渐进性咬合紊乱对大鼠髁突软骨碱性成纤维细胞生长因子表达的影响

目的探讨渐进性咬合紊乱大鼠髁突软骨中碱性成纤维细胞生长因子（bFGF）表达的变化规律。方法建立大鼠渐进性咬合紊乱动物模型,应用免疫组织化学SABC方法检测髁突软骨中bFGF的表达,并通过计算阳性细胞密度来探讨bFGF在不同实验时间点的表达变化情况。结果bFGF主要表达在大鼠髁突软骨的增殖层、过渡层和肥大层。对照组大鼠髁突软骨中从6周龄到10周龄bFGF的表达逐渐增强,10周龄后降低并保持在一个稳定的水平;成年实验组和幼年实验组在实验2、6、8周时,bFGF的表达均高于各自的对照组,具有统计学意义（P<0.05）,而实验4周时与对照组之间无统计学差异。结论bFGF参与了大鼠渐进性咬合紊乱所致髁突软骨的改建活动。     

sIL-1R基因重组质粒在山羊颞下颌关节内的表达

目的:观察脂质体携带的可溶性白介素-1受体(sIL-1RI)基因重组质粒注入山羊颞下颌关节(TMJ)上腔后,在TMJ髁突软骨及滑膜组织中的表达,以及在全身其他重要器官组织中的表达,为施行基因治疗TMJ骨关节病(OA)奠定基础。方法:将脂质体携带的sIL-1RI基因重组质粒分别注入6只山羊TMJ的关节上腔。注射后第2、4周,分别处死3只动物。抽取TMJ关节滑液,用ELISA法检测关节滑液中的蛋白表达量。采用q检验,对实验数据进行方差分析和均数间的两两比较。切取完整关节标本以及肝脏、心脏、肾脏和脑组织,免疫组化法检测重组质粒在这些脏器组织中的表达情况。结果:重组质粒pcDNA3/sIL-1RI在山羊TMJ的滑膜细胞、软骨细胞及软骨下组织均有表达。ELISA方法检测结果显示,实验组表达量明显高于对照组(P<0.05)。在山羊肝脏、心脏、肾脏和脑等全身重要组织器官中未见表达。结论:脂质体携带的重组质粒pcDNA3/sIL-1RI,可在山羊TMJ有效地表达,关节外组织未见表达,因此是安全、可行的。     

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.     

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。     

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.     

Expression of matrix metalloproteinase-2 in osteoarthritic fibrocartilage from human mandibular condyle

Matrix metalloproteinase (MMP)-2 is expressed in osteoarthritic cartilage and synovial fluid and is thought to be involved in the degradation of cartilage extracellular matrix. However, MMP-2 expression and osteoarthritic changes in internal derangement of the temporomandibular joint are unknown. In the present study, we have examined the histological relationship between osteoarthritic changes on articular cartilage with or without articular disc perforation, and MMP-2 expression, in 85 mandibular condyles from cadavers. The expression and tissue immunolocalization of MMP-2 in fibrocartilages from these condyles was examined histochemically. The Mankin grade of histological criteria for specimens with disc perforation was significantly higher than that of specimens without perforation. MMP-2 immunostaining was positive in the cytoplasm of chondrocytes and in their surrounding matrix. There was a linear correlation between MMP-2-positive cell rates and Mankin grade. Our data suggest that MMP-2 plays an important role in fibrocartilage degradation in internal derangement of the temporomandibular joint.     

