RESUMO
BACKGROUND: Recent studies suggested that genetic variants associated with monogenic bone disorders were involved in the pathogenesis of atypical femoral fractures (AFF). Here, we aim to identify rare genetic variants by whole exome sequencing in genes involved in monogenic rare skeletal diseases in 12 women with AFF and 4 controls without any fracture. RESULTS: Out of 33 genetic variants identified in women with AFF, eleven (33.3%) were found in genes belonging to the Wnt pathway (LRP5, LRP6, DAAM2, WNT1, and WNT3A). One of them was rated as pathogenic (p.Pro582His in DAAM2), while all others were rated as variants of uncertain significance according to ClinVar and ACMG criteria. CONCLUSIONS: Osteoporosis, rare bone diseases, and AFFs may share the same genes, thus making it even more difficult to identify unique risk factors.
Assuntos
Sequenciamento do Exoma , Fraturas do Fêmur , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Humanos , Feminino , Fraturas do Fêmur/genética , Fraturas do Fêmur/patologia , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Pessoa de Meia-Idade , Idoso , Predisposição Genética para Doença , Proteína Wnt1/genética , Proteína Wnt3A/genética , Via de Sinalização Wnt/genética , Osteoporose/genética , Osteoporose/patologia , Doenças Ósseas/genética , Estudos de Casos e ControlesRESUMO
This study explores the genetic factors associated with atypical femoral fractures (AFF), rare fractures associated with prolonged anti-resorptive therapy. AFF are fragility fractures that typically appear in the subtrochanteric or diaphyseal regions of the femur. While some cases resemble fractures in rare genetic bone disorders, the exact cause remains unclear. This study investigates 457 genes related to skeletal homeostasis in 13 AFF patients by exome sequencing, comparing the results with osteoporotic patients (n = 27) and Iberian samples from the 1000 Genomes Project (n = 107). Only one AFF case carried a pathogenic variant in the gene set, specifically in the ALPL gene. The study then examined variant accumulation in the gene set, revealing significantly more variants in AFF patients than in osteoporotic patients without AFF (p = 3.7 × 10-5), particularly in ACAN, AKAP13, ARHGEF3, P4HB, PITX2, and SUCO genes, all of them related to osteogenesis. This suggests that variant accumulation in bone-related genes may contribute to AFF risk. The polygenic nature of AFF implies that a complex interplay of genetic factors determines the susceptibility to AFF, with ACAN, SUCO, AKAP13, ARHGEF3, PITX2, and P4HB as potential genetic risk factors. Larger studies are needed to confirm the utility of gene set analysis in identifying patients at high risk of AFF during anti-resorptive therapy.
Assuntos
Conservadores da Densidade Óssea , Doenças Ósseas , Fraturas do Fêmur , Humanos , Fraturas do Fêmur/genética , Fêmur/patologia , Diáfises , DifosfonatosRESUMO
The mechanisms underlying AFF remain unclear, with hypotheses including bone turnover suppression and morphological variation. Recent studies have suggested a potential genetic susceptibility to AFF. A scoping review was conducted using PubMed to identify studies published since 2016. Twenty-one studies were identified, focusing on histological and genetic analysis of AFF patients and Bisphosphonates users. Biopsies and imaging modalities were used to assess histological and morphometric parameters, while genetic sequencing was performed to identify variants in target genes. Genetic studies identified variants in geranylgeranyl diphosphate synthase 1 (GGPS1) and CYP1A1 genes, which play roles in osteoclast function and drug metabolism, respectively. Functional analysis revealed reduced enzymatic activity in mutant variants of these genes, which could be further inhibited by BP use. Other genes, such as ATRAID, ALPL, and COL1A2, were also associated with AFF. Histomorphometric studies supported the hypothesis of bone turnover suppression in AFF, with alterations in tissue mechanical properties and microarchitecture observed, particularly in cortical bone. The findings suggest a potential genetic susceptibility to AFF, with variants in GGPS1 and CYP1A1 genes affecting osteoblast and osteoclast function. Bone turnover suppression and altered tissue properties contribute to the pathogenesis of AFF.
