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1.
Chin J Dent Res ; 21(2): 89-100, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29808172

RESUMEN

Over the past few decades, scientific research into neural crest-derived stem cells has progressed rapidly. The migration and differentiation of neural crest-derived stem cells has been an interesting area of research. Stem cells within teeth originating from the embryonic neural crest have attracted increasing attention in clinical and scientific research because they are easy to obtain and have superb stemness. The stem cells within the teeth include dental pulp stem cells (DPSCs), dental follicle stem cells (DFSCs), stem cells from apical papilla (SCAPs), stem cells from human exfoliated deciduous teeth (SHEDs), and periodontal ligament stem cells (PDLSCs). To date, there have been several interesting studies focusing on dental pulp regeneration, neural regeneration and the revascularization for therapeutic applications.


Asunto(s)
Cresta Neural/citología , Células Madre , Diente/citología , Humanos , Regeneración , Ingeniería de Tejidos
2.
Chin J Dent Res ; 21(2): 101-111, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29808173

RESUMEN

OBJECTIVE: To explore graphene's effects on the gene expression profile of mesenchymal stem cells, and to reveal the mechanisms of graphene-guided osteogenic differentiation. METHODS: Human adipose-derived mesenchymal stem cells (hASCs) were cultured on single-layer graphene-coated titanium disks or titanium disks in proliferation medium (control) or osteoinduction medium for 7 days before RNA extraction. After library construction and RNA sequencing, identification of differentially expressed genes was performed through Limma package of R platform, with a cut-off value of log fold change (logFC) > = |1|. Pathway and Gene ontology (GO) analyses were conducted on DAVID Bioinformatics Resources 6.8 (NIAID/NIH). Network analyses were performed by the Ingenuity Pathways Analysis (IPA). RESULTS: Signalling pathway analysis revealed the top five pathways - cytokine-cytokine receptor interaction, neuroactive-ligand receptor interaction, calcium signalling pathway, PI3K-Akt signalling pathway and cell adhesion molecules. GO analyses demonstrated significant changes on cell adhesion, calcium signalling, and epigenetic regulation. IPA network analyses demonstrated that inflammation-related pathways were influenced by graphene, while the downstream factors of histone H3 and H4 were also altered especially under the existence of osteoinduction medium. CONCLUSION: Graphene promotes osteogenic differentiation of hASCs mainly by influencing cell adhesion, cytokine-cytokine receptor interactions, inflammatory responses, and potentially influences histone H3 and H4 through epigenetic regulation.


Asunto(s)
Diferenciación Celular/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Osteogénesis/genética , Transcriptoma , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica , Grafito/farmacología , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos
3.
Int J Oral Sci ; 9(12): e6, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32987969

RESUMEN

Early childhood caries (ECC) is a considerable pediatric and public health problem worldwide. Preceding studies have focused primarily on bacterial diversity at the taxonomic level. Although these studies have provided significant information regarding the connection between dental caries and oral microbiomes, further comprehension of this microbial community's ecological relevance is limited. This study identified the carbon source metabolic differences in dental plaque between children with and without ECC. We compared the microbial community functional diversity in 18 caries-free subjects with 18 severe ECC patients based on sole carbon source usage using a Biolog assay. The anaerobic microbial community in the ECC patients displayed greater metabolic activity than that of the control group. Specific carbon source metabolism differed significantly between the two groups. Subjects from the two groups were well distinguished by cluster and principal component analyses based on discriminative carbon sources. Our results implied that the microbial functional diversity between the ECC patients and healthy subjects differed significantly. In addition, the Biolog assay furthered our understanding of oral microbiomes as a composite of functional abilities, thus enabling us to identify the ecologically relevant functional differences among oral microbial communities.

4.
PLoS One ; 11(6): e0157214, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27332814

RESUMEN

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.


Asunto(s)
Tejido Adiposo/citología , Bioimpresión/métodos , Diferenciación Celular , Osteogénesis , Impresión Tridimensional , Células Madre/citología , Andamios del Tejido/química , Alginatos/farmacología , Western Blotting , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Coristoma/patología , Técnica del Anticuerpo Fluorescente , Gelatina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Glucurónico/farmacología , Ácidos Hexurónicos/farmacología , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Osteogénesis/efectos de los fármacos , Porosidad , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Madre/efectos de los fármacos , Células Madre/ultraestructura
5.
Beijing Da Xue Xue Bao Yi Xue Ban ; 48(1): 37-44, 2016 Feb 18.
Artículo en Chino | MEDLINE | ID: mdl-26885906

