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1.
Nat Commun ; 15(1): 4575, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38834586

RESUMO

Bone regeneration requires a well-orchestrated cellular and molecular response including robust vascularization and recruitment of mesenchymal and osteogenic cells. In femoral fractures, angiogenesis and osteogenesis are closely coupled during the complex healing process. Here, we show with advanced longitudinal intravital multiphoton microscopy that early vascular sprouting is not directly coupled to osteoprogenitor invasion during calvarial bone regeneration. Early osteoprogenitors emerging from the periosteum give rise to bone-forming osteoblasts at the injured calvarial bone edge. Microvessels growing inside the lesions are not associated with osteoprogenitors. Subsequently, osteogenic cells collectively invade the vascularized and perfused lesion as a multicellular layer, thereby advancing regenerative ossification. Vascular sprouting and remodeling result in dynamic blood flow alterations to accommodate the growing bone. Single cell profiling of injured calvarial bones demonstrates mesenchymal stromal cell heterogeneity comparable to femoral fractures with increase in cell types promoting bone regeneration. Expression of angiogenesis and hypoxia-related genes are slightly elevated reflecting ossification of a vascularized lesion site. Endothelial Notch and VEGF signaling alter vascular growth in calvarial bone repair without affecting the ossification progress. Our findings may have clinical implications for bone regeneration and bioengineering approaches.


Assuntos
Regeneração Óssea , Células-Tronco Mesenquimais , Neovascularização Fisiológica , Osteogênese , Crânio , Animais , Regeneração Óssea/fisiologia , Camundongos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Masculino , Receptores Notch/metabolismo , Receptores Notch/genética , Camundongos Endogâmicos C57BL , Transdução de Sinais , Feminino , Angiogênese
2.
Biol Proced Online ; 26(1): 7, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38504200

RESUMO

BACKGROUND: Osteoclasts are the tissue-specific macrophage population of the bone and unique in their bone-resorbing activity. Hence, they are fundamental for bone physiology in health and disease. However, efficient protocols for the isolation and study of primary human osteoclasts are scarce. In this study, we aimed to establish a protocol, which enables the efficient differentiation of functional human osteoclasts from monocytes. RESULTS: Human monocytes were isolated through a double-density gradient from donor blood. Compared to standard differentiation schemes in polystyrene cell culture dishes, the yield of multinuclear osteoclasts was significantly increased upon initial differentiation of monocytes to macrophages in fluorinated ethylene propylene (FEP) Teflon bags. This initial differentiation phase was then followed by the development of terminal osteoclasts by addition of Receptor Activator of NF-κB Ligand (RANKL). High concentrations of RANKL and Macrophage colony-stimulating factor (M-CSF) as well as an intermediate cell density further supported efficient cell differentiation. The generated cells were highly positive for CD45, CD14 as well as the osteoclast markers CD51/ITGAV and Cathepsin K/CTSK, thus identifying them as osteoclasts. The bone resorption of the osteoclasts was significantly increased when the cells were differentiated from macrophages derived from Teflon bags compared to macrophages derived from conventional cell culture plates. CONCLUSION: Our study has established a novel protocol for the isolation of primary human osteoclasts that improves osteoclastogenesis in comparison to the conventionally used cultivation approach.

3.
Nat Commun ; 13(1): 571, 2022 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-35091558

RESUMO

Developmental osteogenesis, physiological bone remodelling and fracture healing require removal of matrix and cellular debris. Osteoclasts generated by the fusion of circulating monocytes degrade bone, whereas the identity of the cells responsible for cartilage resorption is a long-standing and controversial question. Here we show that matrix degradation and chondrocyte phagocytosis are mediated by fatty acid binding protein 5-expressing cells representing septoclasts, which have a mesenchymal origin and are not derived from haematopoietic cells. The Notch ligand Delta-like 4, provided by endothelial cells, is necessary for septoclast specification and developmental bone growth. Consistent with the termination of growth, septoclasts disappear in adult and ageing bone, but re-emerge in association with growing vessels during fracture healing. We propose that cartilage degradation is mediated by rare, specialized cells distinct from osteoclasts. Our findings have implications for fracture healing, which is frequently impaired in aging humans.


