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Objective To explore the effects of fistular onion bulb extract (FOB) on myocardial ischemia / reperfusion (I / R) injury and possible regulation mechanism. Methods The left anterior descending (LAD) artery of 36 adult rat heart was ligated for 30 minutes and subsequently released for 120 minutes to establish the I / R injury model in vivo. Cultured neonatal rat cardiomyocytes were subjected to 6 hours of hypoxia followed by 18 hours of reoxygenation to establish the hypoxia / reoxygenation (H / R) model in vitro. The rats or cardiomyocytes were randomly divided into the control group, I / R group (or H / R group) and FOB pretreatment+I / R group (or H / R group). The left ventricular (LV) function was measured using PowerLab multichannel physiological recorder; the infarct size was detected by 2, 3, 5-triphenyl tetrazolium chlorid(TTC) staining. The apoptosis rate was detected by flow cytometry, Western blotting and Real-time PCR were used to detect the expression of apoptosis-related proteins and mRNA (Bcl-2, Bax and Caspase-3); Immunofluorescence was used to detect the expression of cytochrome C (Cyt-C). Results In the animal experiments, compared with the control group, the left ventricular systolic and diastolic functions were deteriorated (P<0. 05); compared with the I / R group, the pretreatment of FOB significantly improved the left ventricular systolic and diastolic function, and decreased the infarct size (P<0. 05). In cultured cardiomyocytes, compared with the control group, the apoptosis rate was significantly increased (P<0. 01), the protein and mRNA expression of Bcl-2 decreased, meanwhile the expression of Bax, Caspase-3 and Cyt-C increased in the H / R group (P< 0. 05); Compared with the H / R group, the pretreatment of FOB significantly reduced the apoptosis rate (P < 0. 05), increased the protein and mRNA expression of Bcl-2, at the same time, decreased the expression of Bax, Caspase-3 and Cyt-C(P<0. 05). Conclusion Fistular onion bulb extract has the protective effect on myocardial ischemia / reperfusion injury, and its possible mechanism is to reduce cardiomyocyte apoptosis through mitochondrial pathway.
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STUDY DESIGN: In Vitro cell culture study. PURPOSE: This study aims to investigate the impact of transforming growth factor-beta1 (TGF-β1) and lovastatin on differentiating human mesenchymal stem cells (MSCs) toward nucleus pulposus (NP)-like phenotype. OVERVIEW OF LITERATURE: MSCs offer a cell source to the cell-based therapy for intervertebral disc degeneration. TGF-β1 is used to induce MSCs to differentiate into NP-like cells; however, an undesired expression of collagen type I has been reported. Statins reportedly stimulate expression of bone morphogenetic protein-2 (BMP-2) and promote the chondrogenic phenotype to NP cells. However, the effects of statins with or without TGF-β1 on the differentiation of MSCs into NP-like cells remain unclear. METHODS: Human MSCs were treated with TGF-β1 alone, lovastatin alone, and simultaneous or sequential treatment with TGF-β1 and lovastatin. After the proposed stimulation, the total RNA was extracted to assess the expression profile of NP cells-specific genes. Hematoxylin–eosin staining was used for examining the microscopic morphology. Furthermore, we detected the syntheses of S-100 protein, aggrecan, and collagen type II in the extracellular matrix using immunohistochemical staining. RESULTS: Simultaneous or sequential treatment of TGF-β1 and lovastatin could further augment the BMP-2 overexpression compared with lovastatin-alone treatment. However, the mRNA expression of aggrecan and collagen type II was not compatible with the expression level of BMP-2. Immunohistochemical studies revealed compatible production of aggrecan, collagen type II, and S-100 protein in all three groups treated with lovastatin. Cells in groups treated with lovastatin were less populated than that in the group treated with TGF-β1 alone. CONCLUSIONS: This study demonstrates a promising role of lovastatin in inducing human MSCs into NP-like cells. However, further optimization of cell density before lovastatin treatment, treatment duration, and combination with TGF-β1 are warranted to attain better stimulatory effects.
