Effects of estrogen on chondrocyte proliferation and collagen synthesis in skeletally mature articular cartilage.

PURPOSE: Estrogen has been shown to have a modulating effect on cartilage thickness. This investigation was performed to determine the effects of estrogen supplementation on cartilage thickness, cellular proliferation, and type II and X collagen production in skeletally mature rat cartilage, both in an organ culture and cell culture system. MATERIALS AND METHODS: Mandibular condyles were harvested from 8-week-old female Sprague Dawley rats and placed into tissue culture plates containing culture media with or without 17beta-estradiol supplementation. Organ cultures were labeled with 5-bromo-2'-deoxyuridine on culture day 2 or 4 to determine the effects of estrogen supplementation on the cellular mitotic index. Histomorphometric analysis of the organ culture sections was used to determine the thickness (microm) of the various cartilage zones, as well as the total cartilage thickness following estrogen exposure. Type X collagen was immunohistochemically identified in the ECM of hypertrophic chondrocytes using a rabbit anti-rat collagen type X antibody raised against the NCl domain. The reaction was visualized with an avidin-biotin peroxidase detection system (Vector Laboratories, Burlingame, CA). In a separate experiment, articulating cartilage chondrocytes were harvested by collagenase digestion and cultured at 5 x 10(5) cells per 35 mm tissue culture plate. Second subculture chondrocytes were divided into 2 groups: controls and [10(-8) M] 17beta-estradiol (E(2)-10(-8) M) and grown to confluence. The cell cultures were used to establish growth curves for each group using cell counts at 2-day intervals. RESULTS: In the organ culture experiment, 17beta-estradiol-treated condyles had a significant decrease in total cartilage thickness after 4 days in culture (P < .05). Estrogen supplementation resulted in a significant reduction in the mitotic index as early as culture day 2 (P < .05). Type X collagen deposition into the extracellular matrix was visibly increased in the hypertrophic chondrocyte zone for the estrogen-supplemented group on experimental days 2 and 4 compared with the control group. In the cell culture system, 17beta-estradiol [10(-8) M] decreased chondrocyte proliferation during logarithmic growth (P < .05) and at confluence (P < .05). CONCLUSION: These data show that estrogen decreased cartilage thickness by inhibition of chondrocyte proliferation and increased chondrocyte maturation. These observed effects showed the potential role of estrogen in the modulation of skeletally mature cartilage.     

Involvement of PI3K/Akt pathway in rat condylar chondrocytes regulated by PTHrP treatment

Objective

The aim of this study was to explore the mutual communication of the parathyroid hormone-related peptide (PTHrP) and phosphatidylinositol 3-kinase/threonine protein kinase (PI3K/Akt) pathway on the proliferation and differentiation of condylar chondrocytes from Sprague-Dawley (SD) rats.

Methods

Condylar chondrocytes from the condylar cartilage were cultured and an organ culture system of mandibular condyles was employed. The distribution of PI3K, phospho-Akt (p-Akt), and PTHrP in condylar cartilage was detected by either immunohistochemistry or immunofluorescence. The second passage chondrocytes and condyle specimens in the organ culture system were treated with PTHrP, LY294002, PTHrP and LY294002 in combination, or dimethyl sulfoxide (DMSO), separately. The mRNA and protein levels of type II (Col II) and type X collagen (Col X) were investigated by real-time polymerase chain reaction (PCR) and Western blot analysis. The condyle growth in organ culture system was analysed by haematoxylin–eosin staining.

Results

PTHrP, PI3K, and p-Akt were mainly located in the proliferative and hypertrophic zones. PTHrP promoted the proliferation of condylar chondrocytes, while LY294002 limited this effect. The mRNA and protein levels of Col II and Col X in these cells were reduced by PTHrP and enhanced by LY294002. Organ culture showed a significant enhancement of condyle elongation with PTHrP treatment or a combination of PTHrP and LY294002 treatment. After treatment with LY294002, the length of condyles was reduced compared with the samples treated with DMSO.

Conclusions

We conclude that the PI3K/Akt pathway plays an essential role in the proliferation and differentiation of condylar chondrocytes and is a potential target for PTHrP in regulating chondrocyte differentiation at condylar cartilage.     

