Shi-Bin Yu

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.

Occlusal Effects on Longitudinal Bone Alterations of the Temporomandibular Joint

The pathological changes of subchondral bone during osteoarthritis (OA) development in the temporomandibular joint (TMJ) are poorly understood. In the present study, we investigated the longitudinal alterations of subchondral bone using a rat TMJ-OA model developed in our laboratory. Changes in bone mass were examined by micro-CT, and changes in osteoblast and osteoclast activities were analyzed by real-time PCR, immunohistochemistry, and TRAP staining. Subchondral bone loss was detected from 8 weeks after dental occlusion alteration and reached the maximum at 12 weeks, followed by a repair phase until 32 weeks. Although bone mass increased at late stages, poor mechanical structure and lower bone mineral density (BMD) were found in these rats. The numbers of TRAP-positive cells were increased at 12 weeks, while the numbers of osteocalcin-expressing cells were increased at both 12 and 32 weeks. Levels of mRNA expression of TRAP and cathepsin K were increased at 12 weeks, while levels of ALP and osteocalcin were increased at both 12 and 32 weeks. These findings demonstrated that there is an active bone remodeling in subchondral bone in TMJs in response to alteration in occlusion, although new bone was formed with lower BMD and poor mechanical properties.

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 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.

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.

Age- and sex-related changes of mandibular condylar cartilage and subchondral bone: A histomorphometric and micro-CT study in rats

OBJECTIVE To quantify the age- and sex-related changes in the rat condylar cartilage and subchondral bone. METHODS SD rats were obtained at the ages of 2, 3, 4, 5, 6 and 7 months. For each sex, the temporomandibular joints tissue blocks from four rats were subjected to histological assessment of cartilage thickness and subchondral bone architecture; for the remaining three rats, the mandibular condyles were delivered for gross measurement and evaluation of the mineralization and architecture properties of the subchondral bone by means of micro-CT. RESULTS Rapid decrease of cartilage thickness but increase of subchondral bone density occurred respectively from 2 to 3 and 3 to 4 months old in female and 2 to 4 and 3 to 5 months old in male (P<0.05), whereas rapid changes of subchondral bone architecture occurred from 3 to 4 months old in both sexes (P<0.05). The significant enlargement of condyle size occurred at 4 or 5 months old in female but at 5 or 6 months in male (P<0.05). CONCLUSION This study demonstrated that the rapid developmental changes of rat condylar cartilage and subchondral bone primarily occurred before 4 months of age, resulting in thinner cartilage but larger and thicker subchondral bone, and they were followed by rapid growth in condylar size. Sex differences were identified that the endochondral ossification of fibrocartilage and formation of subchondral bone were faster in female than in male rats, leading to the earlier enlargement of condyle in female than in male.

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.

The effects of age and sex on the expression of oestrogen and its receptors in rat mandibular condylar cartilages

OBJECTIVE Oestrogen expression may indicate a difference in resistance potential to mechanical strain. The purpose of this study was to investigate the expression of oestrogen and oestrogen receptors in mandibular condylar cartilages in male and female Sprague-Dawley rats at different ages. MATERIALS AND METHODS One-hundred SD rats at the age of 2, 4, 8 weeks and 4, 12 months in both sexes, 10 in each age-sex group, were enrolled in this study. The expression of oestradiol, ERalpha and ERbeta was detected in mandibular condylar cartilages by the method of immunohistochemistry, and enzyme-linked immunosorbent assay or western blot. RESULTS Oestradiol and ERs immunoreactivity were obvious in mandibular condylar cartilages of SD rats. Oestradiol and ERalpha were observed in hypertrophic and mature layers, while ERbeta only in hypertrophic layer. There was no sex difference of same age (except 8-week age group) in the expression of oestradiol. The expression of both ERs, however, was usually higher in male than in age-matched female rats (P<0.05), except that the 8-week-old female rats showed a higher ERalpha expression and the 4- and 8-week-old female rats showed a higher ERbeta expression than the age-matched male ones in western blot results (P<0.05). CONCLUSIONS The results that oestradiol, ERalpha and ERbeta are co-expressed in rat mandibular condylar cartilage, indicate that mandibular condylar cartilage is a target for oestrogen. The age and sex related differences in ERs expression may indicate a difference in potential to resist mechanical loading between genders at different ages.

Locally synthesized estrogen plays an important role in the development of TMD

Estrogens have been suggested to play an important role in the development of temporomandibular disorders (TMD). However, a growing body of epidemiological, clinical and experimental researches focusing on the relationship between TMD and exogenous estrogen or serum estrogen has produced conflicting results. Recently, locally synthesized estrogens have been found and proved to contribute greatly to the function of cartilage. We hypothesize that estrogens synthesized locally in condylar cartilage have a profound effect on the development of TMD. Future investigation of local estrogen in condylar cartilage may give, at least partially, valuable evidences for the etiology and treatment strategy of TMD. In our opinion, regulating the amount and effect of locally synthesized estrogen seems to hold interesting future prospects for the treatment of TMD.

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.

Changes of temporomandibular joint and semaphorin 4D/Plexin-B1 expression in a mouse model of incisor malocclusion.

AIMS To investigate the changes in condylar cartilage and subchondral bone of the temporomandibular joint (TMJ) in a mouse model of incisor malocclusion. METHODS By bonding a single (single group) or a pair (pair group) of metal tube(s) to the left incisor(s), a crossbite-like relationship was created between left-side incisors in mice. The morphological changes in the TMJ condyles were examined by hematoxylin and eosin and toluidine blue staining. Indices of osteoclastic activity, including tartrate-resistant acid phosphatase (TRAP) staining and macrophage colony stimulating factor (M-CSF) were investigated by histochemistry or real-time polymerase chain reaction (PCR). The osteoblastic activity was indexed by osteocalcin expression. Expressions of semaphorin 4D and its receptor, Plexin-B1, were detected by real-time PCR. Two-way analysis of variance was used to assess the differences between groups. RESULTS One week and 3 weeks after bonding the metal tube(s), cartilage degradation and subchondral bone loss were evident histologically. Both indices of osteoclastic activity (TRAP and M-CSF) were significantly increased in cartilage and subchondral bone after bonding the metal tube(s). Osteocalcin expression in cartilage was significantly increased at week 3, while its expression in subchondral bone was significantly increased at week 1 but decreased at week 3. The semaphorin 4D expression in cartilage and subchondral bone was significantly decreased at week 1 but significantly increased at week 3. For Plexin-B1 expression, a significant increase was detected in subchondral bone at week 3. CONCLUSION Bonding a single or a pair of metal tube(s) to left incisor(s) is capable of inducing remodeling in the TMJ, which involved cartilage degradation and alteration of osteoclastic and osteoblastic activity.