Immunohistochemical Localization and Distribution of Proliferating Cell Nuclear Antigen in the Rabbit Mandibular Condyle Following Experimental Induction of Anterior Disk Displacement

The purpose of the present study was to identify proliferating cells in control versus experimental condyles two weeks following experimental Induction of anterior disk displacement (ADD) in the rabbit craniomandibular joint (CMJ). The right joint of 15 rabbits was exposed surgically and all diskal attachments were severed except for the posterior attachment. The disk was then repositioned anteriorly and sutured to the zygomatic arch. The left joint served as a sham-operated control. Ten additional joints were used as nonoperated controls. Mandibular condyles were excised two weeks following surgery and processed for proliferating cell nuclear antigen (PCNA) immunostaining. In control and sham operated condyles, PCNA was localized in the nuclei of chondroblasts of the reserve cell layer, chondrocytes of the upper hypertrophic layer and bone marrow cells of the subchondral bone. In contrast to control joints, the PCNA positive cells of the experimental joints were located throughout the osteoarthritic condylar cartilage. In addition, the percentage of PCNA positive cells of the osteoarthritic condylar cartilage was statistically significantly higher when compared to the control group, p < 0.05. It was concluded that surgical induction of ADD in the rabbit CMJ leads to an increase in mitosis of chondrocytes, which lead to cell proliferation and subsequent hyperplasia of the condylar cartilage.     

Experimental induction of anterior disk displacement of the rabbit craniomandibular joint: an immuno-electron microscopic study of collagen and proteoglycan occurrence in the condylar cartilage

BACKGROUND: Results from our previous studies suggest that surgical induction of anterior disk displacement (ADD) in the rabbit craniomandibular joint (CMJ) leads to histopathological alterations consistent with osteoarthritis. In addition, molecular changes in collagens and glycosaminoglycans (GAGs) were observed using immunohistochemistry. The purpose of the present study was to further characterize those molecular changes in collagens and GAGs using immuno-electron microscopy. METHODS: The right joint of 15 rabbits was exposed surgically and all discal attachments were cut except for the posterior attachment (the bilaminar zone). The disc was then repositioned anteriorly and sutured to the zygomatic arch. The left joint was used as a sham-operated control. Ten additional joints were used as non-operated controls. Mandibular condyles were removed 2 weeks following surgery and processed for light and immuno-electron microscopy using colloidal gold-labeled antibodies against collagen type I, II, VI and IX and against keratan sulfate, chondroitin-4 and -6-sulfate, and link protein. RESULTS: Light microscopic results showed osteoarthritic changes. Immuno-electron microscopy of osteoarthritic cartilage demonstrated a decline in type II collagen, the abnormal presence of type I collagen and loss of type VI and IX collagens. Quantitative colloidal gold immuno-electron microscopy confirmed the depletion of keratan sulfate, chondroitin-4 and -6-sulfate, and link protein in osteoarthritic cartilage. CONCLUSION: Anterior disk displacement leads to molecular alterations in both the collagen and the proteoglycans of rabbit condylar cartilage characteristic of osteoarthritis in other synovial joints. These alterations are consistent with loss of the shock absorber function of the cartilage and injury of the underlying bone.     

Histopathological changes in rabbit craniomandibular joint associated with experimentally induced anterior disk displacement (ADD)

Several studies have shown that anterior disk displacement (ADD) of human temporomandibular joint (TMJ) can lead to cellular and extracellular alterations in the disk proper, bilaminar zone (BZ), condyle, articular eminence and synovial membrane. Due to lack of an animal model for this disease, it is not known whether the mechanical displacement of the disk could lead to the observed histopathological changes. The purpose of this experiment was to investigate the histopathological changes that occur in the rabbit craniomandibular joint (CMJ) following surgical induction of ADD. The right CMJ was exposed surgically and the discal attachments were severed except for the BZ attachments. Then the disk was displaced anteriorly and sutured to the zygomatic arch. The left joint served as surgical control. The CMJs were removed after 24 h, 1 week, 2 weeks or 6 weeks and stained with H&E or modified Masson stain. The results showed neovascularization, cell clustering and fibrillation of the displaced disk. The BZ showed marked fibrosis. The condyle showed subchondral hemorrhage and fibrosis followed by osteoarthritic changes in the articular cartilage. The articular eminence showed chondrocytic clustering and an increase in the amount of chon-droid bone. Synovial membrane exhibited marked hyperplasia. We concluded that surgical induction of ADD in the rabbit CMJ leads to cellular and extracellular alterations in the disk proper, BZ, condyle, articular eminence and synovial membrane similar to those described previously in human ADD. It appears that the mechanical trauma resulting from ADD could lead to a cascade of reparative and degenerative changes of the affected joints similar to those described for osteoarthritis.     

