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.     

周期性单轴压力对大鼠髁突软骨细胞结缔组织生长因子表达的影响

目的 探讨在髁突软骨细胞受到周期性单轴压力后,结缔组织生长因子(connective tissuegrowth factor, CTGF)表达的影响,为正畸治疗中髁突软骨受力后的改建提供生物学依据。方法 选取1周龄SD大鼠,提取并培养髁突软骨细胞,免疫组化鉴定。利用四点弯曲细胞力学加载仪对第3代细胞进行力值为2000u strain、0.5Hz的体外周期性单轴压力加载,分别在加力0min、30min、60min和120min后继续培养24h,应用蛋白印迹法检测在不同加力时间CTGF蛋白表达的变化。应用SPSS18.0软件对数据进行统计学分析。结果 CTGF的相对蛋白量在加力0min、30min、60min和120min后,分别为0、1.59、2.34和3.16,随着加载时间的增加,表达呈逐渐上升趋势。且组间差异具有统计学意义(P<0.05) 结论 周期性单轴压力可刺激大鼠髁突软骨细胞CTGF的表达。     

Effect of decreased loading on the metabolic activity of the mandibular condylar cartilage in the rat

The aim of this study was to measure the effect of decreased temporomandibular loading on the proliferative activity and the level of matrix production of the condylar cartilage. The effect of reduced joint loading on the activity of stromelysin-1 (MMP-3), which has been associated with conditions of articular cartilage matrix breakdown, was also examined. Eighty 14-day-old female rats were assigned to two groups. Following weaning at 20 days, the experimental group was fed a soft diet and the incisors were shortened regularly to keep them out of occlusion. The controls were fed a hard diet. The activity of tritiated thymidine incorporation and the incorporation of radiolabelled sulphur were measured 2, 6, 12, 24 and 48 hours after initiation of the experiment. The radiolabelled sulphur intake was significantly lower in the condylar cartilage of the experimental group 6-24 hours after initiation of the experiment, and tritiated thymidine activity was lower after 12-24 hours, indicating lower proliferation and matrix production. The cartilage in the experimental group showed marked immunostaining against MMP-3 in all cartilage layers 9 days after initiation of the experiment. In the control group, the staining was clearly seen only in the superficial fibrous layer and in the erosion front. A marked reduction in proliferative activity and proteoglycan synthesis in mandibular condylar cartilage was found after a continuous soft diet and suppressed incisal mastication in the rat. The results show that sufficient loading is important for condylar cartilage growth, to maintain both ideal proliferation and matrix chondrocyte production.     

An experimental study on the growth of condylar cartilage, using a new vascularized mandible heterotopic transplant model.

PURPOSE: To clarify the influence of external and dynamic factors on the growth of mandibular condyle, we developed vascularized mandible heterotopic transplant (VMHT) model. In this report, we discuss histologic changes in the mandibular condylar cartilage in the absence of external factors, using VMHT models. MATERIALS AND METHODS: Thirty VMHT model using male Lewis rats aged 6 weeks were produced. Six VMHT rats were randomly selected for death at 1, 2, 4, 7, and 14 days after transplantation. We histologically investigated the mandibular condyles of the grafts and controls, the left mandibles of recipient rats. RESULTS: In the transplanted mandibular condyle of VMHT models, the cell arrangement became increasingly irregular with the passage of time, with a concomitant, irregular thickening or thinning of the cartilage layer. There was heterotopic fibrous ossification, extending horizontally under the proliferative cell layer in these regions of thin cartilage. After these events, cartilage cells had disappeared almost completely by 14 days after transplantation. CONCLUSIONS: This study indicated that external and dynamic factors are not necessary for the differentiation and proliferation of the condylar cartilage cells. Instead, these factors affect the maintenance of the orderly growth of the cartilage cells and may serve a critical role in the differentiation of undifferentiated mesenchymal cells into chrondroblasts.     

