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1.
Hum Mol Genet ; 32(17): 2681-2692, 2023 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-37364051

RESUMO

Orofacial clefts, including cleft lip and palate (CL/P) and neural tube defects (NTDs) are among the most common congenital anomalies, but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesized that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) causes not only spinal NTDs in Axial defects (Axd) mice but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate and abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4 kb LTR retrotransposon insertion that disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesize that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans.


Assuntos
Anormalidades Múltiplas , Fenda Labial , Fissura Palatina , Defeitos do Tubo Neural , Disrafismo Espinal , Animais , Humanos , Camundongos , Anormalidades Múltiplas/genética , Fenda Labial/genética , Fissura Palatina/genética , Encefalocele/genética , Mutação , Defeitos do Tubo Neural/genética , Disrafismo Espinal/genética
2.
J Anat ; 242(6): 1037-1050, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36772893

RESUMO

Worldwide research groups and funding bodies have highlighted the need for imaging biomarkers to predict osteoarthritis (OA) progression and treatment effectiveness. Changes in trabecular architecture, which can be detected with non-destructive high-resolution CT imaging, may reveal OA progression before apparent articular surface damage. Here, we analysed the tibial epiphyses of STR/Ort (OA-prone) and CBA (healthy, parental control) mice at different ages to characterise the effects of mouse age and strain on multiple bony parameters. We isolated epiphyseal components using a semi-automated method, and measured the total epiphyseal volume; cortical bone, trabecular bone and marrow space volumes; mean trabecular and cortical bone thicknesses; trabecular volume relative to cortical volume; trabecular volume relative to epiphyseal interior (trabecular BV/TV); and the trabecular degree of anisotropy. Using two-way ANOVA (significance level ≤0.05), we confirmed that all of these parameters change significantly with age, and that the two strains were significantly different in cortical and trabecular bone volumes, and trabecular degree of anisotropy. STR/Ort mice had higher cortical and trabecular volumes and a lower degree of anisotropy. As the two mouse strains reflect markedly divergent OA predispositions, these parameters have potential as bioimaging markers to monitor OA susceptibility and progression. Additionally, significant age/strain interaction effects were identified for total epiphyseal volume, marrow space volume and trabecular BV/TV. These interactions confirm that the two mouse strains have different epiphyseal growth patterns throughout life, some of which emerge prior to OA onset. Our findings not only propose valuable imaging biomarkers of OA, but also provide insight into ageing 3D epiphyseal architecture bone profiles and skeletal biology underlying the onset and development of age-related OA in STR/Ort mice.


Assuntos
Osteoartrite , Camundongos , Animais , Camundongos Endogâmicos CBA , Osteoartrite/diagnóstico por imagem , Tíbia/diagnóstico por imagem , Biomarcadores , Epífises/diagnóstico por imagem
3.
Cell Biochem Funct ; 41(2): 189-201, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36540015

RESUMO

The administration of intermittent parathyroid hormone (iPTH) is anabolic to the skeleton. Recent studies with cultured osteoblasts have revealed that the expression of PHOSPHO1, a bone-specific phosphatase essential for the initiation of mineralisation, is regulated by PTH. Therefore, this study sought to determine whether the bone anabolic response to iPTH involves modulation of expression of Phospho1 and of other enzymes critical for bone matrix mineralisation. To mimic iPTH treatment, primary murine osteoblasts were challenged with 50 nM PTH for 6 h in every 48 h period for 8 days (4 cycles), 14 days (7 cycles) and 20 days (10 cycles) in total. The expression of both Phospho1 and Smpd3 was almost completely inhibited after 4 cycles, whereas 10 cycles were required to stimulate a similar response in Alpl expression. To explore the in vivo role of PHOSPHO1 in PTH-mediated osteogenesis, the effects of 14- and 28-day iPTH (80 µg/kg/day) administration was assessed in male wild-type (WT) and Phospho1-/- mice. The expression of Phospho1, Alpl, Smpd3, Enpp1, Runx2 and Trps1 expression was enhanced in the femora of WT mice following iPTH administration but remained unchanged in the femora of Phospho1-/- mice. After 28 days of iPTH administration, the anabolic response in the femora of WT was greater than that noted in Phospho1-/- mice. Specifically, cortical and trabecular bone volume/total volume, as well as cortical thickness, were increased in femora of iPTH-treated WT but not in iPTH-treated Phospho1-/- mice. Trabecular bone osteoblast number was also increased in iPTH-treated WT mice but not in iPTH-treated Phospho1-/-  mice. The increased levels of Phospho1, Alpl, Enpp1 and Smpd3 in WT mice in response to iPTH administration is consistent with their contribution to the potent anabolic properties of iPTH in bone. Furthermore, as the anabolic response to iPTH was attenuated in mice deficient in PHOSPHO1, this suggests that the osteoanabolic effects of iPTH are at least partly mediated via bone mineralisation processes.


