Effects Of High-fat Diet-induced Obesity On Alveolar Bone Remodeling In Rats During Orthodontic Tooth Movement

Obesity is a common metabolic syndrome that is caused by excessive fat accumulation due to abnormal metabolism.According to the latest data from the World Health Organization,more than 1.9 billion adults worldwide were overweight and more than 650 million people were obese in 2016.These figures are twice as high as in 1980.Obesity has been traditionally linked to good bone health that might be expected to protect the skeleton of adults.However,more and more evidence suggests that obesity is harmful to bone health.High-fat diet-induced obesity has detrimental effects on bone mass and bone microstructure by promoting osteoclast formation and increasing bone resorption.A recent study on rats showed that HFD increased the number of osteoclasts and decreased the number of osteoblasts,and the disruption of alveolar bone microarchitecture and horizontal alveolar bone loss was greater in the obese rats than that in the normal rats.The study also indicates that mature adipocytes co-culture with osteoclasts resulted in augmented osteoclastogenesis in vitro.At the same time,the high-fat diet regulated the expression of 30 inflammatory genes in alveolar bone,such as Fam3 c,InhBa,Tnfs11,Ackr2,Pxmp2,and Chil3.These data suggests that the mechanisms by which an high-fat diet affects bone involve the formation of osteoclasts and the expression of inflammatory genes.Orthodontic tooth movement is the process of signal transduction,cellular response,and tissue remodeling of the tooth-alveolar bone complex triggered by mechanical stimulation.The alveolar bone around the root of the tooth will be reconstructed to achieve a new balance under orthodontic force.Osteoblasts and osteoclasts play important roles in this process.Some hormones and cytokines have synergistic effects on this process.The effects of obesity on alveolar bone in response to orthodontic force and its underlying mechanisms remain unknown.The aim of the study was to investigate the effect of obesity on alveolar bone in response to orthodontic force and its underlying mechanisms by establishing animal models of obesity and orthodontic tooth movement in rats.To study the changes of alveolar bone microstructure parameters and tooth movement rate in obese rats on both the compression and tension sides.The expression of cathepsin K,RANKL,OPG,Runx2 and the number of TRAP-positive osteoclasts in periodontal tissues and the mRNA expression of TNF-?,cathepsin K,RANKL,OPG on the compression side of gingival tissues were observed in obese rats.To provide some clues for clinical orthodontic treatment in patients with obesity and overweight.PART 1: A combined rat model of high-fat diet-induced obesity and orthodontic tooth movement was establishedObjective: To establish a combined model of obesity and orthodontic tooth movement in rats.To measure the tooth movement distance in each group.Methods: Eighty male Sprague-Dawley rats(3 weeks old)were randomly divided into two groups,including the normal diet(ND)group(n=32)and high-fat diet(HFD)group(n=48).They were fed with a normal diet or a high-fat diet,respectively.Body weight was recorded every week.After a 8-week obesity induction period,the body weight of rats in the HFD group exceeded 20% of the average weight of the ND group was classified as obese rats.Finally,32 rats in both the ND and the HFD groups were obtained.Each group was subdivided into four groups(n=8)according to 1,3,7 and 14 days of orthodontic tooth movement.Orthodontic?applications?were?placed between the left upper first molar and incisors.The alveolar bone of the right maxillary first molar(without orthodontic appliance)was considered as the control.Blood samples were collected from the internal iliac vein in rats to measure blood lipids,serum TRAP,and TNF-?.After the rats were sacrificed,the maxillae around the maxillary molars were harvested bilaterally.Epididymal fat and perirenal fat were removed and weighed.The adiposity index was calculated using the formula in the literature.Body weight was recorded,and the naso-anal length was measured to calculate the Lee index.Maxillary bones from ND and HFD rats at different time points(n = 8 in each group)were scanned with a micro-CT device.The tooth movement distance in each group was measured.Results: The obese rat model was established after eight weeks of dietary intervention;increased body weight and body weight gain were observed in the HFD group compared with the ND group(P<0.05 for all).In comparison with the ND group,the HFD group had a significantly greater Lee index and adiposity index,higher serum levels of TRAP,TNF-?,triglycerides,total cholesterol,and low-density lipoprotein cholesterol,and a significantly lower level of high-density lipoprotein cholesterol(P<0.05 for all).Micro-CT analysis showed that the amount of orthodontic tooth movement was significantly greater in the HFD group than in the ND group on days 1,3 and 7(P<0.05 for all).Conclusion: The obese rat model was successfully established after eight weeks of dietary intervention.A combined model of