Paracrine and endocrine actions of bone——the functions of secretory proteins from osteoblasts, osteocytes, and osteoclasts

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.

The dependences of osteocyte network on bone compartment, age, and disease

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.

Elevated Intracellular Ca^(2+) Signals by Oxidative Stress Activate Connexin 43 Hemichannels in Osteocytes

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.

Osteocyte Remodeling of the Perilacunar and Pericanalicular Matrix

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？

Ex vivo 3D osteocyte network construction with primary murine bone cells

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.

Osteocyte viability and bone density in cadmium chloride-induced osteoporosis ameliorated with Pilostigma thonningii stem bark-extracted D-3-O-methy-chiroinositol

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.

Low-level laser therapy enhances the number of osteocytes in calvaria bone defects of ovariectomized rats

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.

Irisin Attenuates Osteoarthritis by Inhibiting Apoptosis of Osteocytes Through Activating Erk Signaling Pathway

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.

Combined OVX and Concurrent Mechanical Disuse Induced Osteocytes Morphological Alteration, and Mitigation by Mechanobiology and Sclerostin Antibody

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.

Osteocyte pericellular and perilacunar matrices as markers of bone-implant mechanical integrity

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.

Modified enzymatic collagen digestion-mediated isolation of osteocytes

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.

Effect of Osteocytes Subjected to Fluid Flow on Osteoblasts

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.

The role of primary cilia in mechanical transmission of osteocyte based on a 3D finite element model

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.

A novel decellularized matrix of Wnt signaling-activated osteocytes accelerates the repair of critical-sized parietal bone defects with osteoclastogenesis, angiogenesis, and neurogenesis

Cell source is the key to decellularized matrix(DM)strategy.This study compared 3 cell types,osteocytes with/without dominant active Wnt/β-catenin signaling(daCO and WTO)and bone marrow stromal cells(BMSCs)for their DMs in bone repair.Decellularization removes all organelles and>95%DNA,and retained>74%collagen and>71%GAG,maintains the integrity of cell basement membrane with dense boundaries showing oval and honeycomb structure in osteocytic DM and smooth but irregular shape in the BMSC-DM.DM produced higher cell survival rate(90%)and higher proliferative activity.In vitro,daCO-DM induces more and longer stress fibers in BMSCs,conducive to cell adhesion,spreading,and osteogenic differentiation.8-wk after implantation of the critical-sized parietal bone defect model,daCO-DM formed tight structures,composed of a large number of densely-arranged type-I collagen under polarized light microscope,which is similar to and integrated with host bone.BV/TV(>54%)was 1.5,2.9,and 3.5 times of WTO-DM,BMSC-DM,and none-DM groups,and N.Ob/T.Ar(3.2×10^(2)/mm^(2))was 1.7,2.9,and 3.3 times.At 4-wk,daCO-DM induced osteoclastogenesis,2.3 times higher than WTO-DM;but BMSC-DM or none-DM didn't.daCO-DM increased the expression of RANKL and MCSF,Vegfa and Angpt1,and Ngf in BMSCs,which contributes to osteoclastogenesis,angiogenesis,and neurogenesis,respectively.daCO-DM promoted H-type vessel formation and nerve markersβ3-tubulin and NeuN expression.Conclusion:daCO-DM produces metabolic and neurovascularized organoid bone to accelerate the repair of bone defects.These features are expected to achieve the effect of autologous bone transplantation,suitable for transformation application.

Multi-scale mechanotransduction of the poroelastic signals from osteon to osteocyte in bone tissue

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.

Nanostructure and mechanical properties of the osteocyte lacunar-canalicular network-associated bone matrix revealed by quantitative nanomechanical mapping

Osteocytes are the main bone cells embedded in the bone matrix where they form a large surface-area network called the lacunar-canalicular network （LCN）, interconnecting their resident spaces with the lacunae by the canaliculi. Increasing evidence points toward osteocytes playing a pivotal role in maintaining bone quality. On the one hand, osteocytes transmit mechanical strain and microenvironmental signals through the LCN to regulate the activity of osteoblasts and osteoclasts; on the other hand, osteocytes are suggested to be able to remodel the LCN-associated bone matrix. However, due to the challenges involved in the assessment and characterization of the LCN-associated bone matrix, little is known about its structure and the corresponding mechanical properties. In this work, we used quantitative nanomechanical mapping, backscattered electron imaging, and nanoindentation to characterize the LCN-associated bone matrix. The results show that the techniques can be used to probe the LCN-associated bone matrix. Nanoindentation and quantitative mechanical mapping reveal spatially inhomogeneous mechanical properties of the bone matrix associated with the osteocyte lacunae and canaliculi. The obtained nano-topography and corresponding nano-mechanical maps reveal altered mechanical properties in the immediate vicinity of the osteocyte lacunae and canaliculi, which cannot be explained solely by the topographic change.

