Osteoclasts and foreign body giant cells (FBGCs) are multinucleated cells derived from monocytes, but they have distinct functions. Osteoclasts resorb bone while FBGCs form in response to foreign material. Regarding bone implants, osteoclasts are responsible for implant integration, but also for bone resorption associated to implant loosening, while FBGCs play a role in the immune response to the foreign material. Little is known about which proteins in the local environment fine-tune the multinucleation of osteoclasts or FBGCs. One candidate is Activin A (ActA). It has been shown to induce larger, more active osteoclasts, but its effect on FBGC differentiation is unknown.We investigated the effect of ActA on the differentiation of osteoclasts and FBGCs from human CD14-positive monocytes. The number of multinucleated cells and the cell area was measured. qPCR was performed to assess the effect of ActA on gene expression. ActA's influence on osteoclast and FBGC formation was studied on plastic, bone and hydroxyapatite coated Titanium discs (ALD-HA).ActA induced fewer, but bigger and more active osteoclasts on plastic and bone. In contrast, ActA did not have an effect on FBGC number. On ALD-HA, ActA reduced the number of FBGCs, but did not influence osteoclast numbers. qPCR showed that ActA upregulated the expression of several genes such as TRAcP, CIITA, OLR1, RHOBTB1 and ALK4, but mainly in osteoclasts. These results show that ActA has a different effect on osteoclasts compared to FBGCs. This difference could be caused by a difference in the expression in the canonical ActA receptor ALK4.

This study aimed to evaluate the effects of the atomic layer deposited hydroxyapatite (ALD-HA) coating of the titanium (Ti) surface on human gingival keratinocyte (HGK) cell adhesion, spreading, viability, and hemidesmosome (HD) formation. Grade 2 square-shaped Ti substrates were used ( n = 62). Half of the substrates were ALD-HA coated, while the other half were used as non-coated controls (NC). The ALD-HA surface was characterized with scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) analysis. The initial cell adhesion and HD formation of HGKs were evaluated after a 24-h cultivation period. The cell proliferation was assessed by cultivating cells for 1, 3, and 7 d. The expression levels of the integrin mediating cell adhesion were detected with the Western Blot method. In addition, cell spreading and expression of the proteins mediating cell adhesion were imaged using a confocal microscope. SEM-EDS analysis demonstrated the formation of HA on the ALD-HA surfaces. The relative cell attachment was significantly higher ( p < .05) on the ALD-HA compared to the NC surface after 1 and 3 d of cell culture. No significant difference was found in integrin α6 or β4 expression. The microscope evaluation showed significantly increased cell spreading with peripheral HD expression on ALD-HA compared to the NC surfaces ( p = .0001). Moreover, laminin γ2 expression was significantly higher on the ALD-HA than on the NC surfaces ( p < .001). Compared to the NC Ti surface, the ALD-HA coating has favorable effects on HGK proliferation, growth, and cell spreading. This indicates that the ALD-HA coating has good potential for improving mucosal attachment on implant surfaces.

This study investigated the impact of the nanocrystalline atomic layer-deposited hydroxyapatite (ALD-HA) coating of titanium (Ti) surface on the attachment and proliferation of human gingival fibroblasts (HGFs). Ti discs were divided into ALD-HA-coated and non-coated (NC) controls. HGFs were harvested from gingival biopsies of patients subjected to extraction of their third molar. The cells were cultivated on the Ti discs for 2 and 24 h to evaluate the initial cell attachment using confocal microscopy. Spreading of cells and the signals of focal adhesion proteins were measured. Moreover, the adhesion proteins vinculin and paxillin expression levels were evaluated using Western blot after 3 d of cultivation. In addition, the proliferation of HGF was assessed by cultivating the cells on Ti discs for 1, 3, and 7 d. Fibroblast spreading was significantly greater on ALD-HA surfaces than on NC surfaces after 2 h (p < 0.001). In addition, the signals of vinculin and paxillin were significantly higher on the ALD-HA than on the NC surfaces at 2 and 24 h. The confocal microscope analysis also revealed significantly higher expression of focal adhesion molecules on ALD-HA surfaces at both time points. Furthermore, the cell proliferation rate was significantly higher at d 3 (p = 0.022) and d 7 (p < 0.001) on the ALD-HA compared to the NC surfaces. These findings indicate that ALD-HA coating enhances focal adhesion formation, cell spreading, and proliferation on Ti surfaces, suggesting its potential to improve gingival tissue attachment to Ti implant surfaces.

