【Abstract】 Objective Sonic hedgehog (Shh) signaling pathway is involved in an important part of regulating angiogenesis. To investigate the effects of recombinant Shh N-terminant (rShh-N) on the expression and secretion of angiogenesis-related factor—vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods Bone marrow mesenchymal stem scells (BMSCs) were isolated from 3-day-old healthy Sprague Dawley rats and cultured to passage 3 in vitro. rShh-N at the concentrations of 0, 10, 100, and 200 ng/mL were applied to culture BMSCs in groups A, B, C, and D, respectively. At 12, 24, 48, and 72 hours of culture, the expressions of VEGF and bFGF mRNA and the levels of VEGF and bFGF in supernatant were measured with real-time quantitative PCR and ELISA, respectively. Results At the gene level, compared with group A, the expressions of VEGF and bFGF mRNA were enhanced in group D (P lt; 0.05) and the upregulation was more significant at 12 and 48 hours than 24 and 72 hours (P lt; 0.01). In group C, bFGF mRNA expression was substantially promoted at 12-72 hours (P lt; 0.05) and VEGF mRNA level was upregulated at 24-72 hours (P lt; 0.05), and both reached peak at 72 hours (P lt; 0.01). In group B, VEGF mRNA expression was inhibited at 12 hours (P lt; 0.05), but the level increased at 48 and 72 hours (P lt; 0.05); bFGF mRNA expression was obviously promoted at 12-48 hours (P lt; 0.05) and the maximum appeared at 48 hours (P lt; 0.01). At the protein level, the secretion of VEGF and bFGF in group D was significantly increased at 12-72 hours, as compared with group A (P lt; 0.05). In group C, VEGF and bFGF secretion was increased at 24-72 hours (P lt; 0.05). The secretion of VEGF in group B was inhibited at 12 and 48 hours (P lt; 0.05) and was promoted at 24 hours (P lt; 0.05); bFGF secretion was up-regulated at 24 and 48 hours (P lt; 0.05). The secretion of VEGF and bFGF in supernatant at 【Abstract】 Objective Sonic hedgehog (Shh) signaling pathway is involved in an important part of regulating angiogenesis. To investigate the effects of recombinant Shh N-terminant (rShh-N) on the expression and secretion of angiogenesis-related factor—vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods Bone marrow mesenchymal stem scells (BMSCs) were isolated from 3-day-old healthy Sprague Dawley rats and cultured to passage 3 in vitro. rShh-N at the concentrations of 0, 10, 100, and 200 ng/mL were applied to culture BMSCs in groups A, B, C, and D, respectively. At 12, 24, 48, and 72 hours of culture, the expressions of VEGF and bFGF mRNA and the levels of VEGF and bFGF in supernatant were measured with real-time quantitative PCR and ELISA, respectively. Results At the gene level, compared with group A, the expressions of VEGF and bFGF mRNA were enhanced in group D (P lt; 0.05) and the upregulation was more significant at 12 and 48 hours than 24 and 72 hours (P lt; 0.01). In group C, bFGF mRNA expression was substantially promoted at 12-72 hours (P lt; 0.05) and VEGF mRNA level was upregulated at 24-72 hours (P lt; 0.05), and both reached peak at 72 hours (P lt; 0.01). In group B, VEGF mRNA expression was inhibited at 12 hours (P lt; 0.05), but the level increased at 48 and 72 hours (P lt; 0.05); bFGF mRNA expression was obviously promoted at 12-48 hours (P lt; 0.05) and the maximum appeared at 48 hours (P lt; 0.01). At the protein level, the secretion of VEGF and bFGF in group D was significantly increased at 12-72 hours, as compared with group A (P lt; 0.05). In group C, VEGF and bFGF secretion was increased at 24-72 hours (P lt; 0.05). The secretion of VEGF in group B was inhibited at 12 and 48 hours (P lt; 0.05) and was promoted at 24 hours (P lt; 0.05); bFGF secretion was up-regulated at 24 and 48 hours (P lt; 0.05). The secretion of VEGF and bFGF in supernatant at
ObjectiveTo investigate the effect of transforming growth factorβ1 (TGF-β1) and basic fibroblast growth factor 1 (bFGF-1) on the cellular activities, prol iferation, and expressions of ligament-specific mRNA and proteins in bone marrow mesenchymal stem cells (BMSCs) and ligament fibroblasts (LFs) after directly co-cultured.
