Relationships between matrix mineralization, oxidative metabolism, and mitochondrial structure during ATDC5 murine chondroprogenitor cell line differentiation.

The mechanistic relationships between the progression of growth chondrocyte differentiation, matrix mineralization, oxidative metabolism, and mitochondria content and structure were examined in the ATDC5 murine chondroprogenitor cell line. The progression of chondrocyte differentiation was associated with a statistically significant (p ≤ 0.05) ~2-fold increase in oxidative phosphorylation. However, as matrix mineralization progressed, oxidative metabolism decreased. In the absence of mineralization, cartilage extracellular matrix mRNA expression for Col2a1, Aggrecan, and Col10a1 were statistically (p ≤ 0.05) ~2-3-fold greater than observed in mineralizing cultures. In contrast, BSP and Phex that are associated with promoting matrix mineralization showed statistically (p ≤ 0.05) higher ~2-4 expression, while FGF23 phosphate regulatory factor was significantly lower (~50%) in mineralizing cultures. Cultures induced to differentiate under both nonmineralizing and mineralizing media conditions showed statistically greater basal oxidative metabolism and ATP production. Maximal respiration and spare oxidative capacity were significantly elevated (p ≤ 0.05) in differentiated nonmineralizing cultures compared to those that mineralized. Increased oxidative metabolism was associated with both an increase in mitochondria volume per cell and mitochondria fusion, while mineralization diminished mitochondrial volume and appeared to be associated with fission. Undifferentiated and mineralized cells showed increased mitochondrial co-localization with the actin cytoskeletal. Examination of proteins associated with mitochondria fission and apoptosis and mitophagy, respectively, showed levels of immunological expression consistent with the increasing fission and apoptosis in mineralizing cultures. These results suggest that chondrocyte differentiation is associated with intracellular structural reorganization, promoting increased mitochondria content and fusion that enables increased oxidative metabolism. Mineralization, however, does not need energy derived from oxidative metabolism; rather, during mineralization, mitochondria appear to undergo fission and mitophagy. In summary, these studies show that as chondrocytes underwent hypertrophic differentiation, they increased oxidative metabolism, but as mineralization proceeds, metabolism decreased. Mitochondria structure also underwent a structural reorganization that was further supportive of their oxidative capacity as the chondrocytes progressed through their differentiation. Thus, the mitochondria first underwent fusion to support increased oxidative metabolism, then underwent fission during mineralization, facilitating their programed death.

Alterations in compositional and cellular properties of the subchondral bone are linked to cartilage degeneration in hip osteoarthritis.

OBJECTIVE: The subchondral bone is an emerging regulator of osteoarthritis (OA). However, knowledge of how specific subchondral alterations relate to cartilage degeneration remains incomplete. METHOD: Femoral heads were obtained from 44 patients with primary OA during total hip arthroplasty and from 30 non-OA controls during autopsy. A multiscale assessment of the central subchondral bone region comprising histomorphometry, quantitative backscattered electron imaging, nanoindentation, and osteocyte lacunocanalicular network characterization was employed. RESULTS: In hip OA, thickening of the subchondral bone coincided with a higher number of osteoblasts (controls: 3.7 ± 4.5 mm-1, OA: 16.4 ± 10.2 mm-1, age-adjusted mean difference 10.5 mm-1 [95% CI 4.7 to 16.4], p < 0.001) but a similar number of osteoclasts compared to controls (p = 0.150). Furthermore, higher matrix mineralization heterogeneity (CaWidth, controls: 2.8 ± 0.2 wt%, OA: 3.1 ± 0.3 wt%, age-adjusted mean difference 0.2 wt% [95% CI 0.1 to 0.4], p = 0.011) and lower tissue hardness (controls: 0.69 ± 0.06 GPa, OA: 0.67 ± 0.06 GPa, age-adjusted mean difference -0.05 GPa [95% CI -0.09 to -0.01], p = 0.032) were detected. While no evidence of altered osteocytic perilacunar/canalicular remodeling in terms of fewer osteocyte canaliculi was found in OA, specimens with advanced cartilage degeneration showed a higher number of osteocyte canaliculi and larger lacunocanalicular network area compared to those with low-grade cartilage degeneration. Multiple linear regression models indicated that several subchondral bone properties, especially osteoblast and osteocyte parameters, were closely related to cartilage degeneration (R2 adjusted = 0.561, p < 0.001). CONCLUSION: Subchondral bone properties in OA are affected at the compositional, mechanical, and cellular levels. Based on their strong interaction with cartilage degeneration, targeting osteoblasts/osteocytes may be a promising therapeutic OA approach. DATA AND MATERIALS AVAILABILITY: All data are available in the main text or the supplementary materials.

