RESUMO
PURPOSE: To identify, characterize, and compare the resident progenitor cell populations within the red-red, red-white, and white-white (WW) zones of freshly harvested human cadaver menisci and to characterize the vascularity of human menisci using immunofluorescence and 3-dimensional (3D) imaging. METHODS: Fresh adult human menisci were harvested from healthy donors. Menisci were enzymatically digested, mononuclear cells isolated, and characterized using flow cytometry with antibodies against mesenchymal stem cell surface markers (CD105, CD90, CD44, and CD29). Cells were expanded in culture, characterized, and compared with bone marrow-derived mesenchymal stem cells. Trilineage differentiation potential of cultured cells was determined. Vasculature of menisci was mapped in 3D using a modified uDisco clearing and immunofluorescence against vascular markers CD31, lectin, and alpha smooth muscle actin. RESULTS: There were no significant differences in the clonogenicity of isolated cells between the 3 zones. Flow cytometry showed presence of CD44+CD105+CD29+CD90+ cells in all 3 zones with high prevalence in the WW zone. Progenitors from all zones were found to be potent to differentiate to mesenchymal lineages. Larger vessels in the red-red zone of meniscus were observed spanning toward red-white, sprouting to smaller arterioles and venules. CD31+ cells were identified in all zones using the 3D imaging and co-localization of additional markers of vasculature (lectin and alpha smooth muscle actin) was observed. CONCLUSIONS: The presence of resident mesenchymal progenitors was evident in all 3 meniscal zones of healthy adult donors without injury. In addition, our results demonstrate the presence of vascularization in the WW zone. CLINICAL RELEVANCE: The existence of progenitors and presence of microvasculature in the WW zone of the meniscus suggests the potential for repair and biologic augmentation strategies in that zone of the meniscus in young healthy adults. Further research is necessary to fully define the functionality of the meniscal blood supply and its implications for repair.
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Menisco/irrigação sanguínea , Células-Tronco Mesenquimais/citologia , Cadáver , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Menisco/citologia , Células-Tronco/citologia , Adulto JovemRESUMO
The aim of this study was to investigate the combination effect of exercise training and eugenol supplementation on the hippocampus apoptosis induced by CPF. 64 adult male albino rats were randomly selected and devided into eight groups of eight including: control, exercise (EXE), chlorpyrifos (CPF), Control + Oil (Co + Oil), Control + DMSO (Co + DMSO), chlorpyrifos + eugenol (CPF + Sup), chlorpyrifos + exercise (CPF + Exe) and, chlorpyrifos + exercise + eugenol (CPF + Exe + Eu). Four experimental groups received intraperitoneal injection (5 days a week) of 3.0 mg/kg body weight CPF in DMSO for 6 consecutive weeks. The exercise groups performed aerobic 5 days per week over 4 weeks. Eugenol were administered by gavage. Finally, the animals were sacrificed using CO2 gas (a half of the rats were anesthetized with ketamine and xylazine and then perfused) to evaluate hippocampus histology and parameters. The results of this study showed that CPF injection significantly decreased BDNF, AChE and ATP in CA1 area of the hippocampus (p Ë 0.05). Also, CA1 apoptosis by tunnel assay, it was found that CPF receiving groups with different dosage, showed a significant increase compared to other groups, which was confirmed by increasing cytochrome C and procaspase-3 in CPF groups (p Ë 0.05). The result of this study show that 4 weeks of exercise training and eugenol supplementation does not improve the destructive effects of CPF in CA1 area of the hippocampus. As a result, it is recommended that future studies longer periods for treatment with exercise and eugenol supplementation.
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Apoptose/efeitos dos fármacos , Clorpirifos/toxicidade , Eugenol/uso terapêutico , Terapia por Exercício , Hipocampo/efeitos dos fármacos , Intoxicação por Organofosfatos/terapia , Condicionamento Físico Animal , Acetilcolinesterase/análise , Trifosfato de Adenosina/análise , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/análise , Caspase 3/análise , Terapia Combinada , Citocromos c/análise , Modelos Animais de Doenças , Eugenol/administração & dosagem , Hipocampo/enzimologia , Hipocampo/patologia , Masculino , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/patologia , Transtornos da Memória/terapia , Proteínas do Tecido Nervoso/análise , Intoxicação por Organofosfatos/tratamento farmacológico , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
BACKGROUND Intervertebral disc (IVD) degeneration is a common cause of lower back pain, which carries substantial morbidity and economic cost. Omega-3 fatty acids (n-3 FA) are known to reduce inflammatory processes with a relatively benign side effect profile. This study aimed to investigate the effect of n-3 FA supplementation on IVD degeneration. MATERIAL AND METHODS Two non-contiguous lumbar discs of 12 Sprague Dawley rats were needle-punctured to induce disc degeneration. Post-surgery, rats were randomly assigned to either a daily n-3 FA diet (530 mg/kg/day of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in a 2: 1 ratio, administered in sucrose solution) or control diet (sucrose solution only), which was given for the duration of the study. After 1 month, blood serum arachidonic acid/eicosapentaenoic acid (AA/EPA) ratios were analyzed. After 2 months, micro-MRI (magnetic resonance imaging) analysis and histological staining of disc explants were performed to analyze the IVD. RESULTS A reduction of blood AA/EPA ratios from 40 to 20 was demonstrated after 1 month of daily supplementation with n-3 FA. Micro-MRI analysis showed an injury-induced reduction of IVD hydration, which was attenuated in rats receiving n-3 FA. Histological evaluation demonstrated the destruction of nucleus pulposus tissue in response to needle puncture injury, which was less severe in the n-3 FA diet group. CONCLUSIONS The results of this study suggest that n-3 FA dietary supplementation reduces systemic inflammation by lowering AA/EPA ratios in blood serum and has potential protective effects on the progression of spinal disc degeneration, as demonstrated by reduced needle injury-induced dehydration of intervertebral discs and reduced histological signs of IVD degeneration.