生长期山羊囊内骨折复位术中保留髁突软骨的重要性及组织学研究

目的：通过组织学观察,探讨髁突囊内骨折内固定术中保留和切除髁突软骨对髁突生长发育的影响。方法：6个月龄山羊12只,随机分为实验组（n=8）和对照组（n=4）。实验组双侧髁突造成囊内骨折并同期行手术复位固定．一侧保留髁突软骨,另一侧切除髁突软骨。术后3个月、6个月处死动物,切取髁突标本行石蜡切片和硬组织切片观察骨折愈合和髁突生长情况。结果：实验组髁突骨折愈合良好,钛板被新生骨组织覆盖;HE染色显示．保留髁突软骨组,髁突软骨结构清晰,与正常对照髁突相同,软骨成骨活跃,髁突生长发育正常;切除髁突软骨组,髁突软骨层消失,表面为成熟的骨细胞覆盖,直接与关节盘的纤维组织相连,新生骨组织少见：硬组织切片显示,钛板与骨组织直接结合．未见组织渗出和排异反应。结论：手术复位髁突囊内骨折时保留髁突软骨,不会影响髁突的生长发育;损伤髁突软骨．会造成髁突与关节盘黏连．引起髁突生长发育障碍。     

血管内皮生长因子及其受体在大鼠下颌骨髁突软骨细胞中的表达和意义

目的：研究VEGF及其受体(Fh—1和Flk—1)在大鼠下颌骨髁突软骨细胞中的表达，探讨其对大鼠下颌骨髁突软骨生长发育的影响。方法：用免疫组化方法，对VEGF及其受体(Fh—1和Flk—1)在大鼠下颌骨髁突软骨细胞中的表达进行检测。结果：大鼠下颌骨髁突软骨的增殖层、肥大层、矿化和钙化层均有表达，而纤维层没有表达。结论：大鼠下颌骨髁突软骨的增殖层、肥大层及矿化和钙化层软骨细胞可产生并分泌VEGF及其受体(Fh—1和Flk—1)，VEGF可能在大鼠下颌骨髁突软骨生长发育中发挥作用。     

Microscopic changes in the condyle and disc in response to distraction osteogenesis of the minipig mandible.

PURPOSE: Unilateral mandibular distraction osteogenesis (DO) has been shown to cause gross changes in the mandibular condyle and articular disc. The purpose of this study was to correlate histologic findings with these gross changes in a minipig distraction model. MATERIALS AND METHODS: Semiburied distractors were placed via submandibular incisions in 15 minipigs. Two unoperated animals served as controls. The protocol consisted of 0-day latency and rates of 1, 2, or 4 mm/day for a 12-mm gap. After the minipigs were killed (at 0, 24, or 90 days), ipsilateral and contralateral condyles and discs were harvested, decalcified, prepared for standard paraffin embedding, and evaluated to determine changes in 1) morphology and thickness of the articular cartilage and subchondral bone and 2) morphology of the disc. RESULTS: In control animals, there were no degenerative changes in the articular cartilage and underlying condylar bone; there were no significant differences in the mean articular cartilage thickness. The temporomandibular joint discs were normal. In experimental animals, distracted condyles showed increasing degenerative changes and mean articular cartilage thickness as the DO rate increased. The discs were thinner. These changes were present, but to a lesser degree, in the contralateral condyles. After 90 days, degenerative changes in the condyles and discs were reduced, after remodeling, except in the 4 mm/day DO group. CONCLUSIONS: Histologic changes in the condyles and temporomandibular joint discs in response to mandibular DO correlated with previously reported gross changes. These changes were greater at higher distraction rates and remodeling back to normal occurred in mandibular condyles distracted at 1 mm/day.     

