Effects Of Estrogen On Functional Status Of Rat Masticatory Muscle In Vitro

Temporomandibular disorder (TMD) is a collective term that embraces a number of clinical conditions that involve the masticatory musculature or temporomandibular joints (TMJ) and other associated structures. Extensive literatures suggest that TMD is more prevalent in women than in men. The severity of symptoms is also related to the age of the patients. Disorders onset tend to occur after puberty, and peaks in the reproductive years. The gender and age distribution of TMD suggests a possible link between its pathogenesis and estrogen.The effects of estrogen are mediated by estrogen receptors (ERs). It has been reported that ERs could be found in TMJ condyle, cartilage, disc and capsule, making it possible that estrogen could influenc the development, restitution and metabolism of the TMJ and associated structures. But the effects of estrogen on masticatory musculature function are unclear. Masticatory muscles play an important role in influencing jaw position and excursive jaw movements and a role in the aetiology of TMD. The present study was to investigate the effect of estrogen on properties and functional status of masticatory muscles in vitro.To establish the masticatory muscle myoblasts culture model in vitro, myoblasts were obtained from five to seven days Sprague-Dawley rats maxillofacial regions using the method of two-step digestion (including colla genase and trypsin) combined with the method of mechanical cell dissociation. Cells were purified using the different speed-attaching method. Morphorlogical characteristics and growth curve were observed. The myoblasts were identified by immunocytochemical stain. It was observed that myoblasts were positively stained by anti-desmin immunocytochemical stain. The percentage of positive count of anti-desmin stained cells was more than 95%. So the rat masticatory muscle myoblasts can be used as in vitro culture model for studying effect of estrogen on myoblasts. To observe the expression of ERs in masticatory muscles, 17-βestradiol at different concentrations was added to cultures of myoblasts and ERs were detected by using immunocytochemical stain. The result showed that both ERαand ERβwere expressed in myoblasts. The expression of ERa is stronger than ERβ. ERa displayed a predominant nuclear distribution pattern and ERβdisplayed a predominant cytoplasm distribution pattern. 17-βestradiol could enhance the expression of ERαand ERβin dose-dependent manner between 10^-10 to 10^-8mol/L. The results indicate that masticatory muscles express functional ERs and the expression of ERs can be regulated by estrogen. Masticatory muscle might be an oestrogen-responsive tissue.To explore the effects of estrogen on proliferation of masticatory muscle myoblasts, 17β-estradiol was added at different concentrations to cultures of the cells and the proliferation was determined by ³H-TdR incorporation. It was observed that although there was a fluctuation at the concentration of 10^-8mol/L, in general, 17β-estradiol with the increased concentration decreased ³H-TdR incorporation in myoblasts in a dose manner, peaking at the concentration of 10^-6mol/L. The findings suggest that estrogen may inhibit the proliferation of masticatory muscle myoblasts which are important to development and regeneration of masticatory muscles.To investigate the effects of estrogen on the mRNA expression level of different myosin heavy chain isoforms of masticatory muscle myoblasts, 17β-estradiol was added at different concentrations to cultures of the cells. The semi-quantitative reverse transcription PCR technology was employed to determine gene expression. The results showed that estrogen increased mRNA expression of MHCâ… and decreased mRNA expression of MHC IIb significantly. The effects of estrogen on MHCâ…¡a andâ…¡x were not significant. The expression and composition of each MHC isoforms within a muscle determines its property. The findings suggest that estrogen can alter MHC expression and influence masticatory muscle properties.To observe the effects of estrogen on the intracellular calcium of masticatory muscles myoblasts, Fluo-4-AM as the Ca²⁺ indicator and the laser confocal microscope system were used to determine the changes of the cytoplasmic Ca²⁺ concentration. In the resting condition, when 17β-estradiol(10^-9mol/L, 10^-8mol/L and 10^-7mol/L) were added to cells cytoplasmic Ca²⁺ immediately increased then decreased right away, and in the end came into a new Ca²⁺ homeostasis in the base line. In the acid condition(PH=6.7) which mimicked muscle fatigue state, 17β-estradiol(10^-9mol/L, 10^-8mol/L and 10^-7mol/L) made the cytoplasmic Ca²⁺decreased immediately then came into a new Ca²⁺ homeostasis under the base line. Cytoplasmic Ca²⁺ plays an important role in cell normal function and cytoplasmic Ca²⁺ accumulation may be an key factor that causes skeletal muscle fatigue and injury. The results suggest that in acid condition, estrogen may maintain the skeletal cytoplasmic Ca²⁺ concentration in a lower level and reduce the cytoplasmic Ca²⁺ accumulation to keep the normal functions of masticatory muscles myoblasts.In conclusion, the present study established the masticatory muscle myoblasts culture model in vitro successfully. The ERs are expressed in masticatory muscle myoblasts. Masticatory muscles are estrogen target tissues. Estrogen inhibits proliferation of myoblasts. The mRNA expression of MHC isoforms in myoblasts can be regulated by estrogen. And estrogen can reduce the myoblasts cytoplasmic Ca²⁺ accumulation in acid condition. The results of this study in combination with the above evidences suggest that the effects of estrogen on masticatory muscle are complex and nonlinear. Estrogen may play a role in the pathogenesis of masticatory muscle parafunction through the influence on the development, regeneration, properties and function status of masticatory muscles.