Experimentally induced unilateral tooth loss: histochemical studies of the temporomandibular joint

Occlusal abnormality may contribute to osteoarthrosis of the temporomandibular joint (TMJ). Since mechanical force may induce changes in the extracellular matrix, we tested the hypothesis that unilateral removal of teeth and the resulting unilateral mastication change the content of glycosaminoglycans (GAGs) in the TMJ condyle and disc. Lower-right-side teeth were extracted from 12 adult male rabbits, which were killed 3 or 6 weeks later. Three normal rabbits served as controls. Sections were analyzed for morphological changes and levels of sulfated GAGs in the condyle and disc. Unilateral removal of teeth led to thickening of the condylar cartilage, alterations in the morphology of chondrocyte nuclei in the condylar cartilage and disc, and increases in levels of negatively charged ions in the hypertrophic layer of condylar cartilage. Small differences were observed, after unilateral removal of teeth, between the functional and non-functional sides of the TMJ. The results suggest that in response to mechanical stress, chondrocytes alter sulfated glycosaminoglycan (GAGs) synthesis and degradation rates, resulting in an elevated level of sulfated GAGs in the condylar cartilage.     

颞下颌关节盘前移位后髁突软骨细胞基质基因表达的变化

目的　探讨颞下颌关节盘前移位后髁突软骨细胞基质基因表达的变化。方法　建立40只大白兔颞下颌关节盘前移位动物模型,用地高辛标记cRNA探针原位杂交技术,检测术后不同病变时期髁突软骨细胞Ⅱ型胶原和蛋白多糖聚合体基因表达的改变。结果　正常髁突的增殖层深层、肥大层和钙化层细胞胞浆内可见大量Ⅱ型胶原和蛋白多糖聚合体mRNA表达。术后1周蛋白多糖聚合体开始下调,至2周达低谷,6周后恢复至正常水平。Ⅱ型胶原4周后开始持续下降,其表达下降程度较蛋白多糖聚合体显著,8周后逐渐恢复正常。结论　关节盘前移位后髁突软骨的基质基因表达发生有序、协调的变化,这种变化意味着适应性改建的启动。     

髁突骨上组织改建中Ⅰ、Ⅱ型胶原的免疫组织化学研究

目的：研究髁突骨上组织改建中Ⅰ、Ⅱ型胶原的变化特征,增加对颞颌关节改建的进一步认识。方法：用免疫组织化学的方法检测拔除成年家兔左侧下凳磨牙后２周、１月和３月时Ⅰ、Ⅱ型的分布及含量的变化。结果：术后２周Ⅰ、Ⅱ型胶原的表达均明显减少,１月和３月其表达有所回升,并且１型胶原出现不均衡分布,拔牙侧与非拔牙侧相比,前者１型胶原的表达较后者稍强,而２型胶原的表达则较弱。结论：成年家兔颞颌关节的改建能力有限,超     

小型猪一侧失牙及义齿修复后颞下颌关节的组织病理学观察

目的：观察一侧失牙及义齿修复对小型猪颞下颌关节（temporomandibular joint，TMJ）组织结构的影响。方法：11只小型猪随机分为空白组（2只）、拔牙组（4只）、修复组（5只），将拔牙组和修复组左侧后牙全部拔除，修复组并于拔牙后3个月进行义齿修复。小型猪一侧失牙6个月时处死，解剖TMJ并制作光镜（HE染色）和透射电镜标本，观察其组织结构改变。结果：拔牙组和修复组小型猪双侧TMJ均发生不同程度的退行性改变。髁突软骨表面带玻璃样变性、断裂、剥脱；肥大带下陷成乳突样，软骨细胞成簇；骨小梁破坏呈蚕蚀状。关节盘胶原纤维变性、紊乱、断裂，盘后组织出现明显软骨化生。髁突软骨瘀斑、凹陷、剥脱的超微病理特征分别是细胞变性、凋亡、坏死。结论：长期一侧失牙可导致双侧TMJ退行性变，及时义齿修复可减轻TMJ的病理损伤。     

Recombinant adeno-associated virus serotype 2 (rAAV2)—An efficient vector for gene delivery in condylar cartilage, glenoid fossa and TMJ disc in an experimental study in vivo

Objective

To elaborate whether rAAV2 can be used for future TMJ gene therapy, we examined the infection efficiencies of rAAV2 in vitro, and the transgene expression pattern mediated by rAAV2 in glenoid fossa, TMJ disc and condylar cartilage in vivo.

