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1.
Anticancer Res ; 41(10): 4781-4787, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34593427

RESUMO

BACKGROUND: Hypoxia can happen during solid tumor growth including osteosarcoma. This study investigated the relationship of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) on osteosarcoma cell growth and apoptosis under hypoxic conditions. MATERIALS AND METHODS: Human osteosarcoma cells were cultured under normal or hypoxic conditions. Inhibitors of HIF-1α and VEGF were applied to the cells separately or in combination to block the respective proteins. Cell proliferation and apoptosis were examined by MTT and TUNEL assays, and real-time PCR and ELISA were performed for mRNA and protein expression. RESULTS: There was a dramatic decrease of cell proliferation and an elevation of apoptosis under hypoxia. Blockage of HIF-1α and VEGFR enhanced the cell growth retardation and promoted apoptotic changes. Moreover, blockage of HIF-1α significantly eliminated the expression of VEGF in the cell culture media, and vice versa. CONCLUSION: HIF-1α and VEGF work closely in regulating osteosarcoma cell growth under hypoxic conditions and blockage of either of them may subsequently influence the presence of the other.


Assuntos
Apoptose , Proliferação de Células , Osteossarcoma/patologia , Hipóxia Tumoral , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Osteossarcoma/metabolismo , Hipóxia Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Bioengineering (Basel) ; 8(10)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34677205

RESUMO

We have recently identified a population of cells within the peripheral nerves of adult rodent animals (mice and rats) that can respond to Bone Morphogenetic Protein-2 (BMP-2) exposure or physical injury to rapidly proliferate. More importantly, these cells exhibited embryonic differentiation potentials that could be induced into osteoblastic and endothelial cells in vitro. The current study examined human nerve specimens to compare and characterize the cells after BMP-2 stimulation. Fresh pieces of human nerve tissue were minced and treated with either BMP-2 (750 ng/mL) or a PBS vehicle for 12 h at 37 °C, before being digested in 0.2% collagenase and 0.05% trypsin-EDTA. Isolated cells were cultured in a restrictive stem cell medium. Significantly more cells were obtained from the nerve pieces with the BMP-2 treatment in comparison with the PBS vehicle controls. Cell colonies started to form at Day 3. Expressions of the four transcription factors, namely, Klf4, c-Myc, Sox2, and Oct4, were confirmed at both the transcriptional and translational levels. The cells can be maintained in the stem cell culture medium for at least 6 weeks without changing their morphology. When the cells were transferred to a fibroblast growth medium, dispersed spindle-shaped motile cells were noted and became fibroblast activated protein-α (FAP) positive with immunocytochemistry staining. The data suggest that human peripheral nerve tissue also contains a population of cells that can respond to BMP-2 and express Klf4, Sox2, cMyc, and Oct4-the four transcription factors driving cell pluripotency. These cells are able to differentiate into FAP-positive fibroblasts. In summary, in human peripheral nerves also reside a population of quiescent cells with pluripotency potential that may be the same cells as rodent nerve-derived adult stem (NEDAPS) cells. It is proposed that these cells are possibly at the core of a previously unknown natural mechanism for healing an injury.

3.
Oxid Med Cell Longev ; 2021: 5579736, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484565

RESUMO

Although intervertebral disc degeneration (IDD) can be described as different stages of change through biological methods, this long and complex process cannot be defined in stages by single or simple combination of biological techniques. Under the background of the development of nuclear magnetic resonance (NMR) technology and the emerging metabonomics, we based on animal models and expanded to the study of clinical human degeneration models. The characteristics of different stages of IDD were analyzed by omics. Omics imaging combined with histology, cytology, and proteomics was used for screening of the intervertebral disc (IVD) of research subjects. Furthermore, mass spectrometry nontargeted metabolomics was used to explore profile of metabolites at different stages of the IDD process, to determine differential metabolic pathways and metabolites. NMR spectroscopy was used to qualitatively and quantitatively identify markers of degeneration. NMR was combined with mass spectrometry metabolomics to explore metabolic pathways. Metabolic pathways were determined through protein molecular biology and histocytology of the different groups. Distinguishing advantages of magnetic resonance spectroscopy (MRS) for analysis of metabolites and effective reflection of structural integrity and water molecule metabolism through diffusion tensor imaging (DTI) were further used to verify the macrometabolism profile during degeneration. A corresponding model of in vitro metabolomics and in vivo omics imaging was established. The findings of this study show that a series of metabolic pathways associated with the glycine-serine-threonine (Gly-Ser-Thr) metabolic axis affects carbohydrate patterns and energy utilization efficiency and ultimately delays disc degeneration through antioxidant effects.