Assuntos
Conservadores da Densidade Óssea , Fraturas do Fêmur , Humanos , Fraturas do Fêmur/diagnóstico por imagem , Fraturas do Fêmur/genética , Fraturas do Fêmur/patologia , Citocromo P-450 CYP1A1/genética , Difosfonatos , Osso e Ossos/patologia , Predisposição Genética para DoençaRESUMO
Bone fractures are the most common form of musculoskeletal trauma worldwide. Numerous microRNAs (miRNAs) have been suggested to be participants in regulating bone-related diseases. Recent studies revealed the regulatory role of miR-22-3p in osteogenic differentiation, but its role in fracture healing has not been investigated previously. Here, a rat femoral fracture model was established, Bone marrow mesenchymal stem cells (BMSCs) were isolated to detect the specific function and underlying mechanisms of miR-22-3p. MiR-22-3p and sclerostin domain-containing 1 (SOSTDC1) expression was determined by RT-qPCR and immunohistochemistry staining. The levels of proteins associated with osteogenic differentiation were assessed by western blotting. Flow cytometry was conducted to identify the isolated rat BMSCs. Alizarin red staining, alkaline phosphatase staining and Oil Red O staining were used to evaluate the osteogenic and adipogenic differentiation of rat BMSCs. The interaction between miR-22-3p and SOSTDC1 was verified using a luciferase reporter assay. Haematoxylin and Eosin (H&E) staining of the bone tissues was performed to analyse the effect of miR-22-3p on histopathological changes in vivo. MiR-22-3p was downregulated in the callus tissues of rat femoral fracture, while the expression of SOSTDC1 was upregulated. The isolated rat BMSCs had the capacity for both osteogenic and adipogenic differentiation. The differentiation capacity of BMSCs into osteoblasts was increased by miR-22-3p overexpression. MiR-22-3p activated the PI3K/AKT pathway by targeting SOSTDC1. SOSTDC1 overexpression and PI3K/AKT signalling inhibitor LY294002 abolished the enhancing effect of miR-22-3p overexpression on the osteogenesis of BMSCs. Thus MiR-22-3p facilitated the femoral fracture healing in rats. MiR-22-3p overexpression promoted fracture healing via the activation of PI3K/AKT pathway by targeting SOSTDC1.
Assuntos
Fraturas do Fêmur , Células-Tronco Mesenquimais , MicroRNAs , Animais , Humanos , Ratos , Proteínas Adaptadoras de Transdução de Sinal/genética , Diferenciação Celular , Células Cultivadas , Fraturas do Fêmur/genética , Fraturas do Fêmur/metabolismo , Fraturas do Fêmur/patologia , Consolidação da Fratura , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Atypical femur fractures (AFFs), considered rare associations of bisphosphonates, have also been reported in patients with monogenic bone disorders without bisphosphonate use. The exact association between AFFs and monogenic bone disorders remains unknown. Our aim was to determine the prevalence of monogenic bone disorders in a Dutch AFF cohort. AFF patients were recruited from two specialist bone centers in the Netherlands. Medical records of the AFF patients were reviewed for clinical features of monogenic bone disorders. Genetic variants identified by whole-exome sequencing in 37 candidate genes involved in monogenic bone disorders were classified based on the American College of Medical Genetics and Genomics (ACMG) classification guidelines. Copy number variations overlapping the candidate genes were also evaluated using DNA array genotyping data. The cohort comprises 60 AFF patients (including a pair of siblings), with 95% having received bisphosphonates. Fifteen AFF patients (25%) had clinical features of monogenic bone disorders. Eight of them (54%), including the pair of siblings, had a (likely) pathogenic variant in either PLS3, COL1A2, LRP5, or ALPL. One patient carried a likely pathogenic variant in TCIRG1 among patients not suspected of monogenic bone disorders (2%). In total, nine patients in this AFF cohort (15%) had a (likely) pathogenic variant. In one patient, we identified a 12.7 Mb deletion in chromosome 6, encompassing TENT5A. The findings indicate a strong relationship between AFFs and monogenic bone disorders, particularly osteogenesis imperfecta and hypophosphatasia, but mainly in individuals with symptoms of these disorders. The high yield of (likely) pathogenic variants in AFF patients with a clinical suspicion of these disorders stresses the importance of careful clinical evaluation of AFF patients. Although the relevance of bisphosphonate use in this relationship is currently unclear, clinicians should consider these findings in medical management of these patients. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Assuntos