Materials and methods

Different dosages of rAAV2-eGFP (MOI: 5 × 10⁴, 1 × 10⁴, 5 × 10³) were applied to primary cultured condylar chondrocytes of rats. Infection efficiencies were analysed by FACSCalitur at different time points. Vastatin, a molecule not naturally expressed in TMJ, was used as a reporter for detection of rAAV2 mediated transgene expression in vivo. Thirty SD rats were injected with either rAAV2-sec-Vastatin (experimental group) or rAAV2-eGFP (control group) into both sides of TMJ. They were sacrificed at the indicated time (7, 14, 21, 30 and 60 days of injection) and the TMJ samples were collected for RT-PCR and immunostaining analysis.

Results

High dosage (MOI 5 × 10⁴) of rAAV2-eGFP can achieve desirable transduction efficiencies in vitro after 5 days. Transgene expression of rAAV-sec-Vastatin persisted for about 21 days in glenoid fossa, around 7 days in TMJ disc and at least 60 days in condylar cartilage in vivo. In condylar cartilage, transgene expression was found in the proliferative layer and chondroblast layer (day 7), chondrocyte layer (day 14), pre-hypertrophic and hypertrophic layer (day 21), hypertrophic layer and deep hypertrophic layer (day 30 and 60).

Conclusion

Recombinant AAV2 could be considered as a promising vector for gene therapy in TMJ which can mediate therapeutic gene expression in glenoid fossa, articular disc and condylar cartilage in vivo.     

Cartilage changes link retrognathic mandibular growth to TMJ disc displacement in a rabbit model

Recent experimental research demonstrated that non-reducing temporomandibular joint (TMJ) disc displacement in growing rabbits impaired mandibular growth. TMJ disc displacement is also shown to induce histological changes of the condylar cartilage. The authors hypothesized that the severity of these changes would correlate to the magnitude of mandibular growth. Bilateral non-reducing TMJ disc displacement was surgically created in 10 growing New Zealand White rabbits. Ten additional rabbits constituted a sham operated control group. Aided by tantalum implants, growth was cephalometrically determined for each mandibular side during a period equivalent to childhood and adolescence in man. At the end of the growth period, histologically classified cartilage features were correlated with the assessed ipsilateral mandibular growth. Non-reducing displacement of the TMJ disc during the growth period induced histological reactions of the condylar cartilage in the rabbit model. The severity of cartilage changes was inversely correlated to the magnitude and the direction of mandibular growth, which resulted in a retrognathic growth pattern.     

生长期兔颞下颌关节盘前移位对髁突软骨内Ihh和PTHrP表达的影响

目的：研究颞下颌关节盘前移位对生长期兔髁突软骨印度豪猪蛋白（Ihh）和甲状旁腺相关蛋白（PTHrP）表达的影响,探讨关节盘前移位与下颌骨髁突生长发育之间的关系。方法：取3个月龄日本大耳白兔21只．建立颞下颌关节盘前移位动物模型,分别于建模后4、8、12周处死取材,观察髁突软骨组织学变化,并通过免疫组织化学方法检测Ihh、PTHrP在髁突软骨中的表达与定位。结果：关节盘移位后4周,髁突软骨结构轻度紊乱;8周时,髁突前部软骨形态结构发生显著改变;12周时．软骨正常结构丧失进一步加剧。关节盘移位后4周,可见Ihh、PTHrP在增殖带深层细胞内明显表达,8周、12周时在髁突深层软骨细胞内表达。结论：颞下颌关节盘前移位导致髁突软骨结构进行性病理性损害．Ihh和PTHrP在盘移位后髁突软骨病理性改变的过程中发挥重要作用．提示关节盘前移位可能通过Ihh-PTHrP负反馈信号通路对生长期髁突软骨内成骨过程产生影响。     