下颌持续前导对成年大鼠髁突软骨骨形态发生蛋白表达及超微结构的影响

目的 观察成年期大鼠在下颌持续前导作用下髁突软骨的改建以及超微结构的变化。方法 将30只9周龄SD大鼠随机分为实验组和对照组,实验组佩戴上颌斜面导板,对照组不做任何处理,分别在第3、7、14、21、30天处死动物并取材,免疫组织化学染色检测骨形态发生蛋白2（BMP-2）在髁突中的表达及分布,透射电镜观察髁突软骨细胞的超微结构,Micro CT分析髁突骨质的变化。结果 与对照组相比,实验组髁突中部和后部软骨增生明显,软骨细胞BMP-2阳性细胞率及灰度值在第7天开始增多,随时间延长而增强,髁突软骨细胞的超微结构出现细胞核固缩,核周微丝变少、脂滴变小、内质网腔隙肿胀、细胞外基质增宽变多等。Micro-CT显示实验组的新生骨和骨小梁厚度、骨小梁数量和分离度随时间延长而增加。结论 在下颌持续前导下,成年期大鼠髁突软骨出现增生性改建,并存在BMP-2的高表达。     

Changes in growth and morphology of the condyle following mandibular distraction in minipigs: overloading or underloading?

OBJECTIVE: Loading of temporomandibular tissues during mandibular distraction may cause changes in condylar growth and cartilage thickness. This study examines the effects of distraction on the condyle in a large animal model by explicitly measuring growth and in vivo loading. DESIGN: Unilateral mandibular distraction was carried out on 20 growing minipigs divided into three groups. One group underwent distraction but not consolidation, whereas the other two groups were allowed a period of consolidation of either 1 or 2 weeks. Animals received fluorochrome and 5-bromo-2'-deoxyuridine (BrdU) labelling and masticatory strain was measured from the condylar neck. Condylar strain was also recorded in an age-matched sample of eight animals that received no distraction surgery. Immunohistochemical procedures were used to identify dividing prechondroblasts and histological analysis was used to measure mineral apposition rate, count dividing cells, and measure the thickness of condylar cartilage. RESULTS: Strain magnitude, particularly compressive strain, was much larger on the non-distraction side compared to the distraction side condyle. Compared to normal loading levels, the distraction side condyle was underloaded whereas the condyle on the intact side was overloaded. Mineral apposition and cartilage thickness were greater on the distraction side condyle compared to the opposite side. Differences between the sides were most pronounced in the group with no consolidation and became progressively reduced with consolidation time. CONCLUSIONS: Increased mineralisation and cartilage thickness on the distraction side condyle are associated with reduced, not increased loading, perhaps because of disruption of the distraction side masseter muscle.     

Static and dynamic loading of mandibular condyles and their positional changes after bilateral sagittal split advancement osteotomies

This study analysed the effects of change of direction of masseter (MAS) and medial pterygoid muscles (MPM) and changes of moment arms of MAS, MPM and bite force on static and dynamic loading of the condyles after surgical mandibular advancement. Rotations of the condyles were assessed on axial MRIs. 16 adult patients with mandibular hypoplasia were studied. The mandibular plane angle (MPA) was <39° in Group I (n=8) and >39° in Group II (n=8). All mandibles were advanced with a bilateral sagittal split osteotomy (BSSO). In Group II, BSSO was combined with Le Fort I osteotomy. Pre and postoperative moment arms of MAS, MPM and bite force were used in a two-dimensional model to assess static loading of the condyles. Pre and postoperative data on muscle cross-sectional area, volume and direction were introduced in three-dimensional dynamic models of the masticatory system to assess the loading of the condyles during opening and closing. Postsurgically, small increases of static condylar loading were calculated. Dynamic loading decreased slightly. Minor rotations of the condyles were observed. The results do not support the idea that increased postoperative condylar loading is a serious cause for condylar resorption or relapse.     

微RNA在下颌髁突软骨生长发育中作用的研究进展

微RNA(micro RNA,miRNA)是一类小的非编码单链RNA,通过与信使RNA结合发挥其生物学作用。研究显示miRNA可能在调节下颌髁突软骨的生长发育中起重要作用。本文就miRNA的产生及其作用机制,对下颌髁突软骨生长发育相关的miRNA研究进展进行综述,以期助于进一步研究下颌髁突软骨生长发育。     

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     

TGF-β在下颌髁突软骨的发育和改建中作用研究进展

［摘要］ 转化生长因子β(transforming growth factor beta,TGF-β)是一种多功能的细胞因子,广泛参与细胞的多种生物学过程,如细胞增殖分化、胞外基质的合成和组织修复等。近年来的研究发现,髁突软骨的发育必须有TGF-β的参与,TGF-β可介导局部的功能刺激作用,参与髁突软骨的适应性改建,与髁突软骨退行性病变相关。本文就TGF-β在髁突软骨发育、改建和退行性关节病中的作用作一综述。     

Origin of mandibular condylar cartilage in mice,rats, and humans: Periosteum or separate blastema?

