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1.

Introduction

As one group of periodontal ligament (PDL) cells, human periodontal ligament stem cells (hPDLSCs) have been isolated and identified as mesenchymal adult stem cells (MSCs) since 2004. It has been well accepted that PDL sensitively mediates the transmission of stress stimuli to the alveolar bone for periodontal tissue remolding. Besides, the direction of MSCs differentiation has been verified regulated by mechanical signals. Therefore, we hypothesized that tensile strain might act on hPDLSCs differentiation, and the early response to mechanical stress should be investigated.

Material and methods

The hPDLSCs were cultured in vitro and isolated via a magnetic activated CD146 cell sorting system. After investigation of surface markers and other experiments for identification, hPDLSCs were subjected to cyclic tensile strain at 3,000 µstrain for 3 h, 6 h, 12 h, and 24 h, without addition of osteogenic supplements. In the control groups, the cells were cultured in similar conditions without mechanical stimulation. Then osteogenic related genes and proteins were analyzed by RT-PCR and western blot.

Results

Cyclic tensile strain at 3,000 µstrain of 6 h, 12 h, and 24 h durations significantly increased mRNA and protein expressions of Satb2, Runx2, and Osx, which were not affected in unloaded hPDLSCs.

Conclusions

We indicate that hPDLSCs might be sensitive to cyclic tensile strain. The significant increase of Runx2, Osx and Satb2 expressions may suggest an early response toward osteogenic orientation of hPDLSCs.  相似文献   

2.

Introduction

Adipose tissue is a source of multipotent adult stem cells. Most studies on human adipose-derived stem cells (ASC) have been on the early passages. Studies in extensive expansion have not been well established yet. In this study, we aim to investigate the effects of extensive expansion on the adipogenic differentiation capability of ASC.

Material and methods

The ability of ASC to undergo adipogenic differentiation in extensive expansion was evaluated by morphological changes, differentiation assay by using Oil Red O staining and changes in the genes expression levels of adipogenic genes, osteogenic genes and stemness genes using quantitative polymerase chain reaction (qPCR) after induction.

Results

Morphological study showed that the formation of lipid droplets can be observed at all passages but decreased at P20 after induction. Data from qPCR showed that most adipogenicgenes expression increased significantlyat P5, P10 and P15 but decreased at P20 after induction. On the other hand, osteogenic genes showed no significant changes after adipogenic induction indicating low potentiality of adipogenic-induced ASC to become osteogenic cells. While stemness genes expression levels showed a decrease or no significant changes after adipogenic induction except Nanog3, which showed a significant increase at P15 and P20.

Conclusions

The ability of ASC to differentiate into mature adipogenic cells decreased after P10 and the decrease in the osteogenics gene expression level during adipogenic induction suggested that the osteogenesis and adipogenesis are not parallel events.  相似文献   

3.

OBJECTIVE

To evaluate the effects of the petroleum ether extract of Cissus quadrangularis on the proliferation rate of bone marrow mesenchymal stem cells, the differentiation of marrow mesenchymal stem cells into osteoblasts (osteoblastogenesis) and extracellular matrix calcification. This study also aimed to determine the additive effect of osteogenic media and Cissus quadrangularis on proliferation, differentiation and calcification.

METHODS

MSCs were cultured in media with or without Cissus quadrangularis for 4 weeks and were then stained for alkaline phosphatase. Extracellular matrix calcification was confirmed by Von Kossa staining. marrow mesenchymal stem cells cultures in control media and osteogenic media supplemented with Cissus quadrangularis extract (100, 200, 300 μg/mL) were also subjected to a cell proliferation assay (MTT).

RESULTS

Treatment with 100, 200 or 300 μg/mL petroleum ether extract of Cissus quadrangularis enhanced the differentiation of marrow mesenchymal stem cells into ALP-positive osteoblasts and increased extracellular matrix calcification. Treatment with 300 μg/mL petroleum ether extract of Cissus quadrangularis also enhanced the proliferation rate of the marrow mesenchymal stem cells. Cells grown in osteogenic media containing Cissus quadrangularis exhibited higher proliferation, differentiation and calcification rates than did control cells.

CONCLUSION

The results suggest that Cissus quadrangularis stimulates osteoblastogenesis and can be used as preventive/ alternative natural medicine for bone diseases such as osteoporosis.  相似文献   

4.

Background and Aims:

Sepsis management remains a great challenge for intensive care medicine. The aim of this study was to evaluate the effect of adding dobutamine versus epinephrine to norepinephrine in treating septic shock patients refractory to fluid therapy.

