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Mechanical stress is essential for bone development. Mechanical stimuli are transduced to biochemical signals that regulate proliferation, differentiation, and cytoskeletal reorganization in osteoblasts. In this study, we used proteomics to evaluate differences in the protein expression profiles of untreated Saos-2 osteoblast cells and Saos-2 cells subjected to mechanical stress loading. Using 2-D electrophoresis, MALDI-TOF mass spectroscopy, and bioinformatics, we identified a total of 26 proteins differentially expressed in stress loaded cells compared with control cells. Stress loaded Saos-2 cells exhibited significant upregulation of 17 proteins and significant downregulation of 9 proteins compared with control cells. Proteins that were most significantly upregulated in mechanically loaded cells included those regulating osteogenesis, energy metabolism, and the stress response, such as eukaryotic initiation factor 2 (12-fold), mitochondrial ATP synthase (8-fold), and peptidylprolyl isomerase A (cyclophilin A)-like 3 (6.5-fold). Among the proteins that were significantly downregulated were those involved in specific signaling pathways and cell proliferation, such as protein phosphatase regulatory (inhibitor) subunit 12B (13.8-fold), L-lactate dehydrogenase B (9.4-fold), Chain B proteasome activator Reg (Alpha) PA28 (7.7-fold), and ubiquitin carboxyl-terminal esterase L1 (6.9-fold). Our results provide a platform to understand the molecular mechanisms underlying mechanotransduction. Key words: bone, osteoblasts, mechanotransduction, proteomics, mechanical loading. Le stress mecanique est essentiel au developpement de l'os. Des stimuli mecaniques sont transduits en signaux biochimiques qui regulent la proliferation, la differenciation et la reorganisation du cytosquelette des osteoblastes. Dans cette etude, nous avons utilise la proteomique afin d'evaluer les differences des profils d'expression proteique chez des osteoblastes Saos-2 non traites ou soumis a un stress mecanique. Nous avons identifie par electrophorese 2D, spectrometrie de masse en MALDI-TOF et par bioinformatique, un total de 26 proteines exprimees de maniere differentielle par les cellules soumises a un stress mecanique comparativement aux cellules controles. Chez les cellules Saos- 2 soumises au stress mecanique, 17 proteines etaient significativement regulees a la hausse et l'expression de 9 proteines etait significativement diminuee comparativement aux cellules controles. Les proteines qui etaient le plus significativement accrues chez les cellules soumises a un stress mecanique comprenaient celles qui regulent l'osteogenese, le metabolisme energetique et la reponse au stress comme le facteur d'initiation eucaryote eIF2 (12 fois), l'ATP synthase mitochondriale (8 fois) et l'isomerase PPIL-3 (peptidyl prolyl isomerase A (cyclophiline A)-like 3) (6.5 fois). Parmi les proteines dont l'expression etait significativement diminuee se trouvaient celles qui sont impliquees dans des voies de signalisation specifiques et la proliferation cellulaire comme la sous-unite regulatrice (inhibitrice) 12B de la proteine phosphatase 1 (13.8 fois), la L-lactate deshydrogenase B (9.4 fois), la chaine B de l'activateur du proteasome Reg (alpha) PA28 (7.7 fois) et l'esterase carboxy-terminale de l'ubiquitine L1 (6.9 fois). Nos resultats fournissent une base pour comprendre les mecanismes moleculaires qui sous-tendent la mecano-transduction. [Traduit par la Redaction] Mots-cles: os, osteoblastes, mecano-transduction, proteomique, charge mecanique., Introduction Mechanical stress is essential for bone development (Watanabe et al. 2004). Prolonged bed rest or microgravity causes mechanical unloading, which has been shown to increase the risk of bone [...]