Conservadores da Densidade Óssea , Fraturas do Fêmur , ATPases Vacuolares Próton-Translocadoras , Humanos , Prevalência , Variações do Número de Cópias de DNA , Fraturas do Fêmur/epidemiologia , Fraturas do Fêmur/genética , Difosfonatos/uso terapêutico , Fêmur , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
OBJECTIVE: Atypical femur fractures (AFFs) are rare fragility fractures originating at the lateral cortex of the femur, affecting the subtrochanteric or diaphyseal area of thebone with a transverse morphology. Occurrence of AFF is specifically associated with a small number of rare monogenic congenital metabolic bone disorders, such as hypophosphatasia, and with long-term treatment with antiresorptiondrugs. The exact pathogenesis of these fractures remains poorly understood and, except for cases of diagnosed HPP or other AFF-causing bone diseases, it is not possible to assess which patients are at higher riskof developing AFFs as a consequence of anti-resorption therapy. DESIGN: We genetically screened 25 unrelated patients who had developed at least one AFF. INTERVENTION: Genetic screening was performed through a nextgeneration sequencing analysis with a customized panel containing 76 human genes involved in the regulation of the mineralization processWe genetically screened 25 unrelated patients who had developed at least one AFF. RESULTS: We found a relatively high frequency (32.0%) of heterozygous rare variants inthe SLC34A1 and SLC9A3R1 genes, two genes whose heterozygous inactivating mutations have been respectively associated with autosomal dominant hypophosphatemic nephrolithiasis/osteoporosis types 1 and 2 (NPHLOP1and NPHLOP2). Other heterozygous rare variants were found in the BMPR1B, CYP27B1, FBN1, MEPE, PIGO, and PHOSPHO1 genes, each in a single AFF case (4.0%). CONCLUSIONS AND RELEVANCE: Our findings suggest that rarevariants of SLC34A1 and SLC9A3R1 could represent a possible genetic risk factor for the occurrence of AFFs. On the other hand, AFFs could represent an unsuspected clinical manifestation and/or an anti-resorption therapycorrelatedadverse event in patients with NPHLOP disorders.
Assuntos
Fraturas do Fêmur , Trocadores de Sódio-Hidrogênio , Humanos , Conservadores da Densidade Óssea/uso terapêutico , Difosfonatos/uso terapêutico , Fraturas do Fêmur/genética , Fêmur/patologia , Osteoporose/tratamento farmacológico , Radiografia , Fatores de Risco , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
Atypical femoral fractures (AFF) are rare fragility fractures in the subtrocantheric or diaphysis femoral region associated with long-term bisphosphonate (BP) treatment. The etiology of AFF is still unclear even though a genetic basis is suggested. We performed whole exome sequencing (WES) analysis of 12 patients receiving BPs for at least 5 years who sustained AFFs and 4 controls, also long-term treated with BPs but without any fracture. After filtration and prioritization of rare variants predicted to be damaging and present in genes shared among at least two patients, a total of 272 variants in 132 genes were identified. Twelve of these genes were known to be involved in bone metabolism and/or AFF, highlighting DAAM2 and LRP5, both involved in the Wnt pathway, as the most representative. Afterwards, we intersected all mutated genes with a list of 34 genes obtained from a previous study of three sisters with BP-related AFF, identifying nine genes. One of these (MEX3D) harbored damaging variants in two AFF patients from the present study and one shared among the three sisters. Gene interaction analysis using the AFFNET web suggested a complex network among bone-related genes as well as with other mutated genes. BinGO biological function analysis highlighted cytoskeleton and cilium organization. In conclusion, several genes and their interactions could provide genetic susceptibility to AFF, that along with BPs treatment and in some cases with glucocorticoids may trigger this so feared complication.
