Role Of 1,25 (OH) ₂ D Deficiency In The Development Of Temporomandibular Joint Osteoarthrosis And Its Mechanism

During the past decade there has been lots of evidence showing that 1,25(OH)2D insufficiency appears to be associated with not only the metabolism of calcium and phosorus, but also several age-related diseases including osteoporosis,cancers, hypertension, cardiovascular diseases, diabetes, neural degenerative diseases and so on. Results from epidemiologic investigations showed that vitamin D deficiency is associated with higher mortality. The length of telomere was shorter in blood white cells of vitamin D deficient women with shorter life span. These evidence indicates that vitamin D may play an important role in the ageing process. Some epidemiological studies indicated that insufficient levels of serum 25-hydroxyvitamin D(25-OHD), a stable form of 1,25(OH)2D, influence the joint cartilage leading to development and progression of OA, but underlying biological mechanisms remain unclear.Osteoarthritis(OA) is the most common cause of chronic disability in elderly adults. OA is a multifactorial condition but the pathological changes seen in osteoarthritic joints have common features no matter what the cause(s) of the condition in a given individual. These features include degradation of the articular cartilage starting at the joint surface and progressing to full thickness loss. Temporomandibular joint(TMJ) OA is a degenerative disease involving all TMJ tissues and leads to anatomical changes and severe pain.To investigate the mechanism of 1,25(OH)2D deficiency in the evolopment and progression of OA, we employed 25-hydroxyvitamin D 1α-hydroxylase knockout [1α(OH)ase-/-] mice model. In the current study, maintained 1α(OH)ase-/- mice and their wild-type littermates on a “rescue” diet containing 2% calcium, 1.25% phosphorus, and 20% lactose from weaning until 18-months of age, to exclude possible effects of extracellular calcium and/or phosphorus and/or PTH, while investigating the role and possible mechanisms of 1,25(OH)2D deficiency in the development and progression of TMJ OA by imageology, histomorphology and immunohistochemistry. Based on the findings, to investigated the role of oxidative stress in TMJ OA inducd by 1,25(OH)2D deficiency. 1α(OH)ase-/- mice were maintained on a diet of regular mouse chow containing 1% calcium and 0.67% phosphorus after weaning, or a rescue diet, or a rescue diet with N-acetyl-L-cysteine(NAC) solution(1g/L), or subcutaneous injection of 1,25(OH)2D 1ug/kg qod, and their wild-type littermates were fed on a diet of regular mouse chow. The phenotype and the probable mechanism were analysed by methods of imageology, histomorphology and immunohistochemistry.Part I: Effect of 1,25(OH)2D deficiency on the TMJ of aging miceDuring the past decade there has been lots of evidence showing that 1,25(OH)2D insufficiency appears to be associated with not only the metabolism of calcium and phosorus, but also several age-related diseases. Some epidemiological studies indicated that insufficient levels of serum 25-hydroxyvitamin D(25-OHD), a stable form of 1,25(OH)2D, influence the joint cartilage leading to development and progression of OA, but underlying biological mechanisms remain unclear. TMJ OA as one type of OA, it also has the major pathological characteristics.To investigated the influence of 1,25(OH)2D deficiency on the age-related disease of bone, OA, maintained 1α(OH)ase-/- mice and their wild-type littermates on a rescue diet from weaning until 18-months of age, while investigating the role and possible mechanisms of 1,25(OH)2D deficiency in the development and progression of TMJ OA by imageology, histomorphology and immunohistochemistry.The results show that serum calcium and phosphorus levels were normal, whereas serum 1,25(OH)2D levels were undetectable in 18-month-old 1α(OH)ase-/-mice. There was increased radiolucency in TMJ subchondral bone of 1α(OH)ase-/-mice compared with their wild-type littermates. Collapse of the articular surface and more subchondral spaces and reduced subchondral bone volume were detected in 1α(OH)ase-/- mice. The thickness of the articular cartilage was decreased with more acellular area, and the type II collagen and glycosaminoglycan abundance were reduced significantly in 18-month-old 1α(OH)ase-/- mice, which are the typical