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Bone histomorphometry is a well-established approach to assessing skeletal pathology, providing a standard evaluation of the cellular components, architecture, mineralization, and growth of bone tissue. However, it depends in part on the subjective interpretation of cellular morphology by an expert, which introduces bias. In addition, diseases like osteogenesis imperfecta (OI) and fibrous dysplasia are accompanied by changes in the morphology and function of skeletal tissue and cells, hindering consistent evaluation of some morphometric parameters and interpretation of the results. For instance, traditional histomorphometry combined with collagen turnover markers suggested that reduced bone formation in classical OI is accompanied by increased bone resorption. In contrast, the well-documented postpubertal reduction in fractures would be easier to explain by reduced bone resorption after puberty, highlighting the need for less ambiguous measurements. Here we propose an approach to histomorphometry based on in situ mRNA hybridization, which uses Col1a1 as osteoblast and Ctsk as osteoclast markers. This approach can be fully automated and eliminates subjective identification of bone surface cells. We validate these markers based on the expression of Bglap, Ibsp, and Acp5. Comparison with traditional histological and tartrate-resistant acid phosphatase staining of the same sections suggests that mRNA-based analysis is more reliable. Unlike inconclusive traditional histomorphometry of mice with α2(I)-Gly610 to Cys substitution in the collagen triple helix, mRNA-based measurements reveal reduced osteoclastogenesis in 11-wk-old animals consistent with the postpubertal catch-up osteogenesis observed by microCT. We optimize the technique for cryosections of mineralized bone and sections of paraffin-embedded decalcified tissue, simplifying and broadening its applications. We illustrate the application of the mRNA-based approach to human samples using the example of a McCune-Albright syndrome patient. By eliminating confounding effects of altered cellular morphology and the need for subjective morphological evaluation, this approach may provide a more reproducible and accessible evaluation of bone pathology.
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Huesos , Colágeno Tipo I , Modelos Animales de Enfermedad , Osteogénesis Imperfecta , Osteogénesis Imperfecta/patología , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/genética , Animales , Ratones , Huesos/patología , Huesos/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , ARN Mensajero/metabolismo , ARN Mensajero/genética , Osteoclastos/metabolismo , Osteoclastos/patología , Pubertad , Osteoblastos/metabolismo , Osteoblastos/patología , Biomarcadores/metabolismo , OsteogénesisRESUMEN
Fibrous dysplasia (FD) is a rare, disabling skeletal disease for which there are no established treatments. Growing evidence supports inhibiting the osteoclastogenic factor receptor activator of nuclear kappa-B ligand (RANKL) as a potential treatment strategy. In this study, we investigated the mechanisms underlying RANKL inhibition in FD tissue and its likely indirect effects on osteoprogenitors by evaluating human FD tissue pre- and post-treatment in a phase 2 clinical trial of denosumab (NCT03571191) and in murine in vivo and ex vivo preclinical models. Histological analysis of human and mouse tissue demonstrated increased osteogenic maturation, reduced cellularity, and reduced expression of the pathogenic Gαs variant in FD lesions after RANKL inhibition. RNA sequencing of human and mouse tissue supported these findings. The interaction between osteoclasts and mutant osteoprogenitors was further assessed in an ex vivo lesion model, which indicated that the proliferation of abnormal FD osteoprogenitors was dependent on osteoclasts. The results from this study demonstrated that, in addition to its expected antiosteoclastic effect, denosumab reduces FD lesion activity by decreasing FD cell proliferation and increasing osteogenic maturation, leading to increased bone formation within lesions. These findings highlight the unappreciated role of cellular crosstalk between osteoclasts and preosteoblasts/osteoblasts as a driver of FD pathology and demonstrate a novel mechanism of action of denosumab in the treatment of bone disease.TRIAL REGISTRATION: ClinicalTrials.gov NCT03571191.
