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 observe the effect of BMSCs on the cardiac function in diabetes mellitus (DM) rats through injecting BMSCs into the ventricular wall of the diabetic rats and investigate its mechanism. Methods BMSCs isolated from male SD rats (3-4 months old) were cultured in vitro, and the cells at passage 5 underwent DAPI label ing. Thirty clean grade SD inbred strain male rats weighing about 250 g were randomized into the normal control group (group A), the DM group (group B), and the cell transplantation group (group C). The rats in groups B and C received high fat forage for 4 weeks and the intraperitoneal injection of 30 mg/kg streptozotocin to made the experimental model of type II DM. PBS and DAPI-labeledpassage 5 BMSCs (1 × 105/μL, 160 μL) were injected into the ventricular wall of the rats in groups B and C, respectively. After feeding those rats with high fat forage for another 8 weeks, the apoptosis of myocardial cells was detected by TUNEL, the cardiac function was evaluated with multi-channel physiology recorder, the myocardium APPL1 protein expression was detected by Western blot and immunohistochemistry test, and the NO content was detected by nitrate reductase method. Group C underwent all those tests 16 weeks after taking basic forage. Results In group A, the apoptosis rate was 6.14% ± 0.02%, the AAPL1 level was 2.79 ± 0.32, left ventricular -dP/dt (LV-dP/dt) was (613.27 ± 125.36) mm Hg/s (1 mm Hg=0.133 kPa), the left ventricular end-diastol ic pressure (LVEDP) was (10.06 ± 3.24) mm Hg, and the NO content was (91.54 ± 6.15) nmol/mL. In group B, the apoptosis rate was 45.71% ± 0.04%, the AAPL1 level 1.08 ± 0.24 decreased significantly when compared with group A, the LVdP/ dt was (437.58 ± 117.58) mm Hg/s, the LVEDP was (17.89 ± 2.35) mm Hg, and the NO content was (38.91±8.67) nmol/mL. In group C, the apoptosis rate was 27.43% ± 0.03%, the APPL1 expression level was 2.03 ± 0.22, the LV -dP/dt was (559.38 ± 97.37) mm Hg/ s, the LVEDP was (12.55 ± 2.87) mm Hg, and the NO content was (138.79 ± 7.23) nmol/ mL. For the above mentioned parameters, there was significant difference between group A and group B (P lt; 0.05), and between group B and group C (P lt; 0.05). Conclusion BMSCs transplantation can improve the cardiac function of diabetic rats. Its possible mechanismmay be related to the activation of APPL1 signaling pathway and the increase of NO content.
Objective To explore the label ing efficiency and cellular viabil ity of rabbit BMSCs labeled with different concentrations of superparamagnetic iron oxide (SPIO) particles, and to determine the feasibil ity of magnetically labeled stem cells with MR imaging. Methods The BMSCs were collected from il iac marrow of 10 adult rabbits (weighing 2.5-3.0 kg) and cultured. The SPIO-poly-L-lysine compound by different ratios mixed with medium, therefore, the final concentration of Fe2+ was 150 (group A), 100 (group B), 50 (group C) and 25 μg (group D) per mL, respectively, the 3rd generation BMSCs culture edium was added to lable; non-labeled cells served as a control (group E). MR imaging of cell suspensions was performed by using T1WI and T2WI sequences at a cl inical 1.5 T MRI system. Results BMSCs were efficiently labeled with SPIO, labeled SPIO particles were stained in all cytoplasms of groups A, B, C and D. With the increasing of Fe2+ concentration, blue dye particles increased. The staining result was negative in group E. The cell viabil ity in groups A, B, C, D and E was 69.20% ± 6.11%, 80.41% ± 2.42%, 94.32% ± 0.67%, 96.24% ± 0.34% and 97.43% ± 0.33%, respectively. There were statistically significant differences between groups A, B and groups C, D and E (P lt; 0.05), and between group A and group B (P lt; 0.05). T1WI images had no specific difference among 5 groups, T2WI images decreased significantly in groups A, B, C, decreased sl ightly in group D, and had l ittle change in group E. The T2WI signal intensities of groups A, B, C, D and E were 23.37 ± 6.21, 26.73 ± 3.60, 29.63 ± 2.82, 45.03 ± 6.76 and 783.15 ± 7.38, respectively, showing significant difference between groups A, B, C, D and group E (P lt; 0.05), and between groups A, B, C and group D (Plt; 0.05). Conclusion BMSCs can be easily and efficiently labeled by SPIO without interference on the cell viabil ity in labled concentration of 20-50 μg Fe2+ per mL. MRI visual ization of SPIO labeled BMSCs is feasible, which may be critical for future experimental studies.
【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 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 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.
