Effect of energy level of photobiomodulation therapy on bone repair in rats / Efecto del nivel de energía de la terapia de fotobiomodulación sobre la reparación ósea en ratas

Abstract The aim of this experimental study was to determine the effect of photobiomodulation therapy on bone repair in a rat tibia osteotomy model at 15 and 30 days. The sample consisted of 36 male Holtzman rats that were randomized into 6 equal groups. Groups A1 and A2: osteotomy + 1 J laser energy. Groups B1 and B2: osteotomy + 3 J laser energy. Groups C1 and C2 (controls): osteotomy only. The bone repair was analyzed by histological evaluation of osteoblasts and osteocytes both at 15 days (groups A1, B1, and C1) and at 30 days (groups A2, B2, and C2). Within the results, in all groups a greater number of osteoblasts was found at 15 days vs 30 days (p<0.05), and a greater number of osteocytes in B1 and C2 vs B2 and C1, respectively (p<0.05). When evaluating the 3 groups worked up to 15 days, more osteoblasts were found in A1 and C1 vs B1 (p<0.001); and osteocytes predominated in A1 and B1 vs C1 (p<0.001). At 30 days there was a greater quantity of osteoblasts in C2 vs A2 and B2 (p<0.05) and of osteocytes in C2 vs B2 (p<0.05). It is concluded that 1 J photobiomodulation therapy improved bone repair at 15 days; however, this improvement was not observed at 30 days because there were no differences between the irradiated groups and the control.

Resumen El objetivo de este estudio experimental fue determinar el efecto de terapia de fotobiomodulación sobre la reparación ósea en un modelo de osteotomía de tibia de rata a los 15 y 30 días. La muestra estuvo compuesta por 36 ratas Holtzman macho que se aleatorizaron en 6 grupos iguales. Grupos A1 y A2: osteotomía + energía láser de 1 Joule. Grupos B1 y B2: osteotomía + energía láser 3 Joule. Grupos C1 y C2 (controles): solo osteotomía. La reparación ósea fue analizada por evaluación histológica de osteoblastos y osteocitos tanto a los 15 días (grupos A1, B1 y C1) como a los 30 días (grupos A2, B2 y C2). Como resultados se encontró que en todos los grupos hubo mayor número de osteoblastos a los 15 días vs. 30 días (p<0,05), y mayor número de osteocitos en B1 y C2 vs B2 y C1, respectivamente (p<0,05). Al evaluar a los animales a los 15 días, se observó mayor número de osteoblastos en A1 y C1 vs B1 (p<0.001); y mayor número de osteocitos en A1 y B1 vs C1 (p<0,001). Al evaluar a los ratones a los 30 días hubo mayor cantidad de osteoblastos en C2 vs A2 y B2 (p<0,05) y de osteocitos en C2 vs B2 (p<0,05). Se concluye que la terapia de fotobiomodulación con 1 Joule mejoró la reparación ósea a los 15 días; sin embargo, dicha mejora no se observó a los 30 días porque no hubo diferencias entre los grupos irradiados y el control.

In vitro regulation of fibroblast growth factor 23 by 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D synthesized by osteocyte-like MC3T3-E1 cells.

Fibroblast growth factor 23 (FGF23) is produced and secreted by osteocytes and is essential for maintaining phosphate homeostasis. One of the main regulators of FGF23, 1,25-dihydroxyvitamin D (1,25(OH)2D3), is primarily synthesized in the kidney from 25-hydroxyvitamin D (25(OH)D) by 1α-hydroxylase (encoded by CYP27B1). Hitherto, it is unclear whether osteocytes can convert 25(OH)D and thereby allow for 1,25(OH)2D3 to induce FGF23 production and secretion locally. Here, we differentiated MC3T3-E1 cells toward osteocyte-like cells expressing and secreting FGF23. Treatment with 10-6 M 25(OH)D resulted in conversion of 25(OH)D to 150 pmol/L 1,25(OH)2D3 and increased FGF23 expression and secretion, but the converted amount of 1,25(OH)2D3 was insufficient to trigger an FGF23 response, so the effect on FGF23 was most likely directly caused by 25(OH)D. Interestingly, combining phosphate with 25(OH)D resulted in a synergistic increase in FGF23 expression and secretion, likely due to activation of additional signaling pathways by phosphate. Blockage of the vitamin D receptor (VDR) only partially abolished the effects of 25(OH)D or 25(OH)D combined with phosphate on Fgf23, while completely inhibiting the upregulation of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1), encoding for 24-hydroxylase. RNA sequencing and in silico analyses showed that this could potentially be mediated by the nuclear receptors Retinoic Acid Receptor ß (RARB) and Estrogen Receptor 2 (ESR2). Taken together, we demonstrate that osteocytes are able to convert 25(OH)D to 1,25(OH)2D3, but this is insufficient for FGF23 activation, implicating a direct effect of 25(OH)D in the regulation of FGF23, which occurs at least partially independent from its cognate VDR. Moreover, phosphate and 25(OH)D synergistically increase expression and secretion of FGF23, which warrants investigating consequences in patients receiving a combination of vitamin D analogues and phosphate supplements. These observations help us to further understand the complex relations between phosphate, vitamin D, and FGF23.

