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1.
JBMR Plus ; 8(8): ziae080, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38989259

RESUMO

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.

2.
JBMR Plus ; 8(8): ziae081, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39045128

RESUMO

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.

3.
J Bone Miner Res ; 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38982734

RESUMO

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.

4.
Connect Tissue Res ; : 1-17, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38982804

RESUMO

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.

5.
Cancers (Basel) ; 16(11)2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38893090

RESUMO

(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.

6.
J Cell Physiol ; 239(8): e31285, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38860464

RESUMO

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.


Assuntos
Diferenciação Celular , Condrócitos , Matriz Extracelular , Mitocôndrias , Animais , Camundongos , Diferenciação Celular/fisiologia , Condrócitos/metabolismo , Mitocôndrias/metabolismo , Matriz Extracelular/metabolismo , Linhagem Celular , Calcificação Fisiológica , Fosforilação Oxidativa , Condrogênese/fisiologia , Dinâmica Mitocondrial/fisiologia , Trifosfato de Adenosina/metabolismo
7.
Int J Mol Sci ; 25(9)2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38731947

RESUMO

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.


Assuntos
Estradiol , Osteoblastos , Transdução de Sinais , Animais , Camundongos , Estradiol/farmacologia , Osteoblastos/metabolismo , Osteoblastos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Linhagem Celular , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/genética , Estrogênios/farmacologia , Estrogênios/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética
8.
JBMR Plus ; 8(6): ziae057, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38764790

RESUMO

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.

9.
Osteoarthritis Cartilage ; 32(5): 535-547, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38403152

RESUMO

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.


Assuntos
Doenças das Cartilagens , Cartilagem Articular , Osteoartrite do Quadril , Humanos , Osteoblastos , Osteócitos
10.
Arch Biochem Biophys ; 750: 109821, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37979903

RESUMO

The metastases of breast cancer to bone often cause osteolytic lesions not only by stimulating osteoclasts to resorb the bone but also by inhibiting osteoblasts from bone formation. Although tumor cell-derived extracellular vesicles (EVs) promote osteoclast differentiation and bone resorption, their roles in osteoblast differentiation and functions have not been elucidated. In this study, we investigated the effects of breast cancer cell-derived EVs on osteoblast differentiation and functions in vitro. We found that upon osteogenic induction, 4T1 bone metastatic mouse mammary tumor cell-derived EVs (4T1-EVs) were inhibited matrix mineralization of ST2 mouse bone marrow stromal cells. Temporal expression analysis of osteoblast marker genes, including runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), collagen type I (Col1a1), bone sialoprotein (Bsp), and osteocalcin (Bglap) revealed that 4T1-EVs decreased their expression during the late stage of osteoblast differentiation. Elevated levels of c-Jun N-terminal kinase (JNK) phosphorylation, upon osteogenic induction, were diminished by 4T1-EVs, significantly. In contrast, the nullification of reduced JNK phosphorylation by anisomycin, a potent JNK activator, increased the expression levels of osteoblast differentiation markers. Overall, our data indicated that 4T1-EVs affect osteoblast maturation, at least partially, through the regulation of JNK activity, which provides novel insights into the pathological impact of osteolytic bone metastasis and the role of EVs in osteoblast differentiation.


Assuntos
Neoplasias Ósseas , Vesículas Extracelulares , Animais , Camundongos , Osso e Ossos , Diferenciação Celular , Osteoblastos , Osteogênese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo
11.
Curr Osteoporos Rep ; 21(6): 787-805, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37897675

RESUMO

PURPOSE OF REVIEW: Metabolic and genetic bone disorders affect not only bone mass but often also the bone material, including degree of mineralization, matrix organization, and lacunar porosity. The quality of juvenile bone is moreover highly influenced by skeletal growth. This review aims to provide a compact summary of the present knowledge on the complex interplay between bone modeling and remodeling during skeletal growth and to alert the reader to the complexity of bone tissue characteristics in children with bone disorders. RECENT FINDINGS: We describe cellular events together with the characteristics of the different tissues and organic matrix organization (cartilage, woven and lamellar bone) occurring during linear growth. Subsequently, we present typical alterations thereof in disorders leading to over-mineralized bone matrix compared to those associated with low or normal mineral content based on bone biopsy studies. Growth spurts or growth retardation might amplify or mask disease-related alterations in bone material, which makes the interpretation of bone tissue findings in children complex and challenging.


