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Myeloid angiogenic cells (MACs) and pericyte-like cells, derived from peripheral blood mononuclear cells (MNCs) by in vitro culturing, are suggested as relevant cell types for angiogenesis and tissue repair. However, the in vivo existence and relevance of these cells has so far remained unknown. Our aim was thus to study, if MACs and pericyte-like cells exist in circulation during the wound healing of skin graft patients, and to evaluate the cellular features of wound repair. MNCs were isolated from blood samples of healthy controls (n = 4) and patients with a traumatic full thickness skin defect (n = 4) before skin grafting and on postoperative days 1 and 6. The numbers of circulating CD14+ CD45+ CD31+ CD34- MACs and CD14+ CD45+ NG2+ pericyte-like cells were assessed by flow cytometry, and gene expression of various pro-angiogenic factors was analysed by qPCR. Wound bed biopsies were taken on postoperative days 6 and 14, and MAC (CD31, CD14 and CD45) and pericyte-related markers (NG2 and PDGFRß) were histologically studied. MACs and pericyte-like cells were detected in both healthy controls and in patients. Before reconstruction, on average 18% of all circulating MNCs represented MACs and 2% pericyte-like cells in wound patients. Number of MACs significantly increased 1.1-1.7-fold in all patients 1 day after skin grafting (p < 0.01). In addition, histological analysis demonstrated effective vascularization of skin grafts, as well as presence of pericytes, and CD14 and CD45 expressing myeloid cells during wound healing. In conclusion, our data shows, for the first time, the presence and mobilisation of MACs and pericyte-like cells in human circulation.
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Pericitos , Transplante de Pele , Humanos , Pericitos/metabolismo , Leucócitos Mononucleares , Cicatrização , Células MieloidesRESUMO
This report identifies human skeletal diseases associated with mutations in WNT1. In 10 family members with dominantly inherited, early-onset osteoporosis, we identified a heterozygous missense mutation in WNT1, c.652TâG (p.Cys218Gly). In a separate family with 2 siblings affected by recessive osteogenesis imperfecta, we identified a homozygous nonsense mutation, c.884CâA, p.Ser295*. In vitro, aberrant forms of the WNT1 protein showed impaired capacity to induce canonical WNT signaling, their target genes, and mineralization. In mice, Wnt1 was clearly expressed in bone marrow, especially in B-cell lineage and hematopoietic progenitors; lineage tracing identified the expression of the gene in a subset of osteocytes, suggesting the presence of altered cross-talk in WNT signaling between the hematopoietic and osteoblastic lineage cells in these diseases.
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Mutação , Osteogênese Imperfeita/genética , Osteoporose/genética , Proteína Wnt1/genética , Adolescente , Adulto , Idade de Início , Idoso , Animais , Criança , Feminino , Genes Dominantes , Genes Recessivos , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Linhagem , Proteína Wnt1/metabolismo , Adulto JovemRESUMO
Background and purpose - Bisphosphonates are widely used in the treatment of bone loss, but they might also have positive effects on osteoblastic cells and bone formation. We evaluated the effect of in vivo zoledronic acid (ZA) treatment and possible concomitant effects of ZA and fracture on the ex vivo osteogenic capacity of rat mesenchymal stromal cells (MSCs). Methods - A closed femoral fracture model was used in adult female rats and ZA was administered as a single bolus or as weekly doses up to 8 weeks. Bone marrow MSCs were isolated and cultured for in vitro analyses. Fracture healing was evaluated by radiography, micro-computed tomography (µCT), and histology. Results - Both bolus and weekly ZA increased fracture-site bone mineral content and volume. MSCs from weekly ZA-treated animals showed increased ex vivo proliferative capacity, while no substantial effect on osteoblastic differentiation was observed. Fracture itself did not have any substantial effect on cell proliferation or differentiation at 8 weeks. Serum biochemical markers showed higher levels of bone formation in animals with fracture than in intact animals, while no difference in bone resorption was observed. Interestingly, ex vivo osteoblastic differentiation of MSCs was found to correlate with in vivo serum bone markers. Interpretation - Our data show that in vivo zoledronic acid treatment can influence ex vivo proliferation of MSCs, indicating that bisphosphonates can have sustainable effects on cells of the osteoblastic lineage. Further research is needed to investigate the mechanisms.
