Objective To investigate the feasibility of fabricating an oriented scaffold combined with chondrogenic-induced bone marrow mesenchymal stem cells (BMSCs) for enhancement of the biomechanical property of tissue engineered cartilage in vivo. Methods Temperature gradient-guided thermal-induced phase separation was used to fabricate an oriented cartilage extracellular matrix-derived scaffold composed of microtubules arranged in parallel in vertical section. No-oriented scaffold was fabricated by simple freeze-drying. Mechanical property of oriented and non-oriented scaffold was determined by measurement of compressive modulus. Oriented and non-oriented scaffolds were seeded with chondrogenic-induced BMSCs, which were obtained from the New Zealand white rabbits. Proliferation, morphological characteristics, and the distribution of the cells on the scaffolds were analyzed by MTT assay and scanning electron microscope. Then cell-scaffold composites were implanted subcutaneously in the dorsa of nude mice. At 2 and 4 weeks after implantation, the samples were harvested for evaluating biochemical, histological, and biomechanical properties. Results The compressive modulus of oriented scaffold was significantly higher than that of non-oriented scaffold (t=201.099, P=0.000). The cell proliferation on the oriented scaffold was significantly higher than that on the non-oriented scaffold from 3 to 9 days (P lt; 0.05). At 4 weeks, collagen type II immunohistochemical staining, safranin O staining, and toluidine blue staining showed positive results in all samples, but negative for collagen type I. There were numerous parallel giant bundles of densely packed collagen fibers with chondrocyte-like cells on the oriented-structure constructs. Total DNA, glycosaminoglycan (GAG), and collagen contents increased with time, and no significant difference was found between 2 groups (P gt; 0.05). The compressive modulus of the oriented tissue engineered cartilage was significantly higher than that of the non-oriented tissue engineered cartilage at 2 and 4 weeks after implantation (P lt; 0.05). Total DNA, GAG, collagen contents, and compressive modulus in the 2 tissue engineered cartilages were significantly lower than those in normal cartilage (P lt; 0.05). Conclusion Oriented extracellular matrix-derived scaffold can enhance the biomechanical property of tissue engineered cartilage and thus it represents a promising approach to cartilage tissue engineering.
Objective Construction of viable tissue engineered bone is one of the most important research fields in the cl inical appl ication of bone tissue engineering, to investigate the function of nerve factors in bone tissue engineering by celldetection in vitro and construction of neurotization tissue engineered bone in vivo. Methods Fifty-four healthy New Zealandwhite rabbits, male or female, weighing 2-3 kg, were involved in this study. Bone marrow mesenchymal stem cells (BMSCs) from the bone marrow of white rabbits were cultured. The second passage of BMSCs were treated with sensory nerve or motor nerve homogenates, using the LG-DMEM complete medium as control. The prol iferation and osteogenic differentiation of the cells were observed and tested by the MTT assay, alkal ine phosphatase (ALP) stain, and collagen type I immunocytochemistry identification. The osteogenic induced BMSCs were inoculated in β tricalcium phosphate (β-TCP) biomaterial scaffold and cultured for 72 hours, then the β-TCP loaded with seed cells was implanted in the rabbit femur with 15 mm bone and periosteum defects. Fifty-four New Zealand white rabbits were randomly divided into three groups (n=18): sensory nerve bundle (group A) or motor nerve bundle (group B) were transplanted into the side groove of β-TCP scaffold, group C was used as a control without nerve bundle transplantation. X-ray detection was performed at the 4th, 8th, and 12th weeks after operation.
