The Effects Of Osteoblasts On Osteoclasts' Formation And Activation Mediated By 1α, 25-(OH)₂D₃

Bone metabolism is the basic process of life to maintain bone tissue updating continuously and to keep vitality of life, and this process depends on the bone remodeling. In various skeletal disease associated with bone loss, such as bone malnutrition, increased osteoclastic bone resorption exceeds formation resulting in low bone mass, skeletal fragility and increased risk of fracture. Recent researchs have showed that many regulating factors for bone metabolism can regulate osteocalsts'formation and activation indirectly by effecting osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) expressed in osteoblasts. Vitamin D and its active metabolite is one of the important regulators for calcium and phosphorus metabolism. However, the therapeutic effect of calcium, phosphorus and vitamin D praeparatum are variable. Therefore, further understanding of the exact mechanisms of vitamin D in bone physiology and pathology is important to prevent metabolic bone disease. To elucidate the mechanism of bone metabolism accommodated by vitamin D, we investigated the effects on osteoblasts'proliferation, differentiation and the expression of RANKL, OPG in osteoblasts treated by different concentrations of 1α,25-(OH)₂D₃, meanwhile the influences of RANKL on osteoclasts'formation and activation detected by histochemistry staining for Tartrate-resistant acid phosphatase (TRAP) and so on.1. The effects of 1α,25-(OH)₂D₃ on proliferation, differentiation and cell cycle of osteoblasts in vitroTo study the influence of 1α,25-(OH)₂D₃ on proliferation, differentiation and Cell Cycle of Osteoblasts (OB) in vitro. OB were isolated from calvaria bone, then dealt with various concentration of 1α,25-(OH)₂D₃ (0, 10^-9, 10^-8, 10^-7 mol/L). After 24, 48, 72 h cultivation, the proliferation and the activity of alkali phosphatase (ALP) of OB was observed. 48 h incubation later, the changes of cell phase were analyzed using flow cytometer. Compared with the control group, 10^-9 mol/L 1α,25-(OH)₂D₃ promoted proliferation of OB in vitro significantly, and inhibited the ALP activity very significantly. The group with 10^-8, 10^-7 mol/L 1α,25-(OH)₂D₃ had lower proliferation rate of OB than group with 10^-9 mol/L 1α,25-(OH)₂D₃ significantly or very significantly, but stimulated the ALP activity significantly or very significantly within 48 h. At 72 h, 10^-7 mol/L 1α,25-(OH)₂D₃ had the lowest proliferation rate of OB, and the highest ALP activity. 10^-8, 10^-7 mol/L 1α,25-(OH)₂D₃ caused G2/M arrest significantly or very significantly. These results showed that low dosage of 1α,25-(OH)₂D₃ can promote proliferation and inhibit differentiation, while higher dosage of 1α,25-(OH)₂D₃ can inhibit proliferation, promote differentiation and cause G2/M arrest.2. The effects of 1α,25-(OH)₂D₃ on cytoskeleton, GJIC and [Ca²⁺]_i of osteoblasts in vitroTo study the influence of 1α,25-(OH)₂D₃ on cytoskeleton, gap junction intercellular communication (GJIC) and intracellular Ca2+ ([Ca²⁺]_i) in Osteoblasts in vitro. OB were isolated from calvaria bone. After 20 min and 24 h treated by 1α,25-(OH)₂D₃ (0, 10^-9, 10^-8, 10^-7 mol/L), [Ca²⁺]_i was evaluated. 24 and 48 h incubation later, F-actin and GJIC were observed. Compared with the control group, [Ca²⁺]_i in group with 1α,25-(OH)₂D₃ all increased significantly (P<0.05) at time 20 min. 24 h incubation later, [Ca²⁺]_i in the group with 10^-9 mol/L 1α,25-(OH)₂D₃ was the lowest (P<0.05). OB in the group with 10^-8 and 10^-7 mol/L 1α,25-(OH)₂D₃ were applanation, stress fibers formed. 