Objective To investigate the effects of intermittent negative pressure on the mRNA expression of osteoprotegerin (OPG) and osteoprotegerin l igand (OPGL) in human BMSCs cultured in vitro. Methods BMSCs were isolated from adult marrow donated by 2 hip osteoarthritis patients with prosthetic replacement in January 2008 and cultured in vitro. The third passage cells were divided into experimental group and control group. The experimental group was induced by negative pressure intermittently for 2 weeks (pressure: 50 kPa, 30 minutes each time, twice per day) and the control groupwas routinely cultured. After 2 weeks of culture, cell morphology was observed by inverted phase contrast microscope, and the mRNA expressions of OPG and OPGL in BMSCs were analyzed by real-time PCR. Results The cell prol iferation speed of the experimental group was slower than that of the control group. The cell morph changed from shuttle to megagon with some prominences in experimental group and the cell morph kept shuttle in the control. The mRNA expression of OPG in experimental group increased significantly (P lt; 0.01) and the mRNA expression of OPGL in experimental group decreased significantly compared with control group (P lt; 0.01) 2 weeks later. Conclusion Intermittent negative pressure is capable of promoting the expression of OPG, while inhibiting the expression of OPGL in human BMSCs.
Objective To investigate the feasibil ity of inducing canine BMSCs to differentiate into epithel ial cells in vitro with epithel ial cell conditioned medium (ECCM). Methods Five mL BMSCs were obtained from il iac spine of a healthy adult male canine with weighing 10 kg, and then isolated and cultured. The oral mucosa was harvested and cut into 4 mm × 4 mm after the submucosa tissue was el iminated; ECCM was prepared. BMSCs of the 2nd passage were cultured and divided into two groups, cultured in ECCM as experimental group and in L-DMEM as control group. The cell morphological characteristics were observed and the cell growth curves of two groups were drawn by the continual cell counting. The cells were identified by immunohistochemical staining through detecting cytokeratin 19 (CK-19) and anti-cytokeratin AE1/AE3 on the21st day of induction. The ultra-structure characteristics were observed under transmission electron microscope. Results The cells of two groups showed long-fusiform in shape and distributed uniformly under inverted phase contrast microscope. The cell growth curves of two groups presented S type. The cell growth curve of the experimental group was right shifted, showing cell prol iferation inhibition in ECCM. The result of immunohistochemical staining for CK-19 and anti-cytokeratin AE1/AE3 was positive in the experimental group, confirming the epithel ial phenotype of the cells; while the result was negative in the control group. The cells were characterized by tight junction under transmission electron microscope. Conclusion The canine ECCM can induce allogenic BMSCs to differentiate into epithel ial cells in vitro.
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 make a comparative study on the effects of whole bone marrow culture method and density gradient centrifugation method in isolating hBMSCs. Methods hBMSCs were obtained from healthy adult volunteers and isolated by whole bone marrow culture method and density gradient centrifugation method. Primary cell morphology was observed using inverted phase contrast microscope and the cells in the 2nd passage were stained with HE after being cultured for 7 days. And then, the generation time of the primary, 2nd and 3rd passage hBMSCs was comparedbetween two methods and the surface markers were detected by flow cytometer. In addition, the ALP expression inosteoinductive hBMSCs were evaluated by ALP activity kit at 3, 6 and 9 days and ALP staining was used for osteoinductivehBMSCs with Kaplow method at 9 days. Results Primary cells isolated with whole bone marrow culture method showedaggregation growth while cells isolated with density gradient centrifugation method showed diffusion growth. HE stainingshowed no significant difference in the morphology of the 2nd passage cells between these two methods. The generationtime of primary cells isolated by whole bone marrow culture method (15.36 ± 1.67) days was significantly shorter than that of cells isolated by density gradient centrifugation method [(18.57 ± 1.05) days] (P lt; 0.01), while the generation time of the 2nd and 3rd passage cells showed no statistically significant differences between these methods (P gt; 0.05). The concent of positive surface markers (CD29, CD44, CD71, CD105, CD166) and negative surface marker CD34 in the 2nd cells showed no significant difference between these two isolation methods (P gt; 0.05); however, negative markers CD14 and CD45 showed significant difference (P lt; 0.01). The ALP expression in osteoinductive cells showed no statistical significant (P gt; 0.05) at 3, 6 and 9 days; and the ALP staining positive cell ratio of whole bone marrow culture method was basically in accordance with that of density gradient centrifugation method at 9 days. Conclusion hBMSCs could be isolated by whole bone marrow culture method, and the cell isolation effects of whole bone marrow culture method are equivalent with density gradient centrifugation method.