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This study aimed to examine the clinical and radiographic outcomes of primary total knee arthroplasy (TKA) with use of NexGen® Legacy® Constrained Condylar Knee (CCK) prosthesis for severe knee deformity. Clinical data of 46 patients (48 knees in total, aged 61 years on average) with severe knee deformity who underwent TKA with NexGen® Legacy® CCK prosthesis between December 2007 and February 2012 were retrospectively analyzed. There were 34 knees with severe valgus with incompetent medial collateral ligament, 11 knees with severe flexion contracture with inability to achieve knee balancing in flexion and extension by posterior soft tissue release, 2 knees with Charcot arthritis with severe varus and bone loss, and 1 with traumatic osteoarthritis with severe varus and ligamentous instability. The mean duration of follow-up was 71 months (range 40-90 months). The New Knee Society scoring (NKSS) system and the Hospital for Special Surgery (HSS) score were used to evaluate the functional and clinical outcomes. Visual Analogue Scale (VAS) was used for pain measurement and Knee Society criteria for evaluation of radiological images. The results showed that, in the total 48 knees, 1 case of loosening due to short-stem tibial component at 3 months post-operatively underwent revision. The 6-year prosthesis survival rate in this cohort was 97.9%. There was no component infection occurring within 6 years. Significant post-operative improvements were found in NKSS and HSS scores. Patient satisfaction was significantly increased. Pain score was decreased significantly. Total functional score was improved from 31.46±11.43 to 86.42±8.87, range of motion (ROM) from 42.42°±23.57° to 95.31°±23.45° and the flexion contracture from 5.31°±7.87° to 0.92°±1.80°. Preoperative radiographic study showed excessive valgus (≥7°) in 37 knees, and varus deformity in 3 knees. Post-operative femorotibial alignment was valgus 3.88°±1.76° in 48 knees. Antero/posterior (A/P) view of X-ray films showed 4 radiolucent lines (RLL) in 48 tibial components. It was concluded that TKA with CCK is effective for the treatment of the severe unstable knee that cannot be balanced by soft tissue.
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Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Artroplastia de Reemplazo de Rodilla , Métodos , Gota , Cirugía General , Huesos de la Pierna , Cirugía General , Osteoartritis , Cirugía General , Dolor , Satisfacción del Paciente , Complicaciones Posoperatorias , Prótesis e Implantes , Infección de HeridasRESUMEN
In order to investigate the promoting effect of low-intensity treadmill exercise on rat dorsal wound healing and the mechanism, 20 Sprague-Dawley rats were randomly divided into two groups: exercise group (Ex) and non-exercise group (non-ex). The rats in Ex group were given treadmill exercise for one month, and those in non-ex group raised on the same conditions without treadmill exercise. Both groups received dorsal wound operation with free access to food and water. By two-week continuous observation and recording of the wound area, the healing rate was analyzed. The blood sample was collected at day 14 post-operation via cardiac puncture for determination of the number of endothelial progenitor cells (EPCs) by flow cytometry, and the concentrations of relevant cytokines such as basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by ELISA. The skin tissue around the wound was dissected to observe the vascular density under the microscope after HE staining, to detect the mRNA level of VEGFR2 and angiopoietin-1 (Ang-1) receptor using RT-qPCR, and protein expression of a-smooth muscle actin (αSMA) and type III collagen (ColIII) using Western blotting. It was found that the wound area in Ex group was smaller at the same time point than in non-ex group. The number of circulating EPCs was greater and the concentrations of vasoactive factors such as VEGF, eNOS and bFGF were higher in Ex group than in non-ex group. HE staining displayed a higher vessel density in Ex group than in non-ex group. Moreover, the mRNA expression of VEGFR2 and Ang-1 detected in the wound tissue in Ex group was higher than in non-ex group. Meanwhile, the protein expression of αSMA and ColIII was more abundant in Ex group than in non-ex group. Conclusively, the above results demonstrate Ex rats had a higher wound healing rate, suggesting low-intensity treadmill exercise accelerates wound healing. The present work may provide some hint for future study of treating refractory wound.
