异。 结论 使用本方法培养兔髁突软骨细胞简单有效。髁突软骨细胞体外培养存在去分化现象,但P2代以内的髁突软骨细胞生物学特性相对稳定,适合用于后期实验研究,P3代以后的髁突软骨细胞逐渐丧失原有细胞的特性。     

The immunolocalisation of VEGF in the articular cartilage of sheep mandibular condyles.

INTRODUCTION: Vascular endothelial growth factor (VEGF) has recently been found to be essential for hypertrophic chondrocyte apoptosis and angiogenesis at the growth plate of long bones, indicating a central role in endochondral ossification. VEGF has more recently, also been shown to be expressed in articular cartilage chondrocytes in human osteoarthritic and rheumatoarthritic joints but not healthy adult joints. To investigate the role of VEGF in the fibrocartilage of the temporomandibular joint, this study aimed to document the presence and distribution of VEGF in the condylar articular cartilage of sheep temporomandibular joints. METHODS: Mandibular condyles of the temporomandibular joints of five 18-month old Wether sheep were fixed, decalcified, paraffin embedded and sectioned. The sections were analyzed using immunohistochemistry for VEGF. RESULTS: VEGF was found to be localised predominantly to the proliferative and maturing layers of chondrocytes in the condylar fibrocartilage of the temporomandibular joints. Articular cartilage is an avascular and alymphatic tissue. As such, the localisation of VEGF to the articular cartilage of normal temporomandibular joint condyles suggests a role for VEGF other than angiogenesis. CONCLUSION: VEGF is shown here for the first time to be present in mandibular condylar cartilage, leading us to propose a possible role in non-angiogenic extracellular matrix remodeling.     

骨髓基质细胞诱导分化修复髁突软骨面缺失的实验研究

目的:采用骨髓基质细胞(BMSCs)体内修复髁突软骨全层缺失。方法:15只山羊,9只作为实验组,将BMSCs和少量软骨细胞(7:3比例混合)按5×107/mL与生物可降解材料复合后,植入山羊髁突软骨全层缺失处;对照组6只山羊,髁突软骨全层缺失区植入支架材料,分别于术后4、8、12周每个时间段取材3只实验动物,2只对照组动物;2组分别用HE染色、Ⅱ型胶原分泌的免疫组化法进行评价。结果:实验组术后4周,山羊髁突软骨缺失区能形成成熟的软骨组织,12周时软骨未退变。对照组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,可以修复山羊颞下颌关节髁突软骨面全层缺失。     

软骨细胞移植修复髁突软骨损伤的实验研究

目的 研究软骨细胞移植修复髁突软骨损伤后的效果及修复组织的性质。方法 选用成年雄性白免５３只,分成５组。第１组：细胞移植组;第２组：单纯胶原膜植入组;第３组：在实验动物有髁突前斜面形成直径２ｍｍ的全层损伤后,未植入任何物品;第４组：空白手术对照组;第５组：正常对照组。结果 细胞移植组动物的髁突软骨损伤区形成软骨组织修复。结论 异体兔髁突软骨细胞移植能够形成类似正常的关节软骨修复髁突软骨缺损。     

Interleukin-1 beta affects cyclooxygenase-2 expression and cartilage metabolism in mandibular condyle

Extracellular matrix degradation in mandibular condylar cartilage is mediated by various cytokines in the temporomandibular joint (TMJ). Interleukin-1 beta (IL-1β) is detected in joint structures with pathologic status, and participates in catabolic action in the extracellular matrix. The purpose of this study was to investigate the effects of IL-1β on cyclooxygenase-2 (COX-2) expression and cartilage metabolism using cultured chondrocytes from mandibular condyle. Articular chondrocytes from the porcine mandibular condylar cartilage around the surface were cultured and treated with 0–10 ng/ml IL-1β or 0–1000 ng/ml prostaglandin (PGE₂) for 0–24 h. The mRNA levels of COX-2, MMP-1, -3, and -13 were evaluated by real-time PCR analysis. The protein levels of PGE₂ and MMPs were examined by ELISA and Western blot analysis, respectively. The expression levels of COX-2 and PGE₂ were enhanced by exogenous IL-1β in chondrocytes. The mRNA levels of MMP-1, -3, and -13 were up-regulated by PGE₂ treatment dose-dependently. It is shown that the expression of COX-2/PGE₂ was enhanced by IL-1β in articular chondrocytes from mandibular condyle, and that MMP-1, -3, and -13 were induced by PGE₂, suggesting that IL-1β-induced COX-2/PGE₂ play a crucial role in catabolic processes of mandibular condylar cartilage under inflammatory conditions.     