obesity and orthodontic tooth movement in rats was established by employing orthodontic force application in the left maxillae of obese rats.Our results indicate that the rate of orthodontic tooth movement in the HFD group was faster than that in the ND group.PART 2: Effects of high-fat diet-induced obesity on alveolar bone in rats without force applicationObjective: To investigate the effects of high-fat diet-induced obesity on alveolar bone microstructure parameters,osteoblastogenesis and osteoclastogenesis in rats without force application.Methods: Trabecular bone on the mesial side of the distobuccal root and distal side of the mesial root of the right maxillary first molars were measured by Micro-CT.The expression of cathepsin K,RANKL,OPG and Runx2 on both the mesial and distal sides of periodontal tissues were analysed by immunohistochemistry staining.The number of osteoclasts on both the mesial and distal sides were observed?by TRAP staining.The mRNA expression of TNF-?,cathepsin K,RANKL,OPG on the mesial side of gingival tissues were analysed by RT-PCR.Results: The obesity induced osteopenic effect on the alveolar bone was demonstrated by significantly decreased BMD and Tb.Th and increased Tb.Sp on the mesial side(P<0.05 for all).No significant difference in the number of TRAP-positive osteoclasts was observed between the HFD and ND groups.However,the diet-induced obesity upregulated the expression of the RANKL/OPG ratio and downregulated the expression of Runx2 on the mesial side of periodontal tissues(P<0.05 for all).Compared with the ND group,the mRNA expression levels of cathepsin K,RANKL,and RANKL/OPG were significantly increased on the mesial side of gingival tissues in the HFD group,while the mRNA expression levels of OPG were significant decreased(P<0.05 for all).No significant difference in the mRNA expression of TNF-? was observed between the HFD and ND groups.Compared with the ND rats,the HFD rats exhibited an osteoporotic phenotype in alveolar bone,with significantly decreased BV/TV and Tb.N and increased Tb.Sp on the distal side(P<0.05 for all).The number of osteoclasts on the distal side was increased in HFD rats compared with the ND rats(P<0.05).There was a reduction in the expression of osteoblastic markers(Runx2)in HFD rats on the distal side of periodontal tissues(P<0.05).No significant change in RANKL/OPG was detected between the ND and HFD groups.Compared with the HFD group,very little cathepsin K immunoreactivity was observed in the ND group on both the mesial and distal sides.Conclusion: Long-term intake of a high-fat diet can disrupt the bone mass and bone microstructure of alveolar bone in rats.High-fat diet-induced obesity accelerates alveolar bone resorption by increasing the number of osteoclasts,promoting osteoclast activity and inhibiting osteoblast activity.PART 3: Effects of high-fat diet-induced obesity on alveolar bone remodeling in rats under orthodontic forceObjective: To investigate the effects of high-fat diet-induced obesity on the alveolar bone microstructure parameters,osteoblastogenesis and osteoclastogenesis on both the compression and tension sides in rats.Methods: Trabecular bone on both the compression and tension sides were measured by Micro-CT after 1,3,7 and 14 days of orthodontic tooth movement.The expression of cathepsin K,RANKL,OPG and Runx2 on both the compression and tension sides of periodontal tissues were analysed by immunohistochemistry staining.The number of TRAP-positive osteoclasts on both the compression and tension sides were observed? by TRAP staining.The mRNA expression of TNF-?,cathepsin K,RANKL,OPG on the compression side of gingival tissues at each time points were analysed by RT-PCR.Results: On the compression side,the BV/TV,Tb.N and Tb.Th were significantly lower and the Tb.Sp was significantly higher in the HFD group than these in the ND group.On the tension side,the HFD group showed significantly lower BMD,BV/TV,Tb.N,and Tb.Th and significantly higher Tb.Sp than the ND group(P<0.05).Histologic analysis revealed an increased expression of osteoclastic markers(cathepsin K and RANKL/OPG ratio)and a decreased expression of osteoblastic markers(Runx2)on both the compression and tension sides of periodontal tissues in HFD rats compared with that in the ND rats.The number of TRAP-positive osteoclasts increased on both the compression and tension sides in the HFD group(P<0.05).The qPCR analysis indicated that the mRNA levels of TNF-?,cathepsin K,RANKL RANKL/OPG on the compression side were significantly increased in HFD rats compared with that in the ND rats.The mRNA expression levels of OPG were significant decreased in HFD rats compared with that in the ND rats.Conclusion: Obesity induced by high-fat diet can further accelerate alveolar bone remodeling and disruption of bone microstructure under orthodontic force by reducing osteoblastogenesis,increasing osteoclastogenesis and regulating inflammatory cytokine expression.The increased alveolar bone remodeling in the obese rats lead to an accelerated OTM.New bone formation was reduced in the tension area will reduce the safety and stability of orthodontic treatment.