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Animales , Masculino , Ratas , Actinas , Metabolismo , Colágeno Tipo III , Metabolismo , Citocinas , Sangre , Células Progenitoras Endoteliales , Biología Celular , Óxido Nítrico Sintasa de Tipo III , Sangre , Esfuerzo Físico , ARN Mensajero , Sangre , Ratas Sprague-Dawley , Receptor TIE-1 , Metabolismo , Carrera , Factor A de Crecimiento Endotelial Vascular , Sangre , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Sangre , Cicatrización de HeridasRESUMEN
<p><b>OBJECTIVE</b>To review the recent developments in the mechanisms of epithelium sodium channels (ENaCs) induced bone formation and regulation.</p><p><b>DATA SOURCES</b>Studies written in English or Chinese were searched using Medline, PubMed and the index of Chinese-language literature with time restriction from 2005 to 2014. Keywords included ENaC, bone, bone formation, osteonecrosis, estrogen, and osteoporosis. Data from published articles about the structure of ENaC, mechanism of ENaC in bone formation in recent domestic and foreign literature were selected.</p><p><b>STUDY SELECTION</b>Abstract and full text of all studies were required to obtain. Studies those were not accessible and those did not focus on the keywords were excluded.</p><p><b>RESULTS</b>ENaCs are tripolymer ion channels which are assembled from homologous α, β, and γ subunits. Crystal structure of ENaCs suggests that ENaC has a central ion-channel located in the central symmetry axis of the three subunits. ENaCs are protease sensitive channels whose iron-channel activity is regulated by the proteolytic reaction. Channel opening probability of ENaCs is regulated by proteinases, mechanical force, and shear stress. Several molecules are involved in regulation of ENaCs in bone formation, including nitride oxide synthases, voltage-sensitive calcium channels, and cyclooxygenase-2.</p><p><b>CONCLUSION</b>The pathway of ENaC involved in shear stress has an effect on stimulating osteoblasts even bone formation by estrogen interference.</p>
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Humanos , Canales de Calcio , Fisiología , Canales Epiteliales de Sodio , Química , Fisiología , Estrógenos , Farmacología , Osteogénesis , FisiologíaRESUMEN
This study aimed to examine the clinical and radiographic outcomes of primary total knee arthroplasy (TKA) with use of NexGen® Legacy® Constrained Condylar Knee (CCK) prosthesis for severe knee deformity. Clinical data of 46 patients (48 knees in total, aged 61 years on average) with severe knee deformity who underwent TKA with NexGen® Legacy® CCK prosthesis between December 2007 and February 2012 were retrospectively analyzed. There were 34 knees with severe valgus with incompetent medial collateral ligament, 11 knees with severe flexion contracture with inability to achieve knee balancing in flexion and extension by posterior soft tissue release, 2 knees with Charcot arthritis with severe varus and bone loss, and 1 with traumatic osteoarthritis with severe varus and ligamentous instability. The mean duration of follow-up was 71 months (range 40-90 months). The New Knee Society scoring (NKSS) system and the Hospital for Special Surgery (HSS) score were used to evaluate the functional and clinical outcomes. Visual Analogue Scale (VAS) was used for pain measurement and Knee Society criteria for evaluation of radiological images. The results showed that, in the total 48 knees, 1 case of loosening due to short-stem tibial component at 3 months post-operatively underwent revision. The 6-year prosthesis survival rate in this cohort was 97.9%. There was no component infection occurring within 6 years. Significant post-operative improvements were found in NKSS and HSS scores. Patient satisfaction was significantly increased. Pain score was decreased significantly. Total functional score was improved from 31.46±11.43 to 86.42±8.87, range of motion (ROM) from 42.42°±23.57° to 95.31°±23.45° and the flexion contracture from 5.31°±7.87° to 0.92°±1.80°. Preoperative radiographic study showed excessive valgus (≥7°) in 37 knees, and varus deformity in 3 knees. Post-operative femorotibial alignment was valgus 3.88°±1.76° in 48 knees. Antero/posterior (A/P) view of X-ray films showed 4 radiolucent lines (RLL) in 48 tibial components. It was concluded that TKA with CCK is effective for the treatment of the severe unstable knee that cannot be balanced by soft tissue.