An immunohistochemical study of collagen types III, VI and IX in rabbit craniomandibular joint tissues following surgical induction of anterior disk displacement

The purpose of this study was to determine the effect of surgical induction of anterior disk displacement (ADD) on type-Ill. VI and IX collagens of the rabbit craniomandibular joint (CMJ) tissues using an immunohistochemical technique. The right joint was exposed surgically, all discal attachments were severed except for the posterior discal attachment (bilaminar zone). The disk was then repositioned anteriorly and sutured to the zygomatic arch. The left joint served as a sham-operated control. Ten additional joints were used as non-operated controls. Deeply anesthetized rabbits were perfused with 2% buffered formalin 2 weeks (10 rabbits) or 6 weeks (10 rabbits) following surgery. The articular disk, bilaminar zone, mandibular condyle and articular eminence were excised. The last two were decalcified in EDTA. All tissues were then sectioned at 10 um in a cryostat. Sections were incubated with monoclonal antibodies directed against type-Ill. VI or IX collagens. Following incubation in the appropriate FITC-fabelled secondary antibodies, all sections were studied under the fluorescence microscope. The results showed a reduction in immunostaining for type-VI and IX collagens in the condylar cartilage, disk and articular eminence at 2 weeks, followed by an increase in their immunostaining at 6 weeks and the appearance of a de novo type-Ill collagen in the condylar cartilage and the articular eminence. It is concluded that surgical induction of ADD in the rabbit CMJ leads to alterations in its type-III. VI and IX collagens.     

Histological changes in the rabbit condyle following posterolateral disk perforation

PURPOSE: To examine changes in condylar cartilage following perforation in the posterolateral region of the articular disk of the craniomandibular joint of a rabbit. MATERIAL AND METHODS: The circular perforation in the left joint of 25 female Japanese white rabbits measured precisely one-sixteenth of a disk. Histological examination, including the immunohistochemical proliferating cell nuclear antigen (PCNA) procedure, was performed on separate sets of five rabbits each 2, 4, 8, 12, and 24 weeks postoperatively. RESULTS: Microscopic examination revealed hypertrophy of the condylar cartilage with osteophyte formation up to 8 weeks after perforation. Proliferative activity then decreased near the condylar surface as the perforation flattened. Twenty-four weeks postoperatively, the condylar surface was found to be fibrous and flattened. Positive PCNA results in cartilage cells indicated proliferation following disk perforation which peaked in the 4th week and then decreased. CONCLUSION: Disk perforation was followed initially by hypertrophy of condylar cartilage, and later by degeneration of the condylar surface. Although osteoarthritic cartilage was found 24 weeks after perforation, degeneration decreased over time. This suggests that remodelling took place after the perforation.     

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。     

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.     

颞下颌关节盘前移位关节区组织病理变化的实验研究

目的通过建立关节盘前移位实验动物模型的方法,来研究人类关节盘前移位病变早期阶段的发展过程和病理学改变。方法１２只新西兰大白兔左颞下颌关节经实验诱导为关节盘前移位模型,右侧为手术对照组。于术后２４ｈ、１周、２周、３周、４周、１０周各处死２只,切取关节组织,ＨＥ染色,光镜观察。结果早期：关节结节、髁突关节软骨增生明显,髁突软骨下出血,骨组织内血管网消失;后期：髁突关节软骨、骨组织及滑膜出现骨关节炎（ＯＡ）样改变。结论实验诱导关节盘前移后关节区的病理学变化与人类相似;关节盘前移位关节区的创伤可引起关节骨关节炎样改变。     

Immunohistochemical analysis of Bcl-2 and Bax oncoproteins in rabbit craniomandibular joint

The purpose of this study was to elucidate the expression of proto-oncogene Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) in fibrocartilage of the disc and hyaline cartilage of the condyle in the rabbit craniomandibular joint (CMJ). Ten New Zealand white rabbit heads were used. Sections were processed by the immunohistochemical techniques using mouse anti-Bcl-2 and anti-Bax antibodies. Intensity levels of immunostaining in condylar cartilage were quantified by a computer-image system. Immunoreactivity for Bcl-2 was mainly observed in the cytoplasm of the reserve cell and chondrocytic cell layers. A mild heterogeneous Bax expression was detected in the cytoplasm of chondrocytes of the upper hypertrophic layer and a few cells of the chondrocytic layer. The cytoplasm of chondrocytes in the disc exhibited a high intensity for Bcl-2, while Bax activity was only sporadically observed. We have shown that Bcl-2 and Bax proteins are present in CMJ cartilage and their expression patterns suggest that these oncoproteins are involved in chondrocyte survival or death via apoptotic pathways.     