Assuntos
Fosfatase Alcalina , Hormônio Paratireóideo , Masculino , Camundongos , Animais , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/farmacologia , Osso e Ossos/metabolismo , Osteoblastos/metabolismo , Osteogênese , Densidade Óssea , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielina Fosfodiesterase/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo
4.
Development ; 146(14)2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358536

RESUMO

In human, mutations of the protocadherins FAT4 and DCHS1 result in Van Maldergem syndrome, which is characterised, in part, by craniofacial abnormalities. Here, we analyse the role of Dchs1-Fat4 signalling during osteoblast differentiation in mouse. We show that Fat4 and Dchs1 mutants mimic the craniofacial phenotype of the human syndrome and that Dchs1-Fat4 signalling is essential for osteoblast differentiation. In Dchs1/Fat4 mutants, proliferation of osteoprogenitors is increased and osteoblast differentiation is delayed. We show that loss of Dchs1-Fat4 signalling is linked to increased Yap-Tead activity and that Yap is expressed and required for proliferation in osteoprogenitors. In contrast, Taz is expressed in more-committed Runx2-expressing osteoblasts, Taz does not regulate osteoblast proliferation and Taz-Tead activity is unaffected in Dchs1/Fat4 mutants. Finally, we show that Yap and Taz differentially regulate the transcriptional activity of Runx2, and that the activity of Yap-Runx2 and Taz-Runx2 complexes is altered in Dchs1/Fat4 mutant osteoblasts. In conclusion, these data identify Dchs1-Fat4 as a signalling pathway in osteoblast differentiation, reveal its crucial role within the early Runx2 progenitors, and identify distinct requirements for Yap and Taz during osteoblast differentiation.


Assuntos
Caderinas/fisiologia , Osteoblastos/fisiologia , Osteogênese/genética , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Células Cultivadas , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Deformidades Congênitas do Pé/genética , Deformidades Congênitas do Pé/patologia , Deformidades Congênitas da Mão/genética , Deformidades Congênitas da Mão/patologia , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Instabilidade Articular/genética , Instabilidade Articular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Gravidez , Transdução de Sinais/genética
5.
J Anat ; 241(4): 875-895, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35866709

RESUMO

Articular calcified cartilage (ACC) has been dismissed, by some, as a remnant of endochondral ossification without functional relevance to joint articulation or weight-bearing. Recent research indicates that morphologic and metabolic ACC features may be important, reflecting knee joint osteoarthritis (OA) predisposition. ACC is less investigated than neighbouring joint tissues, with its component chondrocytes and mineralised matrix often being either ignored or integrated into analyses of hyaline articular cartilage and subchondral bone tissue respectively. Anatomical variation in ACC is recognised between species, individuals and age groups, but the selective pressures underlying this variation are unknown. Consequently, optimal ACC biomechanical features are also unknown as are any potential locomotory roles. This review collates descriptions of ACC anatomy and biology in health and disease, with a view to revealing its structure/function relationship and highlighting potential future research avenues. Mouse models of healthy and OA joint ageing have shown disparities in ACC load-induced deformations at the knee joint. This raises the hypothesis that ACC response to locomotor forces over time may influence, or even underlie, the bony and hyaline cartilage symptoms characteristic of OA. To effectively investigate the ACC, greater resolution of joint imaging and merging of hierarchical scale data will be required. An appreciation of OA as a 'whole joint disease' is expanding, as is the possibility that the ACC may be a key player in healthy ageing and in the transition to OA joint pathology.