Growth stimulation of mandibular condyles and femoral heads of newborn rats by IGF-I

Primary and secondary cartilage differ in embryonic origin and are generally considered to have a different mode of growth. However, few experimental studies exist that directly compare the two types of cartilage and their growth regulation. The regulation of cartilage growth is a complex mechanism involving growth factors like insulin-like growth factor-I (IGF-I). The purpose of this study was to compare the growth of mandibular condyles of 4-day-old rats with that of femoral heads in vitro and to analyze the effects of IGF-I. Explants were cultured for up to 2 weeks with 0, 5, and 25 ng/ml IGF-1. Both, 5 and 25 ng/ml IGF-I significantly stimulated growth of the mandibular condyles while only 25 ng/ml IGF-I stimulated growth of the femoral heads. IGF-I increased glycosaminoglycan synthesis of both condylar and femoral cartilage. However, only the DNA synthesis of the mandibular condyles was significantly increased by IGF-I while that of the femoral heads was not affected. It is concluded that IGF-I stimulates growth of both secondary condylar cartilage and primary femoral cartilage. The mandibular condyle appears to be more sensitive to IGF-I than the femoral head, which may partly be due to the different developmental stage.     

Histologic examination of the temporomandibular joint after mandibular advancement with and without rigid fixation: An experimental investigation in adult Macaca mulatta

This study evaluated the histologic response of the temporomandibular joint (TMJ) following mandibular advancement using rigid and nonrigid fixation in monkeys. Twelve adult female rhesus monkeys underwent sagittal ramus osteotomies with advancement. Six of them were placed into maxillomandibular fixation (MMF); six underwent bicortical bone-screw fixation without MMF. Changes in condylar position were quantified using lateral cephalograms with the aid of bone markers. The animals were killed at 6 weeks and the TMJs were prepared for histologic analysis. Three measures of condylar cartilage thickness were obtained for each animal and were correlated to changes in position of the condyle. Animals who underwent MMF showed a tendency for anterior movement of the condyles; animals who underwent rigid fixation showed a tendency for posterior condylar position. Thicker cartilage layers were found in the MMF animals. Animals who had posterior displacement of the condyles showed evidence of resorption of the posterior surface of the condyle and anterior surface of the postglenoid spine. There was a significant correlation between a change in the horizontal position of the condyle and the thickness of the posterior aspect of the condylar cartilage. The results of this study indicate that alterations in condylar position may induce remodeling changes within the TMJ.     

荧光组织化学法检测髁突软骨中雌激素受体

目的：了解髁突软骨中雌激素受体（ＥＲ）的分布特点及细胞内定位，探讨ＥＲ与髁突软骨增生、分化的关系。方法：应用荧光组织化学方法对青春期大鼠髁突软骨中的ＥＲ进行检测分析。结果：大鼠髁突软骨中存在ＥＲ，且主要位于细胞浆中，胞核中分布较少；ＥＲ主要分布于软骨的生发层细胞中，且在髁突软骨的生长处于增生旺盛时分布较多。结论：髁突软骨中存在雌激素—受体的作用机制，且ＥＲ与髁突软骨的增生、分化关系密切。     

Histologic examination of the temporomandibular joint after mandibular advancement with and without rigid fixation: an experimental investigation in adult Macaca mulatta

This study evaluated the histologic response of the temporomandibular joint (TMJ) following mandibular advancement using rigid and nonrigid fixation in monkeys. Twelve adult female rhesus monkeys underwent sagittal ramus osteotomies with advancement. Six of them were placed into maxillomandibular fixation (MMF); six underwent bicortical bone-screw fixation without MMF. Changes in condylar position were quantified using lateral cephalograms with the aid of bone markers. The animals were killed at 6 weeks and the TMJs were prepared for histologic analysis. Three measures of condylar cartilage thickness were obtained for each animal and were correlated to changes in position of the condyle. Animals who underwent MMF showed a tendency for anterior movement of the condyles; animals who underwent rigid fixation showed a tendency for posterior condylar position. Thicker cartilage layers were found in the MMF animals. Animals who had posterior displacement of the condyles showed evidence of resorption of the posterior surface of the condyle and anterior surface of the postglenoid spine. There was a significant correlation between a change in the horizontal position of the condyle and the thickness of the posterior aspect of the condylar cartilage. The results of this study indicate that alterations in condylar position may induce remodeling changes within the TMJ.     

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.     