4.
Oxid Med Cell Longev ; 2021: 5542241, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34136064

RESUMO

Objective: Low back pain (LBP) is one of the top three causes of disability in developed countries, and intervertebral disc degeneration (IDD) is a major contributor to LBP. In the process of IDD, there is a gradual decrease in nucleus pulposus cells (NPCs) and extracellular matrix (ECM). Exosomes are important exocrine mediators of stem cells that can act directly on cells for tissue repair and regeneration. In this study, we determined the antisenescence, cell proliferation promotion, and ECM modulation effects of human urine-derived stem cell (USC) exosomes (USC-exos) on degenerated intervertebral discs and explored the underlying mechanism. Methods and Materials: USCs were identified by multipotent differentiation and flow cytometry for mesenchymal stem cell- (MSC-) specific surface protein markers. USC-exos were isolated from the conditioned medium of USCs by ultracentrifugation and then analyzed by transmission electron microscopy (TEM), particle size analysis, and western blotting (WB) for exosome marker proteins. The effects of USC-exos on NPC proliferation and ECM synthesis were assessed by Cell Counting Kit-8 (CCK-8), WB, and immunofluorescence (IF) analyses. The protein differences between normal and degenerative intervertebral discs were mined, and the temporal and spatial variations in matrilin-3 (MATN3) content were determined by WB and IF in the intervertebral disc tissues. The candidate molecules that mediated the function of USC-exos were screened out and confirmed by multiple assays. Meanwhile, the mechanism underlying the candidate protein in USC-exos-induced cell proliferation and regulation of ECM synthesis promoting the activities of NPCs was explored. In addition, the effects of USC-exos on ameliorating intervertebral disc degeneration (IVD) in mice were examined by assessing computed tomography (CT), magnetic resonance imaging (MRI), and histological analyses. Results: The flow cytometry results showed that USCs were positive for CD29, CD44, and CD73, which are USC surface-specific markers, but negative for CD34 and CD45. In addition, USCs showed osteogenic, adipogenic, and chondrogenic differentiation potential. USC-exos exhibited a cup-shaped morphology, with a mean diameter of 49.7 ± 7.3 nm, and were positive for CD63 and TSG101 and negative for calnexin. USC-exos could promote NPC proliferation and ECM synthesis. The protein content of the matrilin family was significantly reduced in degenerative intervertebral discs, and the decrease in MATN3 was the most significant. USC-exos were found to be rich in MATN3 protein, and exosomal MATN3 was required for USC-exos-induced promotion of NPC proliferation and ECM synthesis, as well as alleviation of intervertebral disc degeneration in IVD rats. In addition, the effects of MATN3 in USC-exos were demonstrated to be achieved by activating TGF-ß, which elevated the phosphorylation level of SMAD and AKT. Conclusions: Our study suggests that reduced MATN3 can be considered a characteristic of intervertebral disc degeneration. USC-exos may represent a potentially effective agent for alleviating intervertebral disc degeneration by promoting NPC proliferation and ECM synthesis by transferring the MATN3 protein.

5.
J Orthop Res ; 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33991009

RESUMO

Long noncoding RNAs (lncRNAs) have been demonstrated to play critical regulatory roles in posttranscriptional and transcriptional regulation in eukaryotic cells. However, the characteristics of many lncRNAs, particularly their expression patterns in the lesion epicenter of spinal tissues following subacute spinal cord injury (SCI), remain unclear. In this study, we determined the expression profiles of lncRNAs in the lesion epicenter of spinal tissues after traumatic SCI and predicted latent regulatory networks. Standard Allen's drop surgery was conducted on mice, and hematoxylin and eosin staining was used to observe the damaged area. High-throughput sequencing was performed to identify the differential expression profiles of lncRNAs. Quantitative real-time polymerase chain reaction was conducted to evaluate the quality of the sequencing results. Bioinformatics analyses, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, coexpression analysis, and protein-protein interaction analysis, were performed. Targeted binding of lncRNA-miRNA-mRNA was predicted by TargetScan and miRanda. A total of 230 differentially expressed lncRNAs were identified and preliminarily verified, and some potential regulatory networks were constructed. These findings improve our understanding of the mechanisms underlying subacute SCI; differentially expressed lncRNAs are closely involved in pathophysiological processes by regulating multiple pathways. Further studies are essential for revealing the exact mechanism underlying competing endogenous RNA pathways in vivo and in vitro.