Assuntos
Cartilagem Articular , Osteoartrite , Animais , Cartilagem Articular/patologia , Condrócitos/patologia , Cartilagem Hialina/patologia , Articulação do Joelho/patologia , Camundongos , Osteoartrite/patologia
6.
FASEB J ; 35(4): e21451, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33683776

RESUMO

Osteoarthritis (OA) etiopathogenesis is complex with strong environmental/lifestyle determinants that, in laboratory animals, extend to social context and stress levels. This study seeks to identify whether colony housing of rats exerts a social impact on locomotion behaviors to influence alignment between symptomatic (gait) and structural (bone micro-CT measures, cartilage morphometry, and histology) OA outcome measures. Rats were randomly allocated to conventional (type IV; n = 48) or rat colony cage (RCC; n = 30) housing, further randomized to OA surgical models (ACLT + tMx, MMT or DMM) or no surgery (control), and maintained for 19 weeks during which multiple gait recordings were made. Standard histological grading and bone micro-CT data were collected at necropsy. Principal component analysis was used to summarize the variation in gait, micro-CT or histology. Linear mixed effects model or two-way ANOVA was employed to evaluate the impact of the housing system, surgery and time on gait, or micro-CT and histology components Analyses reveal that RCC exaggerates trends in gait change via a combined effect of the housing system and surgery. Intriguingly, RCC-housed nonoperated control rats showed similar gait changes to rats subjected to surgery; the latter exhibited significant structural joint changes in both systems. Stronger correlation between histological and micro-CT bone changes were found in medial and lateral tibia joint compartments of rats housed in RCC system. This study has established that rat social housing exaggerates outcomes in traditional histological measures of OA, generates stronger links between histology and micro-CT bone changes and removes gait differences as a variable in their etiology.


Assuntos
Osso e Ossos/metabolismo , Marcha , Abrigo para Animais , Osteoartrite/patologia , Microtomografia por Raio-X , Animais , Biomarcadores/metabolismo , Masculino , Osteoartrite/etiologia , Ratos , Organismos Livres de Patógenos Específicos
7.
Cell Biochem Funct ; 40(7): 683-693, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35924674

RESUMO

Mouse strains can have divergent basal bone mass, yet this phenotype is seldom reflected in the design of studies seeking to identify new modulators of bone resorption by osteoclasts. Sulforaphane exerts inhibitory effects on in vitro osteoclastogenesis in cells from C57BL/6 mice. Here, we explore whether a divergent basal bone mass in different mouse strains is linked both to in vitro osteoclastogenic potential and to SFX-01 sensitivity. Accordingly, osteoclasts isolated from the bone marrow (BM) of C57BL/6, STR/Ort and CBA mice with low, high, and intermediate bone mass, respectively, were cultured under conditions to promote osteoclast differentiation and resorption; they were also treated with chemically stabilised sulforaphane (SFX-01) and respective sensitivity to inhibition evaluated by counting osteoclast number/resorption activity on dentine discs. We observed that osteoclastogenesis exhibited different macrophage colony-stimulating factor/receptor activator of nuclear factor kappa-Β ligand sensitivity in these mouse strains, with cells from C57BL/6 and CBA generating higher osteoclast numbers than STR/Ort; the latter formed only half as many mature osteoclasts. We found that 100 nM SFX-01 exerted a potent and significant reduction in osteoclast number and resorptive activity in cells derived from C57BL/6 mice. In contrast, 10-fold higher SFX-01 concentrations were required for similar inhibition in CBA-derived cells and, strikingly, a further 2.5-fold greater concentration was required for significant restriction of osteoclast formation/function in STR/Ort. These data are consistent with the notion that the BM osteoclast precursor population contributes to the relative differences in mouse bone mass and that mice with higher bone mass exhibit lower in vitro osteoclastogenic potential as well as reduced sensitivity to inhibition by SFX-01.