The myosin and RhoGAP MYO9B influences osteocyte dendrite growth and responses to mechanical stimuli.

Introduction: Myosin IXB (MYO9B) is an unconventional myosin with RhoGAP activity and thus is a regulator of actin cytoskeletal organization. MYO9B was previously shown to be necessary for skeletal growth and health and to play a role in actin-based functions of both osteoblasts and osteoclasts. However, its role in responses to mechanical stimulation of bone cells has not yet been described. Therefore, experiments were undertaken to determine the role of MYO9B in bone cell responses to mechanical stress both in vitro and in vivo. Methods: MYO9B expression was knocked down in osteoblast and osteocyte cell lines using RNA interference and the resulting cells were subjected to mechanical stresses including cyclic tensile strain, fluid shear stress, and plating on different substrates (no substrate vs. monomeric or polymerized collagen type I). Osteocytic cells were also subjected to MYO9B regulation through Slit-Robo signaling. Further, wild-type or Myo9b -/- mice were subjected to a regimen of whole-body vibration (WBV) and changes in bone quality were assessed by micro-CT. Results: Unlike control cells, MYO9B-deficient osteoblastic cells subjected to uniaxial cyclic tensile strain were unable to orient their actin stress fibers perpendicular to the strain. Osteocytic cells in which MYO9B was knocked down exhibited elongated dendrites but were unable to respond normally to treatments that increase dendrite length such as fluid shear stress and Slit-Robo signaling. Osteocytic responses to mechanical stimuli were also found to be dependent on the polymerization state of collagen type I substrates. Wild-type mice responded to WBV with increased bone tissue mineral density values while Myo9b -/- mice responded with bone loss. Discussion: These results demonstrate that MYO9B plays a key role in mechanical stress-induced responses of bone cells in vitro and in vivo.

Deletion of the sodium/hydrogen exchanger 6 causes low bone volume in adult mice.

The sodium/hydrogen exchanger 6 (NHE6) localizes to recycling endosomes, where it mediates endosomal alkalinization through K+/H+ exchange. Mutations in the SLC9A6 gene encoding NHE6 cause severe X-linked mental retardation, epilepsy, autism and corticobasal degeneration in humans. Patients with SLC9A6 mutations exhibit skeletal malformations, and a previous study suggested a key role of NHE6 in osteoblast-mediated mineralization. The goal of this study was to explore the role of NHE6 in bone homeostasis. To this end, we studied the bone phenotype of NHE6 knock-out mice by microcomputed tomography, quantitative histomorphometry and complementary ex vivo and in vitro studies. We detected NHE6 transcript and protein in both differentiated osteoclasts and mineralizing osteoblasts. In vitro studies with osteoclasts and osteoblasts derived from NHE6 knock-out mice demonstrated normal osteoclast differentiation and osteoblast proliferation without an impairment in mineralization capacity. Microcomputed tomography and bone histomorphometry studies showed a significantly reduced bone volume and trabecular number as well as an increased trabecular space at lumbar vertebrae of 6 months old NHE6 knock-out mice. The bone degradation marker c-terminal telopeptides of type I collagen was unaltered in NHE6 knock-out mice. However, we observed a reduction of the bone formation marker procollagen type 1 N-terminal propeptide, and increased circulating sclerostin levels in NHE6 knock-out mice. Subsequent studies revealed a significant upregulation of sclerostin transcript expression in both primary calvarial cultures and femora derived from NHE6 knock-out mice. Thus, loss of NHE6 in mice causes an increase of sclerostin expression associated with reduced bone formation and low bone volume.