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In order to investigate the promoting effect of low-intensity treadmill exercise on rat dorsal wound healing and the mechanism, 20 Sprague-Dawley rats were randomly divided into two groups: exercise group (Ex) and non-exercise group (non-ex). The rats in Ex group were given treadmill exercise for one month, and those in non-ex group raised on the same conditions without treadmill exercise. Both groups received dorsal wound operation with free access to food and water. By two-week continuous observation and recording of the wound area, the healing rate was analyzed. The blood sample was collected at day 14 post-operation via cardiac puncture for determination of the number of endothelial progenitor cells (EPCs) by flow cytometry, and the concentrations of relevant cytokines such as basic fibroblast growth factor (bFGF), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by ELISA. The skin tissue around the wound was dissected to observe the vascular density under the microscope after HE staining, to detect the mRNA level of VEGFR2 and angiopoietin-1 (Ang-1) receptor using RT-qPCR, and protein expression of a-smooth muscle actin (αSMA) and type III collagen (ColIII) using Western blotting. It was found that the wound area in Ex group was smaller at the same time point than in non-ex group. The number of circulating EPCs was greater and the concentrations of vasoactive factors such as VEGF, eNOS and bFGF were higher in Ex group than in non-ex group. HE staining displayed a higher vessel density in Ex group than in non-ex group. Moreover, the mRNA expression of VEGFR2 and Ang-1 detected in the wound tissue in Ex group was higher than in non-ex group. Meanwhile, the protein expression of αSMA and ColIII was more abundant in Ex group than in non-ex group. Conclusively, the above results demonstrate Ex rats had a higher wound healing rate, suggesting low-intensity treadmill exercise accelerates wound healing. The present work may provide some hint for future study of treating refractory wound.
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This study aimed to examine the diagnostic accuracy and clinical efficacy of initial CT-guided percutaneous biopsy of the vertebral lesions. A total of 305 percutaneous biopsies of the vertebral lesions were performed under either CT guidance (n=127) or C-arm guidance (n=178). The diagnostic accuracy rate was evaluated by comparing the histopathological diagnosis with the ultimate diagnosis. The histopathological diagnosis was consistent with the ultimate diagnosis in 108 (85.0%, 108/127) cases of CT-guided biopsy and in 135 (75.8%, 135/178) cases of C-arm guided biopsy and there was a significant difference. The accuracy of diagnosis based on biopsies varied with different diseases, including primary benign or malignant tumors, metastatic tumors, inflammatory lesions and fractures. A second biopsy or further examinations were required for patients with negative result obtained in the initial biopsy. The complication rate was 3.1% (4/127) in CT-guided biopsy and 7.3% (13/178) in C-arm guided biopsy. In conclusion, CT-guided percutaneous biopsy is an accurate and safe technique for biopsy of the vertebral lesions.
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Humanos , Biopsia con Aguja , Métodos , Diagnóstico Diferencial , Radiografía Intervencional , Métodos , Reproducibilidad de los Resultados , Estudios Retrospectivos , Sensibilidad y Especificidad , Enfermedades de la Columna Vertebral , Diagnóstico , Tomografía Computarizada por Rayos X , MétodosRESUMEN
Traumatic gas gangrene is a fatal infection mainly caused by Clostridium perfringens. It is a challenge to manage gas gangrene in open wounds and control infection after debridement or amputation. The aim of the present study was to use vacuum sealing drainage (VSD) with continuous irrigation of potassium permanganate to manage infective wounds of gas gangrene and observe its clinical efficacy. A total of 48 patients with open traumatic gas gangrene infection were included in this study. Amputations were done for 27 patients, and limb salvage procedures were performed for the others. After amputation or aggressive debridement, the VSD system, including polyvinyl alcohol (PVA) foam dressing and polyurethane (PU) film, with continuous irrigation of 1:5000 potassium permanganate solutions, was applied to the wounds. During the follow-up, all the patients healed without recurrence within 8-18 months. There were four complications. Cardiac arrest during amputation surgery occurred in one patient who suffered from severe septic shock. Emergent resuscitation was performed and the patient returned to stable condition. One patient suffered from mixed infection of Staphylococcal aureus, and a second-stage debridement was performed. One patient suffered from severe pain of the limb after the debridement. Exploratory operation was done and the possible reason was trauma of a local peripheral nerve. Three cases of crush syndrome had dialysis treatment for concomitant renal failure. In conclusion, VSD can convert open wound to closed wound, and evacuate necrotic tissues. Furthermore, potassium permanganate solutions help eliminate anaerobic microenvironment and achieve good therapeutic effect on gas gangrene and mixed infection. VSD with continuous irrigation of potassium permanganate is a novel, simple and feasible alternative for severe traumatic open wounds with gas gangrene infection.