Assuntos
Reabsorção Óssea , Osteoclastos , Animais , Reabsorção Óssea/tratamento farmacológico , Diferenciação Celular , Células Cultivadas , Isotiocianatos , Ligantes , Fator Estimulador de Colônias de Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Ligante RANK/farmacologia , Sulfóxidos
8.
Int J Mol Sci ; 22(24)2021 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-34948015

RESUMO

Mechanical cues play a vital role in limb skeletal development, yet their influence and underpinning mechanisms in the regulation of endochondral ossification (EO) processes are incompletely defined. Furthermore, interactions between endochondral growth and mechanics and the mTOR/NF-ĸB pathways are yet to be explored. An appreciation of how mechanical cues regulate EO would also clearly be beneficial in the context of fracture healing and bone diseases, where these processes are recapitulated. The study herein addresses the hypothesis that the mTOR/NF-ĸB pathways interact with mechanics to control endochondral growth. To test this, murine embryonic metatarsals were incubated ex vivo in a hydrogel, allowing for the effects of quasi-static loading on longitudinal growth to be assessed. The results showed significant restriction of metatarsal growth under quasi-static loading during a 14-day period and concentration-dependent sensitivity to hydrogel-related restriction. This study also showed that hydrogel-treated metatarsals retain their viability and do not present with increased apoptosis. Metatarsals exhibited reversal of the growth-restriction when co-incubated with mTOR compounds, whilst it was found that these compounds showed no effects under basal culture conditions. Transcriptional changes linked to endochondral growth were assessed and downregulation of Col2 and Acan was observed in hydrogel-treated metatarsi at day 7. Furthermore, cell cycle analyses confirmed the presence of chondrocytes exhibiting S-G2/M arrest. These data indicate that quasi-static load provokes chondrocyte cell cycle arrest, which is partly overcome by mTOR, with a less marked interaction for NF-ĸB regulators.


Assuntos
Ossos do Metatarso/embriologia , Ossos do Metatarso/crescimento & desenvolvimento , NF-kappa B/metabolismo , Técnicas de Cultura de Órgãos/métodos , Agrecanas/genética , Animais , Fenômenos Biomecânicos , Colágeno Tipo II/genética , Meios de Cultura , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Hidrogéis , Ossos do Metatarso/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
9.
Int J Mol Sci ; 22(18)2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34575887

RESUMO

The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.


Assuntos
Antígeno CD146/metabolismo , Matriz Extracelular/metabolismo , Laminina/metabolismo , Traumatismos dos Tendões/metabolismo , Tendões/metabolismo , Tendão do Calcâneo/metabolismo , Tendão do Calcâneo/patologia , Animais , Antígeno CD146/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Imunofluorescência , Expressão Gênica , Ligantes , Ligação Proteica , Ratos , Traumatismos dos Tendões/etiologia , Traumatismos dos Tendões/patologia , Tendões/patologia
10.
Biol Proced Online ; 22: 13, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32624710

RESUMO

BACKGROUND: Three-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure utilising fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species. RESULTS: Using fluorescent immunolabelling and optical clearing, we visualised the expression of the novel cross-species marker of tendon basement membrane, laminin-α4 in 3D throughout whole rat Achilles tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex network of laminin-α4 within the tendon core that predominantly localises to the interfascicular matrix compartment. Furthermore, we implemented a chemical drying process capable of creating contrast densities enabling visualisation and quantification of both fascicular and interfascicular matrix volume and thickness by x-ray micro-computed tomography. We also demonstrated that both modalities can be combined using reverse clarification of fluorescently labelled tissues prior to chemical drying to enable bimodal imaging of a single sample. CONCLUSIONS: Whole-mount imaging of tendon allowed us to identify the presence of an extensive network of laminin-α4 within tendon, the complexity of which cannot be appreciated using traditional 2D imaging techniques. Creating contrast for x-ray micro-computed tomography imaging of tendon using chemical drying is not only simple and rapid, but also markedly improves on previously published methods. Combining these methods provides the ability to gain spatio-temporal information and quantify tendon substructures to elucidate the relationship between morphology and function.

