Endoplasmic reticulum stress regulates rat mandibular cartilage thinning under compressive mechanical stress.

Compressive mechanical stress-induced cartilage thinning has been characterized as a key step in the progression of temporomandibular joint diseases, such as osteoarthritis. However, the regulatory mechanisms underlying this loss have not been thoroughly studied. Here, we used an established animal model for loading compressive mechanical stress to induce cartilage thinning in vivo. The mechanically stressed mandibular chondrocytes were then isolated to screen potential candidates using a proteomics approach. A total of 28 proteins were identified that were directly or indirectly associated with endoplasmic reticulum stress, including protein disulfide-isomerase, calreticulin, translationally controlled tumor protein, and peptidyl-prolyl cis/trans-isomerase protein. The altered expression of these candidates was validated at both the mRNA and protein levels. The induction of endoplasmic reticulum stress by mechanical stress loading was confirmed by the activation of endoplasmic reticulum stress markers, the elevation of the cytoplasmic Ca(2+) level, and the expansion of endoplasmic reticulum membranes. More importantly, the use of a selective inhibitor to block endoplasmic reticulum stress in vivo reduced the apoptosis observed at the early stages of mechanical stress loading and inhibited the proliferation observed at the later stages of mechanical stress loading. Accordingly, the use of the inhibitor significantly restored cartilage thinning. Taken together, these results demonstrated that endoplasmic reticulum stress is significantly activated in mechanical stress-induced mandibular cartilage thinning and, more importantly, that endoplasmic reticulum stress inhibition alleviates this loss, suggesting a novel pharmaceutical strategy for the treatment of mechanical stress-induced temporomandibular joint diseases.

Proteomic analysis of early-response to mechanical stress in neonatal rat mandibular condylar chondrocytes.

The objectives of this study were to investigate the early response to mechanical stress in neonatal rat mandibular chondrocytes by proteomic analysis. To evaluate its molecular mechanism, chondrocytes were isolated and cultured in vitro, then loaded mechanical stress by four-point bending system on different patterns. Morphological observation, flow cytometric analysis, and MTT assays indicated that 4,000 microstrain loading for 60 min was an appropriate mechanical stimulus for the following proteome analysis, which produced a transient but obvious inhibitory effect on the cell cycle. Therefore, we took a proteomic approach to identify significantly differential expression proteins in chondrocytes under this mechanical stress. Using 2-DE and MALDI-TOF, we identified seven differentially expressed proteins including the MAPK pathway inhibitor RKIP, cytoskeleton proteins, actin and vimentin, and other selected proteins. Some differentially expressed proteins were validated by both Western blot analysis and fluorescent staining of cytoskeleton at different loading times. The vimentin and RKIP responsive expression were also proven in vivo in oral orthopedic treatment rats, which was in line with the result in vitro. The histological changes in cartilage also showed the inhibition effect. Furthermore, the expressional level of phosphorylated ERK was increased, which demonstrates the changes in MAPK activity. Taken together, these data indicate that mechanical stress resulted in vimentin expression changes first and then led to the subsequent changes in actin expression, MAPK pathway regulated by RKIP and heat shock protein GRP75. All those changes contributed to the cytoskeleton remolding and cell cycle inhibition, finally led to condylar remodeling.

[Functional analysis of novel mutations in PAX9 associated with familial oligodontia].

OBJECTIVE: To gain new insights into the molecular pathogenesis of the 109(InsG) and 139(C--> T) mutations and their roles in familial oligodontia. METHODS: The region of PAX9 paired domain (PAX9PD) was amplified and the expression plasmids were constructed in pGEXlambda -1T by PCR-based cloning. PAX9PD proteins were prepared on the basis of GST instruction. The binding of wild type and two novely mutant PAX9 paired domain to double-stranded DNA targets were analyzed by gel mobility shift assay. RESULTS: Wild type PAX9PD protein bind to the high affinity paired domain recognition sequences, CD19-2(A-ins) and Pax6CON, the 109(InsG) and 139(C--> T) mutant PAX9PD protein were unable to bind to these cognate DNA-binding sites. CONCLUSION: The functional defects in DNA binding of mutant 109(InsG) PAX9 and 139(C--> T) PAX9, as well as loss-of-function of PAX9 most likely result in its haploinsufficiency during the patterning of dentition and the subsequent loss of posterior teeth.