Assuntos
Densidade Óssea/efeitos dos fármacos , Difosfonatos/farmacologia , Fraturas do Fêmur/terapia , Imidazóis/farmacologia , Células-Tronco Mesenquimais/citologia , Animais , Conservadores da Densidade Óssea/farmacologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fraturas do Fêmur/patologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Ratos , Microtomografia por Raio-X , Ácido ZoledrônicoRESUMO
Bone is increasingly recognized as a target for diabetic complications. In order to evaluate the direct effects of high glucose on bone, we investigated the global transcriptional changes induced by hyperglycemia in osteoblasts in vitro. Rat bone marrow-derived mesenchymal stromal cells were differentiated into osteoblasts for 10 days, and prior to analysis, they were exposed to hyperglycemia (25â mM) for the short-term (1 or 3 days) or long-term (10â days). Genes and pathways regulated by hyperglycemia were identified using mRNA sequencing and verified with qPCR. Genes upregulated by 1-day hyperglycemia were, for example, related to extracellular matrix organization, collagen synthesis and bone formation. This stimulatory effect was attenuated by 3 days. Long-term exposure impaired osteoblast viability, and downregulated, for example, extracellular matrix organization and lysosomal pathways, and increased intracellular oxidative stress. Interestingly, transcriptional changes by different exposure times were mostly unique and only 89 common genes responding to glucose were identified. In conclusion, short-term hyperglycemia had a stimulatory effect on osteoblasts and bone formation, whereas long-term hyperglycemia had a negative effect on intracellular redox balance, osteoblast viability and function.
Assuntos
Regulação da Expressão Gênica , Glucose , Osteoblastos , Osteoblastos/metabolismo , Osteoblastos/efeitos dos fármacos , Animais , Glucose/metabolismo , Ratos , Regulação da Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Hiperglicemia/metabolismo , Hiperglicemia/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Transcriptoma , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Sobrevivência Celular/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Células Cultivadas , Estresse Oxidativo/efeitos dos fármacosRESUMO
Recent research has revealed several important pathways of epigenetic regulation leading to transcriptional changes in bone cells. Rest Corepressor 2 (Rcor2) is a coregulator of Lysine-specific histone demethylase 1 (Lsd1), a demethylase linked to osteoblast activity, hematopoietic stem cell differentiation and malignancy of different neoplasms. However, the role of Rcor2 in osteoblast differentiation has not yet been examined in detail. We have previously shown that Rcor2 is highly expressed in mesenchymal stromal cells (MSC) and particularly in the osteoblastic lineage. The role of Rcor2 in osteoblastic differentiation in vitro was further characterized and we demonstrate here that lentiviral silencing of Rcor2 in MC3T3-E1 cells led to a decrease in osteoblast differentiation. This was indicated by decreased alkaline phosphatase and von Kossa stainings as well as by decreased expression of several osteoblast-related marker genes. RNA-sequencing of the Rcor2-downregulated MC3T3-E1 cells showed decreased repression of Rcor2 target genes, as well as significant upregulation of majority of the differentially expressed genes. While the heterozygous, global loss of Rcor2 in vivo did not lead to a detectable bone phenotype, conditional deletion of Rcor2 in limb-bud mesenchymal cells led to a moderate decrease in cortical bone volume. These findings were not accentuated by challenging bone formation by ovariectomy or tibial fracture. Furthermore, a global deletion of Rcor2 led to decreased white adipose tissue in vivo and decreased the capacity of primary cells to differentiate into adipocytes in vitro. The conditional deletion of Rcor2 led to decreased adiposity in fracture callus. Taken together, these results suggest that epigenetic regulation of mesenchymal stromal cell differentiation is mediated by Rcor2, which could thus play an important role in defining the MSC fate.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , Osteoblastos , Animais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Diferenciação Celular/genética , Camundongos , Osteoblastos/metabolismo , Osteoblastos/citologia , Osteogênese/genética , Osteogênese/fisiologia , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Linhagem CelularRESUMO
Musculoskeletal research should synergistically investigate bone and muscle to inform approaches for maintaining mobility and to avoid bone fractures. The relationship between sarcopenia and osteoporosis, integrated in the term 'osteosarcopenia', is underscored by the close association shown between these two conditions in many studies, whereby one entity emerges as a predictor of the other. In a recent workshop of Working Group (WG) 2 of the EU Cooperation in Science and Technology (COST) Action 'Genomics of MusculoSkeletal traits Translational Network' (GEMSTONE) consortium (CA18139), muscle characterization was highlighted as being important, but currently under-recognized in the musculoskeletal field. Here, we summarize the opinions of the Consortium and research questions around translational and clinical musculoskeletal research, discussing muscle phenotyping in human experimental research and in two animal models: zebrafish and mouse.