Assuntos
Cartilagem/metabolismo , Consolidação da Fratura/fisiologia , Células-Tronco Mesenquimais/metabolismo , Osteoclastos/metabolismo , Osteogênese/fisiologia , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Osso e Ossos/ultraestrutura , Cartilagem/citologia , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Consolidação da Fratura/genética , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Imunoeletrônica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Osteoclastos/citologia , Osteogênese/genética , RNA-Seq/métodos
4.
Sci Rep ; 10(1): 20510, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239699

RESUMO

The heparan sulfate proteoglycan Syndecan-1, a mediator of signals between the extracellular matrix and cells involved is able to interact with OPG, one of the major regulators of osteoclastogenesis. The potential of osteoblasts to induce osteoclastogenesis is characterized by a switch of OPG (low osteoclastogenic potential) towards RANKL production (high osteoclastogenic potential). In the present study, we investigated the influence of endogenous Syndecan-1 on local bone-cell-communication via the RANKL/OPG-axis in murine osteoblasts and osteoclasts in wild type and Syndecan-1 lacking cells. Syndecan-1 expression and secretion was increased in osteoblasts with high osteoclastogenic potential. Syndecan-1 deficiency led to increased OPG release by osteoblasts that decreased the availability of RANKL. In co-cultures of Syndecan-1 deficient osteoblasts with osteoclast these increased OPG in supernatant caused decreased development of osteoclasts. Syndecan-1 and RANKL level were increased in serum of aged WT mice, whereas Syndecan-1 deficient mice showed high serum OPG concentration. However, bone structure of Syndecan-1 deficient mice was not different compared to wild type. In conclusion, Syndecan-1 could be regarded as a new modulator of bone-cell-communication via RANKL/OPG axis. This might be of high impact during bone regeneration or bone diseases like cancer where Syndecan-1 expression is known to be even more prevalent.


Assuntos
Osso e Ossos/citologia , Comunicação Celular , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Sindecana-1/metabolismo , Envelhecimento/sangue , Animais , Animais Recém-Nascidos , Desenvolvimento Ósseo , Diferenciação Celular , Camundongos Endogâmicos C57BL , Modelos Biológicos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese , Osteoprotegerina/sangue , Sindecana-1/sangue , Sindecana-1/deficiência
5.
Sci Rep ; 10(1): 16238, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004928

RESUMO

Over the last years, murine in vivo magnetic resonance imaging (MRI) contributed to a new understanding of tissue composition, regeneration and diseases. Due to artefacts generated by the currently used metal implants, MRI is limited in fracture healing research so far. In this study, we investigated a novel MRI-compatible, ceramic intramedullary fracture implant during bone regeneration in mice. Three-point-bending revealed a higher stiffness of the ceramic material compared to the metal implants. Electron microscopy displayed a rough surface of the ceramic implant that was comparable to standard metal devices and allowed cell attachment and growth of osteoblastic cells. MicroCT-imaging illustrated the development of the callus around the fracture site indicating a regular progressing healing process when using the novel implant. In MRI, different callus tissues and the implant could clearly be distinguished from each other without any artefacts. Monitoring fracture healing using MRI-compatible implants will improve our knowledge of callus tissue regeneration by 3D insights longitudinal in the same living organism, which might also help to reduce the consumption of animals for future fracture healing studies, significantly. Finally, this study may be translated into clinical application to improve our knowledge about human bone regeneration.


Assuntos
Consolidação da Fratura , Fraturas Ósseas/fisiopatologia , Animais , Parafusos Ósseos , Interface Osso-Implante , Cerâmica , Modelos Animais de Doenças , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/metabolismo , Fraturas do Fêmur/fisiopatologia , Fixação Intramedular de Fraturas , Fraturas Ósseas/diagnóstico por imagem , Fraturas Ósseas/metabolismo , Imageamento Tridimensional , Imageamento por Ressonância Magnética , Camundongos , Microscopia Eletrônica de Varredura , Zircônio
6.
Aging Cell ; 19(11): e13244, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33085187