Objectives: To detect whether early growth response 1 (EGR1) in peripheral blood leucocytes (PBLs) indicates temporomandibular joint (TMJ) osteoarthritis (OA) lesions. Materials and Methods: Egr1 mRNA expression levels in PBLs were detected in eight malocclusion patients without temporomandibular disorder (TMD) signs and 16 malocclusion patients with clinical TMD signs with (eight) or without (eight) imaging signs of TMJ OA. Twelve 6‐week‐old rats were randomized to a control group and a unilateral anterior crossbite (UAC) group and were sampled at 4 weeks. The Egr1 mRNA expression levels in PBLs and protein expression levels in different orofacial tissues were measured. Results: Patients with TMD signs with/without TMJ OA diagnosis showed lower Egr1 mRNA expression levels in PBLs than patients without TMD signs. The lower Egr1 mRNA expression was also found in the PBLs of UAC rats, which were induced to exhibit early histo‐morphological signs of TMJ OA lesions. In subchondral bone of UAC rats, EGR1 protein expression was decreased, co‐localization of EGR1 with osterix or dentin matrix protein‐1 was identified, and the number of EGR1 and osterix double‐positive cells was reduced (all p < .05). Conclusion: Egr1 reduction in PBLs potentially indicates subchondral bone OA lesions at an early stage. [ABSTRACT FROM AUTHOR]

PURPOSE: To investigate the effect of the experimentally created malocclusion (ECDO) on the expression of IL-1β, IL-6 and TNF-α in mandibular condylar cartilage of Sprague Dawley (SD) rats. METHODS: In the experimental group, the malocclusion was created by moving the first amt third molars 0.8 mm away. HE and immunohistochemical staining were performed on the mandibular condyle at the end of the 8th or 12th week. Gene expression was analyzed by real-time PCR. The data was processed using SPSS12.0 software package. RESULTS: Degenerative changes were found in the experimental group, the level of IL-1β was not significantly different among the groups (P>0.05), while the expression of IL-6 in 8-week ECDO subgroup was significantly higher than that in 8-week control subgroup (P=0.009), and the expression of TNF-α in 8-week ECDO subgroup was significantly higher than that in 8-week control subgroup (P=0.001) and 12-week ECDO subgroup (P=0.004). CONCLUSIONS: At the early period, IL-6 and TNF-α, but not IL-1β, play an important role in the degenerative remodeling procedure in rat condylar cartilage induced by ECDO. [ABSTRACT FROM AUTHOR]

Aim:To investigate the effect of genistein on bone homeostasis in mandibular subchondral bone of rats.Methods:Female SD rats were administered with genistein (10 and 50 mg/kg) or placebo by oral gavage for 6 weeks. Then the animals were sacrificed, and histomorphology and micro-structure of mandibular condyle were examined using HE staining and micro-CT analysis, respectively. The expression levels of alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG), the receptor activator of nuclear factor κB ligand (RANKL) and estrogen receptors (ERs) in mandibular condyle were detected using real-time PCR. Cultured osteoblasts were prepared from rat mandibular condyle for in in vitro study. The cells were treated with genistein (10−7 or 10−4 mol/L) for 48 h. The expression of the bone homeostasis-associated factors and estrogen receptors (ERs) was detected using real-time PCR, and ER silencing was performed.Results:At both the low- and high-doses, genistein significantly increased the bone mineral density (BMD) and bone volume, and resulted in thicker subchondral trabecular bone in vivo. In both in vivo and in vitro study, the low-dose genistein significantly increased the expression of ALP, OC and OPG, but decreased the expression of RANKL and the RANKL/OPG ratio. The high-dose genistein decreased the expression of all these bone homeostasis-associated factors. Both the low and high doses of genistein significantly increased the expression of ERβ, while ERα expression was increased by the low dose genistein and decreased by the high dose genistein. ERβ silencing abrogated most of the effects of genistein treatment.Conclusion:In rat mandibular condylar subchondral bone, low-dose genistein increases bone formation and inhibit bone resorption, while excess genistein inhibits both bone formation and resorption. The effects of genistein were predominantly mediated through ERβ. [ABSTRACT FROM AUTHOR]