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Fenótipo , Animais , Humanos , Músculo Esquelético/metabolismo , Peixe-Zebra , Camundongos , Sarcopenia/metabolismo , Sarcopenia/fisiopatologia , Doenças Musculoesqueléticas/fisiopatologia , Doenças Musculoesqueléticas/genética , Osteoporose/metabolismo , Osteoporose/patologiaRESUMO
Different biomaterials have been clinically used as bone filling materials, although the mechanisms behind the biological effects are incompletely understood. To address this, we compared the effects of five different biomaterials: two bioactive glasses (45S5 and S53P4), hydroxyapatite (HAP), carbonated apatite (CAP), and alumina on the in vitro migration and viability of pre-osteoblastic cells. In addition, we studied the effects of biomaterials' calcium release on cell migration, viability and differentiation. We found differences between the materials as the bioactive glasses promoted rapid pre-osteoblastic cell migration. In contrast, CAP decreased cell migration, which was also associated with lower activity of migration related kinases. Bioactive glasses released significant amounts of calcium into the media, while CAP decreased the calcium concentration. The response of cells to calcium was mechanistically studied by blocking calcium sensing receptor (CaSR) and ATP-gated ion channel P2X7, but this had no effect on cell migration. Surprisingly, HAP and CAP initially decreased cell viability. In summary, bioactive glasses 45S5 and S53P4 had significant and long-lasting effects on the pre-osteoblastic cell migration, which could be related to the observed calcium dissolution. Additionally, bioactive glasses had no negative effects on cell viability, which was observed with HAP and CAP.
Assuntos
Cálcio , Durapatita , Durapatita/farmacologia , Materiais Biocompatíveis , Vidro , Apatitas , Cálcio da Dieta , Movimento CelularRESUMO
Chondrocyte differentiation is a principal progress in endochondral ossification and in the formation of secondary ossification center (SOC) during the long bone development. We have previously reported that targeted deletion of Wnt1 in mesenchymal progenitors (Wnt1Prrx-/-) leads to spontaneous fractures and severe osteopenia in mouse long bones, suggesting that Wnt1 is a key regulator of bone metabolism. However, the effect of Wnt1 on the regulation of cartilage development and chondrocyte differentiation remained unknown. In this study, WNT1 protein expression was observed in lateral superficial cartilage and growth plate pre-hypertrophic chondrocytes in mice. Wnt1 mRNA expression was detected in epiphyseal cartilage from E16.5 to 3 month-old mice. Detailed histological analyses revealed that the average thickness and chondrocyte density of proximal tibial articular cartilage and growth plate were unchanged between Wnt1Prrx-/- and control mice. However, µCT analysis of tibial epiphyses showed that the subchondral bone mass was reduced in Wnt1Prrx-/- mice compared to control mice, as demonstrated by decreased bone volume, trabecular number, trabecular thickness, and increased trabecular separation in Wnt1Prrx-/- mice. Mechanistically, histomorphometric analyses showed that the reduced subchondral bone mass in Wnt1Prrx-/- mice was due to impaired bone formation and enhanced bone resorption. In vitro, exogenous Wnt1 inhibited chondrogenesis and chondrocyte hypertrophy in both cell autonomous and juxtacrine manners, while matrix mineralization and the expression of Mmp13, Mmp9 and Opn were induced in a juxtacrine manner. Taken together, mesenchymal cell-derived Wnt1 is an important regulator of subchondral bone remodeling, although it has no effect on the regulation of growth plate or articular cartilage.