Assuntos
Conservadores da Densidade Óssea/efeitos adversos , Difosfonatos/efeitos adversos , Fraturas do Fêmur/etiologia , Fraturas do Fêmur/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Osteoporose Pós-Menopausa/tratamento farmacológico , Idoso , Estudos de Casos e Controles , Feminino , Fraturas do Fêmur/genética , Perfilação da Expressão Gênica , Humanos , Osteoporose Pós-Menopausa/patologiaRESUMO
CONTEXT: Atypical femoral fractures (AFFs) are very rare atraumatic or mild trauma fractures in the subtrochanteric region or femoral shaft. Some unique genetic variants in Asian populations might confer susceptibility to AFF, since the incidence of AFFs is higher in Asian populations. OBJECTIVE: Because rare variants have been found to be causative in some diseases and the roles of osteomalacia causative genes have not been reported, we investigated rare variants in genes causing abnormal mineralization. METHODS: Exome sequencing was performed to detect variants in gene coding and boundary regions, and the frequencies of deleterious rare alleles were compared between Japanese patients with AFF (nâ =â 42) and controls of the 4.7KJPN panel of Tohoku Medical Megabank by whole genome sequencing (nâ =â 4773). RESULTS: The frequency of the deleterious rare allele of ENPP1 was significantly increased in AFF (Pâ =â .0012, corrected P [Pc]â =â .0155, OR 4.73, 95% CI 2.15-10.40). In multigene panel analysis, the frequencies of deleterious rare alleles of candidate genes were increased in AFF (Pâ =â .0025, OR 2.72, 95% CI 1.49-4.93). Principal component analysis of bone metabolism markers identified a subgroup of patients with AFF with higher frequencies of deleterious rare alleles in ENPP1 (Pâ =â 4.69 × 10-5, Pcâ =â .0006, OR 8.47, 95% CI 3.76-19.09) and the candidate genes (Pâ =â 1.08 × 10-5, OR 5.21, 95% CI 2.76-9.86). CONCLUSION: AFF is associated with genes including ENPP1 that cause abnormal mineralization, suggesting that osteomalacia is an underlying condition predisposing to AFF and that higher incident rates of AFFs in Asian populations might be explained by the genetic risk factors including ENPP1.
Assuntos
Conservadores da Densidade Óssea , Doenças Ósseas , Raquitismo Hipofosfatêmico Familiar , Fraturas do Fêmur , Osteomalacia , Alelos , Conservadores da Densidade Óssea/efeitos adversos , Doenças Ósseas/genética , Difosfonatos/efeitos adversos , Raquitismo Hipofosfatêmico Familiar/complicações , Feminino , Fraturas do Fêmur/epidemiologia , Fraturas do Fêmur/genética , Humanos , Masculino , Osteomalacia/genéticaRESUMO
We previously identified transient brown adipocyte-like cells associated with heterotopic ossification (HO). These ancillary cells support new vessel synthesis essential to bone formation. Recent studies have shown that the M2 macrophage contributes to tissue regeneration in a similar way. To further define the phenotype of these brown adipocyte-like cells they were isolated and characterized by single-cell RNAseq (scRNAseq). Analysis of the transcriptome and the presence of surface markers specific for macrophages suggest that these cells are M2 macrophages. To validate these findings, clodronate liposomes were delivered to the tissues during HO, and the results showed both a significant reduction in these macrophages as well as bone formation. These cells were isolated and shown in culture to polarize towards either M1 or M2 similar to other macrophages. To confirm that these are M2 macrophages, mice received lipopolysacheride (LPS), which induces proinflammation and M1 macrophages. The results showed a significant decrease in this specific population and bone formation, suggesting an essential role for M2 macrophages in the production of bone. To determine if these macrophages are specific to HO, we isolated these cells using fluorescence-activated cell sorting (FACS) from a bone defect model and subjected them to scRNAseq. Surprisingly, the macrophage populations overlapped between the two groups (HO-derived versus callus) suggesting that they may be essential ancillary cells for bone formation in general and not selective to HO. Of further note, their unique metabolism and lipogenic properties suggest the potential for unique cross talk between these cells and the newly forming bone.