Materials and Methods:

Sixty adult patients with the diagnosis of septic shock were included in this study. Norepinephrine infusion was started at a dose of 0.05 μg/kg/min, and increased gradually up to 0.1 μg/kg/min. Upon reaching this dose, patients with mean arterial pressure <70 mmHg were further divided randomly into two equal groups. In group I: the patients continued on norepinephrine and dobutamine was added at a starting dose of 3 μg/kg/min and increased in increments of 2 μg/kg/min up to 20 μg/kg/min. In group II: the patients continued on norepinephrine and epinephrine was added in a starting dose of 0.05 μg/kg/ min and increased in increments of 0.03 μg/kg/min up to 0.3 μg/kg/min.

Results:

Group II patients developed significantly better cardiovascular parameters, lower arterial pH and higher serum lactate and urine output; however, the 28-day mortality and major adverse effects were comparable in both groups.

Conclusions:

The addition of epinephrine to norepinephrine has positive effects on the cardiovascular parameters but negative results on the serum lactate concentration and systemic pH compared with the addition of dobutamine to norepinephrine.  相似文献   

5.

Introduction

Mechanical forces play critical roles in the development and remodelling process of bone. As an alternative cell source for bone engineering, adipose-derived stem cells (ASCs) should be fully investigated for their responses to mechanical stress and the mechanisms responsible for osteogenic induction in response to mechanical signals.

Material and methods

We hypothesized that appropriate application of uniaxial cyclic tensile strain to ASCs could increase bone morphogenetic protein-2 (BMP-2) expression and improve osteogenesis of ASCs. To test our hypothesis, ASCs from the same flask of the same donor were subjected to tensile strain with different patterns in order to eliminate the difference of donor site and passage. After surface markers investigation, the osteo-induced ASCs were subjected to uniaxial cyclic tensile stretch with the following two loading patterns: long duration continuous pattern (6 h, 1 HZ, 2000 µɛ) and short duration consecutive pattern (17 min every day for 10 consecutive days, 1 HZ, 2000 µɛ). Then osteogenic related genes were analysed by real-time PCR.

Results

The ASCs were positive for the markers STRO-1, CD90 and CD44 and negative for CD34. Cyclic tensile strain of 6 continuous h’ duration significantly increased gene expressions of BMP-2 and Runx2, and depressed OCN mRNA expression. In contrast, mechanical loading of 17 min every day did not significantly affect gene expression of BMP-2, Runx2, OCN or ALP.

Conclusions

We indicate that ASCs may sense mechanical loading in a duration-dependent manner and cyclic tensile stretch may modulate the osteogenic differentiation of ASCs via the BMP-2 signalling pathway.  相似文献   

6.
Overview: Periostin (POSTN) is critical to bone and dental tissue morphogenesis, postnatal development, and maintenance; however, its roles in tissue repair and regeneration mediated by human periodontal ligament mesenchymal stem cells (PDLSCs) remain unclear. The present study was designed to evaluate the effects of POSTN on hPDLSCs in vitro. Materials and Methods: hPDLSCs were isolated and characterized by their expression of the cell surface markers CD44, CD90, CD105, CD34, and CD45. Next, 100 ng/mL recombinant human POSTN protein (rhPOSTN) was used to stimulate the hPDLSCs. Lentiviral POSTN shRNA was used to knockdown POSTN. The cell counting kit-8 (CCK8) and scratch assay were used to analyze cell proliferation and migration, respectively. Osteogenic differentiation was investigated using an alkaline phosphatase (ALP) activity assay, alizarin staining, and quantitative calcium analysis and related genes/protein expression assays. Results: Isolated hPDLSCs were positive for CD44, CD90, and CD105 and negative for CD34 and CD45. In addition, 100 ng/mL rhPOSTN significantly accelerated scratch closure, and POSTN-knockdown cells presented slower closure at 24 h and 48 h. Furthermore, the integrin inhibitor Cilengitide depressed the scratch closure that was enhanced by POSTN at 24 h. The CCK8 assay showed that 100 ng/mL rhPOSTN promoted hPDLSC proliferation. Moreover, 100 ng/mL rhPOSTN increased the expression of RUNX2, OSX, OPN, OCN, and VEGF and enhanced ALP activity and mineralization. POSTN silencing decreased the expression of RUNX2, OSX, OPN, OCN, and VEGF and inhibited ALP activity and mineralization. Conclusions: POSTN accelerated the migration, proliferation, and osteogenic differentiation of hPDLSCs.  相似文献   

7.