[Study on the method of two dimensional polycrylamide gel electrophoresis on rat condylar chondrocyte].

PURPOSE: To investigate the protein profile by two dimensional polycrylamide gel electrophoresis on the rat condylar chondrocyte in vitro. METHODS: The third-passage chondrocytes were harvested from the mandibular condyles of 2-day-old rats in this study. The protein profile of the rat mandibular condylar chondrocytes was examined by two dimensional polycrylamide gel electrophoresis (2-DE-PAGE). The 2-DE gel maps on different pH gradients were obtained. The result of modified coomassi blue-sliver staining and sliver staining was compared using Pdquest 7.1 image analysis software. RESULTS: The results showed that the good protein profile of the condylar chondrocytes was obtained by standard Bio-Rad manual. The protein was mainly in the field from pH4 to pH7. The 1203±86 protein points were examined on 2-DE gel map by modified coomassi blue-sliver staining, and 1769±97 protein points was examined by sliver staining. The silver staining map showed more distinctly but higher background than modified coomassi blue-sliver staining. CONCLUSIONS: The protein profile of the condylar chondrocytes enriches the proteomic database and gives evidence to further proteomic research. The 2-DE map obtained by modified coomassi blue-sliver staining is more suitable for MALDI-TOF mass identification. Supported by National Natural Science Foundation of China (Grant No. C30700963), China Postdoctoral Science Foundation(Grant No.20090461088), Jiangsu Provincial Postdoctoral Science Foundation (Grant No.0802003C) and Nanjing City's Science and Technology Foundation (Grant No.200905011).

[Effect of asymmetry elastic on the maturation of bone collagen and mineralization in condyle of adult SD rat].

PURPOSE: The aim of this investigation was to study the osteogenesis activation following asymmetric inter-maxillary elastic on the subchondral bone of adult SD rats. METHODS: Twenty SD ten-week old rats were used in this study (eight rats loading 0.39 N elastic force, another eight rats loading 1.18 N elastic force, the other four rats without loading as control group). The extra-joint device was fixed on the right side by surgery. Tetracycline, calcein and xylenol orange were intraperitoneally injected on one day before surgery, one day before device removal and one day before sacrifice. Masson trichrome staining and in vivo fluorescence technique were used to detect the osteogenesis. RESULTS: The results of bone collagen maturation and velocity of osteogenesis were different from the control group after force loading. Bone collagen showed better maturity in force-loading side and light force induced more active osteogenesis. CONCLUSIONS: The results suggest that even in the adult SD individuals, the subchondral bone still shows remodeling ability to adapt the various mechanical environments. Stress increases the maturity of bone collagen while rotation inhibits maturity. Mechanic force promotes the osteogenesis, especially the light force.

[Effect of asymmetry traction on the expression of type II collagen in adult rat condyle].

