Osteogenesis imperfecta(OI)is a group of diseases caused by defects in type I collagen processing which result in skeletal fragility.While these disorders have been regarded as defects in osteoblast function,the role ...Osteogenesis imperfecta(OI)is a group of diseases caused by defects in type I collagen processing which result in skeletal fragility.While these disorders have been regarded as defects in osteoblast function,the role of matrix-embedded osteocytes in OI pathogenesis remains largely unknown.Homozygous human SP7(c.946 C>T,R316C)mutation results in a recessive form of OI characterized by fragility fractures,low bone mineral density and osteocyte dendrite defects.To better understand how the OI-causing R316C mutation affects the function of SP7,we generated Sp7^(R342C)knock-in mice.Consistent with patient phenotypes,Sp7^(R342C/R342C)mice demonstrate increased cortical porosity and reduced cortical bone mineral density.Sp7^(R342C/R342C)mice show osteocyte dendrite defects,increased osteocyte apoptosis,and intracortical bone remodeling with ectopic intracortical osteoclasts and elevated osteocyte Tnfsf11 expression.展开更多
Age-related osteoporosis poses a significant challenge in musculoskeletal health;a condition characterized by reduced bone density and increased fracture susceptibility in older individuals necessitates a better under...Age-related osteoporosis poses a significant challenge in musculoskeletal health;a condition characterized by reduced bone density and increased fracture susceptibility in older individuals necessitates a better understanding of underlying molecular and cellular mechanisms.Emerging evidence suggests that osteocytes are the pivotal orchestrators of bone remodeling and represent novel therapeutic targets for age-related bone loss.Our study uses the prematurely aged PolgD257A/D257A(PolgA)mouse model to scrutinize age-and sex-related alterations in musculoskeletal health parameters(frailty,grip strength,gait data),bone and particularly the osteocyte lacuno-canalicular network(LCN).Moreover,a new quantitative in silico image analysis pipeline is used to evaluate the alterations in the osteocyte network with aging.Our findings underscore the pronounced degenerative changes in the musculoskeletal health parameters,bone,and osteocyte LCN in PolgA mice as early as 40 weeks,with more prominent alterations evident in aged males.Our findings suggest that the PolgA mouse model serves as a valuable model for studying the cellular mechanisms underlying age-related bone loss,given the comparable aging signs and age-related degeneration of the bone and the osteocyte network observed in naturally aging mice and elderly humans.展开更多
Osteocytes,the primary cells in bone,play a crucial role in sensing external load environments and regulating other bone cells.Due to the piezoelectric effect of the mineralized matrix and collagen that make up bone,t...Osteocytes,the primary cells in bone,play a crucial role in sensing external load environments and regulating other bone cells.Due to the piezoelectric effect of the mineralized matrix and collagen that make up bone,the mechanical stimulus received is converted into an electrical stimulus to affect the reconstruction of bone.Despite the importance of osteocyte,many studies have focused on the mechanical loading and fluid flow of it,there is still a gap in the study of the piezoelectric effects of various mechanosensors on the microscale.In this paper,we developed a finite element model of osteocytes that incorporates the piezoelectric bone matrix.This model is comprehensive,comprising the osteocyte cell body enclosed by lacuna,osteocyte processes enclosed by canaliculi,and the interposed charged ionic fluid.Additionally,it features mechanosensors such as collagen hillocks and primary cilia.In our study,we subjected the piezoelectric bone matrix model to triaxial displacement,subsequently assessing the electrical signal variations across different mechanosensors within the osteocyte.The observed disparities in mechanical perception by various mechanosensors were primarily attributable to greater liquid velocity changes in the polarization direction as opposed to other directions.Collagen hillocks showed insensitivity to piezoelectric signals,serving predominantly to mechanically transmit signals through solid-to-solid contact.In contrast,processes and primary cilia were highly responsive to piezoelectric signals.Interestingly,the processes oriented in the direction of the electric field demonstrated a differential piezoelectric signal perception compared to those in other directions.Primary cilia were especially sensitive to fluid flow pressure changes,which were influenced both by loading rates and external piezoelectric effects.Overall,our findings illuminate the complexity of mechanical perception within osteocytes in a piezoelectric environment.This adds a new dimension to our understanding and suggests avenues for future research in bone reconstruction and cellular mechanical behavioral transmission.展开更多
The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenes...The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin（SOST） that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influencea A osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or ＂osteokines＂from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin（OCN） secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2（LCN2） is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain.We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.展开更多
Osteocytes, the most abundant bone cells, form an interconnected network in the lacunar-canalicular pore system （LCS） buried within the mineralized matrix, which allows osteocytes to obtain nutrients from the blood ...Osteocytes, the most abundant bone cells, form an interconnected network in the lacunar-canalicular pore system （LCS） buried within the mineralized matrix, which allows osteocytes to obtain nutrients from the blood supply, sense external mechanical signals, and communicate among themselves and with other cells on bone surfaces. In this study, we examined key features of the LCS network including the topological parameter and the detailed structure of individual connections and their variations in cortical and cancellous compa~ tments, at different ages, and in two disease conditions with altered mechanosensing （perlecan deficiency and diabetes）. LCS network showed both topological stability, in terms of conservation of connectivity among osteocyte lacunae （similar to the ＂nodes＂ in a computer network）, and considerable variability the pericellular annular fluid gap surrounding lacunae and canaliculi （similar to the ＂bandwidth＂ of individual links in a computer network）. Age, in the range of our study （15-32 weeks）, affected only the pericellular fluid annulus in cortical bone but not in cancellous bone. Diabetes impacted the spacing of the lacunae, while the perlecan deficiency had a profound influence on the pericellular fluid annulus. The LCS network features play important roles in osteocyte signaling and regulation of bone growth and adaptation.展开更多
In order to quantify the poroelastic mechanical signals conduction and evaluate the biomechanical effectiveness of functional units(osteocyte processes,canaliculi and lacuna)in lacunar-canalicular system(LCS),a multis...In order to quantify the poroelastic mechanical signals conduction and evaluate the biomechanical effectiveness of functional units(osteocyte processes,canaliculi and lacuna)in lacunar-canalicular system(LCS),a multiscale poroelastic finite element model was established by using the Comsol Multiphysics software.The poroelastic mechanical signals(pore pressure,fluid velocity,von-Mises stress,strain)were analyzed inside the osteon-osteocyte system.The effects of osteocyte(OCY)’s shape(ellipse and circle),long axis directions(horizontal and vertical)and mechanical properties(Elastic modulus and permeability)on its poroelastic responses were examined.It is found that the OCY processes is the best mechanosensor compared with the OCY body,lacunae and canaliculi.The mechanotransduction ability of the elliptic shaped OCY is stronger than that of circular shaped.The pore pressure and flow velocity around OCYs increase as the elastic modulus and permeability of OCY increase.The established model can be used for studying the mechanism of bone mechanotransduction at the multiscale level.展开更多