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Denosumab , Displasia Fibrosa Ósea , Animales , Humanos , Ratones , Denosumab/farmacología , Displasia Fibrosa Ósea/tratamiento farmacológico , Ligandos , Osteoblastos/metabolismo , Osteogénesis/genéticaRESUMEN
Elucidating a basic blueprint of osteoclast-osteoblast coordination in skeletal remodeling and understanding how this coordination breaks down with age and disease is essential for addressing the growing skeletal health problem in our aging population. The paucity of simple, activatable, biologically relevant models of osteoclast-osteoblast coordination has hindered our understanding of how skeletal remolding is regulated. Here, we describe an inducible ex vivo model of osteoclast-osteoblast progenitor coordination. Induction activates the release of osteoclastogenic factors from osteoprogenitors, which elicits the differentiation and fusion of neighboring preosteoclasts. In turn, multinucleated osteoclasts release soluble coupling factors, RANK+ extracellular vesicles and promote osteoprogenitor proliferation, recapitulating aspects of perturbed coordination in diseases underpinned by excessive osteoclast formation. We expect this model to expedite the investigation of cell-cell fusion, osteoclast-osteoblast progenitor coordination, and extracellular vesicle signaling during bone remodeling and offer a powerful tool for evaluating signaling cascades and novel therapeutic interventions in osteoclast-linked skeletal disease.
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Cutaneous skeletal hypophosphatemia syndrome (CSHS) is a mosaic RASopathy characterized by the association of dysplastic skeletal lesions, congenital skin nevi of epidermal and/or melanocytic origin, and FGF23-mediated hypophosphatemia. The primary physiological source of circulating FGF23 is bone cells. However, several reports have suggested skin lesions as the source of excess FGF23 in CSHS. Consequently, without convincing evidence of efficacy, many patients with CSHS have undergone painful removal of cutaneous lesions in an effort to normalize blood phosphate levels. This study aims to elucidate whether the source of FGF23 excess in CSHS is RAS mutation-bearing bone or skin lesions. Toward this end, we analyzed the expression and activity of Fgf23 in two mouse models expressing similar HRAS/Hras activating mutations in a mosaic-like fashion in either bone or epidermal tissue. We found that HRAS hyperactivity in bone, not skin, caused excess of bioactive intact FGF23, hypophosphatemia, and osteomalacia. Our findings support RAS-mutated dysplastic bone as the primary source of physiologically active FGF23 excess in patients with CSHS. This evidence informs the care of patients with CSHS, arguing against the practice of nevi removal to decrease circulating, physiologically active FGF23.
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Hipofosfatemia , Nevo , Neoplasias Cutáneas , Animales , Ratones , Modelos Animales de Enfermedad , Factores de Crecimiento de Fibroblastos/genética , Hipofosfatemia/genética , Hipofosfatemia/patología , Nevo/genética , Neoplasias Cutáneas/patología , SíndromeRESUMEN
Fibrous dysplasia/McCune-Albright syndrome (FD/MAS) is a rare mosaic bone and endocrine disorder. Although most variants affect the GNAS R201 codon, obtaining a genetic diagnosis is difficult because not all cells harbor the variant, and an invasive biopsy may be required. We explored the presence of GNAS p.R201 variants in blood circulating cell free DNA (ccfDNA) using sensitive techniques of digital droplet polymerase chain reaction (PCR) (ddPCR) and competitive allele-specific TaqMan PCR (castPCR) in an effort to improve the genetic diagnosis of FD/MAS. We isolated ccfDNA from the plasma of 66 patients with a wide range of disease severity and performed both ddPCR and castPCR mutation analysis to search for GNAS p.R201H or R201C variants. We detected R201 variants in ccfDNA samples of 41 of 66 (62.1%) patients by either castPCR or ddPCR, and 45 of 66 (68.2%) of patients if the techniques were combined. Variant detection was more likely in patients with more severe disease. Skeletal disease burden score (SBS) was significantly higher in patients who had detectable variants, and SBS was a predictor of variant allele frequency. By ddPCR analysis, patients aged ≤30 years had higher detection rates, and higher variant allele frequencies, independent of disease burden. We detected variant DNA in only one patient with monostotic FD by ddPCR only. In summary, we have demonstrated that ccfDNA containing variant GNAS can be isolated from the plasma of patients with FD/MAS and that ddPCR and castPCR methods have similar variant detection rates. This methodology represents an important potential advancement in diagnosis for patients with FD/MAS, especially those younger than 30 years or with more severe disease. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.
