Mandibular functional positioning only in vertical dimension contributes to condylar adaptation evidenced by concomitant expressions of L-Sox5 and type II collagen

OBJECTIVE: Concerted expressions of L-Sox5 and type II collagen play an important part in osteogenic transition in epiphyseal cartilage. This study was designed to elucidate the role of mandibular vertical functional positioning in condylar adaptive remodelling by examining L-Sox5 and type II collagen expressions in condylar cartilage. DESIGN: 40 female Sprague-Dawley rats at age of 5 weeks were randomly divided into the experimental (n=20) and control groups (n=20). Bite plates were fitted on the upper posterior teeth of the experimental animals to induce functional repositioning of mandible in vertical dimension. The animals in both experimental and matched control groups were sacrificed on days 3, 6, 9 and 12, respectively. Tissue sections were cut in the sagittal plane through the mandibular condyles and processed with histomorphological examination for cellular response and immunohistochemical test for expressions of L-Sox5 and type II collagen. Quantitative assessment was conducted with computer-assisted imaging system to reveal the correlation between these two factors. RESULTS: (1) Both L-Sox5 and type II collagen were expressed in prechondroblastic cells and chondroblastic cells. (2) When mandible was downward positioned, the amount of L-Sox5 expression was significantly higher by 16.1% (day 9) and 24.2% (day 12) than that of the control (P<0.05); Similarly, type II collagen expression in the experimental group was also significantly stronger by 9.3% (day 9) and 12.3% (day 12) than control group (P<0.05), indicating an enhanced osteogenic transition occurring in condylar cartilage. (3) There was a similarity in temporospatial patterns between the expressions of these two factors, indicating their integral functions in facilitating condylar adaptation. CONCLUSIONS: It is suggested that L-Sox5 plays a key role in adaptive remodelling of condylar cartilage resulting from downward positioning of the mandible. Integration with type II collagen enables L-Sox5 to induce osteogenic transition and consequently to encourage endochondral ossification.     

Functional appliance therapy accelerates and enhances condylar growth.

The present study was designed to quantitatively assess the temporal pattern of expression of Sox 9, the regulator of chondrocyte differentiation and type II collagen, the major component of the cartilage matrix during forward mandibular positioning, and compare it with the expression during natural growth. Female Sprague-Dawley rats, 5 weeks old, were used. Results showed that the expression of Sox 9 and type II collagen are accelerated and enhanced when the mandible is positioned forward. Furthermore, we monitored the amount of new bone formation during mandibular advancement and after the removal of bite-jumping appliances. A substantial increase was observed in the amount of newly formed bone when the mandible was positioned forward. No significant difference in new bone formation could be found after the appliance was removed when compared with natural growth. Thus, functional appliance therapy accelerates and enhances condylar growth by accelerating the differentiation of mesenchymal cells into chondrocytes, leading to an earlier formation and increase in amount of cartilage matrix. This enhancement of growth did not result in a subsequent pattern of subnormal growth for most of the growth period; this indicates that functional appliance therapy can truly enhance condylar growth.     

Identification of temporal pattern of mandibular condylar growth: a molecular and biochemical experiment

OBJECTIVES: Based on the phenomenon that expression of type X collagen and capillary endothelium correlates with endochondral ossification, the prime aim of this study was to establish the temporal pattern of condylar growth in Sprague-Dawley rats by biochemically identifying the expression of these two factors. DESIGN: Sprague-Dawley rats were divided into five groups representing five different stages during somatic pubertal growth. In situ hybridization and immunoperoxidase were performed to examine expression of type X collagen in hypertrophic zone and capillary endothelium in erosive zone of condylar cartilage. Computer-assisted imaging analyses were conducted to allow for a quantitative assessment of the expression of these two factors, from which the temporal pattern of condylar growth was inferred. RESULTS: (1) Synthesis of type X collagen and emergence of capillary endothelium were critical factors during the transition of condylar cartilage from chondrogenesis into osteogenesis, a biological pathway that leads to endochondral bone formation, the mode through which the condyle grows. (2) Quantitative analyses revealed the temporal pattern of the expression of these two factors, indicating that the thrust of natural growth of the condyle in the rats occurred in concomitance with somatic pubertal growth, featured by an acceleration starting from day 38, a maximum growth rate on day 56, followed by a decrease afterwards. CONCLUSION: It is suggested that the biochemical examination of growth markers, such as type X collagen, might be a new approach to accurately depict temporal pattern of condylar growth which is too delicate to be reflected by gross measurement not only in Sprague-Dawley rats but potentially also in other species.     