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。     

颞下颌关节骨关节病髁突软骨细胞及基质合成代谢特性的研究

目的 探讨颞下颌关节骨关节病(TMJOA)软骨细胞的细胞生物学特性。方法 通过手术切除部分关节盘的方法,制造TMJOA的兔动物模型;从模型动物处于骨关节病病变增殖修复期的髁突软骨组织中获取细胞体外培养,并采用RT-PCR法比较病变软骨细胞与正常对照细胞对软骨基质蛋白、基质胶原酶和内源性生长因子的表达差异。结果 从TMJOA模型的髁突软骨组织中获得细胞经体外培养、鉴定,确认为软骨细胞;骨关节病髁突软骨细胞对软骨基质蛋白的表达不平衡,对内源性基质胶原酶的表达水平和内源性生长因子TGF-β1、IGF-1的表达明显增强。结论 本研究从细胞生物学角度证实了骨关节病早期关节软骨内出现合成代谢活跃的组织学特点,并发现该阶段组织细胞的修复代偿反应可致软骨基质成分合成的不平衡,最终可导致软骨基质环境改变。     

软骨细胞增殖与凋亡在人重组白细胞介素-1β介导的颞下颌关节炎症损伤中的意义

目的:探讨细胞增殖与凋亡在人重组白细胞介素-1B介导的颞颌关节炎症损伤中的作用。方法:选用SD大鼠颞下颌关节腔内反复多次注射rhIL-1B,造成炎症损伤,利用免疫组织化学染色及原位末端标记法,观察炎症损伤过程中髁突软骨细胞增殖及凋亡的变化。结果:注射后1~30 d,实验侧髁突软骨增殖层PCNA阳性指数均较对照侧低,以1~7 d最明显;实验侧髁突软骨细胞凋亡数较对照侧多,主要分布于软骨增殖层及前肥厚层中,以1~7 d 最明显,15 d后凋亡细胞数明显减少,仅见个别凋亡细胞散在分布于软骨浅层。结论:rhIL-1B关节内注射不仅使髁突软骨细胞增殖受抑制,而且细胞凋亡也可能是rhIL-1B致关节损伤的途径之一,细胞增殖与凋亡协同参与了颞下颌关节炎症损伤的病理过程。     

Distribution of plasma-membrane Ca2+ pump in mandibular condyles from growing and adult rabbits

Chondrocytes may control the mineralization of the extracellular matrix of condylar cartilage by several mechanisms including the release of microvesicles involved in the initial nucleation, the creation or modification of the local matrix to help propagate or restrict mineralization, and the regulation of the ionic environment at the calcifying foci within the matrix. The plasma membrane Ca2+-Mg2+ ATPase (Ca2+ pump) is known to play a part in the vectorial efflux of calcium in a variety of cells including chondrocytes. The purpose here was to study the distribution of Ca2+-pump protein in mandibular condyles from growing and adult rabbits, and compare the expression of that protein in progressively differentiating chondrocytes whose final stage is associated with a mineralized extracellular matrix. Ca2+-pump antigen was identified immunohistochemically in six growing and six adult rabbit mandibular condyles with a Ca2+ pump-specific monoclonal antibody. The presence of Ca2+-pump antigen was established in hypertrophic chondrocytes, and in osteoblasts and osteoclasts of subchondral bone. Slot-blot analysis of nitrocellulose-immobilized chondrocyte homogenates showed that the amount of Ca2+ pump in growing cartilage was more than twice that in adult cartilage (p < 0.05). The demonstration of Ca2+-pump antigen in the hypertrophic chondrocytes of growing rabbit condyles is consistent with a role for the plasma-membrane Ca2+ pump in the calcification of mandibular condylar cartilage.     

升高咬合对青春期大鼠髁突软骨中增殖细胞核抗原表达变化的影响

目的：探讨升高咬合后髁突软骨中增殖细胞核抗原（PCNA）的变化情况及意义。方法：40只5周龄雄性SD大鼠,随机分为对照组和实验组（双侧后牙板升高咬合）。分别于术后7、14、21及28d,取其右侧髁突,应用免疫组织化学SABC法检测大鼠髁突软骨中PCNA的表达变化,并作图像分析和统计学处理。结果：与对照组相比,实验组髁突软骨PCNA阳性细胞表达在实验第7、14d明显减少,第28d明显增多（P〈0.05）。结论：咬合升高可以引起大鼠髁突软骨的适应性改建。     

