Simvastatin Inhibits Osteocyte Apoptosis And Accelerates Bone Regeneration

BackgroundBone is a mineralized connective tissue that exhibits four types of cells: osteoblast, osteoclast, bone lining cells, and osteocyte. Despite its inert appearance, bone is a highly dynamic organ that is continuously resorbed by osteoclasts and neoformed by osteoblasts. To put it simple, bone remodeling is the process of replacing old bone with new bone. Actually bone remodeling is a highly complex and coordinating cycle comprised of three phases:(1) initiation of bone resorption by osteoclasts, (2) the transition from resorption to new bone formation, and (3) the bone formation by osteoblasts. This process occurs due to coordinated actions of osteoclasts, osteoblasts, osteocytes, and bone lining cells which together form the temporary anatomical structure called basic multicellular unit. There is evidence that osteocytes act as mechanosensors and orchestrators of bone remodeling process. Normal bone remodeling is necessary for fracture healing and skeleton adaptation to mechanical use, as well as for calcium homeostasis. On the other hand, an imbalance of bone resorption and bone formation results in several bone diseases. For example, excessive resorption by osteoclasts leads to bone loss and osteoporosis, whereas the contrary may result in osteopetrosis. Thus, the equilibrium between bone formation and resorption is necessary and depends on the action of several local and systemic factors including hormones, cytokines, chemokines, and biomechanical stimulation.Fracture healing disorder, including delayed union and non-union, remains to be the most common and devastating complication after fracture treatment, usually re-hospitalization and reoperation is needed. Besides the prolonged treatment duration, the outcome is usually unpredictable. To the patients and the health care system, it’s a heavy burden, and to the physicians it’s a serious challenge. Hence, prevention and cure for these disorders has always been the focus of the orthopedic researches. There is a "Diamond concept" in the treatment of fracture non-union, it emphasizes that when dealing with fracture non-union, one should provide growth factors, scaffold and osteoblasts at the same time, and change the fixation device when necessary. We believe in the idea of the category of growth factor should be generalized, not only refers to the growth factors, it should also include all bioactive agents such as hormones, medications. Among them, one promising group is statins.Statins are the main drugs in the treatment of hypercholesterolemia, however, In addition to the cholesterol-lowering effect, statins have a series of pleiotropic effects, including bone anabolic, vasodilative, antithrombotic, antioxidant, anti-inflammatory, and immunosuppressive actions. Among these diverse effects, bone anabolism has been the focus of researchers, and the most often studied statin in this field is simvastatin. There is enough evidence to prove that simvastatin promotes bone regeneration through promoting the proliferation and differentiation of osteoblast, inhibiting osteoblast apoptosis and inhibiting osteoclastogenesis. However, it remains unclear whether it has any impacts on the most abundant cell type in bone, namely, osteocytes.Osteocytes are important receptors, integrators and sensors in bone, which has long been considered a less active cells. However, it has been reported that osteocytes will suffer apoptosis in the state of high bone turnover like bone modeling and metabolic bone disease. This finding has rekindled the passion of studying osteocytes. Over the past decade, we have dramatically expended our understanding over osteocyte, due to the identification of some osteocyte specific makers such as dentin matrix protein 1 (DMP1) and sclerostin. Almost exclusively secreted by osteocytes, sclerostin is a negative regulator of bone regeneration, mainly through inhibiting the proliferation and differentiation of osteoblasts. The expression of sclerostin is regulated by various factors, including mechanical stress, cytokines and endocrine. Plenty of researches have confirmed that downregulation of sclerostin enhances bone formation. Clinically, sclerostin monoclonal antibody was used to treat menopausal osteoporosis, which significantly reduces the sclerostin levels and restores bone mineral density. Receptor activator for nuclear factor-κB ligand (RANKL) is a type Ⅱ transmembrane protein and also a member of the tumor necrosis factors superfamily, which is found to be the only factor who induces osteoclastogenesis. Research has shown that unloading could increase osteocyte apoptosis, as well as the expression level of RANKL. In addition, osteocyte is the main source of RANKL, inhibiting osteocytes apoptosis will downregulate RANKL expression. It seems that osteocyte is more like the "commander cell" of bone modeling and remodeling, influencing osteoblasts and osteoclasts