6.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 4055-4058, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-33018889

RESUMO

Recent mobile and wearable electroencephalogram (EEG)-sensing technologies have been demonstrated to be effective for measuring rapid changes of spatio-spectral EEG correlates of brain and cognitive functions of interest with more ecologically natural settings. However, commercial EEG products are available commonly with a fixed headset in terms of the number of electrodes and their locations to the scalp practically constrains their generalizability for different demands of EEG and brain-computer interface (BCI) study. While most progress focused on innovation of sensing hardware and conductive electrodes, less effort has been done to renovate mechanical structures of an EEG headset. Recently, an electrode-holder assembly infrastructure was designed to be capable of unlimitedly (re)assembling a desired n-channel electrode headset through a set of primary elements (i.e., LEGO-like headset). The present work empirically demonstrated one of its advantage regarding coordinating the homogeneous or heterogeneous sensors covering the target regions of the brain. Towards this objective, an 8-channel LEGO headset was assembled to conduct a simultaneous event-related potential (ERP) recording of the wet- and dry-electrode EEG systems and testify their signal quality during standing still versus treadmill walking. The results showed that both systems returned a comparable P300 signal-to-noise ratio (SNR) for standing, yet the dry system was more susceptible to the movement artifacts during slow walking. The LEGO headset infrastructure facilitates a desired benchmark study, e.g., comparing the signal quality of different electrodes on non-stationary subjects conducted in this work, or a specific EEG and BCI application.


Assuntos
Interfaces Cérebro-Computador , Caminhada , Eletrodos , Eletroencefalografia , Potenciais Evocados
7.
Materials (Basel) ; 13(17)2020 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-32858861

RESUMO

Meniscal tears are often seen in orthopedic practice. The current strategy for meniscal repair has only had limited success with a relatively high incidence of re-operative rate. This study evaluates the therapeutic effects of Bone morphogenetic protein-2 (BMP-2) soaked sutures for cartilage repair, using a rat model of xyphoid healing. Vicryl-resorbable sutures were presoaked in BMP-2 solutions prior to animal experimentation. Rat xyphoid process (an avascular hyaline cartilage structure) was surgically ruptured followed by repair procedures with regular suture or with sutures that were pre-soaked in BMP-2 solutions. In vitro assessment indicated that presoaking the Vicryl-resorbable sutures with 10 µg/mL BMP-2 resulted in a sustained amount of the growth factor release up to 7 days. Histological analysis suggested that application of this BMP-2 soaked suture on the rat xyphoid process model significantly improved the avascular cartilage healing compared to non-soaked control sutures. In conclusion, data here confirm that the rat xyphoid process repair is a reproducible and inexpensive animal model for meniscus and other cartilage repair. More importantly, coating of BMP-2 on sutures appears a potential avenue to improve cartilage repair and regeneration. Further study is warranted to explore the molecular mechanisms of this strategy.

8.
J Orthop Case Rep ; 9(6): 6-10, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32548018

RESUMO

Introduction: Medial dislocation of the long head of the biceps tendon (LHBT) is classically known as a pathognomonic finding for a subscapularis or at least a rotator cuff (RC) injury. However, this case report outlines a young active individual with symptomatic medial dislocation of the long head of the biceps with associated posterior instability, without a corresponding RC injury. Case Report: An 18-year-old male complained of the left shoulder pain and crepitus after a shoulder injury while playing hockey a year prior. Magnetic resonance imaging demonstrated medial dislocation of the LHBT without subscapularis or supraspinatus tendon injury. The patient complained of instability in the shoulder and exam findings supported posterior instability of the glenohumeral joint. Arthroscopic debridement of the glenohumeral joint with arthroscopic posterior capsulorrhaphy and open biceps tenodesis was performed. The patient regained full and painless range of motion at 2-year follow-up. Conclusion: Medial dislocation of the LHBT can occur without injury to the subscapularis tendon. Furthermore, a capsulorrhaphy with open biceps tenodesis and closure of the rotator interval may provide a successful outcome for this rare injury pattern.