Objective To explore the impact of basic fibroblast growth factor (bFGF) and parathyroid hormone-related protein (PTHrP) on early and late chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) induced by transforming growth factor β1 (TGF-β1). Methods BMSCs were isolated from 3 healthy Japanese rabbits (2-month-old, weighing 1.6-2.1 kg, male or female), and were clutured to passage 3. The cells were put into pellet culture system and were divided into 5 groups according to different induce conditions: TGF-β1 group (group A), TGF-β1/bFGF group (group B), TGF-β1/21 days bFGF group (group C), TGF-β1/PTHrP group (group D), and TGF-β1/21 days PTHrP group (group E). At the beginning, TGF-β1 (10 ng/mL) was added to all groups, then bFGF and PTHrP (10 ng/mL) were added to groups B and D respectively; bFGF and PTHrP (10 ng/mL) were added to groups C and E at 21 days respectively. The gene expressions of collagen type I (Col I), Col II, Col X, matrix metalloproteinases (MMP)-13, and alkaline phosphatase (ALP) activity were detected once every week for 6 weeks. The 1, 9-dimethylmethylene blue (DMMB) staining was used to observe the extracellular matrix secretion at 6 weeks. Results The expression of Col I in groups C and E showed a significant downward trend after 3 weeks; the expression in group A was significantly higher than that in groups C and E at 4 and 5 weeks (P lt; 0.05), and than that in groups B and D at 3-6 weeks (P lt; 0.05); and significant differences were found between groups B and C at 3 and 4 weeks, and between groups D and E at 3 weeks (P lt; 0.05). After 3 weeks, the expressions of Col II and Col X in groups C and E gradually decreased, and were significantly lower than those in group A at 4-6 weeks (P lt; 0.05). Groups B and D showed no significant difference in the expressions of Col II and Col X at all time points, but there was significant difference when compared with group A (P lt; 0.05). MMP-13 had no obvious expression at all time points in group A; significant differences were found between group B and groups A, C at 3 weeks (P lt; 0.05); and the expression was significantly higher in group D than in groups A and E (P lt; 0.05). ALP activity gradually increased with time in group A; after 4 weeks, ALP activity in groups C and E obviously decreased, and was significantly lower than that in group A (P lt; 0.05); there were significant differences between groups B and C, and between groups D and E at 2 and 3 weeks (P lt; 0.05). DMMB staining showed more cartilage lacuna in group A than in the other groups at 6 weeks. Conclusion bFGF and PTHrP can inhibit early and late chondrogenic differentiation of BMSCs by changing synthesis and decomposition of the cartilage extracellular matrix. The inhibition is not only by suppressing Col X expression, but also possibly by suppressing other chondrogenic protein.
【Abstract】 Objective To investigate the effect of allogeneic bone marrow-derived mesenchymal stem cells ( BMSCs) transplantation on the airway inflammation and airway remodeling in chronic asthmatic mice. Methods Forty female BALB/c mice were equally randomized into four groups, ie. a normal control group, a BMSCs control group, an asthma model group, and a BMSCs transplantation group. BMSCs were generated from male donor mice, then the mice in the asthma model group and the BMSCs transplantation group were sensitized and challenged with OVA to establish chronic asthmatic mice model. Hematoxylin and eosin staining and Alcian blue-periodic acid-Schiff staining were used to analyze the effects on airway inflammation and airway remodeling after BMSC engraftment. The number of CD4 + CD25 + regulatory T cells in spleen was detected by flow cytometry. Results In lungs of the asthmamodel group, there were intensive inflammatory cells infiltration around airway and blood vessels, goblet cell proliferation, epithelial desquamation, patchy airway occlusion by hyperviscous mucus, and hypertrophy of airway smooth muscle.Airway inflammation and airway remodeling were significantly relieved in the BMSCs transplantation group.There was no obvious inflammatory cells infiltration in the airway and airway remodeling both in the normal control group and the BMSCs control group. The number of CD4 + CD25 + regulatory T cells in spleensignificantly decreased in the asthma model group compared with the two control groups ( P lt; 0. 05) , and significantly increased in the BMSCs transplantation group compared with the asthma model group ( P lt;0. 05) . There was no significant difference in the number of CD4 + CD25 + regulatory T cells in spleen betweenthe control groups and the BMSCs transplantation group. Conclusion BMSCs engraftment can up-regulate CD4 + CD25 + regulatory T cells and relieve airway inflammation and airway remodeling in asthmatic mice.