Elevated oxidative stress （OS） during aging leads to bone loss. OS increases intracellular Ca2＋ （[Ca2＋]i）, resulting in cellular damage and death. We show earlier that Cx43 hemichannels open in response to OS, w...Elevated oxidative stress （OS） during aging leads to bone loss. OS increases intracellular Ca2＋ （[Ca2＋]i）, resulting in cellular damage and death. We show earlier that Cx43 hemichannels open in response to OS, which serves as a protective mechanism for osteocytes. However, the underlying mechanism is unknown. Here, we found that treatment with H202 increased [Ca2＋]i in osteocytes with [Ca2＋]i being primarily derived from an extracellular Ca2~ source. Hemichannel opening induced by OS was inhibited by the depletion of [Ca2＋]i with BAPTA-AM, a Ca2＋ chelator, suggesting that [Ca2＋]i influenced the activity of Cx43 hemichannels. Conversely, blockade of hemichannels had no effect on [Ca2＋]i. A biotinylation assay showed that cell surface-expressed Cx43 was increased by OS, which could be inhibited by BAPTA-AM, suggesting that [Ca2＋]i is necessary for Cx43 migration to the cell surface in response to OS. Together, these data suggest that increased hemichannel activity induced by OS was likely to be caused by elevated [Ca2＋]i through increased Cx43 on the cell surface.展开更多
With additional functions of osteocytes being identified, the concept that osteocytes are just ＂static lacunar-dwelling cells＂ is no longer accepted. We reviewed most of the relevant literature on osteocyte＇s funct...With additional functions of osteocytes being identified, the concept that osteocytes are just ＂static lacunar-dwelling cells＂ is no longer accepted. We reviewed most of the relevant literature on osteocyte＇s function in the direct remodeling of the perilucunar matrix, discussing the advantages and disadvantages. Special attention was paid to how the negative researchers argue about the ＂osteocytic osteolysis＂ principle, and how the positive side addressed the arguments. We also discussed the newly found data of osteocytic remodeling function from our group. With more biotechnology in hand, there is increased excitement in the prospect of now being able to answer the two important questions： do osteocytes have the capability to remove mineral from the perilacunar matrix and if so what are the molecular and cellular mechanisms？ do osteocytes have the capability to deposit new mineral on the perilacunar matrix and if so what are the cellular and molecular mechanisms？展开更多
Osteocytes reside as three-dimensionally（3D） networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of os...Osteocytes reside as three-dimensionally（3D） networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because：（1） current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and（2） primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to：（1） construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and（2） reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to：（1） distribute and entrap cells within the interstitial spaces between the microbeads and（2） maintain average cell-to-cell distance to be about 19 mm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions（SOST and FGF23） and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of：（1） studying flow-induced shear stress on the mechanotransduction function of primary osteocytes,（2） studying physiological functions of 3D-networked osteocytes with in vitro convenience,and（3） developing clinically relevant human bone disease models.展开更多
Background : This study examined the ameliorative effect of D-3-O-methylchiroinositol, isolated from the stem bark of Piliostigma thonningii, on cadmium chloride-induced osteoporosis in male Wistar rats. Methods : Thi...Background : This study examined the ameliorative effect of D-3-O-methylchiroinositol, isolated from the stem bark of Piliostigma thonningii, on cadmium chloride-induced osteoporosis in male Wistar rats. Methods : Thirty-six rats were assigned to three treatment groups(n = 12). Group A(2 mL distilled water), group B:(2.5 mg/kg b.w. CdCl_2) and group C:(2.5 mg/kg b.w. CdCl_2 and D-3-O-methyl-chiroinositol 2 mg/kg b.w.). Bone ash, calcium, phosphate, magnesium, and zinc content, as well as bone histological changes were determined at the end of months 1, 2, and 3. Results : There were significant differences( P ≤ 0.05) in the weight of the cervical, tibia, and femoral bones in all groups. The serum concentration of CdCl_2 was significantly different across the three groups with time. There was significant variation( P < 0.005) in the mean bone ash across groups. The concentration of OH-proline was significantly different( P < 0.0001) across groups. There were significant differences( P < 0.0001) in bone calcium, magnesium, zinc, and phosphorus concentrations. Histology revealed high levels of bone mineralisation in the CdCl_2-treated group, indicative of osteoporosis with hypertrophied osteocytes, while the femur of Wistar rats treated with D-3-O-methyl-chiroinositol showed bone trabeculae and viable osteocytes. Conclusion : The study concluded that D-3-O-methyl-chiroinositol extract from Piliostigma thionningii stem bark ameliorated cadmium chloride-induced osteoporosis in male Wistar rats.展开更多
Background : Osteoporosis can make bone repair difficult. Low-level laser therapy( LLLT) has been shown to be a promising tool for bone neoformation. This study aimed to analyze the effect of LLLT on calvaria bone def...Background : Osteoporosis can make bone repair difficult. Low-level laser therapy( LLLT) has been shown to be a promising tool for bone neoformation. This study aimed to analyze the effect of LLLT on calvaria bone defects of ovariectomized rats using stereology. Methods : Fifty-four Wistar rats were subjected to bilateral ovariectomy, and bone defects were created in calvaria after 150 days. The animals were divided into nine groups(n = ?6 per group), and 24 hours after the bone defects were created they received three, six or 12 sessions of LLLT at 0, 20 or 30 J/cm 2, using a 780-nm low-intensity GaAlAs laser. One-way ANOVA followed by Tukey ' s post hoc test was used for data processing. A difference of P < 0.05 was considered statistically significant. The parameters evaluated were osteocyte density( Nv_(ost)), total osteocyte number( Nto ost), trabecular surface density( Sv_t), and trabecular surface area( Sa_t). Results : Data obtained showed that Nto ost, Sv t, and Sa t in group G2 rats were significantly different from G1(0 J/cm^2)( P < 0.05). Compared to group G4, G5 presented higher values for the parameters Sv t and Sa t, and G6 presented significantly higher values for almost all the analyzed parameters( Nv _(ost), Nto_(ost), Sv_t, and Sa t)( P < 0.05). Compared to group G7, G8 showed a higher value only for the parameter Sa t, and G9 showed significantly higher values for parameters Nv ost, Nto ost, Sv_t, and Sa_t. Conclusion : We conclude that LLLT stimulated bone neoformation and contributed to an increase in the total number of osteocytes, especially with a laser energy density of 30 J/cm^2 given for six and 12 sessions.展开更多
Mechanosensors are the most important organelles for osteocytes to perceive the changes of surrounding mechanical environment.To evaluate the biomechanical effectiveness of collagen hillock,cell process and primary...Mechanosensors are the most important organelles for osteocytes to perceive the changes of surrounding mechanical environment.To evaluate the biomechanical effectiveness of collagen hillock,cell process and primary·cilium in lacunar-canalicular system(LCS),we developed pressure-electricity-structure interaction models by using the COMSOL Multiphysics software to characterize the deformation of collagen hillocks-and primary cilium-based mechanosensors in osteocyte under fluid flow and electric field stimulation.And mechanical signals(pore pressure,fluid velocity,stress,deformation)were analyzed in LCS.The effects of changes in the elastic modulus of collagen hillocks,the number and location of cell processes,the length and location of primary cilia on the mechanosensitivity and the overall poroelastic responses of osteocytes were studied.These models predict that the presence of primary cilium and collagen hillocks resulted in significant stress amplifications(one and two orders of magnitude larger than osteocyte body)on the osteocyte.The growth of cell process along the long axis could stimulate osteocyte to a higher level than along the short axis.The Mises stress of the basal body of primary cilia near the top of osteocyte is 8 Pa greater than that near the bottom.However,the presence of collagen hillocks and primary cilium does not affect the mechanical signal of the whole osteocyte body.The established model can be used for studying the mechanism of bone mechanotransduction at the multiscale level.展开更多