9.
Kans J Med ; 13: 71-76, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32337003

RESUMO

Introduction: Recent studies have shown an increase in post-operative orthopaedic complications associated with pre-operative opioid use. It is, therefore, important to know if patients use opioids before scheduled surgery. The purpose of this study was to determine if urine drug screening (UDS) is an effective screening tool for detecting opioid and illicit drug use prior to joint arthroplasty (JA) procedures. Methods: This retrospective chart review was performed with IRB approval on 166 out of 172 consecutive patients in a community-based practice. All the patients had a pre-operative UDS prior to primary or revision JA by a fellowship trained orthopaedic surgeon between March 2016 and April 2017. Patient demographics documented opioid and illicit drug use, co-morbid diagnosis, and UDS results were collected from clinical charts. Statistical analysis was conducted using Pearson Chi-square, Fisher's exact, McNemar test, and t-tests with IBM SPSS Statistics, ver. 23. Significant differences were p < 0.05. Results: Sixty-four of 166 patients (38.6%) tested positive for opioids. Among them, 55.0% (35/64) had no history of prescription opioid use. Significant differences were observed when comparing the test results of the UDS with the patient reported history of prescribed opioids (p = 0.001). Conclusion: With a significant number of patients testing positive for opioids without evidence of a previous prescription, UDS may be beneficial for initial risk assessment for patients undergoing JA procedures.

10.
Oxid Med Cell Longev ; 2020: 6697577, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33488928

RESUMO

Objective: This study is aimed at determining the effects of human urine-derived stem cell-derived exosomes (USCs-exos) on pressure-induced nucleus pulposus cell (NPC) apoptosis and intervertebral disc degeneration (IDD) and on the ERK and AKT signaling pathways. Methods: The NPCs were obtained from patients with herniated lumbar discs. Western blot analysis (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine endoplasmic reticulum (ER) stress levels of NPCs under stress. Human USCs were identified using an inverted microscope, three-line differentiation experiments, and flow cytometry. A transmission microscope, nanoparticle size analysis, and WB procedures were used to identify the extracted exosomes and observe NPC uptake. A control group, a 48 h group, and a USCs-exos group were established. The control group was untreated, and the 48 h group was pressure-trained for 48 h, while the USCs-exos group was pressure-trained for 48 h and treated with USCs-exos. WB, qRT-PCR, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis were used to determine the ER stress levels in stress conditions and after exosomal treatment. The AKT and ERK pathways were partially detected. Magnetic Resonance Imaging (MRI) and computed tomography (CT) were used to evaluate cell degeneration while exosomal effects on the intervertebral disc (IVD) tissue were determined by hematoxylin and eosin (HE) staining, Safranin O-fast green staining, immunohistochemical staining (IHC), nuclear magnetic resonance (NMR), spectrometric detection, and total correlation spectroscopy (TOCSY). IVD metabolites were also identified and quantified. Results: After pressure culture, ER stress markers (GRP78 and C/EBP homologous protein (CHOP)) in the NPCs were significantly elevated with time (p < 0.05). Human USCs are short and spindle-shaped. They can successfully undergo osteogenic, adipogenic, and chondrogenic differentiation. In this study, these stem cells were found to be positive for CD29, CD44, and CD73. The exosomes were centrally located with a diameter of 50-100 nm. CD63 and Tsg101 were highly expressed while the expression of Calnexin was suppressed. The exosomes can be ingested by NPCs. USCs-exos significantly improved ER stress responses and inhibited excessive activation of the unfolded protein response (UPR) as well as cell apoptosis and disc degeneration through the AKT and ERK signaling pathways (p < 0.05). Conclusion: Through the AKT and ERK signaling pathways, USCs-exos significantly inhibit ER stress-induced cell apoptosis and IDD under pressure conditions. It is, therefore, a viable therapeutic strategy.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Exossomos/metabolismo , Degeneração do Disco Intervertebral/terapia , Células-Tronco/citologia , Urina/citologia , Adulto , Animais , Diferenciação Celular , Humanos , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Masculino , Núcleo Pulposo/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Resposta a Proteínas não Dobradas , Urina/química
11.
J Surg Case Rep ; 2019(11): rjz305, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31723404

