[Research progress of necroptosis on bone related diseases].

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.

Conditional deletion of Adrb2 in mesenchymal stem cells attenuates osteoarthritis-like defects in temporomandibular joint.

ß2-adrenergic signal transduction in mesenchymal stem cells (MSCs) induces subchondral bone loss in osteoarthritis (OA) of temporomandibular joints (TMJs). However, whether conditional deletion of ß2-adrenergic receptor (Adrb2) in nestin+ MSCs can alleviate TMJ-OA development remains unknown. In this study, nestin-Cre mice were crossed with Adrb2 flox mice to generate mice lacking Adrb2 expression specifically in the nestin+ MSCs (Adrb2-/-), and TMJ-OA development in such mice was investigated. Adrb2 flox mice (Adrb2+/+) and Adrb2-/- mice were subjected to unilateral anterior crossbite (UAC), while mice in the control group were subjected to sham operation. Adrb2+/+ and Adrb2-/- mice in the control group showed no distinguishable phenotypic changes in body weight and length, mandibular condylar size, and other histomorphological parameters of the condylar subchondral bone. A significant increase in subchondral bone loss and cartilage degradation was observed in Adrb2+/+ UAC mice; the former was characterized by decreased bone mineral density, bone volume fraction, and trabecular plate thickness, and increased trabecular separation, osteoclast number and osteoclast surface, and pro-osteoclastic factor expression; the latter was characterized by decreased cartilage thickness, chondrocyte density, proteoglycan area, and collagen II and aggrecan expression, but increased matrix metalloproteinase and alkaline phosphatase expression and percentage area of calcified cartilage. Adrb2 deletion in nestin+ MSCs largely attenuated UAC-induced increase in condylar subchondral bone loss, cartilage degradation, and aberrant calcification at the osteochondral interface. Thus, Adrb2-expressing MSCs in the condylar subchondral bone play an important role in TMJ-OA progression and may serve as novel therapeutic targets for TMJ-OA.

Injury responses of Sprague-Dawley rat jaw muscles to an experimental unilateral anterior crossbite prosthesis.

OBJECTIVE: Dental occlusion are frequently changed in clinic. Molecular responses in jaw muscles to aberrant dental occlusion are changes are attractive, yet remain are obscure. DESIGN: Unilateral anterior crossbite (UAC) prostheses were applied to Sprague-Dawley rats and then ceased after two weeks to detect the reactions of the masseter, a representative jaw elevator, and the lateral pterygoid muscle (LPM), a representative jaw depressor. RESULTS: Two weeks of UAC elicited mild injury of the two muscles. Myogenesis and protective reactions were detected as increases in αB-crystallin expression in the masseter after 3 days and in the LPM after 2 weeks, and increases in desmin expression in both muscles after 2 weeks. A switch in fibre types from IIb to IIx occurred in the LPM but not in the masseter. Inflammatory responses, shown by the infiltration of inflammatory cells and increases in TNF-α mRNA expression, and fibrosis responses, shown by increased mRNA expression of Type I and III collagens, appeared very mild in the two muscles. These responses were partially recovered by the cessation of UAC. During the whole process, no obvious changes were observed in mitochondrial function, as indicated by the levels of proliferator-activated receptor γ coactivator 1α, mitofusin-2 and voltage-dependent anion channel. CONCLUSIONS: UAC causes injury and very limited inflammatory and fibrosis adaption in the masseter and LPM. Both muscles respond with myogenesis and protective activity. The LPM responds also with muscle fibre isoform alternations. These alterations were partially recovered by the cessation of dental stimulation at an early stage.

Early growth response 1 reduction in peripheral blood involving condylar subchondral bone loss.

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.

Molecular changes in peripheral blood involving osteoarthritic joint remodelling.