RESUMEN

OBJECTIVE: To investigate the role of bone morphogenetic protein 2/7 heterodimer (BMP-2/7) in the osteogenesis of human adipose-derived stem cells (hASCs). METHODS: hASCs were exposed to three different treatments in vitro: osteogenic medium with 150 µg/L BMP-2/7 (experimental group), osteogenic medium alone (OM group) and proliferation medium (PM group). After 1, 4 and 7 days of osteogenic induction, the amount of cellular DNA was measured to investigate the cytotoxicity. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 21 and 28 days, the calcification deposition was determined by Alizarin Red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on days 1, 4, 7 and 14. In the in vivo study, 6 nude mice were used and 4 groups were set and implanted subcutaneously into the back of nude mice: (1) ß-TCP scaffold only (scaffold control group); (2) ß-TCP scaffold with hASCs cultured by PM in vitro for 1 week (PM control group); (3) ß-TCP scaffold with hASCs cultured by OM in vitro for 1 week (OM control group); (4) ß-TCP scaffold with hASCs cultured by OM with 150 µg/L BMP-2/7 in vitro for 1 week (test group). After 4 weeks of implantation, histological staining was performed to evaluate the in vivo osteogenesis of hASCs. RESULTS: After induction for 1 day, there was no significant difference between the experimental group and the PM group on the cellular DNA content (P>0.05). After 4 days, the cellular DNA content increased under the stimulation of BMP-2/7 (P<0.05). On day 7, there was no significant difference among the three groups (P>0.05). ALP activity was higher by the induction of BMP-2/7 than in OM alone and PM (P<0.05). More mineralization deposition and more expressions of osteoblast-related genes such as Runx2, ALP, COL-1A1 and OC were determined in the experimental group at different time points (P<0.05). HE staining showed that, in the test group and OM control group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the OM control group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test group. Masson's trichrome staining showed that more expression of collagen could be observed in ECM in the test group compared with the other groups. There was small amount of expression of collagen in the OM and PM control groups. No obvious positive results were found in the scaffold group. CONCLUSION: BMP-2/7 heterodimer plays a significant role in the osteogenesis of hASCs and is able to enhance the osteogenic differentiation of hASCs in vitro and in vivo.


Asunto(s)
Tejido Adiposo/citología , Proteína Morfogenética Ósea 2/farmacología , Proteína Morfogenética Ósea 7/farmacología , Diferenciación Celular , Osteogénesis , Células Madre/citología , Animales , Fosfatos de Calcio/química , Células Cultivadas , Colágeno/metabolismo , Humanos , Ratones , Ratones Desnudos , Osteoblastos/citología , Osteoblastos/metabolismo
6.
Beijing Da Xue Xue Bao Yi Xue Ban ; 48(1): 138-42, 2016 Feb 18.
Artículo en Chino | MEDLINE | ID: mdl-26885924

RESUMEN

OBJECTIVE: To explore a method of constructing universal 3-dimensional (3D) colorized digital dental model which can be displayed and edited in common 3D software (such as Geomagic series), in order to improve the visual effect of digital dental model in 3D software. METHODS: The morphological data of teeth and gingivae were obtained by intra-oral scanning system (3Shape TRIOS), constructing 3D digital dental models. The 3D digital dental models were exported as STL files. Meanwhile, referring to the accredited photography guide of American Academy of Cosmetic Dentistry (AACD), five selected digital photographs of patients'teeth and gingivae were taken by digital single lens reflex camera (DSLR) with the same exposure parameters (except occlusal views) to capture the color data. In Geomagic Studio 2013, after STL file of 3D digital dental model being imported, digital photographs were projected on 3D digital dental model with corresponding position and angle. The junctions of different photos were carefully trimmed to get continuous and natural color transitions. Then the 3D colorized digital dental model was constructed, which was exported as OBJ file or WRP file which was a special file for software of Geomagic series. For the purpose of evaluating the visual effect of the 3D colorized digital model, a rating scale on color simulation effect in views of patients'evaluation was used. Sixteen patients were recruited and their scores on colored and non-colored digital dental models were recorded. The data were analyzed using McNemar-Bowker test in SPSS 20. RESULTS: Universal 3D colorized digital dental model with better color simulation was constructed based on intra-oral scanning and digital photography. For clinical application, the 3D colorized digital dental models, combined with 3D face images, were introduced into 3D smile design of aesthetic rehabilitation, which could improve the patients' cognition for the esthetic digital design and virtual prosthetic effect. CONCLUSION: Universal 3D colorized digital dental model with better color simulation can be constructed assisted by 3D dental scanning system and digital photography. In clinical practice, the communication between dentist and patients could be improved assisted by the better visual perception since the colorized 3D digital dental models with better color simulation effect.


Asunto(s)
Modelos Dentales , Imagenología Tridimensional , Fotograbar , Color , Estética Dental , Cara , Humanos , Programas Informáticos , Diente
7.
Beijing Da Xue Xue Bao Yi Xue Ban ; 48(1): 170-4, 2016 Feb 18.
Artículo en Chino | MEDLINE | ID: mdl-26885930