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Cartilagem Articular , Lâmina de Crescimento , Animais , Remodelação Óssea , Condrócitos , Camundongos , Proteína Wnt1RESUMO
OBJECTIVES: A tribochemical silica-coating (TSC) method has been developed to improve the adhesion of dental resin composites to various substrates. The method utilizes airborne-particle abrasion using particles having a silica surface and an alumina core. The impact of the TSC method has been extensively studied but less attention has been paid to the characterization of the silica-modified alumina particles. Due to the role of silicate ions in cell biology, e.g. osteoblast function and bone mineralization, silica-modified alumina particles could also be potentially used as a biomaterial in scaffolds of tissue regeneration. Thus, we carried out detailed physicochemical characterization of the silica-modified alumina particles. METHODS: Silica-modified alumina particles (Rocatec, 3 M-ESPE) of an average particle size of 30 µm were studied for the phase composition, spectroscopic properties, surface morphology, dissolution, and the capability to modify the pH of an immersion solution. The control material was alumina without silica modification. Pre-osteoblastic MC3T3-E1 cells were used to assess cell viability in the presence of the particles. Cell viability was tested at 1, 3, 7 and 10 days of culture with various particle quantities. Multivariate ANOVA was used for statistical analyses. RESULTS: Minor quantities of silica enrichment was verified on the surface of alumina particles and the silica did not evenly cover the alumina surface. In the dissolution test, no change in the pH of the immersion solution was observed in the presence of the particles. Minor quantities of silicate ions were dissolved from the particles to the cell culture medium but no major differences were observed in the viability of pre-osteoblastic cells, whether the cells were cultured with silica-modified or plain alumina particles. SIGNIFICANCE: Characterization of silica-modified alumina particles demonstrated differences in the particle surface structure compared to control alumina. Dissolution of silica layer in Tris buffer or SBF solution varied from that of cell culture medium: minor quantities of dissolved Si were observed in cell culture test medium. The cell viability test did not shown significant differences between control alumina and its silica-modified counterpart.
Assuntos
Óxido de Alumínio , Colagem Dentária , Óxido de Alumínio/química , Resinas Compostas/química , Colagem Dentária/métodos , Teste de Materiais , Cimentos de Resina/química , Silicatos/química , Dióxido de Silício/química , Propriedades de Superfície , Zircônio/químicaRESUMO
Fracture healing is a complex process with multiple overlapping metabolic and differentiation phases. Small non-coding RNAs are involved in the regulation of fracture healing and their presence in circulation is under current interest due to their obvious value as potential biomarkers. Circulating microRNAs (miRNAs) have been characterized to some extent but the current knowledge on tRNA-derived small RNA fragments (tsRNAs) is relatively scarce, especially in circulation. In this study, the spectrum of circulating miRNAs and tsRNAs was analysed by next generation sequencing to show their differential expression during fracture healing in vivo. Analysed tsRNA fragments included stress-induced translation interfering tRNA fragments (tiRNAs or tRNA halves) and internal tRNA fragments (i-tRF), within the size range of 28-36 bp. To unveil the expression of these non-coding RNAs, genome-wide analysis was performed on two months old C57BL/6 mice on days 1, 5, 7, 10, and 14 (D1, D5, D7, D10, and D14) after a closed tibial fracture. Valine isoacceptor tRNA-derived Val-AAC 5'end and Val-CAC 5'end fragments were the major types of 5'end tiRNAs in circulation, comprising about 65 % of the total counts. Their expression was not affected by fracture. After a fracture, the levels of two 5'end tiRNAs Lys-TTT 5' and Lys-CTT 5' were decreased and His-GTG 5' was increased through D1-D14. The level of miR-451a was decreased on the first post-fracture day (D1), whereas miR-328-3p, miR-133a-3p, miR-375-3p, miR-423-5p, and miR-150-5p were increased post-fracture. These data provide evidence on how fracture healing could provoke systemic metabolic effects and further pinpoint the potential of small non-coding RNAs as biomarkers for tissue regeneration.