Biomarkers of temporomandibular joint (TMJ) osteoarthritis (OA) remain unknown. The objective was to detect whether molecular biomarkers from peripheral blood leucocytes (PBLs) engage in TMJ OA lesions. Thirty-four six-week-old Sprague Dawley rats were used. The top upregulated gene ontology categories and gene-fold changes in PBLs were detected by a microarray analysis comparing rats that received 20-week unilateral anterior crossbite (UAC) treatment with age-matched controls (n = 4). Twenty weeks of UAC treatment had been reported to induce TMJ OA-like lesions. The other twenty-four rats were randomly placed in the UAC and control groups at 12- and 20-week time points (n = 6). The mRNA expression levels of the selected biomarkers derived from the microarray analysis and their protein expression in the alveolar bone and TMJ were detected. The microarray analysis indicated that the three most highly involved genes in PBLs were Egr1, Ephx1 and Il10, which were confirmed by real-time PCR detection. The increased protein expression levels of the three detected molecules were demonstrated in cartilage and subchondral bone (P < 0.05), and increased levels of EPHX1 were reported in discs (P < 0.05); however, increased levels were not present in the alveolar bone. Immunohistochemistry revealed the increased distribution of EGR1-positive, EXPH1-positive and IL10-positive cells predominantly in the osteochondral interface, with EXPH1 also present in TMJ discs. In conclusion, the increased mRNA expression of Egr1, Ephx1 and Il10 in PBLs may serve as potential biomarkers for developed osteoarthritic lesions relating to osteochondral interface hardness changes induced by dental biomechanical stimulation.

Bilateral anterior elevation prosthesis boosts chondrocytes proliferation in mice mandibular condyle.

OBJECTIVE: We aimed to develop a mouse model predominating in a proliferative response in the articular cartilage of the temporomandibular joints. MATERIALS AND METHODS: Bilateral anterior elevation of occlusion was developed by installing metal tubes onto the incisors of mice with edge-to-edge relation to prevent tooth wear, leading to an increase in the vertical height of the dental occlusion with time. Morphological changes and expression changes in Cyclin D1, Aggrecan, and type II and type X collagen in the mandibular condylar cartilage were detected. In addition, cells were isolated from the mandibular condylar cartilage and exposed to cyclic tensile strain (CTS). RESULTS: Compared with age-matched controls, the tooth length was longer at 3 weeks, 7 weeks, and 11 weeks in BAE mice (p < 0.05), with increased condylar cartilage thickness, matrix amount, and cell number (p < 0.05). Compared with the deep zone cells, CTS stimulated the superficial zone cells to express a higher level of proliferating cell nuclear antigen, Cyclin D1, Aggrecan, and type II collagen but a lower level of type X collagen and alkaline phosphatase. CONCLUSION: Bilateral anterior elevation stimulated the proliferative response in the mandibular condylar cartilage, offering a new therapeutic strategy for cartilage degeneration.

Proteomics based detection of differentially expressed proteins in human osteoblasts subjected to mechanical stress.

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.

Enhancement of chondrocyte autophagy is an early response in the degenerative cartilage of the temporomandibular joint to biomechanical dental stimulation.

Autophagy is a cell protective mechanism for maintaining cellular homeostasis. The present study aimed to investigate whether autophagy is enhanced in the biomechanically induced degenerative cartilage of the temporomandibular joint (TMJ) and the potential role of mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) and mammalian Target of rapamycin (mTOR) in this observation. To induce degenerative changes in the TMJs, rats were subjected to biomechanical dental stimulation by moving 4 molars away from their original position as we previously reported. The ultrastructure of autophagosome was observed by transmission electron microscopy. The number of lysosomes was analyzed by flow cytometry. The expression levels of Beclin1 and LC3 and the involvement of MAP4K3 activity were detected by immunohistochemistry, real-time PCR and western blot. The activity of the mTOR pathway indicated by p-mTOR and p-p70S6 K was assayed by western blot. TMJ degeneration, characterized by irregular cell arrangement and cell-free area, was induced in the experimental groups. Under transmission electron microscopy, we observed the presence of autophagosomes, small patches of condensed chromatin, abundant rough endoplasmic reticulum and Golgi apparatus. The number of lysosomes and the expression levels of Beclin1 and LC3 increased, while the activity of mTOR and the expression level of MAP4K3 decreased in the experimental groups. Cartilage in TMJ which was induced to be degenerative biomechanically exhibited autophagy accompanied by reduced mTOR and MAP4K3 activity.