ObjectiveTo investigate if mandibular condylar cartilage is derived from the periosteum of the ossifying mandible or from a separate, programmed blastema.Materials and methodsFetal mice at E14.0–16.0, fetal rats at E16.0–18.0, and human embryos at 9 and 10 wks of gestation were used. The initial formation of rat condylar cartilage was investigated by using serial sections and enzyme-histochemistry to detect alkaline phosphatase activity. Histological observations of serial sections of human fetuses as well as 3D-reconstruction models were also analyzed. The expression of collagen type mRNA in developing rat condylar cartilage was directly compared with that in mice by performing in situ hybridization.ResultsAn anlage of the rat condylar process (condylar anlage) was clearly identified in the posterior position of the ossifying mandible and was continuous with it at E16.0. Newly formed rat condylar cartilage was observed at E16.5 and was continuous with the ossifying mandible. Mesenchymal cells in the condylar anlage at E16.0 showed alkaline phosphatase activity and chondrocytes in the newly formed condylar cartilage also showed enzymatic activity. Thus, rat mandibular condylar cartilage that derives from alkaline phosphatase-positive periosteum-like cells is continuous with the ossifying mandible, as previously demonstrated in mice, but rapid differentiation into hypertrophic chondrocytes in rats is not remarkable compared to that in mice. The condylar anlage and the newly formed cartilage were also continuous with the ossifying mandible in human embryos.ConclusionsMammalian mandibular condylar cartilage derives from the periosteum of the ossifying mandible in mice, rats, and humans.     

富血小板血浆修复生长期兔髁突软骨损伤后下颌骨生长发育的三维评估研究

目的　建立生长期髁突软骨及软骨下骨损伤动物模型,利用Materialise Proplan CMF 3.0软件对下颌骨进行三维测量,探讨富血小板血浆（PRP）对生长期髁突软骨损伤修复及维持下颌骨生长发育的作用。方法　生长期新西兰大白兔30只随机分为3组。PRP修复组（10只）建立髁突软骨及软骨下骨损伤动物模型后置入激活后的PRP;手术组（10只）建立髁突软骨及软骨下骨损伤动物模型;假手术组（10只）除不造成软骨及软骨下骨损伤外,其余与手术组相同。术后6周、12周对动物头颅进行螺旋CT扫描,采集SPCC格式数据。利用Materialise Proplan CMF 3.0软件对硬组织进行三维重建并分离上下颌骨,完整显露髁突的形态,确定标志点、参考平面及相应的测量指标,对测量结果进行统计学分析。结果　各个时间点手术组动物各项测量指标的测量值均小于假手术组与PRP修复组动物的（P < 0.05）;术后6周,PRP修复组动物的下颌升支高度、髁突长度、髁突宽度测量值均小于假手术组的（P < 0.05）;PRP修复组动物的下颌体长度、髁突高度测量值与假手术组动物的相比差异无统计学意义（P > 0.05）;术后12周PRP组动物的下颌升支高度、下颌体长度测量值均小于假手术组动物的（P < 0.05）,PRP修复组动物的髁突高度、髁突长度、髁突宽度测量值与假手术组动物的差异无统计学意义（P > 0.05）。结论　生长期髁突软骨损伤后,下颌骨生长发育相对停滞,髁突发生吸收。激活后的PRP可有效修复生长期髁突软骨损伤,维持下颌骨的正常形态,促进下颌骨及髁突的正常生长发育。     

The effects of gradually induced backward movement of the mandible by a Twin Inclined Plane Device in rats