ObjectiveTo investigate the effect of diammonium glycyrrhizinate (DG) plus bone marrow mesenchymal stem cells (MSCs) transplantation in the treatment of acute exacerbation of pulmonary fibrosis induced by bleomycin (BLM) in rats.MethodsMSCs were isolated from male Wistar rats and cultured in vitro. Twenty-four female Wistar rats were randomly divided into 4 groups. The NC group was intratracheally injected with normal saline; the BLM group, the MSC group and the DGMSC group were intratracheally injected with BLM for 7 days; then the MSC group was injected with 0.5 mL of MSCs solution (2.5×106 cells) into the tail vein; the DGMSC group was intraperitoneally injected with DG for 21 days in a dose of 150 mg·kg–1·d–1 on the base of the MSCs injection. The rats were sacrificed on the 28th day and the lung tissue was extracted. Pathological examination was performed to determine the degree of alveolitis and pulmonary fibrosis. Immunofluorescence was used to detect the number and distribution of alveolar type Ⅱ epithelial cells. Alkali hydrolysis method was used to determine the content of hydroxyproline (HYP) in lung tissue; thiobarbituric acid method was used to measure the content of malondialdehyde (MDA) in lung tissue; colorimetric method was used to determine the superoxide dismutase activity (SOD) and total antioxidant capacity (T-AOC); enzyme linked immunosorbent assay was used to detect the expression levels of tumor necrosis factor-α (TNF-α ) and transforming growth factor-β1 (TGF-β1) in lung tissue homogenates.ResultsThe DG combined with MSCs injection can reduce the degree of alveolitis and pulmonary fibrosis in BLM model rats. The content of HYP and TGF-β1 in lung tissue homogenate of the DGMSC group were significantly lower than those in the MSC group (P<0.05). Meanwhile, DG combined with MSCs injection significantly increased the antioxidant capacity of the BLM model rats. MDA content decreased, SOD activity and T-AOC ability improved significantly in the DGMSC group compared with the MSC group (P<0.05). The alveolar type Ⅱ epithelial cells were significantly increased and the cell morphology was maintained in the DGMSC group compared with the MSC group.ConclusionsDG has a synergistic effect with MSCs in treatment of acute exacerbation of pulmonary fibrosis. The mechanism may be related to reducing inflammatory factors during pulmonary fibrosis, attenuating oxidative stress and promoting MSCs migration into lung tissue and transformation to alveolar type Ⅱ epithelial cells.
Objective To investigate tissue engineered spinal cord which was constructed of bone marrow mesenchymal stem cells (BMSCs) seeded on the chitosan-alginate scaffolds bridging the both stumps of hemi-transection spinal cord injury (SCI) in rats to repair the acute SCI. Methods BMSCs were separated and cultured from adult male SD rat. Chitosan-alginate scaffold was produced via freeze drying, of which the structure was observed by scanning electron microscope (SEM) and the toxicity was determined through leaching l iquor test. Tissue engineered spinal cord was constructed by seeding second passage BMSCs on the chitosan-alginate scaffolds (1 × 106/mL) in vitro and its biocompatibil ity was observed under SEM at 1, 3, and 5 days. Moreover, 40 adult female SD rats were made SCI models by hemi-transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). Tissue engineered spinal cord or chitosan-alginate scaffolds or BMSCs were implanted in groups A, B, and C, respectively. Group D was blank control whose spinal dura mater was sutured directly. After 1, 2, 4, and 6 weeks of surgery, the functional recovery of the hindl imbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. Other indexes were tested by wheat germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing, HE staining and immunofluorescence staining after 6 weeks of surgery. Results Chitosan-alginate scaffold showed three-dimensional porous sponge structure under SEM. The cells adhered to and grew on the surface of scaffold, arranging in a directional manner after 3 days of co-culture. The cytotoxicity of chitosan-alginate scaffold was in grade 0-1. At 2, 4, and 6 weeks after operation, the BBB score was higher in group A than in other groups and was lower in group D than in other groups; showing significant differences (P lt; 0.05). At 4 and 6 weeks, the BBB score was higher in group B than in group C (P lt; 0.05). After 6 weeks of operation, WGA-HRP retrograde tracing indicated that there was no regenerated nerve fiber through the both stumps of SCI in each group. HE and immunofluorescence staining revealed that host spinal cord and tissue engineering spinal cord l inked much compactly, no scar tissue grew, and a large number of neurofilament 200 (NF-200) positive fibers and neuron specitic enolase (NSE) positive cells were detected in the lesioned area in group A. In group B, a small quantity of scar tissue intruded into non-degradative chitosan-alginate scaffold at the lesion area edge, and a few of NSE flourescence or NF-200 flourescence was observed at the junctional zone. The both stumps of SCI in group C or group D were filled with a large number of scar tissue, and NSE positive cells or NF-200 positive cells were not detected. Otherwise, there were obviously porosis at the SCI of group D. Conclusion The tissue engineered spinal cord constructed by multi-channel chitosan-alginate bioscaffolds and BMSCs would repair the acute SCI of rat. It would be widely appl ied as the matrix material in the future.