Expression of osteoprotegerin in synovial tissue and degradation of articular cartilage: comparison with arthroscopic findings of temporomandibular joint disorders

OBJECTIVE: The aim of this study was to investigate the correlations among the expression of osteoprotegerin (OPG) in synovial tissue and the degree of synovitis, the degeneration of articular cartilage, and the adhesions in patients with internal derangement and osteoarthritis of the temporomandibular joint (TMJ).Study design The expression of OPG, which was detected immunohistochemically, and the degree of arthroscopy of 31 patients with internal derangement and osteoarthritis of the TMJ were assessed and the correlations between them were analyzed statistically. RESULTS: OPG was expressed in the cytoplasm of the endothelial cells, synovial lining cells, and fibroblast cells. TMJs with osteoarthritis had a higher degree of articular cartilage degeneration than did TMJs with internal derangement. There was a correlation between the expression of OPG in the endothelial cells and the degree of the articular cartilage degeneration (P <.01). CONCLUSION: The expression of OPG might be associated with the development of degenerative changes of articular cartilage.     

Roles of hypoxia inducible factor-1α in the temporomandibular joint

ObjectiveTemporomandibular joint osteoarthritis (TMJ-OA) is a degenerative disease characterized by permanent cartilage loss. Articular cartilage is maintained in a low-oxygen environment. The chondrocyte response to hypoxic conditions involves expression of hypoxia inducible factor 1α (HIF-1α), which induces chondrocytes to increase expression of vascular endothelial growth factor (VEGF). Here, we investigated the role of HIF-1α in mechanical load effects on condylar cartilage and subchondral bone in heterozygous HIF-1α-deficient mice (HIF-1α^+/−).DesignMechanical stress was applied to the TMJ of C57BL/6NCr wild-type (WT) and HIF-1α^+/− mice with a sliding plate for 10 days. Histological analysis was performed by HE staining, Safranin-O/Fast green staining, and immunostaining specific for articular cartilage homeostasis.ResultsHIF-1α^+/− mice had thinner cartilage and smaller areas of proteoglycan than WT controls, without and with mechanical stress. Mechanical stress resulted in prominent degenerative changes with increased expression of HIF-1α, VEGF, and the apoptosis factor cleaved Caspase-3 in condylar cartilage.ConclusionOur results indicate that HIF-1α may be important for articular cartilage homeostasis and protective against articular cartilage degradation in the TMJ under mechanical stress condition, therefore HIF-1α could be an important new therapeutic target in TMJ-OA.     

骨保护素基因敲除对老年小鼠关节软骨的影响

目的:研究骨保护素(osteoprotegerin,OPG)在老年小鼠关节软骨增龄性变化中的作用及意义。方法 :取鼠龄为1年的骨保护素基因敲除(opg-knockout,Opg-KO)型小鼠和野生型(wildtype,WT)小鼠的长骨组织,分别进行X-ray、micro-CT检查,并行组织学染色观察关节软骨组织形态学的改变;行甲苯胺蓝染色,观察关节软骨蛋白聚糖的改变;行免疫组化染色,观察关节软骨Ⅱ型胶原表达情况。结果:与WT小鼠相比,Opg-KO小鼠关节表面粗糙,边缘性骨赘增多,软骨下骨的骨密度增高,关节软骨表面扁平塌陷,软骨细胞明显减少,排列紊乱,蛋白聚糖显著丢失,Ⅱ型胶原表达明显减少。结论:Opg-KO小鼠发生了严重骨关节炎,骨保护素对于维持老年小鼠关节软骨结构的完整有重要作用。     

Molecular cloning, expression of rat Msx-1 and Msx-2 during early embryo genesis and roles for mandibular chondrogenesis]

Quail-chick chimera experiments have shown a contribution of carnial neural crest cells to the craniofacial skeletal elements. Moreover, tissue interactions between epithelial-mesenchymal interaction during early facial process development are required for both skeletal differentiation and morphogenesis. In this study, it was observed that Msx homeobox containing genes expressed in the facial process were important molecules of cartilage morphogenesis. Rat cDNAs were isolated and encoded by Msx-1 and -2, and then the expression patterns using in situ hybridization were investigated during early rat face development. These genes were correlatively expressed in the cranial neural crest forming area (E 9.5 dpc) and the facial process (E 12.5 dpc). Antisence inhibition of Msx genes in the E 12.5 mandibular process exhibited the alteration of their gene expression and cartilage patterns. Antisence inhibition of Msx-1 induced lack of the medial portion of cartilage, and antisence inhibition of Msx-2 enhanced chondrogenesis of mandibular process under the organ culture condition. Thus it was concluded that expression of Msx genes during mandibular process development comprises important signals of chondrogenesis.     