RESUMO
Compelling clinical data together with genetically modified mouse models have demonstrated that Wnt1 is a key Wnt ligand in bone metabolism, regulating both osteoblast activity and osteoclast differentiation. We have previously shown that deletion of Wnt1 in limb mesenchymal cells leads to severe ostepenic bone phenotype and spontaneous fractures very early after birth. However, the function of Wnt1 in mature skeleton remained unknown. To investigate the role of Wnt1 specifically in adult bone metabolism, we generated an osteoblast lineage-targeted inducible Wnt1 knockout mouse model using tetracycline-controlled Osterix-Cre mouse line (Osx-Cre). In this model, the Cre recombinase expression is suppressed by administering doxycycline (Dox) in drinking water. As expected, Wnt1Osx-/- mice without Dox developed spontaneous fractures early by 3 weeks of age due to severe trabecular and cortical osteopenia. Administration of Dox to Wnt1Osx-Dox-/- and control mice until 4 weeks of age suppressed Wnt1 deletion and completely prevented the fractures. Withdrawal of Dox led to deletion in Wnt1 allele but the fracture incidence progressively decreased in Wnt1Osx-Dox-/- mice at 8 or 12 weeks of age (4 and 8 weeks after Dox withdrawal). Interestingly, deletion of Wnt1 at 4 weeks of age resulted only in a modest and transient trabecular osteopenia that was more pronounced in females and was normalized by 12 weeks of age. However, diaphyseal cortical bone mass and cortical thickness in the femurs were significantly decreased in Wnt1Osx-Dox-/- mice of both genders. Mechanisticly, this was due to impaired periosteal bone formation. Based on our data, in addition to its essential role in early skeletal growth, Wnt1 is an important regulator of modeling-based bone formation and cortical thickness in adult mice.
Assuntos
Células-Tronco Mesenquimais , Osteogênese , Animais , Osso e Ossos , Feminino , Masculino , Camundongos , Camundongos Knockout , Osteoblastos , Proteína Wnt1/genéticaRESUMO
Mesenchymal stromal cells (MSCs) are known to stimulate the survival and growth of endothelial cells (ECs) by producing paracrine signals, as well as to differentiate into pericytes and thereby support blood vessel formation and stability. On the other hand, cells with an EC-like phenotype have been found within the CD14+ and CD34+ cell populations of peripheral blood (PB) mononuclear cells (MNCs). The aim of this study was to investigate the proangiogenic differentiation potential of human MSC-MNC co-cultures. Bone marrow-derived MSCs (2,500 cells/cm2) were co-cultured with MNCs (50,000 cells/cm2), which were isolated from the PB of healthy donors. MSCs and MNCs cultured alone at same cell densities were used as controls. Cells in MNC fraction and in co-cultures were isolated for CD14, CD34, and CD31 surface markers with magnetic-activated cell sorting. Co-cultures were analyzed for cell proliferation and morphology, as well as for the expression of various hematopoietic, endothelial, and pericyte markers by immunocytochemistry, quantitative PCR (qPCR), and flow cytometry. Vascular endothelial growth factor (VEGF) expression and secretion was measured with qPCR and enzyme-linked immunosorbent assay, respectively. Our results show that in co-cultures with MSCs, CD14+CD45+ MNCs differentiated into spindle-shaped, nonproliferative, EC-like, myeloid angiogenic cells (MACs) expressing CD31, but also into pericyte-like cells expressing neural/glial antigen 2 (NG2) and CD146. Functionality of the isolated MACs was demonstrated in co-cultures with human umbilical vein endothelial cells, where they supported the formation of tube-like structures. NG2+ cells of MNC-origin were found among both CD34-CD14+ and CD34-CD14- cell populations, indicating the existence of different subtypes of pericyte-like cells. In addition, VEGF was shown to be secreted in MSC-MNC co-cultures, mainly by MSCs. In conclusion, MSCs were shown to possess proangiogenic capacity in MSC-MNC co-cultures as they supported the differentiation of functional MACs, as well as the differentiation of pericyte-like cells of MNC origin. This phenomenon was mediated at least partially via secreted VEGF.