OBJECTIVE: The aim of this investigation was to study the expression of collagen type II in the cartilage of mandibular condyle following asymmetric inter-maxillary traction. METHODS: Two hundred and twenty SD rats were used in this study (one hundred and four rats loading 0.39 N elastic force, another one hundred and four rats loading 1.18 N elastic force, while twelve rats for control). The extra-joint device was fixed on the right side by surgery. Half of the experimental group was killed at 3, 7, 14, 28 days. The devices were removed at the 28th day in the rest rats, and the rats were sacrificed at 3, 7, 14, 28 days after removing the device. The type II collagen expression levels of all the joints were measured using immunohistochemical techniques. RESULTS: The positive expression of the type II collagen was mainly observed in the cytoplasm of chondrocyte, especially in maturative and hypertrophic layer. The expression intensity was different in different stages and different sides. Both of the two experimental groups showed the same tendency, while the changes in the light force group were more obviously than the heavy force group. In the right side (force-loading side), the type II collagen expression decreased at the early force-loading period. After the device was removed, the expressions increased immediately but then reach the lowest level. The expression almost recovered to normal level at the end of experiment. In the left side (none force-loading side), the expression remained increasing after force-loading and reached the peak at the 14th day. CONCLUSION: These results suggest that even in the adult individuals, the chondrocyte showed reaction to the mechanical force by altering type II collagen expression patterns and it may be the cause of the cartilage remolding after asymmetric inter-maxillary traction. A forward elastic force showed a depressant effect in matrix synthesis, and heavy force had stronger effect. But the rotation of condyle accelerated the matrix synthesis.

[Early effects of the cyclic uniaxial compressive stress on Actin and Vimentin of the rat condylar chondrocytes].

OBJECTIVE: To investigate the early effects of the cyclic uniaxial compressive stress on Actin and Vimentin of the rat condylar chondrocyte. METHODS: The third-passage chondrocyte were harvested from the mandibular condyles of 2-day-old rats, and a cellular compressive stress device was used to apply stress on cells at 4 000 microstrain for 15, 30, 60, 120, 240 min. The early effects of the cyclic uniaxial compressive stress on Actin and Vimentin of the rat mandibular condylar chondrocytes were examined by laser scanning confocal microscope (LSCM), immunofluorescence technique and Western blot. RESULTS: The expression of fluorescent light of cys-toskeleton protein changed obviously with 4000 microstrain compressive stress loading. The expression of Actin significantly decreased in 60 min, and the expression of Vimentin decreased in 30 min. Then the expression of these two protein recovered in 120 min. CONCLUSION: There are time-responsiveness between the 4000 microstrain compressive stress stimulate and Actin, Vimentin. It shows the expression of Actin and Vimentin down-regulated at first under the compressive stress, then increased by feedback. It hints that there are "self-regulate" mechanisms in the cell response to mechanics stimulate.

[Early-response of the condylar chondrocyte under cyclic uniaxial compressive stress].

OBJECTIVE: To investigate the protein profile after treatment of the cyclic uniaxial compressive stress on the rat condylar chondrocyte in vitro. METHODS: The third-passage chondrocytes were harvested from the mandibular condyles of 2-day-old rats, and a cellular compressive stress device (self-made four-point bending system) was used to apply stress on cells at 2000 microstrain and 4000 microstrain (0.5 Hz frequency) for 60 min. The early effects of cyclic uniaxial compressive stress on the protein profile of the rat mandibular condylar chondrocytes were examined by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser-desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: The results showed that the protein profile of the condylar chondrocyte did not change statistically in 2000 microstrain group. In 4000 microstrain group, the protein profile of the condylar chondrocyte was changed. Three new proteins appeared. Five proteins disappeared. Twenty-two proteins were down-regulated and 7 proteins were up-regulated (P < 0.05). The eight different protein spots were identified by MALDI-TOF-MS. It included cytoskeleton protein (gamma-actin and vimentin), glycometabolism protein (alpha enolase and stress-70 protein) and signal transduction protein (Raf kinase inhibited protein, RKLP). CONCLUSIONS: There were significant alternations of the protein profile in the rat condylar chondrocyte after the 4000 microstrain cyclic uniaxial compressive stress loading for 60 min. These different proteins might take part in the early response to the cyclic uniaxial compressive stress.

[Early effects of the cyclic uniaxial compressive stress on Stress70/GRP75 in the rat condylar chondrocytes].