Membrane-initiated estrogen receptor α (mERα) signaling has been shown to affect bone mass in murine models. However, it remains unknown which cell types mediate the mERα-dependent effects on bone. In this study, we generated a novel mouse model with a conditional C451A mutation in Esr1, which enables selective knockout of the palmitoylation site essential for the membrane localization of ERα (C451Af/f). First, we used Runx2-Cre mice to generate Runx2-C451Af/f mice with conditional inactivation of mERα signaling in Runx2-expressing osteoblast lineage cells. No significant changes were observed in body weight, weights of estrogen-responsive organs, or serum concentrations of estradiol between female Runx2-C451Af/f and homozygous C451Af/f littermate controls. High-resolution microcomputed tomography analysis showed a consistent decrease in cortical bone mass in the tibia, femur, and vertebra L5 of Runx2-C451Af/f mice and three-point bending analysis of humerus revealed an impaired mechanical bone strength in Runx2-C451Af/f female mice compared to controls. Additionally, primary osteoblast cultures from mice lacking mERα signaling showed impaired differentiation compared to controls. In contrast, conditional inactivation of mERα signaling in hematopoietic cells, by transplantation of bone marrow from mice lacking mERα signaling in all cells to adult wildtype female mice, did not result in any skeletal alterations. In conclusion, this study demonstrates that mERα signaling in osteoblast lineage cells plays a crucial role in the regulation of cortical bone in female mice and shows that mERα inactivation in hematopoietic cells of adult female mice is dispensable for bone regulation.

Previous studies have shown that the gut microbiota regulates bone mass and that certain strains of Bifidobacterium longum prevent bone loss in ovariectomized (ovx) mice. A novel strain of Bifidobacterium longum (B. longum subsp. longum DSM 32947; BL) with a broad carbohydrate degradation capacity and the ability to stimulate certain lactobacilli was recently identified. In the present study, we tested if BL improves bone health in gonadal intact and ovx female mice. Ten-week-old C57BL/6 J female mice were subjected to ovx or sham surgery. One week after surgery, mice were treated with arabinoxylan oligosaccharides (AXOS; veh) or a combination of AXOS and BL for five weeks. BL treatment increased BL abundance in the cecal content. Dual-energy X-ray absorptiometry showed that BL increased total body bone mineral density in both sham and ovx mice compared with veh-treated mice (p < 0.01). Computed tomography analyses showed that BL increased trabecular bone volume fraction of the L4 vertebra, mainly due to increased trabecular thickness in both sham and ovx mice (p < 0.05). In addition, BL increased the mid-diaphyseal cortical bone area of the femur (p < 0.05) and improved its strength (p = 0.05). In conclusion, treatment with BL increases parameters for bone health in female mice.

Air particle abrasion (APA) using bioactive glass (BG) effectively decontaminates titanium (Ti) surface biofilms and the retained glass particles on the abraded surfaces impart potent antibacterial properties against various clinically significant pathogens. The objective of this study was to investigate the effect of BG APA and simulated body fluid (SBF) immersion of sandblasted and acid-etched (SA) Ti surfaces on osteoblast cell viability. Another goal was to study the antibacterial effect against Streptococcus mutans. Square-shaped 10 mm diameter Ti substrates (n = 136) were SA by grit blasting with aluminum oxide particles, then acid-etching in an HCl-H2SO4 mixture. The SA substrates (n = 68) were used as non-coated controls (NC-SA). The test group (n = 68) was further subjected to APA using experimental zinc-containing BG (Zn4) and then mineralized in SBF for 14 d (Zn4-CaP). Surface roughness, contact angle, and surface free energy (SFE) were calculated on test and control surfaces. In addition, the topography and chemistry of substrate surfaces were also characterized. Osteoblastic cell viability and focal adhesion were also evaluated and compared to glass slides as an additional control. The antibacterial effect of Zn4-CaP was also assessed against S. mutans. After immersion in SBF, a mineralized zinc-containing Ca-P coating was formed on the SA substrates. The Zn4-CaP coating resulted in a significantly lower Ra surface roughness value (2.565 μm; p < 0.001), higher wettability (13.35°; p < 0.001), and higher total SFE (71.13; p < 0.001) compared to 3.695 μm, 77.19° and 40.43 for the NC-SA, respectively. APA using Zn4 can produce a zinc-containing calcium phosphate coating that demonstrates osteoblast cell viability and focal adhesion comparable to that on NC-SA or glass slides. Nevertheless, the coating had no antibacterial effect against S. mutans.