function by sclerostin and RANKL. So far, however, there is still a lack of information refer to the relationship between osteocyte apoptosis and the expression of sclerostin and RANKL.Objective1. To explore the impact of simvastatin on repairing of rat open tibia osteotomy by the means of medical imaging, histopathology and molecular biology, with special focus on the simvastatin-induced inhibition of osteocyte apoptosis, aiming to provide theoretical basis for subsequent research.2. To verify the relationships between simvastatin, osteocyte apoptosis and expression of sclerostin and RANKL via the isolation and culture of primary osteocyte of rat.MethodsIn vivo1. Preparation of simvastatin coated implant160 mg simvastatin was dissolved in the solution composed of 1.5 ml ethyl aceta and 100 mg PDLLA, yielding a 10% w/w concentration of simvastatin working solution. K-wire with the length of 30mm and diameter of 1.0mm were dipped twice in the simvastatin working solution and them dried under laminar condition, kept in-20° for use.2. Modeling and grouping72 female Sprague Dawley rats were randomly divided into simvastatin treating group and control group. Open tibia osteotomy was performed according to the published method by Christine et al. The simvastatin coated implants were use as intramedullary fixation devices for the experiment group, while the one for control group there’s only PDLLA coating.3. Radiographic examination2 weeks,4 weeks and 8 weeks after surgery, the animal were sedated and subjected to radiographic examination. The modified Lane-Sandhu radiographic score was used to evaluate the healing status of both groups.4. Histopathological examination2 weeks,4 weeks and 8 weeks after surgery, animal were sacrificed and the tibiae were dissected and subjected to the process of decalcification, dehydration, tissue transparent, infiltration paraffin, embedding and finally sectioned into 5μm slices. HE staining and safranin O-fast green staining were used for histopathological examination.5. Western blot assay2 weeks,4 weeks and 8 weeks after surgery, routine western blotting assay were performed to detect the expression of sclerostin and RANKL within fracture calluses of both group.6. Real-time PCR assayReal-time PCR assay was performed at 2 weeks,4 weeks and 8 weeks post-surgery to evaluate the mRNA level of Bcl-2, caspase-3 and sclerostin in fracture calluses. The PCR procedures were performed according to the TaKaRa RNA PCR Kit(AMV) Ver.3.0 instruction.7. Immunohistochemical stainingImmunohistochemical staining was performed at 4 weeks after surgery to evaluate the expression of caspase-3, sclerostin and a-SMA protein.8. TUNEL assayTUNEL in situ cell apoptosis detection assay was performed at 4 weeks after surgery to assess the apoptosis of osteocytes in the samples from both groups. The procedures were performed under the instruction of the manufacturer.9. Biomechanics testAt 8 weeks after surgery, three-point bending test was performed to biomechanical property of the tibia samples from each group. Ultimate load and elasticity modulus were recorded. The results were expressed as percentage to the contralateral side.In vitro1. Isolation and identification of primary osteocyte from SD ratThe long bone and calvarias were dissected from 3 infantile rats, and the periosteum and other attached soft tissue were removed thoroughly. Bone marrow were flushed out with Hank’s solution. Consecutively digest the bone particles with collagenase and 0.02% EDTA solution. Cells were collected at the final digestion and seeded in to rat tail collagen coated dish and cultured with α-MEM and supplemented with 5% FBS and 5% calf serum. The 5th to 10th passages were used in the present study. The distinguishing features of osteocytes include (1) morphology:osteocytes are usually star shape or branch-like with multiple long dendrites which connects to the surrounding cells. (2) low alkaline phosphatase activity compared to osteoblasts. (3) higher level of osteocalcin expression when compared to osteoblasts.2. Establishment of osteocyte apoptosis modelTNF-α was used to induce apoptosis of osteocytes. In order to obtain the optimal concentration of TNF-α stimulation, a serial concentration of TNF-α was used to stimulate osteocyte and detect the corresponding apoptosis rate with FlowCytometric.3. Detection of osteocyte apoptosisTUNEL assay were performed to detect apoptosis of osteocyte under deferent conditions. All procedures were performed under the instruction of the manufacturer. Assess the result with fluorescent microscope, set the excitation wavelength for 460 nm and the emission wavelength for 545 nm.4. Reactive oxygen species detectionIntracellular reactive oxygen species was detected in osteocytes using the Cellular Reactive Oxygen Species Detection Assay Kit (Deep Red Fluorescence). Briefly, the ROS assay solution (100μl/well) was added to the treated or untreated cells and incubated in a 5% CO2,37℃ incubator for 1 