MethodsBMSCs from 3-month-old Sprague Dawley rats were isolated and cultured using intensity gradient centrifugation. LFs were isolated using collagenase. The cells at passage 3 were divided into 6 groups: non-induced BMSCs group (group A), non-induced LFs group (group B), non-induced co-cultured BMSCs and LFs group (group C), induced BMSCs group (group D), induced LFs group (group E), and induced co-cultured BMSCs and LFs group (group F). The cellular activities and prol iferation were examined by inverted contrast microscope and MTT; the concentrations of collagen type Ⅰ and type Ⅲ were determined by ELISA; and mRNA expressions of collagen types I andⅢ, fibronectin, tenascin C, and matrix metalloproteinase 2 (MMP-2) were measured by real-time fluorescent quantitative PCR.
ResultsA single cell layer formed in the co-cultured cells under inverted contrast microscope. Group F had fastest cell fusion ( > 90%). The MTT result indicated that group F showed the highest absorbance (A) value, followed by group D, and group B showed the lowest A value at 9 days after culture, showing significant difference (P < 0.05). Moreover, the result of ELISA showed that group F had the highest concentration of collagen type Ⅰ and type Ⅲ (P < 0.05); the concentration of collagen type Ⅲ in group E was significantly higher than that in group D (P < 0.05), but no significant difference was found in the concentration of collagen type Ⅰ between 2 groups (P > 0.05). The ratios of collagen type Ⅰ to type Ⅲ were 1.17, 1.19, 1.10, 1.25, 1.17, and 1.18 in groups A-F; group D was higher than the other groups. The real-time fluorescent quantitative PCR results revealed that the mRNA expressions of collagen type Ⅰ and type Ⅲ and fibronectin were highest in group F; the expression of tenascin C was highest in group D; the expression of MMP-2 was highest in group E; and all differencs were significant (P < 0.05).
ConclusionDirectly co-cultured BMSCs and LFs induced by TGF-β1 and bFGF-1 have higher cellular activities, proliferation, and expressions of ligament-specific mRNA and protein, which can be used as a potential source for ligament tissue engineering.
Objective To investigate the feasibility of cartilaginous implantscontaining bone marrow stromal cells(MSCs) derived from chondrocytes in biological resurfacing procedures for repairing articular cartilage defect. Methods MSCs derived from chondrocytes were obtained with high initial cell density subculture. An implant was constructed by dispersing the chondrocytes in a acid soluble type Ⅰ collagen gel(5×106cells/ml, final cell concentration). A fullthickness defect 3 mm×5 mm was created in the trochlear groove of femur in 36 rabbits. A piece of cotton soaked in 0.5% trypsin was laid into the defect for 5 minutes, then the defect was filled with MSC/collagen gel implant on one side(n=36), filledwith a plain collagen gel on the other side(n=18),and left empty as controls on the other side(n=18). The animals were sacrificed at 4, 8, 12, 24, 32,and 48 weeks. The repaired tissue was examined and evaluated with Pineda gradingscale. Results In MSCs group, the implanted cells resembled well differentiated chondrocytes and were surrounded by metachromatic matrix and the reparative tissue resembled hyaline cartilage after 4 weeks; bone was formed at the base of the defects, the thickness of new cartilage was larger than tht of normal one after 8 weeks; the thickness was reduced proximally, approximating to that of normal cartilage, and chondrocyte columns was formed and subchondral bone and tidemark reappeared after 12 weeks; the thickness of the new tissue was about 55% of the normal tissue, with smooth surface and there were hypertrophic chondrocytes near the tidemark after 24 weeks; no hypertrophic chondrocytes were observed, indicating cessation of endochondral ossification after 32 weeks; the tissue architecture was the same as that at 32 weeks, hyaline-like cartilage persisting, with subchondral bone and tidemark in continuity after 48 weeks. The four layer cell orientation was not as clear as that of normal cartilage. The defects were partially filled with fibrous tissue in controls. At 32 weeks, erosive cartilage, naked subchondralbone and proliferative synovial membrane indicated the presence of osteoarthrosis. There were no statistical difference according to Pineda tissue scales in the specimens from the MSCs group between 24, 32, and 48 weeks, but there was significant difference between 4 weeks and 24, 32 and 48 weeks (Plt;0.05). The joint function recovered after 2 weeks in MSCs group, while it deteriorated progressively incontrols. Conclusion MSCs derived from chondrocytes improve repair of largefullthickness defect in articular cartilage. The reparative hyaline-like cartilage is stable differentiation after 24 weeks, maintains good joint function after 48 weeks.