Osteoarthritis(OA)is an inflammatory disease involving the joints that is prevalent in the global aging population.The purpose of this study is to determine whether irisin can attenuate osteoarthritis(OA)progression i...Osteoarthritis(OA)is an inflammatory disease involving the joints that is prevalent in the global aging population.The purpose of this study is to determine whether irisin can attenuate osteoarthritis(OA)progression in anterior cruciate ligament transection(ACLT)mice models and the mechanism of irisin therapy effect on OA by increase the resistance of apoptosis in MLO-Y4 cells induced by mechanical stretch in vitro.Methods For in vivo study,3-month-old male C57BL/6 J mice were randomized to three groups,sham-operated,anterior cruciate ligament transection(ACLT)-operated treated with vehicle,and ACLT-operated treated with irisin by intraperitoneal injection once a week.Cartilage erosion was observed by HE staining.Osteoarthritis Research Society International(OARSI)scores were evaluated according to the safranin O stai-ning.The microstructure of tibia cortical bone,trabecular bone,and subchondral bone was analyzed by micro-CT and the bone histomorphometry has been administrated including mineral apposition rate(MAR).Edu staining and cck-8 were used for the detection of the proliferation of MLO-Y4 cells.For mechanical stress,cells were seeded on the collagen-I coated chamber subjected with a peak biaxial stretch of 20%at 1 Hz for 16 hours to induce apoptosis.Flow cytometry was used for the detection of apoptosis and cell cycle.TUNNEL was used for staining the apoptotic cells and rt-PCR was applied for quantifying the expression of mRNA such as Bax,Bcl-2,SOST,c-myc,Opg.Western blot was utilized to confirm the mechanism of how irisin decrease the osteocyte apoptosis.Results In vivo,irisin can attenuate articular cartilage degeneration.Irisin maintains the proportion of hyaline cartilage and calcified cartilage and keep fewer cartilage erosions in ACLT-operated mice.For immunohistochemical(IHC)staining,irisin reduced the expression of caspase3,Bax and matrix metalloproteinase-13 in both cartilage and subchondral bone.Irisin-treated ACLT group shows higher Trabecular number(Tb.N)and bone volume fraction(BV/TV)compared to the vehicle-treated ACLT group.In vitro, irisin significantly increased the proliferation of MLO-Y4 cells detected by Edu and Ki67 staining,and irisin can protect the cells from both mechanical stretchinduced apoptosis detected by FITC-PI flow cytometry and maintain the cell activity by regulating the expression of Bax,Bcl-2,and c-myc.Transcriptome sequencing shows that irisin significantly activates the MAPK signaling pathway and we confirm the result by western blot:irisin effectively activates the Erk signaling pathway through phosphorylation and has a certain activation effect on p38 signaling pathway,no activation was observed for FAK signaling pathway.Conclusions Irisin can attenuate the progression of OA by decrease the apoptosis of osteocyte,which can improve the microarchitecture of subchondral bone.Erk pathway activation plays an important role in reducing the apoptosis of osteocyte.展开更多
Osteoporosis and osteopenia are major health issues that mainly affect elderly people,women after menopause and immobilized patients.Our previous studies have proved that sclerostin antibody(Scl-Ab)can dramatically en...Osteoporosis and osteopenia are major health issues that mainly affect elderly people,women after menopause and immobilized patients.Our previous studies have proved that sclerostin antibody(Scl-Ab)can dramatically enhance bone formation and reduce bone resorption in a severe osteoporosis rat model with the combination of ovariectomy(OVX)and hindlimb immobilization(HLS).However,the mechanism in the cellular level is unclear.The objective of this study is to assess the effect of Scl-Ab on osteocytic morphology change in a combined OVX and HLS rat model via quantification of long-and short-axis and the ratio and osteocyte volume in midshaft cortical bone.Four-month-old virgin female SD rats were divided into 7 groups(n=11 per group):Sham+Veh,Sham+HLS+Veh,Sham+HLS+Scl-Ab,OVX+Veh,OVX+Scl-Ab,OVX+HLS+Veh,OVX+HLS+Scl-Ab.HLS was performed 2 weeks after sham or OVX surgery;and treatment was initiated at the time of HLS.Scl-Ab(25 mg/kg)or vehicle was subcutaneously injected twice per week for 5 weeks.Femurs were harvested at the end of study and embedded in PMMA and polished for SEM imaging.Cortical bone mid shaft osteocyte number per bone area was quantified under 1K magnification;the ratios between long axis and short axis of osteocytes were quantified under 2K magnification;osteocyte dendrite number and surface area were quantified under 5K magnification.Osteocyte dendrites width was quantified using 10K magnification.All the quantification was done by ImageJ.We have reported that multiple morphological and structural changes in osteocytes,including a decreased osteocyte density and reduced osteocyte dendrite number in HLS,OVX or the combination group and Scl-Ab’s ability to abolish these unfavorable alterations.We continued our SEM analysis on osteocytes and discovered that the oval shape of osteocyte under HLS,OVX or HLS+OVX has been distorted toward a spindle-like shape,with relatively longer long axis and shorter short axis,assuming osteocyte has a perfect spheroid shape.The ratio between long-and short-axis showed an increased trend in OVX and HLS condition,but Scl-Ab inhibited these increases(P<0.001,P<0.01,respectively).The volume decreased in HLS,OVX group,but Scl-Ab maintained osteocytes’volume in HLS condition(P<0.001).It indicates that cortical bone responds to HLS and/or OVX and Scl-Ab treatment via multiple cellular mechanisms,including density of osteocyte,dendrite number and osteocyte shape.The change of osteocyte shape may imply an altered cytoskeleton system within osteocyte and a subsequent disruption of mechanosensing ability for osteocyte,which lead to bone loss macroscopically.These data suggest Scl-Ab’s therapeutic potential could be related with its ability to maintain osteocyte’s morphologic and structural changes induced by OVX,HLS or both.展开更多
To develop durable bone healing strategies through improved control of bone repair,it is of critical importance to understand the mechanisms of bone mechanical integrity when in contact with biomaterials and implants....To develop durable bone healing strategies through improved control of bone repair,it is of critical importance to understand the mechanisms of bone mechanical integrity when in contact with biomaterials and implants.Bone mechanical integrity is defined here as the adaptation of structural properties of remodeled bone in regard to an applied mechanical loading.Accordingly,the authors present why future investigations in bone repair and regeneration should emphasize on the matrix surrounding the osteocytes.Osteocytes are mechanosensitive cells considered as the orchestrators of bone remodeling,which is the biological process involved in bone homeostasis.These bone cells are trapped in an interconnected porous network,the lacunocanalicular network,which is embedded in a bone mineralized extracellular matrix.As a consequence of an applied mechanical loading,the bone deformation results in the deformation of this lacunocanalicular network inducing a shift in interstitial fluid pressure and velocity,thus resulting in osteocyte stimulation.The material environment surrounding each osteocyte,the so called perilacunar and pericellular matrices properties,define its mechanosensitivity.While this mechanical stimulation pathway is well known,the laws used to predict bone remodeling are based on strains developing at a tissue scale,suggesting that these strains are related to the shift in fluid pressure and velocity at the lacunocanalicular scale.While this relationship has been validated through observation in healthy bone,the fluid behavior at the bone-implant interface is more complex.The presence of the implant modifies fluid behavior,so that for the same strain at a tissue scale,the shift in fluid pressure and velocity will be different than in a healthy bone tissue.In that context,new markers for bone mechanical integrity,considering fluid behavior,have to be defined.The viewpoint exposed by the authors indicates that the properties of the pericellular and the perilacunar matrices have to be systematically investigated and used as structural markers of fluid behavior in the course of bone biomaterial development.展开更多