Ginkgonitroside, a new nitrophenyl glycoside and bioactive compounds from Ginkgo biloba leaves controlling adipocyte and osteoblast differentiation.

Eight compounds (1-8) including one novel nitrophenyl glycoside, ginkgonitroside (1) were isolated from the leaves of Ginkgo biloba, a popular medicinal plant. The structure of the new compound was characterized using extensive spectroscopic analyses via 1D and 2D NMR data interpretations, HR-ESIMS, and chemical transformation. To the best of our knowledge, the present study is the first to report the presence of nitrophenyl glycosides, which are relatively unique phytochemicals in natural products, in G. biloba. The isolated compounds (1-8) were examined for their effects on the regulation of mesenchymal stem cell (MSC) differentiation. Compounds 1-3 and 8 were able to suppress MSC differentiation toward adipocytes. In contrast, compounds 5 and 8 showed activity promoting osteogenic differentiation of MSCs. These findings demonstrate that the active compounds showed regulatory activity on MSC differentiation between adipocytes and osteocytes.

Amelioration of bone fragility by pulsed electromagnetic fields in type 2 diabetic KK-Ay mice involving Wnt/ß-catenin signaling.

Type 2 diabetes mellitus (T2DM) results in compromised bone microstructure and quality, and subsequently increased risks of fractures. However, it still lacks safe and effective approaches resisting T2DM bone fragility. Pulsed electromagnetic fields (PEMFs) exposure has proven to be effective in accelerating fracture healing and attenuating osteopenia/osteoporosis induced by estrogen deficiency. Nevertheless, whether and how PEMFs resist T2DM-associated bone deterioration remain not fully identified. The KK-Ay mouse was used as the T2DM model. We found that PEMF stimulation with 2 h/day for 8 wk remarkably improved trabecular bone microarchitecture, decreased cortical bone porosity, and promoted trabecular and cortical bone material properties in KK-Ay mice. PEMF stimulated bone formation in KK-Ay mice, as evidenced by increased serum levels of bone formation (osteocalcin and P1NP), enhanced bone formation rate, and increased osteoblast number. PEMF significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. PEMF exerted beneficial effects on osteoblast- and osteocyte-related gene expression in the skeleton of KK-Ay mice. Nevertheless, PEMF exerted no effect on serum biomarkers of bone resorption (TRAcP5b and CTX-1), osteoclast number, or osteoclast-specific gene expression (TRAP and cathepsin K). PEMF upregulated gene expression of canonical Wnt ligands (including Wnt1, Wnt3a, and Wnt10b), but not noncanonical Wnt5a. PEMF also upregulated skeletal protein expression of downstream p-GSK-3ß and ß-catenin in KK-Ay mice. Moreover, PEMF-induced improvement in bone microstructure, mechanical strength, and bone formation in KK-Ay mice was abolished after intragastric administration with the Wnt antagonist ETC-159. Together, our results suggest that PEMF can improve bone microarchitecture and quality by enhancing the biological activities of osteoblasts and osteocytes, which are associated with the activation of the Wnt/ß-catenin signaling pathway. PEMF might become an effective countermeasure against T2DM-induced bone deterioration.NEW & NOTEWORTHY PEMF improved trabecular bone microarchitecture and suppressed cortical bone porosity in T2DM KK-Ay mice. It attenuated T2DM-induced detrimental consequence on trabecular and cortical bone material properties. PEMF resisted bone deterioration in KK-Ay mice by enhancing osteoblast-mediated bone formation. PEMF also significantly suppressed osteocytic apoptosis and sclerostin expression in KK-Ay mice. The therapeutic potential of PEMF on T2DM-induced bone deterioration was associated with the activation of Wnt/ß-catenin signaling.

In vivo performance of Al2O3-Ti bone implants in the rat femur.