RESUMEN

OBJECTIVE: Human adipose-derived stem cells (hASCs) are a highly attractive source in bone tissue engineering. To generate a luciferase reporter system that could be used to quantitatively and rapidly examine osteogenic differentiation potential of human adipose-derived stem cells (hASCs) in vitro, and eventually make it possible to monitor the osteogenic differentiation of transplanted cells in vivo. METHODS: The genomic DNA harboring promotor regions of osteocalcin and DNA sequences encoding luciferase genes were amplified by PCR and cloned into the pLVX-pTRE-puro vector to generate the OC(pro)-Luc-Puro construct. Then, the OC(pro)-Luc-Puro construct together with three assistant vectors: pMDLg/pRRE, pRSV-REV, and pVSVG, were transiently transfected into HEK293T cells followed by viral supernatants collection, filtration and concentration. Next, the hASCs stably expressing luciferase reporter gene driven by osteocalcin promotor were created with the lentivirus carrying OC(pro)-Luc-Puro cassette under puromycin selection. The OC(pro)-Luc-hASCs were then cultured in the absence or presence of osteogenic differentiation medium. On the 7th and 14th days, after osteogenic induction, cellular extracts were collected and analyzed by luciferase reporter assay. Meanwhile, alizarin red staining and quantification as well as quantitative reverse transcription PCR (qRT-PCR) analysis of osteogenic associated genes osteocalcin (OC), runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) were used to assess the osteogenic differentiation ability of OC(pro)-Luc-hASCs. RESULTS: OC(pro)-Luc-Puro plasmid and OCpro-Luc-hASCs were successfully generated. On the 7th and 14th days after osteogenic induction, the luciferase activity of the cellular extracts from OC(pro)-Luc-hASCs was dramatically increased. Consistently, the extracellular matrix mineralization, as shown by Alizarin red S (ARS) staining and quantification was also markedly intensified and a marked increase of the mRNA expression levels of OC, Runx2 and ALP, although to variable extent, was observed upon osteogenic differentiation. These results indicated that the observations from traditional experiments examining hASCs osteogenic differentiation were largely in agreement with that of our luciferase reporter assay in OC(pro)-Luc-hASCs. CONCLUSION: We established a luciferase reporter system that could be used to rapidly, quantitatively and specifically determine osteogenic differentiation ability of hASCs. Comparing with the traditional time-consuming methods, the system we generated here was highly effective. This system not only can be used to examine ostogenic differentiation of hASCs in a high throughput manner, but also provides a way to monitor ostogenic differentiation of cells in vivo.


Asunto(s)
Tejido Adiposo/citología , Genes Reporteros , Osteogénesis , Células Madre/citología , Fosfatasa Alcalina/genética , Huesos , Diferenciación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Células HEK293 , Humanos , Luciferasas , Osteocalcina/genética , Regiones Promotoras Genéticas , Ingeniería de Tejidos
8.
Beijing Da Xue Xue Bao Yi Xue Ban ; 47(5): 878-82, 2015 Oct 18.
Artículo en Chino | MEDLINE | ID: mdl-26474635

RESUMEN

In this article, different methods to deal with teeth fractures were discussed by presenting a case of traumatic crown-root fracture in the anterior esthetic zone. The traumatic crown-root fracture is a common problem in clinic. When a fracture line locates in close proximity to or below the alveolar bone crest, the fracture most likely involve the junctional epithelium and the connective tissue attachment. This type of fracture becomes a challenge for restorative dentists because it involves biologic, functional, and esthetic considerations, especially when the fracture occurs in an esthetic area. In this case, a young patient presented with two fractured upper anterior teeth to the Department of Periodontics, Peking University School and Hospital of Stomatology. After the comprehensive clinical evaluation, the right central incisor was decided to extract for implant therapy and the right lateral incisor was decided to retain by one modified crown lengthening surgery. The most common technique applied to save a retained root is a clinical crown lengthening procedure. However, the aggressive alveolar bone resection of both target and adjacent teeth to reestablish the bone width and periodontal health may compromise functional and esthetic outcomes. To reduce loss of excessive osseous tissue during osteotomy procedure, the modified crown lengthening of the right lateral incisor was performed, including minor bone resection and root reshaping. Regarding the right central incisor, the retained root was all located below the alveolar bone crest. The extraction and implant procedure, combined with guided bone graft were performed to avoid the damage to neighbor teeth during traditional restorative therapy and to reshape a preferable buccal contour. At the last visit, the patient was recalled with healthy periodontium, normal tooth function and favorable esthetic results.


Asunto(s)
Fracturas de los Dientes/terapia , Proceso Alveolar , Beijing , Estética Dental , Fracturas Óseas , Humanos , Incisivo , Raíz del Diente
9.
Beijing Da Xue Xue Bao Yi Xue Ban ; 47(1): 47-51, 2015 Feb 18.
Artículo en Chino | MEDLINE | ID: mdl-25686328

RESUMEN

OBJECTIVE: To construct and evaluate a novel tissue-engineered bone composed of murine stromal cell-derived factor 1(mSDF-1), simvastatin (SIM) and collagen scaffold (Bio-Oss®), serving as a cell-homing approach for bone formation. METHODS: In the study, 32 ICR mice were randomly divided into 4 groups,each group including 8 mice. The drug-loaded collagen scaffolds were implanted subcutaneously onto the cranium of each mouse according to the groups: (1) 1:50 (volume ratio) dimethyl sulfoxide (DMSO)/phosphate-buffered saline (PBS) solution + collagen scaffold (blank control group); (2) 10⁻³ mol/L SIM solution + collagen scaffold (SIM group); (3) 200 mg/L mSDF-1 solution + collagen scaffold (mSDF-1 group); and (4) 10® mol/L SIM +200 mg/L mSDF-1 solution + collagen scaffold (SIM + mSDF-1 group). One week after implantation, the mice were treated by injecting the same drug solution mentioned above around the scaffold once a day for two days. The specimens were harvested 6 weeks after implantation and the bone formation was evaluated by soft X-ray analysis, HE staining and immunohistochemical staining. Angiogenesis of each group was checked by calculation of vessels in each tissue section. RESULTS: Six weeks after implantation, the collagen scaffolds were retrieved. The value of gray scale for the SIM+mSDF-1 group [(421 836.5 ± 65 425.7)pixels] was significantly higher than that of the blank control group[(153 345.6 ± 45 222.2) pixels, P<0.01], the SIM group [(158 119.2 ± 100 284.2)pixels, P<0.01], and the mSDF-1 group[(255 529.5 ± 152 142.4)pixels, P<0.05]; HE staining analysis revealed that significant bone formation was achieved in the SIM + mSDF-1 group; The immunohistochemical staining showed the existence of osteopontin and osteocalcin in the SIM + mSDF-1 group; There were more vessels in the SIM+mSDF-1 group[(46 ± 8)vessels/mm²] than in the blank control group [(23 ± 7) vessels/mm2, P<0.01], and the SIM group[(24 ± 6) vessels/mm2, P<0.01]. CONCLUSION: The novel tissue-engineered bone composed of mSDF-1, SIM and collagen scaffolds has the potential to form bone subcutaneously in vivo. It represents a novel method of in vivo bone re-generation without seed cell delivery.