Changes of myelinated nerve and myelin basic protein expression in rats' periodontal ligaments after experimental tooth movement.

INTRODUCTION: Information about the effect of tooth movement on the myelinated nerve in the periodontal ligament is limited. In this study, we aimed to investigate what responses of the periodontal myelinated nerve can be evoked during experimental tooth movement. METHODS: In experimental-I group, the maxillary left and mandibular right third molars were moved distally. In experimental-II group, the maxillary left third molar but not the right one was moved, and the bilateral mandibular third molars were extracted. The ultrastructures of the myelinated nerve in the periodontal ligament of the bilateral maxillary third molars were observed under a transmission electron microscope. The expression of myelin basic protein was evaluated by immunohistochemistry. RESULTS: Degenerative ultrastructural changes of the myelinated nerve in the periodontal ligament were noticed mainly in the myelin sheath; these were observed earlier and were recoverable in the experimental-I group. In contrast, the ultrastructural changes of the myelinated nerve occurred mainly in the axons, were observed later, and were unrecoverable in the experimental-II group. A concomitant decrease of myelin basic protein expression was observed in both groups. CONCLUSIONS: Both experimental tooth movement and occlusal changes accompanying it caused changes of the myelinated nerve in the periodontal ligament.

Degenerative changes in rat condylar cartilage induced by non-matching occlusion created by scattered orthodontic teeth-moving.

The effect of occlusion on the temporomandibular joint (TMJ) is debated. By inserting rubber-bands that were replaced by self-curing resin one week later, the left maxillary and the right mandibular first-molars were moved and kept mesially in Sprague-Dawley (SD) rats in both experimental I (EXP-I) and II (EXP-II) groups, aiming to establish a non-matching cusp-to-fossa occlusal relation. Four weeks later, the left maxillary and the right mandibular third-molars were moved and kept distally in the EXP-II group. Degenerative changes, typically as a cell-free area, were observed in TMJs of the EXP groups. Binary logistical analysis indicated that the odds ratio of EXP group, EXP-II vs. EXP-I, on the incidence of a cell-free area, was 2.8 (p=.036). Time point, gender, and side did not have such effects (p>0.05). The results indicate that the persistence of more scattered non-matching cusp-to-fossa occlusion is more harmful to the condylar cartilage in terms of the incidence of degenerative changes.

Effect of estrogen and dietary loading on condylar cartilage.

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.

Excess genistein suppresses the synthesis of extracellular matrix in female rat mandibular condylar cartilage.

AIM: To investigate the effect of excess genistein on the extracellular matrix in mandibular condylar cartilage of female rats in vivo. METHODS: Female SD rats were administered through oral gavage with genistein (50 mg/kg) or placebo daily for 6 weeks. The morphological changes of temporomandibular joints were studied with HE staining. The expression of cartilage matrix compounds (aggrecan and collagen type II), estrogen-related molecules (aromatase, estradiol, ERα and ERß) and proliferating cell nuclear antigen (PCNA) in mandibular condylar cartilage was detected using immunohistochemistry, ELISA and real-time PCR. RESULTS: The genistein treatment significantly reduced the thickness of the posterior and middle regions of mandibular condylar cartilage, and decreased the expression of collagen type II, aggrecan and PCNA. Compared with the control group, the estradiol content and expression levels of the key estradiol-synthesizing enzyme aromatase in the genistein-treatment group were significantly decreased. The genistein treatment significantly increased the expression of ERß, but decreased the expression of ERα. CONCLUSION: Excess genistein suppresses extracellular matrix synthesis and chondrocytes proliferation, resulting in thinner mandibular condylar cartilage. These effects may be detrimental to the ability of mandibular condylar cartilage to adapt to mechanical loads.

