RESUMO
Osteoporotic vertebral compression fractures (OVCFs) are the most prevalent fractures among patients with osteoporosis, leading to severe pain, deformities, and even death. This study explored the use of ectopic embryonic calvaria derived mesenchymal stem cells (EE-cMSCs), which are known for their superior differentiation and proliferation capabilities, as a potential treatment for bone regeneration in OVCFs. We evaluated the impact of EE-cMSCs on osteoclastogenesis in a RAW264.7 cell environment, which was induced by the receptor activator of nuclear factor kappa-beta ligand (RANKL), using cytochemical staining and quantitative real-time PCR. The osteogenic potential of EE-cMSCs was evaluated under various hydrogel conditions. An osteoporotic vertebral body bone defect model was established by inducing osteoporosis in rats through bilateral ovariectomy and creating defects in their coccygeal vertebral bodies. The effects of EE-cMSCs were examined using micro-computed tomography (µCT) and histology, including immunohistochemical analyses. In vitro, EE-cMSCs inhibited osteoclast differentiation and promoted osteogenesis in a 3D cell culture environment using fibrin hydrogel. Moreover, µCT and histological staining demonstrated increased new bone formation in the group treated with EE-cMSCs and fibrin. Immunostaining showed reduced osteoclast activity and bone resorption, alongside increased angiogenesis. Thus, EE-cMSCs can effectively promote bone regeneration and may represent a promising therapeutic approach for treating OVCFs.
Assuntos
Diferenciação Celular , Modelos Animais de Doenças , Células-Tronco Mesenquimais , Osteogênese , Osteoporose , Crânio , Animais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Ratos , Crânio/patologia , Camundongos , Osteoporose/patologia , Osteoporose/metabolismo , Osteoporose/terapia , Feminino , Células RAW 264.7 , Osteoclastos/metabolismo , Regeneração Óssea , Ratos Sprague-Dawley , Transplante de Células-Tronco Mesenquimais/métodos , Corpo Vertebral/metabolismo , Microtomografia por Raio-X , Fraturas por Osteoporose/terapia , Fraturas por Osteoporose/metabolismo , Fraturas por Osteoporose/patologiaRESUMO
Biodegradable polymers have been used with various systems for tissue engineering. Among them, poly(lactic-co-glycolic) acid (PLGA) has been widely used as a biomaterial for bone regeneration because of its great biocompatibility and biodegradability properties. However, there remain substantial cruxes that the by-products of PLGA result in an acidic environment at the implanting site, and the polymer has a weak mechanical property. In our previous study, magnesium hydroxide (MH) and bone extracellular matrix are combined with a PLGA scaffold (PME) to improve anti-inflammation and mechanical properties and osteoconductivity. In the present study, the development of a bioactive nanocomplex (NC) formed along with polydeoxyribonucleotide and bone morphogenetic protein 2 (BMP2) provides synergistic abilities in angiogenesis and bone regeneration. This PME hybrid scaffold immobilized with NC (PME/NC) achieves outstanding performance in anti-inflammation, angiogenesis, and osteogenesis. Such an advanced PME/NC scaffold suggests an integrated bone graft substitute for bone regeneration.
RESUMO
In most avian species, the early embryo suspends development when the ambient temperature is too low; the resultant dormant state is called cold torpor. However, very little is known about dormant avian embryos at the cellular level. To investigate the molecular processes that occur in the chicken blastoderm during cold torpor, we performed transcriptome analysis and investigated cellular responses in dormant embryos. In embryos stored at low temperature, we observed up-regulation of genes and proteins related to endoplasmic reticulum stress and stress-activated protein kinase signaling. In addition, the proportion of early apoptotic cells rose dramatically during torpor, whereas the proportion of late apoptotic cells was unchanged. Cell cycle analysis revealed that mitotic arrest occurred at the G2 phase in a DNA damage-independent manner and that the arrest was alleviated after incubation at 37°C. Our data suggest that the dormant avian embryo tolerates cold stress by inducing stress-tolerance pathways, inhibiting late apoptosis, and triggering cell cycle arrest at the G2 phase.-Ko, M. H., Hwang, Y. S., Rim, J. S., Han, H. J., Han, J. Y. Avian blastoderm dormancy arrests cells in G2 and suppresses apoptosis.