RESUMO

Bone loss is one of the consequences of aging, leading to diseases such as osteoporosis and increased susceptibility to fragility fractures and therefore considerable morbidity and mortality in humans. Here, we identify microRNA-146a (miR-146a) as an essential epigenetic switch controlling bone loss with age. Mice deficient in miR-146a show regular development of their skeleton. However, while WT mice start to lose bone with age, animals deficient in miR-146a continue to accrue bone throughout their life span. Increased bone mass is due to increased generation and activity of osteoblasts in miR-146a-deficient mice as a result of sustained activation of bone anabolic Wnt signaling during aging. Deregulation of the miR-146a target genes Wnt1 and Wnt5a parallels bone accrual and osteoblast generation, which is accompanied by reduced development of bone marrow adiposity. Furthermore, miR-146a-deficient mice are protected from ovariectomy-induced bone loss. In humans, the levels of miR-146a are increased in patients suffering fragility fractures in comparison with those who do not. These data identify miR-146a as a crucial epigenetic temporal regulator which essentially controls bone homeostasis during aging by regulating bone anabolic Wnt signaling. Therefore, miR-146a might be a powerful therapeutic target to prevent age-related bone dysfunctions such as the development of bone marrow adiposity and osteoporosis.


Assuntos
MicroRNAs/genética , Osteoporose/genética , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Diferenciação Celular/fisiologia , Epigênese Genética , Feminino , Masculino , Camundongos , MicroRNAs/metabolismo , Osteoblastos/citologia , Osteoporose/patologia , Proteína Wnt-5a/metabolismo , Proteína Wnt1/metabolismo
7.
Calcif Tissue Int ; 106(6): 655-664, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32140760

RESUMO

Enhanced osteoclast formation and function is a fundamental cause of alterations to bone structure and plays an important role in several diseases impairing bone quality. Recent work revealed that TRP calcium channels 3 and 6 might play a special role in this context. By analyzing the bone phenotype of TRPC6-deficient mice we detected a regulatory effect of TRPC3 on osteoclast function. These mice exhibit a significant decrease in bone volume per tissue volume, trabecular thickness and -number together with an increased number of osteoclasts found on the surface of trabecular bone. Primary bone marrow mononuclear cells from TRPC6-deficient mice showed enhanced osteoclastic differentiation and resorptive activity. This was confirmed in vitro by using TRPC6-deficient RAW 264.7 cells. TRPC6 deficiency led to an increase of TRPC3 in osteoclasts, suggesting that TRPC3 overcompensates for the loss of TRPC6. Raised intracellular calcium levels led to enhanced NFAT-luciferase reporter gene activity in the absence of TRPC6. In line with these findings inhibition of TRPC3 using the specific inhibitor Pyr3 significantly reduced intracellular calcium concentrations and normalized osteoclastic differentiation and resorptive activity of TRPC6-deficient cells. Interestingly, an up-regulation of TRPC3 could be detected in a cohort of patients with low bone mineral density by comparing micro array data sets of circulating human osteoclast precursor cells to those from patients with high bone mineral density, suggesting a noticeable contribution of TRP calcium channels on bone quality. These observations demonstrate a novel regulatory function of TRPC channels in the process of osteoclastic differentiation and bone loss.


Assuntos
Osteoclastos , Osteoporose/metabolismo , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6/metabolismo , Animais , Cálcio/metabolismo , Osso Esponjoso/metabolismo , Humanos , Camundongos , Osteoclastos/metabolismo , Células RAW 264.7
8.
BMC Musculoskelet Disord ; 15: 184, 2014 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-24885217

RESUMO

BACKGROUND: The overexpression of tumor necrosis factor (TNF)-α leads to systemic as well as local loss of bone and cartilage and is also an important regulator during fracture healing. In this study, we investigate how TNF-α inhibition using a targeted monoclonal antibody affects fracture healing in a TNF-α driven animal model of human rheumatoid arthritis (RA) and elucidate the question whether enduring the anti TNF-α therapy after trauma is beneficial or not. METHODS: A standardized femur fracture was applied to wild type and human TNF-α transgenic mice (hTNFtg mice), which develop an RA-like chronic polyarthritis. hTNFtg animals were treated with anti-TNF antibody (Infliximab) during the fracture repair. Untreated animals served as controls. Fracture healing was evaluated after 14 and 28 days of treatment by clinical assessment, biomechanical testing and histomorphometry. RESULTS: High levels of TNF-α influence fracture healing negatively, lead to reduced cartilage and more soft tissue in the callus as well as decreased biomechanical bone stability. Blocking TNF-α in hTNFtg mice lead to similar biomechanical and histomorphometrical properties as in wild type. CONCLUSIONS: High levels of TNF-α during chronic inflammation have a negative impact on fracture healing. Our data suggest that TNF-α inhibition by an anti-TNF antibody does not interfere with fracture healing.