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Ácidos Nucleicos Libres de Células , Displasia Fibrosa Ósea , Displasia Fibrosa Poliostótica , Humanos , Displasia Fibrosa Poliostótica/genética , Mutación , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Cromograninas/genética , Displasia Fibrosa Ósea/genética , Ácidos Nucleicos Libres de Células/genéticaRESUMEN
Fibroblast growth factor-23 (FGF23) measurement is a critical tool in the evaluation of patients with disordered phosphate homeostasis. Available laboratory reference ranges for blood FGF23 were developed using samples from normophosphatemic individuals. Reliance on such values can lead to misdiagnosis in patients with FGF23-mediated hypophosphatemia, such as X-linked hypophosphatemia (XLH) and tumor-induced osteomalacia (TIO), in whom pathology-driving FGF23 levels can be in the "normal range." To determine FGF23 levels that are diagnostic for the identification of patients with FGF23-mediated hypophosphatemic disorders, we studied 149 patients with various disorders of FGF23-mediated and FGF23-independent hypophosphatemia and defined cut-off levels for both intact FGF23 (iFGF23) and C-terminal FGF23 (cFGF23) that can accurately distinguish between FGF23-mediated and FGF23-independent hypophosphatemia. In addition, to demonstrate the relationship between FGF23 and phosphate across the spectrum of human physiology, we assessed blood levels of FGF23 and phosphate in 434 patients with various forms of hypophosphatemia, hyperphosphatemia, and normophosphatemia. An intact FGF23 cut point of 27 pg/mL was 100% sensitive and specific in distinguishing FGF23-mediated from FGF23-independent hypophosphatemia, and a cFGF23 cut point of 90 RU/mL was 100% sensitive and specific in distinguishing specifically TIO from FGF23-independent hypophosphatemia. There was overlap in the cFGF23 range of 45-90 RU/mL between genetic forms of FGF23 excess and FGF23-independent hypophosphatemia, substantiating the superiority of iFGF23 over cFGF23 in making the diagnosis of FGF23-mediated hypophosphatemia. In this cohort, using the laboratory upper limit of normal for cFGF23 (180 RU/mL) would result in a misdiagnosis in more than half of patients with FGF23-mediated hypophosphatemia. In this, the largest study of FGF23 in chronic hypophosphatemia to date, we established iFGF23 and cFGF23 cut-off values to assist in the evaluation and diagnosis of hypophosphatemic conditions. © 2022 American Society for Bone and Mineral Research (ASBMR). This article has been contributed to by US Government employees and their work is in the public domain in the USA.
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Raquitismo Hipofosfatémico Familiar , Factores de Crecimiento de Fibroblastos , Hipofosfatemia , Osteomalacia , Humanos , Raquitismo Hipofosfatémico Familiar/diagnóstico , Factores de Crecimiento de Fibroblastos/sangre , Hipofosfatemia/diagnóstico , Osteomalacia/diagnóstico , FosfatosRESUMEN
BACKGROUND: Fibrous dysplasia (FD) is an uncommon bone disease characterized by the replacement of normal bone architecture with abnormal fibro-osseous connective tissue. Here, we discuss 2 cases of craniofacial FD, with malignant sarcomatous degeneration - a rare and morbid complication of the disease. CASE HISTORY: Two cases of craniofacial FD with malignant degeneration are presented. In the first, a 68-year-old male with a history of FD presented with acutely worsening left-sided facial pain and V2 and V3 hypoesthesia. Imaging findings suggested a large infratemporal fossa mass with biopsy demonstrating sarcomatous degeneration. Radical craniofacial resection achieved a gross total resection with likely microscopic disease. The patient was unable to tolerate adjuvant chemotherapy or radiation and succumbed to his disease 13 months following surgery.In the second case, a 36-year-old male with McCune-Albright Syndrome and craniofacial FD presented with acutely worsening left-sided headaches and midface hypoesthesia. Imaging revealed a heterogenous and expansile lesion with erosive changes in the left nasal cavity and infratemporal fossa. Pathology was suggestive of low grade sarcomatous degeneration. Given the extensive involvement of the skull base, the tumor was deemed unresectable, and the patient soon died following initiation of chemotherapy. CLINICAL RELEVANCE: Malignant sarcomatous transformation is a rare and challenging complication of craniofacial FD. Indolent onset, advanced spread at time of presentation, and close relationship with vital neurovascular structures are all hurdles for the treating clinician. The entity poses a diagnostic dilemma, as pathological analysis can be equivocal and may mimic nonmalignant processes, such as locally aggressive FD.