RESUMO

Lumbar spine fusion has become a common and effective procedure in orthopedic practice, and a spinal subdural hygroma development is a rare complication following this procedure. We report here the case of a revision lumbar spine fusion at levels L4-5, L5-S1, where the patient subsequently developed cauda equina syndrome 2 days post-operatively. Magnetic resonance imaging (MRI) showed a subdural, extra-arachnoid fluid collection from T12-L2, cephalad to the site of spine fusion. It appears the first case reported a subdural hygroma developed cephalad to the site of spine fusion. When a patient complains of radicular pain along with urinary retention and neurologic deficits post-lumbar spine surgery, cauda equina syndrome possibly caused by subdural hygroma should be considered. This warrants immediate MRI and emergent reoperation to relieve the pressure on the spinal cord may be necessary.

12.
BMJ Case Rep ; 12(10)2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640983

RESUMO

A man in his mid-80s presented with bilateral posterior fracture dislocations of the humerus after suffering a seizure. He had Parskinson's disease and lived with his wife at home. His left shoulder was not felt to be reconstructable. The initial treatment plan was to perform reverse total shoulder arthroplasty (rTSA) on the left and non-operatively reduce his right shoulder. A left rTSA was performed, but his right shoulder was unstable due to a glenoid fracture and soft tissue instability. In order to preserve the patient's quality of life, a right rTSA was performed 4 days later. In the follow-up period, the patient was able to regain enough pain-free range of motion on activities of daily living. The patient died from complications of Parkinson's disease 10 months postoperatively.


Assuntos
Artroplastia do Ombro/métodos , Fratura-Luxação/cirurgia , Lesões do Ombro/cirurgia , Acidentes por Quedas , Idoso de 80 Anos ou mais , Fratura-Luxação/etiologia , Humanos , Masculino , Doença de Parkinson , Convulsões/complicações , Lesões do Ombro/etiologia
13.
Regen Med ; 14(3): 199-211, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30761943

RESUMO

Aim: This study investigated a coordinated strategy of revitalizing bone allograft with circulating multipotent stromal cells (MSCs). Materials & methods: After chemotactic and releasing assessments, stromal cell-derived factor 1 and platelet-derived growth factor BB in copolymers were coated on the bone allograft (AlloS-P). Allograft coated with copolymers alone (Allo), as controls, or AlloS-P was implanted into the femur of athymic mice, which received intravenous injections of human MSCs or saline at weeks 1, 2 and 3. Results: At week 8, the total callus volume (both cartilaginous and bony callus) around the allograft was the largest in the AlloS-P + MSC group (p < 0.05). Conclusion: Coating bone allograft with stromal cell-derived factor 1 and platelet-derived growth factor BB and intravenous injections of MSCs improved allograft incorporation.


Assuntos
Transplante Ósseo , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Osteogênese , Células Estromais/citologia , Cicatrização , Administração Intravenosa , Aloenxertos , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Nus
14.
J Biomed Mater Res A ; 107(1): 187-194, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30358096