This study established a method for isolating large numbers of high-purity osteocytes from high-density bone.Bone fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA...This study established a method for isolating large numbers of high-purity osteocytes from high-density bone.Bone fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA nine times,and the digested cells and bone chips(BC)were cultured,digested,and passaged when cells were fully grown.The types of cells obtained were identified by morphology,viable cell counts,alkaline phosphatase staining,and biochemical activity analyses,and specific osteocyte and osteoblast markers were evaluated by quantitative real-time polymerase chain reaction.Our results showed that among the cells obtained from the third digestion(fractions 7–9)of wild mice tibias and femurs and the remaining BCs,85%–90%of the cells were osteocytes.Moreover,their morphology was approximately one-tenth to one-fifth the size of osteoblasts,star-shaped or polygonal,with a dendritic structure,negative for alkaline phosphatase staining,and showed a high expression of dmp1 and sclerostin.Ninety percent of the cells in fractions 1–3 were osteoblasts,and were fusiform or polygonal shape.The activity of osteoblast-specific alkaline phosphatase and mRNA expression were high in this fraction,while the expression of osteocyte-specific dmp1 and sclerostin was not detected.In the second portion(fractions 4–6),a large number were osteoblasts,mixed with a small number of osteocytes,and had high alkaline phosphatase activity and osteocyte mRNA levels,a specific level of the osteocyte marker dmp1,and no sclerostin was detected.Osteocytes in daβcatot mice were also successfully isolated by this method,and we found that Wnt signaling increased the proliferation of these osteocytes.The proposed method can be used to culture osteocytes and osteoblasts of high purity and can be used for isolation and culture of these two kinds of cells from high-density bone,which provides an avenue for the study of osteocyte function in vitro.展开更多
Osteocytes act as mechanosensors in bone, which can send mechanical signals directly to osteoblasts through gap junctions. However, under physiological conditions the number of gap junctions is limited because of the ...Osteocytes act as mechanosensors in bone, which can send mechanical signals directly to osteoblasts through gap junctions. However, under physiological conditions the number of gap junctions is limited because of the quantity variance of the two kinds of cells. In this study, the possibility of indirect interaction between these two cells was investigated. A new flow champer was designed in which osteocytes and osteoblasts were cocultured in two champers separately. 0steocytes were exposed to fluid flow and then the ALP activity, osteocalcin and osteopotin of osteoblasts were determined. The results showed that either ALP activity or production of osteocalcin and osteopotin in osteoblasts that were cocuhured with sheared osteocytes increased, which indicated that osteocytes could regulate osteoblasts indirectly through some soluble factors.展开更多
Changes in osteocyte spatial arrangement and orientation that are associated with aging and certain bone diseases have attracted much attention.The purpose of the current study is to demonstrate effects of osteocyte o...Changes in osteocyte spatial arrangement and orientation that are associated with aging and certain bone diseases have attracted much attention.The purpose of the current study is to demonstrate effects of osteocyte orientation on the deflection of fluid flow in bone by modeling osteocytes rotated by 0°,30°,45°,60°,and 90°relative to the bone fluid flow axis.The lacunocanalicular network was assumed to be regularly arranged and uniformly distributed and the osteon was defined as a representative cubic periodic unit cell(CPUC)at the microscale level.Calculation of canaliculi number and distribution around the osteocyte enabled estimation of osteon microstructural parameters toward the establishment of an osteon poroelastic finite element model to investigate specific loading-induced interstitial fluid flow and nutrient transport parameters in the bone under different boundary conditions and loading types.The results showed that osteocyte orientation under loading conditions approximating normal physiological loads markedly influenced predicted osteon maximum fluid pressure(p),fluid velocity(v),and fluid shear stress(τ)values.Moreover,results showing the nonuniform distribution of p andτvalues within the osteon wall indicated that osteocyte orientation and canaliculi three-dimensional distribution were important parameters for predicting the degree of anisotropy of lacuno-canalicular system permeability,of anisotropy of lacuno-canalicular system permeability,while also demonstrating that osteocyte orientation had little effect on nutrient transport.Furthermore,loading type and lacunocanalicular tortuosity effects on osteon fluid flow were greater than osteocyte orientation-associated effects.The results of this study may help researchers accurately quantify bone fluid flow behavior to enhance understanding of mechanotransduction mechanisms in bone.展开更多
Bone adapts to mechanical loading by changing its shape and mass.Osteocytes,as major mechanosensors,are critical for bone modeling/remodeling in response to mechanical stimuli.Intracellular calcium oscillation is one ...Bone adapts to mechanical loading by changing its shape and mass.Osteocytes,as major mechanosensors,are critical for bone modeling/remodeling in response to mechanical stimuli.Intracellular calcium oscillation is one of the early responses in osteocytes,and this further facilitates bone cell communication through released biochemical signals.Our previous study has found that mechanically induced calcium oscillations in osteocytes enhance the release of extracellular vesicles(EVs),and those released EVs can elevate bone formation activity.However,the mechanism of mechanically stimulated EVs’regulation of bone formation and resorption is still unclear.Here,using in vitro studies,we exposed OCY454 cells,with relatively high sclerostin expression,to steady fluid flow(SFF)and characterized the functions of rapidly released EVs in osteoblast and osteoclast regulation.Our study demonstrates that SFF stimulates intracellular calcium response in OCY454 cells and further induces sclerostin,osteoprotegerin(OPG),receptor activator of NF-κB ligand(RANKL)inside or outside EVs to regulate osteoblast and osteoclast activities.This load-induced protein and EVs release is load-duration dependent.Moreover,stimulated osteocytes rapidly regulate osteoclast maturation through EVs capsulated RANKL.In contrast,other regulating proteins,OPG,and sclerostin,are mainly released directly into the medium without EV capsulation.展开更多
The primary cilium,as a mechanical receptor of osteocytes,participates in the regulation of osteocyte mechanosensitivity.However,how the length of osteocyte primary cilia changes with fluid shear stress(FSS)are unclea...The primary cilium,as a mechanical receptor of osteocytes,participates in the regulation of osteocyte mechanosensitivity.However,how the length of osteocyte primary cilia changes with fluid shear stress(FSS)are unclear,and how the mechanical transmission within osteocytes altered by primary cilia is not well understood yet.Therefore,the ciliary length changes of osteocyte under 15dyn/cm2 of FSS were experimentally detected,and then 3D finite element models of osteocyte primary cilia containing the basal body and axoneme were built.The results showed that(1)The ciliary length of the CON group,FSS 1h,and FSS 6h were 3.71±1.34μm,3.79±1.04μm,and 1.24±0.73μm respectively,indicating the different durations of FSS might lead to the adaptive changes of cilium length.The calculations showed(2)when the ciliary length became shorter with the ciliary angle stayed the same,the deformation and stress of the cell membrane and membrane skeleton was increased.However,the deformation and stress of the cilia membrane,basal body,the rotation angles of basal body were decreased,and those of cytoplasm,cytoskeleton,actin cortex and nucleus were also decreased;(3)With the decrease of the ciliary angle,the deformation and stress of the cilia membrane,basal body,as well as the rotation angles of basal body were increased.Those of the cytoplasm,cytoskeleton,actin cortex,and nucleus were also increased except the cell membrane and membrane skeleton.The calculation results suggested the length and angle of the primary cilia,the deformation and stress of intracellular structures in osteocyte were altered with ciliary basal body,indicated the connection between the basal body and cytoskeleton may be a key factor that affected the mechanical transport in osteocytes across the cell membrane.This finally promoted the adaptive change of ciliary length under FSS.展开更多
基金support from the National Institute of Health(K99AR081897,R00AR081897)M.N.W.acknowledges funding support from the National Institute of Health(P01DK011794,R01DK116716)+1 种基金the Smith Family Foundation Odyssey Award,and the Chen Institute Massachusetts General Hospital Research Scholar(2024-2029)awardμCT and bone histomorphometry were performed by the Center for Skeletal Research at Massachusetts General Hospital,a NIH-funded program(P30AR066261 and AR075042)led by Mary Bouxsein and Marie Demay).