Magnetic resonance imaging of the temporomandibular joint in the rat compared with low-powered light microscopy

Objective

High magnetic field magnetic resonance imaging (MRI) was applied to the temporomandibular joint (TMJ) in the rat. The purpose of this study was the depiction of the internal structure of the TMJ, including the articular disc, articular cartilage, and the upper and lower joint cavities. We also proposed MRI settings and slices suitable for imaging the TMJ in the rat.

Methods

Temporomandibular joints from one female and eight male Sprague Dawley rats (5–8 weeks old) and four male Wistar–Hamamatsu rats (7–8 weeks old) were used. Using scout images, the horizontal plane was defined as being parallel to the body of the basisphenoid bone underneath the base of the brain. The coronal plane was defined as a slice vertical to the horizontal plane and vertical to the longitudinal fissure of the cerebrum. The sagittal plane was defined as a slice vertical to the horizontal plane and parallel to the longitudinal fissure of the cerebrum.

Results

T₁-weighted MR images with a spatial resolution of 75 μm were obtained for 5 min. The temporal bone and mandibular condyle were depicted as lower signal intensity images and the articular disc was depicted as an intermediate signal intensity image. In accordance with Gd-DTPA-enhanced MR or T₂-weighted MR images, the articular disc, articular cartilage, and the upper and lower joint cavities could be assigned clearly.

Conclusion

These MRI findings closely agreed with those observed with haematoxylin–eosin staining under light microscopy, suggesting that MRI is a useful method for analyzing the complex structure of the TMJ in the rat.     

鼠颞颌关节中弹力纤维的电镜观察

为了进一步观察鼠颞颌关节各部位的有胶原纤维和弹力纤维，作者应用计算机图象分析系统和电镜来研究鼠颞颌关节中三种弹力纤维的分布与形态。结果表明：在关节各部位中的弹力纤维以微原纤维和前弹力纤维为主。关节盘表面的微原纤维排列方向与胶原纤维几乎成直角。在关节盘表层下方和关节其它部位的弹力纤维排列上未见方向性。弹力纤维在关节盘和关节囊中形成一个广泛的纤维网。     

Growth hormone and insulin-like growth factor I receptors in the temporomandibular joint of the rat.

While there are numerous investigations on hormonal control of long bone epiphyseal growth, corresponding knowledge is sparse concerning the condylar cartilage. We investigated the distribution of growth hormone (GH) and insulin-like growth factor I (IGF-I) receptors in the temporomandibular joint (TMJ), especially the condyle, and compared the findings with information of long bone epiphyseal plates. The localization of the receptors was examined in vivo by immunohistochemical methods in one- to 21-day-old rats. GH receptors were detected in various components of the TMJ, but not in the fibrous articular surface or in the cartilage layers of the condyle. IGF-I receptors were found in the fibrous articular surface of the condyle and particularly in the superior and posterosuperior regions of the condylar cartilage, the depth of the labeled cell layer increasing significantly with age. It is evident that the expression of GH and IGF-I receptors is area-specific in the TMJ. Early post-natal growth and development of the mandibular condylar cartilage seem to be IGF-I-dependent but not directly dependent on GH.     