ß-glycerophosphate, not low magnitude fluid shear stress, increases osteocytogenesis in the osteoblast-to-osteocyte cell line IDG-SW3.

AIM: As osteoblasts deposit a mineralized collagen network, a subpopulation of these cells differentiates into osteocytes. Biochemical and mechanical stimuli, particularly fluid shear stress (FSS), are thought to regulate this, but their relative influence remains unclear. Here, we assess both biochemical and mechanical stimuli on long-term bone formation and osteocytogenesis using the osteoblast-osteocyte cell line IDG-SW3. METHODS: Due to the relative novelty and uncommon culture conditions of IDG-SW3 versus other osteoblast-lineage cell lines, effects of temperature and media formulation on matrix deposition and osteocytogenesis were initially characterized. Subsequently, the relative influence of biochemical (ß-glycerophosphate (ßGP) and ascorbic acid 2-phosphate (AA2P)) and mechanical stimulation on osteocytogenesis was compared, with intermittent application of low magnitude FSS generated by see-saw rocker. RESULTS: ßGP and AA2P supplementation were required for mineralization and osteocytogenesis, with 33°C cultures retaining a more osteoblastic phenotype and 37°C cultures undergoing significantly higher osteocytogenesis. ßGP concentration positively correlated with calcium deposition, whilst AA2P stimulated alkaline phosphatase (ALP) activity and collagen deposition. We demonstrate that increasing ßGP concentration also significantly enhances osteocytogenesis as quantified by the expression of green fluorescent protein linked to Dmp1. Intermittent FSS (~0.06 Pa) rocker had no effect on osteocytogenesis and matrix deposition. CONCLUSIONS: This work demonstrates the suitability and ease with which IDG-SW3 can be utilized in osteocytogenesis studies. IDG-SW3 mineralization was only mediated through biochemical stimuli with no detectable effect of low magnitude FSS. Osteocytogenesis of IDG-SW3 primarily occurred in mineralized areas, further demonstrating the role mineralization of the bone extracellular matrix has in osteocyte differentiation.

17ß-Estradiol (E2) Activates Matrix Mineralization through Genomic/Nongenomic Pathways in MC3T3-E1 Cells.

Estrogen plays an important role in osteoporosis prevention. We herein report the possible novel signaling pathway of 17ß-estradiol (E2) in the matrix mineralization of MC3T3-E1, an osteoblast-like cell line. In the culture media-containing stripped serum, in which small lipophilic molecules such as steroid hormones including E2 were depleted, matrix mineralization was significantly reduced. However, the E2 treatment induced this. The E2 effects were suppressed by ICI182,780, the estrogen receptor (ER)α, and the ERß antagonist, as well as their mRNA knockdown, whereas Raloxifene, an inhibitor of estrogen-induced transcription, and G15, a G-protein-coupled estrogen receptor (GPER) 1 inhibitor, had little or no effect. Furthermore, the E2-activated matrix mineralization was disrupted by PMA, a PKC activator, and SB202190, a p38 MAPK inhibitor, but not by wortmannin, a PI3K inhibitor. Matrix mineralization was also induced by the culture media from the E2-stimulated cell culture. This effect was hindered by PMA or heat treatment, but not by SB202190. These results indicate that E2 activates the p38 MAPK pathway via ERs independently from actions in the nucleus. Such activation may cause the secretion of certain signaling molecule(s), which inhibit the PKC pathway. Our study provides a novel pathway of E2 action that could be a therapeutic target to activate matrix mineralization under various diseases, including osteoporosis.

Bone matrix properties in adults with osteogenesis imperfecta are not adversely affected by Setrusumab - a sclerostin neutralizing antibody.