Assuntos
Adipócitos Marrons/metabolismo , Fraturas do Fêmur/metabolismo , Fêmur/metabolismo , Macrófagos/metabolismo , Ossificação Heterotópica/metabolismo , Osteogênese , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/patologia , Animais , Plasticidade Celular , Células Cultivadas , Ácido Clodrônico/farmacologia , Modelos Animais de Doenças , Fraturas do Fêmur/genética , Fraturas do Fêmur/patologia , Fêmur/patologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos Transgênicos , Ossificação Heterotópica/genética , Ossificação Heterotópica/patologia , Fagocitose , Fenótipo , Receptores Adrenérgicos beta 3/metabolismo , TranscriptomaRESUMO
Osteoporosis is the most common metabolic bone disorder and nitrogen-containing bisphosphonates (BP) are a first line treatment for it. Yet, atypical femoral fractures (AFF), a rare adverse effect, may appear after prolonged BP administration. Given the low incidence of AFF, an underlying genetic cause that increases the susceptibility to these fractures is suspected. Previous studies uncovered rare CYP1A1 mutations in osteoporosis patients who suffered AFF after long-term BP treatment. CYP1A1 is involved in drug metabolism and steroid catabolism, making it an interesting candidate. However, a functional validation for the AFF-associated CYP1A1 mutations was lacking. Here we tested the enzymatic activity of four such CYP1A1 variants, by transfecting them into Saos-2 cells. We also tested the effect of commonly used BPs on the enzymatic activity of the CYP1A1 forms. We demonstrated that the p.Arg98Trp and p.Arg136His CYP1A1 variants have a significant negative effect on enzymatic activity. Moreover, all the BP treatments decreased CYP1A1 activity, although no specific interaction with CYP1A1 variants was found. Our results provide functional support to the hypothesis that an additive effect between CYP1A1 heterozygous mutations p.Arg98Trp and p.Arg136His, other rare mutations and long-term BP exposure might generate susceptibility to AFF.
Assuntos
Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Fraturas do Fêmur/genética , Fraturas do Fêmur/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Citocromo P-450 CYP1A1/química , Difosfonatos/uso terapêutico , Fraturas do Fêmur/enzimologia , Humanos , Incidência , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Filogenia , Alinhamento de SequênciaRESUMO
Fracture healing is a complicated process affected by many factors, such as inflammatory responses and angiogenesis. Omentin-1 is an adipokine with anti-inflammatory properties, but whether omentin-1 affects the fracture healing process is still unknown. Here, by using global omentin-1 knockout (omentin-1-/-) mice, we demonstrated that omentin-1 deficiency resulted in delayed fracture healing in mice, accompanied by increased inflammation and osteoclast formation, and decreased production of platelet-derived growth factor-BB (PDGF-BB) and osteogenesis-promoting vessels that are strongly positive for CD31 and Endomucin (CD31hiEmcnhi) in the fracture area. In vitro, omentin-1 treatment suppressed the ability of the tumor necrosis factor-α (TNF-α)-activated macrophages to stimulate multi-nuclear osteoclast formation, resulting in a significant increase in the generation of mono-nuclear preosteoclasts and PDGF-BB, a pro-angiogenic protein that is abundantly secreted by preosteoclasts. PDGF-BB significantly augmented endothelial cell proliferation, tube formation and migration, whereas direct treatment with omentin-1 did not induce obvious effects on angiogenesis activities of endothelial cells. Our study suggests a positive role of omentin-1 in fracture healing, which may be associated with the inhibition of inflammation and stimulation of preosteoclast PDGF-BB-mediated promotion of CD31hiEmcnhi vessel formation.
Assuntos
Citocinas/genética , Fraturas do Fêmur/genética , Consolidação da Fratura , Proteínas Ligadas por GPI/genética , Lectinas/genética , Sialoglicoproteínas/metabolismo , Animais , Movimento Celular , Modelos Animais de Doenças , Feminino , Fraturas do Fêmur/etiologia , Fraturas do Fêmur/imunologia , Técnicas de Inativação de Genes , Camundongos , Osteoclastos/metabolismo , Osteogênese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Células RAW 264.7 , Microtomografia por Raio-XRESUMO
PURPOSE OF REVIEW: Atypical femur fractures (AFFs) are rare subtrochanteric or diaphyseal fractures regarded as side effects of bisphosphonates (BPs), possibly with a genetic background. Here, we summarize the most recent knowledge about genetics of AFFs. RECENT FINDINGS: AFF has been reported in 57 patients with seven different monogenic bone disorders including hypophosphatasia and osteogenesis imperfecta; 56.1% had never used BPs, while 17.5% were diagnosed with the disorder only after the AFF. Gene mutation finding in familial and sporadic cases identified possible AFF-related variants in the GGPS1 and ATRAID genes respectively. Functional follow-up studies of mutant proteins showed possible roles in AFF. A recent small genome-wide association study on 51 AFF cases did not identify significant hits associated with AFF. Recent findings have strengthened the hypothesis that AFFs have underlying genetic components but more studies are needed in AFF families and larger cohorts of sporadic cases to confirm previous results and/or find novel gene variants involved in the pathogenesis of AFFs.