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Ácidos Graxos Ômega-3/farmacologia , Degeneração do Disco Intervertebral/tratamento farmacológico , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos Ômega-3/metabolismo , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Dor Lombar/patologia , Imageamento por Ressonância Magnética/métodos , Imageamento por Ressonância Magnética/veterinária , Masculino , Núcleo Pulposo/citologia , Ratos , Ratos Sprague-DawleyRESUMO
The Revised Fibromyalgia Impact Questionnaire (FIQR), an updated version of the Fibromyalgia Impact Questionnaire (FIQ) achieved a better balance among different domains (i.e., function, overall impact, and symptom severity) and attempts to address the limitations of FIQ. As there is no Persian version of the FIQR available, we aimed to investigate the validity and reliability of a Persian translation of the FIQR in Iranian patients. After translating the FIQR into Persian, it was administered to 77 female patients with fibromyalgia syndrome. All of the patients filled out the questionnaire together with a Persian version of the FIQ, short form-12 (SF-12). The tender-point count was also calculated. One week later, FM patients filled out the Persian FIQR at their second visit. Reliability was analyzed by internal consistency and reproducibility including Cronbach's α coefficient and intra-class correlation coefficient. Construct validity was evaluated by Spearman's correlation coefficient and Pearson's correlation coefficient. Statistical analysis was performed using SPSS for Windows version 17.0. All patients included in this study were female, and the mean age was 38.23 ± 10.68 years. The total scores of the FIQR and FIQ were 49.77 ± 18.27 and 54.05 ± 14.00 that were closely correlated (r = 0.63, p < 0.01), and each of the three domains of the Persian FIQR was also correlated well with the three related FIQ domains (r = 0.36-0.63, p < 0.01). Also some significant inverse correlations of FIQR with quality-of-life (assessed by SF-12) domains and items were found. Cronbach's α was 0.87 for FIQR in the first visit. The Persian FIQR showed adequate reliability and validity. This instrument can be used in the clinical evaluation of Iranian patients with fibromyalgia.
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Fibromialgia/diagnóstico , Perfil de Impacto da Doença , Inquéritos e Questionários , Atividades Cotidianas , Adulto , Efeitos Psicossociais da Doença , Características Culturais , Feminino , Fibromialgia/fisiopatologia , Fibromialgia/psicologia , Humanos , Irã (Geográfico) , Pessoa de Meia-Idade , Medição da Dor , Valor Preditivo dos Testes , Qualidade de Vida , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , TraduçãoRESUMO
Replacement of lost cranial bone (partly mesodermal and partly neural crest-derived) is challenging and includes the use of nonviable allografts. To revitalize allografts, bone marrow-derived mesenchymal stromal cells (mesoderm-derived BM-MSCs) have been used with limited success. We hypothesize that coating of allografts with induced neural crest cell-mesenchymal progenitor cells (iNCC-MPCs) improves implant-to-bone integration in mouse cranial defects. Human induced pluripotent stem cells were reprogramed from dermal fibroblasts, differentiated to iNCCs and then to iNCC-MPCs. BM-MSCs were used as reference. Cells were labeled with luciferase (Luc2) and characterized for MSC consensus markers expression, differentiation, and risk of cellular transformation. A calvarial defect was created in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and allografts were implanted, with or without cell coating. Bioluminescence imaging (BLI), microcomputed tomography (µCT), histology, immunofluorescence, and biomechanical tests were performed. Characterization of iNCC-MPC-Luc2 vs BM-MSC-Luc2 showed no difference in MSC markers expression and differentiation in vitro. In vivo, BLI indicated survival of both cell types for at least 8 weeks. At week 8, µCT analysis showed enhanced structural parameters in the iNCC-MPC-Luc2 group and increased bone volume in the BM-MSC-Luc2 group compared to controls. Histology demonstrated improved integration of iNCC-MPC-Luc2 allografts compared to BM-MSC-Luc2 group and controls. Human osteocalcin and collagen type 1 were detected at the allograft-host interphase in cell-seeded groups. The iNCC-MPC-Luc2 group also demonstrated improved biomechanical properties compared to BM-MSC-Luc2 implants and cell-free controls. Our results show an improved integration of iNCC-MPC-Luc2-coated allografts compared to BM-MSC-Luc2 and controls, suggesting the use of iNCC-MPCs as potential cell source for cranial bone repair.