Assuntos
Diferenciação Celular/fisiologia , Células Endoteliais/citologia , Leucócitos Mononucleares/citologia , Células-Tronco Mesenquimais/citologia , Pericitos/citologia , Adulto , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Feminino , Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Pericitos/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto JovemRESUMO
OBJECTIVE: Clinically used bioceramics have been characterized previously with different kinds of methods and comparison of results have proven to be difficult due to varieties of the material properties of interest. Therefore, in this study we compared clinically commonly used bioceramics of hydroxyapatite and carbonate apatite, two bioactive glasses 45S5 and S53P4, and alumina with respect of properties which according to the present knowledge are significant for bone biology. METHODS: Physicochemical properties of the materials were characterized by various methods. Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) was used to analyze the material vibrational features. X-ray Power Diffraction (XRD) was used to characterize the material crystal structure and scanning electron microscopy-energy-dispersive x-ray analysis (SEM-EDXA) was used to evaluate the morphology and size of the materials and to calculate their oxide content. The dissolution behavior of the materials, ion release and pH changes in Tris buffer in a continuous flow-through reaction for 24-hours were determined. The change of the surface of the bioactive glasses by interfacial reaction during the Tris immersion was examined and the thickness of the surface reaction layer of the materials was studied. RESULTS: SEM examination showed that the particle morphology of BG 45S5, BG S53P4 and alumina particle's surface was smooth. The surface of HAP was porous, but also CAP showed some surface porosity. An increase in the pH of the immersion solution was observed especially for BG 45S5 and BG S53P4. HAP, CAP and alumina caused only a minor increase in pH. BGs 45S5 and S53P4 showed a rapid initial release of sodium and calcium ions, followed by the release of silicon species. Minor release of sodium ions was registered for HAP, CAP and alumina. Calcium ion release was low but constant over the experimental time while only a minor initial dissolution was measured for HAP. SIGNIFICANCE: The in vitro study showed differences in the materials' properties, which are considered to be important for biological suitability and in clinical applications, such as materials tomography, ion release and pH changes.
Assuntos
Materiais Biocompatíveis , Cerâmica , Durapatita , Vidro , Porosidade , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Material-induced ossification is suggested as a suitable approach to heal large bone defects. Fiber-reinforced composite-bioactive glasses (FRC-BGs) display properties that could enhance the ossification of calvarial defects. Here, we analyzed the healing processes of a FRC-BG implant in vivo from the perspective of material-induced ossification. Histological analysis of the implant, which was removed 5 months after insertion, showed the formation of viable, noninflammatory mesenchymal tissue with newly-formed mineralized woven bone, as well as nonmineralized connective tissue with capillaries and larger blood vessels. The presence of osteocytes was detected within the newly generated bone matrix. To expand our understanding on the osteogenic properties of FRC-BG, we cultured human adipose tissue-derived mesenchymal stromal cells (AD-MSCs) in the presence of two different BGs (45S5 and S53P4) and Al2 O3 control. AD-MSCs grew and proliferated on all the scaffolds tested, as well as secreted abundant extracellular matrix, when osteogenic differentiation was appropriately stimulated. 45S5 and S53P4 induced enhanced expression of COL2A1, COL10A1, COL5A1 collagen subunits, and pro-osteogenic genes BMP2 and BMP4. The concomitant downregulation of BMP3 was also detected. Our findings show that FRC-BG can support the vascularization of the implant and the formation of abundant connective tissue in vivo. Specifically, BG 45S5 and BG S53P4 are suited to evoke the osteogenic potential of host mesenchymal stromal cells. In conclusion, FRC-BG implant demonstrated material-induced ossification both in vitro and in vivo.
Assuntos
Materiais Biocompatíveis/administração & dosagem , Osteogênese/efeitos dos fármacos , Próteses e Implantes , Humanos , Masculino , Pessoa de Meia-Idade , Crânio/lesões , Crânio/metabolismoRESUMO
BACKGROUND: Osteoradionecrosis (ORN) of the mandible is a severe complication of radiotherapy for head and neck cancer and is arduously difficult to manage. Current treatment options carry risks with some patients remaining incurable. Mesenchymal stromal/stem cell (MSC) therapy has shown promising results supporting osteogenesis and regeneration of radiotherapy-damaged tissues. The aim of this study was to systematically review the literature on the safety and efficacy of MSCs in treating ORN. METHODS: A systematic search was performed on MEDLINE, Embase, Cochranes Library online databases, and clinicaltrials.gov to identify preclinical and clinical studies examining the effect of MSCs on osseous healing of ORN. The preclinical studies were assessed according to the SYRCLEs guidelines and risk of bias tool. RESULTS: Six studies (n = 142) from 5 countries were eligible for analysis. Of these four were preclinical studies and two clinical case studies. Preclinical studies found MSC treatment to be safe, demonstrating bone restorative effects and improved soft tissue regeneration. In the clinical cases, healing of bone and soft tissue was reported with no serious adverse events. CONCLUSION: The evidence from the included studies suggests that MSCs may have beneficial regenerative effects on the healing of ORN. None of the studies reported adverse events with the use of MSCs. More carefully controlled studies with well-identified cells are however needed to demonstrate the efficacy of MSCs in a clinical setting. Graphical abstract.