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Adolescente , Adulto , Anciano , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Drenaje , Gangrena Gaseosa , Terapéutica , Terapia de Presión Negativa para Heridas , Métodos , Permanganato de Potasio , Usos Terapéuticos , Estudios Retrospectivos , Irrigación Terapéutica , Resultado del TratamientoRESUMEN
In this study we present our experiences with the reverse sural fasciocutaneous flap to reconstruct the distal lower limb soft tissue defects caused by traumatic injuries. These flap graftings were carried out from Oct. 2010 to Dec. 2012 in our department. The series consisted of 36 patients, including 21 men and 15 women with an average age of 46.2 years (14-83 years) and with a medium follow-up period of 18 months (12-24 months). Of all the cases of acute trauma, there were 10 cases of trauma of distal tibia, 9 cases of trauma of perimalleolus, and 17 cases of trauma of midfoot and forefoot. Related risk factors in the patients were diabetes (2 cases), advanced age (>65 years, 3 cases) and cigarette smoking (6 cases). The reverse flow sural island flap irrigation depended on lower perforators of the peroneal artery. The fasciocutaneous pedicle was 3-4 cm in width and the anatomical structures consisted of the superficial and deep fascia, the sural nerve, short saphenous vein, superficial sural artery together with an islet of subcutaneous cellular tissue and skin. The most proximal border of the flap was only 1.5 cm away from the popliteal skin crease and the pivot point was 5-7 cm above the tip of the lateral malleolus. All the flaps survived. No arterial crisis occurred in any case. The venous congestion occurred in 2 cases and got better after raising the limbs and bloodletting. Only in an old man, 1.5 cm necrosis of distal margin of his flap occurred and finally healed after continuous dressing change. One-stage skin grafting was performed, and all the donor sites were sutured and successfully healed. It was concluded that the reverse sural fasciocutaneous flap is safe and reliable to extend to the proximal third even near the popliteal skin crease. We also concluded this flap can be safely and efficiently used to treat patients with large and far soft-tissue defects from the distal leg to the forefoot with more versatility and it is easier to reach the recipient sites.
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Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Fascia , Trasplante , Supervivencia de Injerto , Extremidad Inferior , Heridas y Lesiones , Cirugía General , Procedimientos de Cirugía Plástica , Métodos , Vena Safena , Trasplante , Trasplante de Piel , Traumatismos de los Tejidos Blandos , Cirugía General , Nervio Sural , Trasplante , Colgajos Quirúrgicos , Resultado del TratamientoRESUMEN
In this study we present our experiences with the reverse sural fasciocutaneous flap to reconstruct the distal lower limb soft tissue defects caused by traumatic injuries. These flap graftings were carried out from Oct. 2010 to Dec. 2012 in our department. The series consisted of 36 patients, including 21 men and 15 women with an average age of 46.2 years (14-83 years) and with a medium follow-up period of 18 months (12-24 months). Of all the cases of acute trauma, there were 10 cases of trauma of distal tibia, 9 cases of trauma of perimalleolus, and 17 cases of trauma of midfoot and forefoot. Related risk factors in the patients were diabetes (2 cases), advanced age (>65 years, 3 cases) and cigarette smoking (6 cases). The reverse flow sural island flap irrigation depended on lower perforators of the peroneal artery. The fasciocutaneous pedicle was 3-4 cm in width and the anatomical structures consisted of the superficial and deep fascia, the sural nerve, short saphenous vein, superficial sural artery together with an islet of subcutaneous cellular tissue and skin. The most proximal border of the flap was only 1.5 cm away from the popliteal skin crease and the pivot point was 5-7 cm above the tip of the lateral malleolus. All the flaps survived. No arterial crisis occurred in any case. The venous congestion occurred in 2 cases and got better after raising the limbs and bloodletting. Only in an old man, 1.5 cm necrosis of distal margin of his flap occurred and finally healed after continuous dressing change. One-stage skin grafting was performed, and all the donor sites were sutured and successfully healed. It was concluded that the reverse sural fasciocutaneous flap is safe and reliable to extend to the proximal third even near the popliteal skin crease. We also concluded this flap can be safely and efficiently used to treat patients with large and far soft-tissue defects from the distal leg to the forefoot with more versatility and it is easier to reach the recipient sites.
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Seventy-three patients with spinal nerve sheath tumor who were surgically treated in our hospital during the years 2004-2010 were retrospectively reviewed with respect to recovery of neurological function, recurrence of the tumor and occurrence of kyphotic deformities. Preoperative clinical manifestations, imaging data, surgical records and follow-up results were comprehensively analyzed. The follow-up duration was 12-60 months with an average of 32.0 months. Out of the 73 cases enrolled, 69 had gradual recovery of sensation, motor and sphincter functions 1 week to 3 months after operation. Forty-six cases had incomplete paralysis, whose American Spinal Injury Association (ASIA) grades, however, were gradually increased during the follow-up period, 4 cases had no significant improvement of the clinical symptoms and no change in ASIA grades during the follow-up period. Two cases had postoperative recurrence of the tumor. There were no deaths, no spinal instability, and no kyphotic malformations found in any cases. Our study indicated that complete removal of the tumor is important for good recovery, and an ideal surgical method may reduce the recurrence of the tumor or the occurrence of complications.