RESUMO

This study investigated the interactive behavior of the particulate and ion forms of cobalt-chromium (Co-Cr) alloy challenged preosteoblasts during the process of prosthetic implant loosening. Preosteoblasts were challenged with Co-Cr particles or Co(II) ions for 72 h, followed by the proliferation and PCR assays. For in vivo test, a titanium pin was implanted into proximal tibia of SCID mice to mimic knee replacement. Co-Cr particles or Co(II) ion challenged preosteoblasts (5 × 105 ) were intra-articularly injected into the implanted knee. The animals were sacrificed 5 weeks post-op, and the prosthetic knees were harvested for biomechanical pin-pullout testing, histological evaluations, and microCT assessment. In vitro study suggested that Co-Cr particles and Co(II) ions significantly suppressed the proliferation of preosteoblasts in a dose-dependent manner. RT-PCR data on the challenged cells indicated overexpression of receptor activator of nuclear factor kappa-B ligand (RANKL) and inhibited osteoprotegerin (OPG) gene expression. Introduction of the differently challenged preosteoblasts to the pin-implant mouse model resulted in reduced implant interfacial shear strength, thicker peri-implant soft-tissue formation, more TRAP+ cells, lower bone mineral density, and bone volume fraction. In conclusion, both Co-Cr particles and Co(II) ions interfered with the growth, maturation, and functions of preosteoblasts, and provides evidence that the metal ions as well play an important role in effecting preosteoblasts in the pathogenesis of aseptic loosening. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 187-194, 2019.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Ligas de Cromo/toxicidade , Implantes Experimentais/efeitos adversos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteólise/metabolismo , Material Particulado/toxicidade , Falha de Prótese/efeitos adversos , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos SCID , Osteoblastos/patologia , Osteoclastos/patologia , Osteólise/patologia
15.
Neurochem Res ; 43(8): 1641-1649, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29934690

RESUMO

To assess the therapeutic effects of microRNA-21 (miR-21) knockdown (KD) for acute thoracic spinal cord contusion using a mouse model. Forty C57/BL6 mice were randomly divided into four groups: mice in the sham-operated (Sham) group received surgical procedure without spinal cord contusion; the spinal cord injury (SCI) group mice underwent spinal cord contusion without treatment; mice in the miR-21 KD group underwent spinal cord contusion followed by a single dose subdural injection of miR-21 KD vectors (1 × 107 TU); and the negative control (NC) group mice were given subdural injection of comparable amount of NC vectors (1 × 107 TU) after spinal cord contusion. The Basso Mouse Scale (BMS) was employed to assess hindlimb motor functions. Hematoxylin-eosin and Luxol fast blue staining were performed to evaluate pathologic changes in spinal cord tissues. Peripheral blood serum levels of tumor necrosis factor α (TNFα), transforming growth factor ß (TGF-ß) and interleukin-1ß (IL-1ß) were determined by the enzyme-linked immunosorbent assay, and mRNA expression of Brain derived neurotrophic factor (BDNF) was examined by reverse transcription-polymerase chain reaction (RT-PCR). Western blotting was performed to analyze the AKT signaling pathway. KD of miRNA-21 effectively improved the BMS scores at day 14 post-surgery compared with the SCI group (p < 0.01). The spinal cord tissue in the miR-21 KD group displayed the most overt histologic signs of recovery, with axonal regeneration and the recovery of neuronal morphology at day 14 post-surgery. Significantly alleviation of TGF-ß1, TNF-α and IL-1ß was also found in sera from the miR-21 inhibition group in comparison to others, whereas BDNF gene expression was upregulated following miR-21 KD (p < 0.01). Further, significantly decreased AKT phosphorylation activity was illustrated in the miR-21 KD group (p < 0.001). The data suggest that miR-21 KD significantly reduces the inflammatory response at the damaged spinal cord site and promotes motor functional recovery. The treatment also elevated expression of BDNF, a neurotrophin participating in nerve regeneration.


Assuntos
MicroRNAs/genética , Regeneração Nervosa/genética , Traumatismos da Medula Espinal/terapia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Terapia Genética/métodos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Transdução de Sinais/fisiologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/patologia
16.
Cancer Med ; 7(6): 2518-2529, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29659181