Introduction

Aging people''s bone regeneration potential is always impaired. Bone marrow stromal cells (MSCs) contain progenitors of osteoblasts. Donor age may affect MSCs’ proliferation and differentiation potential, but the genomic base is still unknown. Due to recent research''s indication that a core circadian component, brain and muscle ARNT-like 1 protein (BMAL1), has a role in premature aging, we investigated the normal aging mechanism in mice with their MSCs and Bmal1 gene/protein level.

Material and methods

1, 6 and 16 month old C57BL/6 mice were used and the bone marrow stromal cells were gained and cultured at early passage. Bmal1 gene and protein level were detected in these cells. Marrow stromal cells were also induced to differentiate to osteoblasts or adipocytes. Three groups of mice MSCs were compared on proliferation by flow cytometry, on cell senescence by SA-β-gal expression and after osteo-induction on osteogenic potential by the expression of osterix (Osx), alkaline phosphatase (ALP) and osteocalcin (OCN).

Results

Bmal1 gene and protein level as well as S-phase fraction of the cell cycle decreased in MSCs along with the aging process. At the same time, SA-β-gal+ levels increased, especially in the aged mice MSCs. When induced to be osteogenic, Osx gene expression and ALP activity declined in the mid-age and aged mice MSCs, while OCN protein secretion deteriorated in the aged mice MSCs.

Conclusions

These findings demonstrate that mouse MSCs changed with their proliferation and osteo-differentiation abilities at different aging stages, and that Bmal1 is related to the normal aging process in MSCs.  相似文献   

8.
9.

Background

Mesenchymal stem cells (MSCs) hold promise for application in adult stem cell-mediated regenerative medicine in bone remodeling and fracture repair. MSCs in vitro can be directed to osteogenic lineage by dexamethasone (DEX); however, the use of DEX is not practical in clinical settings because of adverse side effects such as glucocorticoid-induced osteoporosis. For identifying substances that facilitate osteogenesis, a monitoring system, which detects the osteogenic differentiation stage of MSCs accurately and easily, is required.

Methods

By focusing on the human osteocalcin (OC) gene whose expression profile is described along with osteogenic differentiation, we constructed the luciferase (Luc) reporter gene driven by the enhancer/promoter sequence of the human OC gene (OC-Luc) utilizing a mammalian artificial chromosome. Mammalian artificial chromosome is a suitable platform for loading reporter constructs, because of its stable episomal maintenance in host cells, transferability into any cell and assurance of long-term physiological transgene expression. We loaded the OC-Luc on a mammalian artificial chromosome vector engineered from mouse chromosome (designated as mouse artificial chromosome, MAC) in Chinese hamster ovary cells (OC-Luc/MAC) and transferred this into human MSC cells via chromosome transfer.

Results

OC-Luc/MAC in human MSC cells are responsive to positive and negative stimulation by 1 alpha,25-dihydroxyvitamin D3 and DEX in differentiation stage of MSCs to osteoblasts, reflecting the manner of physiological expression.

Conclusion

The OC-Luc/MAC reporter system may contribute not only to monitoring the osteogenic differentiation stage from MSC but also to identify novel osteogenic drugs.  相似文献   

10.

Purpose

Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs).

Materials and Methods

Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates.

Results

ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)γ expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARγ, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARγ agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types.

Conclusion

The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.  相似文献   

11.

Context:

Central venous catheter (CVC) insertion induces pain and discomfort to a conscious patient despite application of a local anesthetic (LA) field block and this pain can be greatly lessened by using additional analgesics.

Aim:

The aim of this study is to evaluate the efficacy of fentanyl along with LA field infiltration in controlling pain and discomfort associated with CVC insertion.

Settings and Design:

A prospective, randomized, double-blind, placebo-controlled trial was conducted at tertiary referral center.

Materials and Methods:

Fifty-four patients scheduled for planned CVC were randomly assigned to receive either fentanyl (2 μg/kg) or 0.9% normal saline. Pain and discomfort using a verbal numeric rating pain scale at 5 times points during CVC insertion were assessed and analyzed.

Results:

The median interquartile range pain score is worst for placebo group after LAI (5 [3-6]) and in the immediate postprocedure period (5 [4-5]) which was significantly attenuated by addition of fentanyl (3.5 [2-5] and 3 [2-4]) (P = 0.009 and 0.001 respectively). Overall, fentanyl and placebo group were not statistically different with median discomfort score except at T10 (P = 0.047).

Conclusions:

Preprocedural bolus fentanyl infusion provides adequate analgesia and can be safely used for alleviating pain during CVC insertion in conscious patients.  相似文献   

12.