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Displasia Fibrosa Craneofacial , Displasia Fibrosa Ósea , Displasia Fibrosa Poliostótica , Sarcoma , Adulto , Anciano , Displasia Fibrosa Craneofacial/complicaciones , Displasia Fibrosa Ósea/complicaciones , Displasia Fibrosa Poliostótica/diagnóstico , Humanos , Hipoestesia , Masculino , Sarcoma/complicacionesRESUMEN
Micro-computed tomography (µCT) has become essential for analysis of mineralized as well as nonmineralized tissues and is therefore widely applicable in the life sciences. However, lack of standardized approaches and protocols for scanning, analyzing, and reporting data often makes it difficult to understand exactly how analyses were performed, how to interpret results, and if findings can be broadly compared with other models and studies. This problem is compounded in analysis of the dentoalveolar complex by the presence of four distinct mineralized tissues: enamel, dentin, cementum, and alveolar bone. Furthermore, these hard tissues interface with adjacent soft tissues, the dental pulp and periodontal ligament (PDL), making for a complex organ. Drawing on others' and our own experience analyzing rodent dentoalveolar tissues by µCT, we introduce techniques to successfully analyze dentoalveolar tissues with similar or disparate compositions, densities, and morphological characteristics. Our goal is to provide practical guidelines for µCT analysis of rodent dentoalveolar tissues, including approaches to optimize scan parameters (filters, voltage, voxel size, and integration time), reproducibly orient samples, define regions and volumes of interest, segment and subdivide tissues, interpret findings, and report methods and results. We include illustrative examples of analyses performed on genetically engineered mouse models with phenotypes in enamel, dentin, cementum, and alveolar bone. The recommendations are designed to increase transparency and reproducibility, promote best practices, and provide a basic framework to apply µCT analysis to the dentoalveolar complex that can also be extrapolated to a variety of other tissues of the body. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.
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CONTEXT: Fibrous dysplasia/McCune-Albright syndrome (FD/MAS) is a rare bone and endocrine disorder resulting in fractures, pain, and disability. There are no targeted or effective therapies to alter the disease course. Disease arises from somatic gain-of-function variants at the R201 codon in GNAS, replacing arginine by either cysteine or histidine. The relative pathogenicity of these variants is not fully understood. OBJECTIVE: This work aimed 1) to determine whether the most common GNAS variants (R201C and R201H) are associated with a specific clinical phenotype, and 2) to determine the prevalence of the most common GNAS variants in a large patient cohort. METHODS: This retrospective cross-sectional analysis measured the correlation between genotype and phenotype characterized by clinical, biochemical, and radiographic data. RESULTS: Sixty-one individuals were genotyped using DNA extracted from tissue or circulating cell-free DNA. Twenty-two patients (36.1%) had the R201C variant, and 39 (63.9%) had the R201H variant. FD skeletal disease burden, hypophosphatemia prevalence, fracture incidence, and ambulation status were similar between the 2 groups. There was no difference in the prevalence of endocrinopathies, ultrasonographic gonadal or thyroid abnormalities, or pancreatic involvement. There was a nonsignificant association of cancer with the R201H variant. CONCLUSION: There is no clear genotype-phenotype correlation in patients with the most common FD/MAS pathogenic variants. The predominance of the R201H variant observed in our cohort and reported in the literature indicates it is likely responsible for a larger burden of disease in the overall population of patients with FD/MAS, which may have important implications for the future development of targeted therapies.