hour. The fluorescence signal was monitored at an excitation wavelength of 650 nm and an emission wavelength of 675nm. Untreated cells were used to determine the background fluorescence.5. Real-time PCRReal-time PCR assay was performed to evaluate the mRNA level of IL-1α、 TNF-α、NF-κB in osteocyte under different conditions. The PCR procedures were performed according to the TaKaRa RNA PCR Kit(AMV) Ver.3.0 instruction.6. Western blot assayWestern blot assay was performed to evaluate the expression level of sclerostin, RANKL protein, as well as the apoptotic gene bax, bcl-2 and caspase-3 in osteocyte. Besides, the activation status of the MAPKs and NF-κB pathways were also assessed with western blot assay.ResultsIn vivo1. Radiographic examinationTwo weeks after surgery, the radiographs revealed that there’s no obvious difference was noticed between both group, no apparent callus formation was presented. At 4 weeks and 8 weeks after surgery, in regard to fracture callus formation and healing quality, simvastatin treatment group was superior to the control group.The Lane-Sandhu radiographic score revealed that 2 weeks postoperatively, the score of the experimental group was (0.26±0.86) points, and the control group was (0.35±1.00) points, no statistical difference presented (P>0.05). At 4 weeks post operation, the score of experimental group was (4.92±1.44) points, while the control group is (3.58±2.06) points, the statistical analysis revealed a results of P<0.05, the experimental group is superior to the control group. At 8 weeks after surgery, the Lane-Sandhu score of the experimental group was (7.92±2.53) points, while the control group is (5.17±1.94) points, yielding a statistical results of P<0.05, indicated that the experimental group is better than the control group.2. Histopathology examinationHE staining revealed that at 2 weeks after surgery, the fracture gap of both groups was filled with inflammatory tissue, no obvious fibrous calluses were noticed. 4 weeks after fracture fixation, callus formation was easily noticed in both groups, the amount of callus in experimental group was greater than the control group, with a large number of new blood vessels, and callus fiber callus was gradually replaced by cartilaginous callus. At 8 weeks after surgery, the staining showed that the cartilaginous callus in experiment group was gradually replaced with neoformed trabecular bone, and the Bone trabecular thickness was uniform and more orderly when compared to the time point of 4 weeks after surgery. Thus it can be seen that the callus remodeling was initiated in the experiment group. In contrast, the amount of callus in the control group was fewer, with retarded cartilaginous ossification. Also, the trabecular bone in the control group was presented with uneven thickness and disordered arrangement. The safranin O-fast green staining revealed a similar result, there’s more cartilaginous tissue in the control group, indicated that the cartilaginous ossification process was faster in experiment.3. Results of western blot assayThe results of western blot assay demonstrated that the protein expression level of sclerostin and RANKL in experiment group was significantly lower than the control group. When compared these 2 parameters within one group, it showed a trend of decline.4. Results of Real-time PCRThe results of western blot assay demonstrated that at 2 weeks after surgery, the caspase-3, sclerostin and RANKL mRNA level in experiment group was significantly lower than the control group (p<0.05), while the mRNA level of Bcl-2 was higher in the experiment group. At 4 weeks after surgery, the caspase-3, sclerostin and RANKL mRNA level in experiment group was still significantly lower than the control group (p<0.05), and the mRNA level of Bcl-2 was also higher in the experiment group. At 8 weeks after surgery, there’s no statistically significant difference exist between the caspase-3 and sclerostin mRNA level in both groups, while the mRNA level of Bcl-2 was still higher in the experiment group (p<0.05), the mRNA RANKL level in experiment group was higher than the control group. Except for RANKL, all other showed a trend of decline.5. Results of TUNEL assayThe TUNEL assay revealed that at 4 weeks after surgery, there were more TUNEL positive stain osteocytes in the control group than in the experiment group (t=3.648,p=0.004).6. Results of Immunohistochemical stainingAt 4 weeks after surgery, the IHC staining demonstrated that there were significantly more caspase-3 and sclerostin positive stained osteocytes in the control group. However, the micro vessel density was greater in experiment group (p<0.05).7. Biomechanical Test resultsAt 8 weeks after surgery, the three-point bending test revealed that the ultimate load and elasticity modulus of the experiment group were significantly higher than of the control group (p<0.05).In vitro1. SD rat primary osteocyte isolation and identificationAt the early stage of osteocyte