Ⅰ、Ⅱ、Ⅹ型胶原及碱性磷酸酶在鼠胚髁突软骨发生中的意义

目的　探讨髁突软骨发生中Ⅰ、Ⅱ、Ⅹ型胶原及碱性磷酸酶(ALP)的组织学分布特征及髁突软骨内成骨的 分子机制。方法　取14~18 d鼠胚,分别行Ⅰ、Ⅱ、Ⅹ型胶原及ALP抗体免疫组织化学染色。结果　胚胎第14天, 髁突形成的位置可见间充质细胞聚集并与骨膜相连,间充质细胞及骨膜中Ⅰ型胶原及ALP阳性;第15天,肥大软 骨细胞中Ⅹ型胶原表达阳性,其周围的间充质细胞中Ⅰ、Ⅱ型胶原阳性,ALP在两种细胞中均呈阳性;第16天,软骨 膜、纤维层间充质细胞至肥大软骨细胞上层中Ⅰ型胶原表达阳性,多形细胞层下方至肥大软骨细胞下层中Ⅱ型胶 原表达阳性,Ⅹ型胶原仅表达于肥大软骨细胞,ALP在软骨膜及肥大软骨细胞中呈阳性,但在多形细胞层呈阴性或 弱阳性。结论　髁突软骨的发生机制与长骨不同,其软骨内成骨的早期Ⅰ、Ⅱ、Ⅹ型胶原均有表达,可能始发于ALP 阳性的下颌骨膜间充质细胞。     

后牙缺失引起关节盘改建的组织学及免疫组化的研究

目的 :研究一侧后牙缺失后双侧颞下颌关节关节盘细胞及细胞外基质的变化规律。方法 :拔除成年家兔左侧下颌磨牙 ,用免疫组化方法检测Ⅰ、Ⅱ、Ⅲ型胶原在术后 2周、1个月和 3个月双侧关节盘的分布及含量变化。结果 :非拔牙侧关节盘出现钙化、纤维化、血管侵入、形变等 ,而拔牙侧只有钙化、纤维走行改变 ;双侧Ⅰ、Ⅱ型胶原的表达均明显减少 ,并且Ⅰ型胶原出现不均衡分布 ;Ⅲ型胶原的表达术后明显增多 ,以非拔牙侧更明显。结论 :后牙缺失过久会导致成年家兔关节盘抗压抗拉特性下降 ,脆性增加 ;单侧后牙缺失时 ,有牙侧关节盘的病变更显著     

Effects of growth factors and glucosamine on porcine mandibular condylar cartilage cells and hyaline cartilage cells for tissue engineering applications

Temporomandibular joint (TMJ) condylar cartilage is a distinct cartilage that has both fibrocartilaginous and hyaline-like character, with a thin proliferative zone that separates the fibrocartilaginous fibrous zone at the surface from the hyaline-like mature and hypertrophic zones below. In this study, we compared the effects of insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF), transforming growth factor beta1 (TGF-β1), and glucosamine sulphate on porcine TMJ condylar cartilage and ankle cartilage cells in monolayer culture. In general, TMJ condylar cartilage cells proliferated faster than ankle cartilage cells, while ankle cells produced significantly greater amounts of glycosaminoglycans (GAGs) and collagen than TMJ condylar cartilage cells. IGF-I and bFGF were potent stimulators of TMJ cell proliferation, while no signals statistically outperformed controls for ankle cell proliferation. IGF-I was the most effective signal for GAG production with ankle cells, and the most potent upregulator of collagen synthesis for both cell types. Glucosamine sulphate promoted cell proliferation and biosynthesis at specific concentrations and outperformed growth factors in certain instances. In conclusion, hyaline cartilage cells had lower cell numbers and superior biosynthesis compared to TMJ condylar cartilage cells, and we have found IGF-I at 100 ng/mL and glucosamine sulphate at 100 μg/mL to be the most effective signals for these cells under the prescribed conditions.     

Disc-like changes and type II collagen mRNA expression in the bilaminar zone of rabbit temporomandibular joint following disc displacement]

OBJECTIVE: To study the adaptive alteration in bilaminar zone of rabbits' temporomandibular joint following disc displacement. METHODS: Twenty-six Japanese white rabbits were used in this study. Among these rabbits,6 were used as controls. The right discs of other 20 rabbits were displaced anteriorly by operation. Four of these rabbits were killedatn 1, 2, 4, 6 and 8 weeks respectively after surgery. The TMJS were studied by HE staining, Alcin bluen staining and in situ detection of type II collagen mRNA expression. RESULTS: There appeared cartilage metaplasia after one week following disc displacement. Typical chondrocytes could be found in the bilaminar zone. The new chondrocytes expressed type II collagen. CONCLUSIONS: The bilaminar zone of TMJ will be remodeled following disc displacement and become a disc-like tissue to function as a disc.     