文摘Osteogenesis imperfecta(OI)is a group of diseases caused by defects in type I collagen processing which result in skeletal fragility.While these disorders have been regarded as defects in osteoblast function,the role of matrix-embedded osteocytes in OI pathogenesis remains largely unknown.Homozygous human SP7(c.946 C>T,R316C)mutation results in a recessive form of OI characterized by fragility fractures,low bone mineral density and osteocyte dendrite defects.To better understand how the OI-causing R316C mutation affects the function of SP7,we generated Sp7^(R342C)knock-in mice.Consistent with patient phenotypes,Sp7^(R342C/R342C)mice demonstrate increased cortical porosity and reduced cortical bone mineral density.Sp7^(R342C/R342C)mice show osteocyte dendrite defects,increased osteocyte apoptosis,and intracortical bone remodeling with ectopic intracortical osteoclasts and elevated osteocyte Tnfsf11 expression.
基金the European Research Council(ERC Advanced MechAGE-ERC-2016-ADG-741883)the Swiss National Science Foundation(no.188522).
文摘Age-related osteoporosis poses a significant challenge in musculoskeletal health;a condition characterized by reduced bone density and increased fracture susceptibility in older individuals necessitates a better understanding of underlying molecular and cellular mechanisms.Emerging evidence suggests that osteocytes are the pivotal orchestrators of bone remodeling and represent novel therapeutic targets for age-related bone loss.Our study uses the prematurely aged PolgD257A/D257A(PolgA)mouse model to scrutinize age-and sex-related alterations in musculoskeletal health parameters(frailty,grip strength,gait data),bone and particularly the osteocyte lacuno-canalicular network(LCN).Moreover,a new quantitative in silico image analysis pipeline is used to evaluate the alterations in the osteocyte network with aging.Our findings underscore the pronounced degenerative changes in the musculoskeletal health parameters,bone,and osteocyte LCN in PolgA mice as early as 40 weeks,with more prominent alterations evident in aged males.Our findings suggest that the PolgA mouse model serves as a valuable model for studying the cellular mechanisms underlying age-related bone loss,given the comparable aging signs and age-related degeneration of the bone and the osteocyte network observed in naturally aging mice and elderly humans.
基金supported by the National Natural Science Foundation of China(Grant Nos.12272250,12372310,and 82172503)China Postdoctoral Science Foundation(Grant No.2020M680913)+1 种基金Shanxi Scholarship Council of China(Grant No.2022081)Shanxi Province Graduate Education Innovation Program(Grant Nos.2022Y278 and 2023-125).
文摘Osteocytes,the primary cells in bone,play a crucial role in sensing external load environments and regulating other bone cells.Due to the piezoelectric effect of the mineralized matrix and collagen that make up bone,the mechanical stimulus received is converted into an electrical stimulus to affect the reconstruction of bone.Despite the importance of osteocyte,many studies have focused on the mechanical loading and fluid flow of it,there is still a gap in the study of the piezoelectric effects of various mechanosensors on the microscale.In this paper,we developed a finite element model of osteocytes that incorporates the piezoelectric bone matrix.This model is comprehensive,comprising the osteocyte cell body enclosed by lacuna,osteocyte processes enclosed by canaliculi,and the interposed charged ionic fluid.Additionally,it features mechanosensors such as collagen hillocks and primary cilia.In our study,we subjected the piezoelectric bone matrix model to triaxial displacement,subsequently assessing the electrical signal variations across different mechanosensors within the osteocyte.The observed disparities in mechanical perception by various mechanosensors were primarily attributable to greater liquid velocity changes in the polarization direction as opposed to other directions.Collagen hillocks showed insensitivity to piezoelectric signals,serving predominantly to mechanically transmit signals through solid-to-solid contact.In contrast,processes and primary cilia were highly responsive to piezoelectric signals.Interestingly,the processes oriented in the direction of the electric field demonstrated a differential piezoelectric signal perception compared to those in other directions.Primary cilia were especially sensitive to fluid flow pressure changes,which were influenced both by loading rates and external piezoelectric effects.Overall,our findings illuminate the complexity of mechanical perception within osteocytes in a piezoelectric environment.This adds a new dimension to our understanding and suggests avenues for future research in bone reconstruction and cellular mechanical behavioral transmission.
基金supported in part by grants from 973 Program from the Chinese Ministry of Science and Technology (MOST) (2014CB964704 and 2015CB964503)the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB19000000)the National Natural Science Foundation of China (NSFC) (31371463, 81672119, and 81725010)
文摘The skeleton is a dynamic organ that is constantly remodeled. Proteins secreted from bone cells, namely osteoblasts, osteocytes,and osteoclasts exert regulation on osteoblastogenesis, osteclastogenesis, and angiogenesis in a paracrine manner. Osteoblasts secrete a range of different molecules including RANKL/OPG, M-CSF, SEMA3A, WNT5A, and WNT16 that regulate osteoclastogenesis. Osteoblasts also produce VEGFA that stimulates osteoblastogenesis and angiogenesis. Osteocytes produce sclerostin（SOST） that inhibits osteoblast differentiation and promotes osteoclast differentiation. Osteoclasts secrete factors including BMP6, CTHRC1, EFNB2, S1P, WNT10B, SEMA4D, and CT-1 that act on osteoblasts and osteocytes, and thereby influencea A osteogenesis. Osteoclast precursors produce the angiogenic factor PDGF-BB to promote the formation of Type H vessels, which then stimulate osteoblastogenesis. Besides, the evidences over the past decades show that at least three hormones or ＂osteokines＂from bone cells have endocrine functions. FGF23 is produced by osteoblasts and osteocytes and can regulate phosphate metabolism. Osteocalcin（OCN） secreted by osteoblasts regulates systemic glucose and energy metabolism, reproduction, and cognition. Lipocalin-2（LCN2） is secreted by osteoblasts and can influence energy metabolism by suppressing appetite in the brain.We review the recent progresses in the paracrine and endocrine functions of the secretory proteins of osteoblasts, osteocytes, and osteoclasts, revealing connections of the skeleton with other tissues and providing added insights into the pathogenesis of degenerative diseases affecting multiple organs and the drug discovery process.
基金supported partially by funds from the NIH (RO1AR054385, P30GM103333)
文摘Osteocytes, the most abundant bone cells, form an interconnected network in the lacunar-canalicular pore system （LCS） buried within the mineralized matrix, which allows osteocytes to obtain nutrients from the blood supply, sense external mechanical signals, and communicate among themselves and with other cells on bone surfaces. In this study, we examined key features of the LCS network including the topological parameter and the detailed structure of individual connections and their variations in cortical and cancellous compa~ tments, at different ages, and in two disease conditions with altered mechanosensing （perlecan deficiency and diabetes）. LCS network showed both topological stability, in terms of conservation of connectivity among osteocyte lacunae （similar to the ＂nodes＂ in a computer network）, and considerable variability the pericellular annular fluid gap surrounding lacunae and canaliculi （similar to the ＂bandwidth＂ of individual links in a computer network）. Age, in the range of our study （15-32 weeks）, affected only the pericellular fluid annulus in cortical bone but not in cancellous bone. Diabetes impacted the spacing of the lacunae, while the perlecan deficiency had a profound influence on the pericellular fluid annulus. The LCS network features play important roles in osteocyte signaling and regulation of bone growth and adaptation.
基金supported by the National Natural Science Foundation of China(Grants 11972242,11702183,11632013,and 11572213)the Scientific and Technological Innovation Projects of Colleges and Universities in Shanxi Province(Grant 2017135)Philosophy and Social Sciences Research of Higher Learning Institutions of Shanxi(Grant 2017313).