Objective To compare the growth and extracellular matrix biosynthesis of nucleus pulposus cells (NPCs)and bone marrow mesenchymal stem cells (BMSCs) in thermo-sensitive chitosan hydrogel and to choose seed cells for injectable tissue engineered nucleus pulposus. Methods NPCs were isolated and cultured from 3-week-old New Zealand rabbits (male or female, weighing 150-200 g). BMSCs were isolated and cultured from bone marrow of 1-month-old New Zealand rabbits (male or female, weighing 1.0-1.5 kg). The thermo-sensitive chitosan hydrogel scaffold was made of chitosan, disodium β glycerophosphate, and hydroxyethyl cellulose. Then, NPCs at the 2nd passage or BMSCs at the 3rd passage were mixed with chitosan hydrogel to prepare NPCs or BMSCs-chitosan hydrogel complex as injectable tissue engineered nucleus pulposus. The viabil ities of NPCs and BMSCs in the chitosan hydrogel were observed 2 days after compound culture. The shapes and distributions of NPCs and BMSCs on the scaffold were observed by scanning electron microscope (SEM) 1 week after compound culture. The histology and immunohistochemistry examination were performed. The expressions of aggrecan and collagen type II mRNA were analyzed by RT-PCR 3 weeks after compound culture. Results The thermo-sensitive chitosan hydrogel was l iquid at room temperature and sol idified into gel at37 (after 15 minutes) due to crossl inking reaction. Acridine orange/propidium iodide staining showed that the viabil ity rates of NPCs and BMSCs in chitosan hydrogel were above 90%. The SEM observation demonstrated that the NPCs and BMSCs distributed in the reticulate scaffold, with extracellular matrix on their surfaces. The results of HE, safranin O histology and immunohistochemistry staining confirmed that the NPCs and BMSCs in chitosan hydrogel were capable of producing extracellular matrix. RT-PCR results showed that the expressions of collagen type II and aggrecan mRNA were 0.564 ± 0.071 and 0.725 ± 0.046 in NPCs culture with chitosan hydrogel, and 0.713 ± 0.058 and 0.852 ± 0.076 in BMSCs culture with chitosan hydrogel; showing significant difference (P lt; 0.05). Conclusion The thermo-sensitive chitosan hydrogel has good cellular compatibil ity. BMSCs culture with chitosan hydrogel maintains better cell shape, prol iferation, and extracellular matrix biosynthesis than NPCs.
Objective
To observe the effect of bone marrow mesenchymal stem cells (BMSCs) conditioned medium on microglia (MGs) and its secretion of arginase 1 (Arg1).
Methods
The BMSCs separated through differential adhesion method from the femur and tibia marrow of 4-week-old Sprague Dawley (SD) rats were cultured and identified by Vimentin immunofluorescence staining; whereas MGs separated through trypsin digestion method from the brain of 3-day-old SD rats were cultured and identified by Iba1 immunofluorescence staining. The primary MGs were cultured with DMEM/F12 medium containing BMSCs conditioned medium (experimental group) and with single DMEM/F12 medium (control group), respectively. After 48 hours of culture, the morphology of MGs was observed by inverted phase contrast microscope, the activated state of MGs was detected by using Iba1 immunofluorescence staining, and Arg1 expression of MGs was assessed by Iba1-Arg1 double-labelling immunofluorescence staining and Western blot method.
Results
Inverted phase contrast microscope observation showed that BMSCs entered logarithmic growth phase at 14 days after culture, and more than 98% cells were positive to Vimentin immunofluorescence staining; whereas MGs entered logarithmic growth phase at 21 days after culture, and around 80% cells were positive to Iba1 immunofluorescence staining. Inverted phase contrast microscope observation displayed that in the experimental group, MGs were activated with increased size of soma, shortened process, and amoeba change. Immunofluorescence staining displayed that the Iba1 positive cells number in the experimental group was significantly higher than that in the control group (t=0.007, P=0.000); double-labelling immunofluorescence staining revealed that the Iba1-Arg1 positive cells number in the experimental group was significantly higher than that in the control group (t=0.007, P=0.000); and Western blot results elucidated that the relative expression of Arg1 protein in the experimental group was significantly higher than that in the control group (t=0.001, P=0.000).