心理应激后大鼠颞下颌关节系统的病理性改变

目的:观察心理应激对大鼠颞下颌关节(temporomandibularjoint,TMJ)的影响及其病理改变。方法:90只SD大鼠,随机分为3组:对照组,心理应激组和足部电击组。每组分别在1、3、5周后观察大鼠的关节盘,髁突及翼外肌的显微和超微结构,并与正常组动物进行比较。结果:心理应激组动物的TMJ出现明显的病理性改变,扫描电镜显示1周时髁突深面胶原纤维暴露,裂缝周围纤维排列紊乱。颞下颌关节盘及髁突均出现胶原纤维断裂,其中3周组动物病理性改变最为严重,可见翼外肌细胞线粒体水肿,严重者出现空泡样变5周时出现适应性表现。结论:长期的心理应激可引起TMJ发生病理性改变,但随着心理应激时间的延长,关节可能会出现一定程度的修复,这可能与动物的适应性有关。     

Type II collagen and aggrecan mRNA expressions in rabbit condyle following disc displacement

The aim of this investigation was to study the remodelling of cartilage in the mandibular condyle following disc displacement (DD) of the temporomandibular joint (TMJ). Forty adult Japanese white rabbits were used in this study. The right joints of 28 of the 40 rabbits had their discs surgically displaced. Four of the 28 were killed at 4 days or 1, 2, 4, 6, 8 and 12 weeks after surgery. The messenger RNA (mRNA) expression levels of aggrecan and type II collagen in cartilages were measured using in situ hybridization techniques. Results showed that aggrecan mRNA expression reduced in the first week after DD. The expression began to recover after 4 weeks and reached a normal level after 6 weeks. Type II collagen mRNA expression reduced from 4 weeks and the expression recovered after 8 weeks. This suggests that the chondrocyte reacting to the displacement of the TMJ disc, alters its matrix gene expression patterns and it is may be the cause of the shape changes of TMJ after DD.     

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。     

七种不同（牙合）型对颞下颌关节应力影响的三维有限元分析

目的　探讨由形态变化产生的根尖主应力变化对颞下颌关节 (TMJ)应力的影响。方法　取光弹法测得的正中载荷作用下 7种型各牙根尖应力参数 ,在所建下颌骨三维有限元模型上加载 ,比较其TMJ应力。结果　 (1)髁突前斜面及其表面软骨和关节盘中部主要承受压应力 ,髁突后斜面及其表面软骨和关节盘后部主要承受拉应力 ;(2 )与正常相比 ,平面、远中、部分后牙反、缺牙的TMJ应力均有不同程度变化 ,其中平面的变化最明显。单侧上颌第三磨牙伸长、单侧下颌第三磨牙伸长的TMJ应力变化均不明显。结论　由型改变导致的正中咬合时根尖主应力变化 ,对关节应力性质的影响不明显 ,但对应力大小有一定影响。     

下颌前方牙齿缺失修复前后对颞下颌关节应力分布影响的三维有限元研究

目的:研究因前牙缺失导致的根尖主应力变化对颞下颌关节(TM J)应力分布的影响。方法:将光弹法测得的正中咬合时各牙合型牙根主应力数据,在三维有限元模型上加载,比较TM J各部应力变化。结果:1)髁突前斜面及所对应的关节软骨和关节盘的中部主要承受压应力,髁突后斜面及关节盘的后部主要承受拉应力。2)前牙5-5缺失可以导致TM J应力明显减小,修复后可有效的消除这种影响。结论:前牙缺失可引起TM J应力变化,有可能进一步导致关节病的发生,应当及时修复。     

Condyle and mandibular bone change after unilateral condylar neck fracture in growing rats