Asunto(s)
Sustitutos de Huesos/química , Quimiocina CXCL12/farmacología , Minerales/química , Osteogénesis , Simvastatina/farmacología , Animales , Colágeno/química , Ratones , Ratones Endogámicos ICR , Osteocalcina/metabolismo , Osteopontina/metabolismo , Cráneo , Ingeniería de Tejidos
10.
Biomaterials ; 35(15): 4489-98, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24589359

RESUMEN

The purpose of this study was to investigate the cooperative effects of simvastatin (SIM) and stromal cell-derived factor-1α (SDF-1α) on the osteogenic and migration capabilities of mesenchymal stem cells (MSCs), and construct a cell-free bone tissue engineering system comprising SIM, SDF-1α and scaffold. We found that 0.2 µm SIM significantly increased alkaline phosphatase activity (P < 0.05) of mouse bone marrow MSCs with no inhibition of cell proliferation, and enhanced the chemotactic capability of SDF-1α (P < 0.05). Next, we constructed a novel cell-free bone tissue engineering system using PLGA loaded with SIM and SDF-1α, and applied it in critical-sized calvarial defects in mice. New bone formation in the defect was evaluated by micro-CT, HE staining and immunohistochemistry. The results showed that PLGA loaded with SIM and SDF-1α promoted bone regeneration significantly more than controls. We investigated possible mechanisms, and showed that SDF-1α combined with SIM increased MSC migration and homing in vivo, promoted angiogenesis and enhanced the expression of BMP-2 in newly-formed bone tissue. In conclusion, SIM enhanced the chemotactic capability of SDF-1α and the cell-free bone tissue engineering system composed of SIM, SDF-1α and scaffold promoted bone regeneration in mouse critical-sized calvarial defects.


Asunto(s)
Regeneración Ósea/efectos de los fármacos , Quimiocina CXCL12/uso terapéutico , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Simvastatina/uso terapéutico , Cráneo/lesiones , Ingeniería de Tejidos/métodos , Fosfatasa Alcalina/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL12/administración & dosificación , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Simvastatina/administración & dosificación , Cráneo/efectos de los fármacos , Cráneo/patología
11.
Beijing Da Xue Xue Bao Yi Xue Ban ; 46(1): 90-4, 2014 Feb 18.
Artículo en Chino | MEDLINE | ID: mdl-24535356

RESUMEN

OBJECTIVE: To explore a new method of patient-involved digital design, esthetic outcome prediction and fabrication for the esthetic rehabilitation of anterior teeth, and to provide an alternative choice for the restoration of anterior teeth. METHODS: In this study, 32 patients with esthetic problems in their anterior teeth were included and divided into two groups randomly: the experimental group (16 patients) and control group (16 patients). In the experimental group, the dentition and facial images were obtained by intra-oral scanning and Three-dimensional (3D) facial scanning and then calibrated. The design of the rehabilitation and the esthetic outcome prediction were created by computer-aided design (CAD) software. After morphologic modification according to the patients' opinions, prostheses were fabricated according to the final design by computer-aided manufacturing (CAM) equipment. As for the control group, the regular design method was applied to restore their anterior teeth. The time consuming in the first insertion of each restoration in both groups was recorded. The quality of the prostheses was assessed by another prosthedontist. The satisfaction to prostheses and the facial appearance were evaluated by the patients. RESULTS: The process of the patient-involved digital design and outcome anticipation was successfully established. The patients were satisfied with the esthetic effects of the anterior restoration made by the digital technique. The acceptance rate of the patients on the digital rehabilitation in the experimental group was 100%. There was no significant difference of the quality of the prostheses between the two groups. The satisfaction rate of the patients on prostheses and facial appearance was significantly higher in the experimental group than in the control group (P < 0.05). In addition, the time consuming in the first insertion of the experimental group was much shorter than that in the control group (P < 0.01). CONCLUSION: The new method of the patient-involved digital design, esthetic outcome prediction and fabrication for the esthetic rehabilitation of anterior teeth is a practical technique. This method is useful in shortening the time consuming of the restoration of anterior teeth and improving the patient satisfaction with the esthetic outcome.