Conclusion
BMSCs conditioned medium can activate MGs and induce MGs to express Arg1.
ObjectiveTo study the effect of Triton X-100 promoting liposome-mediated bone morphogenetic protein 2 (BMP-2) gene transfection of rat bone marrow mesenchymal stem cells (BMSCs).
MethodsBMSCs were separated and cultured from the femur and tibia of healthy Wistar rats (8-week-old, male). The 3rd passage BMSCs identified by detecting the surface antigen were used to transfect. The optimum concentration of Triton X-100 for liposome mediated gene transfection was determined with ELISA meter by the way of MTT. In optimum concentration of Triton X-100, liposome mediated BMP-2 gene was transfected to BMSCs. The experiment was divided into 3 groups according to types of trasfection agents:BMSCs were transfected with Triton X-100+liposome+BMP-2 (experimental group), with liposome+ BMP-2 (conventional transfection group), and untransfected BMSCs served as blank control group. After 48 hours of transfecting, the green fluorescent protein (GFP) in cells was detected through inverted fluorescence microscope. After 72 hours of transfection, real-time fluorescence quantitative PCR was applied to measure the mRNA expression of BMP-2.
Results0.01% Triton X-100 was determined to be the optimum concentration for not only making the BMSCs maintain vitality, but also achieving a certain effect on BMSCs. After trasfecting for 48 hours, GFP was observed through inverted fluorescence microscope in the experimental group and conventional transfection group, but was not observed in the blank control group. After trasfecting for 72 hours, the relative BMP-2 mRNA expression level was 5.94±0.12 in the experimental group, and was 4.99±0.08 in the conventional transfection group, showing significant difference (t=360.28, P=0.02). The transfection efficiency was increased by 19% in the experimental group.
Conclusion0.010%Triton X-100 can promote the liposome mediated BMP-2 gene transfection of rat BMSCs, and can improve the transfection efficiency.
Objective To explore the pathology and clinical features of bone marrow tuberculosis. Methods 30 cases of bone marrow tuberculosis diagnosed in West China Hospital between January 2004 and December 2010 were recruited in the study. Their pathology and clinical data were retrospectively analyzed. Results The majority of the patients were young and middle-aged. The ratio of male to female was 1.5∶1. Fever was the main symptom, which often accompanied by cough, sputum, fatigue, anorexia, abdominal pain, bloating and diarrhea and other symptoms. Bone marrow tuberculosis involved multiple systems, accompanied by cytopenia and pulmonary tuberculosis. The ratio of sputumsmear positive for acid-fast bacilli was low. Bone marrow biopsy mainly showed granulomatous inflammation with or without caseous necrosis. The mortality could be significantly reduced by anti-tuberculosis treatment in time. Conclusion Bone marrow tuberculosis is serious and often accompanied by multiple systems tuberculosis. Early diagnosis is particularly important. Timely and regular treatment is in great needed.
ObjectiveTo investigate the regulatory effect of simvastatin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at middle/late stages by p38MAPK pathway under condition of osteoinductive environment.
MethodsThe bone marrow of bilateral femur and tibia were harvested from 20 4-week-old female Sprague Dawley rats. BMSCs were isolated and cultured with whole bone marrow culture method; the second generation of cells were randomly divided into 5 groups: control group (complete medium, CM), simvastatin group (simvastatin medium, SIM), osteogenic induction group (osteogenic induction medium, OM), simvastatin and osteogenic induction group (simvastatin+osteogenic induction medium, OM+SIM), and blocker group (SB203580+simvastatin+osteogenic induction medium, OM+SIM+SB). MTT assay was used to detect the cell activity in CM group and SIM group at 2, 3, 4, 5, and 6 days, ELISA method to measure the content of alkaline phosphatase (ALP) in OM group and OM+SIM group at 7 and 14 days. The mRNA and protein expressions of osteocalcin (OCN) were detected by real-time quatitative PCR and Western blot after 1, 12, and 24 hours of osteogenic induction at 21 and 28 days. The protein expressions of phospho-p38 (p-p38) and p38 in OM group, OM+SIM group, and OM+SIM+SB group were detected by Western blot at the best induction time of simvastatin.