兔颞下颌关节盘前移位的X线研究

目的　通过影像学检查了解颞下颌关节盘前移位的病理变化过程,证实关节盘前移位与退行性改变之间的关系。方法　在手术组动物一侧关节区显露颞骨颧突根部,用丝线垂直穿过关节盘前带的延伸部并拉缝线向前并固定,使颞下颌关节盘前移位。手术对照组的手术步骤与手术组相同,但不缝合关节盘前带的延伸部,也不将关节盘位向前方。正常对照组5只,手术组和手术对照组动物术后1、2、4、8、10、12和16周分别处死。拍摄关节X线片,观察38只兔颞下颌关节X线表现。将兔左、右关节区锯成组织块,肉眼观察兔关节盘的位置。结果　手术组关节盘位置与形态均发生了改变,13侧为部分关节盘前移位,完全性关节盘前移位为11侧,关节盘穿孔为9侧。部分关节盘前移位出现关节间隙狭窄或消失,髁状突骨密度增高。完全性关节盘前移位与关节盘穿孔表现为髁状突骨质增生、破坏及肥大,关节结节磨平以及硬化。结论　颞下颌关节盘前移位可导致关节骨质改变。完全性关节盘前移位和关节盘穿孔与骨关节病的关系密切。     

Temporomandibular joint: surgically created disk displacement causes arthrosis in the rabbit.

To document a causal relationship between temporomandibular joint disk displacement and arthrosis, the disk was surgically displaced in one temporomandibular joint in each of three rabbits. The rabbits were sacrificed after 4 weeks and the mandibular condyles were studied radiographically and histologically. All three joints that underwent disk displacement had radiographic and histologic evidence of arthrosis, which included erosion of the bone, irregularity and fissure formation of the articular soft tissue cover, disruption of the subchondral layer of cartilage cells, and chondrocyte proliferation. No radiographic or histologic changes occurred in the joints that were untouched. The results suggest that surgically created disk displacement can cause arthrosis in the temporomandibular joint of the rabbit.     

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。     

兔关节盘前移位后髁突软骨细胞中OPG和OPGL的表达及意义

目的:探讨关节盘前移位后髁突软骨中护骨素(osteoprotegerin,OPG)及其配体(osteoprotegerinligand,OPGL)表达的变化及意义。方法:20只日本大耳白兔,16只用弹力丝牵引关节盘前伸部,建立颞下颌关节盘前移位的动物模型,分别于术后1、2、4、8周处死。2只行模拟手术作为手术对照组,术后2周处死;另2只不行手术,作为空白对照。取颞下颌关节标本,HE染色,观察其镜下结构,SP免疫组化法检测髁突软骨中OPG和OPGL的表达与分布,并对各标本OPG和OPGL的表达进行灰度测定。用单因素方差分析法对所得数据进行统计学分析。结果:各实验组动物术后体重无明显减轻,伤口愈合良好,大体及显微镜下观察,关节盘明显前移位。OPG和OPGL在各组髁突软骨的增殖层及肥大层浅层均有表达,其中关节盘移位组OPG、OPGL染色较强,染色灰度值与对照组均有显著性差异(P<0.05),但OPG/OPGL比率基本稳定。结论:关节盘移位后OPG和OPGL的表达均有升高,可能参与关节盘移位后髁突软骨病变的发展及转归。     

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。     

The effects of elevated fibroblast growth factor 23 on mandibular growth in rats

ObjectiveThe aim of this study is to elucidate the local effects of fibroblast growth factor 23 (FGF23) in on mandibular condylar growth in growing rats.DesignGrowing Sprague–Dawley rats received intra-temporomandibular joint injections of phosphate buffer solution (PBS), adenovirus-mediated green fluorescent protein (Ad-GFP) or adenovirus-mediated fibroblast growth factor 23 (Ad-FGF23), which were marked as groups A, B, and C, respectively. In vitro, we treated rat mandibular cartilage chondrocytes with PBS, Ad-GFP, and Ad-FGF23.ResultsThe mandibular condyles in group C grew smaller sizes than those in the other control groups due to significant differences among the experimental and control groups with the value of C–D, Q–R (P ≤ 0.05), accompanied by diminished bone mass of sub-cartilage condyles via micro CT analysis. Histologically, the length of the hypertrophic zone was diminished and was associated with decreasing chondrocyte proliferation in group C. Quantitative real-time PCR indicated significant decreases in the expression of chondrogenesis marker genes, including Type X collagen (Col X) and SRY-type box 9 (Sox 9). Moreover, elevated Ad-FGF23 suppressed chondrocyte proliferation and the expression of the chondrogenic differentiation markers Col X and Sox 9 of in vitro.ConclusionsLocal injection of FGF23 suppressed the development and decreased the bone mass of condyles through the decreasing the formation of condylar cartilage, specifically by regulating condylar cartilage cell viability and chondrogenesis expression.