As a new type of cell death, necroptosis is initiated by tumor necrosis factor receptor 1(TNFR1), and then activated receptor-interacting protein kinase 1(RIP1) and receptor-interacting protein kinase 3 (RIP3), following by the activation of mixed lineage kinase domain-like protein(MLKL) to deliver cell death signal. When necroptosis happens, damage associated molecular patterns (DAMPs) enter into extracellular area through the ruptured cytomembrane, followed by the disordered tissue hemeostasis. In recent years, many researches showed that necroptosis playimportant roles in a few bone related diseases, such as osteoporosis, osteonecrosis, osteosarcoma, etc. Thus, we try to briefly review the researches in this field.

Aims: To study the effect of estrogen deficiency and altered temporomandibular joint loading on the histomorphology of condylar cartilage and on the expression of types II and X collagen and matrix metalloproteinase-3 (MMP-3)., Methods: Thirty-six female rats were divided into four groups: ovariectomized rats on a normal diet, nonovariectomized control rats on a normal diet, ovariectomized rats on a soft diet, and nonovariectomized control rats on a soft diet. Ovariectomy was performed at the age of 60 days. Repeated-measures ANOVA was used to analyze the data., Results: The condylar cartilage in the ovariectomized normal diet group showed a significantly higher number of cells than in the nonovariectomized control rats (P < .001). The proportional amount of MMP-3 expression was significantly higher in the ovariectomized rats than in the nonovariectomized control rats in both diet groups (P < .001). The area covered by types II and X collagen was significantly higher in the experimental groups than in the control groups (P < .01)., Conclusion: Condylar cartilage is sensitive to both estrogen level and dietary loading.

Aims: To examine the effect of decreased estrogen level and altered diet hardness on condylar cartilage morphology of the rat temporomandibular joint (TMJ) and on the expression of condylar cartilage estrogen receptor alpha (ERa) and matrix metalloproteinase-8 (MMP-8)., Methods: A total of 36 female rats was divided into four groups: ovariectomized rats fed a normal diet, non-ovariectomized controls fed a normal diet, ovariectomized rats fed a soft diet, and non-ovariectomized controls fed a soft diet. Ovariectomy was performed at the age of 60 days. Seven days after the operation, the rats were sacrificed. Repeated measures ANOVA and Duncan's multiple comparison tests were used for statistical analysis., Results: The ovariectomized rats had thicker cartilage layers than the controls, both in the normal diet and soft diet groups. The thinnest cartilage layers were found in the control rats fed with the soft diet. The thickness of the chondroblastic layer was significantly higher (P < .001) in the normal-diet rats than in the soft-diet rats in both ovariectomized and non-ovariectomized groups. The thickness of the proliferative layer was significantly higher (P < .001) in the ovariectomized soft-diet rats than in the soft-diet control rats. The proportional amount of ERa was statistically significantly higher (P < .001) in the condylar cartilage of the ovariectomized rats than in the non-ovariectomized control rats both in the normal- and soft-diet groups. The proportional amount of ERa was statistically significantly higher (P < .001) in the ovariectomized normal-diet rats than in the ovariectomized soft-diet rats. The proportional number of MMP-8-positive cells was statistically significantly higher (P < .001) in the condylar cartilage of ovariectomized rats fed the soft diet than in non-ovariectomized control rats fed the soft diet. Control rats fed with the normal diet had a higher proportional amount of MMP-8 positive cells than control rats fed with the soft diet (P < .05)., Conclusion: The rat TMJ condylar cartilage is sensitive to changes in estrogen levels and altered diet hardness.

