Study On The Role Of Osterix In The Heterotopic Ossification Of Fluoride Bone Injury

Fluoride bone injury is chronic metabolic bone disease caused by long-term excess fluoride intake, which mainly pathological feature is bone metabolism disorder. Heterotopic ossification of fluoride bone injury is a special kind of ectopic osteogenesis, and its mainly clinical manifestation include extensive hyperostosis osteosclerosis, osteomalacia, osteoporosis, and calcification of interosseous membrane, muscle tendon and ligament, and so on, which may be the important factor leading to paralysis and mutilation. The active of osteoblasts and the acceleration of bone turnover are the characteristic lesions of fluoride bone injury according to a wealth of cell culture in vitro and animal experiments. Regulatory effect of the related factors in local bone metabolism network is one of the mechanisms of bone turnover acceleration resulted from excess fluoride.Osterix, a zinc-finger transcription factor, which is connected with osteoblasts and bone formation. It is specifically expressed in the process of osteoblasts differentiation and calcification, and can regulate many important genes expression, such as osteocalcin, bone sialoprotein, collagen type I and so on. Studies found that Osterix could promote the process of osteoblasts differentiation and heterotopic ossification. Core-binding factor-1 (Cbfa1, also known as Runx2) which high-restrictive expression in the bone, is an early and specific marker gene at the beginning of osteoblasts differentiation. Therefore, Osterix and Cbfa1 are the essential transcription factors in the presses of bone formation.Almostly diseases are the interaction between genes and environment. This subject analysis the protein changes of Osterix and Cbfa1 in fluoride exposures, search for the early indexes of bone damage by detecting the serum protein concentration of osterix and Cbfa1; our study also investigate the gene susceptibility of osterix in fluoride bone injury, expound the role of osterix in the process of fluoride bone injury, hoping to provide a new target for the prevention of fluoride bone injury, and providing theory basis for further research.Objective To analysis the changes of Osterix(zinc finger structures transcriptional factor) and Cbfa1 protein levels in the fluoride exposures and heterotopic ossification of fluoride bone injuries, and the correlation between serum fluoride, urinary fluoride and Osterix,Cbfa1 protein levels, to construct Logistic regression model with the associated risk factors of heterotopic ossification of fluoride bone injury, then to study on the susceptibility between heterotopic ossification of fluoride bone injury and Osterix gene, and the role of Osterix in the process of heterotopic ossification in fluoride bone injury by examining the concentrations of Osterix and Cbfa1 protein in serum and the base mutation of 1kb-length-Osterix gene.Methods Male people were chosen who have worked more than five years in one the Aluminum plant in Hubei Province, according to the different work places. Research objects included: 176 workers were chosen from the Potroom, 28 workers were chosen from the Aluminum Rod and Aluminum Strip Departments and 22 workers were chosen from the Organ and Quality Test Departments. Biomaterials were collected under the principle of informed consents. According to the Fluorotic teeth diagnostic criteria and bone X-ray examinationl (pelvic and left radius anterior posterior X-ray examination),the research object will be divided into:①75 workers who had heterotopic ossification of fluoride bone injuries(bone injury group for short), which were from the Potroom; According to the degree of heterotopic ossification in fluoride bone injury, people were divided into two groups: mild-bone injury group (30 workers) and severe-bone injury group (45 workers);②22 workers who just had dental fluorosis but not bone-injury(dental fluorosis group for short),in which 16 worker were from the Potroom, 4 worker were from the Aluminum Rod and Aluminum Strip Departments, and 2 worker were from the Organ and Quality Test Departments;③129 fluoride exposure workers without dental fluorosis or bone-injury, in which 85 worker were from the Potroom(high fluoride group for short), 24 worker were from the Aluminum Rod and Aluminum Strip Departments(low fluoride group for short), and 20 worker were from the Organ and Quality Test Departments(the control group). The concentrations of Cbfa1 and Osterix protein were detected by enzyme-linked immunosorbent assay method. Then used the Logistic regression to construct the model with the associated risk factors of heterotopic ossification of fluoride bone injury. Using HRM and DNA sequencing to detect three SNP sites (located in exon2 141-1042bp)of Osterix gene.Results 1) The concentration of Osterix in the low fluoride group was(1159.20±387.58)ng/ml, but in high fluoride group it was(743.81±492.41)ng/ml, in the control group it was(860.09±410.98)ng/ml, which was higher in the low fluoride group than in the high fluoride group and control group, and the difference had statistical significance(P<0.05);The concentration of Cbfa1 in the low fluoride group was(916.40±360.21)ng/ml, but in high fluoride group it was(629.43±430.47)ng/ml, which was higher in the low fluoride group than in the high fluoride group, and the difference had