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Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Envejecimiento , Patología , Neoplasias de la Vaina del Nervio , Patología , Cirugía General , Neoplasias de la Columna Vertebral , Patología , Cirugía General , Resultado del TratamientoRESUMEN
It is widely known that hypoxia can promote chondrogenesis of human bone marrow derived mesenchymal stem cells (hMSCs) in monolayer cultures. However, the direct impact of oxygen tension on hMSC differentiation in three-dimensional cultures is still unknown. This research was designed to observe the direct impact of oxygen tension on the ability of hMSCs to "self assemble" into tissue-engineered cartilage constructs. hMSCs were cultured in chondrogenic medium (CM) containing 100 ng/mL growth differentiation factor 5 (GDF-5) at 5% (hypoxia) and 21% (normoxia) O2 levels in monolayer cultures for 3 weeks. After differentiation, the cells were digested and employed in a self-assembly process to produce tissue-engineered constructs under hypoxic and normoxic conditions in vitro. The aggrecan and type II collagen expression, and type X collagen in the self-assembled constructs were assessed by using immunofluorescent and immunochemical staining respectively. The methods of dimethylmethylene blue (DMMB), hydroxyproline and PicoGreen were used to measure the total collagen content, glycosaminoglycan (GAG) content and the number of viable cells in each construct, respectively. The expression of type II collagen and aggrecan under hypoxic conditions was increased significantly as compared with that under normoxic conditions. In contrast, type X collagen expression was down-regulated in the hypoxic group. Moreover, the constructs in hypoxic group showed more significantly increased total collagen and GAG than in normoxic group, which were more close to those of the natural cartilage. These findings demonstrated that hypoxia enhanced chondrogenesis of in vitro, scaffold-free, tissue-engineered constructs generated using hMSCs induced by GDF-5. In hypoxic environments, the self-assembled constructs have a Thistological appearance and biochemical parameters similar to those of the natural cartilage.
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Femenino , Humanos , Masculino , Agrecanos , Genética , Metabolismo , Células de la Médula Ósea , Metabolismo , Cartílago , Biología Celular , Metabolismo , Diferenciación Celular , Genética , Hipoxia de la Célula , Células Cultivadas , Condrogénesis , Genética , Colágeno Tipo II , Genética , Metabolismo , Colágeno Tipo X , Metabolismo , Expresión Génica , Glicosaminoglicanos , Metabolismo , Factor 5 de Diferenciación de Crecimiento , Farmacología , Inmunohistoquímica , Células Madre Mesenquimatosas , Metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ingeniería de Tejidos , MétodosRESUMEN
The purpose of this study was to investigate the repair of the osteoarthritis(OA)-induced cartilage injury by transfecting the new TGF-β3 fusion protein (LAP-MMP-mTGF-β3) with targeted therapy function into the bone marrow-derived mesenchymal stem cells (MSCs) in rats. The recombinant of pIRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector pIRES-EGFP. LAP and mTGF-β3 fragments were obtained from rat embryos by RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, so as to construct the recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3. pIRES-EGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs. The genetically modified MSCs were cultured in medium with MMP-1 or not. The transfected MSCs were transplanted in the rat OA models. The OA animal models were surgically induced by anterior cruciate ligament transaction (ACLT). The pathological changes were observed under a microscope by HE staining, Alcian blue, Safranin-fast Green and graded by Mankin's scale. pIRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing, and the mTGF-β3 fusion protein (39 kD) was certified by Western blotting. Those genetically modified MSCs could differentiate into chondrocytes induced by MMP and secrete the relevant-matrix. The transfected MSCs could promote chondrogenesis and matrix production in rat OA models in vivo. It was concluded that a new fusion protein LAP-MMP-mTGF-β3 was constructed successfully by gene engineering, and could be used to repair the OA-induced cartilage injury.