PURPOSE: The purpose of this study was to investigate the early effects of the cyclic uniaxial compressive stress on Stress70/GRP75 in the rat condylar chondrocyte. METHODS: The third-passage chondrocytes were harvested from the mandibular condyles of 2-day-old rats for this study, and a cellular compressive stress device (self-made four-point bending system, Patent No.01129166.4 & 01256849.x) was used to apply stress on cells at 4000 microstrain (0.5 Hz frequency) for 0, 15, 30, 60, 120, 240 minutes respectively. The early effects of the cyclic uniaxial compressive stress on Stress70/GRP75 in the rat mandibular condylar chondrocytes were examined by Western blot and image analysis. One-way ANOVA was used for statistical analysis. RESULTS: The results showed that the expression of Stress70/GRP75 changed significantly with 4000 microstrain compressive stress loading. The gray scale value of protein band at 0 minute was 114.2+/-5.08, significantly decreased to 86.1+/-5.09 (P<0.001) after 30 minutes, and then gradually increased to 104.0+/-4.41 (P<0.01) at 60 minutes. The expression of Stress70/GRP75 up-regulated to 134.5+/-3.74 (P<0.001) at 120 minutes, and continued to increase within 240 minutes. CONCLUSION: There is time-dependent response between the 4000 microstrain compressive stress stimulate and Stress70/GRP75. The expression of Stress70/GRP75 inhibited initially under the compressive stress, and then increased by feedback with the increased loading time.

[The expression of TGF-beta1 mRNA in the maxillary sutures of puberty rhesus loaded with the class III intermaxillary orthopedic force].

OBJECTIVE: The purpose of the study is to detect the expression of TGF-beta1 mRNA in the maxillary sutures of puberty rhesus during different periods of the loading of intermaxillary class III orthopedic force. METHODS: The animal model was established with 6 puberty female rhesus, which were randomly divided into experimental group (wearing class III twin-block magnet appliance, each 2 rhesus for 3 and 6 month respectively) and control group (not wearing any appliance, each 1 rhesus for 3 and 6 month respectively). Tissue sections were obtained perpendicular to the sutures. In situ hybridization was used to the expression of TGF-beta1 mRNA, and the expression intensity was measured and statistics was performed by SPSS 11.0. RESULTS: There were no statistic differences of cellular density between palatomaxillary suture in vertical group and pterygomaxillary suture in horizontal group, but statistic differences were found between other groups. The expression of TGF-beta1 mRNA was detected in the control group, but the expression intensity was obviously increased after the load of intermaxillary class III orthopedic force. Statistically significant differences were found among all groups except the six months experimental group and control group of temporozygomatic sutures and pterygomaxillary sutures. Experimental groups were more intensive than the control group and three months group was more intensive than the six months group. CONCLUSION: The active tissue remodeling happened in the circummaxillary sutures by the effect of class III intermaxillary orthopedic force. Cell proliferation, extracellular matrix synthesis and bone formation were accelerated. During the different remodeling periods, the expression intensities were different, which may be related to the different force loading manners, the different reaction of sutures to the orthopedic force and the different biological function of TGF-beta1 in the different periods.

[The effect of Class III intermaxillary orthopedic force loading on the maxilla of puberty rhesus: a histomorphologic study].