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Cromograninas/genética , Displasia Fibrosa Ósea/genética , Displasia Fibrosa Poliostótica/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Adolescente , Adulto , Sustitución de Aminoácidos , Niño , Preescolar , Estudios Transversales , Femenino , Displasia Fibrosa Ósea/epidemiología , Displasia Fibrosa Ósea/patología , Displasia Fibrosa Poliostótica/epidemiología , Displasia Fibrosa Poliostótica/patología , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Mutación Missense , Prevalencia , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Fibrous dysplasia (FD) is a benign bone disease characterized by expansile lesions that typically stabilize with age. Rarely, FD can undergo malignant transformation, presenting with atypical, rapid growth and destruction of adjacent bone. Other potential causes of rapid FD expansion include secondary lesions, such as aneurysmal bone cysts. We describe a case of an aggressive occipital lesion that presented with pain associated with diplopia and tinnitus, raising concern for malignant transformation. A massive intraosseous arteriovenous fistula was identified giving rise to an anomalous vein coursing to the cavernous sinus with compression of the abducens nerve. The vascular anomaly was mapped and after embolization symptoms resolved; a biopsy with extensive genetic analyses excluded malignancy. The differential diagnosis for expanding FD lesions includes aggressive FD, malignant transformation, and secondary vascular anomalies. In cases when traditional radiographic and histologic assessments are nondescript, use of additional radiographic modalities and genetic analyses are required to make an accurate diagnosis and guide treatment. When vascular anomalies are suspected, detailed angiography with embolization is necessary to define and treat the lesion. However, to rule out malignant transformation, genetic screening is recommended.
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Fístula Arteriovenosa , Quistes Óseos Aneurismáticos , Displasia Fibrosa Ósea , Fístula Arteriovenosa/terapia , Quistes Óseos Aneurismáticos/complicaciones , Displasia Fibrosa Ósea/complicaciones , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Rayos XRESUMEN
Fibrous dysplasia/McCune-Albright Syndrome (FD/MAS), arising from gain-of-function mutations in Gαs, and cutaneous skeletal hypophosphatemia syndrome (CSHS), arising from gain-of-function mutations in the Ras/MAPK pathway, are strikingly complex, mosaic diseases with overlapping phenotypes. Both disorders are defined by mosaic skin and bone involvement, and both are complicated by increased FGF23 production. These similarities have frequently led to mis-diagnoses, primarily in patients with CSHS who are often assumed to have FD/MAS. The intriguing similarities in skeletal involvement in these genetically distinct disorders have led to novel insights into FGF23 physiology, making an understanding of FD/MAS and CSHS relevant to both clinicians and researchers interested in bone and endocrine disorders. This review will give an overview of FD/MAS and CSHS, focusing on the roles of mosaicism and FGF23 in the pathogenesis and clinical presentation of these disorders.
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Factores de Crecimiento de Fibroblastos/sangre , Displasia Fibrosa Poliostótica/diagnóstico , Hipofosfatemia/diagnóstico , Factor-23 de Crecimiento de Fibroblastos , Displasia Fibrosa Poliostótica/sangre , Displasia Fibrosa Poliostótica/genética , Humanos , Hipofosfatemia/sangre , Hipofosfatemia/genética , Mosaicismo , Mutación , Transducción de Señal/genéticaRESUMEN
Osteoarthritic and other types of articular cartilage defects never heal on their own. Medicinal and surgical approaches are often ineffective, and the supply of autologous chondrocytes for tissue engineering is very limited. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested as an adequate cell source for cartilage reconstruction. However, the majority of studies employing BMSCs for cartilage tissue engineering have used BMSCs predifferentiated into cartilage prior to implantation. This strategy has failed to achieve formation of stable, hyaline-like cartilage, resistant to hypertrophy in vivo. We hypothesized that in vitro predifferentiation of BMSCs is not necessary when cells are combined with an adequate scaffold that supports the formation of stable cartilage in vivo. In this study, naïve (undifferentiated) human BMSCs were attached to dehydrothermally crosslinked stable fibrin microbeads (FMBs) without and with other scaffolds and implanted subcutaneously into immunocompromised mice. Optimal formation of abundant, hypertrophy-resistant, ectopic hyaline-like cartilage was achieved when BMSCs were attached to FMBs covalently coated with hyaluronic acid. The cartilage that was formed was of human origin and was stable for at least 28 weeks in vivo. Stem Cells Translational Medicine 2019;8:586-592.