Assuntos
Apoptose/fisiologia , Blastoderma/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Animais , Embrião de Galinha , Retículo Endoplasmático/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Transdução de Sinais/fisiologia , Estresse Fisiológico/fisiologia , TranscriptomaRESUMO
BACKGROUND: Primordial germ cells (PGCs), the precursors of functional gametes, have distinct characteristics and exhibit several unique molecular mechanisms to maintain pluripotency and germness in comparison to somatic cells. They express germ cell-specific RNA binding proteins (RBPs) by modulating tissue-specific cis- and trans-regulatory elements. Studies on gene structures of chicken vasa homologue (CVH), a chicken RNA binding protein, involved in temporal and spatial regulation are thus important not only for understanding the molecular mechanisms that regulate germ cell fate, but also for practical applications of primordial germ cells. However, very limited studies are available on regulatory elements that control germ cell-specific expression in chicken. Therefore, we investigated the intricate regulatory mechanism(s) that governs transcriptional control of CVH. RESULTS: We constructed green fluorescence protein (GFP) or luciferase reporter vectors containing the various 5' flanking regions of CVH gene. From the 5' deletion and fragmented assays in chicken PGCs, we have identified a CVH promoter that locates at -316 to +275 base pair fragment with the highest luciferase activity. Additionally, we confirmed for the first time that the 5' untranslated region (UTR) containing intron 1 is required for promoter activity of the CVH gene in chicken PGCs. Furthermore, using a transcription factor binding prediction, transcriptome analysis and siRNA-mediated knockdown, we have identified that a set of transcription factors play a role in the PGC-specific CVH gene expression. CONCLUSIONS: These results demonstrate that cis-elements and transcription factors localizing in the 5' flanking region including the 5' UTR and an intron are important for transcriptional regulation of the CVH gene in chicken PGCs. Finally, this information will contribute to research studies in areas of reproductive biology, constructing of germ cell-specific synthetic promoter for tracing primordial germ cells as well as understanding the transcriptional regulation for maintaining germness in PGCs.
RESUMO
The adipocytes are the predominant cell types that constitute the bulk of the thymic microenvironment by the fifth decade of life in healthy humans. An age-related increase in thymic adiposity is associated with reduced thymopoiesis and compromised immune surveillance in the elderly. However, the mechanisms regulating the generation of intrathymic adipocytes during aging remain to be elucidated. Here, we report that the CD45- thymic stromal cells (TSCs) are amenable to adipogenesis. We identified that the Wnt inhibitor axin is expressed in the lymphoid as well as stromal cells of the thymus with increased expression in CD45- TSCs of older mice. Knockdown of axin by RNA interference in CD45- primary TSCs led to a marked reduction in adipogenesis with significantly lower expression of adipogenic transcripts peroxisome proliferator-activated receptor 2 (PPAR), adipocyte fatty acid-binding protein (aP2), and perilipin. Age-related elevated axin expression was increased specifically in thymic fibroblasts and medullary thymic epithelial cells (TECs) but not in the cortical TEC or CD45+ cells. Consistent with a role of axin in promoting thymic adipogenesis, axin expression was also colocalized with lipid-expressing adipogenic cells in aging thymus. The prolongevity intervention, caloric restriction (CR), prevented the age-related increase in axin and the adipogenic cell in the thymus together with increase in thymic output. We have recently demonstrated that CR induces ghrelin, which can partially reverse thymic involution. Here, we show that axin expression is not affected by ablation of ghrelin receptors in aging mice, suggesting a ghrelin-independent mechanism for regulation of axin. Our data are consistent with the hypothesis that blocking the specific proadipogenic signals in the thymus may complement the present approaches to rejuvenate thymic function during aging.