Unilateral fracture of the condylar neck in immature subjects might lead to mandible asymmetry and condyle remodelling. A rat model was used to investigate mandibular deviation and condylar remodelling associated with condyle fracture. 72 4-week-old male rats were randomly divided into three groups: an experimental group (unilateral transverse condylar fracture induced surgically), a sham operation group (surgical exposure but no fracture), and a non-operative control group (no operation). The rats were killed at intervals up to 9weeks after surgery, and outcomes were assessed using various measures of mandible deviation, histological and X-ray observation, and immunohistochemical measures of expression levels of connective tissue growth factor (CTGF) and type II collagen (Col II). The fracture led to the degeneration of mandibular size, associated with atrophy of fractured condylar process. Progressive remodelling of cartilage and increasing expression levels of CTGF and Col II were found. The authors conclude that condylar fracture can lead to asymmetries in mandible and condyle remodelling and expression of CTGF and Col II in condylar cartilage on both the ipsilateral and the contralateral sides.     

Matrix metalloproteinase and its inhibitor in temporomandibular joint osteoarthrosis after indirect trauma in young goats

Our aim was to examine the change in expression of matrix metalloproteinases (MMP-13), matrix metalloproteinases-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the articular cartilage of goats with experimentally-induced osteoarthrosis of the temporomandibular joint (TMJ) at various times. Osteoarthrosis was induced in 20 goats in the bilateral TMJ and 5 goats acted as controls. There were 5 goats in each group, and a group was killed at 7 days, and 1, 3, and 6 months postoperatively. The samples were collected, and the joints evaluated histologically. Immunofluorescence was used to detect the presence of MMPs and TIMP-1 in the articular disc and condylar cartilage. The ultrastructure of the articular disc and condylar surface at 1 month was examined with scanning electron microscopy (SEM). Osteoarthrosis of the TMJ progressed gradually over time. MMP-13, MMP-3, and TIMP-1 were expressed strongly in the TMJ soon after injury; MMP-13 became gradually weakened, and MMP-3 strengthened later. None of these were expressed in the normal condyle. After a month the surface of the arthrotic condyle was uneven, and the underlying collagen fibrils were exposed in irregular fissures on the surface. The secretion of TIMP-1 was related closely to the changes of MMPs during osteoarthrosis of the TMJ. The unbalanced ratio between them caused degradation of the matrix of the cartilage and might be the cause of osteoarthrosis of the TMJ.     

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。     

关节盘穿孔致大鼠颞下颌关节骨关节病的形态学及组织病理学观察

目的通过关节盘穿孔建立大鼠颞下颌关节骨关节病(temporomandibular joint osteoarthrosis,TMJOA)模型,并观察颞下颌关节(temporomandibular joint,TMJ)髁突的形态学特征及组织病理学变化。方法8只12周龄雄性SD大鼠购自重庆医科大学,体质量200~250 g,通过随机数表法将其分为1周组和4周组,每组各4只。用齿科慢机球钻在每只大鼠左侧(模型侧)关节盘中后部形成直径1.5 mm的规则圆形穿孔,右侧(对照侧)不予处理。1周组和4周组大鼠分别于处理后1、4周时处死,获取双侧TMJ组织,通过形态学观察、影像学检查分析关节软骨的退化程度及软骨下骨的结构变化。通过免疫组织化学染色、苏木精染色、番红O-固绿染色、抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色,分析大鼠TMJ髁突软骨的组织病理学变化。结果形态学观察显示1周组模型侧关节盘红肿明显,4周组模型侧可见典型的骨关节病鸟嘴样改变,1周组和4周组模型侧髁突的高度[分别为(2.73±0.14)和(2.49±0.25)cm]均显著低于对照侧[分别为(3.30±0.09)和(3.30±0.12)cm](P<0.01);影像学结果显示4周组模型侧骨小梁微观结构破坏明显;番红O-固绿染色结果显示1周组及4周组模型侧的蛋白聚糖均显著低于对照侧(P<0.01);TRAP染色结果显示1周组及4周组模型侧的破骨细胞数目均显著多于对照侧(P<0.01);免疫组织化学染色、苏木精染色结果显示1周组及4周组模型侧的胶原纤维密度较对照侧均显著减少(P<0.01),而基质金属蛋白酶显著增多(P<0.01)。结论大鼠关节盘穿孔后,模型侧髁突软骨出现了典型的TMJOA病理改变。