A Three-Dimensional Mechanical Loading Model of Human Osteocytes in Their Native Matrix.

Osteocytes are mechanosensory cells which are embedded in calcified collagenous matrix. The specific native matrix of osteocytes affects their regulatory activity, i.e., transmission of signaling molecules to osteoclasts and/or osteoblasts, in the mechanical adaptation of bone. Unfortunately, no existing in vitro model of cortical bone is currently available to study the mechanosensory function of human osteocytes in their native matrix. Therefore, we aimed to develop an in vitro three-dimensional mechanical loading model of human osteocytes in their native matrix. Human cortical bone explants containing osteocytes in their three-dimensional native matrix were cultured and mechanically loaded by three-point bending using a custom-made loading apparatus generating sinusoidal displacement. Osteocyte viability and sclerostin expression were measured 1-2 days before 5 min loading and 1 day after loading. Bone microdamage was visualized and quantified by micro-CT analysis and histology using BaSO4 staining. A linear relationship was found between loading magnitude (2302-13,811 µÉ›) and force (1.6-4.9 N) exerted on the bone explants. At 24 h post-loading, osteocyte viability was not affected by 1600 µÉ› loading. Sclerostin expression and bone microdamage were unaffected by loading up to 8000 µÉ›. In conclusion, we developed an in vitro 3D mechanical loading model to study mechanoresponsiveness of viable osteocytes residing in their native matrix. This model is suitable to study the effect of changed bone matrix composition in metabolic bone disease on osteocyte mechanoresponsiveness.

Magnesium prevents vascular calcification in Klotho deficiency.

Klotho knock-out mice are an important model for vascular calcification, which is associated with chronic kidney disease. In chronic kidney disease, serum magnesium inversely correlates with vascular calcification. Here we determine the effects of serum magnesium on aortic calcification in Klotho knock-out mice treated with a minimal or a high magnesium diet from birth. After eight weeks, serum biochemistry and aorta and bone tissues were studied. Protective effects of magnesium were characterized by RNA-sequencing of the aorta and micro-CT analysis was performed to study bone integrity. A high magnesium diet prevented vascular calcification and aortic gene expression of Runx2 and matrix Gla protein found in such mice on the minimal magnesium diet. Differential expression of inflammation and extracellular matrix remodeling genes accompanied the beneficial effects of magnesium on calcification. High dietary magnesium did not affect serum parathyroid hormone, 1,25-dihydroxyvitamin D3 or calcium. High magnesium intake prevented vascular calcification despite increased fibroblast growth factor-23 and phosphate concentration in the knock-out mice. Compared to mice on the minimal magnesium diet, the high magnesium diet reduced femoral bone mineral density by 20% and caused excessive osteoid formation indicating osteomalacia. Osteoclast activity was unaffected by the high magnesium diet. In Saos-2 osteoblasts, magnesium supplementation reduced mineralization independent of osteoblast function. Thus, high dietary magnesium prevents calcification in Klotho knock-out mice. These effects are potentially mediated by reduction of inflammatory and extracellular matrix remodeling pathways within the aorta. Hence magnesium treatment may be promising to prevent vascular calcification, but the risk for osteomalacia should be considered.

Ovariectomy increases RANKL protein expression in bone marrow adipocytes of C3H/HeJ mice.