Assuntos
Anticorpos Monoclonais/farmacologia , Artrite/complicações , Fraturas do Fêmur/fisiopatologia , Consolidação da Fratura/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Monoclonais/uso terapêutico , Artrite/tratamento farmacológico , Artrite Reumatoide , Pinos Ortopédicos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Modelos Animais de Doenças , Feminino , Fraturas do Fêmur/patologia , Fraturas do Fêmur/cirurgia , Fixação Interna de Fraturas , Consolidação da Fratura/fisiologia , Humanos , Inflamação , Infliximab , Camundongos , Camundongos Transgênicos , Estresse Mecânico , Torque , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Suporte de Carga
9.
Bone ; 56(1): 48-54, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23680479

RESUMO

Collagen binding integrins are of essential importance in the crosstalk between cells and the extracellular matrix. Integrin α2ß1 is a major receptor for collagen I, the most abundant protein in bone. In this study we show for the first time that integrin α2 deficiency is linked to collagen type I expression in bone. Investigating the femurs of wild type and integrin α2ß1 deficient mice, we found that loss of integrin α2 results in altered bone properties. Histomorphometric analysis of integrin α2 long bones displayed more trabecular network compared to wild type bones. During age related bone loss the integrin α2ß1 deficient bones retain trabecular structure even at old age. These findings were supported by functional, biomechanical testing, wherein the bones of integrin α2ß1 deficient mice do not undergo age-related alteration of biomechanical properties. These results might be explained by the increased presence of collagen in integrin α2ß1 deficient bone. Collagen type I could be detected in higher quantities in the integrin α2ß1 deficient bones, forming abnormal, amorphous fibrils. This was linked to higher expression levels of collagen type I and other bone formation related proteins as alkaline phosphatase of integrin α2ß1 deficient osteoblasts. Osteoclasts of integrin α2ß1 deficient mice did not show any differences. Consequently these results indicate that the absence of integrin α2ß1 alleviates the effects of age related bone degradation through over-expression of collagen type I and demonstrate a molecular mechanism how collagen binding integrins might directly impact bone aging.


Assuntos
Envelhecimento/patologia , Reabsorção Óssea/patologia , Colágeno Tipo I/metabolismo , Integrina alfa2beta1/deficiência , Envelhecimento/genética , Animais , Biomarcadores/metabolismo , Fenômenos Biomecânicos , Reabsorção Óssea/genética , Calcificação Fisiológica/genética , Contagem de Células , Diferenciação Celular/genética , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Dissecação , Feminino , Fêmur/patologia , Fêmur/fisiopatologia , Fêmur/ultraestrutura , Colágenos Fibrilares/genética , Colágenos Fibrilares/metabolismo , Colágenos Fibrilares/ultraestrutura , Regulação da Expressão Gênica , Integrina alfa2beta1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteoblastos/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
10.
Arthritis Rheum ; 65(3): 743-52, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23233348

RESUMO

OBJECTIVE: Syndecan 4, a heparan sulfate proteoglycan, has been associated with osteoarthritis. The present study was undertaken to analyze the functional role of syndecan 4 in endochondral ossification of mouse embryos and in adult fracture repair, which, like osteoarthritis, involves an inflammatory component. METHODS: Sdc4 promoter activity was analyzed in Sdc4(-/-) lacZ-knockin mice, using ß-galactosidase staining. Endochondral ossification in embryos from embryonic day 16.5 was assessed by histologic and immunohistologic staining. Bone fracture repair was analyzed in femora of adult mice on days 7 and 14 postfracture. To evaluate Sdc2 and Sdc4 gene expression with and without tumor necrosis factor α (TNFα) and Wnt-3a stimulation, quantitative real-time polymerase chain reaction was performed. RESULTS: In Sdc4(-/-) lacZ-knockin animals, syndecan 4 promoter activity was detectable at all stages of chondrocyte differentiation, and Sdc4 deficiency inhibited chondrocyte proliferation. Aggrecan turnover in the uncalcified cartilage of the epiphysis was decreased transiently in vivo, but this did not lead to a growth phenotype at birth. In contrast, among adult mice, fracture healing was markedly delayed in Sdc4(-/-) animals and was accompanied by increased callus formation. Blocking of inflammation via anti-TNFα treatment during fracture healing reduced these changes in Sdc4(-/-) mice to levels observed in wild-type controls. We analyzed the differences between the mild embryonic and the severe adult phenotype, and found a compensatory up-regulation of syndecan 2 in the developing cartilage of Sdc4(-/-) mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt-3a in vitro led to increased expression of syndecan 2, while stimulation with TNFα resulted in up-regulation of syndecan 4 but decreased expression of syndecan 2. TNFα stimulation reduced syndecan 2 expression and increased syndecan 4 expression even in the presence of Wnt-3a, suggesting that inflammation has a strong effect on the regulation of syndecan expression. CONCLUSION: Our results demonstrate that syndecan 4 is functionally involved in endochondral ossification and that its loss impairs fracture healing, due to inhibition of compensatory mechanisms under inflammatory conditions.