OBJECTIVE:

To evaluate the roles of oxidative stress and lipid peroxidation in the ventricular remodeling that is induced by tobacco smoke exposure after myocardial infarction.

METHODS:

After induced myocardial infarction, rats were allocated into two groups: C (control, n=25) and ETS (exposed to tobacco smoke, n=24). After 6 months, survivors were submitted to echocardiogram and biochemical analyses.

RESULTS:

Rats in the ETS group showed higher diastolic (C = 1.52 ± 0.4 mm2, ETS = 1.95 ± 0.4 mm2; p=0.032) and systolic (C = 1.03 ± 0.3, ETS = 1.36 ± 0.4 mm2/g; p=0.049) ventricular areas, adjusted for body weight. The fractional area change was smaller in the ETS group (C = 30.3 ± 10.1 %, ETS = 19.2 ± 11.1 %; p=0.024) and E/A ratios were higher in ETS animals (C = 2.3 ± 2.2, ETS = 5.1 ± 2.5; p=0.037). ETS was also associated with a higher water percentage in the lung (C = 4.8 (4.3–4.8), ETS = 5.5 (5.3–5.6); p=0.013) as well as higher cardiac levels of reduced glutathione (C = 20.7 ± 7.6 nmol/mg of protein, ETS = 40.7 ± 12.7 nmol/mg of protein; p=0.037) and oxidized glutathione (C = 0.3 ± 0.1 nmol/g of protein, ETS = 0.9 ± 0.3 nmol/g of protein; p=0.008). No differences were observed in lipid hydroperoxide levels (C = 0.4 ± 0.2 nmol/mg of tissue, ETS = 0.1 ± 0.1 nmol/mg of tissue; p=0.08).

CONCLUSION:

In animals exposed to tobacco smoke, oxidative stress is associated with the intensification of ventricular remodeling after myocardial infarction.  相似文献   

13.

Background:

Outcome and predictors of survival after cardiopulmonary resuscitation (CPR) in Intensive Care Units (ICUs) have been extensively studied in western world, but data from developing countries is sparse.

Objectives:

To study the outcome and predictors of survival after CPR in a Medical ICU (MICU) of a tertiary level teaching hospital in North India.

Materials and Methods:

A 1-year prospective cohort study.

Results:

Of 105 in-MICU CPRs, forty patients (38.1%) achieved return of spontaneous circulation (ROSC). Only one patient (0.9%) survived up to hospital discharge. The predictors of ROSC were ventricular tachycardia/ventricular fibrillation as first monitored rhythm, intubation during CPR and CPR duration ≤ 10 min. CPR duration > 10 min was a significant factor for resuscitation failure.

Conclusions:

The rate of survival to hospital discharge after in-MICU CPRs is extremely poor. Our data may aid treating physicians, resuscitation teams, and families in understanding the likely outcome of patients after in-MICU CPRs.  相似文献   

14.

OBJECTIVES:

Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction.

MATERIALS AND METHODS:

Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction.

RESULTS:

Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction.

CONCLUSION:

Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adipose-derived stem cells was most prominent after one week of chondrogenic induction.  相似文献   

15.

Introduction

TGF-β1 is a cytokine with many different effects on cell proliferation, differentiation and inflammation and can protect against the development of COPD. This work aims to study the association between COPD and the TGF-β1 gene genotypes.

Material and methods

The study included 70 males: 25 smokers with COPD, 25 resistant smokers, and 20 normal non-smokers as the control. They were subjected to spirometry pre- and post-bronchodilator (FEV1, FEV1/FVC), estimation of serum level of TGF-β1 gene by PCR and RFLP.

Results

The percent of Pro-Leu was 28% in the COPD group, 84% in the resistant smokers group and 85% in the control group. There was a highly significant statistical difference in FEV1% of predicted associated with the distribution of TGF-β1 gene genotypes: 56.9 ±8.4% with Pro-Leu genotype and 35.5 ±8.8% with Leu-Leu genotype in COPD patients, 93.2 ±6.2% with Pro-Leu genotype and 86.7 ±0.9% with Leu-Leu genotype in the resistant smokers group.

Conclusions

The Pro-allele genotype is associated with increased production of TGF-β1, which has a protective role against the development of COPD and is important in preserving the decline of FEV1 in COPD patients.  相似文献   

16.
17.