Abstract Objective: To develop a model of gradually induced backward movement of the mandible under normal masticatory action and to examine morphological changes in the mandible and condylar cartilage in rats. Materials and Methods: The newly developed Twin Inclined Plane Device (TIPD) was composed of upper and lower posterior metal crowns with a long inclined plane on both sides separately and was applied in experimental groups of 6-week-old male Wister rats. After 3, 14, 30, and 60?days, the rats were euthanatized and samples were collected. Various measurements and hematoxylin-and-eosin stains were performed. Results: From day 30 on, the length of the condylar process was shorter in the TIPD groups than in the control groups (P < .05). The angulation of the condylar process axis to the mandibular plane was greater in the TIPD groups (P < .01). The thickness of the condylar cartilage in the posterior part of the posterior region was thinner in the TIPD groups (P < .05) on day 30 and even thinner (P < .01) on day 60; from day 30 on, the thickness in the anterior part of the posterior region was thicker in the TIPD groups (P < .01). Conclusion: TIPD can successfully induce backward movement of the mandible under normal masticatory action. TIPD can cause region-specific changes in condylar cartilage and leads to a continuous remodeling.     

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??Objective??An animal model of condylar cartilage and subchondral bone injury during growing period is established??and three-dimensional measurement of mandibular was performed with Materialise Proplan CMF 3.0 software to investigate the effect of PRP on repairing condylar cartilage and maintaining mandibular growth in growing period. Methods??Thirty growing New Zealand white rabbits were randomly divided into three groups. The PRP group animals ??10?? were implanted in activated PRP after the establishment of the animal model of condylar cartilage and subchondral bone injury.The condylar cartilage and subchondral bone injury models were established in the operation group ??10??. The sham operation group ??10?? was similar to the operation group except for the condylar cartilage and subchondral bone injury. At 6 and 12 weeks after surgery the animals received spiral computed tomography??and SPCC data were collected.Maxillary and mandibular three-dimensional reconstruction was performed using Materialise Proplan CMF 3.0. The upper and lower jaws were separated and the morphology of condylar was completely revealed. The result of measurement are analysed with statistical method after the determining the marking points??reference planes and corresponding measurement indexes. Results??The indexes of the operation group were all smaller than those of the sham operation group and the PRP group at each time point??P < 0.05????6 weeks after surgery??the height of mandibular ramus??the length of condyle and the width of condyle in PRP group were less than those in sham operation group??P < 0.05??. There was no significant difference in mandibular length or condylar height between PRP group and sham operation group ??P > 0.05??. The mandibular ramus height and mandibular body length in PRP group were less than those in sham operation group at 12 weeks after operation??P < 0.05??. There was no significant difference in condylar height??length or width between PRP group and sham operation group ??P > 0.05??. Conclusion??After the injury of mandibular condylar cartilage??the growth and development of mandibular are relatively stagnant??and the mandibular condyle is absorbed. Activated PRP can effectively repair the injury of condylar cartilage during growing period??promote the normal growth and development of mandible and condyle??and maintain the normal shape of mandible.     

前伸下颌后大鼠髁突软骨内明胶酶表达变化的研究

目的研究前伸下颌后明胶酶（MMP- 2、MMP- 9）在大鼠髁突软骨内表达的变化。方法选取5周龄雄性SD大鼠60只,随机等量分成实验组和对照组,实验组大鼠白天配戴自制的上颌斜面导板式活动矫治器引导下颌前伸,对照组大鼠不戴矫治器。在实验的1、2、4周处死动物,免疫组化检测明胶酶在大鼠髁突软骨内表达的变化。结果在正常髁突软骨内,MMP- 2呈中等强度表达,MMP- 9表达很低。前伸下颌后,髁突软骨内MMP- 2的表达无明显变化,而MMP- 9的表达显著增高（P<0.01）。结论明胶酶参与了功能矫形时下颌髁突软骨的适应性改建。     

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.     