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Animales , Ratas , Secuencia de Bases , Western Blotting , Células de la Médula Ósea , Metabolismo , Cartílago Articular , Patología , Cirugía General , Diferenciación Celular , Genética , Células Cultivadas , Condrocitos , Metabolismo , Condrogénesis , Genética , Proteínas Fluorescentes Verdes , Genética , Metabolismo , Metaloproteinasas de la Matriz , Genética , Metabolismo , Trasplante de Células Madre Mesenquimatosas , Métodos , Células Madre Mesenquimatosas , Metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Osteoartritis , Cirugía General , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión , Genética , Metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Factor de Crecimiento Transformador beta3 , Genética , Metabolismo , Resultado del TratamientoRESUMEN
The purpose of the present study was to observe the structure and functional change of the bone-coating-prosthesis interface in vivo and to evaluate the histocompatibility of self-made prosthetic femoral components in the body and the degree of their bonding with the surrounding bone tissues as well as their stability. Six mature beagle dogs underwent bilateral hip replacement with prosthetic femur components. Three groups were established in terms of different coating of prothesis (four joints in each group): atmosphere (A) plasma-sprayed pure titanium (Ti) prosthetic joint with hydroxyapatite (HA) coating (HA+Ti+A group); vacuum (V) plasma-sprayed pure Ti prosthetic joint with HA coating (HA+Ti+V group); vacuum plasma-sprayed pure Ti prosthetic joint with Ti-HA stepped coating (Ti+HAG+Ti+V group). The hip joints were functionally evaluated, and subjected to X-ray examination, biomechanics inspection, and histological examination. As a result, X-ray imaging revealed all prosthetic joints were in a good location and no dislocation of joint was found. Shear strength of interface was significantly higher in Ti+HAG+Ti+V group than in HA+Ti+V group (P<0.05) and HA+Ti+A group (P<0.05) at 28th week. Histological examination showed the amount of newborn bone in Ti+HAG+Ti+V group was more than in HA+Ti+V group and HA+Ti+A group after 28 weeks. It was suggested that vacuum plasma-sprayed pure Ti prosthetic joint with TI-HA stepped coating could improve the bonding capacity of bone-prosthesis, enhance the stability of prosthesis, and increase the fixion of prosthetic femoral components because of better bone growth. This new type of biological material in prosthetic femoral components holds promises for application in clinical practice.
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Animales , Perros , Fenómenos Biomecánicos , Fisiología , Desarrollo Óseo , Fisiología , Materiales Biocompatibles Revestidos , Farmacología , Durapatita , Farmacología , Fémur , Fisiología , Prótesis e Implantes , Titanio , Farmacología , VacioRESUMEN
A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells (ADSCs). The recombinant pIRES-EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFP. LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups: experimental group (MMP group), negative control group (no MMP) and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen (ColII) was detected by using Alcian blue staining and immunohistochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and sequencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differentiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.
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Animales , Femenino , Masculino , Conejos , Tejido Adiposo , Metabolismo , Condrogénesis , Genética , Proteínas Recombinantes de Fusión , Genética , Metabolismo , Células Madre , Metabolismo , Factor de Crecimiento Transformador beta3 , Genética , MetabolismoRESUMEN
It is widely known that hypoxia can promote chondrogenesis of human bone marrow derived mesenchymal stem cells (hMSCs) in monolayer cultures. However, the direct impact of oxygen tension on hMSC differentiation in three-dimensional cultures is still unknown. This research was designed to observe the direct impact of oxygen tension on the ability of hMSCs to "self assemble" into tissue-engineered cartilage constructs. hMSCs were cultured in chondrogenic medium (CM) containing 100 ng/mL growth differentiation factor 5 (GDF-5) at 5% (hypoxia) and 21% (normoxia) O2 levels in monolayer cultures for 3 weeks. After differentiation, the cells were digested and employed in a self-assembly process to produce tissue-engineered constructs under hypoxic and normoxic conditions in vitro. The aggrecan and type II collagen expression, and type X collagen in the self-assembled constructs were assessed by using immunofluorescent and immunochemical staining respectively. The methods of dimethylmethylene blue (DMMB), hydroxyproline and PicoGreen were used to measure the total collagen content, glycosaminoglycan (GAG) content and the number of viable cells in each construct, respectively. The expression of type II collagen and aggrecan under hypoxic conditions was increased significantly as compared with that under normoxic conditions. In contrast, type X collagen expression was down-regulated in the hypoxic group. Moreover, the constructs in hypoxic group showed more significantly increased total collagen and GAG than in normoxic group, which were more close to those of the natural cartilage. These findings demonstrated that hypoxia enhanced chondrogenesis of in vitro, scaffold-free, tissue-engineered constructs generated using hMSCs induced by GDF-5. In hypoxic environments, the self-assembled constructs have a Thistological appearance and biochemical parameters similar to those of the natural cartilage.