Assuntos
Desenvolvimento Ósseo/fisiologia , Fraturas do Fêmur/fisiopatologia , Consolidação da Fratura/fisiologia , Sindecana-4/fisiologia , Animais , Diferenciação Celular/fisiologia , Condrócitos/citologia , Condrócitos/fisiologia , Feminino , Fêmur/citologia , Fêmur/embriologia , Fêmur/fisiologia , Lâmina de Crescimento/citologia , Lâmina de Crescimento/embriologia , Lâmina de Crescimento/fisiologia , Inflamação/fisiopatologia , Óperon Lac/genética , Masculino , Camundongos , Camundongos Knockout , Osteogênese/fisiologia , Gravidez , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Sindecana-2/genética , Sindecana-2/fisiologia , Sindecana-4/genética , Tíbia/citologia , Tíbia/embriologia , Tíbia/fisiologia
11.
J Bacteriol ; 189(7): 2945-8, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17259316

RESUMO

Hydropathy profile analyses of the amino acid sequence of the quorum-sensing hybrid histidine kinase LuxN of Vibrio harveyi predict a periplasmic location of the N terminus. To test this, two-hybrid proteins consisting of LuxN and an N-terminally fused maltose-binding protein with or without a leader sequence were analyzed with regard to the enzymatic activities of LuxN, protease accessibility, and complementation of an Escherichia coli malE mutant. The results strongly support a periplasmic location of the N terminus, implying that LuxN is anchored with nine transmembrane domains in the cytoplasmic membrane.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Quinases/metabolismo , Percepção de Quorum/fisiologia , Fatores de Transcrição/metabolismo , Vibrio/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Histidina Quinase , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
12.
J Biol Chem ; 281(34): 24398-404, 2006 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-16807235

RESUMO

The Gram-negative bacterium Vibrio harveyi produces and responds to three autoinducers, AI-1, AI-2, and CAI-1 to regulate cell density dependent gene expression by a process referred to as quorum sensing. The concentration of the autoinducers is sensed by three cognate hybrid sensor kinases, and information is channeled via the HPt protein LuxU to the response regulator LuxO. Here, a detailed biochemical study on the enzymatic activities of the membrane-integrated hybrid sensor kinase LuxN, the sensor for N-(d-3-hydroxybutanoyl)homoserine lactone (AI-1), is provided. LuxN was heterologously overproduced as the full-length protein in Escherichia coli. LuxN activities were characterized in vitro and are an autophosphorylation activity with an unusually high ATP turnover rate, stable LuxU phosphorylation, and a slow phosphatase activity with LuxU approximately P as substrate. The presence of AI-1 affected the kinase but not the phosphatase activity of LuxN. The influence of AI-1 on the LuxN--> LuxU signaling step was monitored, and in the presence of AI-1, the kinase activity of LuxN, and hence the amount of LuxU approximately P produced, were significantly reduced. Half-maximal inhibition of kinase activity by AI-1 occurred at 20 mum. Together, these results indicate that AI-1 directly interacts with LuxN to down-regulate its autokinase activity and suggest that the key regulatory step of the AI-1 quorum sensing system of Vibrio harveyi is AI-1-mediated repression of the LuxN kinase activity.


Assuntos
4-Butirolactona/análogos & derivados , Proteínas de Bactérias/metabolismo , Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismo , 4-Butirolactona/análise , 4-Butirolactona/química , 4-Butirolactona/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Regulação para Baixo , Ativação Enzimática , Ligação Proteica , Proteínas Quinases/análise , Proteínas Quinases/química , Transdução de Sinais , Fatores de Transcrição/análise , Fatores de Transcrição/química , Vibrio/metabolismo
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