The increased limb bone density documented previously for aquatic tetrapods has been proposed to be an adaptation to overcome buoyancy during swimming and diving. It can be achieved by increasing the amount of bone deposition or by reducing the amount of bone resorption, leading to cortical thickening, loss of medullary cavity, and compaction of trabecular bone. The present study examined the effects of locomotor habit, body size, and phylogeny on the densitometric, cross-sectional, and biomechanical traits of femoral diaphysis and neck in terrestrial, semiaquatic, and aquatic carnivores, and in terrestrial and semiaquatic rodents (12 species) by using peripheral quantitative computed tomography, three-point bending, and femoral neck loading tests. Groupwise differences were analyzed with the univariate generalized linear model and the multivariate linear discriminant analysis supplemented with hierarchical clustering. While none of the individual features could separate the lifestyles or species adequately, the combinations of multiple features produced very good or excellent classifications and clusterings. In the phocid seals, the aquatic niche allowed for lower femoral bone mineral densities than expected based on the body mass alone. The semiaquatic mammals mostly had high bone mineral densities compared to the terrestrial species, which could be considered an adaptation to overcome buoyancy during swimming and shallow diving. Generally, it seems that different osteological properties at the levels of mineral density and biomechanics could be compatible with the adaptation to aquatic, semiaquatic, or terrestrial niches.

Although knee measurements yield high classification rates in metric sex estimation, there is a paucity of studies exploring the knee in artificial intelligence-based sexing. This proof-of-concept study aimed to develop deep learning algorithms for sex estimation from radiographs of reconstructed cadaver knee joints belonging to the Terry Anatomical Collection. A total of 199 knee radiographs were obtained from 100 skeletons (46 male and 54 female cadavers; mean age at death 64.2 years, range 50-102 years) whose tibiofemoral joints were recon-structed in standard anatomical position. The AIDeveloper software was used to train, validate, and test neural network architectures in sex estimation based on image classification. Of the explored algorithms, an MhNet-based model reached the highest overall testing accuracy of 90.3%. The model was able to classify all females (100.0%) and most males (78.6%) correctly. These preliminary findings encourage further research on artificial intelligence-based methods in sex estimation from the knee joint. Combining radiographic data with automated and externally validated algorithms may establish valuable tools to be utilized in forensic anthropology.

This study aimed to evaluate the surface characteristics of a nanocrystalline hydroxyapatite coating made through atomic layer deposition (ALD-HA) on titanium surfaces and to investigate its effect on blood coagulation and platelet adhesion. Grade 2 square titanium discs (0.7 cm, 1 mm thick) were used (n = 108). Half of the substrates (n = 54) were coated with ALD-HA, and the other half were used as the non-coated control. Surface free energy (SFE), contact angle (CA), surface roughness (Ra), and chemical composition were evaluated. Blood thrombogenic properties were assessed on ALD-HA and non-coated surfaces using the kinetic clotting time method. The platelets’ adhesion and morphology were also evaluated. The ALD-HA-coated surfaces demonstrated significantly higher polar SFE (p < 0.001) and lower CA (p < 0.001) values compared to the non-coated surfaces. In addition, the surface roughness was significantly lower for the ALD-HA (p < 0.001) than for the non-coated surfaces. Platelets adhered to both surfaces; however, there was variability in platelet morphologies in different areas with higher platelet density on the ALD-HA surfaces. There was no significant difference in the overall absorbance values of the hemolyzed hemoglobin for both substrates, and the total clotting time was achieved at 60 min. It can be concluded that the ALD-HA coating of titanium can enhance surface wettability, increase surface free energy, and support blood coagulation and platelet adhesion.