Changes in the position of the condyle after bilateral sagittal split ramus osteotomy in patients with mandibular retrusion and protrusion: a new condyle: fossa matching concept

The purpose of this study was to compare the condylar positional changes after bilateral sagittal split ramus osteotomy (BSSRO) in patients with mandibular retrusion and those with mandibular prognathism. We also studied the correlation between the degree of matching of the condyle and fossa, and condylar displacement. Thirty patients with mandibular retrusion (n = 11) or mandibular prognathism (n = 19) who underwent BSSRO were included. The condylar position was assessed from spiral computed tomographic (CT) scans taken preoperatively, during the first postoperative week, and at least 6 months postoperatively. All data were measured by MIMICS 17.0 and analyzed by Student’s t test and Pearson’s correlation analysis. The size of the condyles of patients with mandibular retrusion was significantly less than those of patients with mandibular prognathism (491.5 (172.8) compared with 823.2 (212.0) mm³). The size of the glenoid fossa in those with mandibular retrusion (599.6 (110.4) mm³) and those with prognathism (597.6 (151.6) mm³) did not seem to differ. Postoperatively the condyles moved outwards, backwards, and downwards in both groups of patients. Correlation analysis between the condyle:fossa volume ratio and the condylar positional changes showed that a large condyle:fossa volume ratio correlated with the smaller positional changes in the condyle. The condylar position changed immediately after mandibular advancement and setback, and persisted in the long term. Larger condyles tended to have fewer positional changes.     

Distribution of insulin-like growth factor-I mRNA in the mandibular condyle and rib cartilage of the rat during growth

This study makes a molecular biological comparison of primary and secondary cartilage at an early phase of postnatal development. The distribution of insulin-like growth factor-I (IGF-I) mRNA expression in the mandibular condyle and rib cartilage of 1-28-day-old rats was examined after in situ hybridisation using an oligo probe cocktail for IGF-I mRNA. In the condyle, expression was localised to a narrow strip under the articular layer where the cells are undifferentiated. Essentially, no differences were found in IGF-I synthesis within three samples from the same age group or between different age groups. In rib cartilage, IGF-I mRNA was localised within the germinative, proliferative and early hypertrophic cell layers in 1-28-day-old rats. Again, there were no differences in expression among animals of the same age or as a function of age. This pattern of IGF-I mRNA expression indicates that IGF-I synthesis during growth of the mandibular condylar cartilage is different from that of costal cartilage. The findings shed light on the problem of overgrowth often associated with the use of costochondral grafts to replace defective mandibular condyles.     

体外髁突软骨培养方法的建立

目的:建立体外髁突器官培养系统,明确体外培养髁突软骨的生物学特性,为髁突软骨体外实验提供基础。方法:选用新生SD大鼠的髁突软骨作为器官培养材料,采用格栅式器官培养法,建立体外髁突软骨器官培养模型。分别在取材培养后1~6 d收集样本,制备组织切片并观察。结果:培养早期(4 d以内),髁突软骨层次分明,细胞结构完整,形态良好。培养时间延长至5 d,软骨表层局部出现空腔。结论:体外器官培养的髁突软骨在4d内能维持良好的形态和生长状态,具有生物活性,可以应用于髁突软骨的相关体外实验研究。     

Histological evaluation of condylar hyperplasia model of rabbit following distraction osteogenesis of the condylar neck

Summary Condylar hyperplasia is the excessive unilateral growth of mandibular leading to facial asymmetry, occlusal disturbance, joint pain and dysfunction. The aim of this study is to evaluate the histological presence of temporomandibular joint in model of condylar hyperplasia by lengthening unilateral condylar neck of distraction osteogenesis. An extra oral distractor was employed to achieve unilateral condylar neck distraction (1·0 mm daily for 7 days). The experimental condylar necks were elongated by 7 mm compared to the contralateral. Eleven adult white rabbits were used. Eight rabbits were, respectively, sacrificed after the post‐distraction period (4 or 8 weeks). All animals were evaluated clinically and histomorphometrically. The condyles radiologically showed remodelling, flattening and sclerosis. In 4‐week group, thinning of the cartilage was evident, and the trabeculae were long, not multiply connected. A thin, dense fibrous layer covered all over the surface of cartilage. In 8‐week group, the cartilaginous layer was similar to thickness of the normal cartilage, but still thinner than control. However, the fibrous layers covering condyle manifested slight degenerative changes, and even depressions and erosions were seen in the cartilage and subchondral bone. The trabeculae showed denser and multiply connected. In 8‐week group, the cartilaginous thickness of surgical condyles was significantly thinner than the contralateral. This study indicates that unilateral distraction of condylar neck loads the condyles asymmetrically. Asymmetrical loads affect more on the surgical condyles than the contralateral, and after 8 weeks of the post‐distraction, condyle could recover from asymmetrical loads in some degree.     

