Demonstration Of Mechanical Load-stimulated Osteocytic Osteolysis And Its Mechanism In Bone Remodeling During Tooth Movement

Background:Mechanical force plays important roles in promoting tissue growth and remodeling.Of all tissues,skeletal tissues,including alveolar bone,are especially susceptible to change under mechanical force.Furthermore,behaviors of bone cells,including mesenchymal stem cells(MSCs),osteocytes,osteoblasts,and osteoclasts,are modulated by multiple mechanical stimuli.Osteocytes,the most abundant and long-lived cells in bone,play multiple roles in bone homeostasis and remodeling.Via their extensive cell matrix connections,interconnected dendrites,and the lacuno-canalicular system(LCS)that permeates bone matrix,osteocytes sense and amplify mechanical stimulation and transduce it into biochemical signals,further regulating osteoblast/osteoclast-coupled bone remodeling.In addition to promoting osteoclastic bone resorption,osteocytes can directly resorb bone matrix along the LCS via osteocytic osteolysis.Numerous in vivo and in vitro studies have revealed the role of osteocytic osteolysis in a variety of physiologically challenging conditions such as lactation,vitamin D deficiency,and parathyroid hormone(PTH)treatment.There has been no concluded clear interaction between mechanical loading and ostcocytic osteolysis.Previous study revealed increased lacuna size induced by osteocytic osteolysis in the tibia and lumbar in mice during lactation,while lactation failed to induce osteocytic osteolysis in the calvaria,an unloaded bone,which imply the potential mechanical coupling in lactation induced osteocytic osteolysis.Tooth movement is a mechanically stimulated bone remodeling process.During this process,osteocytes sense compression or tension stimuli and regulate the recruitment,differentiation,and bone remodeling functions of osteoblasts,osteoclasts,and MSCs.Moreover,several mechanisms have been proven to support the important orchestrational roles of osteocytes during tooth movement and bone remodeling,such as secretion of cytokines,cell apoptosis,and necrosis.However,whether osteocytes,as mechanically sensitive cells,sense mechanical stimuli and directly resorb their localized bone matrix via osteocytic osteolysis during tooth movement remains unclear.Purpose:In this study,we used an in vivo model of mechanical load-induced tooth movement in animals and cyclic-stretch loading and continuous compression loading in vitro to learn whether osteocytes sensed mechanical stimuli and directly resorbed their localized bone matrix via osteocytic osteolysis,thereby contributing to alveolar-bone remodeling during tooth movement.Materials and Methods:1.In-vivo experiments:1)We established experimental animal model of mechanical loading in 50 male Wistar rats.For the experiment groups,the right maxillary first molar and the incisors were ligated by nickel-titanium coil springs(5 mm length,509-0010,TOMY,Japan);animals in the control group received the same treatment except for insertion of coil springs.Each coil spring provided approximately 25 g force to move teeth.After 1,3,7 and 14 days of treatment,the rats were sacrificed by overdose of pentobarbital sodium,and the right maxillae were harvested,half of maxillae(n = 5 in each group)were fixed in sodium-phosphate buffered formaldehyde(4%Paraformaldehyde,pH 7.2)for decalcified and embedded in paraffin,followed by transverse sections for Masson staining,Immunohistochemical staining and Tartrate-resistant acid phosphatase(TRAP)staining;the other(n=5 in each group)were fixed in sodium-phosphate buffered formaldehyde(4%formaldehyde,pH 7.0)before transferred to 70%ethanol for micro-computed tomography before further embedded in poly methyl-methacrylate(PMMA),sectioning and examination using backscatter scanning electron microscope(BSEM).2)For further analyze direct bone remodeling of osteocyte on lacunar wall,we used double-fluorochrome labeling to label the mineral being replaced within lacunae on the perilacunar matrix.Calcein green(5 mg/kg ip)and Alizarin red(20 mg/kg ip)were injected at different time points.12 male Wistar rats were divided to 3 groups:Control group received sham surgery and with a 7-day observation period;the osteolysis group received were mechanical loaded and possess a 7-day observation period from the beginning of mechanical loading;the recovery group were mechanical loaded and received a 7-day observation period from 7 days after mechanical loading.Calcein green(5 mg/kg ip)was injected at 2 days before the beginning of observation periods,and Alizarin red(20 mg/kg ip)were injected at 2 days before the ending of the observation periods.After fixed in 4%Paraformaldehyde at 4℃ for 48h,samples were dehydrated in graded ethanol before embedded in PMMA.Sections with thickness about 150 μm were obtained and observed using confocal fluorescent microscopy.3)Next,to analyze the potential effect of osteocytic osteolysis on the bone remodeling during orthodontic tooth movement,we used micro-CT and nano-indication to assess the bone mineral density and the mechanical properties of peri-osteocytic bone matrix.4)For further investigate the mechanism by which mechanical loading induces oetcocytic osteolysis,we assessed the expression of Sclerostin and PTHR1 in osteocytes of the alveolar bone under mechanical loading;the serum Calcium and PTH level were assessed as well.2.In-vitro experiments:Cells were seeded into 6-well plates coated with collagen type I at a density of 2 × 105 cells/well in 2 mL medium.When cell confluence was approximately 80%,1 g/cm2 continuous