下颌前伸后髁突软骨成熟层内X型胶原的表达

目的：检测下颌骨前伸条件下,大鼠髁突内Ｘ型胶原的表达,从而论证髁突新骨的形成状况。方法：１００只同源雌性大鼠５组实验组及５组对照组,实验组动物戴用统一规格的咬合前导矫正器,各实验组及相应对照组动物分别在实验的第３、７、１４、２１及３０ｄ处死。应用分子原位杂交及免疫组织化学技术对髁突软骨成熟慨内的Ｘ型胶原ｍＲＮＡ及其蛋白的表达进行评价。结果：实验组,特别是２１ｄ组动物其髁突软骨成熟层细胞内有强烈的Ｍ     

Forward mandibular positioning enhances condylar adaptation in adult rats

The aim of this investigation was to assess quantitatively the adaptive changes in the condyles of adult rats to forward mandibular positioning. The level of types II and X collagen expressed in the condyles of adult rats was compared with that formed in response to forward mandibular positioning and the levels of expression were correlated to the amount of bone formed in response to mandibular advancement. Seventy-eight 120-day-old female Sprague-Dawley rats were included in this study. The rats were randomly allocated to six groups. Each group consisted of nine rats with bite-jumping devices and four untreated controls. The animals in each group were sacrificed on days 3, 7, 14, 21, 30, and 60. Immunostaining was used for the detection of types II and X collagen, while Alcian blue-PAS was used to observe the extracellular matrix and new bone formation. The results showed that new cartilage was formed in the posterior condyle. The highest level of expression of types II and X collagen were present on day 21, the amount of increase was 247.99 and 540.08 per cent, respectively. The highest level of new bone formation was measured at day 30 of advancement when the amount of increase in new bone formation was 318.91 per cent. These findings indicate that forward mandibular positioning causes changes in the biophysical environment of the temporomandibular joint (TMJ) of adult rats that leads to condylar adaptation.     

Changes in condylar cartilage after anterior mandibular displacement in juvenile pigs

Adaptive remodelling of the mandibular condyle in response to mandibular advancement is the mechanism exploited by orthodontic forward displacement devices.ObjectiveThis work investigated the expression of collagens, matrix metalloproteinases and vascular endothelial growth factor during this process.DesignTwenty juvenile pigs were randomly divided into two experimental groups, where the treatment group was fitted with mandibular advancement splints, and the control group was not. Changes in the mRNA content of condylar cartilage tissue was then were measured by real-time PCR using specific primers after 4 weeks of treatment.ResultsThe temporal pattern of the expression of Col1 and MMP13 during condylar adaptation coincided with that during natural condylar growth. The amount of the expression of Col10 during condylar adaptation was significantly lower (p < 0.05), whereas the expression of Col2, MMP8 and VEGF was significantly higher compared to natural growth (p < 0.05).ConclusionsIt is suggested that condylar adaptation in growing pigs triggered by mandibular forward positioning results not only from passive adaptation of cartilage, but also involves growth affected processes. Our results showed that mechanical strain produced by mandibular advancement induced remodelling and revascularization in the posteriocranial mandibular condyle. These results are mostly consistent with former published histological and histomorphometrical analyses.     

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.     