文摘In order to quantify the poroelastic mechanical signals conduction and evaluate the biomechanical effectiveness of functional units(osteocyte processes,canaliculi and lacuna)in lacunar-canalicular system(LCS),a multiscale poroelastic finite element model was established by using the Comsol Multiphysics software.The poroelastic mechanical signals(pore pressure,fluid velocity,von-Mises stress,strain)were analyzed inside the osteon-osteocyte system.The effects of osteocyte(OCY)’s shape(ellipse and circle),long axis directions(horizontal and vertical)and mechanical properties(Elastic modulus and permeability)on its poroelastic responses were examined.It is found that the OCY processes is the best mechanosensor compared with the OCY body,lacunae and canaliculi.The mechanotransduction ability of the elliptic shaped OCY is stronger than that of circular shaped.The pore pressure and flow velocity around OCYs increase as the elastic modulus and permeability of OCY increase.The established model can be used for studying the mechanism of bone mechanotransduction at the multiscale level.
基金supported by Welch Foundation Grant AQ-1507National Institutes of Health Grant EY012085 to JXJ
文摘Elevated oxidative stress （OS） during aging leads to bone loss. OS increases intracellular Ca2＋ （[Ca2＋]i）, resulting in cellular damage and death. We show earlier that Cx43 hemichannels open in response to OS, which serves as a protective mechanism for osteocytes. However, the underlying mechanism is unknown. Here, we found that treatment with H202 increased [Ca2＋]i in osteocytes with [Ca2＋]i being primarily derived from an extracellular Ca2~ source. Hemichannel opening induced by OS was inhibited by the depletion of [Ca2＋]i with BAPTA-AM, a Ca2＋ chelator, suggesting that [Ca2＋]i influenced the activity of Cx43 hemichannels. Conversely, blockade of hemichannels had no effect on [Ca2＋]i. A biotinylation assay showed that cell surface-expressed Cx43 was increased by OS, which could be inhibited by BAPTA-AM, suggesting that [Ca2＋]i is necessary for Cx43 migration to the cell surface in response to OS. Together, these data suggest that increased hemichannel activity induced by OS was likely to be caused by elevated [Ca2＋]i through increased Cx43 on the cell surface.
基金supported in part by the National Institutes of Health AR-46798
文摘With additional functions of osteocytes being identified, the concept that osteocytes are just ＂static lacunar-dwelling cells＂ is no longer accepted. We reviewed most of the relevant literature on osteocyte＇s function in the direct remodeling of the perilucunar matrix, discussing the advantages and disadvantages. Special attention was paid to how the negative researchers argue about the ＂osteocytic osteolysis＂ principle, and how the positive side addressed the arguments. We also discussed the newly found data of osteocytic remodeling function from our group. With more biotechnology in hand, there is increased excitement in the prospect of now being able to answer the two important questions： do osteocytes have the capability to remove mineral from the perilacunar matrix and if so what are the molecular and cellular mechanisms？ do osteocytes have the capability to deposit new mineral on the perilacunar matrix and if so what are the cellular and molecular mechanisms？
基金the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (1R21AR065032 to W.Y.L and J.Z.)the National Science Foundation (DMR 1409779 to W.Y.L and J.Z.)
文摘Osteocytes reside as three-dimensionally（3D） networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because：（1） current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and（2） primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to：（1） construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and（2） reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to：（1） distribute and entrap cells within the interstitial spaces between the microbeads and（2） maintain average cell-to-cell distance to be about 19 mm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions（SOST and FGF23） and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of：（1） studying flow-induced shear stress on the mechanotransduction function of primary osteocytes,（2） studying physiological functions of 3D-networked osteocytes with in vitro convenience,and（3） developing clinically relevant human bone disease models.
文摘Background : This study examined the ameliorative effect of D-3-O-methylchiroinositol, isolated from the stem bark of Piliostigma thonningii, on cadmium chloride-induced osteoporosis in male Wistar rats. Methods : Thirty-six rats were assigned to three treatment groups(n = 12). Group A(2 mL distilled water), group B:(2.5 mg/kg b.w. CdCl_2) and group C:(2.5 mg/kg b.w. CdCl_2 and D-3-O-methyl-chiroinositol 2 mg/kg b.w.). Bone ash, calcium, phosphate, magnesium, and zinc content, as well as bone histological changes were determined at the end of months 1, 2, and 3. Results : There were significant differences( P ≤ 0.05) in the weight of the cervical, tibia, and femoral bones in all groups. The serum concentration of CdCl_2 was significantly different across the three groups with time. There was significant variation( P < 0.005) in the mean bone ash across groups. The concentration of OH-proline was significantly different( P < 0.0001) across groups. There were significant differences( P < 0.0001) in bone calcium, magnesium, zinc, and phosphorus concentrations. Histology revealed high levels of bone mineralisation in the CdCl_2-treated group, indicative of osteoporosis with hypertrophied osteocytes, while the femur of Wistar rats treated with D-3-O-methyl-chiroinositol showed bone trabeculae and viable osteocytes. Conclusion : The study concluded that D-3-O-methyl-chiroinositol extract from Piliostigma thionningii stem bark ameliorated cadmium chloride-induced osteoporosis in male Wistar rats.
基金FAPESP(Sao Paulo Research Foundation),Grant/Award Number:2011/50686-0National Institute and Technology-Translational Medicine(INCT.TM)CNPq(National Council for Scientific and Technological Development)
文摘Background : Osteoporosis can make bone repair difficult. Low-level laser therapy( LLLT) has been shown to be a promising tool for bone neoformation. This study aimed to analyze the effect of LLLT on calvaria bone defects of ovariectomized rats using stereology. Methods : Fifty-four Wistar rats were subjected to bilateral ovariectomy, and bone defects were created in calvaria after 150 days. The animals were divided into nine groups(n = ?6 per group), and 24 hours after the bone defects were created they received three, six or 12 sessions of LLLT at 0, 20 or 30 J/cm 2, using a 780-nm low-intensity GaAlAs laser. One-way ANOVA followed by Tukey ' s post hoc test was used for data processing. A difference of P < 0.05 was considered statistically significant. The parameters evaluated were osteocyte density( Nv_(ost)), total osteocyte number( Nto ost), trabecular surface density( Sv_t), and trabecular surface area( Sa_t). Results : Data obtained showed that Nto ost, Sv t, and Sa t in group G2 rats were significantly different from G1(0 J/cm^2)( P < 0.05). Compared to group G4, G5 presented higher values for the parameters Sv t and Sa t, and G6 presented significantly higher values for almost all the analyzed parameters( Nv _(ost), Nto_(ost), Sv_t, and Sa t)( P < 0.05). Compared to group G7, G8 showed a higher value only for the parameter Sa t, and G9 showed significantly higher values for parameters Nv ost, Nto ost, Sv_t, and Sa_t. Conclusion : We conclude that LLLT stimulated bone neoformation and contributed to an increase in the total number of osteocytes, especially with a laser energy density of 30 J/cm^2 given for six and 12 sessions.
基金supported by the National Natural Science Foundation of China(Grant Nos.11972242,11632013,11702183)China Postdoctoral Science Foundation(Grant No.2020M680913).