ResultsMTT assay showed that no significant difference was found in absorbance (A) value between CM group and SIM group at each time point (P > 0.05), indicating no effect of 1×10-7 mol/L simvastatin on cell viability. ELISA results showed that ALP content significantly increased in OM+SIM group when compared with OM group at 7 and 14 days; the ALP content was significantly higher at 7 days than 14 days in OM group and OM+SIM group (P < 0.05). OCN mRNA and protein expressions at 12 hours were significantly higher than those at other time points in each group (P < 0.05), and the expressions of OM+SIM group was significantly higher than those of OM group (P < 0.05). The best induction time of simvastatin was 12 hours. At 12 hours after blocking intervention, the p-p38/p38 in OM+SIM+SB group was significantly lower than that in OM group and OM+SIM group (P < 0.05), and the p-p38/p38 in OM+SIM group was significantly higher than that in OM group (P < 0.05).
ConclusionSimvastatin can increase the mRNA and protein expression levels of OCN and the protein of p-p38 in osteogenic differentiation of BMSCs at middle/ late stages, and its best induction time is 12 hours.
Objective
To improve the knowledge of bone marrow tuberculosis by summarizing the risk factors, clinical features, diagnosis, therapeutic response and prognosis.
Methods
The medical records of 62 patients with bone marrow tuberculosis from January 2004 to December 2014 were retrospectively analyzed.
Results
The patients included 34 males and 28 females. Their age ranged from 15 to 80 years with a mean age of 45.3±35.7 years. Among them, 21 cases (33.9%) had one or more risk factors such as advanced kidney disease or liver disease, chronic respiratory disease, rheumatoid immune system disease and diabetes. No specific clinical features were found in patients with bone marrow tuberculosis, and the most common symptom was fever (98.4%). Blood routine examination revealed that variety of hematological abnormalities include leucopenia, thrombocytopenia, anemia or pancytopenia occurred in 59 patients (95.2%). Chest CT scan showed typical disseminated tuberculosis in 22 patients (36.1%), secondary pulmonary tuberculosis in 6 patients (9.8%), and atypical manifestations of tuberculosis in 26 patients (42.6%). The common patterns of abdomen abnormalities on ultrasonic testing or CT scan were hepatosplenomegaly. Bone marrow biopsy was performed in all the 62 patients, among them, the needle-aspirated bone marrow specimens showed granulomatous lesions in 53 patients (85.5%). The acid-fast staining of bone marrow smear was performed in a total of 57 patients, which was positive in 28 patients (49.1%). Twenty-five patients completed phone call follow-up, and 8 patients died from bone marrow tuberculosis and its complications.
Conclusions
Bone marrow tuberculosis is a rare manifestation of disseminated tuberculosis. The common symptom of this disease is pyrexia . The clinical presentations and laboratory examinations of this disease are nonspecific. Bone marrow biopsy is a major method for diagnosis. Part of patients with bone marrow tuberculosis lack of imaging proof for lung lesion. Therefore, we should heighten vigilance for bone marrow tuberculosis in patients with fever of unknown origin.
OBJECTIVE: A rare huge desmoplastic fibroma on thoracic wall in 1 female case of 25 years old was resected, and the accompanying huge thoracic wall defect, ribs and soft tissues were repaired by tissue engineered bone and pedicled flap. The paper aims to explore the clinical results of early stage after operation. METHODS: Autogeneic bone marrow stromal cells (MSC) were obtained from bone marrow puncture of iliac bone and isolated and cultured according to the Houghton’s methods, MSC were directively induced and differentiated to osteoblasts. Allogeneic ribs were made to the bio-derived bone scaffold materials after treatment of decell, deantigen, decalcification and dry freezing. 5 x 10(6)/ml MSC were cocultured with the bio-derived bone for 6 days in vitro. After intact resection of tumor, the diaphragm flap was applied to repair pleural cavity, the three defect ribs were repaired by tissue engineered bone and the soft tissue defect was repaired by transfer of pedicled ipsilateral abdominal flaps. RESULTS: The patient recovered well with first intention. Followed up for 3 months, tissue engineered ribs were matured in vitro and the heart and pulmonary functions were improved markedly. CONCLUSION: The tissue engineered bone constructed by autogeneic MSC is advantageous in individual treatment.