Propulsive appliance stimulates the synthesis of insulin-like growth factors I and II in the mandibular condylar cartilage of young rats

Functional orthopedic appliances correct dental malocclusion partially by exerting indirect mechanical stimulus on the condylar cartilage, modulating growth and the adaptation of orofacial structures. However, the exact nature of the biological responses to this therapy is not well understood. Insulin-like growth factors I and II (IGF-I and IGF-II) are important local factors during growth and differentiation of several tissues, including cartilage. The aim of this study was to verify the mRNA and protein expression of IGF-I and IGF-II in the condylar cartilage of young male Wistar rats that used a mandibular propulsive appliance for 3, 5, 7, 9, 11, 13 or 15 days. For this purpose, sagittal sections of decalcified and paraffin-embedded condyles were submitted to immunohistochemistry and in situ hybridization. IGF-I and IGF-II expression increased with developmental age in the control and treated rats. After 9 days of treatment the positivity for both peptides in the animals that wore the propulsive appliance increased even more, expressively different from the age-matched controls. The expression patterns of both IGFs were similar, although IGF-I labelling was stronger. Furthermore, the enhanced expression of both peptides was in parallel with the proliferating cell nuclear antigen (PCNA) positivity, a proliferation cell marker. The modulation of IGF-I and IGF-II expression in the condylar cartilage in response to the propulsive appliance suggests that both peptides are involved in the mandibular adaptation during this therapy.     

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.     

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.     

Forward deviation of the mandibular condyle enhances endochondral ossification of condylar cartilage indicated by increased expression of type X collagen

OBJECTIVE: Using type X collagen as a marker, this research was designed to examine the alteration of condylar growth in response to mandibular condylar forward positioning. METHODS: One hundred female Sprague-Dawley rats with 5 weeks of age were randomly divided into five experimental and five control groups. In the experimental groups, bite jumping appliances created forward positioning of the condyle. The experimental rats, together with the age-matched controls, were sacrificed on days 3, 7, 14, 21 and 30, respectively. Tissue sections were cut in the sagittal plane through the mandibular condyle and were processed for in situ hybridization and immunostaining of type X collagen and then for quantitative imaging analyses. RESULTS: (1) Both type X collagen mRNA in situ hybridization signals and type X collagen immunostaining were localized within the hypertrophic zone of the condylar cartilage. (2) With condylar forward positioning, the level of type X collagen mRNA signals (8,541 +/- 74 microm(2) at peak) was 300% higher than that in the controls (2,117 +/- 78 microm(2) at peak); type X collagen immunostaining in condylar advancing groups (54,864 +/- 134 microm(2) at peak) was 254% more than that in the controls (15,470 +/- 121 microm(2) at peak). (3) The amount of type X collagen mRNA signals and immunostaining in experimental and control groups reached the highest levels at day 14 and day 21, respectively, indicating that an increase in endochondral ossification occurred 21 days after condylar forward deviation. CONCLUSION: Condylar forward repositioning provokes an enhanced maturation of condylar chondrocytes resulting in increased synthesis of type X collagen, a extracellular protein that attributes to endochondral ossification.     

The adaptive remodeling of condylar cartilage---a transition from chondrogenesis to osteogenesis

Mandibular condylar cartilage is categorized as articular cartilage but markedly distinguishes itself in many biological aspects, such as its embryonic origin, ontogenetic development, post-natal growth mode, and histological structures. The most marked uniqueness of condylar cartilage lies in its capability of adaptive remodeling in response to external stimuli during or after natural growth. The adaptation of condylar cartilage to mandibular forward positioning constitutes the fundamental rationale for orthodontic functional therapy, which partially contributes to the correction of jaw discrepancies by achieving mandibular growth modification. The adaptive remodeling of condylar cartilage proceeds with the biomolecular pathway initiating from chondrogenesis and finalizing with osteogenesis. During condylar adaptation, chondrogenesis is activated when the external stimuli, e.g., condylar repositioning, generate the differentiation of mesenchymal cells in the articular layer of cartilage into chondrocytes, which proliferate and then progressively mature into hypertrophic cells. The expression of regulatory growth factors, which govern and control phenotypic conversions of chondrocytes during chondrogenesis, increases during adaptive remodeling to enhance the transition from chondrogenesis into osteogenesis, a process in which hypertrophic chondrocytes and matrices degrade and are replaced by bone. The transition is also sustained by increased neovascularization, which brings in osteoblasts that finally result in new bone formation beneath the degraded cartilage.     