Assuntos
Doenças Ósseas , Calcinose , Criança , Humanos , Osso e Ossos , Doenças Ósseas/metabolismo , Matriz Óssea/metabolismo , Densidade Óssea , Calcinose/metabolismo
12.
J Bone Miner Res ; 38(12): 1846-1855, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37877440

RESUMO

Postnatally, severe vitamin D deficiency commonly results in rickets as well as potential defects in tooth mineralization. The effects of milder deficiency on oral health outcomes later in life are still unclear. This study used micro-computed tomography (µCT), energy dispersive X-ray analysis (EDX), and Raman spectroscopy to investigate mineral density, total density, and elemental composition of enamel and dentine in 63 exfoliated primary incisors from participants with known 25-hydroxyvitamin D levels (25-OHD) at birth. No differences in mineralization and chemical composition using µCT and EDX analysis were observed irrespective of 25-OHD status. Subtle structural differences were observed via Raman spectroscopy, with more crystalline enamel observed in those with sufficient 25-OHD at birth. Although subtle, the differences seen suggest further attention should be given to children with known milder levels of vitamin D deficiency in early life. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).


Assuntos
Deficiência de Vitamina D , Vitamina D , Criança , Recém-Nascido , Humanos , Microtomografia por Raio-X , Minerais , Dente Decíduo , Densidade Óssea
13.
Front Cell Dev Biol ; 11: 1201200, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37727505

RESUMO

The mineralization of the extracellular matrix (ECM) is an essential and crucial process for physiological bone formation and pathological calcification. The abnormal function of ECM mineralization contributes to the worldwide risk of developing mineralization-related diseases; for instance, vascular calcification is attributed to the hyperfunction of ECM mineralization, while osteoporosis is due to hypofunction. AnnexinA6 (AnxA6), a Ca2+-dependent phospholipid-binding protein, has been extensively reported as an essential target in mineralization-related diseases such as osteoporosis, osteoarthritis, atherosclerosis, osteosarcoma, and calcific aortic valve disease. To date, AnxA6, as the largest member of the Annexin family, has attracted much attention due to its significant contribution to matrix vesicles (MVs) production and release, MVs-ECM interaction, cytoplasmic Ca2+ influx, and maturation of hydroxyapatite, making it an essential target in ECM mineralization. In this review, we outlined the recent advancements in the role of AnxA6 in mineralization-related diseases and the potential mechanisms of AnxA6 under normal and mineralization-related pathological conditions. AnxA6 could promote ECM mineralization for bone regeneration in the manner described previously. Therefore, AnxA6 may be a potential osteogenic target for ECM mineralization.

14.
J Bone Miner Res ; 38(10): 1509-1520, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37493605

RESUMO

Vitamin C (VitC) is essential for bone health, and low VitC serum levels increase the risk for skeletal fractures. If and how VitC affects bone mineralization is unclear. Using micro-computed tomography (µCT), histologic staining, as well as quantitative backscattered electron imaging (qBEI), we assessed the effects of VitC on femoral structure and microarchitecture, bone formation, and bone mineralization density distribution (BMDD) in the VitC incompetent Gulo-/- mouse model and wild-type mice. In particular, VitC-supplemented, 20-week-old mice were compared with age-matched counterparts where dietary VitC intake was excluded from week 15. VitC depletion in Gulo-/- mice severely reduced cortical thickness of the diaphyseal shaft and bone volume around the growth plate (eg, bone volume of the primary spongiosa -43%, p < 0.001). Loss of VitC also diminished the amount of newly formed bone tissue as visualized by histology and calcein labeling of the active mineralization front. BMDD analysis revealed a shift to higher calcium concentrations upon VitC supplementation, including higher average (~10% increase in female VitC deficient mice, p < 0.001) and peak calcium concentrations in the epiphyseal and metaphyseal spongiosa. These findings suggest higher bone tissue age. Importantly, loss of VitC had significantly more pronounced effects in female mice, indicating a higher sensitivity of their skeleton to VitC deficiency. Our results reveal that VitC plays a key role in bone formation rate, which directly affects mineralization. We propose that low VitC levels may contribute to the higher prevalence of bone-degenerative diseases in females and suggest leveraging this vitamin against these conditions. © 2023 American Society for Bone and Mineral Research (ASBMR).


Assuntos
Deficiência de Ácido Ascórbico , Mustelidae , Masculino , Camundongos , Animais , Feminino , Cálcio/farmacologia , Microtomografia por Raio-X , Osso e Ossos/diagnóstico por imagem , Densidade Óssea , Calcificação Fisiológica , Ácido Ascórbico/farmacologia
15.
bioRxiv ; 2023 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-37333124