[Effect of experimentally created disordered occlusion on the expression of IL-1ß, IL-6 and TNF-α in condylar cartilage of rats].

PURPOSE: To investigate the effect of the experimentally created malocclusion (ECDO) on the expression of IL-1ß, 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 and 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-1ß 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-1ß, play an important role in the degenerative remodeling procedure in rat condylar cartilage induced by ECDO.

Osteochondral angiogenesis in rat mandibular condyles with osteoarthritis-like changes.

OBJECTIVE: To investigate angiogenesis at the osteochondral junction and changes in expression of pro- and anti-angiogenic factors in rat mandibular condyles with osteoarthritis-like changes. METHODS: In order to evoke osteoarthritis-like lesions in mandibular condyles, disordered occlusion was created experimentally in rats. Osteochondral vascularity was assessed histologically at 20 and 24 weeks. Protein and mRNA levels of pro-angiogenic factors including vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and matrix metalloproteases 9 (MMP9), and anti-angiogenic factor chondromodulin-I (CHM-I) were investigated by means of immunohistochemical staining and real-time PCR. RESULTS: Osteochondral angiogenesis was demonstrated as increased numbers of vascular channels terminating in the calcified cartilage and non-calcified cartilage in 20- and 24-week experimental groups compared with controls (all P<0.05). In the experimental groups, VEGF, CTGF and MMP9 were highly expressed in the tissues adjacent to the osteochondral junction. However, CHM-I was more expressed in the superior but not deep hypertrophic chondrocytes. Compared to their age-matched controls, the protein levels of VEGF and CTGF were higher in 20-week experimental group, and the protein and mRNA levels of CTGF, MMP-9, and CHM-I increased in the 24-week experimental group (all P<0.05). CONCLUSION: In the present rat model, osteochondral angiogenesis was observed in mandibular condyles with osteoarthritis-like changes, accompanied with local upregulation of VEGF, CTGF and MMP9. Although the increase in CHM-I may moderate pro-angiogenic factors effects in the superior cartilage, the deficiency of deep hypertrophic chondrocytes to express CHM-I may permit vascular invasion into condylar cartilage.

Dose-dependent effects of genistein on bone homeostasis in rats' mandibular subchondral bone.

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 ERß, while ERα expression was increased by the low dose genistein and decreased by the high dose genistein. ERß 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 ERß.

Effect of estrogen and altered diet hardness on the expression of estrogen receptor alpha and matrix metalloproteinase-8 in rat condylar cartilage.

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.

Subchondral bone loss following orthodontically induced cartilage degradation in the mandibular condyles of rats.

Osteoarthritis (OA) is a degenerative joint disease generally characterized by progressive cartilage degradation and subchondral bone changes. Subchondral bone changes have been proposed to initiate or accompany with cartilage degradation in OA. The purpose of this study was to characterize cartilage damage, subchondral bone remodeling, and the possible mechanism involved in these morphological changes in our reported rat model with OA-like lesions in the mandibular condyle. In experimental groups, the dental occlusion was orthodontically disturbed. By histological analysis, transmission electron microscopy (TEM), micro-CT scanning and serum tests, changes in condylar cartilage and subchondral bone were analyzed at 8 and 12 weeks after treatment. The mRNA and protein levels of bone pro-resorptive and pro-formative factors by chondrocytes were investigated. Increased degraded cartilage areas and obvious cartilage calcification were observed in 8- and 12-week treated (EXP) groups compared to the age-matched controls. Subchondral bone loss, characterized as decreased bone mineral density (BMD), bone volume fraction (BV/TV) and trabecular thickness (Tb.Th), but increased trabecular separation (Tb.Sp), was observed in the 12-week but not the 8-week EXP group, respectively, versus their age-matched controls. The subchondral bone loss in the 12-week EXP group was accompanied with decreased new bone formation rate, but increased serum carboxy terminal telopeptides (CTXs), and increased osteoclast numbers and proportion of surface area in the subchondral bone regions. Increased mRNA and protein levels of M-CSF, VEGF, RUNX and RANKL/OPG ratio, but decreased OPG, were found in condylar cartilage in the 12-week EXP group versus its age-matched controls, and those of RANKL/OPG ratios were significantly higher in the 12-week EXP group than the 8-week EXP. In addition, increased mRNA levels of VEGF, RUNX and RANKL/OPG ratio, but decreased OPG, were also found in condylar cartilage in the 8-week EXP group versus its age-matched controls (All P<0.05). This study demonstrated that obvious subchondral bone loss followed cartilage degradation in the mandibular condyles in the present rat models and suggested that the imbalance of chondrocyte-secreted regulatory factors within the degraded cartilage may play a role in the osteoclastogenesis, and thus leading to the subchondral bone loss in OA.