BACKGROUND: Alumina-titanium (Al2O3-Ti) biocomposites have been recently developed with improved mechanical properties for use in heavily loaded orthopedic sites. Their biological performance, however, has not been investigated yet. METHODS: The aim of the present study was to evaluate the in vivo biological interaction of Al2O3-Ti. Spark plasma sintering (SPS) was used to fabricate Al2O3-Ti composites with 25 vol.%, 50 vol.%, and 75 vol.% Ti content. Pure alumina and titanium were also fabricated by the same procedure for comparison. The fabricated composite disks were cut into small bars and implanted into medullary canals of rat femurs. The histological analysis and scanning electron microscopy (SEM) observation were carried out to determine the bone formation ability of these materials and to evaluate the bone-implant interfaces. RESULTS: The histological observation showed the formation of osteoblast, osteocytes with lacuna, bone with lamellar structures, and blood vessels indicating that the healing and remodeling of the bone, and vasculature reconstruction occurred after 4 and 8 weeks of implantation. However, superior bone formation and maturation were obtained after 8 weeks. SEM images also showed stronger interfaces at week 8. There were differences between the composites in percentages of bone area (TB%) and the number of osteocytes. The 50Ti composite showed higher TB% at week 4, while 25Ti and 75Ti represented higher TB% at week 8. All the composites showed a higher number of osteocytes compared to 100Ti, particularly 75Ti. CONCLUSIONS: The fabricated composites have the potential to be used in load-bearing orthopedic applications.

Effects of 3% binahong ( Anredera cordifolia) leaf extract gel on alveolar bone healing in post-extraction tooth socket wound in Wistar rats ( Rattus norvegicus).

Background: Binahong ( Anredera cordifolia (Ten.) STEENIS) is a widely available herbal plant in Indonesia and has been intensely researched for its healing abilities due to its biological activities, but few have studied its capability in accelerating hard tissue healing in post-extraction tooth sockets. The purpose of this study was to analyse the effects of 3% binahong leaf extract gel on alveolar bone healing in post-extraction sockets in Wistar rats. Methods: In this study, 48 male Wistar rats were randomly allocated to twelve groups. After the extraction of the left mandibular incisor, sockets in Group I to IV were given 3% binahong leaf extract gel, group V to VIII were given a control gel, and group IX to XII were given Gengigel ® for 14 days. The residual socket volume (RSV) and fibroblast proliferation were observed on the 3 rd, 7 th, and 14 th day post-extraction, while the osteoblast and osteocyte proliferation were observed on the 7 th, 14 th, and 28 th day post-extraction. The RSV data were analysed using repeated measure ANOVA and one-way ANOVA, while the histopathological data were analysed using one-way ANOVA. Results: The results showed that the binahong group had the lowest RSV and the highest fibroblast proliferation compared to the other groups on the 7th day (p<0.05) and the highest osteoblast and osteocyte proliferation compared to the other groups on the 14 th day (p<0.05). Conclusion: The experiment showed that 3% binahong leaf extract gel could accelerate wound closure, which was characterized by a greater decrease in the RSV value in comparison to the other treatment groups and could enhance alveolar bone healing by increasing the proliferation of fibroblasts, osteoblasts, and osteocytes.

A Novel 3D Osteoblast and Osteocyte Model Revealing Changes in Mineralization and Pro-osteoclastogenic Paracrine Signaling During Estrogen Deficiency.

Recent in vitro studies have revealed that the mechanobiological responses of osteoblasts and osteocytes are fundamentally impaired during estrogen deficiency. However, these two-dimensional (2D) cell culture studies do not account for in vivo biophysical cues. Thus, the objectives of this study are to (1) develop a three-dimensional (3D) osteoblast and osteocyte model integrated into a bioreactor and (2) apply this model to investigate whether estrogen deficiency leads to changes in osteoblast to osteocyte transition, mechanosensation, mineralization, and paracrine signaling associated with bone resorption by osteoclasts. MC3T3-E1s were expanded in media supplemented with estrogen (17ß-estradiol). These cells were encapsulated in gelatin-mtgase before culture in (1) continued estrogen (E) or (2) no further estrogen supplementation. Constructs were placed in gas permeable and water impermeable cell culture bags and maintained at 5% CO2 and 37°C. These bags were either mechanically stimulated in a custom hydrostatic pressure (HP) bioreactor or maintained under static conditions (control). We report that osteocyte differentiation, characterized by the presence of dendrites and staining for osteocyte marker dentin matrix acidic phosphoprotein 1 (DMP1), was significantly greater under estrogen withdrawal (EW) compared to under continuous estrogen treatment (day 21). Mineralization [bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (ALP), calcium] and gene expression associated with paracrine signaling for osteoclastogenesis [receptor activator of nuclear factor kappa-ß ligand (RANKL)/osteoprotegerin OPG ratio] were significantly increased in estrogen deficient and mechanically stimulated cells. Interestingly, BSP and DMP-1 were also increased at day 1 and day 21, respectively, which play a role in regulation of biomineralization. Furthermore, the increase in pro-osteoclastogenic signaling may be explained by altered mechanoresponsiveness of osteoblasts or osteocytes during EW. These findings highlight the impact of estrogen deficiency on bone cell function and provide a novel in vitro model to investigate the mechanisms underpinning changes in bone cells after estrogen deficiency.