48 h later, the expression of F-actin in the control group and group with 10^-9 mol/L 1α,25-(OH)₂D₃ reduced. Compared with the control group, GJIC was weakened after treated with 10^-9 mol/L 1α,25-(OH)₂D₃ very significantly (P<0.01), while GJIC enhanced in the group with 10^-8 and 10^-7 mol/L 1α,25-(OH)₂D₃ very significantly (P<0.01). These results demonstrated that [Ca²⁺]_i can be mediated by 1α,25-(OH)₂D₃, higher dosage of 1α,25-(OH)₂D₃ can maintain the morphous of OB and stimulate the communication among OB, while lower dosage of 1α,25-(OH)₂D₃ can inhibit the expression of F-actin and reduce the communication among OB. 3. The effects of 1α,25-(OH)₂D₃ on the morphous and ultrastructure of osteoblasts in vitroTo investigate the effects on the development and ultrastructure of osteoblasts (OB) under different dosages of 1α,25-(OH)₂D₃ in vitro. The morphous and ultrastructure were observed using scanning electron microscope (SEM) and transmission electron microscope (TEM) independently after cultured for 48 h. Compared with the control group, more microvillus and mitochondria were observed in the group with 10^-9 mol/L 1α,25-(OH)₂D₃. In the group with 10^-8 mol/L 1α,25-(OH)₂D₃, osteoblasts became flatter, and contained abundant slender cytoplasmic processes and endoplasmic reticula. Lots of filiform fibers forming network in the Extracellular Matrix of OB, more vacuole and calcium granule, less organelles were observed in the group with 10^-7 mol/L 1α,25-(OH)₂D₃. In conclusion, the present study verified further morphologically that higher concentration of 1α,25-(OH)₂D₃ had obviously facilitative effects on differentiation and functional expression of osteoblasts cultured in vitro, while lower dosage of 1α,25-(OH)₂D₃ stimulate proliferation.4. The expression of RANKL and OPG in osteoblasts treated by 1α,25-(OH)₂D₃ in vitroTo investigate the expression of RANKL, OPG and RANKL mRNA, OPG mRNA, osteobalsts obtained from Sprague Dawley rats were treated with different concentrations of 1α,25-(OH)₂D₃. The expression of RANKL and OPG was detected by the method of Immunohistochemistry and ELISA. RANKL mRNA and OPG mRNA were determined through FQ-PCR. Compared with the control group and the group with 10^-9 mol/L 1α,25-(OH)₂D₃, 10^-8 and 10^-7 mol/L 1α,25-(OH)₂D₃ can significantly or very significantly induce the expression of RANKL and RANKL mRNA. 10^-9, 10^-8 mol/L 1α,25-(OH)₂D₃ can stimulate the expression of OPG and OPG mRNA significantly or very significantly, while 10^-7 mol/L 1α,25-(OH)₂D₃ can inhibit the expression of OPG mRNA significantly. The ratio of RANKL/OPG in group with 10^-9 mol/L 1α,25-(OH)₂D₃ was higher than control group at the 48th hour. However the expression of RANKL/OPG and RANKL mRNA/OPG mRNA in the group with 10^-8, 10^-7 mol/L were higher than the control group and the group with 10^-9 mol/L 1α,25-(OH)₂D₃ all the time. These results showed that 1α,25-(OH)₂D₃ can enhance bone turnover through facilitating the formation and activity of osteoclasts via enhance RANKL mRNA/OPG mRNA and RANKL/OPG dose dependently.5. The effects of RANKL on osteoclasts'formation and activation in vitroTo investigate the effects of RANKL on osteoclasts'formation and activation in vitro, bone marrow cells were isolated from 5 to 6 weeks old ICR mice. The first step of culture with different cytokines (A: the control group without any cytokines; B: 50 ng/mL RANKL; C: 25 ng/mL M-CSF) was followed by the second step (â… : the control group without any cytokines;â…¡: 25 ng/mL M-CSF;â…¢: 25 ng/mL M-CSF + 50 ng/mL RANKL). The morphology was observed by phase-contrast microscope. Osteoclasts'shap and activation were identified by acid phosphatase (ACP) staing, tartrate resistant acid phosphatase (TRAP) staining and observating of F-actin, detection of resorption lacunae through scanning electron microscopy. The cells at the control group and the group with 50 ng/mL RANKL had no ability of adherence and proliferation, while 25 ng/mL M-CSF could promote cells'adherence and proliferation at the 3rd day of the first step. After 2 days incubation at the second step, there were more mononuclear giant cells treated by 25 ng/mL M-CSF + 50 ng/mL RANKL than that treated by M-CSF alone. However, all the cells formed under 25 ng/mL M-CSF had ACP activity. 9 days incubation later, the number of osteoclasts with three nucleus in the group with 25 ng/mL M-CSF + 50 ng/mL RANKL (10.17±1.55/well) was more than those in the control group (0/well) and the group with 25 ng/mL M-CSF (0.67±0.69/well) very significantly. RANKL could induce the expression of F-actin, and facilitate the formation of bone resorption lacunar. These results demonstrated that RANKL could induce osteoclats'formation and activation at presence of M-CSF, but the number of osteoclasts was still parum.