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Mandíbula/patologia , Transplante de Células-Tronco Mesenquimais , Osteorradionecrose/terapia , Animais , Modelos Animais de Doenças , Humanos , Masculino , Pessoa de Meia-Idade , Osteorradionecrose/patologia , Viés de Publicação , Risco , Fatores de Tempo , CicatrizaçãoRESUMO
Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Osteocytes are the most abundant cells in bone, and their death by microdamage has been suggested to be the major event leading in the initiation of osteoclastic bone resorption. Even though there is increasing evidence that osteocyte density, microcracks and targeted remodeling are related, there still exist several questions. For example, how osteoclasts are targeted to the specific site of microdamage for repair. It has been proposed that apoptotic osteocytes could secrete a specific signal to target osteoclasts. The other question is the nature of this signal. To elucidate the role of microdamage-induced osteocyte cell death in the initiation of targeted remodelling, this paper discusses the potential use of an in vitro model, in which osteocytes can be three-dimensionally cultured and locally damaged. Furthermore, the method enables one to study the osteocyte-derived soluble interactions with bone marrow cells. It was demonstrated that damaged osteocytes locally affect osteoclast precursors by secreting osteoclastogenic factors, and thus can have a role in the initiation of resorption in bone remodelling. This strongly supports the idea that damage to osteocyte cellular network has the potential to stimulate osteoclastic proliferation and therefore the activation of Basic Multicellular Units (BMUs).
Assuntos
Remodelação Óssea/fisiologia , Osso e Ossos/citologia , Osteócitos/fisiologia , Animais , Apoptose/fisiologia , Células da Medula Óssea/citologia , Reabsorção Óssea/patologia , Osso e Ossos/patologia , Diferenciação Celular/fisiologia , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Fraturas Ósseas/patologia , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Osteoclastos/fisiologia , Osteócitos/metabolismo , Osteócitos/patologia , Ligante RANK/metabolismo , Estresse MecânicoRESUMO
Bone is an elementary component in the human skeleton. It protects vital organs, regulates calcium levels and allows mobility. As a result of daily activities, bones are cyclically strained causing microdamage. This damage, in the form of numerous microcracks, can cause bones to fracture and therefore poses a threat to mechanical integrity. Bone is able to repair the microcracks through a process called remodelling which is tightly regulated by bone forming and resorbing cells. However, the manner by which microcracks are detected, and repair initiated, has not been elucidated until now. Here we show that microcrack accumulation causes damage to the network of cellular processes, resulting in the release of RANKL which stimulates the differentiation of cells specialising in repair.
Assuntos
Remodelação Óssea/fisiologia , Osso e Ossos/patologia , Consolidação da Fratura/fisiologia , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Diferenciação Celular/fisiologia , Fraturas de Estresse/metabolismo , Fraturas de Estresse/patologia , Humanos , Osteogênese/fisiologia , Osteopetrose/metabolismo , Osteopetrose/patologia , Osteoprotegerina/metabolismo , Ligante RANK/biossíntese , Ligante RANK/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
Bisphosphonates are widely used anti-resorptive drugs in the adult population. In children, their use has mainly been limited to patients with osteogenesis imperfecta. However, the powerful effects of bisphosphonates on bone turnover have raised concern about their long-term effects on the growing skeleton. We aimed to study the effects of two commonly used bisphosphonates, alendronate (Aln) and pamidronate (Pam) on normal bone growth as well as their potential to prevent glucocorticoid-induced growth retardation. Effects on bone growth were studied in fetal rat metatarsal bones (day E20) that were cultured for 5-47 days and measured every 2-7 days. Cellular mechanisms were investigated in metatarsal bones and also in the human chondrocytic cell line HCS-2/8. Chondrocyte viability (WST-1), proliferation (BrdU incorporation), differentiation (collagen type X immunohistochemistry) and apoptosis (TUNEL and Cell Death ELISA) were determined. At a clinically relevant concentration of bisphosphonates (1 microM), metatarsal bone growth was stimulated by both Aln (p<0.001 for length and p<0.05 for width) and Pam (p<0.05 for both length and width) from day 19 of culture. The growth-stimulatory effect was associated with increased chondrocyte proliferation (+21% with Aln and +24% with Pam), while cell differentiation and apoptosis were not affected. Despite the finding that both Aln and Pam (1 muM) rescued HCS-2/8 cells from undergoing dexamethasone-induced apoptosis, neither of them was able to prevent dexamethasone-induced growth retardation of fetal rat metatarsal bones. Aln and Pam have the capacity to stimulate the growth of cultured fetal rat metatarsal bones; an effect associated with increased proliferation of growth plate chondrocytes. Our experimental data suggest that bisphosphonates are ineffective in preventing glucocorticoid-induced growth retardation. Nevertheless, based on our in vitro data, both Aln and Pam appear safe to use in growing children, at least with regard to their effects on linear bone growth.
