【Abstract】 Objective To investigate the secretion of target gene and differentiation of BMSCs transfected by TGF-β1 and IGF-1 gene alone and together into chondrocytes and to provide a new method for culturing seed cells in cartilage tissue engineering. Methods The plasmids pcDNA3.1-IGF-1 and pcDNA3.1-TGF-β1 were ampl ified and extracted, then cut by enzymes, electrophoresed and analyzed its sequence. BMSCs of Wistar rats were separated and purificated by the density gradient centrifugation and adherent separation. The morphologic changes of primary and passaged cells were observed by inverted phase contrast microscope and cell surface markers were detected by immunofluorescence method. According to the transfect situation, the BMSCs were divided into 5 groups, the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by TGF-β1 (Group C), the group transfected by IGF-1 (Group D) and the group transfected both by TGF-β1 and IGF-1 (Group E). After being transfected, the cells were selected, then the prol iferation activity was tested by MTT and expression levels were tested by RT-PCR and Western blot. Results The result of electrophoresis showedthat sequence of two bands of the target genes, IGF-1 and TGF-β1, was identical with the sequence of GeneBank cDNA. A few adherent cells appeared after 24 hours culture, typical cluster formed on the forth or fifth days, and 80%-90% of the cells fused with each other on the ninth or tenth days. The morphology of the cells became similar after passaging. The immunofluorescence method showed that BMSCs were positive for CD29 and CD44, but negative for CD34 and CD45. A few cells died after 24 hoursof transfection, cell clone formed at 3 weeks after selection, and the cells could be passaged at the forth week, most cells became polygonal. The boundary of some cells was obscure. The cells were round and their nucleus were asymmetry with the particles which were around the nucleus obviously. The absorbency values of the cells tested by MTT at the wavelength of 490 nm were0.432 ± 0.038 in group A, 0.428 ± 0.041 in group B, 0.664 ± 0.086 in group C, 0.655 ± 0.045 in group D and 0.833 ± 0.103 in group E. The differences between groups A, B and groups C, D, E were significant (P lt; 0.01). The differences between groups A and B or between C, D and E were not significant (P gt; 0.05)。RT-PCR and Western blot was served to detect the expression of the target gene and protein. TGF-β1 was the highest in group C, 0.925 0 ± 0.022 0, 124.341 7 ± 2.982 0, followed by group E, 0.771 7 ± 0.012 0, 101.766 7 ± 1.241 0（P lt; 0.01）; The expression of IGF-1 was the highest in group E, 1.020 0 ± 0.026 0, 128.171 7 ± 9.152 0, followed by group D, 0.465 0 ± 0.042 0, 111.045 0 ± 6.248 0 （P lt; 0.01）. And the expression of collagen II was the hignest in group E, 0.980 0 ± 0.034 0, 120.355 0 ± 12.550 0, followed by group C, 0.720 0 ± 0.026 0, 72.246 7 ± 7.364 0（P lt; 0.01）. Conclusion The repairment of cartilage defects by BMSCs transfected with TGF-β1 and IGF-1 gene together hasa good prospect and important significance of cl inic appl ication in cartilage tissue engineering.
【Abstract】 Objective To approach the possibil ity of combination of simvastatin and BMSCs transplantation forsteroid-associated osteonecrosis of femoral head. Methods The BMSCs harvested from 24 rabbits were prepared for cell suspension at a concentration of 1 × 107/mL, and combined with gelatin sponge. Seventy New Zealand white rabbits received one intravenous injection of l ipopolysaccharide (10 μg/ kg). After 24 hours, three injections of 20 mg/kg of methylprednisolone were given intramuscularly at a time interval of 24 hours. Forty-eight rabbits diagnosed as having femoral head necrosis by MRI were divided into 4 groups randomly, group A: no treatment; group B: only decompression; group C: decompression and BMSCs transplantation; and group D: simvastatin drench (10 mg/kg.d) decompression and BMSCs transplantation. The general information of animals were recorded; after 4 and 8 weeks of operation, 6 rabbits of each group were chosen randomly to do MRI scan, and femoral heads were harvested to do histopathology and scanning electron microscope examination. Results After 8 weeks, rabbits became more active than before treatment, and walking way became normal gradually in groups C and D. Fourweeks after operation, the MRI low signal region of all groups had no obvious changes, but 8 weeks later, the necrosis signal region of group A magnified while it reduced obviously in group D. Histopathological observation: 4 weeks after operation, diffuse presence of empty lacunae and pyknotic nuclei of osteocytes were found in the trabeculae, and few newborn micrangium could been seen in group A; lots of empty lacunae and a small quantity of newborn micrangium could been found in group B; and large amounts of osteoblats and newborn micrangium were found around the necrosis regions in groups C and D. The positive ratio of empty lacunae and microvessel density in