Evidence from systematic reviews on photobiomodulation of human bone and stromal cells: Where do we stand?

This study summarizes the available evidence from systematic reviews on the in vitro effects of photobiomodulation on the proliferation and differentiation of human bone and stromal cells by appraising their methodological quality. Improvements for future studies are also highlighted, with particular emphasis on in vitro protocols and cell-related characteristics. Six reviews using explicit eligibility criteria and methods selected in order to minimize bias were included. There was no compelling evidence on the cellular mechanisms of action or treatment parameters of photobiomodulation; compliance with quality assessment was poor. A rigorous description of laser parameters (wavelength, power, beam spot size, power density, energy density, repetition rate, pulse duration or duty cycle, exposure duration, frequency of treatments, and total radiant energy), exposure conditions (methods to ensure a uniform irradiation and to avoid cross-irradiation, laser-cell culture surface distance, lid presence during irradiation) and cell-related characteristics (cell type or line, isolation and culture conditions, donor-related factors where applicable, tissue source, cell phenotype, cell density, number of cell passages in culture) should be included among eligibility criteria for study inclusion. These methodological improvements will maximize the contribution of in vitro studies on the effects of photobiomodulation on human bone and stromal cells to evidence-based translational research.

Uranium Effect on Osteocytic Cells In Vitro.

Once absorbed in the body, natural uranium [U(VI)], a radionucleotide naturally present in the environment, is targeted to the skeleton which is the long-term storage organ. We and others have reported the U(VI) negative effects on osteoblasts (OB) and osteoclasts (OC), the main two cell types involved in bone remodeling. In the present work, we addressed the U(VI) effect on osteocytes (OST), the longest living bone cell type and the more numerous (> 90%). These cells, which are embedded in bone matrix and thus are the more prone to U(VI) long-term exposure, are now considered as the chief orchestrators of the bone remodeling process. Our results show that the cytotoxicity index of OST is close to 730 µM, which is about twice the one reported for OB and OC. However, despite this resistance potential, we observed that chronic U(VI) exposure as low as 5 µM led to a drastic decrease of the OST mineralization function. Gene expression analysis showed that this impairment could potentially be linked to an altered differentiation process of these cells. We also observed that U(VI) was able to trigger autophagy, a highly conserved survival mechanism. Extended X-ray absorption fine structure analysis at the U LIII edge of OST cells exposed to U(VI) unambiguously shows the formation of an uranyl phosphate phase in which the uranyl local structure is similar to the one present in Autunite. Thus, our results demonstrate for the first time that OST mineralization function can be affected by U(VI) exposure as low as 5 µM, suggesting that prolonged exposure could alter the central role of these cells in the bone environment.

Pigment epithelium-derived factor (PEDF) reduced expression and synthesis of SOST/sclerostin in bone explant cultures: implication of PEDF-osteocyte gene regulation in vivo.