Osteogenesis imperfecta (OI) is a skeletal dysplasia characterized by low bone mass and frequent fractures. Children with OI are commonly treated with bisphosphonates to reduce fracture rate, but treatment options for adults are limited. In the Phase 2b ASTEROID trial, setrusumab (a sclerostin neutralizing antibody, SclAb) improved bone density and strength in adults with type I, III and IV OI. Here, we investigate bone matrix material properties in tetracycline-labeled trans-iliac biopsies from three groups: i) control: individuals with no metabolic bone disease, ii) OI: individuals with OI, iii) SclAb-OI: individuals with OI after six months of setrusumab treatment (as part of the ASTEROID trial). In addition to bone histomorphometry, bone mineral and matrix properties were evaluated with nanoindentation, Raman spectroscopy, second harmonic generation imaging, quantitative backscatter electron imaging, and small-angle x-ray scattering. Spatial locations of fluorochrome labels were identified to differentiate inter-label bone of the same tissue age and intra-cortical bone. No difference in collagen orientation was found between the groups. The bone mineral density distribution and analysis of Raman spectra indicate that OI groups have greater mean mineralization, greater relative mineral content, and lower crystallinity than the control group, which was not altered by SclAb treatment. Finally, a lower modulus and hardness were measured in the inter-label bone of the OI-SclAb group compared to the OI group. Previous studies suggest that even though bone from OI has a higher mineral content, the ECM has comparable mechanical properties. Therefore, fragility in OI may stem from contributions from other yet unexplored aspects of bone organization at higher length scales. We conclude that SclAb treatment leads to increased bone mass while not adversely affecting bone matrix properties in individuals with OI.

Individuals with osteogenesis imperfecta (OI), also known as "brittle bone disease," have low bone mass and frequent fractures. Low bone mass occurs due to an imbalance between cells that remove bone and cells that form bone. Pharmaceutical treatments that block removal of bone lead to reduced fracture rates in children with OI. Effective treatment options for adults are limited. Setrusumab is a drug that leads to increased bone mass and strength in adults with OI. Here, we investigate whether Setrusumab alters the bone material in addition to improving bone mass. Three groups are compared: individuals with OI treated with Setrusumab, individuals with OI not treated with Setrusumab, and individuals without OI. A lower modulus and hardness were measured with nanoindentation in the Setrusumab-treated group. However, we did not find any changes in the bone's multi-scale structure. Fragility in OI may stem from other yet unexplored aspects of bone organization. We conclude that Setrusumab treatment leads to increased bone mass while not adversely affecting bone material properties in individuals with OI.

Collagen structures of demineralized bone paper direct mineral metabolism.

Bone is a dynamic mineralized tissue that undergoes continuous turnover throughout life. While the general mechanism of bone mineral metabolism is documented, the role of underlying collagen structures in regulating osteoblastic mineral deposition and osteoclastic mineral resorption remains an active research area, partly due to the lack of biomaterial platforms supporting accurate and analytical investigation. The recently introduced osteoid-inspired demineralized bone paper (DBP), prepared by 20-µm thin sectioning of demineralized bovine compact bone, holds promise in addressing this challenge as it preserves the intrinsic bony collagen structure and retains semi-transparency. Here, we report on the impact of collagen structures on modulating osteoblast and osteoclast-driven bone mineral metabolism using vertical and transversal DBPs that exhibit a uniaxially aligned and a concentric ring collagen structure, respectively. Translucent DBP reveals these collagen structures and facilitates longitudinal tracking of mineral deposition and resorption under brightfield microscopy for at least 3 wk. Genetically labeled primary osteogenic cells allow fluorescent monitoring of these cellular processes. Osteoblasts adhere and proliferate following the underlying collagen structures of DBPs. Osteoblastic mineral deposition is significantly higher in vertical DBP than in transversal DBP. Spatiotemporal analysis reveals notably more osteoblast adhesion and faster mineral deposition in vascular regions than in bone regions. Subsequent osteoclastic resorption follows these mineralized collagen structures, directing distinct trench and pit-type resorption patterns. In vertical DBP, trench-type resorption occurs at an 80% frequency, whereas transversal DBP shows 35% trench-type and 65% pit-type resorption. Our studies substantiate the importance of collagen structures in regulating mineral metabolism by osteogenic cells. DBP is expected to serve as an enabling biomaterial platform for studying various aspects of cellular and extracellular bone remodeling biology.

Matrix first, minerals later: fine-tuned dietary phosphate increases bone formation in zebrafish.