Introduction

Myeloma bone disease (MBD) is the result of the increased activity of osteoclasts (OCs), which is not accompanied by a comparable increase of osteoblast (OB) function, thus leading to enhanced bone resorption. Osteoblasts can also regulate osteoclast activity through expression of cytokines, such as receptor activator of nuclear factor-κB ligand (RANKL), which activates osteoclast differentiation, and osteoprotegerin (OPG), which inhibits RANKL by acting as a decoy receptor.

Material and methods

Based on a series of 21 patients with multiple myeloma (MM) and human osteoblast cell line HFOB1.19, we provide evidence that the bone marrow-derived mesenchymal stem cells (BMMSCs) of patients with MM exhibit normal phenotype, but showed reduced efficiency to differentiate into OBs as compared with normal controls.

Results

In vitro assays showed that MM cells inhibited the potential of osteogenic differentiation of BMMSCs from healthy controls and rendered the OBs sensitive to TRAIL-induced apoptosis. There was no evidence of the formation of tartrate-resistant acid phosphatase positive OCs. The osteogenic differentiation of HFOB1.19 was also inhibited in the presence of RPMI 8266 or XG7 MM cells, as confirmed by von Kossa and ALP staining. Osteoblast s induced from BMMSCs supported survival and proliferation of MM cells, especially when the MM cells were cultured in medium containing rhTRAIL and dexamethasone. Multiple myeloma cells proliferated and grew well in the presence of residual OBs.

Conclusions

Besides OCs, our results demonstrated that OBs and MM cells were dependent upon each other and made a microenvironment suitable for MM cells.  相似文献   

18.
19.

Introduction

Maximizing responses of malignant gliomas is hampered by resistance to temozolomide (TMZ). Increasing efficacy but not toxicity is a key issue when testing drug combinations. The antimyeloma agent bortezomib (BZ) has shown promising results in vitro and is currently being tested in glioblastoma (GBM) patients. In this study we investigate whether reduction of TMZ dosage is feasible without compromising the antitumor effect of TMZ-BZ combination.

Material and methods

U87 GBM cells were treated with increasing doses of TMZ (1, 10, 100, 1000 µM), BZ (0.001, 0.01, 0.1, 1) and the combination during a 48-hour period, and apoptotic or/and necrotic cell death was evaluated by flow cytometry.

Results

Bortezomib alone at a dose as low as 0.001 µM markedly induced cell death, particularly late apoptosis, to a level which was comparable with high TMZ dosage. For combination treatments, the dose of 0.1 µM BZ, which was more potent than the maximal dose of TMZ (1000 µM), was chosen to be added to increasing TMZ concentrations. The combination of 0.1 BZ µM BZ with low doses of TMZ (1, 10 µM) further increased the cell death rate in an additive manner, at levels higher than those induced by high doses of TMZ monotherapy (100, 1000 µM).

Conclusions

Efficacy of TMZ-BZ combination is feasible with low doses of TMZ in vitro.  相似文献   

20.

Background:

This prospective double-blind trial was undertaken to analyze the role of perioperatively administered dexmedetomidine on the occurrence of chronic pain in cases undergoing surgery for breast cancer.

Subjects and Methods:

Eighty-six cases were randomly assigned to two groups to receive either dexmedetomidine (2 μg/ml) in group D or saline in group C, in a loading dose of 0.5 ml/kg, intravenous (IV), 30 min prior to induction, followed by a continuous infusion of 0.25 ml/kg/h IV till the completion of surgery, and then the dose tapered to 0.1 ml/kg/h for up to 24 h. The standardized questionnaires that measured chronic pain (Brief Pain Inventory, BPI; Short Form McGill Pain Questionnaire, SF-MPQ2) and quality of life (Quality of Life Scale, QOLS) were gathered after 3 months of surgery as a primary outcome. Pain (verbal numerical score, VNS), sedation scores (Ramsay scoring), and analgesic requirements were also assessed for 72 h postoperatively.

Results:

In total, 84 cases (n=42) were analyzed for acute pain and 69 (34 in group D and 35 in group C) for chronic pain. The consumption of isoflurane/fentanyl intra-operatively and paracetamol postoperatively was significantly lower in Group D. The sedation scores were non-significant between the groups. The VNS at rest and after movement was significantly lower in group D at corresponding times (except at 60 min) throughout the assessment period. The BPI and SF-MPQ2 scores were significantly lower in group D in most of the factors. The QOLS score was significantly better in group D in all items except for relationships, friends, and learning.

Conclusion:

The perioperative infusion of dexmedetomidine has a pivotal role in attenuating the incidence and severity of chronic pain and improving the quality of life in cases undergoing breast cancer surgery.  相似文献   

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