RESUMO

Understanding the genetic basis of cortical bone traits can allow for the discovery of novel genes or biological pathways regulating bone health. Mice are the most widely used mammalian model for skeletal biology and allow for the quantification of traits that can't easily be evaluated in humans, such as osteocyte lacunar morphology. The goal of our study was to investigate the effect of genetic diversity on multi-scale cortical bone traits of three long bones in skeletally-mature mice. We measured bone morphology, mechanical properties, material properties, lacunar morphology, and mineral composition of mouse bones from two populations of genetic diversity. Additionally, we compared how intra-bone relationships varied in the two populations. Our first population of genetic diversity included 72 females and 72 males from the eight Inbred Founder strains used to create the Diversity Outbred (DO) population. These eight strains together span almost 90% of the genetic diversity found in mice (Mus musculus). Our second population of genetic diversity included 25 genetically unique, outbred females and 25 males from the DO population. We show that multi-scale cortical bone traits vary significantly with genetic background; heritability values range from 21% to 99% indicating genetic control of bone traits across length scales. We show for the first time that lacunar shape and number are highly heritable. Comparing the two populations of genetic diversity, we show each DO mouse does not resemble a single Inbred Founder but instead the outbred mice display hybrid phenotypes with the elimination of extreme values. Additionally, intra-bone relationships (e.g., ultimate force vs. cortical area) were mainly conserved in our two populations. Overall, this work supports future use of these genetically diverse populations to discover novel genes contributing to cortical bone traits, especially at the lacunar length scale.

16.
J Bone Miner Res ; 38(8): 1192-1207, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37191192

RESUMO

Chronic kidney disease (CKD) is characterized by kidney damage and loss of renal function. CKD mineral and bone disorder (CKD-MBD) describes the dysregulation of mineral homeostasis, including hyperphosphatemia and elevated parathyroid hormone (PTH) secretion, skeletal abnormalities, and vascular calcification. CKD-MBD impacts the oral cavity, with effects including salivary gland dysfunction, enamel hypoplasia and damage, increased dentin formation, decreased pulp volume, pulp calcifications, and altered jaw bones, contributing to clinical manifestations of periodontal disease and tooth loss. Underlying mechanisms are not fully understood, and CKD mouse models commonly require invasive procedures with high rates of infection and mortality. We aimed to characterize the dentoalveolar effects of an adenine diet (AD)-induced CKD (AD-CKD) mouse model. Eight-week-old C57BL/6J mice were provided either a normal phosphorus diet control (CTR) or adenine and high-phosphorus diet CKD to induce kidney failure. Mice were euthanized at 15 weeks old, and mandibles were collected for micro-computed tomography and histology. CKD mice exhibited kidney failure, hyperphosphatemia, and hyperparathyroidism in association with porous cortical bone in femurs. CKD mice showed a 30% decrease in molar enamel volume compared to CTR mice. Enamel wear was associated with reduced ductal components, ectopic calcifications, and altered osteopontin (OPN) deposition in submandibular salivary glands of CKD mice. Molar cusps in CKD mice were flattened, exposing dentin. Molar dentin/cementum volume increased 7% in CKD mice and pulp volume decreased. Histology revealed excessive reactionary dentin and altered pulp-dentin extracellular matrix proteins, including increased OPN. Mandibular bone volume fraction decreased 12% and bone mineral density decreased 9% in CKD versus CTR mice. Alveolar bone in CKD mice exhibited increased tissue-nonspecific alkaline phosphatase localization, OPN deposition, and greater osteoclast numbers. AD-CKD recapitulated key aspects reported in CKD patients and revealed new insights into CKD-associated oral defects. This model has potential for studying mechanisms of dentoalveolar defects or therapeutic interventions. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).


Assuntos
Distúrbio Mineral e Ósseo na Doença Renal Crônica , Hiperfosfatemia , Insuficiência Renal Crônica , Camundongos , Animais , Distúrbio Mineral e Ósseo na Doença Renal Crônica/complicações , Adenina , Microtomografia por Raio-X , Hiperfosfatemia/complicações , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/complicações , Fósforo
17.
J Bone Miner Res ; 38(5): 792-807, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36824055

RESUMO

Lipids play a crucial role in signaling and metabolism, regulating the development and maintenance of the skeleton. Membrane lipids have been hypothesized to act as intermediates upstream of orphan phosphatase 1 (PHOSPHO1), a major contributor to phosphate generation required for bone mineralization. Here, we spatially resolve the lipid atlas of the healthy mouse knee and demonstrate the effects of PHOSPHO1 ablation on the growth plate lipidome. Lipids spanning 17 subclasses were mapped across the knee joints of healthy juvenile and adult mice using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), with annotation supported by shotgun lipidomics. Multivariate analysis identified 96 and 80 lipid ions with differential abundances across joint tissues in juvenile and adult mice, respectively. In both ages, marrow was enriched in phospholipid platelet activating factors (PAFs) and related metabolites, cortical bone had a low lipid content, whereas lysophospholipids were strikingly enriched in the growth plate, an active site of mineralization and PHOSPHO1 activity. Spatially-resolved profiling of PHOSPHO1-knockout (KO) mice across the resting, proliferating, and hypertrophic growth plate zones revealed 272, 306, and 296 significantly upregulated, and 155, 220, and 190 significantly downregulated features, respectively, relative to wild-type (WT) controls. Of note, phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, lysophosphatidylethanolamine, and phosphatidylethanolamine derived lipid ions were upregulated in PHOSPHO1-KO versus WT. Our imaging pipeline has established a spatially-resolved lipid signature of joint tissues and has demonstrated that PHOSPHO1 ablation significantly alters the growth plate lipidome, highlighting an essential role of the PHOSPHO1-mediated membrane phospholipid metabolism in lipid and bone homeostasis. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).