Estrogen deficiency induces bone loss by increasing bone resorption, in part through upregulation of receptor activator of nuclear factor-κB ligand (RANKL). RANKL is secreted by osteoblasts and osteocytes, but more recently bone marrow (pre)adipocytes have also been shown to express RANKL. Estrogen deficiency increases bone marrow adipose tissue (BMAT). The aim of this study was to determine the effect of ovariectomy (OVX) on RANKL protein expression by bone marrow adipocytes in C3H/HeJ mice. Fourteen-week-old female C3H/HeJ mice (n = 20) were randomized to sham surgery (Sham) or OVX. After 4 wk animals were euthanized. BMAT volume fraction (BMAT volume/marrow volume) was quantified by polyoxometalate-based contrast-enhanced nano-computed tomography. The percentage of RANKL-positive bone marrow adipocytes (RANKL-positive bone marrow adipocytes/total adipocytes) and the percentage of RANKL-positive osteoblasts covering the bone surface (bone surface covered in RANKL-positive osteoblasts/total bone surface) were quantified in the distal metaphysis of immunohistochemically stained sections of the left femur. The effects of OVX were analyzed by Student's t test or Mann-Whitney U test. RANKL was detected in osteoblasts, osteocytes, and bone marrow adipocytes. OVX significantly increased mean percentage of RANKL-positive bone marrow adipocytes [mean (SD): Sham 42 (18)%; OVX 64 (12)%; P = 0.029] as well as BMAT volume/marrow volume [median (interquartile range): Sham 1.4 (4.9)%; OVX 7.2 (7.3)%; P = 0.008] compared with Sham. We show that OVX increased both the percentage of RANKL-positive bone marrow adipocytes and the total BMAT volume fraction in C3H/HeJ mice. Therefore, RANKL produced by bone marrow adipocytes could be an important contributor to OVX-induced bone loss in C3H/HeJ mice.

Exploration of the skeletal phenotype of the Col1a1 +/Mov13 mouse model for haploinsufficient osteogenesis imperfecta type 1.

Introduction: Osteogenesis Imperfecta is a rare genetic connective tissue disorder, characterized by skeletal dysplasia and fragile bones. Currently only two mouse models have been reported for haploinsufficient (HI) mild Osteogenesis Imperfecta (OI); the Col1a1 +/Mov13 (Mov13) and the Col1a1 +/-365 mouse model. The Mov13 mice were created by random insertion of the Mouse Moloney leukemia virus in the first intron of the Col1a1 gene, preventing the initiation of transcription. Since the development of the Mov13 mice almost four decades ago and its basic phenotypic characterization in the 90s, there have not been many further studies. We aimed to extensively characterize the Mov13 mouse model in order to critically evaluate its possible use for preclinical studies of HI OI. Methods: Bone tissue from ten heterozygous Mov13 and ten wild-type littermates (WT) C57BL/6J mice (50% males per group) was analyzed at eight weeks of age with bone histomorphometry, micro computed tomography (microCT), 3-point bending, gene expression of different collagens, as well as serum markers of bone turnover. Results: The Mov13 mouse presented a lower bone strength and impaired material properties based on our results of 3-point bending and microCT analysis respectively. In contrast, no significant differences were found for all histomorphometric parameters. In addition, no significant differences in Col1a1 bone expression were present, but there was a significant lower P1NP concentration, a bone formation marker, measured in serum. Furthermore, bone tissue of Mov13 mice presented significantly higher expression of collagens (Col1a2, Col5a1 and Col5a2), and bone metabolism markers (Bglap, Fgf23, Smad7, Edn1 and Eln) compared to WT. Finally, we measured a significantly lower Col1a1 expression in heart and skin tissue and also determined a higher expression of other collagens in the heart tissue. Conclusion: Although we did not detect a significant reduction in Col1a1 expression in the bone tissue, a change in bone structure and reduction in bone strength was noted. Regrettably, the variability of the bone phenotype and the appearance of severe lymphoma in adult Mov13 mice, does not favor their use for the testing of new long-term drug studies. As such, a new HI OI type 1 mouse model is urgently needed.

Mapping the Response of Human Osteocytes in Native Matrix to Mechanical Loading Using RNA Sequencing.