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Fibrina/química , Cartílago Hialino/citología , Microesferas , Andamios del Tejido/química , Animales , Diferenciación Celular , Condrogénesis , Humanos , Cartílago Hialino/metabolismo , Ácido Hialurónico/química , Huésped Inmunocomprometido , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ingeniería de Tejidos , Trasplante HeterólogoRESUMEN
Fibrous dysplasia of bone (FD) is a mosaic disease caused by mutations in GNAS. Constitutive activation of the α-subunit of the Gs stimulatory protein (Gαs) leads to dysregulated proliferation of bone marrow stromal cells (BMSCs), generating expansile lesions of fibrotic tissue and abnormal bone. Local bone remodeling regulation by BMSCs is also altered, and FD tissue is characterized by abundant osteoclast-like cells that may be essential for lesion expansion. Animal models show local expression of RANKL in bone lesions, and treatment with the RANKL neutralizing antibody denosumab decreased lesion expansion rate in a patient with aggressive FD. However, the role of RANKL/osteoprotegerin (OPG) in FD pathophysiology is not yet understood. We measured serum levels of RANKL, OPG, and inactive RANKL-OPG complexes in FD patients of known disease burden and in healthy volunteers (HVs). RANK, RANKL, and Ki67 immunohistochemistry were assessed in FD tissue. Cultured FD and HV BMSCs were stimulated with prostaglandin E2 (PGE2 ) and 1,25 vitamin D3 to increase RANKL expression, and media levels of RANKL and OPG were measured. Osteoclastogenic induction by FD or HV BMSCs was assessed in co-cultures with HV peripheral monocytes. FD patients showed a 16-fold increase in serum RANKL compared to HVs. OPG was moderately increased (24%), although RANKL/OPG ratio was 12-fold higher in FD patients than in HVs. These measurements were positively correlated with the skeletal burden score (SBS), a validated marker of overall FD burden. No differences in serum inactive RANKL-OPG complexes were observed. In FD tissue, RANKL+ and Ki67+ fibroblastic cells were observed near RANK+ osteoclasts. High levels of RANKL were released by FD BMSCs cultures, but were undetectable in HV cultures. FD BMSC released less OPG than HV BMSCs. FD, but not HV BMSCs, induced osteoclastogenesis in monocyte co-cultures, which was prevented by denosumab addition. These data are consistent with the role of RANKL as a driver in FD-induced osteoclastogenesis. © 2018 American Society for Bone and Mineral Research.
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Células de la Médula Ósea/metabolismo , Displasia Fibrosa Ósea/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteoprotegerina/metabolismo , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Transducción de Señal , Células de la Médula Ósea/patología , Células Cultivadas , Femenino , Displasia Fibrosa Ósea/patología , Humanos , Masculino , Células Madre Mesenquimatosas/patologíaRESUMEN
Ectopic bone formation in mice is the gold standard for evaluation of osteogenic constructs. By regular procedures, usually only 4 constructs can be accommodated per mouse, limiting screening power. Combinatorial cassettes (combi-cassettes) hold up to 19 small, uniform constructs from the time of surgery, through time in vivo, and subsequent evaluation. Two types of bone tissue engineering constructs were tested in the combi-cassettes: i) a cell-scaffold construct containing primary human bone marrow stromal cells with hydroxyapatite/tricalcium phosphate particles (hBMSCs + HA/TCP) and ii) a growth factor-scaffold construct containing bone morphogenetic protein 2 in a gelatin sponge (BMP2+GS). Measurements of bone formation by histology, bone formation by X-ray microcomputed tomography (µCT) and gene expression by quantitative polymerase chain reaction (qPCR) showed that constructs in combi-cassettes were similar to those created by regular procedures. Combi-cassettes afford placement of multiple replicates of multiple formulations into the same animal, which enables, for the first time, rigorous statistical assessment of: 1) the variability for a given formulation within an animal (intra-animal variability), 2) differences between different tissue-engineered formulations within the same animal and 3) the variability for a given formulation in different animals (inter-animal variability). Combi-cassettes enable a more high-throughput, systematic approach to in vivo studies of tissue engineering constructs.