group D were 19.30 ± 1.52 and 7.08 ± 1.09, showing significant difference compared with other groups (P lt; 0.05). After 8 weeks of treatment, the bone trabecula collapsed in many regions in group A; there was fibra callus formation along the decompression channel in group B; few empty lacunae was in the bone trabecular, but the shape of marrow cavity was not normal in group C; and it showed almost normal appearance in group D. The positive ratio of empty lacunae and microvessel density in group D were 11.31 ± 1.28 and 12.37 ± 1.32, showing significant differences compared with other groups (P lt; 0.05), meanwhile, showing significant difference compared with that of 4 weeks after operation(P lt; 0.05). Scanning electron microscope: 8 weeks after operation, the bone trabecula collapsed in many regions, and few osteoblasts could be found on the surface, a great quantity of fat cells cumulated in the bone marrow in group A; cracked bone trabecula could be found occasionally in group B; the density of bone trabecula was lower than the normal in group C; and the shape of the marrow avity and thedensity of bone trabecula were similar to the normal in group D. Conclusion Simvastatin can promote the differentiation of osteocyte and vascular endothel ial cell from MSCs, the combination of simvastatin and marrow stem cells transplantation for the treatment of steroid-associated osteonecrosis of femoral head have good appl ication prospects.
ObjectiveTo comprehensively analyze and compare the biological difference between bone marrow mesenchymal stem cells (BMSCs) and placenta-derived MSCs (PMSCs) in hypoxia and to extend the knowledge for seed cells selection. MethodsThe domestic and foreign related literature about the effects of hypoxia microenvironment on proliferation, apoptosis, differentiation, paracrine secretion, migration, and homing ability of BMSCs and PMSCs were summarized and analysed. ResultsPMSCs proliferated much faster and more sensitive to the hypoxia than BMSCs; in addition, PMSCs showed stronger survivability. Similar to BMSCs, PMSCs can home to hypoxic-ischemic tissues efficiently, secrete a lot of growth factors and differentiate into tissue-specific cells to stimulate tissue regeneration. ConclusionPMSCs as the seed cells will have broad application prospects in the regenerative medicine.
Objective To introduce growth and differentiation factor 5 (GDF-5) gene into hBMSCs using recombinant adenovirus vector and to investigate the effect of GDF-5 gene expression on hBMSCs osteogenic differentiation. Methods Recombinant adenovirus GDF-5 (Ad-GDF-5) containing green fluorescent protein (GFP) and Ad-GFP were amplifiedand tittered. hBMSCs at passage 3 were infected with two viruses at different titers. At 2 days after intervention, GFP expression was observed using fluorescence microscope, and GDF-5 expression in hBMSCs was detected by RT-PCR. Adherent hBMSCs at passage 3 were randomly divided into 4 groups: experimental group (GDF-5 gene transfection), osteogenic induction group, Ad- GFP infection group, and control group. Cell differentiation was detected by inverted phase contrast microscope observation, fluorescence microscope observation, reverse transcription fluorescence quantitative PCR, immunofluorescence staining, and von Kossa staining at different time points after intervention. Results The titer of Ad-GDF-5 and Ad-GFP was 1.0 × 109 pfu/mL and 1.2 × 109 pfu/mL, respectively. hBMSCs was efficiently infected by Ad-GDF-5 and Ad-GFP, and expressed target gene and GFP gene. At 1-7 days after intervention, morphology and growth pattern of the hBMSCs in the experimental group and the osteogenic induction group were transformed into osteoblast-l ike cells, whereas the cells in the other two groups were still maintained their original morphology and growth pattern. Reverse transcription fluorescence quantitative PCR detection: at 4 days after intervention, GDF-5 expression in the experimental group was obviously higher than that of other groups (P lt; 0.05); ALP, Col I, and OC gene expression in the experimental and the osteogenic induction group were superior to those of theAd-GFP infection and the control group (P lt; 0.05); Col I gene expression in the osteogenic induction group was greater than that of the experimental group (P lt; 0.05). Immunofluorescence staining: at 4 days after intervention, the cells in the osteogenic induction group and the experimental group expressed and secreted Col I, and no expression of Col I was evident in the other two groups. At 10 days after intervention, the cells in the osteogenic induction and the experimental group were positive for von Kossa staining, and the results of the other two groups were negative. Conclusion GDF-5 gene can be transferred into hBMSCs via adenovirus vector and be expressed stably. It can facil itate the osteogenic differentiation of the hBMSCs and lay a foundation for the further study of this kind of gene transferred hBMSCs effect on bone tissue repair.