Asunto(s)
Diseño Asistido por Computadora , Estética Dental , Incisivo , Participación del Paciente , Humanos , Imagenología Tridimensional , Satisfacción del Paciente
12.
Beijing Da Xue Xue Bao Yi Xue Ban ; 44(6): 916-20, 2012 Dec 18.
Artículo en Chino | MEDLINE | ID: mdl-23247458

RESUMEN

OBJECTIVE: To explore the effect of human adipose-derived stromal cells (hASCs) on the osteogenesis during the process of bone formation in vivo, and to lay the foundation of further investigations on the mechanism of in vivo osteogenesis of hASCs. METHODS: hASCs were isolated from adipose tissue by the method of collagenase digestion, and were routinely proliferated and passaged. In the in vivo study 16 nude mice were used and 4 groups were set and implanted subcutaneously into the back of nude mice: (1) blank; (2) ß-tricalcium phosphate (ß-TCP) scaffold only (scaffold control group); (3) ß-TCP scaffold with human fibroblasts (negative cell control group); (4) ß-TCP scaffold with hASCs (test group). After 1 week, 2 weeks, 4 weeks and 6 weeks of implantation, samples from the 4 nude mice were collected at each time point. Scanning electron microscope (SEM) observation and histological staining were performed to evaluate the in vivo osteogenesis of hASCs. RESULTS: SEM images showed that large amount of extracellular matrix (ECM) could be observed around hASCs in test group after 2 weeks of implantation. At the time point of 4 weeks, mineral deposit was found in ECM. At the time point of 6 weeks, the mineral deposit was observed to increase significantly. HE staining showed that the ECM with eosinophilic staining could be observed around hASCs after 2 weeks of implantation. At the time point of 4 weeks, newly-formed bone-like tissue could be found in ECM around the scaffold materials. At the time point of 6 weeks, more bone-like tissues were observed in ECM with typical structure of bone tissue. In comparison, no obvious mineralization and bone-like tissue were found in other groups. CONCLUSION: hASCs play important roles in the process of osteogenesis in vivo, including secretion of large amount of ECM, acceleration of the mineralization of ECM and guidance for the formation of bone-like tissues.


Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/fisiología , Osteogénesis , Células del Estroma/trasplante , Ingeniería de Tejidos/métodos , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Madre Multipotentes/citología , Células Madre Multipotentes/trasplante , Células del Estroma/citología , Andamios del Tejido , Trasplante Heterólogo
13.
Beijing Da Xue Xue Bao Yi Xue Ban ; 44(3): 475-80, 2012 Jun 18.
Artículo en Chino | MEDLINE | ID: mdl-22692324

RESUMEN

OBJECTIVE: To investigate the effect of recombinant human tumor necrosis factor alpha (rhTNF-α) on the osteogenesis potential of the osteo-induced human adipose-derived stromal cells (hASCs) in vitro. METHODS: hASCs at passage 4 were divided into four groups according to culturing conditions: basal medium [BM, DMEM + 10% FBS + antibiotics], BM with 10 µg/L rhTNF-α, osteogenic medium (OM, BM + dexamethasone + L-ascorbate + ß-glycerophosphate) and OM with 10 µg/L rhTNF-α. On days 3, 7, 14 and 21, alkaline phosphatase (ALP) activities were examined. On days 14 and 21, the staining and quantitation of calcium deposition were performed. For the cells under osteogenic induction, osteoblast-related genes, such as core-binding factor α1 (Cbfa1), Osterix (Osx) and osteocalcin (OC) were analyzed with reverse transcription PCR on days 3, 7, 14, and 21, and real time PCR was performed to confirm the effect of rhTNF-α on genes expression on day 3 . RESULTS: rhTNF-α promoted ALP activities of induced hASCs on day 14 (3.527 ± 0.415 vs. 2.345 ± 0.354,P<0.01) and on day 21 (3.106 ± 0.105 vs. 2.442 ± 0.163,P<0.01) and promoted calcium deposition of induced hASCs on day 14 (2.896 ± 0.173 vs. 0.679 ± 0.173,P<0.01) and on day 21 (2.231 ± 0.233 vs. 1.729 ± 0.229, P<0.01). RT-PCR and Real-time PCR assays showed that rhTNF-α augmented the expression of Cbfa1, Osx and OC of these cells. CONCLUSION: The findings indicate that 10 µg/L rhTNF-α can promote the osteogenic potential of osteogenetically induced hASCs in vitro.


Asunto(s)
Adipocitos/citología , Diferenciación Celular/efectos de los fármacos , Osteoblastos/citología , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adulto , Fosfatasa Alcalina/metabolismo , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteogénesis/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Factor de Necrosis Tumoral alfa/genética
14.
Beijing Da Xue Xue Bao Yi Xue Ban ; 44(1): 55-8, 2012 Feb 18.
Artículo en Chino | MEDLINE | ID: mdl-22353901

RESUMEN

OBJECTIVE: To investigate the osteogenic capability of primary human adipose-derived stromal cells (hASCs) in vivo. METHODS: hASCs were isolated from adipose tissue by the method of collagenase digestion. After 7 and 14 days of osteogenic induction, alkaline phosphatase (ALP) staining and Alizarin Red staining were performed to test the osteogenic potential of hASCs in vitro. After 14 days of adipogenic induction, the adipogenic potential of hASCs was assayed by Oil Red O staining.In the in vivo part, 12 nude mice were used. Test group (scaffold with hASCs) and control group (scaffold only) were symmetrically implanted into the back of nude mice. After 4 weeks and 8 weeks of implantation, samples were collected. Histological and immunohistochemical staining were performed to investigate the osteogenic capability of hASCs. RESULTS: Approximately 6×10(7) hASCs could be isolated from 300 mL adipose tissue. ALP, Alizarin Red and Oil Red O staining of hASCs showed positive results after specific inductions. These results demonstrated the osteogenic and adipogenic potentials of hASCs in vitro. Bone-like tissue could be observed in the test group at 4 weeks and 8 weeks after the implantation. Immunohistochemical staining showed that there were positive results of osteocalcin, ALP and anti-human nuclei in the bone-like tissue areas. CONCLUSION: A large number of primary hASCs can be isolated from human adipose tissue; hASCs combined with scaffold show osteogenic capability in vivo.


Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/fisiología , Osteogénesis , Células del Estroma/trasplante , Ingeniería de Tejidos/métodos , Tejido Adiposo/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Proliferación Celular , Humanos , Ratones , Ratones Desnudos , Células Madre Multipotentes/citología , Células Madre Multipotentes/trasplante , Osteogénesis/genética , Células del Estroma/citología , Técnicas de Cultivo de Tejidos , Andamios del Tejido , Trasplante Heterólogo
15.
Beijing Da Xue Xue Bao Yi Xue Ban ; 44(1): 160-2, 2012 Feb 18.
Artículo en Chino | MEDLINE | ID: mdl-22353921

RESUMEN

Human adipose-derived stromal cells (hASCs) can be obtained from adipose tissues that offer an abundant and easily accessible pool of stem cells. Thus, hASCs have become a highly attractive source of seed cells in bone tissue engineering and have promising prospects in bone regeneration. Since 2002, our research group has performed a series of experiments on hASCs and its application in bone tissue engineering, including: to substitute dexamethasone by 1,25 (OH)2 vitamin D3 to induce osteogenic differentiation of hASCs; to explore the effect of epigenetic regulation and to inflammation on the osteogenic differentiation of hASCs; to construct a novel and simple tissue engineered bone system by hASCs and human platelet-rich plasma (hPRP) and to investigate the bone formation capability of this tissue engineered bone and the stimulatory effect of simvastatin. Our results suggested that 1,25 (OH)2 vitamin D3 could replace dexamethasone to induce the osteogenic differentiation of hASCs; retinoblastoma binding protein 2 (RBP2), as one of histone demethylases, could regulate the osteogenic differentiation of hASCs epigenetically while tumor necrosis factor α (TNFα), as a inflammatory factor, could also influence the osteogenic differentiation of hASCs. Moreover, we found that in vivo bone formation could be detected by our novel tissue engineered bone composed with hASCs and hPRP; simvastatin could enhance the bone formation capability of this tissue-engineered structure.


Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/fisiología , Osteogénesis , Células del Estroma/citología , Ingeniería de Tejidos/métodos , Calcitriol/farmacología , Células Cultivadas , Medios de Cultivo/farmacología , Humanos
16.
Emerg Med J ; 29(2): 113-7, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21335580

RESUMEN

BACKGROUND: The aim of this study was to test if Procalcitonin PCT value at the time of admission is a predictor of mortality and/or a diagnostic marker of concomitant infection in exertional heatstroke. METHODS: 68 patients with exertional heatstroke admitted to the multidisciplinary intensive care unit were studied. Serum PCT was detected by means of a specific and ultrasensitive immunoluminometric assay within 2 h of admission. The Acute Physiology and Chronic Health Evaluation (APACHE) II score was evaluated within 24 h of admission. RESULTS: There was no significant difference in PCT levels between concomitant infection and non-infection patients (p=0.712). Elevated PCT level in exertional heatstroke patients was associated with a more critical pathological state. PCT values in patients with multiple organ dysfunction syndrome (MODS) were significantly higher than those without MODS (p=0.007.). PCT values were also positively correlated with APACHE II scores (r=0.588, p=0.016). PCT values in non-survivors were higher than in survivors at univariate regression analysis (p=0.017). After adjusting for confounders, PCT concentration also remained an independent determinant of mortality (OR 2.98; 95% CI 1.02 to 4.41; p=0.039). Receiver operating characteristic curve for PCT concentration was located above the reference line, which shows an association with mortality. The area under the curve for PCT concentration (0.705; 95% CI 0.547 to 0.862) was statistical significantly (p=0.019). As a predictor of mortality, PCT value was inferior to APACHE II score. CONCLUSIONS: PCT value at the time of admission is an independent predictor of mortality, but maybe not a good indicator of concomitant infection in exertional heatstroke.


Asunto(s)
Calcitonina/sangre , Golpe de Calor/sangre , Golpe de Calor/mortalidad , Precursores de Proteínas/sangre , Adulto , Área Bajo la Curva , Biomarcadores/sangre , Temperatura Corporal/fisiología , Péptido Relacionado con Gen de Calcitonina , China , Humanos , Masculino , Esfuerzo Físico/fisiología , Valor Predictivo de las Pruebas , Estudios Prospectivos , Análisis de Regresión , Factores de Riesgo , Sensibilidad y Especificidad , Adulto Joven
17.
Zhonghua Kou Qiang Yi Xue Za Zhi ; 46(3): 148-52, 2011 Mar.
Artículo en Chino | MEDLINE | ID: mdl-21575435