Osteocytes sense mechanical loads and transduce mechanical signals into a chemical response. They are the most abundant bone cells deeply embedded in mineralized bone matrix, which affects their regulatory activity in the mechanical adaptation of bone. The specific location in the calcified bone matrix hinders studies on osteocytes in the in vivo setting. Recently, we developed a three-dimensional mechanical loading model of human osteocytes in their native matrix, allowing to study osteocyte mechanoresponsive target gene expression in vitro. Here we aimed to identify differentially expressed genes by mapping the response of human primary osteocytes in their native matrix to mechanical loading using RNA sequencing. Human fibular bone was retrieved from 10 donors (age: 32-82 years, 5 female, 5 male). Cortical bone explants (8.0 × 3.0 × 1.5 mm; length × width × height) were either not loaded or mechanically loaded by 2000 or 8000 µÉ› for 5 minutes, followed by 0, 6, or 24 hours post-culture without loading. High-quality RNA was isolated, and differential gene expression analysis performed by R2 platform. Real-time PCR was used to confirm differentially expressed genes. Twenty-eight genes were differentially expressed between unloaded and loaded (2000 or 8000 µÉ›) bone at 6 hours post-culture, and 19 genes at 24 hours post-culture. Eleven of these genes were related to bone metabolism, ie, EGR1, FAF1, H3F3B, PAN2, RNF213, SAMD4A, and TBC1D24 at 6 hours post-culture, and EGFEM1P, HOXD4, SNORD91B, and SNX9 at 24 hours post-culture. Mechanical loading significantly decreased RNF213 gene expression, which was confirmed by real-time PCR. In conclusion, mechanically loaded osteocytes differentially expressed 47 genes, of which 11 genes were related to bone metabolism. RNF213 might play a role in mechanical adaptation of bone by regulating angiogenesis, which is a prerequisite for successful bone formation. The functional aspects of the differentially expressed genes in bone mechanical adaptation requires future investigation. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Bone cells from patients with quiescent Crohn's disease show a reduced growth potential and an impeded maturation.

Patients with Crohn's disease (CD) are at increased risk of developing osteoporosis. The mechanism underlying bone loss in CD patients is only partly understood. Inflammation is thought to contribute by causing a disturbed bone remodeling. In this study, we aimed to compare functional characteristics of osteoblasts from CD patients and controls, as osteoblasts are one of the effector cells in bone remodeling. The study included 18 patients with quiescent CD and 18 healthy controls. Bone cells obtained from iliac crest biopsies were cultured in the absence and presence of the inflammatory cytokines IL-1α, IL-1ß, IL-6, TNF-α, IL-10, and TGF-ß. At various time points, cell proliferation and differentiation were analyzed. Bone cells from CD patients showed a prolonged culture period to reach confluence and a decreased cell number at confluence. CD patient-derived bone cell cultures produced higher alkaline phosphatase levels, whereas osteocalcin levels were considerably reduced compared to control cultures. At the proliferation level, the responsiveness to inflammatory cytokines was similar in bone cells from CD patients and controls. At the differentiation level, CD cultures showed an increased responsiveness to IL-6 and a decreased responsiveness to TGF-ß. Responsiveness to the other cytokines tested was unaffected. In summary, we show a reduced growth potential and impeded maturation of bone cells from quiescent CD patients in vitro. These disease-related alterations combined with an unchanged sensitivity of CD patient-derived bone cells to inflammatory cytokines, provide a new insight in the understanding of CD-associated bone loss.

Osteocyte Apoptosis, Bone Marrow Adiposity, and Fibrosis in the Irradiated Human Mandible.

Purpose: To assess the effect of radiation therapy on osteocyte apoptosis, osteocyte death, and bone marrow adipocytes in the human mandible and its contribution to the pathophysiology of radiation damage to the mandibular bone. Methods and Materials: Mandibular cancellous bone biopsies were taken from irradiated patients and nonirradiated controls. Immunohistochemical detection of cleaved caspase-3 was performed to visualize apoptotic osteocytes. The number of apoptotic osteocytes per bone area and per total amount of osteocytes, osteocytes per bone area, and empty lacunae per bone area were counted manually. The percentage fibrotic tissue and adipose tissue per bone marrow area, the percentage bone marrow of total area, and the mean adipocyte diameter (µm) was determined digitally from adjacent Goldner stained sections. Results: Biopsies of 15 irradiated patients (12 men and 3 women) and 7 nonirradiated controls (5 men and 2 women) were assessed. In the study group a significant increase was seen in the number of empty lacunae, the percentage of adipose tissue of bone marrow area, and the adipocyte diameter. There was no significant difference in bone marrow fibrosis nor apoptotic osteocytes between the irradiated group and the controls. Conclusions: Irradiation alone does not seem to induce excessive bone marrow fibrosis. The damage to bone mesenchymal stem cells leads to increased marrow adipogenesis and decreased osteoblastogenic potential. Early osteocyte death resulting in avital persisting bone matrix with severely impaired regenerative potential may contribute to the vulnerability of irradiated bone to infection and necrosis.