11.
Am J Pathol ; 189(4): 753-761, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30664862

RESUMO

Glucocorticoid-induced secondary osteoporosis is the most predictable side effect of this anti-inflammatory. One of the main mechanisms by which glucocorticoids achieve such deleterious outcome in bone is by antagonizing Wnt/ß-catenin signaling. Sclerostin, encoded by Sost gene, is the main negative regulator of the proformative and antiresorptive role of the Wnt signaling pathway in the skeleton. It was hypothesized that the partial inactivation of sclerostin function by genetic manipulation will rescue the osteopenia induced by high endogenous glucocorticoid levels. Sost-deficient mice were crossed with an established mouse model of excess glucocorticoids, and the effects on bone mass and structure were evaluated. Sost haploinsufficiency did not rescue the low bone mass induced by high glucocorticoids. Intriguingly, the critical manifestation of Sost deficiency combined with glucocorticoid excess was sporadic, sudden, unprovoked, and nonconvulsive death. Detailed histopathologic analysis in a wide range of tissues identified peracute hemopericardium and cardiac tamponade to be the cause. These preclinical studies reveal outcomes with direct relevance to ongoing clinical trials that explore the use of antisclerostin antibodies as a treatment for osteoporosis. They particularly highlight a potential for increased cardiovascular risk and may inform improved stratification of patients who might otherwise benefit from antisclerostin antibody treatment.


Assuntos
Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/etiologia , Tamponamento Cardíaco/etiologia , Glucocorticoides/toxicidade , Haploinsuficiência , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/patologia , Tamponamento Cardíaco/metabolismo , Tamponamento Cardíaco/patologia , Modelos Animais de Doenças , Feminino , Marcadores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Via de Sinalização Wnt
12.
BMC Vet Res ; 15(1): 114, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30975149

RESUMO

BACKGROUND: Our understanding of the biology of osteoblasts is important as they underpin bone remodelling, fracture healing and processes such as osseointegration. Osteoblasts isolated from human humeral samples display distinctive biological activity in vitro, which relates to the samples' bone types (subchondral (S), trabecular (T), cortical (C)). Our aim was to isolate primary osteoblast cultures from different bone types from the proximal femur of a clinical population of dogs presented for total hip replacement and compare the behaviour of the osteoblasts derived from different bone types, to identify a preferred bone type for isolation. RESULTS: No differences were found for osteoblast doubling time (median for S = 2.9, T = 3.1 and C = 2.71 days, respectively; p = 0.33), final cell number (median for S = 54,849, T = 49,733, C = 61,390 cells/cm2; p = 0.34) or basal tissue non-specific alkaline phosphatase (TNAP) activity (median for S = 0.02, T = 0.02, C = 0.03 U/min/mg protein; p = 0.81) between bone types after 6 days of culture in basal media. There were no differences in mineralizing TNAP activity (S = 0.02, T = 0.02, C = 0.03 U/min/mg protein, p = 0.84) or in mineralized area (S = 0.05, T = 0.04, C = 0.04%, p = 0.92) among cells from different bone types. CONCLUSIONS: There is no significant difference in mean doubling time, basal or mineralizing TNAP activity or mineralized area in osteoblasts derived from subchondral, cortical, or trabecular bone types from the canine femoral head. However, there appears to be a high level of inter-animal variability in the studied parameters, which was independent of age, body mass, and sex. Trabecular isolate osteoblasts have the least variation of the bone types studied, and therefore should be considered a preferred source for primary osteoblast cultures. The work here provides baselines for canine osteoblast function, which has utility for future comparative studies.