compressive force was applied to the MLO-Y4 using glass layers and aluminium tube caps containing weighed metal balls,cells were continuously compressed for 6,12,or 24 h and collected at the same time point.The control group was maintained under identical culture conditions without compression.mRNA and proteinsamples were collected at the same time point for reverse transcription polymerase chain reaction(RT-PCR)and western blot analysis.Further,cell was also subject to 4%formaldehyde for fixation followed by immunofluorescence staining.3.Analysis:Image-Pro plus software was applied for analysis of Integrated Optical Density from Immunohistochemical staining,and osteocyte lacuna area from Masson staining and BSEM.For western blot analysis,the relative density of three comparable results was measured using ImageJ analysis software.Statistical analysis and data visualization were realized using SPSS software(version 26),OrginPro 9 and Graphpad Prism(version 8.4.2).Results:1.Tooth movement distance was 48.0 ± 3.74 μm on day 1,84.0±12.1 μm on day 3(P<0.001),92.0± 6.6 μm on day 7(P<0.001),and 198.0 ± 22.0 μm on day 14(P<0.001).Therefore,the experimental-animal model of mechanical loading was fit for purpose.2.Immunohistochemical(IHC)staining revealed the expression of osteolysis-specific tartrate resistant proteins acid phosphatase 5(ACP5)and cathepsin K(CTSK).Expression of ACP5 and CTSK was significantly upregulated after force was applied for 3,and 7 days(ACP5,F 1,3,7 d,P<0.001;CTSK,F 1 d,P<0.01;F 3 d,P<0.001;F 7 d,P<0.05 versus control).TRAP staining indicated the percentage of TRAP-positive osteocytes elevated after 3-and 7-day mechanical loading(F 3,7 day,P<0.001 versus control).3.Accessed by backscatter scanning electron microscope(BSEM),the osteocyte lacunar area(Os.La.Ar.)enlarged in alveolar bone under compression.Significant enlargement of lacunar area was found on days 3 after force application(P<0.01 versus control).4.To further evaluate resorption of organic bone matrix around osteocytes,we evaluated changes in bone collagen surrounding the lacunae via Masson staining.In the compressed alveolar bone,significant enlargement of peri-osteocyte void space was found on days 1 and 3 after force application(F 1,3 d,P<0.001 versus control).5.As double fluorochrome labeling shown,two distinctive lines of fluorochrome labeling,Calcein green(first injection)and Alizarin red(second injection),can be found at the bone surface and lacunar wall.In the control group,red labeling was partly on the top of green labeling on the alveolar surface,and lacuna was labeled with green bands.The alveolar surface of osteolysis group was labeled with green without red band,and lacuna was labeled with only thin green band or intermittent red label outside the outline of green label,indicating the remove of bone mineral from the bone surface and lacunar wall.In the recovery group,a continuous red label was observed above thin green label on the bone surface,and lacunae was labeled with both fluorochromes,red label observed inside green label,indicating the mineral deposition on bone surface and inside lacuna.6.In-vitro mechanical loading on MLO-Y4 cells demonstrated elevated expression of osteolysis-specific markers(ACP5,CTSK)in both transcription and expression levels.After static-compression loading,mRNA levels of CTSK were significantly upregulated in the short term(12 h,P<0.001)and continuously upregulated in the long term(24 h,P<0.001),and those of ACP5 were significantly increased at 24 h(P<0.001).Moreover,ACP5 and CTSK protein levels were significantly upregulated at 12 and 24 h after compression loading(ACP5:12 h,P<0.01;24 h,P<0.001;CTSK:12 h,P<0.001;24 h,P<0.01),and this was further confirmed by immunofluorescence(IF)staining.These findings confirmed that MLO-Y4 cells could sense mechanical stimuli and increased the expression of functional proteins in osteocytic osteolysis.7.BMD showed a decreasing trend on days 1,3,and 7,and was almost reversed on day 14(654.3 ± 30.6 mg/cm3 on day 14 vs.675.1 ± 21.8 mg/cm3 on day 0).Nanoindication showed tissue elastic modulus and Vicker’s hardness in bone matrix surrounding lacuna significantly decreased at 1 and 3 days after mechanical loading(elastic modulus,F 1 d,P<0.05,F 3d,P<0.01;Vicker’s hardness,F 1,3 d,P<0.05).8.Serum calcium showed non-significantly alteration after 1,3,7,and 14-day mechanical loading.Parathyroid hormone(PTH)concentration in serum significantly increased at 3 days after mechanical loading(P<0.001).Immunohistochcmical staining revealed significantly elevated expression of Parathyroid hormone type receptor 1(PTHR1)in osteocytes in the compressed alveolar after 1,3 and 7 days of mechanical loading.The expression of sclerostin also significantly increased after 1 day of mechanical loading.After 12h application of compression at 1 gram,expression of PTHR1(P<0.01)instead of sclerostin significantly increased in MLO-Y4 cells.Conclusions:1.Mechanical loading induces the function of osteolysis in osteocytes.2.During mechanical force induced tooth movement,osteocytes directly remove their surrounding mineral and organic bone matrix via osteocytic osteolysis,and thus increase lacuna area of alveolar bone at the compression side.3.The elastic modulus and Vicker’s hardness in alveolar bone matrix surrounding lacuna significantly decreased during mechanical stimulated bone remodeling,which tracked reversely with lacuna area.4.Osteocytic osteolysis induced by compression during orthodontic tooth movement was tightly regulated through the parathyroid hormone(PTH)/PTHrP receptor(PTH1R)signaling pathway.