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Interface Osso-Implante , Células-Tronco Pluripotentes Induzidas , Células-Tronco Mesenquimais , Aloenxertos , Animais , Células da Medula Óssea , Diferenciação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Crista Neural/citologia , Osseointegração , Crânio/diagnóstico por imagem , Microtomografia por Raio-XRESUMO
STUDY DESIGN: Experimental animal model. OBJECTIVE: The purpose of this study was to evaluate the hypothesis that insulin dependent diabetes mellitus (IDDM) will inhibit the formation of a solid bony union after spinal fusion surgery via an alteration of the microenvironment at the fusion site in a rat model. SUMMARY OF BACKGROUND DATA: Previous studies report diabetes mellitus (DM) and specifically IDDM as a risk factor for complications and poor surgical outcomes following spinal fusion. METHODS: Twenty control and 22 diabetic rats were obtained at 5 weeks of age. At 20 weeks of age, all animals underwent posterolateral lumbar fusion surgery using a tailbone autograft with diabetic rats receiving an implantable time release insulin pellet. A subset of rats was sacrificed 1-week postsurgery for growth factor (PDGF, IGF-I, TGF-ß, and VEGF) and proinflammatory cytokine ELISA analysis. All other rats were sacrificed 3-months postsurgery for fusion evaluation via manual palpation and micro CT. Glycated hemoglobin (HbA1c) was measured at surgery and sacrifice on all animals. RESULTS: Compared with healthy rats undergoing spinal fusion, rats with IDDM demonstrated a significant reduction in manual palpation fusion rates (16.7% vs. 43%, p<.05). Average bone mineral density, bone volume, and bone volume fraction were also significantly reduced and negatively correlated to blood glucose levels. IL-1B, IL-5, IL-10, TNF-α, and KC/GRO were significantly elevated in fusion beds of IDDM rats. CONCLUSIONS: This study demonstrates that rats with IDDM demonstrate a reduced rate and quality of spinal fusion with increased local levels of inflammatory cytokines. Targeted modalities are required to improve bone healing in this growing, high-risk population. CLINICAL SIGNIFICANCE: This is the first translational animal model of IDDM to evaluate the rate and quality of spinal fusion while controlling for other surgical and patient-related risk factors. Our findings demonstrate the complex nature by which IDDM impairs bone healing and highlight the need for additional basic science research to further elucidate this mechanism in order to develop more effective therapeutic interventions.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Doenças da Coluna Vertebral , Fusão Vertebral , Animais , Humanos , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/cirurgia , Ratos , Fusão Vertebral/efeitos adversosRESUMO
INTRODUCTION: Synthetic bone grafts are often used to achieve a well-consolidated fusion mass in spinal fusion procedures. These bone grafts function as scaffolds, and ideally support cell function and facilitate protein binding. OBJECTIVE: The aim was to characterize an electrospun, synthetic bone void filler (Reb) for its bone morphogenetic protein (BMP)-2 release properties and support of human mesenchymal stem cell (hMSC) function in vitro, and its efficacy in promoting BMP-2-/bone marrow aspirate-(BMA)-mediated posterolateral spinal fusion (PLF) in vivo. METHODS: BMP-2 release kinetics from Reb versus standard absorbable collagen sponge (ACS) was determined. hMSC adhesion and proliferation on Reb was tested using cell counting, fluorescence microscopy and MTS. Cell osteogenic differentiation was quantified via cellular alkaline phosphatase (ALP) activity. For in vivo analysis, 18 Lewis rats were treated during PLF surgery with the following groups: (I) Reb + BMA, (II) Reb + BMA + BMP-2 and (III) BMA. A safe, minimally effective dose of BMP-2 was used. Fusion consolidation was followed for 3 months using radiography and micro-CT. After sacrifice, fusion rate and biomechanical stiffness was determined using manual palpation, biomechanical tests and histology. RESULTS: In vitro, BMP-2 release kinetics were similar between Reb versus ACS. MSC proliferation and differentiation were increased in the presence of Reb. At 3 months post-surgery, fusion rates were 29% (group I), 100% (group II), and 0% (group III). Biomechanical stiffness was higher in group II versus I. Micro-CT showed an increased bone volume and connectivity density in group II. Trabecular thickness was increased in group I versus II. H&E staining showed newly formed bone in group II only. CONCLUSIONS: Reb possesses a high protein binding affinity and promotes hMSC function. Combination with BMA and minimal dose BMP-2 allowed for 100% bone fusion in vivo. This data suggests that a minimally effective dose of BMP-2 can be used when combined with Reb.