Assuntos
Lipidômica , Monoéster Fosfórico Hidrolases , Camundongos , Animais , Monoéster Fosfórico Hidrolases/metabolismo , Lâmina de Crescimento/metabolismo , Camundongos Knockout , Homeostase , Fosfolipídeos
18.
Eur J Endocrinol ; 188(1)2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36762943

RESUMO

OBJECTIVE: Atypical femur fractures (AFFs) are rare fragility fractures originating at the lateral cortex of the femur, affecting the subtrochanteric or diaphyseal area of thebone with a transverse morphology. Occurrence of AFF is specifically associated with a small number of rare monogenic congenital metabolic bone disorders, such as hypophosphatasia, and with long-term treatment with antiresorptiondrugs. The exact pathogenesis of these fractures remains poorly understood and, except for cases of diagnosed HPP or other AFF-causing bone diseases, it is not possible to assess which patients are at higher riskof developing AFFs as a consequence of anti-resorption therapy. DESIGN: We genetically screened 25 unrelated patients who had developed at least one AFF. INTERVENTION: Genetic screening was performed through a nextgeneration sequencing analysis with a customized panel containing 76 human genes involved in the regulation of the mineralization processWe genetically screened 25 unrelated patients who had developed at least one AFF. RESULTS: We found a relatively high frequency (32.0%) of heterozygous rare variants inthe SLC34A1 and SLC9A3R1 genes, two genes whose heterozygous inactivating mutations have been respectively associated with autosomal dominant hypophosphatemic nephrolithiasis/osteoporosis types 1 and 2 (NPHLOP1and NPHLOP2). Other heterozygous rare variants were found in the BMPR1B, CYP27B1, FBN1, MEPE, PIGO, and PHOSPHO1 genes, each in a single AFF case (4.0%). CONCLUSIONS AND RELEVANCE: Our findings suggest that rarevariants of SLC34A1 and SLC9A3R1 could represent a possible genetic risk factor for the occurrence of AFFs. On the other hand, AFFs could represent an unsuspected clinical manifestation and/or an anti-resorption therapycorrelatedadverse event in patients with NPHLOP disorders.


Assuntos
Fraturas do Fêmur , Trocadores de Sódio-Hidrogênio , Humanos , Conservadores da Densidade Óssea/uso terapêutico , Difosfonatos/uso terapêutico , Fraturas do Fêmur/genética , Fêmur/patologia , Osteoporose/tratamento farmacológico , Radiografia , Fatores de Risco , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa , Trocadores de Sódio-Hidrogênio/metabolismo
19.
JBMR Plus ; 7(1): e10701, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36699640

RESUMO

Relevance of mineralized nodules in two-dimensional (2D) osteoblast/osteocyte cultures to bone biology, pathology, and engineering is a decades old question, but a comprehensive answer appears to be still wanting. Bone-like cells, extracellular matrix (ECM), and mineral were all reported but so were non-bone-like ones. Many studies described seemingly bone-like cell-ECM structures based on similarity to few select bone features in vivo, yet no studies examined multiple bone features simultaneously and none systematically studied all types of structures coexisting in the same culture. Here, we report such comprehensive analysis of 2D cultures based on light and electron microscopies, Raman microspectroscopy, gene expression, and in situ messenger RNA (mRNA) hybridization. We demonstrate that 2D cultures of primary cells from mouse calvaria do form bona fide bone. Cells, ECM, and mineral within it exhibit morphology, structure, ultrastructure, composition, spatial-temporal gene expression pattern, and growth consistent with intramembranous ossification. However, this bone is just one of at least five different types of cell-ECM structures coexisting in the same 2D culture, which vary widely in their resemblance to bone and ability to mineralize. We show that the other two mineralizing structures may represent abnormal (disrupted) bone and cartilage-like structure with chondrocyte-to-osteoblast transdifferentiation. The two nonmineralizing cell-ECM structures may mimic periosteal cambium and pathological, nonmineralizing osteoid. Importantly, the most commonly used culture conditions (10mM ß-glycerophosphate) induce artificial mineralization of all cell-ECM structures, which then become barely distinguishable. We therefore discuss conditions and approaches promoting formation of bona fide bone and simple means for distinguishing it from the other cell-ECM structures. Our findings may improve osteoblast differentiation and function analyses based on 2D cultures and extend applications of these cultures to general bone biology and tissue engineering research. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

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