Bone morphogenetic protein 3 expression pattern in rat condylar cartilage, femoral cartilage and mandibular fracture callus

Mandibular condylar cartilage differs from primary cartilage in morphological organization of the chondrocytes and in responses to biomechanical stress and humoral factors. For the first time, we describe the expression of Bmp3 mRNA in relation to types I, II and X collagen mRNA (as determined by in situ hybridization) in chondrocytes of the rat mandibular condylar cartilage, femoral articular cartilage, femoral growth plate cartilage, and temporal cartilage, which transiently appeared in the reparative response stage of mandibular ramus fracture healing. In all cartilages evaluated, Bmp3 was expressed in proliferating chondrocytes that expressed type I collagen in condylar cartilage, articular cartilage, and temporal cartilage appearing during fracture healing. Bmp3 was also found in hypertrophic chondrocytes that expressed type X collagen mRNA in all cartilages evaluated. Furthermore, in remodeling bone, Bmp3 mRNA was strongly expressed in active osteoblast cells in periosteal reaction layers formed after fracture. These findings suggest that Bmp3 expression in a special layer of typical articular cartilage may be regulated by mechanical stress stimulation. We also found that Bmp3 was expressed in the periosteal layers of the bone segments near the fracture site during fracture healing.     

Incisor disocclusion in rats affects mandibular condylar cartilage at the cellular level

The effect of altered occlusion on the mandibular condylar cartilage remains unclear. OBJECTIVE: This study investigated the effect of unilateral incisor disocclusion on cartilage thickness, on mitotic activity and on chondrocytes maturation and differentiation in the mandibular condylar cartilage of rats. DESIGN: The upper and lower left incisors were trimmed 2mm every second day in five rats. In other five rats, the incisor occlusion was not altered. Condylar tissues from both sides of each mandible were processed and stained for Herovici's stain and immunohistochemistry for bromodeoxyuridine (BrdU), transforming growth factor-beta1 (TGF-beta1), alkaline phosphatase (ALP) and osteocalcin (OCN). Measurements of cartilage thickness and the numbers of immunopositive cells for each antibody were analysed by one-way analysis of variance (ANOVA). RESULTS: No significant differences were observed in cartilage thickness after 7 days of unilateral incisor disocclusion. However, the numbers of immunopositive cells for BrdU as a marker of DNA synthesising cells, TGF-beta1 as a marker of chondrocytes differentiation, and ALP and OCN as markers of chondrocytes maturation, were significant higher in the cartilage cells on both sides when incisor occlusion was unilaterally altered. Interestingly, alkaline phosphatase was highly expressed on the condylar side of incisor disocclusion, whereas osteocalcin was highly expressed on the side opposite to the incisor disocclusion. CONCLUSIONS: It is demonstrated that after 7 days, unilateral incisor disocclusion affects the mandibular condylar cartilage at the cellular level by increasing the mitotic activity and by accelerating chondrocytes maturation. Chondrocytes maturation appears more accelerated on the side opposite to incisor disocclusion.     

软骨细胞外泌体在大鼠颞下颌关节骨关节炎软骨异常钙化中的作用研究

目的 探索软骨细胞外泌体在大鼠颞下颌关节（temporomandibular joint，TMJ）骨关节炎（osteoarthritis，OA）退变软骨异常钙化中的作用。方法 选取48只6周龄雌性SD大鼠，根据实验时间点（2、4、8周）随机分为三大组，每大组再随机分为对照组与单侧前牙反牙合（unilateral anterior crossbite，UAC）实验组，每组8只大鼠。对UAC实验组大鼠施加UAC刺激诱导TMJ OA，于2、4、8周后分别处死各组大鼠，对TMJ髁突软骨进行CD63、基质羧基谷氨酸蛋白（matrix gla protein，MGP）的免疫组化染色和mRNA含量检测，同时透射电镜观察软骨中钙化痕迹及外泌体结构。提取大鼠TMJ髁突软骨细胞体外培养并施加流体剪切力刺激，茜素红染色检测钙化结节形成情况，对软骨细胞和外泌体蛋白进行Western Blot检测。结果 UAC实验组大鼠TMJ髁突软骨内CD63分子高表达，同时在钙化痕迹周围存在大量外泌体样结构，而MGP含量则在4周和8周时显著减少。流体剪切力可显著促进TMJ髁突软骨细胞钙化结节形成，同时外泌体数目增多，但外泌体中MGP蛋白含量显著降低。对正常培养的软骨细胞施加外泌体后可显著促进钙化结节的生成。结论 大鼠TMJ OA软骨中外泌体数目增加，同时外泌体内MGP含量降低，两方面共同促进软骨异常钙化的发生。