雌激素受体与Ⅱ型胶原在人胚髁突软骨中的表达

目的探讨雌激素受体（ER）和Ⅱ型胶原在人胚髁突软骨中的表达情况。方法 采用苏木精-伊红（HE）及免疫组织化学染色法检测ERα、ERβ和Ⅱ型胶原在人胚颞下颌关节髁突软骨各层的表达。结果 Ⅱ型胶原主要表达于髁突软骨的过渡层及肥大层。ERα主要表达于髁突软骨的过渡层及肥大层,均匀分布于细胞内;ERβ集中表达于肥大层,且以细胞核内分布为主。纤维层及增殖层未见Ⅱ型胶原和ER表达,钙化软骨层有少量表达。结论 ER与Ⅱ型胶原在髁突软骨中分布一致,雌激素可选择性地结合不同亚型的ER,从而调节髁突软骨细胞分泌Ⅱ型胶原的能力。     

Mechanical and hypoxia stress can cause chondrocytes apoptosis through over-activation of endoplasmic reticulum stress

ObjectiveTo examine the role of mechanical force and hypoxia on chondrocytes apoptosis and osteoarthritis (OA)-liked pathological change on mandibular cartilage through over-activation of endoplasmic reticulum stress (ERS).MethodsWe used two in vitro models to examine the effect of mechanical force and hypoxia on chondrocytes apoptosis separately. The mandibular condylar chondrocytes were obtained from three-week-old male Sprague–Dawley rats. Flexcell 5000T apparatus was used to produce mechanical forces (12%, 0.5 Hz, 24 h vs 20%, 0.5 Hz, 24 h) on chondrocytes. For hypoxia experiment, the concentration of O₂ was down regulated to 5% or 1%. Cell apoptosis rates were quantified by annexin V and propidium iodide (PI) double staining and FACS analysis. Quantitative real-time PCR and western blot were performed to evaluate the activation of ERS and cellular hypoxia. Then we used a mechanical stress loading rat model to verify the involvement of ERS in OA-liked mandibular cartilage pathological change. Histological changes in mandibular condylar cartilage were assessed via hematoxylin & eosin (HE) staining. Immunohistochemistry of GRP78, GRP94, HIF-1α, and HIF-2α were performed to evaluate activation of the ERS and existence of hypoxia. Apoptotic cells were detected by the TUNEL method.ResultsTunicamycin, 20% mechanical forces and hypoxia (1% O₂) all significantly increased chondrocytes apoptosis rates and expression of ERS markers (GRP78, GRP94 and Caspase 12). However, 12% mechanical forces can only increase the apoptotic sensitivity of chondrocytes. Mechanical stress resulted in OA-liked pathological change on rat mandibular condylar cartilage which included thinning cartilage and bone erosion. The number of apoptotic cells increased. ERS and hypoxia markers expressions were also enhanced. Salubrinal, an ERS inhibitor, can reverse these effects in vitro and in vivo through the down-regulation of ERS markers and hypoxia markers.ConclusionWe confirmed that mechanical stress and local hypoxia both contributed to the chondrocytes apoptosis. Mechanical stress can cause OA-like pathological change in rat mandibular condylar cartilage via ERS activation and hypoxia existed in the meantime. Both mechanical forces and hypoxia can induce ERS and cause chondrocytes apoptosis only if the stimulate was in higher level. Salubrinal can protect chondrocytes from apoptosis, and relieve OA-liked pathological change on mandibular condylar cartilage under mechanical stress stimulation.     

Condylar cartilage response to continuous passive motion in adult guinea pigs: A pilot study.

This article reports on the development and testing of a continuous passive motion device suitable for experimental animals and on the effect of 1 week of continuous passive motion on the thickness of the condylar cartilage in adult guinea pigs. Continuous passive motion is currently used for postsurgical joint therapy and consists of moving a synovial joint passively and constantly through its normal range of motion. In this study continuous passive motion was applied to the temporomandibular joints of two experimental animals for 7 hours per day for 5 days. Two animals were restrained for the same amount of time that the experimental animals were on the continuous passive motion machines (control group 1), and two animals were allowed normal cage activity (control group 2). Six weeks later all animals were sacrificed. Each mandibular condyle was sectioned sagitally, and routine histologic sections were prepared with hematoxylin-eosin stain. Microscopic examination of the mandibular condylar cartilage of the animals placed on continuous passive motion showed a marked increase in thickness compared with the condylar cartilage found on the control animals. The condylar cartilage of the animals placed on continuous passive motion averaged 345 microm, and the controls averaged 232 microm. These findings demonstrate the application of continuous passive motion in an experimental model and provide interesting information regarding the response of the mandibular condyle to increased motion. More studies are necessary to further investigate the effect of continuous passive motion on the condylar cartilage.