Assuntos
Cães/anatomia & histologia , Fêmur/citologia , Osteoblastos/fisiologia , Animais , Calcificação Fisiológica , Osso Esponjoso/citologia , Osso Cortical/citologia , Cães/fisiologia , Feminino , Técnicas In Vitro , Masculino , Osteoblastos/citologia
13.
Curr Osteoporos Rep ; 17(6): 560-569, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31760583

RESUMO

PURPOSE OF REVIEW: Osteoporosis is an age-related disorder characterized by bone loss and increased fracture susceptibility. Whether this is due to reduced loading in less active elderly individuals or inherent modifications in bone cells is uncertain. We suppose that osteoporosis is nonetheless prima facie evidence for impaired mechanoadaptation; either capacity to accrue new bone declines, or the stimulus for such accrual is absent/can no longer be triggered in the aged. Herein, we provide only sufficient background to enable a focus on recent advances which seek to address such dilemmas. RECENT FINDINGS: Recent advances from innovative high-impact loading regimes emphasize the priming of mechanoadaptation in the aged, such that low-to-moderate intensity loading becomes beneficial. These new findings lead us to speculate that aged bone mechanoadaptation is not driven solely by strain magnitude but is instead sensitive to high strain gradients. Impaired mechanoadaptation is a feature of the aged skeleton. Recent advances indicate that novel interventional loading regimes can restore mechanoadaptive capacity, enabling new approaches for retaining bone health in the aged. Innovative exercise paradigms appear to be capable of "hacking" into the osteogenic signal produced by exercise such that low-to-moderate intensity activities may also become more beneficial. Deciphering the underpinning mechanism(s) will also enable new pharmacological intervention for retaining bone health in the aged.


Assuntos
Adaptação Fisiológica/fisiologia , Envelhecimento/fisiologia , Osso e Ossos/fisiologia , Osteoporose/fisiopatologia , Suporte de Carga/fisiologia , Animais , Fenômenos Biomecânicos , Humanos , Osteoporose/terapia , Treinamento Resistido
14.
BMC Musculoskelet Disord ; 20(1): 344, 2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31351471

RESUMO

BACKGROUND: Subchondral bone (SCB) thickening is one of the earliest detectable changes in osteoarthritic joints and is considered a potential trigger for subsequent articular cartilage degeneration. In this manuscript, we examine whether disruption to the SCB osteocyte network contributes to the initiation and pathogenesis of osteoarthritis. METHODS: We examined expression patterns of the glycoprotein E11/podoplanin by immunohistochemical labelling in murine, human and canine osteoarthritis models. We also examined the effects of twice-weekly administration of Bortezomib, a proteasome inhibitor which stabilises osteocyte E11 levels, to C57/BL6 wild-type male mice (1 mg/kg/day) for 8 weeks after surgical destabilisation of the medial meniscus. By inducing osteoarthritis-like changes in the right knee joint of 12-week-old male E11 hypomorphic mice (and corresponding controls) using a post-traumatic joint loading model, we also investigated whether a bone-specific E11 deletion in mice increases joint vulnerability to osteoarthritis. Articular cartilage degradation and osteophyte formation were assessed by histology and in line with the OARSI grading system. RESULTS: Our studies reveal increased E11 expression in osteocytes of human and canine osteoarthritic SCB. We found that Bortezomib administration had no effect on surgically-induced osteoarthritis, potentially due to a lack of the expected stabilisation of E11 in the SCB. We also found, in concordance with our previous work, wild-type mice exhibited significant load-induced articular cartilage lesions on the lateral femoral condyle (p < 0.01) and osteophyte formation. In contrast, E11 hypomorphic mice did not develop osteophytes or any corresponding articular lesions. CONCLUSIONS: Overall, these data suggest that an intact osteocyte network in the SCB contributes to the development of mechanically-driven osteoarthritis. Further, the data presented here indicate that the molecular pathways that preserve the osteocyte network, such as those driven by E11, may be targeted to limit osteoarthritis pathogenesis.