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Sustitutos de Huesos/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Proteína Morfogenética Ósea 2/química , Sustitutos de Huesos/metabolismo , Fosfatos de Calcio/química , Células Cultivadas , Durapatita/química , Femenino , Gelatina/química , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Osteogénesis , Politetrafluoroetileno/química , PorosidadRESUMEN
Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.
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Células de la Médula Ósea/metabolismo , Médula Ósea/metabolismo , Perfilación de la Expresión Génica/métodos , Células Madre Mesenquimatosas/metabolismo , Adiposidad , Animales , Criopreservación/métodos , Hematopoyesis , Humanos , OsteogénesisRESUMEN
Mechanical loading plays a key role in bone formation and maintenance. While unloading induces osteocyte apoptosis and bone loss in vivo, mechanical stimuli prevents osteocyte death through a mechanism involving ß-catenin accumulation and ERK nuclear translocation. Vascular endothelial growth factor (VEGF) has a crucial role in bone formation, but its interaction with osteocytes is not completely understood. Of interest, VEGF receptor 2 (VEGFR2) has recently been shown to mediate the mechanical response of endothelial cells. The present study aimed to evaluate the putative role of the VEGF system in osteocyte mechanosensing. We show that either short (10 min) mechanical stimulus by pulsatile fluid flow (FF) (10 dyn/cm(2), 8 Hz) or exogenous VEGF165 (6 ng/ml) similarly stimulated cell viability, ERK phosphorylation, and ß-catenin membrane translocation. A VEGFR2 antagonist (SU5416) or transfection with specific VEGFR2 siRNAs (siVEGFR2) decreased these events. FF for 10 min increased VEGFR2 phosphorylation at both Tyr-1059 and Tyr-1175; an effect that was mimicked by VEGF165 but was unaffected by a VEGF neutralizing antibody. Subsequently (at 6 h), this mechanical stimulus induced VEGF gene overexpression, which was prevented by siVEGFR2 transfection. Depletion of the structural protein caveolin-1 by using siRNA technology impaired FF-induced VEGFR2 phosphorylation. In conclusion, these in vitro findings point to caveolin-1-dependent VEGFR2 activation as an important mechanism whereby mechanical stimuli promote osteocyte viability.
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Caveolina 1/metabolismo , Células Endoteliales/metabolismo , Mecanotransducción Celular/fisiología , Osteocitos/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Movimiento Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Activación Enzimática , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Osteocitos/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
The tumor suppressor, p53, plays a critical role in suppressing osteosarcoma. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) have been suggested to give rise to osteosarcomas. However, the role of p53 in BMSCs has not been extensively explored. Here, we report that p53 regulates the lineage choice of mouse BMSCs (mBMSCs). Compared to mBMSCs with wild-type p53, mBMSCs deficient in p53 have enhanced osteogenic differentiation, but with similar adipogenic and chondrogenic differentiation. The role of p53 in inhibiting osteogenic lineage differentiation is mainly through the action of Runx2, a master transcription factor required for the osteogenic differentiation of mBMSCs. We find that p53 indirectly represses the expression of Runx2 by activating the microRNA-34 family, which suppresses the translation of Runx2. Since osteosarcoma may derive from BMSCs, we examined whether p53 has a role in the osteogenic differentiation of osteosarcoma cells and found that osteosarcoma cells with p53 deletion have higher levels of Runx2 and faster osteogenic differentiation than those with wild-type p53. A systems biology approach reveals that p53-deficient mBMSCs are more closely related to human osteosarcoma while mBMSCs with wild-type p53 are similar to normal human BMSCs. In summary, our results indicate that p53 activity can influence cell fate specification of mBMSCs, and provide molecular and cellular insights into the observation that p53 loss is associated with increased osteosarcoma incidence.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/fisiología , Proteína p53 Supresora de Tumor/deficiencia , Animales , Línea Celular Tumoral , Células Cultivadas , Humanos , Ratones , Ratones NoqueadosRESUMEN