Objective To compare the molecular phenotype of human intervertebral disc cells and articular chondrocytes and to analyze whether hBMSCs can differentiate into both chondrocytes and nucleus pulposus cells after combined induction of TGF-β3 and BMP-7 in vitro. Methods The cells with the characteristics of hBMSCs were isolated from marrow aspirates of the volunteer donors’ il iac crest. Human bone marrow was removed and fractionated, and adherent cell cultures were establ ished. The 4th passage cells were then translated into an aggregate culture system in a serum-free medium. The pellet cultures of hBMSCs were divided into four groups: 10 ng/mL TGF-β3 group (group A), 200 ng/mL BMP-7 group (group B), combination group of TGF-β3 and BMP-7 (group C) and blank group as the control (group D). Histological observation, RT-PCR and RQ-PCR were appl ied to measure the expressions of collagen type I, II, X, aggrecan and SOX9 on the 4th and 21st day after cell induction, respectively. Results As was shown by histological observation, the induced cells expressed the feature of chondrocytes in morphology and ECM in groups A and C on the 21st day after the culture. And the collagen type II was positive after staining in groups A and C. The cell morphology of the induced cells in groups B and C had no obviouly changed. PCR detection showed that the expressions of SOX9, aggrecan, collagen type I, II in groups A and C at 21st day were more increased than those at 4th day (P lt; 0.05). The only expressions of collagen type I in groups B and D at 21st day were more increased than those at 4th day (P lt; 0.05). The expressions of collagen type X only was positive in group A. Conclusion Combination of TGF-β3 and BMP-7 can make the differentiated cells from hBMSCs much closer to intervertebral disc cells, so it perhaps could provide seed cells for intervertebral disc tissue engineering.
To investigate the effect of BMSCs on the repair of digestive tract injury and its mechanisms.Methods Recent l iterature on the effect of BMSCs on the repair of digestive tract injury was reviewed. Results BMSCs had the potency of self-repl ication, prol iferation and multipotential differentiation, which played an important role in the repair of digestive tract injury. The probable mechanisms included: BMSCs’ abil ity of migrating to the injured tissue and inhibiting the host immune response; BMSCs’ dedifferentiation and redifferentiation; BMSCs’ direct differentiation into the epithel ial cellsor the stem cells of digestive tract; BMSCs’ fusion with the stem cells or the mature epithel ial cells of digestive tract; BMSCs’ participation in the reconstruction of injured microenvironment. Conclusion BMSCs participates in the repair of digestive tract injury and has a bright future in the treatment of digestive system disease.
【Abstract】 Objective To investigate the possibil ity of BMSCs seeded into collagen Ⅰ -glycosaminoglycan (CG)matrices to form the tissue engineered cartilage through chondrocyte inducing culture. Methods Bone marrow aspirate of dogs was cultured and expanded to the 3rd passage. BMSCs were harvested and seeded into the dehydrothemal treatment (DHT)cross-l inked CG matrices at 1×106 cells per 9 mm diameter sample. The samples were divided into experimental group and control group. In the experimental group, chondrogenic differentiation was achieved by the induction media for 2 weeks. Medium was changed every other day in both experimental group and control group. The formation of cartilage was assessed by HE staining and collagen Ⅱ immunohistochemical staining. Results The examinations under the inverted phase contrast microscopeindicated the 2nd and 3nd passage BMSCs had the similar morphology. HE staining showed the BMSCs in the experimental group appeared polygon or irregular morphology in the CG matrices, while BMSCs in the control group appeared fibroblast-l ike spindle or round morphology in the CG matrices. Extracellular matrix could be found around cells in the experimental group. Two weeks after seeded, the cells grew in the CG matrices, and positive collagen Ⅱ staining appeared around the cells in the experimentalgroup. There was no positive collagen Ⅱ staining appeared in the control group. Conclusion It is demonstrated that BMSCs seeded CG matrices can be induced toward cartilage by induction media.