Background Global sclerostin inhibition reduces fracture risk efficiently but has been associated with cardiovascular side effects. The strongest genetic signal for circulating sclerostin is in the B4GALNT3 gene region, but the causal gene is unknown. B4GALNT3 expresses the enzyme beta-1,4-N-acetylgalactosaminyltransferase 3 that transfers N-acetylgalactosamine onto N-acetylglucosaminebeta-benzyl on protein epitopes (LDN-glycosylation). Methods To determine if B4GALNT3 is the causal gene, B4galnt3 / mice were developed and serum levels of total sclerostin and LDN-glycosylated sclerostin were analysed and mechanistic studies were performed in osteoblast-like cells. Mendelian randomization was used to determine causal associations. Findings B4galnt3 / mice had higher circulating sclerostin levels, establishing B4GALNT3 as a causal gene for circulating sclerostin levels, and lower bone mass. However, serum levels of LDN-glycosylated sclerostin were lower in B4galnt3 / mice. B4galnt3 and Sost were co-expressed in osteoblast-lineage cells. Overexpression of B4GALNT3 increased while silencing of B4GALNT3 decreased the levels of LDN-glycosylated sclerostin in osteoblast-like cells. Mendelian randomization demonstrated that higher circulating sclerostin levels, genetically predicted by variants in the B4GALNT3 gene, were causally associated with lower BMD and higher risk of fractures but not with higher risk of myocardial infarction or stroke. Glucocorticoid treatment reduced B4galnt3 expression in bone and increased circulating sclerostin levels and this may contribute to the observed glucocorticoid-induced bone loss. Interpretation B4GALNT3 is a key factor for bone physiology via regulation of LDN-glycosylation of sclerostin. We propose that B4GALNT3-mediated LDN-glycosylation of sclerostin may be a bone-specific osteoporosis target, separating the anti-fracture effect of global sclerostin inhibition, from indicated cardiovascular side effects.Funding Found in acknowledgements.Copyright (c) 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Hydroxyapatite (HA; Ca10(PO4)6(OH)2) coating of bone implants has many beneficial properties as it improves osseointegration and eventually becomes degraded and replaced with new bone. We prepared HA coating on a titanium substrate with atomic layer deposition (ALD) and compared monocyte differentiation and material resorption between ALD-HA and bone. After stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), human peripheral blood monocytes differentiated into resorbing osteoclasts on bovine bone, but non-resorbing foreign body cells were observed on ALD-HA. The analysis of the topography of ALD-HA and bone showed no differences in wettability (water contact angle on ALD-HA 86.2° vs. 86.7° on the bone), but the surface roughness of ALD-HA (Ra 0.713 µm) was significantly lower compared to bone (Ra 2.30 µm). The cellular reaction observed on ALD-HA might be a consequence of the topographical properties of the coating. The absence of resorptive osteoclasts on ALD-HA might indicate inhibition of their differentiation or the need to modify the coating to induce osteoclast differentiation.

Aging is associated with low bone and lean mass as well as alterations in the gut microbiota (GM). In this study, we determined whether the reduced bone mass and relative lean mass observed in old mice could be transferred to healthy young mice by GM transplantation (GMT). GM from old (21-month-old) and young adult (5-month-old) donors was used to colonize germ-free (GF) mice in three separate studies involving still growing 5- or 11-week-old recipients and 17-week-old recipients with minimal bone growth. The GM of the recipient mice was similar to that of the donors, demonstrating successful GMT. GM from old mice did not have statistically significant effects on bone mass or bone strength, but significantly reduced the lean mass percentage of still growing recipient mice when compared with recipients of GM from young adult mice. The levels of propionate in the cecum of mice receiving old donor GM were significantly lower than those in mice receiving young adult donor GM. Bacteroides ovatus was enriched in the microbiota of recipient mice harboring GM from young adult donors. The presence of B. ovatus was not only significantly associated with high lean mass percentage in mice, but also with lean mass adjusted for fat mass in the large human HUNT cohort. In conclusion, GM from old mice reduces lean mass percentage but not bone mass in young, healthy, still growing recipient mice. Future studies are warranted to determine whether GM from young mice improves the musculoskeletal phenotype of frail elderly recipient mice.