Assuntos
Adiposidade , Envelhecimento/metabolismo , Grelina/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Timo/citologia , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/genética , Adulto , Animais , Proteína Axina , Restrição Calórica , Regulação da Expressão Gênica , Saúde , Humanos , Lipídeos , Masculino , Camundongos , Pessoa de Meia-Idade , Células Estromais/citologia , Timo/crescimento & desenvolvimento , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Cold exposure induces brown adipocytes in retroperitoneal fat (RP) of adult A/J mice but not in C57BL/6J (B6) mice. In contrast, induction of the mitochondrial uncoupling protein 1 gene (Ucp1) in interscapular brown adipose tissue (iBAT) shows no strain dependence. We now show that unlike iBAT, in which Ucp1 was expressed in the fetus and continued throughout life, in RP, Ucp1 was transiently expressed between 10 and 30 days of age and then disappeared. Similar to the lack of genetic variation in the expression of Ucp1 in iBAT during cold induction of adult mice, no genetic variation in Ucp1 expression in iBAT was detected during development. In contrast, UCP1-positive multilocular adipocytes, together with corresponding increases in Ucp1 expression, appeared in RP at 10 days of age in A/J and B6 mice, but with much higher expression in A/J mice. At 20 days of age, brown adipocytes represent the major adipocyte present in RP of A/J mice. The disappearance of brown adipocytes by 30 days of age suggested that tissue remodeling occurred in RP. Genetic variability in Ucp1 expression could not be explained by variation in the expression of selective transcription factors and signaling molecules of adipogenesis. In summary, the existence of genetic variability between A/J and B6 mice during the development of brown adipocyte expression in RP, but not in iBAT, suggests that developmental mechanisms for the brown adipocyte differentiation program are different in these adipose tissues.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo Marrom/fisiologia , Tecido Adiposo/fisiologia , Variação Genética , Tecido Adiposo/anatomia & histologia , Tecido Adiposo Marrom/anatomia & histologia , Animais , Peso Corporal , Temperatura Baixa , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos A , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Scarring, the end result of the wound healing process in adult mammals, is a problem of significant clinical importance. We observed that athymic nude-nu mice, similar to mammalian fetuses, are able to restore the structure and integrity of injured skin through a process resembling regeneration, where scar formation is absent. Among the postinjured skin tissues collected from athymic nude-nu, wild-type controls (C57BL/6J), severe-combined immunodeficient, Rag (lack of B and T cells), athymic (thymectomized neonates and adult C57BL/6J), and mice treated with an immunosuppressant (cyclosporin A), only athymic nude-nu mice showed: a lack of scar by histological examination (hematoxylin & eosin and Masson's trichrome staining), low levels of collagen (as determined by hydroxyproline content), high levels of hyaluronic acid, a statistically significant increase in elastic modulus for injured samples over unwounded (biomechanical testing) and low levels of the pro-scarring cytokines platelet-derived growth factor-B and transforming growth factor beta1. Additionally, immunohistochemical and Western blot analyses of postinjured tissues as well as flow cytometry analysis of blood samples showed the presence of CD8-positive cells in all studied animals except nude-nu mice. We conclude that scarless skin healing in athymic nude-nu mice provides a new model to study the influence of the immune system on tissue regeneration.
Assuntos
Cicatriz/imunologia , Pele/imunologia , Cicatrização/imunologia , Animais , Colágeno/metabolismo , Elasticidade , Citometria de Fluxo , Ácido Hialurônico/metabolismo , Hidroxiprolina/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Pele/metabolismo , Pele/patologia , Fenômenos Fisiológicos da Pele , Resistência à TraçãoRESUMO
High phenotypic variation in diet-induced obesity in male C57BL/6J inbred mice suggests a molecular model to investigate non-genetic mechanisms of obesity. Feeding mice a high-fat diet beginning at 8 wk of age resulted in a 4-fold difference in adiposity. The phenotypes of mice characteristic of high or low gainers were evident by 6 wk of age, when mice were still on a low-fat diet; they were amplified after being switched to the high-fat diet and persisted even after the obesogenic protocol was interrupted with a calorically restricted, low-fat chow diet. Accordingly, susceptibility to diet-induced obesity in genetically identical mice is a stable phenotype that can be detected in mice shortly after weaning. Chronologically, differences in adiposity preceded those of feeding efficiency and food intake, suggesting that observed difference in leptin secretion is a factor in determining phenotypes related to food intake. Gene expression analyses of adipose tissue and hypothalamus from mice with low and high weight gain, by microarray and qRT-PCR, showed major changes in the expression of genes of Wnt signaling and tissue re-modeling in adipose tissue. In particular, elevated expression of SFRP5, an inhibitor of Wnt signaling, the imprinted gene MEST and BMP3 may be causally linked to fat mass expansion, since differences in gene expression observed in biopsies of epididymal fat at 7 wk of age (before the high-fat diet) correlated with adiposity after 8 wk on a high-fat diet. We propose that C57BL/6J mice have the phenotypic characteristics suitable for a model to investigate epigenetic mechanisms within adipose tissue that underlie diet-induced obesity.