RESUMEN
The purpose of this study was to investigate the repair of the osteoarthritis(OA)-induced cartilage injury by transfecting the new TGF-β3 fusion protein (LAP-MMP-mTGF-β3) with targeted therapy function into the bone marrow-derived mesenchymal stem cells (MSCs) in rats. The recombinant of pIRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector pIRES-EGFP. LAP and mTGF-β3 fragments were obtained from rat embryos by RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, so as to construct the recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3. pIRES-EGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs. The genetically modified MSCs were cultured in medium with MMP-1 or not. The transfected MSCs were transplanted in the rat OA models. The OA animal models were surgically induced by anterior cruciate ligament transaction (ACLT). The pathological changes were observed under a microscope by HE staining, Alcian blue, Safranin-fast Green and graded by Mankin's scale. pIRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing, and the mTGF-β3 fusion protein (39 kD) was certified by Western blotting. Those genetically modified MSCs could differentiate into chondrocytes induced by MMP and secrete the relevant-matrix. The transfected MSCs could promote chondrogenesis and matrix production in rat OA models in vivo. It was concluded that a new fusion protein LAP-MMP-mTGF-β3 was constructed successfully by gene engineering, and could be used to repair the OA-induced cartilage injury.
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Seventy-three patients with spinal nerve sheath tumor who were surgically treated in our hospital during the years 2004-2010 were retrospectively reviewed with respect to recovery of neurological function, recurrence of the tumor and occurrence of kyphotic deformities. Preoperative clinical manifestations, imaging data, surgical records and follow-up results were comprehensively analyzed. The follow-up duration was 12-60 months with an average of 32.0 months. Out of the 73 cases enrolled, 69 had gradual recovery of sensation, motor and sphincter functions 1 week to 3 months after operation. Forty-six cases had incomplete paralysis, whose American Spinal Injury Association (ASIA) grades, however, were gradually increased during the follow-up period, 4 cases had no significant improvement of the clinical symptoms and no change in ASIA grades during the follow-up period. Two cases had postoperative recurrence of the tumor. There were no deaths, no spinal instability, and no kyphotic malformations found in any cases. Our study indicated that complete removal of the tumor is important for good recovery, and an ideal surgical method may reduce the recurrence of the tumor or the occurrence of complications.
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The purpose of the present study was to observe the structure and functional change of the bone-coating-prosthesis interface in vivo and to evaluate the histocompatibility of self-made prosthetic femoral components in the body and the degree of their bonding with the surrounding bone tissues as well as their stability. Six mature beagle dogs underwent bilateral hip replacement with prosthetic femur components. Three groups were established in terms of different coating of prothesis (four joints in each group): atmosphere (A) plasma-sprayed pure titanium (Ti) prosthetic joint with hydroxyapatite (HA) coating (HA+Ti+A group); vacuum (V) plasma-sprayed pure Ti prosthetic joint with HA coating (HA+Ti+V group); vacuum plasma-sprayed pure Ti prosthetic joint with Ti-HA stepped coating (Ti+HAG+Ti+V group). The hip joints were functionally evaluated, and subjected to X-ray examination, biomechanics inspection, and histological examination. As a result, X-ray imaging revealed all prosthetic joints were in a good location and no dislocation of joint was found. Shear strength of interface was significantly higher in Ti+HAG+Ti+V group than in HA+Ti+V group (P<0.05) and HA+Ti+A group (P<0.05) at 28th week. Histological examination showed the amount of newborn bone in Ti+HAG+Ti+V group was more than in HA+Ti+V group and HA+Ti+A group after 28 weeks. It was suggested that vacuum plasma-sprayed pure Ti prosthetic joint with TI-HA stepped coating could improve the bonding capacity of bone-prosthesis, enhance the stability of prosthesis, and increase the fixion of prosthetic femoral components because of better bone growth. This new type of biological material in prosthetic femoral components holds promises for application in clinical practice.