Insulin-like growth factor-I (IGF-I) levels, which are reduced by age, and cortical bone dimensions are major determinants of fracture risk in elderly subjects. Inactivation of liver-derived circulating IGF-I results in reduced periosteal bone expansion in young and older mice. In mice with lifelong depletion of IGF-I in osteoblast lineage cells, the long bones display reduced cortical bone width. However, it has not previously been investigated whether inducible inactivation of IGF-I locally in bone in adult/old mice affects the bone phenotype. Adult tamoxifen-inducible inactivation of IGF-I using a CAGG-CreER mouse model (inducible IGF-IKO mice) substantially reduced IGF-I expression in bone (-55%) but not in liver. Serum IGF-I and body weight were unchanged. We used this inducible mouse model to assess the effect of local IGF-I on the skeleton in adult male mice, avoiding confounding developmental effects. After tamoxifen-induced inactivation of the IGF-I gene at 9 months of age, the skeletal phenotype was determined at 14 months of age. Computed tomography analyses of tibia revealed that the mid-diaphyseal cortical periosteal and endosteal circumferences and calculated bone strength parameters were decreased in inducible IGF-IKO mice compared with controls. Furthermore, 3-point bending showed reduced tibia cortical bone stiffness in inducible IGF-IKO mice. In contrast, the tibia and vertebral trabecular bone volume fraction was unchanged. In conclusion, inactivation of IGF-I in cortical bone with unchanged liver-derived IGF-I in older male mice resulted in reduced radial growth of cortical bone. This suggests that not only circulating IGF-I but also locally derived IGF-I regulates the cortical bone phenotype in older mice.

Osteoclasts are multinucleated bone resorbing cells that can be differentiated from human monocytes in vitro. There are few studies comparing osteoclastogenesis of different monocyte sources. We compared monocytes from human bone marrow (BM), peripheral blood (PB), and umbilical cord blood (CB) and their osteoclastogenic potential by culturing them with RANKL (20 and 80 ng/ml) and M-CSF (10 ng/ml) for 14 days. We also cultured cells without growth factors, as umbilical cord blood monocytes have been reported to be able to fuse spontaneously into osteoclasts. The data was analysed on d4, d8, d11, and d14. After culture with RANKL and M-CSF, all types of cell cultures developed TRACP -positive multinuclear cells that were able to form resorption pits on human bone slices. Only occasional multinuclear cells and small infrequent resorbed areas could be found in PB and CB-derived cultures without growth factors. BM-derived cells formed greater resorption areas than PB- and CB-derived monocytes. The greatest monocyte population in BM samples were intermediate (CD14++CD16+) and in PB and CB classical monocytes (76.3% and 54.4%, respectively). In conclusion, our data demonstrates that bone resorbing osteoclasts can be differentiated from BM, PB and CB. However, the osteoclast precursor origin can affect the osteoclast properties and function.

Osteoporotic fracture is among the most common and costly of diseases. While reasonably heritable, its genetic determinants have remained elusive. Forearm fractures are the most common clinically recognized osteoporotic fractures with a relatively high heritability. To establish an atlas of the genetic determinants of forearm fractures, we performed genome-wide association analyses including 100,026 forearm fracture cases. We identified 43 loci, including 26 new fracture loci. Although most fracture loci associated with bone mineral density, we also identified loci that primarily regulate bone quality parameters. Functional studies of one such locus, at TAC4, revealed that Tac4-/- mice have reduced mechanical bone strength. The strongest forearm fracture signal, at WNT16, displayed remarkable bone-site-specificity with no association with hip fractures. Tall stature and low body mass index were identified as new causal risk factors for fractures. The insights from this atlas may improve fracture prediction and enable therapeutic development to prevent fractures.