Assuntos
Fraturas do Fêmur/genética , Conservadores da Densidade Óssea/efeitos adversos , Doenças Ósseas/tratamento farmacológico , Doenças Ósseas/genética , Dimetilaliltranstransferase , Farnesiltranstransferase , Fraturas do Fêmur/etiologia , Estudo de Associação Genômica Ampla , Geraniltranstransferase , Humanos , Proteínas de Membrana Transportadoras , MutaçãoRESUMO
Mesenchymal stem cells (MSCs) have the potential to reduce healing time and treat nonunion in fracture patients. In this study, bone marrow MSCs-derived extracellular vesicles (B-EVs) were firstly extracted and identified. CD9-/- and normal mice were enrolled for the establishment of fracture models and then injected with B-EVs. Osteoblast differentiation and fracture recovery were estimated. The levels of osteoblast-related genes were detected, and differentially expressed microRNAs (miRs) in B-EVs-treated normal fracture mice were screened and verified. The downstream mechanisms of miR were predicted and assessed. The loss-of functions of miR-335 in B-EV and gain-of-functions of VapB were performed in animal and cell experiments to evaluate their roles in bone fracture. Collectively, B-EVs promoted bone fracture recovery and osteoblast differentiation by releasing miR-335. miR-335 downregulation in B-EVs impaired B-EV functions in fracture recovery and osteoblast differentiation. miR-335 could target VapB, and VapB overexpression reversed the effects of B-EVs on osteoblast differentiation. B-EV treatment activated the Wnt/ß-catenin pathway in fracture mice and osteoblasts-like cells. Taken together, the study suggested that B-EVs carry miR-335 to promote bone fracture recovery via VapB and the Wnt/ß-catenin pathway. This study may offer insights into bone fracture treatment.
Assuntos
Exossomos/metabolismo , Exossomos/transplante , Fraturas do Fêmur/cirurgia , Fêmur/metabolismo , Consolidação da Fratura , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Células 3T3 , Animais , Diferenciação Celular , Modelos Animais de Doenças , Exossomos/genética , Fraturas do Fêmur/genética , Fraturas do Fêmur/metabolismo , Fraturas do Fêmur/patologia , Fêmur/lesões , Fêmur/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Osteoblastos/patologia , Osteogênese , Tetraspanina 29/genética , Tetraspanina 29/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Via de Sinalização WntAssuntos
Fraturas do Fêmur/enzimologia , Fêmur/enzimologia , Consolidação da Fratura , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Modelos Animais de Doenças , Fraturas do Fêmur/genética , Fraturas do Fêmur/fisiopatologia , Fêmur/lesões , Fêmur/fisiopatologia , Masculino , MicroRNAs/genética , Fosforilação , RNA Interferente Pequeno , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
microRNAs (miRNAs) have recently been recognized as playing an important role in bone-associated diseases. This study investigated whether the reduced miR-155-5p in steroid-associated osteonecrosis of the femoral head (ONFH) attenuated osteogenic differentiation and cell proliferation by targeting GSK3B. Bone marrow was collected from the proximal femurs of patients with steroid-associated ONFH (n = 10) and patients with new femoral neck fracture (n = 10) and mesenchymal stem cells (MSCs) were isolated. The expression profile, the biological function of miR-155-5p, and the interaction between miR-155-5p and GSK3B were investigated by cell viability measurement, western blot, real-time polymerase chain reaction, luciferase reporter assay, and Alizarin Red S (ARS) staining of MSCs. The MSCs that were obtained from the femoral neck fracture group and from the steroid-associated ONFH group were transfected with or without miR-155-5p. We found that, in ONFH samples, the level of mature miR-155-5p was significantly lower than that of control samples. By inhibiting GSK3B, miR-155-5p promoted the nuclear translocation of ß-catenin, increased the expression of osteogenesis-related genes, and facilitated the proliferation and differentiation of MSCs. Restoring the expression of GSK3B in MSCs partially reversed the effect of miR-155-5p. These findings suggest that reduced miR-155-5p in steroid-associated ONFH attenuates osteogenic differentiation and cell proliferation by increased levels of GSK3B and inhibition of Wnt signaling.