Assuntos
Cartilagem Articular/patologia , Glicoproteínas de Membrana/metabolismo , Osteoartrite/patologia , Osteófito/patologia , Animais , Bortezomib/administração & dosagem , Modelos Animais de Doenças , Cães , Humanos , Masculino , Glicoproteínas de Membrana/genética , Meniscos Tibiais/patologia , Camundongos , Camundongos Knockout , Osteoartrite/tratamento farmacológico , Osteoartrite/etiologia , Osteócitos/efeitos dos fármacos , Osteócitos/patologia , Osteófito/tratamento farmacológico , Suporte de Carga
15.
J Cell Physiol ; 233(7): 5334-5347, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29215722

RESUMO

E11/podoplanin is critical in the early stages of osteoblast-to-osteocyte transitions (osteocytogenesis), however, the upstream events which regulate E11 expression are unknown. The aim of this study was to examine the effects of FGF-2 on E11-mediated osteocytogenesis and to reveal the nature of the underlying signaling pathways regulating this process. Exposure of MC3T3 osteoblast-like cells and murine primary osteoblasts to FGF-2 (10 ng/ml) increased E11 mRNA and protein expression (p < 0.05) after 4, 6, and 24 hr. FGF-2 induced changes in E11 expression were also accompanied by significant (p < 0.01) increases in Phex and Dmp1 (osteocyte markers) expression and decreases in Col1a1, Postn, Bglap, and Alpl (osteoblast markers) expression. Immunofluorescent microscopy revealed that FGF-2 stimulated E11 expression, facilitated the translocation of E11 toward the cell membrane, and subsequently promoted the formation of osteocyte-like dendrites in MC3T3 and primary osteoblasts. siRNA knock down of E11 expression achieved >70% reduction of basal E11 mRNA expression (p < 0.05) and effectively abrogated FGF-2-related changes in E11 expression and dendrite formation. FGF-2 strongly activated the ERK signaling pathway in osteoblast-like cells but inhibition of this pathway did not block the ability of FGF-2 to enhance E11 expression or to promote acquisition of the osteocyte phenotype. The results of this study highlight a novel mechanism by which FGF-2 can regulate osteoblast differentiation and osteocyte formation. Specifically, the data suggests that FGF-2 promotes osteocytogenesis through increased E11 expression and further studies will identify if this regulatory pathway is essential for bone development and maintenance in health and disease.


Assuntos
Diferenciação Celular/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glicoproteínas de Membrana/genética , Osteogênese/efeitos dos fármacos , Células 3T3 , Animais , Fator 2 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteogênese/genética
16.
J Cell Physiol ; 232(11): 3006-3019, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28488815

RESUMO

The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone-specific Pdpn knockdown on osteocyte form and function in the post-natal mouse. Extensive skeletal phenotyping of male and female 6-week-old Oc-cre;Pdpnflox/flox (cKO) mice and their Pdpnflox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (p < 0.05). Consistent with this, we observed an increase in stiffness in female cKO mice in comparison to fl/fl mice (p < 0.01). Moreover, analysis of the osteocyte phenotype by phalloidin staining revealed a significant decrease in the dendrite volume (p < 0.001) and length (p < 0.001) in cKO mice in which deletion of Pdpn also modifies the bone anabolic loading response (p < 0.05) in comparison to age-matched fl/fl mice. Together, these data confirm a regulatory role for Pdpn in osteocyte dendrite formation and as such, in the control of osteocyte function. As the osteocyte dendritic network is known to play vital roles in regulating bone modeling/remodeling, this highlights an essential role for Pdpn in bone homeostasis.


Assuntos
Diferenciação Celular , Forma Celular , Deleção de Genes , Glicoproteínas de Membrana/metabolismo , Osteócitos/metabolismo , Osteogênese , Tíbia/metabolismo , Animais , Feminino , Genótipo , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos Knockout , Osteócitos/patologia , Fenótipo , Transdução de Sinais , Tíbia/diagnóstico por imagem , Tíbia/patologia , Microtomografia por Raio-X
17.
J Anat ; 231(2): 298-308, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28737011