culture, a few osteoblast and fibroblast were growing among the osteocyte. After several passages, it became a purer and well growing cell line, with distinguishing features. Osteocyte are star shape or branch-like, usually with more than 4 long dendrites, which connects the surrounding cells. In contrast, osteoblast is usually fusiform or cone in shape with less and slim dendrites. In addition, the alkaline phosphatase activity in osteocytes is lower than in osteoblasts, whereas the osteocalcin level in osteocytes is much higher than in osteoblasts.2. The optimal concentration of TNF-α stimulation and simvastatin administrationAccording to the results of flow cytometry, TNF-α induced osteocyte apoptosis in a dose-dependent manner. Accordingly,20ng/ml was chosen as the concentration of TNF-α stimulation. MTT test demonstrated that the viability of osteocyte decreased as the concentration of simvastatin increased (F=57.04, P<0.000). Accordingly,10-5 mo 1/1 was chosen as the concentration of simvastatin pretreatment.3. Intracellular ROS detectionThe cellular reactive species detection assay revealed that TNF-α induced significant ROS production in osteocyte (P<0.01, vs control), while simvastatin pretreatment could effectively suppress the ROS production (P<0.01 vs TNF-α treatment group).4. Detection of TNF-α-induced apoptosis of osteocytesTUNEL assay demonstrated 20ng/ml of TNF-α significantly induced osteocyte apoptosis (p<0.01, vs control). Simvastatin pretreatment exerted protection effect against TNF-α-induced osteocyte apoptosis. In addition, flow cytometry revealed a similar result, the ROS scavenger NAC was proved to suppress the TNF-α-induced osteocyte apoptosis.5. TNF-α induced upregulation of sclerostin and RANKL expression in osteocyteWestern blot assay demonstrated that 20ng/ml of TNF-α significantly increased the expression of sclerostin and RANKL (p<0.01, vs control), while simvastatin pretreatment significantly inhibited expression of sclerostin and RANKL in a dose-dependent manner.6. Simvastatin inhibited activation of apoptotic factorsWestern blot assay revealed that when exposed to TNF-α, osteocyte increased the expression of Bax and decreased the expression of Bcl-2. However, simvastatin treatment significantly reversed the expression of Bax and Bcl-2. The bcl-2/bax ratio in simvastatin group was significantly higher than in control group. TNF-α stimulation also upregulated the expression of cleaved caspase-3, indicating that caspase-3 is relevant to the TNF-α-induced apoptosis of osteocyte, and simvastatin treatment is able to decrease the expression of cleaved caspase-3.7. Simvastatin inhibits osteocyte apoptosis via inactivation of JNK/NF-κB pathwayWestern blot assay revealed that when exposed to TNF-α, osteocyte increased the phosphorylation of p38, ERK and JNK. However, simvastatin (10-5 M) treatment significantly suppressed the phosphorylation of JNK, not phosphorylation of p38 or ERK. In addition, TNF-α induced the phosphorylation of IKKα/β and simvastatin treatment could reverse this effect. On the other hand, TNF-α induced the expression of NF-κB in a time-dependent manner. The effects of MAPKs inhibitors on the expression of were assessed, the result showed that only JNK inhibitor SP600125 has inhibitory effect on the expression of NF-κB. Moreover, JNK inhibitor SP600125 and NF-κB inhibitor BAY11-7082 exerted suppressive effect on the activation of caspase-3.8. Simvastatin downregulated sclerostin and RANKL expression via inactivation of JNK/NF-κB pathwayWestern blot assay confirmed that JNK inhibitor SP600125 and NF-κB inhibitor BAY11-7082 exerted suppressive effect on the expression of sclerostin and RANKL.9. Simvastatin downregulated mRNA level of IL-1α, TNF-α and NF-κB via inactivation of JNK/NF-κB pathwayReal-time PCR demonstrated that TNF-α induced the expression level of IL-1α, TNF-α and NF-κB mRNA. NF-κB inhibitor BAY 11-7082 exerted suppressive effect on the expression of IL-la and TNF-α mRNA, while JNK inhibitor SP600125 inhibited the expression level of NF-κB mRNA.ConclusionThe present study aimed to explore the biological effects of simvastatin on bone regeneration in vivo and in vitro, with special focus on its effect upon osteocytes. The main findings of the present study are as follows:1. Locally administration of simvastatin accelerated open tibia osteotomy repair and improved fracture healing quality, which was related to inhibited osteocyte apoptosis and downregulation of sclerostin and RANKL expression;2. Through the stimulation of TNF-a, ideal osteocyte apoptosis model was established, providing satisfactory cell model for consecutive research on the biology of osteocyte;3. Simvastatin inhibited the activation of ROS/JNK/NF-κB signaling pathway, further suppressed the apoptotic pathway, finally inhibited osteocyte apoptosis;4. Simvastatin downregulated the expression of sclerostin and RANKL, which was also relevant to the inactivation of ROS/JNK/NF-κB signaling pathway.