Mechanosensation of osteocyte with collagen hillocks and primary cilia under pressure and electric field stimulation

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.

In silico analysis of RNA and small RNA sequencing data from human BM-MSCs and differentiated osteocytes, chondrocytes and tenocytes

Background:Adult stem cells have a remarkable capacity of differentiating into various cell types necessary for tissue and organ regeneration.Multiple studies have focused on the differentiation potential of mesenchymal stem cells(MSCs),however little is known about the molecular characteristics of MSCs and their progenies obtained from donors of different ages.In this study,we analyzed publicly available sequencing data obtained from young(~22-year-old,n=8)and older(~65.5-year-old,n=8)donors of MSCs and their differentiated counterparts:osteocytes,chondrocytes and tenocytes.The raw mRNA and small RNA(non-coding RNA)sequencing data was downloaded from NIH BioProjects and systematically analyzed in order to identify uniquely expressed genes in MSC-derived osteocytes,chondrocytes and tenocytes of younger and older people.Results:We identified many commonly up-and downregulated genes are similar in both groups.However,the young group displayed a greater variety of differentially expressed genes in all analyzed MSC-derived cells.This discrepancy in gene expression profiles between younger and older groups may indicate a greater differentiation potential of MSCs isolated from younger donors.miRNA and mRNA integrated analysis showed key miRNAs that regulate mRNAs in both groups from all differentiated lineages.Conclusions:Our analysis provides additional information to previously reported data for identification of MSC markers of plasticity and engraftment.In addition,our data may shed light upon the molecular mechanisms of age-associated musculoskeletal diseases caused by a decreased capacity of MSCs to regenerate the locomotor system in elderly people.

Androgens and estrogens in skeletal sexual dimorphism

Bone is an endocrine tissue expressing androgen and estrogen receptors as well as steroid metabolizing enzymes. The bioactivity of circulating sex steroids is modulated by sex hormone-binding globulin and local conversion in bone tissue, for example, from testosterone （T） to estradiol （E2） by aromatase, or to dihydrotestosterone by 5（x-reductase enzymes. Our understanding of the structural basis for gender differences in bone strength has advanced considerably over recent years due to increasing use of （high resolution） peripheral computed tomography. These microarchitectural insights form the basis to understand sex steroid influences on male peak bone mass and turnover in cortical vs trabecular bone. Recent studies using Cre/LoxP technology have further refined our mechanistic insights from global knockout mice into the direct contributions of sex steroids and their respective nuclear receptors in osteoblasts, osteoclasts, osteocytes, and other cells to male osteoporosis. At the same time, these studies have reinforced the notion that androgen and estrogen deficiency have both direct and pleiotropic effects via interaction with, for example, insulin-like growth factor 1, inflammation, oxidative stress, central nervous system control of bone metabolism, adaptation to mechanical loading, etc., This review will summarize recent advances on these issues in the field of sex steroid actions in male bone homeostasis.

A fluid flow model in the lacunar-canalicular system under the pressure gradient and electrical field driven loads

The lacunar-canalicular system(LCS)is acknowledged to directly participate in bone tissue remodeling.The fluid flow in the LCS is synergic driven by the pressure gradient and electric field loads due to the electro-mechanical properties of bone.In this paper,an idealized annulus Maxwell fluid flow model in bone canaliculus is established,and the analytical solutions of the fluid velocity,the fluid shear stress,and the fluid flow rate are obtained.The results of the fluid flow under pressure gradient driven(PGD),electric field driven(EFD),and pressure-electricity synergic driven(P-ESD)patterns are compared and discussed.The effects of the diameter of canaliculi and osteocyte processes are evaluated.The results show that the P-ESD pattern can combine the regulatory advantages of single PGD and EFD patterns,and the osteocyte process surface can feel a relatively uniform shear stress distribution.As the bone canalicular inner radius increases,the produced shear stress under the PGD or P-ESD pattern increases slightly but changes little under the EFD pattern.The increase in the viscosity makes the flow slow down but does not affect the fluid shear stress(FSS)on the canalicular inner wall and osteocyte process surface.The increase in the high-valent ions does not affect the flow velocity and the flow rate,but the FSS on the canalicular inner wall and osteocyte process surface increases linearly.In this study,the results show that the shear stress sensed by the osteocyte process under the P-ESD pattern can be regulated by changing the pressure gradient and the intensity of electric field,as well as the parameters of the annulus fluid and the canaliculus size,which is helpful for the osteocyte mechanical responses.The established model provides a basis for the study of the mechanisms of electro-mechanical signals stimulating bone tissue(cells)growth.