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。     

下颌前伸后大鼠髁突软骨增殖细胞核抗原表达昼夜节律

目的 比较昼夜不同时段戴用功能矫治器后,髁突软骨增殖细胞核抗原（ＰＣＮＡ）表达的昼夜变化,为探讨功能矫治昼夜最佳介入时段,提供直接实验依据。方法 采用免疫组织化学法,检测不同时段前伸大鼠下颌后,髁突软内ＰＣＮＡ表在的昼液变化。结果 戴用功能矫治器后,ＰＣＮＡ表达强度增加;白天戴用组明显高于夜晚组。结论 功能矫治器可以刺激髁突软骨增生;在髁突软骨昼夜自发性生长最旺盛时段戴用矫治器,效果最佳。     

PTHrP regulates chondrocyte maturation in condylar cartilage

PTHrP is a key factor regulating the pace of endochondral ossification during skeletal development. Mandibular advancement solicits a cascade of molecular responses in condylar cartilage. However, the pace of cellular maturation and its effects on condylar growth are still unknown. The purpose of this study was to evaluate the pattern of expression of PTHrP and correlate it to cellular dynamics of chondrocytes in condylar cartilage during natural growth and mandibular advancement. We fitted 35-day-old Sprague-Dawley rats with functional appliances. Experimental animals with matched controls were labeled with bromodeoxyuridine 3 days before their death, so that mesenchymal cell differentiation could be traced. Mandibular advancement increased the number of differentiated chondroblasts and subsequently increased the cartilage volume. Higher levels of PTHrP expression in experimental animals coincided with the slowing of chondrocyte hypertrophy. Thus, mandibular advancement promoted mesenchymal cell differentiation and triggered PTHrP expression, which retarded their further maturation to allow for more growth.     

Indian hedgehog: a mechanotransduction mediator in condylar cartilage

Indian hedgehog (Ihh) is a critical mediator transducing mechanical signals to stimulate chondrocyte proliferation. To clarify the cellular signal transduction pathway that senses and converts mechanical signals into tissue growth in mandibular condyle, we evaluated Ihh expression and its relation to the kinetics of replicating mesenchymal cells in condylar cartilage during natural growth and mandibular advancement. Thirty-five-day-old Sprague-Dawley rats were fitted with functional appliances. Experimental animals with matched controls were doubly labeled with iododeoxyuridine and bromodeoxyuridine so that we could evaluate the cycles of the proliferative mesenchymal cells. Mandibular advancement triggered Ihh expression in condylar cartilage. A higher level of Ihh expression coincided with the increase of the replicating mesenchymal cells' population and the shortening of the turnover time. These findings suggested that Ihh acts as a mediator of mechanotransduction that converts mechanical signals resulting from anterior mandibular displacement to stimulate cellular proliferation in condylar cartilage.     

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。     

Expression of extracellular matrix in human mandibular condyle

ObjectivesThe age-related expression of the extracellular matrices in human mandibular condyle was examined.Study designThe distribution patterns of types I to V collagens, laminin, fibronectin, fibronectin receptor, and transforming growth factor β in 34 human mandibular condyles dissected from autopsy specimens were studied by immunohistochemical procedure with special attention on the age-related changes.ResultsType I collagen was detected in the full layer of the condylar cartilage, and a stronger immunoreaction was delineated in the articular and cartilage zone. Types II and III collagen were mainly localized in the fibrocartilage zone. Type IV collagen and laminin were detected not only in the basement membrane of the blood vessels but also in the degenerated lesion where the expression of transforming growth factor β was also detected. Immunostaining of type V collagen and fibronectin was noted in the perichondrocytic area, whereas that of fibronectin receptor was seen in the chondrocytes. In materials from younger cadavers types I, II, IV, and V collagens, fibronectin, its receptor, and laminin showed stronger expression in the degenerative lesions than in the normal portions. In the sections from cadavers over the seventh decade, the immunoreaction of extracellular matrices was weak compared with the younger materials, and no increased reaction of extracellular matrices in the degenerative lesions was detected. In addition, severe osteoarthrosis was frequently seen in the older materials in macroscopic findings.ConclusionsThese results suggest that the expression of extracellular matrices thus seems to be closely related to agingand degenerative changes in the condyle.