Immunotherapy is a promising strategy for the treatment of human epidermal growth factor receptor 2 (HER2)-positive tumors. Previously, we constructed an immuno-carboxy terminal fragment of Bid (immuno-tBid) and reported its specific and effective destruction of HER2-positive tumor cells. In this study, in order to further reduce the immunogenicity of the previous immuno-proapoptotic protein, we constructed a novel immuno-tBid by replacing domain II of Pseudomonas exotoxin A with a short furin cleavage sequence from the diphtheria toxin. In order to explore the possible application of this novel immuno-tBid in the treatment of osteosarcoma, we first examined the expression of the HER2 protein in a subclone of a human osteosarcoma cell line with relatively high metastatic potential (SOSP-9607-E10), as well as in clinical specimens of osteosarcoma. Quantitative real-time PCR and Western blot analysis revealed that the expression of HER2 was up-regulated in the SOSP-9607-E10 cells, while immunohistochemical analysis revealed that HER2 was overexpressed in 37% of the tissue specimens examined. Both HER2-positive SOSP-9607-E10 and SKBR-3 cells, as well as HER2-negative HeLa cells were transiently transfected with the novel immuno-tBid in order to study its specific pro-apoptotic effect. We demonstrate here that this novel immuno-tBid induces the specific destruction of HER2-overexpressing SOSP-9607-E10 cells through the release of cytochrome C. These results suggest that the novel immuno-tBid with a minimized exogenous fragment could represent a competitive approach for the treatment of HER2-positive osteosarcoma.

Objective: To investigate the effect of experimentally created occlusal disorders (ECOD) on the expression of estrogen in rat condylar cartilage., Methods: The model of ECOD was created by moving right upper and left lower first molars anteriorly. The animals in ECOD were sacrificed at 2, 4, 6, 8 weeks later. In removed occlusal disorders group, the moved first molars were extracted at 6 weeks later, and the animals were sacrificed 2 weeks later. The expression of estrogen was detected by SABC technique of immunocytochemistry, and then was analyzed by the density of estrogen-positive chondrocytes., Results: 1) Estrogen was abundant in mature layer and hypertrophic layer of rat mandibular condylar cartilage. 2) In control group, the expression of estrogen decreased gradually from 6-week-old to 16-week-old. 3) In both childhood and puberty rats, the expression of estrogen in experiment group was significantly higher at 2 weeks after treatment, while no difference was found at 4, 6, 8 weeks after treatment. However, the expression in removed occlusal disorder group was higher than that in control group and 8 weeks of ECOD group., Conclusion: In rat condylar cartilage, the expression of estrogen de-creases with age. Induced by ECOD, the expression of estrogen increases in early stage of remodelling activity.

Objective: To examine the expression of molar root patterning gene 1 (Mrp1) and predict the Mrp1 structure by bioinformatics analysis., Methods: A pair of Mrp1-specific PCR primers were designed, and RT-PCR method was used to study the mRNA's expression pattern in rat molar root and other organs. Gene positioning and other protein sequence prediction were carried out by chromosome analysis and other bioinformatics analysis., Results: Mrp1 was expressed not only in the molar but also in the developing pancreas, liver, lung and kidney tissues. Mrp1 was located in the 18q12.3 chromosome of the rats and the Mrp1 amino acids sequence had about 37% homology with a known protein Uroplakin IIIb (p35) which was an urothelial differentiation membrane molecular marker. A trans-membrane structure, 5 PKC phosphorylation sites and 4 CKII phosphorylation sites in Mrp1 were found., Conclusions: Mrp1 has a broad expression in different developing organs, and it may have a important function in the rat tooth root development.