Mechanical stress regulates bone regulatory gene expression independent of estrogen and vitamin D deficiency in rats.

Mechanical stress determines bone mass and structure. It is not known whether mechanical loading affects expression of bone regulatory genes in a combined deficiency of estrogen and vitamin D. We studied the effect of mechanical loading on the messenger RNA (mRNA) expression of bone regulatory genes during vitamin D and/or estrogen deficiency. We performed a single bout in vivo axial loading with 14 N peak load, 2 Hz frequency and 360 cycles in right ulnae of nineteen weeks old female control Wistar rats with or without ovariectomy (OVX), vitamin D deficiency and the combination of OVX and vitamin D deficiency (N = 10/group). Total bone RNA was isolated 6 hours after loading, and mRNA expression was detected of Mepe, Fgf23, Dmp1, Phex, Sost, Col1a1, Cyp27b1, Vdr, and Esr1. Serum levels of 25(OH)D, 1,25(OH)2 D and estradiol were also measured at this time point. The effect of loading, vitamin D and estrogen deficiency and their interaction on bone gene expression was tested using a mixed effect model analysis. Mechanical loading significantly increased the mRNA expression of Mepe, and Sost, whereas it decreased the mRNA expression of Fgf23 and Esr1. Mechanical loading showed a significant interaction with vitamin D deficiency with regard to mRNA expression of Vdr and Esr1. Mechanical loading affected gene expression of Mepe, Fgf23, Sost, and Esr1 independently of vitamin D or estrogen, indicating that mechanical loading may affect bone turnover even during vitamin D deficiency and after menopause.

Effects of different training modalities on phosphate homeostasis and local vitamin D metabolism in rat bone.

OBJECTIVES: Mechanical loading may be an important factor in the regulation of bone derived hormones involved in phosphate homeostasis. This study investigated the effects of peak power and endurance training on expression levels of fibroblast growth factor 23 (FGF23) and 1α-hydroxylase (CYP27b1) in bone. METHODS: Thirty-eight rats were assigned to six weeks of training in four groups: peak power (PT), endurance (ET), PT followed by ET (PET) or no training (control). In cortical bone, FGF23 was quantified using immunohistochemistry. mRNA expression levels of proteins involved in phosphate and vitamin D homeostasis were quantified in cortical bone and kidney. C-terminal FGF23, 25-hydroxyvitamin D3, parathyroid hormone (PTH), calcium and phosphate concentrations were measured in plasma or serum. RESULTS: Neither FGF23 mRNA and protein expression levels in cortical bone nor FGF23 plasma concentrations differed between the groups. In cortical bone, mRNA expression levels of sclerostin (SOST), dental matrix protein 1 (DMP1), phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and matrix extracellular phosphoglycoprotein (MEPE) were lower after PT compared to ET and PET. Expression levels of CYP27b1 and vitamin D receptor (VDR) in tibial bone were decreased after PT compared to ET. In kidney, no differences between groups were observed for mRNA expression levels of CYP27b1, 24-hydroxylase (CYP24), VDR, NaPi-IIa cotransporter (NPT2a) and NaPi-IIc cotransporter (NPT2c). Serum PTH concentrations were higher after PT compared to controls. CONCLUSION: After six weeks, none of the training modalities induced changes in FGF23 expression levels. However, PT might have caused changes in local phosphate regulation within bone compared to ET and PET. CYP27b1 and VDR expression in bone was reduced after PT compared to ET, suggesting high intensity peak power training in this rat model is associated with decreased vitamin D signalling in bone.

Inhibition of TGFß signaling decreases osteogenic differentiation of fibrodysplasia ossificans progressiva fibroblasts in a novel in vitro model of the disease.