RESUMEN

OBJECTIVE: To explore the effect of retinoblastoma binding protein 2 (RBP-2), a histone H3K4 demethylase, on osteogenic differentiation of human adipose-derived stromal cell (hASC). METHODS: According to the GenBank sequence information of RBP-2, four different small interfering RNAs (siRNA) targeting RBP-2 gene were designed and the corresponding short hairpin RNAs (shRNA) were cloned into pLL 3.7 lentivirus RNA interference vector. The lentivirus with RBP-2-siRNA was packaged in 293T cells. The effective sequence was examined and selected by Western blotting and real-time PCR. The lentiviruses with efficient knockdown effects were used to infect hASC. On the 14th day after osteogenic differentiation, alkaline phosphatase (ALP) activities of hASC were quantitatively tested and at the same time, ALP staining and alizarin red staining were performed to assess the difference of osteogenic differentiation between the knockdown group and the control group. RESULTS: The recombinant lentivirus siRNA targeting RBP-2 was successfully constructed and the expression of RBP-2 mRNA and protein were dramatically suppressed by infection with RBP-2-siRNA lentivirus. On the 14th day after osteogenic induction, ALP activity of hASC in the knockdown group [(299.2 ± 22.7), (224.3 ± 17.7) U/g] was much stronger than that in the control group [(129.9 ± 12.9) U/g, P < 0.05] and the same result was achieved for the ALP staining and alizarin red staining. CONCLUSIONS: The constructed RBP-2-siRNA lentivirus could markedly decrease the expression of RBP-2 and promote osteogenic differentiation of hASC. It indicated that RBP-2 can repress the osteogenic differentiation of hASC.


Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular , Osteogénesis , Proteína 2 de Unión a Retinoblastoma/metabolismo , Células del Estroma/citología , Adulto , Fosfatasa Alcalina/metabolismo , Línea Celular Tumoral , Células Cultivadas , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Lentivirus , Osteosarcoma/patología , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteína 2 de Unión a Retinoblastoma/genética , Células del Estroma/metabolismo
18.
Beijing Da Xue Xue Bao Yi Xue Ban ; 41(5): 565-70, 2009 Oct 18.
Artículo en Chino | MEDLINE | ID: mdl-19829676

RESUMEN

OBJECTIVE: To investigate the proliferation and the secretion of vascular endothelial growth factor(VEGF), fibroblast growth factor-2(FGF-2) and insulin-like growth factor-1(IGF-1) of human adipose tissue-derived stromal cells(hADSCs) before and after osteogenic differentiation under the stimuli of recombinant human tumor necrosis factor alpha (rhTNF-alpha). METHODS: hADSCs were obtained from human lipoaspirates. All the cells used were at passage four. The proliferation of hADSCs was measured with MTT assays 48, 72, 96 hours after being treated with 0, 1, 5, 10, 50 or 100 microg/L rhTNF-alpha respectively. The secretion of VEGF, FGF-2 and IGF-1 of the undifferentiated hADSCs under stimuli of rhTNF-alpha with the above 5 concentration grades was observed and the secretion of these 3 growth factors of hADSCs at different stages of osteogenic differentiation under stimuli of 10 microg/L rhTNF-alpha was also observed. All the supernatants were harvested for measuring after 24 hours' incubation with rhTNF-alpha. The secretion of VEGF, FGF-2 and IGF-1 was measured with ELISA, and the values were normalized to the cell number of the corresponding wells. RESULTS: The effect of rhTNF-alpha on the proliferation of hADSCs varied with the concentration and time. Compared with the control(0 microg/L), 10 microg/L rhTNF-alpha showed no suppression or acceleration on proliferation of hADSCs at hour 48, but significantly promoted the proliferation at hour 96 (0.903+/-0.042 vs 0.810+/-0.011, P<0.01), 100 microg/L rhTNF-alpha seemed to suppress the proliferation at hour 48 (0.317+/-0.024 vs 0.458+/-0.046, P<0.01), but appeared to promote it (0.956+/-0.030 vs 0.810+/-0.011, P<0.01) at hour 96. rhTNF-alpha(1, 5, 10, 50 and 100 microg/L) significantly increased VEGF, FGF-2 and IGF-1 production of hADSCs versus the control (0 microg/L) (P<0.01). After osteogenic differention, the secretion of the three growth factors of hADSCs (without rhTNF-alpha treated) was elevated with the days increasing. Under the stimulus of 10 microg/L rhTNF-alpha, the hADSCs after 1 day of osteogenic differentiation significantly increased the secretion of VEGF (P<0.01) compared with the group without rhTNF-alpha treated; after 3 and 7 days of osteogenic differentiation, the hADSCs significantly increased the secretion of VEGF (P<0.01), FGF-2 (P<0.05)and IGF-1 (P<0.05). However, after 14 days of osteogenic differentiation, 10 microg/L rhTNF-alpha appeared to suppress the production of VEGF (P<0.01), FGF-2 (P<0.05) and IGF-1 (P<0.05) of the differentiated hADSCs. CONCLUSION: Within certain concentration range, rhTNF-alpha can promote the proliferation of hADSCs and the production of VEGF, FGF-2 and IGF-1. The effect of 10 microg/L rhTNF-alpha on the production of VEGF, FGF-2 and IGF-1 of the differentiated hADSCs varied at different stages of osteogenic differentiation.