Mutations in Serpinf1 gene which encodes pigment epithelium-derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective matrix mineralization. We reported previously that PEDF reduced expression and synthesis of Sost/Sclerostin as well as other osteocytes genes encoding proteins that regulate matrix mineralization [1]. To determine whether PEDF had an effect on osteocyte gene expression in bone, we used bone explant cultures. First, osteocytes were isolated from surgical waste of bone fragments obtained from patients undergoing elective foot surgeries under approved IRB protocol by Penn State College of Medicine IRB committee. Primary osteocytes treated with PEDF reduced expression and synthesis of Sost/Sclerostin and matrix phosphoglycoprotein (MEPE) as well as dentin matrix protein (DMP-1). On the whole, PEDF reduced osteocyte protein synthesis by 50% and by 75% on mRNA levels. For bone explants, following collagenase digestion, bone fragments were incubated in alpha-MEM supplemented with 250 ng/ml of PEDF or BSA. After 7 days of incubation in a medium supplemented with PEDF, analysis of mRNA by PCR and protein by western blotting of encoded osteocyte proteins showed reduced Sclerostin synthesis by 39% and MEPE by 27% when compared to fragments incubated in medium supplemented with BSA. mRNA expression levels of osteocytes in bone fragments treated with PEDF were reduced by 50% for both SOST and MEPE when compared to BSA-treated bone fragments. Taken together, the data indicate that PEDF has an effect on osteocyte gene expression in bone and encourage further studies to examine effect of PEDF on bone formation indices in animal models and its effect on osteocyte gene expression in vivo following PEDF administration.

Pulsed electromagnetic fields: promising treatment for osteoporosis.

Osteoporosis (OP) is considered to be a well-defined disease which results in high morbidity and mortality. In patients diagnosed with OP, low bone mass and fragile bone strength have been demonstrated to significantly increase risk of fragility fractures. To date, various anabolic and antiresorptive therapies have been applied to maintain healthy bone mass and strength. Pulsed electromagnetic fields (PEMFs) are employed to treat patients suffering from delayed fracture healing and nonunions. Although PEMFs stimulate osteoblastogenesis, suppress osteoclastogenesis, and influence the activity of bone marrow mesenchymal stem cells (BMSCs) and osteocytes, ultimately leading to retention of bone mass and strength. However, whether PEMFs could be taken into clinical use to treat OP is still unknown. Furthermore, the deeper signaling pathways underlying the way in which PEMFs influence OP remain unclear.

Release of CXCL12 From Apoptotic Skeletal Cells Contributes to Bone Growth Defects Following Dexamethasone Therapy in Rats.

Dexamethasone (Dex) is known to cause significant bone growth impairment in childhood. Although previous studies have suggested roles of osteocyte apoptosis in the enhanced osteoclastic recruitment and local bone loss, whether it is so in the growing bone following Dex treatment requires to be established. The current study addressed the potential roles of chemokine CXCL12 in chondroclast/osteoclast recruitment and bone defects following Dex treatment. Significant apoptosis was observed in cultured mature ATDC5 chondrocytes and IDG-SW3 osteocytes after 48 hours of 10-6 M Dex treatment, and CXCL12 was identified to exhibit the most prominent induction in Dex-treated cells. Conditioned medium from the treated chondrocytes/osteocytes enhanced migration of RAW264.7 osteoclast precursor cells, which was significantly inhibited by the presence of the anti-CXCL12 neutralizing antibody. To investigate the roles of the induced CXCL12 in bone defects caused by Dex treatment, young rats were orally gavaged daily with saline or Dex at 1 mg/kg/day for 2 weeks, and received an intraperitoneal injection of anti-CXCL12 antibody or control IgG (1 mg/kg, three times per week). Aside from oxidative stress induction systemically, Dex treatment caused reductions in growth plate thickness, primary spongiosa height, and metaphysis trabecular bone volume, which are associated with induced chondrocyte/osteocyte apoptosis and enhanced chondroclast/osteoclast recruitment and osteoclastogenic differentiation potential. CXCL12 was induced in apoptotic growth plate chondrocytes and metaphyseal bone osteocytes. Anti-CXCL12 antibody supplementation considerably attenuated Dex-induced chondroclast/osteoclast recruitment and loss of growth plate cartilage and trabecular bone. CXCL12 neutralization did not affect bone marrow osteogenic potential, adiposity, and microvasculature. Thus, CXCL12 was identified as a potential molecular linker between Dex-induced skeletal cell apoptosis and chondroclastic/osteoclastic recruitment, as well as growth plate cartilage/bone loss, revealing a therapeutic potential of CXCL12 functional blockade in preventing bone growth defects during/after Dex treatment. © 2018 American Society for Bone and Mineral Research.

PTH (1-34) affects bone turnover governed by osteocytes exposed to fluoride.