细胞凋亡与bcl-2基因在颞颌关节发育中的作用

目的;本研究通过检测胎鼠及出血后早期颞颌关节发育中的细胞凋亡与ｂｃｌ－２基因表达的变化,探讨细胞凋亡与ｂｃｌ－２基因在颞颌关节发发育中的作用。方法：利用原位末端标记法及原位杂交检测ｍＲＮＡ技术,对胚胎及生后１周ＳＤ大鼠颞颌关节发育不同时期细胞凋亡及ｂｃｌ－２基因的表达情况进行观察。     

Age-associated changes in decorin in rat mandibular condylar cartilage

The small proteoglycan decorin strongly binds the fibrils of collagen types I and II; this interaction is thought to play a part in the maintenance of tissue integrity and biomechanical properties. In limb articular cartilage, there is evidence that decorin synthesis increases with age and that it is elevated in response to increased loading or in osteoarthritic cartilage. The aim here was to characterize the presence and relative amount of decorin in the condylar cartilage of the temporomandibular joint (TMJ) with maturation by Western blotting, and to assess its tissue localization by immunohistochemistry. Comparative data were obtained from tibial articular cartilage, which has been extensively studied. Cartilage from the mandibular condyle and tibial plateau was harvested from 24-day-old (growing) and 161-day-old (young adult) female Sprague-Dawley rats. In growing animals, decorin appeared slightly more abundant in the mandibular condylar cartilage than in articular cartilage, whereas in young adult animals the decorin content in the TMJ cartilage was noticeably less than in limb articular cartilage. Although there was an increase in decorin abundance with age at the TMJ, the increase in decorin with age in limb articular cartilage was considerably more pronounced. These data indicate that, although decorin is present in mandibular condylar cartilage, its abundance in adults is less than in limb articular cartilage; thus, maturation-associated changes may be dissimilar in magnitude from those documented for limb articular cartilage.     

Swell1基因在小鼠髁突软骨发育中的时空表达

目的: 探讨Swell1(LRRC8A)基因在小鼠髁突软骨中的时空表达模式。方法: 通过获取胚胎15.5、16.5、18.5天以及新生小鼠的髁突样本,利用H-E染色、免疫荧光染色和qRT-PCR探讨小鼠髁突显微结构及软骨发育相关基因和Swell1基因的时空表达变化。采用SPSS 25.0软件包对数据进行统计学分析。结果: Swell1基因表达于髁突发育过程中,从胚胎16.5天开始,在肥大软骨细胞层表达,之后表达逐渐增强,并在小鼠胚胎发育过程中持续表达,直至小鼠出生仍有明显表达。结论: Swell1在小鼠髁发育过程中主要在肥大软骨细胞中表达,可能参与软骨细胞肥大的调控。     

Tissue regeneration induced by a transgene for bone morphogenetic protein-12 (BMP-12) in a bone defect

BMP-12, other than BMP-2, 4, and 7, is known to induce the formation of tendon/ligament-like tissues. However, the role of the protein in the process of bone or cartilage healing has not been adequately examined. The purpose of the present study was to examine if a plasmid vector encoding BMP-12 would induce tendon/ ligament formation in a rat bone defect model. A lyophilized atelocollagen pellet with 100 microg of a plasmid vector encoding BMP-12 or BMP-2 was implanted in a bone defect of a rat femoral metaphysis. The samples were retrieved at 2, 4 and 8 weeks post-operation and subjected to histology and real-time PCR analysis. BMP-12 transgene indicated prominent fibrogenesis at 2 weeks. Consequently, the tissue was substituted by bone at 8 weeks. The gene expression of collagen type III was up-regulated by BMP-12 gene transfer after 2 weeks, followed by an increase of elastin and Six1 genes after 4 weeks. These genes, which are intimately related to tendon/ligament formation, decreased in expression at 8 weeks in preparation for osteogenesis afterward. On the other hand, the BMP-2 transgene quickly induced the synthesis of bone-related genes such as collagen type I and ALP. It is concluded that BMP-12 can initiate the development of tendon/ligament-like tissue in a rat bone defect at early healing stages. Maintaining such generation of tissue on bone could be useful for modifying periodontal tissue engineering.