【Abstract】 Objective To investigate the in vivo osteogenic feasibil ity of tissue engineered periosteum constructedby porcine SIS and BMSCs in allogenic New Zealand rabbit. Methods The tissue engineered periosteum constructed by SIS scaffold and BMSCs was prepared in vitro .Twelve 2-month-old New Zealand rabbits were used in the experiments. The 1.5-2.0 cm critical bone defects were made in the both sides of radius of the animals. The tissue engineered periosteum was grafted into one side defect randomly, while the other side defect was only grafted SIS. Four weeks after operation, the forearms of all animals were checked by X-ray. Then, animals were sacrificed to harvest the specimen which were treated promptly for HE and Masson staining.The X-ray film and the morphological tissue staining outcome were evaluated qual itatively. Results After operation,all animals had a normal behavior and diet; the incision healed normally; the forearm could move normally for bearing weight.The tissue engineered periosteum constructed by allogenic BMSCs and heterogeneic SIS scaffold could form new bone tissue, andbridged the bone defect which could be confirmed either in X-ray film or histological staining. The newly formed bone tissue had similar bone density to normal bone. A lot of irregular newly formed vessels and medullary cavity inserted in the newly borned tissue. No lymphocytes infiltrated in histological examination. While the control side had no any osteogenesis neithter in X-ray, nor in HE and Masson staining inspecting; the defect space only occupied with some connective tissue. Conc lu sion Tissue engineered periosteum can form new bone in allogenic rabbit and has the feasibil ity to repair the segmental diaphysis defect.
Objective To introduce the research of cell transplantation for treating intervertebral disc degeneration. Methods The original articles in recent years about cell transplantation for treating intervertebral disc degeneration were extensively reviewed, and retrospective and comprehensive analysis was performed. Results Transplantation of intevertebraldisc-derived cells or BMSCs by pure cell transplantation or combined with collagen scaffold into intervertebral disc couldexpress nucleus pulposus-l ike phenotype. All the cells transplanted into intervertebral disc could increase extracellular matrix synthesis and rel ieve or even inhibit further intervertebral disc degeneration. Conclusion Cell transplantation for treating intervertebral disc degeneration may be a promising approach.
Objective To compare the effect of mosaicplasty, mosaicplasty with gene enhanced tissue engineering and mosaicplasty with the gels of non-gene transduced BMSCs in alginate on the treatment of acute osteochondral defects. Methods Western blot test was conducted to detect the expression of hTGF-β1, Col II and Aggrecan in 3 groups, namely hTGF-β1 transduction group, Adv-βgal transduction group and blank control group without transduction. Eighteen 6-month-old Shanghai mascul ine goats weighing 22-25 kg were randomized into groups A, B and C (n=6). BMSCs were isolatedfrom the autologous bone marrow of groups B and C, and were subcultured to get the cells at passage 3. In group B, the BMSCs were transduced with hTGF-β1. For the animals of 3 groups, acute cyl indrical defects 5 mm in diameter and 3 mm in depth were created in the weight bearing area of the medial femoral condyle of hind l imbs. In group A, the autologous osteochondral mosaicplasty was performed to repair the defect; in group B, besides the mosaicplasty, the dead space between the cyl indrical grafts and the host cartilage were injected with the suspension of hTGF-β1 gene transduced autogenous BMSCs in sodium alginate, and CaCl2 was dropped into it to form calcium alginate gels; in group C, the method was the same as the group B, but the BMSCs were not transduced. General condition of the goats after operation was observed, the goats were killed 12 and 24 weeks after operation to receive gross and histology observation, which was evaluated by the histological grading scale of O’Driscoll, Keeley and Salter. Immunohistochemistry and TEM observation were performed 24 weeks after operation. Results Western blot test showed the expression of the hTGF-β1, Col II and the Aggrecan in the hTGF-β1 transduction group were significantly higher than that of the Adv-βgal transduction and the blank control groups. All the goats survived until the end of experiment and all the wounds healed by first intention. Gross observation revealed the boundaries of the reparative tissue in group B were indistinct, with smooth and continuous surfaces of the whole repaired area; while there were gaps in the cartilage spaces of groups A and C. Histology observation showed the dead space between the cyl indrical grafts in group A had fibrocartilage-l ike repair tissue, fill ing of fibrous tissue or overgrowth of the adjacent cartilage; the chondrocytes in group B had regular arrangements, with favorable integrations; while the dead space between the cyl indrical grafts in group C had fibrocartilage-l ike repair tissue, with the existence of gaps. The histology scores of group B at different time points were significantly higher than that of groups A and C, and group C was better than group A (P lt; 0.05); for group B, significant difference was detected between 12 weeks and 24 weeks in the histology score (P lt; 0.05）. Immunohistochemistry staining for Col II 24 weeks after operation showed the chondrocytes and lacuna of the reparative tissue in group B was obviously stained, while groups A and C presented l ight staining. TEM observation showed there were typical chondrocytes in the reparative tissue in group B, while parallel or interlaced arrangement collagen fiber existed in groups A and C. Conclusion Combining mosaicplasty with tissue engineering methods can solve theproblem caused by single use of mosaicplasty, including the poor concrescence of the remnant defect and poor integration with host cartilages.
【Abstract】 Objective To review the recent progress of BMSCs acting as seeding cell for tissue engineeredcartilage. Methods The recent ten years l iterature about BMSCs acting as seeding cell for tissue engineered cartilage was extensively reviewed. Results Scaffold provided an optimal environment for the growth of BMSCs. Cytokine and gene del ivery could promote BMSCs to differentiate toward chondrocytes. All of them played important roles in the field of cartilage tissue engineering. Conclusion The improvement of three-dimensional scaffolds, the rational use of cytokine, and the enhancement of gene del ivery will promote the development of cl inical cartilage reconstruction.