Exposure to fluoride from environmental sources remains an overlooked, but serious public health risk. In this study, we looked into the role osteocytes play on the mechanism underlying fluoride induced osteopathology. We analyzed bone formation and resorption related genes generated by osteocytes that were exposed to varied doses of fluoride with and without PTH in vitro. Correspondingly, osteogenesis and osteoclastogenesis related genes were also investigated in rats exposed to fluoride for 8 weeks, and the PTH(1-34)was applied at the last 3 weeks to observe its role in regulating bone turnover upon fluoride treatment. The data in vitro indicated that fluoride treatment inhibited Sost expression of mRNA and protein and stimulated RANKL mRNA protein expression as well as the RANKL/OPG ratio in the primary osteocytes. Single PTH treatment played the similar role on expression of these genes and proteins. The PTH combined administration enhanced the action of fluoride treatment on RNAKL/OPG and SOST/Sclerostin. The up-regulation of RANKL and decreasing of Sost induced by fluoride and/or PTH treatment was validated in vivo and suggests that osteocytes are a major source of RANKL and Sost, both of which play essential roles in fluoride affecting osteogenesis and osteoclastogenesis. Expression of Wnt/ß-catenin was up-regulated in both in vitro osteocytes treated with high dose of fluoride and bone tissue of rats in the presence of fluoride and PTH. In vivo, fluoride and single PTH stimulated bone turnover respectively, furthermore, PTH combined with low dose of fluoride treatment reinforced the osteogenesis and osteoclastogenesis genes expression, however, co-treatment of PTH reversed the effect of high dose of fluoride on osteogenesis and osteoclastogenensis related factors. In conclusion, this study demonstrated that osteocytes play a key role in fluoride activated bone turnover, and PTH participates in the process of fluoride modulating SOST/Sclerostin and RANKL expression.

The anti-metastatic micro-environment of the bone: Importance of osteocyte Cx43 hemichannels.

Bone metastases of tumor cells are a common and life-threatening feature of a variety of late-stage cancers, including breast cancers. However, until now, much less has been known about the intrinsic anti-metastatic properties of the bones and how these could be exploited to prevent or treat bone metastases. Very recently, native Cx43 hemichannels present in osteocytes have been identified as important anti-metastatic signaling complexes by establishing high local extracellular ATP levels. Moreover, bisphosphonate drugs, applied as adjuvant therapies in the treatment of breast cancer patients and bone diseases, are known to display anti-metastatic properties. Now, it became clear that these compounds exert their effects through osteocyte Cx43 hemichannels, thereby triggering their opening and promoting ATP release in the extracellular micro-environment. Hence, endogenous osteocyte Cx43 hemichannels emerge as important and promising therapeutic targets for the prevention of bone metastases and/or clinical treatment of bone-metastasized breast cancers.

Immobilization and long-term recovery results in large changes in bone structure and strength but no corresponding alterations of osteocyte lacunar properties.

The ability of osteocytes to demineralize the perilacunar matrix, osteocytic osteolysis, and thereby participate directly in bone metabolism, is an aspect of osteocyte biology that has received increasing attention during the last couple of years. The aim of the present work was to investigate whether osteocyte lacunar properties change during immobilization and subsequent recovery. A rat cortical bone model with negligible Haversian remodeling effects was used, with temporary immobilization of one hindlimb induced by botulinum toxin. Several complementary techniques covering multiple length scales enabled correlation of osteocyte lacunar properties to changes observed on the organ and tissue level of femoral bone. Bone structural parameters measured by µCT and mechanical properties were compared to sub-micrometer resolution SR µCT data mapping an unprecedented number (1.85 million) of osteocyte lacunae. Immobilization induced a significant reduction in aBMD, bone volume, tissue volume, and load to fracture, as well as the muscle mass of rectus femoris. During the subsequent recovery period, the bone structural and mechanical properties were only partly regained in spite of a long-term (28weeks) study period. No significant changes in osteocyte lacunar volume, density, oblateness, stretch, or orientation were detected upon immobilization or subsequent recovery. In conclusion, the bone architecture and not osteocyte lacunar properties or bone material characteristics dominate the immobilization response as well as the subsequent recovery.

Sclerostin antibody (Scl-Ab) improves osteomalacia phenotype in dentin matrix protein 1(Dmp1) knockout mice with little impact on serum levels of phosphorus and FGF23.