BACKGROUND:The increasing demand for bone implants with improved osseointegration properties has prompted researchers to develop various coating types for metal implants. Atomic layer deposition (ALD) is a method for producing nanoscale coatings conformally on complex three-dimensional surfaces. We have prepared hydroxyapatite (HA) coating on titanium (Ti) substrate with the ALD method and analyzed the biocompatibility of this coating in terms of cell adhesion and viability.METHODS:HA coatings were prepared on Ti substrates by depositing CaCO3 films by ALD and converting them to HA by wet treatment in dilute phosphate solution. MC3T3-E1 preosteoblasts were cultured on ALD-HA, glass slides and bovine bone slices. ALD-HA and glass slides were either coated or non-coated with fibronectin. After 48h culture, cells were imaged with scanning electron microscopy (SEM) and analyzed by vinculin antibody staining for focal adhesion localization. An 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test was performed to study cell viability.RESULTS:Vinculin staining revealed similar focal adhesion-like structures on ALD-HA as on glass slides and bone, albeit on ALD-HA and bone the structures were thinner compared to glass slides. This might be due to thin and broad focal adhesions on complex three-dimensional surfaces of ALD-HA and bone. The MTT test showed comparable cell viability on ALD-HA, glass slides and bone.CONCLUSION:ALD-HA coating was shown to be biocompatible in regard to cell adhesion and viability. This leads to new opportunities in developing improved implant coatings for better osseointegration and implant survival.

Introduction: All animals, including humans, are exposed to heavy metals which are known to accumulate in different tissues, especially in bone. During pregnancy, the maternal bone turnover is increased and the metals in the mother's body can be mobilized into the bloodstream. Heavy metals in maternal blood are known to pass through the placenta to the fetal blood and finally, deposited to bone tissue. However, there are no studies on the concentration of metals in the fetal solid tissues and until now, the rate of metal transfer from mother to fetus is not exactly known. Materials and methods: Samples of the blood, liver, placenta, and three different bones were collected from 17 pregnant ewes and their 27 fetuses. The animals had no known exposure to heavy metals. The concentrations of Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, Sb, Sn, Sr, Te, Ti, Tl, V, and Zn were analyzed using ICP-MS. Results and discussion: The concentration of Sb, Sn, Te, and Tl were under the detection limit in all the samples. The other metals were found in all maternal and fetal tissues, suggesting that all detectable metals cross the placenta. Blood concentrations were low compared to solid tissue concentrations. The concentrations of essential elements varied between maternal and fetal tissues, which could be explained by biological differences. The differences in concentrations of non-essential elements between the ewe and fetuses were smaller. The most significant differences were between maternal and fetal concentrations of Ba and Sr, which is at least partly explained by the mineralization degree of the bone. Conclusion: Heavy metals accumulate in fetal solid tissues in sheep that are not directly exposed to heavy metals. Because of the differences in anatomy between human and sheep placenta, the accumulation in the tissue of human fetuses should be extrapolated cautiously. However, there might be some clinical relevance for fertile aged women who are exposed to heavy metals, such as women who work in the metal industry or who have undergone joint replacement surgery.

Osteoporosis is an age-dependent serious skeletal disease that leads to great suffering for the patient and high social costs, especially as the global population reaches higher age. Decreasing estrogen levels after menopause result in a substantial bone loss and increased fracture risk, whereas estrogen treatment improves bone mass in women. RSPO3, a secreted protein that modulates WNT signaling, increases trabecular bone mass and strength in the vertebrae of mice, and is associated with trabecular density and risk of distal forearm fractures in humans. The aim of the present study was to determine if RSPO3 is involved in the bone-sparing effect of estrogens. We first observed that estradiol (E2) treatment increases RSPO3 expression in bone of ovariectomized (OVX) mice, supporting a possible role of RSPO3 in the bone-sparing effect of estrogens. As RSPO3 is mainly expressed by osteoblasts in the bone, we used a mouse model devoid of osteoblast-derived RSPO3 (Runx2-creRspo3flox/flox mice) to determine if RSPO3 is required for the bone-sparing effect of E2 in OVX mice. We confirmed that osteoblast-specific RSPO3 inactivation results in a substantial reduction in trabecular bone mass and strength in the vertebrae. However, E2 increased vertebral trabecular bone mass and strength similarly in mice devoid of osteoblast-derived RSPO3 and control mice. Unexpectedly, osteoblast-derived RSPO3 was needed for the full estrogenic response on cortical bone thickness. In conclusion, although osteoblast-derived RSPO3 is a crucial regulator of vertebral trabecular bone, it is required for a full estrogenic effect on cortical, but not trabecular, bone in OVX mice. Thus, estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.NEW & NOTEWORTHY Osteoblast-derived RSPO3 is known to be a crucial regulator of vertebral trabecular bone. Our new findings show that RSPO3 and estrogen regulate trabecular bone independent of each other, but that RSPO3 is necessary for a complete estrogenic effect on cortical bone.