Assuntos
Necrose da Cabeça do Fêmur/induzido quimicamente , Necrose da Cabeça do Fêmur/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Osteogênese/genética , Esteroides/efeitos adversos , Sequência de Bases , Núcleo Celular/metabolismo , Proliferação de Células/genética , Feminino , Fraturas do Fêmur/genética , Fraturas do Fêmur/patologia , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Transporte Proteico , Transdução de Sinais , beta Catenina/metabolismoRESUMO
BACKGROUND: In the stabilization of femoral fractures in mono- and polytrauma, clinical practice has shown better care through intramedullary nailing. However, the reason why this is the case is not fully understood. In addition to concomitant injuries, the immunological aspect is increasingly coming to the fore. Neutrophil granulocytes (PMNL), in particular next to other immunological cell types, seem to be associated with the fracture healing processes. For this reason, the early phase after fracture (up to 72 h after trauma) near the fracture zone in muscle tissue was investigated in a pig model. MATERIAL AND METHODS: A mono- and polytrauma pig model (sole femur fracture or blunt thoracic trauma, hemorrhagic shock, liver laceration, and femur fracture) was used to demonstrate the immunological situation through muscle biopsies and their analysis by histology and qRT-PCR during a 72 h follow-up phase. Two stabilization methods were used (intramedullary nail vs. external fixator) and compared with a nontraumatized sham group. RESULTS: Monotrauma shows higher PMNL numbers in muscle tissue compared with polytrauma (15.52 ± 5.39 mono vs. 8.23 ± 3.36 poly; p = 0.013), regardless of the treatment strategy. In contrast, polytrauma shows a longer lasting invasion of PMNL (24 h vs. 72 h). At 24 h in the case of monotrauma, the fracture treated with external fixation shows more PMNL than the fracture treated with intramedullary nailing (p = 0.026). This difference cannot be determined in polytrauma probably caused by a generalized immune response. Both monotrauma and polytrauma show a delayed PMNL increase in the muscle tissue of the uninjured side. The use of intramedullary nailing in monotrauma resulted in a significant increase in IL-6 (2 h after trauma) and IL-8 (24 and 48 h after trauma) transcription. CONCLUSION: The reduction of PMNL invasion into the nearby muscle tissue of a monotrauma femur fracture stabilized by intramedullary nailing supports the advantages found in everyday clinical practice and therefore underlines the usage of nailing. For the polytrauma situation, the fixation seems to play a minor role, possibly due to a generalized immune reaction.
Assuntos
Modelos Animais de Doenças , Fraturas do Fêmur/cirurgia , Fixação de Fratura/métodos , Granulócitos/patologia , Traumatismo Múltiplo/cirurgia , Músculos/patologia , Animais , Pinos Ortopédicos , Citocinas/genética , Fixadores Externos , Fraturas do Fêmur/genética , Fixação de Fratura/instrumentação , Fixação Intramedular de Fraturas/instrumentação , Fixação Intramedular de Fraturas/métodos , Consolidação da Fratura/genética , Expressão Gênica , Humanos , Traumatismo Múltiplo/genética , Músculos/metabolismo , SuínosRESUMO
BACKGROUND: An enzymatic crosslinking strategy using hydrogen peroxide and horseradish peroxidase is receiving increasing attention for application with in situ-formed hydrogels (IFHs). Several studies have reported the application of IFHs in cell delivery and tissue engineering. IFHs may also be ideal carrier materials for bone repair, although their potential as a carrier for bone morphogenetic protein (BMP)-2 has yet to be examined. Here, we examined the effect of an IFH made of hyaluronic acid (IFH-HA) containing BMP-2 in promoting osteogenesis in a mouse refractory fracture model. METHODS: Immediately following a fracture procedure, animals either received no treatment (control) or an injection of IFH-HA/PBS or IFH-HA containing 2 µg BMP-2 (IFH-HA/BMP-2) into the fracture site (n = 16, each treatment). RESULTS: Fracture sites injected with IFH-HA/BMP-2 showed significantly greater bone volume, bone mineral content, and bone union compared with sites receiving no treatment or treated with IFH-HA/PBS alone (each n = 10). Gene expression levels of osteogenic markers, Alpl, Bglap, and Osx, were significantly raised in the IFH-HA/BMP-2 group compared to the IFH-HA/PBS and control groups (each n = 6). CONCLUSION: IFH-HA/BMP-2 may contribute to the treatment of refractory fractures.