RESUMO

Investigate whether rAAV-anginex gene therapy combined with radiotherapy could decrease growth and pulmonary metastasis of osteosarcoma in mice and examine the mechanisms involved in this therapeutic strategy. During in vitro experiment, multiple treatment regimes (rAAV-eGFP, radiotherapy, rAAV-anginex, combination therapy) were applied to determine effects on proliferation of endothelial cells (ECs) and G-292 osteosarcoma cells. During in vivo analysis, the same multiple treatment regimes were applied to osteosarcoma tumor-bearing mice. Use microcomputed tomography to evaluate tumor size. Eight weeks after tumor cell inoculation, immunohistochemistry was used to assess the therapeutic efficacy according to microvessel density (MVD), proliferating cell nuclear antigen (PCNA), and terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assays. Metastasis of lungs was also evaluated by measuring number of metastatic nodules and wet weight of metastases. The proliferation of ECs and the tumor volumes in combination therapy group were inhibited more effectively than the other three groups at end point (P < 0.05). Cell clone assay showed anginex had radiosensitization effect on ECs. Immunohistochemistry showed tumors from mice treated with combination therapy exhibited the lowest MVD and proliferation rate, with highest apoptosis rate, as confirmed by IHC staining for CD34 and PCNA and TUNEL assays (P < 0.05). Combination therapy also induced the fewest metastatic nodules and lowest wet weights of the lungs (P < 0.05). rAAV-anginex combined with radiotherapy induced apoptosis of osteosarcoma cells and inhibited tumor growth and pulmonary metastasis on the experimental osteosarcoma models. We conclude that the primary mechanism of this process may be due to sensitizing effect of anginex to radiotherapy.


Assuntos
Neoplasias Ósseas/genética , Terapia Genética , Osteossarcoma/genética , Peptídeos/genética , Radiação Ionizante , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Terapia Combinada , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Humanos , Camundongos , Metástase Neoplásica , Osteossarcoma/diagnóstico por imagem , Osteossarcoma/patologia , Osteossarcoma/terapia , Radiossensibilizantes , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Mol Med Rep ; 16(4): 5555-5560, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28849116

RESUMO

Scutellarin is the major effective constituent of the commonly used Chinese medicine Erigeron breviscapus. It has been applied in the clinic to treat various diseases, and is characterized by high content, a stable source, controllable quality, high efficiency and low toxicity. In addition, its potential pharmacological effects have been increasingly identified and elucidated. The present study was performed to examine the role of scutellarin on collagen­induced arthritis (CIA). Mice were injected subcutaneously with bovine collagen type II and administered scutellarin for 2 weeks by gavage 20 mg/kg/day. ELISA kits were used to measure the levels of interleukin (IL)­1ß, IL­6, tumor necrosis factor­α (TNF­α), oxidative stress markers [superoxide dismutase (SOD) and malondialdehyde (MDA)] and caspase­3/-9 activity. Bax, Bcl­2, toll like receptor 4 (TLR4) and nuclear factor (NF)­κB protein expression was analyzed using western immunoblot analyses. The present study demonstrated that scutellarin prevented CIA, and inhibited the expression of inflammation factors, IL­1ß, IL­6 and TNF­α. In addition, scutellarin reduced the levels of oxidative stress markers, SOD and MDA, as well as intercellular adhesion molecule­1 and monocyte chemoattractant protein 1 in CIA mice. Caspase­3/-9, Bax/Bcl­2, TLR4 and NF­κB protein expression were reduced in CIA mice following scutellarin treatment. The results of the current study suggest a novel effect of scutellarin involving the inhibition of TLR4/NF­κB­mediated inflammation.


Assuntos
Anti-Inflamatórios/farmacologia , Apigenina/farmacologia , Artrite Experimental/etiologia , Artrite Experimental/metabolismo , Glucuronatos/farmacologia , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Anti-Inflamatórios/química , Apigenina/química , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Biomarcadores , Bovinos , Quimiocina CCL2/metabolismo , Colágeno/efeitos adversos , Colágeno Tipo II/efeitos adversos , Modelos Animais de Doenças , Glucuronatos/química , Molécula 1 de Adesão Intercelular/metabolismo , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos
19.
J Orthop Translat ; 8: 5-11, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30035088