Unlike treatments for most rickets, the treatment using 1,25-(OH)2 vitamin D3 has little efficacy on patients with hypophosphatemic rickets, a set of rare genetic diseases. Thus, understanding the local cause for osteomalacia in hypophosphatemic rickets and developing an effective treatment to restore mineralization in this rare disease has been a longstanding goal in medicine. Here, we used Dmp1 knockout (KO) mice (whose mutations led to the same type of autosomal recessive hypophosphatemic rickets in humans) as the model in which the monoclonal antibody of sclerostin (Scl-Ab) was tested in two age groups for 8weeks: the prevention group (starting at age 4weeks) and the treatment group (starting at age 12weeks). Applications of Scl-Ab greatly improved the osteomalacia phenotype (>15%) and the biomechanical properties (3-point bending, ~60%) in the treated long-bone group. Our studies not only showed improvement of the osteomalacia in the alveolar bone, which has the highest bone metabolism rate, as well as the long bone phenotypes in treated mice. All these improvements attributed to the use of Scl-Ab are independent of the change in serum levels of phosphorus and FGF23, since Scl-Ab had little efficacy on those parameters. Finally, we propose a model to explain how Scl-Ab can improve the Dmp1 KO osteomalacia phenotype, in which the sclerostin level is already low.

Organ and tissue level properties are more sensitive to age than osteocyte lacunar characteristics in rat cortical bone.

Modeling and remodeling induce significant changes of bone structure and mechanical properties with age. Therefore, it is important to gain knowledge of the processes taking place in bone over time. The rat is a widely used animal model, where much data has been accumulated on age-related changes of bone on the organ and tissue level, whereas features on the nano- and micrometer scale are much less explored. We investigated the age-related development of organ and tissue level bone properties such as bone volume, bone mineral density, and load to fracture and correlated these with osteocyte lacunar properties in rat cortical bone. Femora of 14 to 42-week-old female Wistar rats were investigated using multiple complementary techniques including X-ray micro-computed tomography and biomechanical testing. The body weight, femoral length, aBMD, load to fracture, tissue volume, bone volume, and tissue density were found to increase rapidly with age at 14-30 weeks. At the age of 30-42 weeks, the growth rate appeared to decrease. However, no accompanying changes were found in osteocyte lacunar properties such as lacunar volume, ellipsoidal radii, lacunar stretch, lacunar oblateness, or lacunar orientation with animal age. Hence, the evolution of organ and tissue level properties with age in rat cortical bone is not accompanied by related changes in osteocyte lacunar properties. This suggests that bone microstructure and bone matrix material properties and not the geometric properties of the osteocyte lacunar network are main determinants of the properties of the bone on larger length scales.

The resistance of cortical bone tissue to failure under cyclic loading is reduced with alendronate.

Bisphosphonates are the most prescribed preventative treatment for osteoporosis. However, their long-term use has recently been associated with atypical fractures of cortical bone in patients who present with low-energy induced breaks of unclear pathophysiology. The effects of bisphosphonates on the mechanical properties of cortical bone have been exclusively studied under simple, monotonic, quasi-static loading. This study examined the cyclic fatigue properties of bisphosphonate-treated cortical bone at a level in which tissue damage initiates and is accumulated prior to frank fracture in low-energy situations. Physiologically relevant, dynamic, 4-point bending applied to beams (1.5 mm × 0.5 mm × 10 mm) machined from dog rib (n=12/group) demonstrated mechanical failure and micro-architectural features that were dependent on drug dose (3 groups: 0, 0.2, 1.0mg/kg/day; alendronate [ALN] for 3 years) with cortical bone tissue elastic modulus (initial cycles of loading) reduced by 21% (p<0.001) and fatigue life (number of cycles to failure) reduced in a stress-life approach by greater than 3-fold with ALN1.0 (p<0.05). While not affecting the number of osteons, ALN treatment reduced other features associated with bone remodeling, such as the size of osteons (-14%; ALN1.0: 10.5±1.8, VEH: 12.2±1.6, ×10(3) µm2; p<0.01) and the density of osteocyte lacunae (-20%; ALN1.0: 11.4±3.3, VEH: 14.3±3.6, ×10(2) #/mm2; p<0.05). Furthermore, the osteocyte lacunar density was directly proportional to initial elastic modulus when the groups were pooled (R=0.54, p<0.01). These findings suggest that the structural components normally contributing to healthy cortical bone tissue are altered by high-dose ALN treatment and contribute to reduced mechanical properties under cyclic loading conditions.