Objective To investigate the therapeutic effect of BMSCs- chitosan hydrogel complex transplantation on intervertebral disc degeneration and to provide experimental basis for its cl inical appl ication. Methods Two mill il iter of bone marrow from 6 healthy one-month-old New Zealand rabbits were selected to isolate and culture BMSCs. Then, BMSCs at passage 3 were labeled by 5-BrdU and mixed with chitosan hydrogel to prepare BMSCs- chitosan hydrogel complex. Six rabbitswere selected to establ ish the model of intervertebral disc degeneration and randomized into 3 groups (n=2 per group): control group in which intervertebral disc was separated and exposed but without further processing; transplantation group in which 30 μL of autogenous BMSCs- chitosan hydrogel complex was injected into the center of defected intervertebral disc; degeneration group in which only 30 μL of 0.01 mol/L PBS solution was injected. Animals were killed 4 weeks later and the repaired discs were obtained. Then cell 5-BrdU label ing detection, HE staining, aggrecan safranin O staining, Col II immunohistochemical staining and gray value detection were conducted. Results Cell label ing detection showed that autogenous BMSCs survived and prol iferated after transplantation, forming cell clone. HE staining showed that in the control and transplantation groups, the intervertebral disc had a clear structure, a distinct boundary between the central nucleus pulposus and the outer anulus fibrosus, and the obviously stained cell nuclear and cytochylema; while the intervertebral disc in the degeneration group had a deranged structure and an indistinct division between the nucleus pulposus and the outer anulus fibrosus. Aggrecan safarine O stainning notified that intervertebral disc in the control and transplantation groups were stained obviously, with a clear structure; while the intervertebral disc in the degeneration group demonstrated a deranged structure with an indistinct division between the nucleus pulposus and the anulus fibrosus. Col II immunohistochemical staining showed that the tawny-stained region in the control group was located primarily in the central nucleus pulposus with a clear structure of intervertebral disc, the central nucleus pulposus in the transplantation group was positive with obvious tawny-stained intercellular substances and a complete gross structure, while the stained color in the degeneration group was l ighter than that of other two groups, with a indistinct structure.Gray value assay of Col II immunohistochemical staining section showed that the gray value of the control, the ransplantation and the degeneration group was 223.84 ± 3.93, 221.03 ± 3.53 and 172.50 ± 3.13, respectively, indicating there was no significant difference between the control and the transplantation group (P gt; 0.05), but a significant difference between the control and transplantation groups and the degeneration group (P lt; 0.05). Conclusion The rabbit BMSCs-chitosan hydrogel complex can repair intervertebral disc degeneration, providing an experimental foundation for the cl inical appl ication of injectable tissue engineered nucleus pulposus complex to treat intervertebral disc degeneration.
Objective To investigate the expression levels of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B l igand (RANKL) mRNAs in BMSCs in patients suffering glucocorticoid-induced necrosis of the femoral head (GNFH), and to discuss the relationshi p between OPG/RANKL system and GNFH. Methods The bone tissue and BMSCs of femoral head were collected from 35 patients suffering GNFH (experimental group) and from 21 patients suffering fracture of femoral neck (control group). The ratio of men to women was 4 ∶ 3 in two groups, aged 41 to 70 years (mean 55.34years in the experimental group and mean 55.33 years in the control group). The patients of experimental group received over 3 weeks’ glucocorticoid treatment or more than 1 week’s high-dose glucocorticoid therapy in recent 2 years, but patients of the control group did not receive more than 1 week’s hormone therapy. In 2 groups, the microstructure of bone tissue of femoral head was detected by HE staining. The BMSCs were isolated and cultured by adherent-wall method; the expression levels of OPG and RANKL mRNAs were examined by real-time quantitative polymerase chain reaction and the ratio of OPG mRNA to RANKL mRNA was caculated. Results Bone trabeculae and bone units were replaced by interrupted bone fragments, which were surrounded by inflammation and granulation tissue and few osteocytes were seen in bone lacunae in the experimental group. In control group, bone trabeculae and bone units were made by complete lamellar bone which surrounded blood vessels and osteocytes were seen in lacunae. The expression levels of OPG mRNA in the experimental group (0.37 ± 0.12) was significantly lower than that in the control group (0.47 ± 0.13), and the levels of RANKL mRNA in the experimental group (1.12 ± 0.39) was significantly higher than that in the control group (0.84 ± 0.24), showing statistically significant difference (P lt; 0.05). The ratio of OPG mRNA to RANKL mRNA in the experimental group (0.37 ± 0.17) was significantly lower than that in the control group (0.61 ± 0.26, P lt; 0.05). Conclusion The GNFH may be related to the expression levels of OPG mRNA and RANKL mRNA in BMSCs.
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 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.