Assuntos
Proteína Morfogenética Óssea 2/administração & dosagem , Proteína Morfogenética Óssea 2/farmacologia , Fraturas do Fêmur/tratamento farmacológico , Fraturas do Fêmur/fisiopatologia , Fêmur/metabolismo , Fêmur/patologia , Hidrogéis/administração & dosagem , Osteogênese/efeitos dos fármacos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Densidade Óssea , Modelos Animais de Doenças , Fraturas do Fêmur/genética , Fraturas do Fêmur/patologia , Fêmur/fisiopatologia , Expressão Gênica/efeitos dos fármacos , Injeções Intralesionais , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Osteogênese/genética , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismoRESUMO
Systemic and local posttraumatic responses are often monitored on mRNA expression level using quantitative real-time PCR (qRT-PCR), which requires normalisation to adjust for confounding sources of variability. Normalisation requests reference (housekeeping) genes stable throughout time and divergent experimental conditions in the tissue of interest, which are crucial for a reliable and reproducible gene expression analysis. Although previous animal studies analysed reference genes following isolated trauma, this multiple-trauma gene expression analysis provides a notable study analysing reference genes in primarily affected (i.e. bone/fracture callus and hypothalamus) and secondarily affected organs (i.e. white adipose tissue, liver, muscle and spleen), following experimental long bone fracture and traumatic brain injury. We considered tissue-specific and commonly used top-ranked reference candidates from different functional groups that were evaluated applying the established expression stability analysis tools NormFinder, GeNorm, BestKeeper and RefFinder. In conclusion, reference gene expression in primary organs is highly time point as well as tissue-specific, and therefore requires careful evaluation for qRT-PCR analysis. Furthermore, the general application of Ppia, particularly in combination with a second reference gene, is strongly recommended for the analysis of systemic effects in the case of indirect trauma affecting secondary organs through local and systemic pathophysiological responses.
Assuntos
Lesões Encefálicas Traumáticas/genética , Fraturas do Fêmur/genética , Perfilação da Expressão Gênica/normas , Transcriptoma , Animais , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/metabolismo , Feminino , Fraturas do Fêmur/complicações , Fraturas do Fêmur/metabolismo , Perfilação da Expressão Gênica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Padrões de ReferênciaRESUMO
The expression profile and specific roles of microRNAs (miRNAs) in regulation of atrophic bone nonunion are not fully understood. Here, we present evidence that miRNAs are involved in regulation of several osteogenic genes and may contribute to the development of atrophic bone nonunion.The miRNA expression profile of repairing tissues in atrophic bone nonunion patients (group A) and in callus tissues from patients with healed fractures (group B) were quantitatively measured. microRNA microarrays were used to identify differentially expressed miRNAs, and the bioinformatics methods were used to predict the potential target genes. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and dual-luciferase reporter assay were performed in human bone marrow stromal cells (hBMSCs) to validate the microarray results.Nine miRNAs in group A were up-regulated 1.5 times compared to group B, while the other 9 miRNAs in group A were down-regulated 1.5 times. Several target regions of these miRNAs were identified in the osteogenic genes, as well as in the other genes in their families or related regulatory factors. Four miRNAs (hsa-miR-149, hsa-miR-221, hsa-miR-628-3p, and hsa-miR-654-5p) could play important roles in regulating bone nonunion development. hBMSCs transfected with these miRNAs significantly decreased mRNA levels of alkaline phosphatase, liver/bone/kidney (ALPL), platelet derived growth factor subunit A (PDGFA), and bone morphogenetic protein 2 (BMP2). Lower protein expression levels were observed using western blotting, confirming that ALPL, PDGFA, and BMP2 were directly targeted by hsa-miR-149, hsa-miR-221, and hsa-miR-654-5p, respectively.In summary, hsa-miR-149, hsa-miR-221, and hsa-miR-654-5p may play important biological roles by repressing osteogenic target genes ALPL, PDGFA, and BMP2, and, therefore, contributing to progression of atrophic bone nonunion.