Asunto(s)
Tejido Adiposo/metabolismo , Inductores de la Angiogénesis/metabolismo , Osteogénesis , Células del Estroma/enzimología , Factor de Necrosis Tumoral alfa/farmacología , Tejido Adiposo/citología , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas Recombinantes/farmacología , Células del Estroma/citología , Células del Estroma/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo
19.
Zhongguo Wei Zhong Bing Ji Jiu Yi Xue ; 21(3): 147-50, 2009 Mar.
Artículo en Chino | MEDLINE | ID: mdl-19278583

RESUMEN

OBJECTIVE: To analyze clinical effect of immuno-modulatory therapy with ulinastatin and thymosin alpha1 on patients with sepsis. METHODS: Two hundred and forty-two septic patients admitted to Guangzhou General Hospital of Guangzhou Military Command intensive care unit (ICU) during 2004.10-2008.6 were included, and they were randomly divided into treatment group (128 cases) and control group (114 cases). The patients in control group were given regular conventional treatment according to Surviving Sepsis Campaign (SSC) in 2004, including early fluid resuscitation, antibiotic therapy, mechanical ventilation (MV) and blood purification. The treatment group received conventional treatment plus immuno-modulation therapy including ulinastatin (first 200 kU injection intravenous twice a day for 4 days and 100 kU for another 6 days) and thymosin alpha1 (1.6 mg subcutaneous twice a day for 4 days, followed by 1.6 mg per day subcutaneous for another 6 days). The total treatment course was 10 days. General demographics were observed, and acute physiology and chronic health evaluation II (APACHE II) scores were recorded. Serum interleukin-6 (IL-6), IL-10 levels of peripheral blood were detected by enzyme linked immunosorbent assay (ELISA). Peripheral blood CD14(+) monocyte human leucocyte antigen DR (HLA-DR) expression, and ratio of helper T lymphocyte 1 (Th1) cytokines interferon-gamma (CD4(+)IFN-gammaww(+)), and Th2 cytokines (CD4(+) IL-4(+)) were assessed with flow cytometer. Duration of infection and MV, length of ICU stay, rate of development of multiple organ dysfunction syndrome (MODS) and mortality rate on 28 days were observed as end-point. RESULTS: Before treatment, there was no difference in all biomarkers between two groups (all P>0.05). After treatment, peripheral blood CD14ww+ monocyte HLA-DR expression and the ratio of CD4(+)IFN-gamma (+)/CD4(+) IL-4(+) increased significantly in the treatment group (both P<0.05), with serum IL-6, IL-10 levels and APACHE II scores all reduced remarkably (all P<0.05). The values showed significant differences compared with those of control group (all P<0.05). The MODS development rate in the treatment group was much lower than that of control group (21% vs. 47%, P<0.05), and the length of use of MV was significantly reduced [(6.08+/-2.46) days vs. (8.23+/-3.47) days, P<0.05]. There was no difference in the infection duration and length of ICU stay (both P>0.05). The mortality rate on 28 days in the treatment group was much lower than that in control group (20% vs. 33%, P<0.05). CONCLUSION: The immuno-modulation therapy of ulinastatin and thymosin alpha1 can remarkably improve the duration of MV and the development rate of MODS and mortality rate on 28 days in the patients with sepsis, probably due to its effect in ameliorating the immuno-imbalance state of the patients. However, the duration of infection and length of ICU stay are not effected.


Asunto(s)
Glicoproteínas/uso terapéutico , Sepsis/tratamiento farmacológico , Timosina/análogos & derivados , Adulto , Anciano , Femenino , Antígenos HLA-DR/metabolismo , Humanos , Interferón gamma/metabolismo , Interleucina-10/sangre , Interleucina-4/metabolismo , Interleucina-6/sangre , Receptores de Lipopolisacáridos , Masculino , Persona de Mediana Edad , Sepsis/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Timalfasina , Timosina/uso terapéutico
20.
Clin Biochem ; 42(10-11): 1025-31, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19324026

RESUMEN

OBJECTIVE: This purpose of this study was to determine if serum procalcitonin (PCT) concentration at the time of admission to the ICU is a predictor of all-cause short-term mortality. DESIGN AND METHODS: This prospective cross-sectional study was conducted over a 16-month period with 86 consecutive critically ill patients. The semi-quantitative PCT-Q test was performed and APACHE II scores and C-reactive protein (CRP) concentrations were determined within 24 h of admission. RESULTS: PCT-Q test value was a better predictor of all-cause short-term mortality than CRP value or APACHE II score. PCT > or = 10 ng/mL was highly and independently correlated with mortality. Use of PCT-Q > or = 10 ng/mL was superior to use of APACHE II > or = 25 or CRP > or = 10 mg/dL as a predictor of poor outcome. CONCLUSIONS: A PCT-Q value > or = 10 ng/mL obtained at the time of admission to the ICU is a strong predictor of short-term mortality.


Asunto(s)
Calcitonina/sangre , Unidades de Cuidados Intensivos/estadística & datos numéricos , Admisión del Paciente , Precursores de Proteínas/sangre , Sepsis/mortalidad , APACHE , Proteína C-Reactiva/análisis , Péptido Relacionado con Gen de Calcitonina , Enfermedad Crítica/mortalidad , Demografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Curva ROC , Sepsis/sangre , Sepsis/microbiología , Análisis de Supervivencia , Factores de Tiempo , Resultado del Tratamiento
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