statistical significance(P<0.05); 2) The concentration of Osterix in dental fluorosis group was(1216.20±368.89)ng/ml, but in bone injury group it was(802.61±516.55)ng/ml, in the control group it was(860.09±410.98)ng/ml, which was higher in the dental fluorosis group than in the bone injury group and control group, and the difference had statistical significance(P<0.05); the concentration of Cbfa1 in dental fluorosis group was(1087.00±287.53)ng/ml, but in bone injury group it was(753.28±486.40)ng/ml, which was higher in the dental fluorosis group than in the bone injury group, and the difference had statistical significance(P<0.05); the protein concentrations of Osterix in mild-bone injury and severe-bone injury group was(797.88±486.32)ng/ml and(806.72±516.44)ng/ml separately, which lower than its concentrations in the dental fluorosis group, and the difference had statistical significance(P<0.05); the protein concentrations of Cbfa1 in mild-bone injury and severe-bone injury group was(726.38±494.43)ng/ml and (776.68±491.79)ng/ml separately, which lower than its concentrations in the dental fluorosis group, and the difference had statistical significance (P<0.05); 3) In the bone-injury group, correlation coefficient (r) between serum fluoride and Osterix, Cbfa1 protein concentration was -0.434 and -0.469 separately(P<0.05), correlation coefficient (r) between urine fluoride and Cbfa1 protein concentration was -0.436(P<0.05). But in the control group, correlation coefficient (r) between serum fluoride, urine fluoride and Osterix, Cbfa1 protein concentration was -0.227, -0.274, -0.075, -0.058 separately, and difference had no statistical significance(P>0.05). Correlation coefficient (r) between Osterix and Cbfa1 protein concentration in bone-injury and control group was 0.897and 0.878 separately(P<0.05); 4) Logistic regression model showed that: compared with the high concentration of serum fluorine(serum fluorine>0.20mg/L), the low concentration of serum fluorine(serum fluorine≤0.17 mg/L and 0.17<serum fluorine≤0.20 mg/L) had statistical significance, OR was 0.244 and 0.264 separately(P=0.039,0.033), B was -1.409 and -1.333 separately; compared with the high concentration of urine fluorine (urine fluorine>4.00mg/L), the low concentration of urine fluorine(urine fluorine≤2.00 mg/L and 2.00<urine fluorine≤4.00mg/L) had statistical significance, OR was 0.051 and 0.086 separately(P=0.000, 0.000), B was -2.978 and-2.451 separately; compared with the high concentration of Cbfa1(Cbfa1>1200.00ng/ml), the low concentration of Cbfa1(800.00ng/ml<Cbfa1≤1200.00ng/ml) had statistical significance, OR was 0.151 (P=0.016), B was -1.892; compared with the high concentration of Osterix (Osterix >1200.00ng/ml), the low concentration of Osterix(400.00ng/ml<Osterix≤800.00ng/ml and 800.00ng/ml<Osterix≤1200.00ng/ml) had statistical significance, OR was 18.148 and 5.746 separately(P=0.044, 0.030), B was 2.899 and1.749 separately; 5) The gene types of the three SNP sites of Osterix gene were CC,GG,CC, and the genotypes were same between the bone-injury and control groups, which were homozygotes, so there was no difference between two groups(P>0.05).Conclusion 1) Osterix and Cbfa1 protein concentration increased with low fluoride exposure and fluoride-loaded, but decreased with high fluoride exposure and high fluoride-loaded, that is to say, Osterix and Cbfa1 protein concentration decreased with the increasing of fluoride exposure and fluoride-loaded; 2) Osterix and Cbfa1 protein concentration increased when the body had dental fluorosis, however, it decreased after bone-injury. In other words, Osterix and Cbfa1 protein concentration decreased with the happening and worse of heterotopic ossification in fluoride bone injuries, so the decreasing turning point of Osterix protein concentration might be the time for the starting of heterotopic ossification in fluoride bone injuries; 3) In the bone-injury group, the correlation between Osterix, Cbfa1 protein concentration and serum fluorine, urine fluorine was negative, so Osterix and Cbfa1 protein concentration reduced with the increase of serum fluorine and urine fluorine; the correlation between Osterix and Cbfa1 was positive, so Osterix protein concentration increased with the rising of Cbfa1; 4) High-level serum fluorine(serum fluorine>0.20mg/L), urine fluorine(urine fluorine>4.00mg/L), Cbfa1 protein concentration(Cbfa1>1200ng/ml), and when Osterix protein concentration was between 400ng/ml and 1200ng/ml, the happening risk of heterotopic ossification of fluoride bone injuries was rising; 5) The SNP sites and gene mutation of Osterix(located in exon2,141-1042bp) were not found in the patients of the heterotopic ossification in fluorine bone injury. So the susceptibility between susceptibility and heterotopic ossification of fluoride bone injury still need further study. To sum up, Osterix and Cbfa1 protein concentration increased with low fluoride exposure, fluoride-loaded and when the body occurs dental fluorosis, but decreased with high fluoride exposure, high fluoride-loaded and after bone-injury, so the decreasing turning point of Osterix protein concentration might be the time for the starting of heterotopic ossification in fluoride bone injuries. So we think Osterix protein concentration can be used as monitoring index of early bone damage in heterotopic ossification of fluoride bone injuries.