RESUMO

Introduction: Wear particles produced from prosthetic joints may play critical roles in periprosthetic inflammatory reactions and osteolysis. The objective of this study was to quantify and compare the response to wear debris from different biomaterials at the bone-implant interface in a rat knee model. Methods: Sixty rats were divided into titanium alloy (Ti-6Al-4V), cobalt chromium (Co-Cr), ceramic (Al2O3), ultrahigh molecular weight polyethylene (UHMWPE), and control (phosphate buffered saline) groups with 12 animals per group. A nonweight-bearing titanium rod was implanted into the right distal femur of each rat followed by intra-articular injections of the biomaterial particles to the surgical knees for up to 16 weeks. Micro-computed tomography scanning was performed monthly and at the time of sacrifice to determine bone densities around the bone-implant interface. Histological evaluations were executed to quantify local inflammatory reactions and osteoclastogenesis. Results: Co-Cr particles resulted in the most severe reductions in bone density. UHMWPE and ceramic particles resulted in a rapid reduction in bone density followed by a recovery. Inflammatory pseudo-membranes were ubiquitously present close to the femoral condyle and pin insertion site. Ceramic particles significantly promoted periprosthetic tissue formation compared with the other groups (p < 0.05). Cathepsin K positive cells were dominantly present at the peri-implant site following challenges of metallic alloy and ceramic particles. Conclusion: Different biomaterials in particulate form exert different forms of adverse effects in terms of the amount of osteolysis and inflammatory reactions on bone tissue at the bone-implant interface. It provides information for engineering more appropriate materials for arthroplasty components.

20.
Spine J ; 17(2): 252-259, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27664339

RESUMO

BACKGROUND: The clinical use of recombinant human bone morphogenic protein 2 (rhBMP-2, Infuse) has been associated with nerve-related complications including new-onset sciatica, and retrograde ejaculation. PURPOSE: To better understand the interaction of rhBMP-2 and peripheral nerves with the intent of making procedures safer. STUDY DESIGN/SETTING: Using a mouse model to examine the direct effect of diluted rhBMP-2 (Infuse) on murine sciatic nerves. METHODS: Animal studies were approved by the Institutional Animal Care and Use Committee. Balb/c mouse sciatic nerves were surgically exposed and 60 ng (in 10 µL) of rhBMP-2 was applied to the nerve. In separate experiments, the sciatic nerves were subjected to mechanical compression using forceps (and not exposed to rhBMP-2). The third group of mice received direct injection of the same amount of rhBMP-2, or sterile saline as a control, into the hamstring area of the posterior thigh without surgery. Mouse limbs with intact sciatic nerve were collected at 24, 48, or 72 hours after treatment for histology processing. A separate set of identically treated sciatic nerves were retrieved from mice at the same time points and cells were isolated by collagenase and trypsin digestion. The isolated cells were cultured in a stem cell medium containing 20% knockout serum and human leukemia inhibitory factor. Immunohistochemical or immunofluorescent cell stains against KLF4, Sox2, c-Myc, and Oct4 were performed on the mouse tissue sections and cell culture slides. In addition, real-time polymerase chain reaction (PCR) was performed to quantify the mRNA expression profiles of the stem cell marker genes in cultured cells. RESULTS: Profound morphological changes of the mouse sciatic nerves were noted after exposure to rhBMP-2, with a rapid and robust cell proliferation within the nerves followed by migration of these cells into surrounding tissue. Immunohistochemical stain revealed strong nuclear stains of KLF4, Sox2, Oct4, and c-Myc on the overwhelming majority of these proliferating cells in the nerve. Intramuscular injections of rhBMP-2 or willful physical compression of the nerves showed similar cell proliferation effects as the direct application of Infuse to the sciatic nerve. The cells in stem cell culture medium grew steadily without feeder cells and appeared fairly uniform. They were adherent to substrate and were motile. Double fluorescent staining on the cells indicated colocalizationof all pairs of the four stem cell markers in the cell nuclei. Real-time PCR confirmed the strong mRNA expressions of KLF4, Sox2, Oct4, and c-Myc in these isolated cells. CONCLUSION: Exposure to BMP-2 causes a marked proliferation of previously quiescent cells within peripheral nerves. These cells simultaneously express KLF4, Sox2, Oct4, and c-Myc, the transcription factors that confer embryonic pluripotency. Work described in the companion paper reveals some of the differentiation capacity of the cells and their likely clinical significance. In addition, the effects of direct exposure of nerves to rhBMP-2 as described here should clearly illuminate the mechanism of BMP-2-related nerve complications. We would suggest that the use of this agent in proximity to known neural structures should only be done with extreme caution.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Proliferação de Células , Células-Tronco Pluripotentes/efeitos dos fármacos , Nervo Isquiático/citologia , Animais , Diferenciação Celular , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fator 3 de Transcrição de Octâmero , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Nervo Isquiático/lesões
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