文摘Mechanosensors are the most important organelles for osteocytes to perceive the changes of surrounding mechanical environment.To evaluate the biomechanical effectiveness of collagen hillock,cell process and primary·cilium in lacunar-canalicular system(LCS),we developed pressure-electricity-structure interaction models by using the COMSOL Multiphysics software to characterize the deformation of collagen hillocks-and primary cilium-based mechanosensors in osteocyte under fluid flow and electric field stimulation.And mechanical signals(pore pressure,fluid velocity,stress,deformation)were analyzed in LCS.The effects of changes in the elastic modulus of collagen hillocks,the number and location of cell processes,the length and location of primary cilia on the mechanosensitivity and the overall poroelastic responses of osteocytes were studied.These models predict that the presence of primary cilium and collagen hillocks resulted in significant stress amplifications(one and two orders of magnitude larger than osteocyte body)on the osteocyte.The growth of cell process along the long axis could stimulate osteocyte to a higher level than along the short axis.The Mises stress of the basal body of primary cilia near the top of osteocyte is 8 Pa greater than that near the bottom.However,the presence of collagen hillocks and primary cilium does not affect the mechanical signal of the whole osteocyte body.The established model can be used for studying the mechanism of bone mechanotransduction at the multiscale level.
基金supported by the National Natural Science Foundation of China ( 31670957)
文摘Osteoarthritis(OA)is an inflammatory disease involving the joints that is prevalent in the global aging population.The purpose of this study is to determine whether irisin can attenuate osteoarthritis(OA)progression in anterior cruciate ligament transection(ACLT)mice models and the mechanism of irisin therapy effect on OA by increase the resistance of apoptosis in MLO-Y4 cells induced by mechanical stretch in vitro.Methods For in vivo study,3-month-old male C57BL/6 J mice were randomized to three groups,sham-operated,anterior cruciate ligament transection(ACLT)-operated treated with vehicle,and ACLT-operated treated with irisin by intraperitoneal injection once a week.Cartilage erosion was observed by HE staining.Osteoarthritis Research Society International(OARSI)scores were evaluated according to the safranin O stai-ning.The microstructure of tibia cortical bone,trabecular bone,and subchondral bone was analyzed by micro-CT and the bone histomorphometry has been administrated including mineral apposition rate(MAR).Edu staining and cck-8 were used for the detection of the proliferation of MLO-Y4 cells.For mechanical stress,cells were seeded on the collagen-I coated chamber subjected with a peak biaxial stretch of 20%at 1 Hz for 16 hours to induce apoptosis.Flow cytometry was used for the detection of apoptosis and cell cycle.TUNNEL was used for staining the apoptotic cells and rt-PCR was applied for quantifying the expression of mRNA such as Bax,Bcl-2,SOST,c-myc,Opg.Western blot was utilized to confirm the mechanism of how irisin decrease the osteocyte apoptosis.Results In vivo,irisin can attenuate articular cartilage degeneration.Irisin maintains the proportion of hyaline cartilage and calcified cartilage and keep fewer cartilage erosions in ACLT-operated mice.For immunohistochemical(IHC)staining,irisin reduced the expression of caspase3,Bax and matrix metalloproteinase-13 in both cartilage and subchondral bone.Irisin-treated ACLT group shows higher Trabecular number(Tb.N)and bone volume fraction(BV/TV)compared to the vehicle-treated ACLT group.In vitro, irisin significantly increased the proliferation of MLO-Y4 cells detected by Edu and Ki67 staining,and irisin can protect the cells from both mechanical stretchinduced apoptosis detected by FITC-PI flow cytometry and maintain the cell activity by regulating the expression of Bax,Bcl-2,and c-myc.Transcriptome sequencing shows that irisin significantly activates the MAPK signaling pathway and we confirm the result by western blot:irisin effectively activates the Erk signaling pathway through phosphorylation and has a certain activation effect on p38 signaling pathway,no activation was observed for FAK signaling pathway.Conclusions Irisin can attenuate the progression of OA by decrease the apoptosis of osteocyte,which can improve the microarchitecture of subchondral bone.Erk pathway activation plays an important role in reducing the apoptosis of osteocyte.
文摘Osteoporosis and osteopenia are major health issues that mainly affect elderly people,women after menopause and immobilized patients.Our previous studies have proved that sclerostin antibody(Scl-Ab)can dramatically enhance bone formation and reduce bone resorption in a severe osteoporosis rat model with the combination of ovariectomy(OVX)and hindlimb immobilization(HLS).However,the mechanism in the cellular level is unclear.The objective of this study is to assess the effect of Scl-Ab on osteocytic morphology change in a combined OVX and HLS rat model via quantification of long-and short-axis and the ratio and osteocyte volume in midshaft cortical bone.Four-month-old virgin female SD rats were divided into 7 groups(n=11 per group):Sham+Veh,Sham+HLS+Veh,Sham+HLS+Scl-Ab,OVX+Veh,OVX+Scl-Ab,OVX+HLS+Veh,OVX+HLS+Scl-Ab.HLS was performed 2 weeks after sham or OVX surgery;and treatment was initiated at the time of HLS.Scl-Ab(25 mg/kg)or vehicle was subcutaneously injected twice per week for 5 weeks.Femurs were harvested at the end of study and embedded in PMMA and polished for SEM imaging.Cortical bone mid shaft osteocyte number per bone area was quantified under 1K magnification;the ratios between long axis and short axis of osteocytes were quantified under 2K magnification;osteocyte dendrite number and surface area were quantified under 5K magnification.Osteocyte dendrites width was quantified using 10K magnification.All the quantification was done by ImageJ.We have reported that multiple morphological and structural changes in osteocytes,including a decreased osteocyte density and reduced osteocyte dendrite number in HLS,OVX or the combination group and Scl-Ab’s ability to abolish these unfavorable alterations.We continued our SEM analysis on osteocytes and discovered that the oval shape of osteocyte under HLS,OVX or HLS+OVX has been distorted toward a spindle-like shape,with relatively longer long axis and shorter short axis,assuming osteocyte has a perfect spheroid shape.The ratio between long-and short-axis showed an increased trend in OVX and HLS condition,but Scl-Ab inhibited these increases(P<0.001,P<0.01,respectively).The volume decreased in HLS,OVX group,but Scl-Ab maintained osteocytes’volume in HLS condition(P<0.001).It indicates that cortical bone responds to HLS and/or OVX and Scl-Ab treatment via multiple cellular mechanisms,including density of osteocyte,dendrite number and osteocyte shape.The change of osteocyte shape may imply an altered cytoskeleton system within osteocyte and a subsequent disruption of mechanosensing ability for osteocyte,which lead to bone loss macroscopically.These data suggest Scl-Ab’s therapeutic potential could be related with its ability to maintain osteocyte’s morphologic and structural changes induced by OVX,HLS or both.
文摘To develop durable bone healing strategies through improved control of bone repair,it is of critical importance to understand the mechanisms of bone mechanical integrity when in contact with biomaterials and implants.Bone mechanical integrity is defined here as the adaptation of structural properties of remodeled bone in regard to an applied mechanical loading.Accordingly,the authors present why future investigations in bone repair and regeneration should emphasize on the matrix surrounding the osteocytes.Osteocytes are mechanosensitive cells considered as the orchestrators of bone remodeling,which is the biological process involved in bone homeostasis.These bone cells are trapped in an interconnected porous network,the lacunocanalicular network,which is embedded in a bone mineralized extracellular matrix.As a consequence of an applied mechanical loading,the bone deformation results in the deformation of this lacunocanalicular network inducing a shift in interstitial fluid pressure and velocity,thus resulting in osteocyte stimulation.The material environment surrounding each osteocyte,the so called perilacunar and pericellular matrices properties,define its mechanosensitivity.While this mechanical stimulation pathway is well known,the laws used to predict bone remodeling are based on strains developing at a tissue scale,suggesting that these strains are related to the shift in fluid pressure and velocity at the lacunocanalicular scale.While this relationship has been validated through observation in healthy bone,the fluid behavior at the bone-implant interface is more complex.The presence of the implant modifies fluid behavior,so that for the same strain at a tissue scale,the shift in fluid pressure and velocity will be different than in a healthy bone tissue.In that context,new markers for bone mechanical integrity,considering fluid behavior,have to be defined.The viewpoint exposed by the authors indicates that the properties of the pericellular and the perilacunar matrices have to be systematically investigated and used as structural markers of fluid behavior in the course of bone biomaterial development.