Osteocytes have a major role in the control of bone remodeling. Mechanical stimulation decreases osteocyte apoptosis and promotes bone accrual, whereas skeletal unloading is deleterious in both respects. PTH1R ablation or overexpression in osteocytes in mice produces trabecular bone loss or increases bone mass, respectively. The latter effect was related to a decreased osteocyte apoptosis. Here, the putative role of PTH1R activation in osteocyte protection conferred by mechanical stimulation was assessed. Osteocytic MLO-Y4 cells were subjected to mechanical stimuli represented by hypotonic shock (216 mOsm/kg) or pulsatile fluid flow (8 Hz, 10 dynes/cm(2)) for a short pulse (10 min), with or without PTH1R antagonists or after transfection with specific PTHrP or PTH1R siRNA. These mechanical stimuli prevented cell death induced within 6 hours by etoposide (50 µM), related to PTHrP overexpression; and this effect was abolished by the calcium antagonist verapamil (1 µM), a phospholipase C (PLC) inhibitor (U73122; 10 µM), and a PKA activation inhibitor, Rp-cAMPS (25 µM), in these cells. Each mechanical stimulus also rapidly induced ß-catenin stabilization and nuclear ERK translocation, which were inhibited by the PTH1R antagonist PTHrP(7-34) (1 µM), or PTH1R siRNA, and mimicked by PTHrP(1-36) (100 nM). Mechanical stretching by hypotonic shock did not affect cAMP production but rapidly (<1 min) stimulated Ca(i)(2+) transients in PTH1R-overexpressing HEK-293 cells and in MLO-Y4 cells, in which calcium signaling was unaffected by the presence of a PTHrP antiserum or PTHrP siRNA but inhibited by knocking down PTH1R. These novel findings indicate that PTH1R is an important component of mechanical signal transduction in osteocytic MLO-Y4 cells, and that PTH1R activation by PTHrP-independent and dependent mechanisms has a relevant role in the prosurvival action of mechanical stimulus in these cells.
Asunto(s)
Mecanotransducción Celular , Osteocitos/citología , Osteocitos/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Soluciones Hipotónicas/farmacología , Mecanotransducción Celular/efectos de los fármacos , Ratones , Modelos Biológicos , Osteocitos/efectos de los fármacos , Proteína Relacionada con la Hormona Paratiroidea/farmacología , beta Catenina/metabolismoRESUMEN
Matrix proteoglycans such as biglycan (Bgn) dominate skeletal tissue and yet its exact role in regulating bone function is still unclear. In this paper we describe the potential role of (Bgn) in the fracture healing process. We hypothesized that Bgn could regulate fracture healing because of previous work showing that it can affect normal bone formation. To test this hypothesis, we created fractures in femurs of 6-week-old male wild type (WT or Bgn+/0) and Bgn-deficient (Bgn-KO or Bgn-/0) mice using a custom-made standardized fracture device, and analyzed the process of healing over time. The formation of a callus around the fracture site was observed at both 7 and 14 days post-fracture in WT and Bgn-deficient mice and immunohistochemistry revealed that Bgn was highly expressed in the fracture callus of WT mice, localizing within woven bone and cartilage. Micro-computed tomography (µCT) analysis of the region surrounding the fracture line showed that the Bgn-deficient mice had a smaller callus than WT mice. Histology of the same region also showed the presence of less cartilage and woven bone in the Bgn-deficient mice compared to WT mice. Picrosirius red staining of the callus visualized under polarized light showed that there was less fibrillar collagen in the Bgn-deficient mice, a finding confirmed by immunohistochemistry using antibodies to type I collagen. Interestingly, real time RT-PCR of the callus at 7 days post-fracture showed a significant decrease in relative vascular endothelial growth factor A (VEGF) gene expression by Bgn-deficient mice as compared to WT. Moreover, VEGF was shown to bind directly to Bgn through a solid-phase binding assay. The inability of Bgn to directly enhance VEGF-induced signaling suggests that Bgn has a unique role in regulating vessel formation, potentially related to VEGF storage or stabilization in the matrix. Taken together, these results suggest that Bgn has a regulatory role in the process of bone formation during fracture healing, and further, that reduced angiogenesis could be the molecular basis.