A novel recombinant immuno-tBid with a furin site effectively suppresses the growth of HER2-positive osteosarcoma cells in vitro.

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.

Changes in the expression of MMP-3, MMP-9, TIMP-1 and aggrecan in the condylar cartilage of rats induced by experimentally created disordered occlusion.

OBJECTIVE: To investigate the effects of experimentally created disordered occlusion on the mandibular condylar cartilage in terms of histological morphology and expression of MMP-3, MMP-9, TIMP-1 and aggrecan. MATERIALS AND METHODS: Eighty 8-week-old Sprague-Dawley rats were randomly divided into two experimental (Exp) and two control (Con) groups, with equal sex and number distribution as subgroups. In the Exp group, the disordered occlusion was created by orthodontically moving the first and third molars 0.8mm away. Hematoxylin-eosin and immunohistochemical staining were performed on the mandibular condyles at the end of the 8th or 12th week. Gene expression was analysed by real-time PCR. RESULTS: Osteoarthritis-like lesions, typically seen as a cell-free area, were detected in the Exp group, predominantly in females. In the cell-free area, the immunopositive expression of MMP-3, MMP-9, TIMP-1 and aggrecan were absent. Hyper-proliferation changes, typically seen as conjunctive invaginations of chondrocytes, were also observed where immunopositive expression of the tested materials was strong. There were sex and time point related differences in gene expression. In the 8-week subgroup, the expression of MMP-3 decreased, while aggrecan increased in males; however, both MMP-9 and TIMP increased in the female group (P<0.05). In the 12-week subgroup, the expression of MMP-3 increased, while TIMP, MMP-9 (male only) and aggrecan (female only) decreased (P<0.05). CONCLUSIONS: The present results indicate that the experimentally created disordered occlusion led to osteoarthritis-like lesions accompanied by changes in the expression of MMP-3, MMP-9, TIMP-1 and aggrecan in mandibular condyle cartilage with gender differences.

Mandibular condylar cartilage response to moving 2 molars in rats.

INTRODUCTION: The purpose of this study was to investigate the responses of mandibular condylar cartilage to moving 2 molars in different combinations. METHODS: Rats were assigned to male and female control and experimental groups (each, n = 5). Elastic rubber bands were used to move medially the maxillary left and the mandibular right first molars in experimental group I. The same method was used to distally move the maxillary left and the mandibular right third molars, 2 mandibular third molars, and 2 maxillary third molars in experimental groups II, III, and IV, respectively. At the end of the eighth week, all condyles were examined histologically. The areas of histologic change as a percentage of total cartilage area were compared by using the Mann-Whitney U test. RESULTS: Cartilage degenerative remodeling was observed in experimental groups II, III, and IV. The percentage areas of degenerative remodeling were higher in female experimental groups II and III than in the female control group, and in female experimental group II than in female experimental group IV and male experimental group II (all, P <0.05). CONCLUSIONS: The mandibular condylar cartilage of female rats responded variously to different combinations of molar movement; the most obvious remodeling was observed in groups in which the maxillary left and mandibular right third molars were moved.