Effect of low masticatory function on condylar growth: a morphometric study in the rat.

The aim of this study was to estimate the influence of functional alterations on the size of the mandibular condyle and to elucidate in detail, by means of histomorphometric analysis, the effect of changing the consistency of the diet on different portions of the condylar cartilage in growing rats. Forty growing rats were randomly divided into 2 groups. One group received the normal hard diet for rats; the other group received a standardized soft diet. The experimental period was 28 days. Ten animals from each group were used for gross morphometric analysis; the other 10 animals were used for histologic analysis of the condyle. The morphometric analysis of the condylar cartilage was based on the 25th, 50th, and 75th percentiles of the mediolateral sections of the condyles. The sections were divided into 3 parts: the anterior, intermediate, and posterior part; 4 measurements were performed in each. Significant differences were found in the condylar length and width between the groups, the soft diet group having a smaller condyle. The histomorphometric analysis of cartilage thickness showed significant differences between the 2 groups, being thinner in the anterior part and thicker in the posterior part of the condyle in the soft diet group. These routine histologic findings cannot explain the gross morphologic differences in the condylar size between the groups; this means that increased condylar cartilage thickness is not necessarily evidence of increased condylar growth. The results from this study indicate that a low masticatory function leads to decreased growth of the condyle and changes in the thickness of the cartilage. This may be the effect of an alteration in the stress distribution in the temporomandibular joint area, because of the absence of large masticatory forces.     

The PI3K/Akt signalling pathway may play an internal role related to abnormal condylar growth: a preliminary study

Developmental deformity of the mandible is one of the most common craniofacial malformations and is closely related to abnormal condylar growth. In this study, the role of PI3K/Akt signalling in the regulation of chondrocyte proliferation and hypertrophic differentiation in the condylar cartilage was studied. Immunohistochemical staining was used to investigate the expression of PI3K and p-Akt in the rat condyle cartilage. Rat condylar chondrocytes were cultured for the investigation of chondrocyte proliferation and hypertrophic differentiation when PI3K/Akt was inhibited. In addition, organ culture of the rat mandibular condyle was performed to evaluate the condyle cartilage growth while PI3K/Akt was inhibited. PI3K-positive cells and p-Akt-positive cells showing cytoplasmic staining were found to be present in the condylar cartilage. Reduced cell proliferation was observed in the culture of rat condylar chondrocytes when PI3K/Akt was inhibited; however, the hypertrophic differentiation level was increased. The proliferative zone thickness of condylar cartilage in the experimental group was less than that in the control group (P = 0.00185), but the hypertrophic zone was greater than that in the control group (P = 0.01048). PI3K/Akt signalling exerts opposite influences on chondrocyte proliferation and hypertrophic differentiation of the condylar cartilage, and these data suggest that PI3K/Akt is a potential intracellular regulation signal pathway in condylar cartilage development.     

透明质酸、TGF-β1对下颌骨髁突软骨增殖分化的影响

目的:研究透明质酸(HA)、TGF-β1因子对下颌骨髁突软骨增殖分化的影响.方法:取新生小鼠的下颌骨髁突软骨体外进行组织培养,按培养液内添加因子不同分为对照组、HA(0.5 mg/ml)、TGF-31(5 ng/ml)组,于培养1、2、4、6、8周后进行形态学观察、软骨面积测量、茜素红染色以及碱性磷酸酶染色研究.结果:对照组中髁突软骨在培养4周后软骨内开始出现高密度光阻射区,茜素红染色、碱性磷酸酶染色提示软骨基质出现了钙化、软骨内成骨的过程;HA组中髁突软骨内未出现高密度光阻射区,而髁突软骨面积却显著增大(P ＜0.05);TGF-β1组中髁突软骨在培养2周后提前出现了高密度光阻射区,然软骨面积无显著改变(P＞0.05).结论:在体外培养下,HA可以促进髁突软骨的增殖,对软骨细胞的肥大分化有一定的抑制作用,TGF-31在早期可显著促进髁突软骨细胞的肥大分化.     

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.