Bone matrix formation and mineralization are two closely related, yet separated processes. Matrix formation occurs first, mineralization is a second step strictly dependent on the dietary intake of calcium and phosphorus (P). However, mineralization is commonly used as diagnostic parameter for bone-related diseases. In this context, bone loss, often characterized as a condition with reduced bone mineral density, represents a major burden for human health, for which increased dietary mineral intake is generally recommended. Using a counterintuitive approach, we use a low-P diet followed by a sufficient-P intake to increase bone volume. We show in zebrafish by histology, qPCR, micro-CT, and enzyme histochemistry that a two-months period of reduced dietary P intake stimulates extensive formation of new bone matrix, associated with the upregulation of key genes required for both bone matrix formation and mineralization. The return to a P-sufficient diet initiates the mineralization of the abundant matrix previously deposited, thus resulting in a striking increase of the mineralized bone volume as proven at the level of the vertebral column, including vertebral bodies and arches. In summary, bone matrix formation is first stimulated with a low-P diet, and its mineralization is later triggered by a sufficient-P dietary intake. In zebrafish, the uncoupling of bone formation and mineralization by alternating low and sufficient dietary P intake significantly increases the bone volume without causing skeletal malformations or ectopic mineralization. A modification of this approach to stimulate bone formation, optimized for mammalian models, can possibly open opportunities to support treatments in patients that suffer from low bone mass.

ß,γ-Methylene-ATP and its metabolite medronic acid affect both arterial media calcification and bone mineralization in non-CKD and CKD rats.

Arterial media calcification or pathological deposition of calcium-phosphate crystals in the vessel wall contributes significantly to the high mortality rate observed in patients with CKD. Extracellular nucleotides (ie, ATP or UTP) regulate the arterial calcification process by interacting with (1) purinergic receptors and (2) breakdown via ecto-nucleotidases, such as ectonucleotide pyrophosphatase/phosphodiesterase NPP1 or NPP3, affecting the local levels of calcification inhibitor, pyrophosphate, and stimulator inorganic phosphate (PPi/Pi ratio). Also, it has been shown that ATP analogs (ie, ß,γ-methylene-ATP [ß,γ-meATP]) inhibit vascular smooth muscle cell calcification in vitro. In the first experiment, daily dosing of ß,γ-meATP (2 mg/kg) was investigated in rats fed a warfarin diet to trigger the development of non-CKD-related arterial medial calcifications. This study showed that ß,γ-meATP significantly lowered the calcium scores in the aorta and peripheral vessels in warfarin-exposed rats. In a second experiment, daily dosing of 4 mg/kg ß,γ-meATP and its metabolite medronic acid (MDP) was analyzed in rats fed an adenine diet to promote the development of CKD-related arterial medial calcification. Administration of ß,γ-meATP and MDP did not significantly decrease aortic calcification scores in this model. Moreover, both compounds induced deleterious effects on physiological bone mineralization, causing an imminent risk for worsening the already compromised bone status in CKD. Due to this, it was not possible to raise the dosage of both compounds to tackle CKD-related arterial calcification. Again, this points out the difficult task of targeting solely ectopic calcifications without negatively affecting physiological bone mineralization. On the other hand, aortic mRNA expression of Enpp1 and Enpp3 was significantly and positively associated with aortic calcification scores, suggesting that normalizing the aortic NPP1/3 activity to control values might be a possible target to treat (CKD-induced) arterial media calcifications.

Quantitative Bone SPECT/CT of Central Cartilaginous Bone Tumors: Relationship between SUVmax and Radiodensity in Hounsfield Unit.

(1) Background: it is challenging to determine the accurate grades of cartilaginous bone tumors. Using bone single photon emission computed tomography (SPECT)/computed tomography (CT), maximum standardized uptake value (SUVmax) was found to be significantly associated with different grades of cartilaginous bone tumor. The inquiry focused on the effect of the tumor matrix on SUVmax. (2) Methods: a total of 65 patients from 2017 to 2022 with central cartilaginous bone tumors, including enchondromas and low-to-intermediate grade chondrosarcomas, who had undergone bone SPECT/CT were retrospectively enrolled. The SUVmax was recorded and any aggressive CT findings of cartilaginous bone tumor and Hounsfield units (HU) of the chondroid matrix as mean, minimum, maximum, and standard deviation (SD) were reviewed on CT scans. Pearson's correlation analysis was performed to determine the relationship between CT features and SUVmax. Subgroup analysis was also performed between the benign group (enchondroma) and the malignant group (grade 1 and 2 chondrosarcoma) for comparison of HU values and SUVmax. (3) Results: a significant negative correlation between SUVmax and HU measurements, including HUmax, HUmean, and HUSD, was found. The subgroup analysis showed significantly higher SUVmax in the malignant group, with more frequent CT aggressive features, and significantly lower HUSD in the malignant group than in the benign group. (4) Conclusions: it was observed that higher SUVmax and lower HUSD were associated with a higher probability of having a low-to-intermediate chondrosarcoma with aggressive features and a less calcified tumor matrix.