PURPOSE: The purpose of this study is to quantitatively study the remodling changes of circummaxillary sutures of puberty rhesus loaded with Class III intermaxillary orthopedic force. METHODS: The animal model was established with 6 puberty female rhesus, which were randomly divided into experimental group (wore Class III Twin-block magnet appliance, each 2 rhesus for 3 and 6 months respectively) and control group(did not wear any appliance, each 1 rhesus for 3 and 6 months respectively ). The specimens were stained by hematoxylin and eosin, and image analysis system (IAS) was used in the quantatitive study of sutures width and cell density. One-way analysis of variance (ANOVA) was employed, and LSD analysis was used when interclass significance was found. RESULTS: Compared to the control group, different changes were shown in different sutures.New bone formation was observed in all experimental animal. There was no significance difference between palatomaxillary suture and pterygomaxillary suture in cellular density (P>0.05). Zygomaticotemporal suture showed no difference between 3-month experimental group and control group;however, 6-month experimental group was significantly higher than 3-month experimental group (P<0.01).For frontomaxillary suture, 3-month experimental group was significantly higher than the control group, and 6-month experimental group was significantly higher than later one (P<0.01).Zygomaticomaxillary suture in the experimental group was significantly higher than the control group (P<0.01);meanwhile, 3-month experimental group was significantly higher than 6-month experimental group (P<0.05). Furthermore, the width of sutures were different in these groups. Palatomaxillary suture became significant narrower in 3-month experimental group (P<0.01),but zygomaticotemporal suture became significant wider (P<0.01). The width of sutures restored at 6th month (P>0.05). The width of pterygomaxillary suture was not different between 3-month experimental group and the control group (P>0.05),but it became significant wider in 6-month experimental group (P<0.05). Frontomaxillary sutures of the experimental group were significantly narrower than the control group, and 6-month experimental group was significantly narrower than 3-month experimental group (P<0.01). For zygomaticomaxillary suture, the experimental groups were significantly wider than the control group;meanwhile, there was no difference between the experimental groups. CONCLUSION: Proper Class III orthopedic force can promote the physiological remodling of maxillary sutures.

[Novel mutations of PAX9 gene in Chinese patients with oligodontia].

OBJECTIVE: To investigate the mutational characteristics of PAX9 gene in Chinese patients with congenital oligodontia and thus to provide a molecular basis for studying the pathogenesis of oligodontia. METHODS: Thirteen individuals with oligodontia and 9 healthy individuals, from 4 unrelated autosomal dominant families, and 16 sporadic patients with hypodontia in China, as well as 196 healthy control individuals (without oligodontia or hypodontia) were screened. Congenital absence of teeth was confirmed by panoramic X-ray analysis. Mutations of PAX9 gene were detected using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. After the finding of abnormal SSCP bands, analysis was carried out with DNA sequencing. RESULTS: PCR-SSCP detected SSCP bands alteration in exon2 of PAX9 gene in two unrelated families. Sequencing of PAX9 gene revealed a novel frameshift mutation (109InsG) and a novel missense mutation (C139T). All the affected members of each family were heterozygous for the mutations. In sporadic patients and the other two families, no similar sequence changes in PAX9 gene were found. CONCLUSIONS: The results extend the spectrum of mutations in PAX9 gene associated with oligodontia. The novel mutations will play an important role in gene diagnosis of oligodontia.

[The expression of TGF-beta 1 mRNA in pubescent rhesus monkeys' condyle under Class III intermaxillary functional orthopedic force].

OBJECTIVE: To investigate the expression of TGF-beta 1 mRNA in pubescent Rhesus monkeys' condylar under Class III intermaxillary functional orthopedic force for different lengths of time. METHODS: Six pubescent Rhesus monkey were divided into two test groups and a control group. Monkeys in the test groups were TMAIII while the control groups did not. Histological method (HE staining) and in situ hybridization were employed in this study. RESULTS: 1. The histological results showed that, compared with the control group, the anterior part of the condyle became thicker while the median part and the posterior part became thinner in 3 months group. However, in 6 months group, the change was similar to 3 months group. 2. The results of in situ hybridization showed that, in control group, TGF-beta 1 mRNA mildly expressed in the anterior part of the condyle while extensively in the median and posterior parts. In 3 months group, TGF-beta 1 mRNA expressed in all parts of condyle; the most intensive expression was in the anterior part. Compared with 3 months group, the expression of TGF-beta 1 mRNA decreased in 6 months group, but the expression in anterior part was stronger than in median and posterior parts. CONCLUSION: Under Class III Orthopedic therapy, TGF-beta 1 mRNA probably participated in the endochrondral bone remodeling in the condyle, and the expression was closely related to loading time. In 3 months group, the expression of TGF-beta 1 mRNA was stronger than that in 6 months group. It was inferred that the remodeling of endochrondral bone was more active in 3 months group.