Fibrodysplasia ossificans progressiva is a rare genetic disorder characterized by progressive heterotopic ossification. FOP patients develop soft tissue lumps as a result of inflammation-induced flare-ups which leads to the irreversible replacement of skeletal muscle tissue with bone tissue. Classical FOP patients possess a mutation (c.617G>A; R206H) in the ACVR1-encoding gene which leads to dysregulated BMP signaling. Nonetheless, not all FOP patients with this mutation exhibit equal severity in symptom presentation or disease progression which indicates a strong contribution by environmental factors. Given the pro-inflammatory role of TGFß, we studied the role of TGFß in the progression of osteogenic differentiation in primary dermal fibroblasts from five classical FOP patients based on a novel method of platelet lysate-based osteogenic transdifferentiation. During the course of transdifferentiation the osteogenic properties of the cells were evaluated by the mRNA expression of Sp7/Osterix, Runx2, Alp, OC and the presence of mineralization. During transdifferentiation the expression of osteoblast markers Runx2 (p<0.05) and Alp were higher in patient cells compared to healthy controls. All cell lines exhibited increase in mineralisation. FOP fibroblasts also expressed higher baseline Sp7/Osterix levels (p<0.05) confirming their higher osteogenic potential. The pharmacological inhibition of TGFß signaling during osteogenic transdifferentiation resulted in the attenuation of osteogenic transdifferentiation in all cell lines as shown by the decrease in the expression of Runx2 (p<0.05), Alp and mineralization. We suggest that blocking of TGFß signaling can decrease the osteogenic transdifferentiation of FOP fibroblasts.

Increased expression of matrix extracellular phosphoglycoprotein (MEPE) in cortical bone of the rat tibia after mechanical loading: identification by oligonucleotide microarray.

Skeletal integrity in humans and animals is maintained by daily mechanical loading. It has been widely accepted that osteocytes function as mechanosensors. Many biochemical signaling molecules are involved in the response of osteocytes to mechanical stimulation. The aim of this study was to identify genes involved in the translation of mechanical stimuli into bone formation. The four-point bending model was used to induce a single period of mechanical loading on the right tibia, while the contra lateral left tibia served as control. Six hours after loading, the effects of mechanical loading on gene-expression were determined with microarray analysis. Protein expression of differentially regulated genes was evaluated with immunohistochemistry. Nine genes were found to exhibit a significant differential gene expression in LOAD compared to control. MEPE, Garnl1, V2R2B, and QFG-TN1 olfactory receptor were up-regulated, and creatine kinase (muscle form), fibrinogen-B beta-polypeptide, monoamine oxidase A, troponin-C and kinesin light chain-C were down-regulated. Validation with real-time RT-PCR analysis confirmed the up-regulation of MEPE and the down-regulation of creatine kinase (muscle form) and troponin-C in the loaded tibia. Immunohistochemistry showed that the increase of MEPE protein expression was already detectable six hours after mechanical loading. In conclusion, these genes probably play a role during translation of mechanical stimuli six hours after mechanical loading. The modulation of MEPE expression may indicate a connection between bone mineralization and bone formation after mechanical stimulation.

Effect of raloxifene treatment on osteocyte apoptosis in postmenopausal women.

Increased osteocyte apoptosis, as the result of estrogen deficiency, could play a role in the decrease of bone mass and bone strength seen in postmenopausal osteoporosis. We investigated whether treatment with raloxifene of postmenopausal women with osteoporosis affects osteocyte apoptosis. Transiliac bone biopsies were obtained from 26 osteoporotic women at baseline and after 2 years of treatment with placebo or raloxifene. Immunohistochemical detection of cleaved caspase-3 was performed on sections from nondecalcified bone biopsies to visualize apoptosis. In the trabecular bone total osteocytes, positively stained osteocytes and empty lacunae were counted and percent positive cells and percent empty lacunae determined. Statistical evaluation was performed by Wilcoxon's paired t-test and Spearman's rank correlations. There was no significant difference in percentage positive osteocytes between baseline and follow-up biopsies in both the placebo and the raloxifene groups. The percentage empty lacunae increased significantly in the placebo group (11.20 +/- 1.43 vs. 9.00 +/- 2.25, P = 0.014) but not in the raloxifene group. At baseline in both groups combined, there was a negative correlation between indices of bone remodeling and the percentage positive osteocytes (bone formation rate/bone volume r = -0.67, P = 0.001). We found no direct evidence for an effect of raloxifene treatment on osteocyte apoptosis, but small effects of raloxifene treatment cannot be excluded. The percent of apoptotic osteocytes was dependent on the level of bone remodeling in an individual.