RESUMO

The phosphatase PHOSPHO1 is involved in the initiation of biomineralisation. Bones in Phospho1 knockout (KO) mice show histological osteomalacia with frequent bowing of long bones and spontaneous fractures: they contain less mineral, with smaller mineral crystals. However, the consequences of Phospho1 ablation on the microscale structure of bone are not yet fully elucidated. Tibias and femurs obtained from wild-type and Phospho1 null (KO) mice (25-32 weeks old) were embedded in PMMA, cut and polished to produce near longitudinal sections. Block surfaces were studied using 20 kV backscattered-electron (BSE) imaging, and again after iodine staining to reveal non-mineralised matrix and cellular components. For 3D characterisation, we used X-ray micro-tomography. Bones opened with carbide milling tools to expose endosteal surfaces were macerated using an alkaline bacterial pronase enzyme detergent, 5% hydrogen peroxide and 7% sodium hypochlorite solutions to produce 3D surfaces for study with 3D BSE scanning electron microscopy (SEM). Extensive regions of both compact cortical and trabecular bone matrix in Phospho1 KO mice contained no significant mineral and/or showed arrested mineralisation fronts, characterised by a failure in the fusion of the calcospherite-like, separately mineralising, individual micro-volumes within bone. Osteoclastic resorption of the uncalcified matrix in Phospho1 KO mice was attenuated compared with surrounding normally mineralised bone. The extent and position of this aberrant biomineralisation varied considerably between animals, contralateral limbs and anatomical sites. The most frequent manifestation lay, however, in the nearly complete failure of mineralisation in the bone surrounding the numerous transverse blood vessel canals in the cortices. In conclusion, SEM disclosed defective mineralising fronts and extensive patchy osteomalacia, which has previously not been recognised. These data further confirm the role of this phosphatase in physiological skeletal mineralisation.


Assuntos
Osso e Ossos/patologia , Osso e Ossos/ultraestrutura , Osteomalacia/patologia , Monoéster Fosfórico Hidrolases/deficiência , Animais , Calcificação Fisiológica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
18.
Calcif Tissue Int ; 100(6): 631-640, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28236102

RESUMO

Tissue inhibitor of metalloproteinases-3 (TIMP-3) maintains a healthy extracellular matrix by regulating matrix metalloproteinases (MMP), disintegrin-metalloproteinases (ADAM), and disintegrin-metalloproteinases with ThromboSpondin-like motifs (ADAMTS) activity. Currently, there is a need for a comprehensive understanding of the effects of TIMP-3 on the bone quality and integrity. In this study, we examined the mechanical, morphological, and compositional properties of TIMP-3 knock out (Timp-3 -/-) mouse bone. We hypothesize that the lack of TIMP-3 plays an important role in maintaining the overall bone integrity. Mechanical properties of humeri, lumbar vertebrae, and femurs from Timp-3 -/- mice were determined using 3-point bending, compression, and notched 3-point bending, respectively. Morphological properties of the humeral cortical and trabecular bone and the caudal vertebrae cortical bone were evaluated using micro-computed tomography, while the composition of the femoral cortical and trabecular bone was examined using Fourier transform infrared spectroscopic imaging. Our results revealed that the integrity of the Timp-3 -/- bone is compromised due to changes in its composition, structure, and mechanics. Reductions in the yield and ultimate load and stress capacity, and loss in bone fracture toughness were attributed to reduced density and thickness, and increased porosity of cortical bone. Thin trabeculae were dense, highly connected, and closely packed in Timp-3 -/- bone. Furthermore, altered cortical and trabecular bone mineralization and increased compositional heterogeneity were found in Timp-3 -/- bone, all being indicative of high bone remodeling. In conclusion, this study suggests that the lack of TIMP-3 is detrimental to bone development and maintenance.


Assuntos
Densidade Óssea/fisiologia , Osso e Ossos/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Animais , Feminino , Fraturas Ósseas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidor Tecidual de Metaloproteinase-3/deficiência
19.
Cell Biochem Funct ; 35(1): 56-65, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28083967

RESUMO

Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load-bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18ß-glycyrrhetinic acid, we also demonstrated that this osteocyte-related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial-derived primary osteoblasts. We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain-related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity.


Assuntos
Junções Comunicantes/metabolismo , Osteoblastos/citologia , Osteócitos/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Galinhas , Técnicas de Cocultura , Junções Comunicantes/efeitos dos fármacos , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacologia , Osteoblastos/metabolismo , Osteócitos/metabolismo , Fenótipo , Crânio/citologia , Tíbia/citologia
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