基金supported by the National Natural Science Foundation of China(No.8167090813).
文摘This study established a method for isolating large numbers of high-purity osteocytes from high-density bone.Bone fragments derived from mice tibia and femurs were alternately digested with type I collagenase and EDTA nine times,and the digested cells and bone chips(BC)were cultured,digested,and passaged when cells were fully grown.The types of cells obtained were identified by morphology,viable cell counts,alkaline phosphatase staining,and biochemical activity analyses,and specific osteocyte and osteoblast markers were evaluated by quantitative real-time polymerase chain reaction.Our results showed that among the cells obtained from the third digestion(fractions 7–9)of wild mice tibias and femurs and the remaining BCs,85%–90%of the cells were osteocytes.Moreover,their morphology was approximately one-tenth to one-fifth the size of osteoblasts,star-shaped or polygonal,with a dendritic structure,negative for alkaline phosphatase staining,and showed a high expression of dmp1 and sclerostin.Ninety percent of the cells in fractions 1–3 were osteoblasts,and were fusiform or polygonal shape.The activity of osteoblast-specific alkaline phosphatase and mRNA expression were high in this fraction,while the expression of osteocyte-specific dmp1 and sclerostin was not detected.In the second portion(fractions 4–6),a large number were osteoblasts,mixed with a small number of osteocytes,and had high alkaline phosphatase activity and osteocyte mRNA levels,a specific level of the osteocyte marker dmp1,and no sclerostin was detected.Osteocytes in daβcatot mice were also successfully isolated by this method,and we found that Wnt signaling increased the proliferation of these osteocytes.The proposed method can be used to culture osteocytes and osteoblasts of high purity and can be used for isolation and culture of these two kinds of cells from high-density bone,which provides an avenue for the study of osteocyte function in vitro.
基金National Natural Science Foundation of China grant number: 10872024
文摘Osteocytes act as mechanosensors in bone, which can send mechanical signals directly to osteoblasts through gap junctions. However, under physiological conditions the number of gap junctions is limited because of the quantity variance of the two kinds of cells. In this study, the possibility of indirect interaction between these two cells was investigated. A new flow champer was designed in which osteocytes and osteoblasts were cocultured in two champers separately. 0steocytes were exposed to fluid flow and then the ALP activity, osteocalcin and osteopotin of osteoblasts were determined. The results showed that either ALP activity or production of osteocalcin and osteopotin in osteoblasts that were cocuhured with sheared osteocytes increased, which indicated that osteocytes could regulate osteoblasts indirectly through some soluble factors.
基金supported by the Department of Science and Technology of Jilin Province(Grant No.YDZJ202201-ZYTS568)the National Natural Science Foundation of China(Grant No.82172593)the Doctoral Program Foundation of Jilin Medical University(Grant No.JYBS2021025LK).
文摘Changes in osteocyte spatial arrangement and orientation that are associated with aging and certain bone diseases have attracted much attention.The purpose of the current study is to demonstrate effects of osteocyte orientation on the deflection of fluid flow in bone by modeling osteocytes rotated by 0°,30°,45°,60°,and 90°relative to the bone fluid flow axis.The lacunocanalicular network was assumed to be regularly arranged and uniformly distributed and the osteon was defined as a representative cubic periodic unit cell(CPUC)at the microscale level.Calculation of canaliculi number and distribution around the osteocyte enabled estimation of osteon microstructural parameters toward the establishment of an osteon poroelastic finite element model to investigate specific loading-induced interstitial fluid flow and nutrient transport parameters in the bone under different boundary conditions and loading types.The results showed that osteocyte orientation under loading conditions approximating normal physiological loads markedly influenced predicted osteon maximum fluid pressure(p),fluid velocity(v),and fluid shear stress(τ)values.Moreover,results showing the nonuniform distribution of p andτvalues within the osteon wall indicated that osteocyte orientation and canaliculi three-dimensional distribution were important parameters for predicting the degree of anisotropy of lacuno-canalicular system permeability,of anisotropy of lacuno-canalicular system permeability,while also demonstrating that osteocyte orientation had little effect on nutrient transport.Furthermore,loading type and lacunocanalicular tortuosity effects on osteon fluid flow were greater than osteocyte orientation-associated effects.The results of this study may help researchers accurately quantify bone fluid flow behavior to enhance understanding of mechanotransduction mechanisms in bone.
文摘Bone adapts to mechanical loading by changing its shape and mass.Osteocytes,as major mechanosensors,are critical for bone modeling/remodeling in response to mechanical stimuli.Intracellular calcium oscillation is one of the early responses in osteocytes,and this further facilitates bone cell communication through released biochemical signals.Our previous study has found that mechanically induced calcium oscillations in osteocytes enhance the release of extracellular vesicles(EVs),and those released EVs can elevate bone formation activity.However,the mechanism of mechanically stimulated EVs’regulation of bone formation and resorption is still unclear.Here,using in vitro studies,we exposed OCY454 cells,with relatively high sclerostin expression,to steady fluid flow(SFF)and characterized the functions of rapidly released EVs in osteoblast and osteoclast regulation.Our study demonstrates that SFF stimulates intracellular calcium response in OCY454 cells and further induces sclerostin,osteoprotegerin(OPG),receptor activator of NF-κB ligand(RANKL)inside or outside EVs to regulate osteoblast and osteoclast activities.This load-induced protein and EVs release is load-duration dependent.Moreover,stimulated osteocytes rapidly regulate osteoclast maturation through EVs capsulated RANKL.In contrast,other regulating proteins,OPG,and sclerostin,are mainly released directly into the medium without EV capsulation.
基金National Natural Science Foundation of China(11972068,12002026).
文摘The primary cilium,as a mechanical receptor of osteocytes,participates in the regulation of osteocyte mechanosensitivity.However,how the length of osteocyte primary cilia changes with fluid shear stress(FSS)are unclear,and how the mechanical transmission within osteocytes altered by primary cilia is not well understood yet.Therefore,the ciliary length changes of osteocyte under 15dyn/cm2 of FSS were experimentally detected,and then 3D finite element models of osteocyte primary cilia containing the basal body and axoneme were built.The results showed that(1)The ciliary length of the CON group,FSS 1h,and FSS 6h were 3.71±1.34μm,3.79±1.04μm,and 1.24±0.73μm respectively,indicating the different durations of FSS might lead to the adaptive changes of cilium length.The calculations showed(2)when the ciliary length became shorter with the ciliary angle stayed the same,the deformation and stress of the cell membrane and membrane skeleton was increased.However,the deformation and stress of the cilia membrane,basal body,the rotation angles of basal body were decreased,and those of cytoplasm,cytoskeleton,actin cortex and nucleus were also decreased;(3)With the decrease of the ciliary angle,the deformation and stress of the cilia membrane,basal body,as well as the rotation angles of basal body were increased.Those of the cytoplasm,cytoskeleton,actin cortex,and nucleus were also increased except the cell membrane and membrane skeleton.The calculation results suggested the length and angle of the primary cilia,the deformation and stress of intracellular structures in osteocyte were altered with ciliary basal body,indicated the connection between the basal body and cytoskeleton may be a key factor that affected the mechanical transport in osteocytes across the cell membrane.This finally promoted the adaptive change of ciliary length under FSS.