Assuntos
Alendronato/farmacologia , Dexametasona/farmacologia , Difosfonatos/farmacologia , Ossos do Pé/efeitos dos fármacos , Ossos do Pé/crescimento & desenvolvimento , Animais , Densidade Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Ossos do Pé/citologia , Humanos , Pamidronato , Ratos , Ratos Sprague-Dawley , Técnicas de Cultura de TecidosRESUMO
Endothelial progenitors found among the peripheral blood (PB) mononuclear cells (MNCs) are interesting cells for their angiogenic properties. Mesenchymal stromal cells (MSCs) in turn can produce proangiogenic factors as well as differentiate into mural pericytes, making MSCs and MNCs an attractive coculture setup for regenerative medicine. In this study, human bone marrow-derived MSCs and PB-derived MNCs were cocultured in basal or osteoblastic medium without exogenously supplied growth factors to demonstrate endothelial cell, pericyte and osteoblastic differentiation. The expression levels of various proangiogenic factors, as well as endothelial cell, pericyte and osteoblast markers in cocultures were determined by quantitative polymerase chain reaction. Immunocytochemistry for vascular endothelial growth factor receptor-1 and α-smooth muscle actin as well as staining for alkaline phosphatase were performed after 10 and 14 days. Messenger ribonucleic acid expression of endothelial cell markers was highly upregulated in both basal and osteoblastic conditions after 5 days of coculture, indicating an endothelial cell differentiation, which was supported by immunocytochemistry for vascular endothelial growth factor receptor-1. Stromal derived factor-1 and vascular endothelial growth factor were highly expressed in MSC-MNC coculture in basal medium but not in osteoblastic medium. On the contrary, the expression levels of bone morphogenetic protein-2 and angiopoietin-1 were significantly higher in osteoblastic medium. Pericyte markers were highly expressed in both cocultures after 5 days. In conclusion, it was demonstrated endothelial cell and pericyte differentiation in MSC-MNC cocultures both in basal and osteoblastic medium indicating a potential for neovascularization for tissue engineering applications.
Assuntos
Indutores da Angiogênese/metabolismo , Diferenciação Celular , Células Endoteliais/citologia , Leucócitos Mononucleares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Pericitos/citologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Pericitos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
Sperm flagellar protein 2 (SPEF2) is essential for motile cilia, and lack of SPEF2 function causes male infertility and primary ciliary dyskinesia. Cilia are pointing out from the cell surface and are involved in signal transduction from extracellular matrix, fluid flow and motility. It has been shown that cilia and cilia-related genes play essential role in commitment and differentiation of chondrocytes and osteoblasts during bone formation. Here we show that SPEF2 is expressed in bone and cartilage. The analysis of a Spef2 knockout (KO) mouse model revealed hydrocephalus, growth retardation and death prior to five weeks of age. To further elucidate the causes of growth retardation we analyzed the bone structure and possible effects of SPEF2 depletion on bone formation. In Spef2 KO mice, long bones (tibia and femur) were shorter compared to wild type, and X-ray analysis revealed reduced bone mineral content. Furthermore, we showed that the in vitro differentiation of osteoblasts isolated from Spef2 KO animals was compromised. In conclusion, this study reveals a novel function for SPEF2 in bone formation through regulation of osteoblast differentiation and bone growth.