Background Rheumatoid arthritis (RA) and osteoarthritis (OA) are common joint diseases associated with changes in local, as well as systemic bone structure and osteoclast function. We investigated how the different soluble inflammatory stimuli in these diseases can affect osteoclastogenesis and bone resorption in vitro. Methods. Human peripheral blood mononuclear cell-derived osteoclasts were cultured on bone slices with serum from treatment-naïve RA patients and healthy controls and with synovial fluid samples acquired from RA and OA patients. The concentrations of 29 different cytokines and related proteins, including RANKL and OPG, were analyzed in the fluids tested. Results RA serum and synovial fluid increased both osteoclastogenesis and bone resorption. Osteoclastogenesis and activity increased more in the cultures containing OA than RA synovial fluid. The osteoclasts cultured in different culture media exhibited different phenotypes, especially the cells cultured with OA synovial fluid were generally larger and had more nuclei. A general increase in proinflammatory cytokines in RA synovial fluid and serum was found. Surprisingly, OA synovial fluid showed lower levels of osteoclastogenesis inhibiting cytokines, such as IL-4 and IL-10, than RA synovial fluid, which at least partly explains more pronounced osteoclastogenesis. No significant difference was found in RANKL or OPG levels. Conclusion The proinflammatory stimulus in OA and RA drives the monocyte differentiation towards inflammatory osteoclastogenesis and altered osteoclast phenotype.

Osteoporosis is a common skeletal disease, with increased risk of fractures. Currently available osteoporosis treatments reduce the risk of vertebral fractures, mainly dependent on trabecular bone, whereas the effect on nonvertebral fractures, mainly dependent on cortical bone, is less pronounced. WNT signaling is a crucial regulator of bone homeostasis, and the activity of WNTs is inhibited by NOTUM, a secreted WNT lipase. We previously demonstrated that conditional inactivation of NOTUM in all osteoblast lineage cells increases the cortical but not the trabecular bone mass. The aim of the present study was to determine if NOTUM increasing cortical bone is derived from osteoblast precursors/early osteoblasts or from osteocytes/late osteoblasts. First, we demonstrated Notum mRNA expression in Dmp1-expressing osteocytes and late osteoblasts in cortical bone using in situ hybridization. We then developed a mouse model with inactivation of NOTUM in Dmp1-expressing osteocytes and late osteoblasts (Dmp1-creNotumflox/flox mice). We observed that the Dmp1-creNotumflox/flox mice displayed a substantial reduction of Notum mRNA in cortical bone, resulting in increased cortical bone mass and decreased cortical porosity in femur but no change in trabecular bone volume fraction in femur or in the lumbar vertebrae L5 in Dmp1-creNotumflox/flox mice as compared with control mice. In conclusion, osteocytes and late osteoblasts are the principal source of NOTUM in cortical bone, and NOTUM derived from osteocytes/late osteoblasts reduces cortical bone mass. These findings demonstrate that inhibition of osteocyte/late osteoblast-derived NOTUM might be an interesting pharmacological target to increase cortical bone mass and reduce nonvertebral fracture risk.NEW & NOTEWORTHY NOTUM produced by osteoblasts is known to regulate cortical bone mass. Our new findings show that NOTUM specifically derived by DMP1-expressing osteocytes and late osteoblasts regulates cortical bone mass and not trabecular bone mass.