Assuntos
Regulação da Expressão Gênica , Obesidade/genética , Obesidade/patologia , Proteínas Adaptadoras de Transdução de Sinal , Tecido Adiposo , Ração Animal , Animais , Comportamento Animal , Peso Corporal , Modelos Animais de Doenças , Metabolismo Energético , Comportamento Alimentar , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , FenótipoRESUMO
Induction of brown adipocytes in white fat depots by adrenergic stimulation is a complex genetic trait in mice that affects the ability of the animal to regulate body weight. An 80-fold difference in expression of the mitochondrial uncoupling gene (Ucp1) at the mRNA and protein levels between A/J and C57BL/6J (B6) mice is controlled by allelic interactions among nine quantitative trait loci (QTLs) on eight chromosomes. Overlapping patterns of these QTLs also regulate expression levels of Pgc-1alpha, Pparalpha, and type 2 deiodinase. Independent validation that PPARalpha is associated with Ucp1 induction was obtained by treating mice with the PPARalpha agonist clofibrate, but not from the analysis of PPARalpha knockout mice. The most upstream sites of regulation for Ucp1 that differed between A/J and B6 were the phosphorylation of p38 mitogen-activated protein kinase and CREB and then followed by downstream changes in levels of mRNA for PPARgamma, PPARalpha, PGC-1alpha, and type 2 deiodinase. However, compared to Ucp1 expression, the two- to fourfold differences in the expression of these regulatory components are very modest. It is proposed that small variations in the levels of several transcriptional components of the Ucp1 enhanceosome interact synergistically to achieve large differences in Ucp1 expression.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Tecido Adiposo/citologia , Animais , Cromossomos , Clofibrato/farmacologia , Temperatura Baixa , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Hipolipemiantes/farmacologia , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Canais Iônicos , Camundongos , Camundongos Knockout , Proteínas Mitocondriais , PPAR alfa/agonistas , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Locos de Características Quantitativas , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição , Transcrição Gênica , Proteína Desacopladora 1 , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Iodotironina Desiodinase Tipo IIRESUMO
Adipocytes arise from multipotent stem cells of mesodermal origin, which also give rise to the muscle, bone, and cartilage lineages. However, signals and early molecular events that commit multipotent stem cells into the adipocyte lineage are not well established mainly due to lack of an adequate model system. We have identified a novel source of adult stem cells from the external murine ears referred to here as an ear mesenchymal stem cells (EMSC). EMSC have been isolated from several standard and mutant strains of mice. They are self-renewing, clonogenic, and multipotent, since they give rise to osteocytes, chondrocytes, and adipocytes. The in vitro characterization of EMSC indicates very facile adipogenic differentiation. Morphological, histochemical, and molecular analysis after the induction of differentiation showed that EMSC maintain adipogenic potentials up to fifth passage. A comparison of EMSC to the stromal-vascular (S-V) fraction of fat depots, under identical culture conditions (isobutyl-methylxanthine, dexamethasone, and insulin), revealed much more robust and consistent adipogenesis in EMSC than in the S-V fraction. In summary, we show that EMSC can provide a novel, easily obtainable, primary culture model for the study of adipogenesis.