Abstract： Objective To investigate the effects of hepatocyte growth factor（HGF）gene transfected bone marrow mesenchymal stem cells （MSCs）transplantation in pigs with chronic ischemic heart disease. Methods MSCs were isolated from pig bone marrow by density gradient centrifugation and adherent cell culture, purified, and determined by cellsurface antigens（CD34, CD44, CD71, Ⅷ factor and desmin）. MSCs were transfected by adenovirus expressing hepatocyte growth factor（AdHGF）, and the influence of HGF on the biological characteristics of MSCs was tested. The pig model of chronic myocardial ischemia was established by placing Ameroid ring inside the left circumflex coronary artery via leftthoracotomy. A total of 40 pigs were randomly divided into 5 groups （n=8） and were injected 5×106/ml MSCs＋ 4×109 pfu 200 μl AdHGF （MSCs＋ AdHGF group）, 4×109 pfu 200 μl AdHGF （AdHGF group）, 5×106/ml MSCs 200 μl（MSCs group），4×109 pfu 200 μl AdNull （AdNull group）and 1 ml saline（control group） into the ischemic myocardiumrespectively. Echocardiogram, digital subtraction angiography （DSA） of coronary artery, single photon emission computed tomography（SPECT） myocardial perfusion imaging and cardiomyocyte apoptosis were examined after 4 weeks. Results Positive CD44 and CD71 and negative CD34, Ⅷ factorand desmin were detected in MSCs by flow cytometer. HGF had a b influence on stimulating the proliferation and differentiation of MSCs. Echocardiogram examination showed that left ventricular end-diastolic volume（LVEDV），left ventricular ejection fraction（LVEF），fractional shortening（FS）of MSCs＋ AdHGF group were significantly increased after treatment （P＜ 0.05）. DSA detection showed that ischemic neovascularization of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group （P＜ 0.05）. SPECT showed that the left ventricular myocardium of MSCs＋ AdHGF group appeared thickened,myocardial perfusion was significantly improved and the myocardial motion was significantly increased （P＜ 0.05）. Vascular density of MSCs＋ AdHGF group was significantly higher than those of AdHGF group and MSCs group by HE stain of myocardium ［（39.4±1.2）/ HPF vs. （36.5±1.4）/ HPF and（34.5±1.7）/ HPF，P＜ 0.05］. Cardiomyocyte apoptosis rate of MSCs＋ AdHGF group was significantly lower than those of AdHGF group and MSCs group by TUNEL stain （P＜ 0.05）. Conclusion Combination transplantation can promote the angiogenesis of chronic ischemic myocardium, inhibit cardiomyocyte apoptosis and improve heart function in pigs with chronic ischemic heart disease. The effect of HGF gene transfected MSCs transplantation is better than that of MSCs or HGF transplantation alone.
Objective To investigate the effects of R-spondin 2 (Rspo2) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and bone mineral content in ovariectomized mice. Methods BMSCs were extracted from the bone marrow of the long bones of 7 4-week-old female C57BL/6 mice using whole bone marrow culture and passaged. After the cell phenotype was identified by flow cytometry, the 3rd generation cells were co-cultured with 10, 20, 40, 80, and 100 nmol/L Rspo2. Then, the cell activity and proliferative capacity were determined by cell counting kit 8 (CCK-8), and the intervention concentration of Rspo2 was screened for the subsequent experiments. The osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase (ALP) staining, and the mRNA levels of osteogenesis-related genes [RUNX family transcription factor 2 (Runx2), collagen type Ⅰ alpha 1 (Col1), osteocalcin (OCN)] were detected by real-time fluorescence quantitative PCR (RT-qPCR). In addition, 18 10-week-old female C57BL/6 mice were randomly divided into sham operation group (sham group), ovariectomy group (OVX group), and OVX+Rspo2-intervention group (OVX+Rspo2 group), with 6 mice in each group. The sham group only underwent bilateral back incision and suturing, while the other two groups established osteoporosis mouse models by bilateral ovarian castration. Then, the mice were given a weekly intraperitoneal Rspo2 (1 mg/kg) treatment in OVX+Rspo2 group and saline at the same dosage in sham group and OVX group. After 12 weeks of treatment, the body mass and uterus mass of the mice were weighed in the 3 groups to assess whether the OVX model was successfully prepared; the tibia bones were stained with HE and immunohistochemistry staining to observe the changes in tibial bone mass and the expression level of Runx2 protein in the bone tissues. Blood was collected to detect the expressions of bone metabolism markers [ALP, OCN, type Ⅰ procollagen amino-terminal peptide (PINP)] and bone resorption marker [β-collagen degradation product (β-CTX)] by ELISA assay. Micro-CT was used to detect the bone microstructure changes in the tibia, and three-dimensional histomorphometric analyses were performed to analyze the trabeculae thickness (Tb.Th), trabeculae number (Tb.N), trabeculae separation (Tb.Sp), and bone volume fraction (BV/TV). Results