phenotype of OA. The degenerative alterations of the mandibular condyles that were observed in 1,25(OH)2D deficient mice were associated with DNA damage andsenesence in chondrocytes and osteoblasts in 18-month-old 1α(OH)ase-/- mice. Increased cellular senescence could induce more senescence-associated inflammatory cytokine production in the chondrocytes including Interleukin(IL)-1α, IL-1β, IL-6, matrix metalloprotein(MMP)-3 and MMP-13, which are play an important role in the development and progression of OA.These findings suggested that 1,25(OH)2D deficiency resulted in senescence phenotype of chondrocytes. The senescent cells accompanied by genotoxic damage led to SASP and some factors involved in senescence-associated secretory phenotype(SASP) also participated in the progression of OA.Part II: Effect of 1,25(OH)2D deficiency on the TMJ developmentTo investigate the further effect of 1,25(OH)2D deficiency on TMJ, 1α(OH)ase-/-mice were maintained on a diet of regular mouse chow after weaning, or a rescue diet, or a rescue diet with N-acetyl-L-cysteine(NAC) solution(1g/L), or subcutaneous injection of 1,25(OH)2D 1ug/kg qod, and their wild-type littermates were fed on a diet of regular mouse chow. The mice were sacrificed at 10-week-old. The results were analyzed by imageology, histomorphology immunohistochemistry and biochemical detection.The results show that mineral density were reduced dramatically in the subchondral bone of 1α(OH)ase-/- mice with regular diet, compared with their wild-type littermates, given the rescue diet, can slightly increase the mineral dentisy of subchondral bone, and the mineral dentisy of 1α(OH)ase-/- mice with a rescue diet with NAC increased compared with the 1α(OH)ase-/- mice given a rescue diet. There was no difference of mineral dentisy between 1α(OH)ase-/- mice with subcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates fed on a diet of regular mouse chow. The ALP-positive area increased in the subchondral bone of 1α(OH)ase-/- mice with regular diet, while the ALP-positive area of the mice with a rescue diet decreased dramtically, compared with their wild-type littermates. 1α(OH)ase-/- mice with a rescue diet with NAC can partly rescue the ALP-positivearea compared with the 1α(OH)ase-/- mice given a rescue diet. There was no difference of ALP-positive area between 1α(OH)ase-/- mice with subcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates fed on a diet of regular mouse chow.The type II collagen abundance were also reduced in 1α(OH)ase-/- mice with regular diet, given the rescue diet the abundance could be slightly increased, and the rescue diet with NAC also could further increase the abundance, compared with the 1α(OH)ase-/- mice just fed on rescue diet. There was also no difference of the abundance between 1α(OH)ase-/- mice with subcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates. The osteoclast numbers were increased in 1α(OH)ase-/- mice with regular diet, but the activity were low. While the number of the mice with a rescue diet decreased dramtically, compared with their wild-type littermates, and the rescue diet with NAC also could further increase the number and activity, compared with the 1α(OH)ase-/- mice just fed on rescue diet. There was also no difference of the number and activity between 1α(OH)ase-/- mice with subcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates.The increased content of H2O2 and MDA, decreased concentration of T-SOD and GSH-Px were detected in TMJ of 1α(OH)ase-/- mice compared with their wild-type littermates, given the rescue diet the content of H2O2 and MDA could be slightly reduced, and the rescue diet with NAC also could further decreased the content of H2O2 and MDA, compared with the 1α(OH)ase-/- mice just fed on rescue diet. There was also no difference of the content of H2O2 and MDA between 1α(OH)ase-/- mice with subcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates.These results demonstrated that 1,25(OH)2D deficiency contributes to the TMJ bone and cartilage formation disorders, which maight be associated with the increased levels of oxidative-stress.PartⅢ: The role of oxidative stress in 1,25(OH)2D deficiency induced TMJ OATo investigate the further effect of oxidative stress in 1,25(OH)2D deficiency induced TMJ OA, 1α(OH)ase-/- mice were maintained on a diet of regular mouse