Objective To establ ish a two-dimensional biological printing technique of hBMSCs so as to control the cell transfer process and keep cell viabil ity after printing. Methods Bone marrow (5 mL) was obtained from healthy volunteer. The hBMSCs were regularly subcultured to harvest cells at passage 2, which were adjusted to the single cell suspensionat a density of 1 × 106/mL. The experiment was divided into 3 groups: printing group 1 in which cells underwent propidium iodide (PI) fluorescent label ing, then were transferred by rapid prototype biological printer (interval in x-axis 300 μm, interval in y-axis 1 500 μm), and laser scanning confocal microscope was appl ied to observe cell fluorescence; printing group 2 in which cells received no PI label ing and were cultured for 2 hours after transfer, Live/Dead viabil ity Kit was adopted to detect cell viabil ity and laser scanning confocal microscope was appl ied to observe cell fluorescence; half of the cells in printing group receiving no Live/Dead viabil ity Kit detection were cultured for 7 days, then inverted microscope was used to observe cell morphology, routine culture was conducted after the adherence of cells, the growth condition of cells was observed dynamically; control group in which steps were the same as the printing group 2 except that cell suspension received no printing. Results Laser scanning confocal microscope observation on the cells in printing group 1 revealed the “cell ink droplets” were distributed regularly and evenly in the two-dimensional layer and each contained 15-35 cells, meeting the requirement of designing two-dimensional cell printing. The cells in printing group 2 went through cell viabil ity test, laser scanning confocal microscope observation showed the fluorescence of cells 30 minutes after cell incubation. There was no significant difference between the control group and the printing groups in terms of cell viabil ity. The printed cells presented normal adherence, good morphology and good growth state 7 days after routine culture. Conclusion Biological printing technique can real ize the oriented, quantificational and regulardistribution of hBMSCs in the two-dimensional plane and lays the foundation for the construction of three-dimensional cellprinting or even organ printing system.
Objective To review researches of BMSCs in tumor therapy. Methods The recent relevant l iterature was extensively reviewed. The tropism of BMSCs to cancer, the effect of BMSCs on tumor growth and the appl ication of BMSCs in tumor therapy were summarized. Results BMSCs has the tropism to tumor and may inhibit or enhance growth of tumor. BMSCs as gene-del ivery vehicle for gene therapy had obtained certain therapeutic efficacy. However, BMSCs can become tumorigenic. Conclusion BMSCs is a good gene-del ivery vehicle for gene therapy. The relationship of BMSCs and tumorcells should be studied deeply for enhance the safety of BMSCs in gene therapy of tumor.
Objective To supply references to tissue-engineered skin cl inical appl ications with autogenic BMSCs composited collagen membrane to repair swine full-thickness cutaneous deficiency. Methods Twenty mL bone marrow were obtained respectively from 4 swine, autogenic BMSCs were cultured and passed to the 3rd passage. The fresh bovine tendontreated by means of chemically cross-l inked was made 5 cm diameter collagen I (Col I) membrane. The 2 × 107/mL P3 swine autogenic BMSCs labeled DAPI were planted to sterile Col I membrane for 24 hours incubation, then the tissue-engineered skin was constructed. The five full-thickness skin defect of 5 cm diameter was excised to the muscle from forward to backward on the back midl ine two sides of swine. The tissue-engineered skin were implanted in the experimental group, while Col I membrane was implanted in control group. After 3 and 8 weeks of implantation, the two swine wound surface heal ing circumstance was observed and further evaluated with histology analysis and TEM. After 3 weeks of implantation, the experimental group were observed with fluorescence microscopy and staining for glycogen. Results After 3 weeks of implantation, the wound surface of control group were observed nigrescence, scab and putrescence, and after 8 weeks of implantation, also evident putrescence and scar. The wound surface of experiment group was al ive after 3 weeks implantation, appearance was leveled off and flexible without evident scar. The wound surface recovered well after 8 weeks of implantation, wound surface heal ing rate was significantly difference between the two groups (P lt; 0.01). After 3 weeks of implantation, control group were observed acestoma hyperplasia and no epidermal coverage by histology analysis. The experimental group was showed integrity epidermis and dermis structure. The basal layer was crimson and continuously positive with glycogen staining. After 8 weeks of implantation, the experimental group and control group were emerged normal skin structure. After 3 weeks of implantation in control group, a lot of neutrophil ic granulocytes and fibroblasts were noticed, but no epidermal structure was observed under TEM. In the experimental group, a lot of epidermal cells were observed, dermatome connection among epidermal cells and hemidermosome connection between basilar membrane cells and basal membrane were observed in epidermis. In the dermis experimental group, blood capillary endothel ial cells were noticed. Furthermore, considerable collagen fiber deposit was found in the surrounding tissue of fibroblasts. After 3 weeks of implantation, BMSCs labeled with DAPI were located reconstructed epidermal basement membrane and dermis by fluorescence microscopy. Conclusion Tissue-engineered skin which is composited with autogenic BMSCs as seed cells and collagen membrane were potential prospects in appl ication of repairing swine full-thickness cutaneous deficiency.