CCK-8 assay showed that Rspo2 concentrations below 80 nmol/L were not cytotoxic (P>0.05), and the cell viability of 20 nmol/L Rspo2 group was significantly higher than that of the control group (P<0.05). Based on the above results, 10, 20, and 40 nmol/L Rspo2 were selected for subsequent experiments. ALP staining showed that the positive cell area of each concentration of Rspo2 group was significantly larger than that of the control group (P<0.05), with the highest showed in the 20 nmol/L Rspo2 group. The expression levels of the osteogenesis-related genes (Runx2, Col1, OCN) significantly increased, and the differences were significant between Rspo2 groups and control group (P<0.05) except for Runx2 in the 40 nmol/L Rspo2 group. In animal experiments, all groups of mice survived until the completion of the experiment, and the results of the body mass and uterus mass after 12 weeks of treatment showed that the OVX model was successfully prepared. Histological and immunohistochemical staining showed that the sparseness and connectivity of bone trabecula and the expression of Runx2 in the OVX group were lower than those in the sham group, whereas they were reversed in the OVX+Rspo2 group after treatment with Rspo2, and the differences were significant (P<0.05). ELISA assay showed that compared with the sham group, the serum bone metabolism markers in OVX group had an increase in ALP and a decrease in PINP (P<0.05). After Rspo2 intervention, PINP expression significantly reversed and increased, with significant differences compared to the sham group and OVX group (P<0.05). The bone resorption marker (β-CTX) was significantly higher in the OVX group than in the sham group (P<0.05), and it was significantly decreased in the OVX+Rspo2 group when compared with the OVX group (P<0.05). Compared with the sham group, Tb.Th, Tb.N, and BV/TV significantly decreased in the OVX group, while Tb.Sp significantly increased (P<0.05); after Rspo2 intervention, all of the above indexes significantly improved in the OVX+Rspo2 group (P<0.05) except Tb.Th. Conclusion Rspo2 promotes differentiation of BMSCs to osteoblasts, ameliorates osteoporosis due to estrogen deficiency, and promotes bone formation in mice.
ObjectiveTo evaluate the combination of lipopolysaccharide-amine nanopolymersomes (LNPs), as a gene vector, with target gene and the transfection in bone marrow mesenchymal stem cells (BMSCs) so as to provide a preliminary experiment basis for combination treatment of bone defect with gene therapy mediated by LNPs and stem cells. MethodsPlasmid of bone morphogenetic protein 2 (pBMP-2)-loaded LNPs (pLNPs) were prepared. The binding ability of pLNPs to pBMP-2 was evaluated by a gel retardation experiment with different ratios of nitrogen to phosphorus elements (N/P). The morphology of pLNPs (N/P=60) was observed under transmission electron microscope (TEM) and atomic force microscope (AFM). The size and Zeta potential were measured by dynamic light scattering (DLS). The resistance of pLNPs against DNase I degradation over time was explored. The viability of BMSCs, transfection efficiency, and expression of target protein were investigated after transfection by pLNPs in vitro. ResultsAt N/P≥1.5, pLNPs could completely retard pBMP-2; at N/P of 60, pLNPs was uniform vesicular shape under AFM; TEM observation demonstrated that pLNPs were spherical nano-vesicles with the diameter of (72.07±11.03) nm, DLS observation showed that the size of pLNPs was (123±6) nm and Zeta potential was 20 mV; pLNPs could completely resist DNase I degradation within 4 hours, and such protection capacity to pBMP-2 decreased slightly at 6 hours. The cell survival rate first increased and then decreased with the increase of N/P, and reached the maximum value at N/P of 45; the cytotoxicity was in grade I at N/P≤90, which meant no toxicity for in vivo experiment. While the transfection efficiency of pLNPs increased with the increase of N/P, and reached the maximum value at N/P of 60. So it is comprehensively determined that the best N/P was 60. At 4 days, transfected BMSCs expressed BMP-2 continuously at a relatively high level at N/P of 60. ConclusionLNPs can compress pBMP-2 effectively to form the nanovesicles complex, which protects the target gene against enzymolysis. LNPs has higher transfection efficiency and produces more amount of protein than polyethylenimine 25k and Lipofectamine 2000.
ObjectiveTo investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice.MethodsMale and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry.ResultsWestern blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 (P<0.05), and in group B1 than in groups A and B (P<0.05); but no significant difference was found between groups A and B (P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ (P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ (P>0.05).ConclusionGIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.