Objective To access the possibil ity of CPC as a suitable scaffold for tissue engineering artificial rib by morphologic observation, adhesion experiments and cellar prol iferation experiments. Methods The 5 mm × 5 mm × 5 mm CPCs were prepared and the structure and components of CPC were compared with those of the normal human bone by micro-CT and scanning electron microscope. Bone marrow aspirates were harvested from the young pig and monuclear cells were separated. The first passage cells were collected and re-suspended in the culture media at a density of 6 × 105 cells/mL. There was 150 μL suspension which was incoluated on the CPC, and then cells were recollected and counted 4, 12 and 24 hours after inoculation. MTT was used to examine the growth condition of BMSCs on the surface of CPC. The scanning electron microscope was used to observe the CPC scaffold 7 days after inoculation, and comparison was made with CPC and the normal human bone. Results The adhesion rate of CPC was 28.00% ± 0.98%, 46.70% ± 1.14% and 48.50% ± 1.18%, respectively 4, 12 and 24 hours after compound culture. The prol iferation rate of CPC was 1.103 ± 0.214, 1.557 ± 0.322, 1.920 ± 0.178, 2.564 ± 0.226, 2.951 ± 0.415 and 3.831 ± 0.328, respectively 1, 2, 3, 4, 5 and 6 days after compound culture, with an obvious rising trend. The micro-CT demonstrated that the content of hydroxyapatite of porous phosphate calcium was (1 101.222 8 ± 0.618 4) mg/ ccm while that of the normal human bone was (1 072.552 3 ± 0.744 2) mg/ccm, and the porosity of porous phosphate calcium was 70.26% ± 0.45% while that of the normal human bone was 72.82% ± 0.51%, and there was no significant difference (P gt; 0.05). The experiment of cell prol iferation showed that the cell which was cultivated with porous phosphate calcium prol iferated rapidly. Through the inverted phase contrast microscope, it was found that the cells grew well and there was no dead cell, which indicated that the material had no toxicity. The rate of the cell adhesion to CPC was less than 50%. Conclusion The structure and components of CPC are similar to those of the normal human bone, and BMSCs grow well on the surface of it, so it is asuitable scaffold for tissue engineering artificial rib. However, the cell adhesion abil ity is to be further improved.
【Abstract】 Objective To explore the interventional effect of platelet lysate (PL) on osteogenic differentiation ofBMSCs by induction in rats in vitro. Methods Twenty-four clean-grade adult Wistar rats, weighing from 250 g to 300 g, maleor female, were included in this study. PL was obtained through three times of centrifugation and repeated freeze-thaw for the blood aspirated from cardiac cavities in 16 Wistar rats. ELISA assay was conducted to detect the concentration of growth factors PDGF, TGF-β1, IGF-1 and VEGF in PL. The BMSCs harvested by flushing femurs of 8 adult Wistar rats were isolated, cultivated and expanded in vitro. The cells at the 4 passage were performed for osteogenic differentiation by induction in three groups of A (5% PL of final concentration in basic induction medium), B (1% PL of final concentration in basic induction medium), and C (no presence of PL in basic induction medium as a control). The morphological changes of the cells were dynamically observed with inverted phase contrast microscope during the whole period. At different time-points, ALP staining (7 days) and ALP/TP (2, 8, 12 days) of the cells were detected to evaluate ALP activity, and the mineral formation in extracellular martrix was examined with Al izarin red staining which provided quantitative analysis of mineral deposits. Results ELISA assay showed that the content of PDGF, TGF-β1, IGF-1 and VEGF in PL reached (300 ± 30), (140 ± 25), (80 ± 35), (70 ± 20) pg/mL, respectively. Morphological observation displayed BMSCs in group A or B gradually turned from spindle-shape to square- or polygon-shape as the morphorlogical type of osteoblast-l ike cells at 7 days. The cells in group A showed slower shape changesbut higher prol iferation than that in group B or C. Moreover, at the 20 days, the cells in group A still displayed dense gro wth and produced obviously decreased amount of mineral deposits in ECM when compared with group B or C. At the 7 days, the cells ofgroup A showed smaller amount of granules positive for ALP staining in cytoplasm when compared with groups B and C, and displayed marked reduction in ALP activity assay at the 2, 8, and 10 days compared with that of groups B and C (P lt; 0.05). At the 20 days, Al izarin red staining showed the number of mineral deposits in groups A, B and C were 7.67 ± 1.10, 12.87 ± 0.81 and 15.59 ± 0.25, respectively, while the area of mineral deposits were (161 778.70 ± 44 550.80), (337 349.70 ± 56 083.24), and (415 921.70 ± 71 725.39) pixels, respectively. The number of mineral deposits and the area of mineral deposits in group A were smaller than those in groups B and C (P lt;0.05). But there was no statistically significant difference between groups B and C (P gt; 0.05). Conclusion PL is a kind of system carrying various growth factors. Exposure of PL inhibits both ALP activity and mineral formation of BMCs in a dose-dependent way under the osteogenic induction environment.