chow after weaning, or a rescue diet, or a rescue diet with N-acetyl-L-cysteine(NAC) solution(1g/L), or subcutaneous injection of 1,25(OH)2D 1ug/kg qod, and their wild-type littermates were fed on regular diet. The mice were sacrificed at the initial stage of OA(6-month-old). The results were analyzed by imageology, histomorphology, immunohistochemistry and biochemical detection.The results show that mineral density were reduced dramatically in the subchondral bone of 1α(OH)ase-/- mice with regular diet, compared with their wild-type littermates. Given the rescue diet, can slightly increase the mineral dentisy of subchondral bone, and the mineral dentisy of 1α(OH)ase-/- mice with a rescue diet with NAC increased compared with the 1α(OH)ase-/- mice given a rescue diet, but it still lower than that of their wild-type littermates. There was no difference of mineral dentisy between 1α(OH)ase-/- mice with subcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates fed on a diet of regular mouse chow.The type II collagen and glycosaminoglycan abundance were also reduced in 1α(OH)ase-/- mice with regular diet. Given the rescue diet the type II collagen and glycosaminoglycan abundance could be slightly increased, and the rescue diet with NAC also could further increase the abundance, compared with the 1α(OH)ase-/- mice just fed on rescue diet, but still decreased compared with their wild-type littermates. There was also no difference of the abundance between 1α(OH)ase-/- mice with subcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates. The osteoclast numbers were increased in 1α(OH)ase-/- mice with regular diet compared with their wild-type littermates, but the activity were still low. While both the number and activity of the ostoclast of the mice with a rescue diet increased dramtically, compared with their wild-type littermates, and the rescue diet with NAC restrained the number and activity of the osteoclasts, compared with the 1α(OH)ase-/-mice just fed on rescue diet. There was no difference of the number and activity among 1α(OH)ase-/- mice with the rescue diet with NAC, 1α(OH)ase-/- mice withsubcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates.The increased content of H2O2 and MDA, decreased concentration of T-SOD and GSH-Px were detected in TMJ of 1α(OH)ase-/- mice compared with their wild-type littermates. Given the rescue diet the content of H2O2 and MDA could be slightly reduced, and the rescue diet with NAC also could further decreased, compared with the 1α(OH)ase-/- mice just fed on rescue diet. There was also no difference of the content of H2O2 and MDA between 1α(OH)ase-/- mice with subcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates.The unmbers of DNA damage and senesence in chondrocytes were incresed in 1α(OH)ase-/- mice with regular diet. Given the rescue diet the DNA damage and senesence in chondrocytes could be slightly decreased, and the rescue diet with NAC also could further decrease the number, compared with the 1α(OH)ase-/- mice just fed on rescue diet, but still increased compared with their wild-type littermates. There was also no difference of the abundance between 1α(OH)ase-/- mice with subcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates.To further determine whether 1,25(OH)2D deficiency induced TMJ OA through NF-κB signal pathway. The expressions of p-P65, p-IκBα and IL-1α were detected by immunohistochemistry and Western blot. The deta shoued that the expression of p-P65 and p-IκBα were increased in 1α(OH)ase-/- mice with regular diet. Given the rescue diet the expressions were slightly reduced. the rescue diet with NAC also could further decrease the number, compared with the 1α(OH)ase-/- mice just fed on rescue diet, but still increased compared with their wild-type littermates. There was also no difference of the expressions between 1α(OH)ase-/- mice with subcutaneous injection of 1,25(OH)2D 1ug/kg qod and their wild-type littermates.These results demonstrated that 1,25(OH)2D deficiency resulted in higher levels of oxidative stress in TMJ, which could induce the DNA damage and accelerate the senescence progression of chondrocytes. Then activated the NF-κB signal pathway, which resulted in the SASP, and the secretion of senescence-associated inflammatory cytokines played an important role in the development and progression of TMJ OA.