Objective: This article was to study the effect of induced occlusal disorders and removed occlusal disorders on the expression of bone morphogenetic protein-2 (BMP-2) of condylar cartilage., Methods: Young and adult female Sprague-Dawley rats were divided respectively into induced occlusal disorders group, removed occlusal disorders group and control group, 3 rats every group. For induced occlusal disorders rats, the elastic rubbers were inserted between the first and second molar in the left upper side and right lower side to form the disordered occlusion. They were killed under anaesthesia 8 weeks after the treatment. For removed occlusal disorders rats, the first molars that caused disordered occlusion were extracted 6 weeks after forming disordered occlusion. 2 weeks later, they were killed under anaesthesia. For normal rats, they were killed at the same time with experimental rats. Hibateral temporomandibular joints of each rat were removed and stained with HE and monoclone antibody of BMP-2. The thickness of condylar cartilage was measured. The expression of BMP-2 in condylar cartilage was detected by half-quantity immunohistochemical analysis., Results: For adult induced occlusal disorders group, the thickness of cartilage in intermediate part of condyle decreased. However, it increased in the posterior part. After removing occlusal disorders, the thickness of posterior condylar cartilage returned to normal level. But it was still thinner than control group in the intermediate part. The expression of BMP-2 in anterior, intermediate, posterior part of condylar cartilage of young induced occlusal disorders group was higher than that of young removed occlusal disorders group expression of BMP-2 showed induced occlusal disorders group was higher than removed occlusal disorders group, which was higher than control group. and control group. No difference of the expression of BMP-2 was found in young removed occlusal disorders group and control group. For the expression of BMP-2 in intermediate part of condylar cartilage, both adult induced and removed occlusal disorders groups were higher than adult control group. For the posterior part of adult condyle cartilage, the expression of BMP-2 showed induced occlusal disorders group was higher than removed occlusal disorders group, which was higher than control group., Conclusion: Induced occlusal disorders can lead higher expression of BMP-2 in condylar cartilage of young and adult rats. Adaptability of condylar cartilage of adult rats is weaker than young rats, especially the intermediate part.

Purpose: To investigate the effect of gradually induced occlusal disorders on the expression of FGFR1 in rat condylar cartilage., Methods: A model of gradually induced occlusal disorders in rat was established.The expression of FGFR1 was detected by SABC immunocytochemistry and analyzed by density of positive cells in condylar cartilage. The data were analyzed using SPSS 11.0 software package., Results: FGFR1-positive chondrocytes were abundant in the maturative layer and hypertrophic layer, but only few FGFR1-positive chondrocytes were found in the proliferative layer. In the control group, the expression of FGFR1 increased from 6-week-old to 10-week-old rats, and then decreased and went stable. In both young and adult group, the expression of FGFR1 in the experimental group was significantly lower at 4, 6 weeks and higher at 8 weeks than that in the control group, especially in the young group(P<0.05). But no difference was found between the experimental group and the control group at 2 weeks in both groups., Conclusion: FGFR1 may play an important role in the remodling of condylar cartilage induced by occlusal disorders.

Aims: To investigate the expression of aromatase in temporomandibular joints(TMJs) of male and female rats of different ages., Methods: The expression of aromatase in terms of protein and mRNA was examined by immunohistochemistry, Western blot, and in situ hybridization in the TMJs of Sprague-Dawley rats. The expression level of aromatase protein was also quantified by the density of aromatase-positive chondrocytes in TMJ condylar cartilage. A tritiated water assay was used to analyze the activity of aromatase in cartilage chondrocytes., Results: Strong aromatase protein and mRNA hybridization signals were detected in hypertrophic and mature chondrocytes in all the cases examined. The density of aromatase-positive chondrocytes was relatively stable at higher levels before 8 weeks of age. The density decreased gradually in females after 8 weeks of age but not in males., Conclusion: These results demonstrate that aromatase is expressed intensely in condylar cartilage and suggest that it may play an important role in pathophysiological mechanisms in condylar cartilage.

Objective: To investigate clinical features of disk displacement during the course of condylar fracture and to explore the techniques of disk reposition and suturation., Methods: 32 patients (10 females and 22 males) who had disk displacements with condylar fractures were followed up. Reduction and reposition of the dislocated disks simultaneously with fixation of fractures were performed. 7 patients underwent intermaxillary fixation with elastic bands for 1 to 2 weeks., Results: The occlusions were satisfactory in all cases but one for the reason of ramus height loss. No TMJ symptom was found when examined 3 months post operation., Conclusions: Anterior disk displacements were most occurred with high condylar process fractures. Surgical reposition and suturation of disk play an important role for the later TMJ-function.