Objective To compare the effect of two different methods of cell seeding on spatial distribution and gene expression of hBMSCs in biocoral scaffold in vitro cultures. Methods The composite of hBMSCs and biocoral scaffold was prepared by traditional seeding (group A) and fibrin glue seeding (group B). The seeding efficiency was measured after 30 minutes of incubation in group B and after 3 hours in group A. At 2, 7, 14 and 21 days after culture, the samples were harvestedand the serial longitudinal sections were cut for each embedded composite. The sections were stained with DAPI and were measured using fluorescence microscope with apotome under serial optical sections. The cell number in every 10 × objective field was automatically measured by AxioVision image analysis software and levels (from seeding surface to bottom L1-L5) or columns (from centre to margin) for comparing cell distribution were set up. The specific osteogenic genes [osteonectin (ON), core binding factor α1 (Cbfα1), osteocalcin (OC)] expression was measured by RT-PCR. Results The seeding efficiency was significantly higher in group B (88.32% ± 4.2%) than in group A (66.51% ± 12.33%, P lt; 0.01). At 2 days after culture, the cell number from L1 to L4 decreased gradully in two groups (P lt; 0.05); in the cell number of different columns, there was no significant difference in group A (Pgt; 0.05) whereas significant difference in group B (P lt; 0.05); there was no significant difference in gene expression between two groups (P gt; 0.05). At 7 days after culture, the cell number was less than that at 2 days in group A and there was significant difference among levels (P lt; 0.05). The cell number and osteogenic gene expression increased sharply and there appeared uniform cell distribution in group B (P gt; 0.05). The gene expression of ON and Cbfα1 in group B was higher than that in group A (Plt; 0.05). At 14 days after culture, the cell number in levels or columns in group A decreased sharply and was less than that at 7 days (P lt; 0.05); whereas the cell number was similar to that at 7 days in group B (P gt; 0.05). The OC gene expression reached the highest level in group B at 14 days. The gene expression was higher in group B than in group A (P lt; 0.05). At 21 days after culture, there was significant difference in the cell number among levels and in the gene expression between group A and group B (P lt; 0.05); there was no significant difference in the cell number among columns in two groups (Pgt; 0.05). In addition, the cell number of most levels and columns in group B was more than that in group A at 7, 14 and 21 days after culture (P lt; 0.05). Conclusion More uniform cell distribution with rapid prol iferation and osteogenic differentiation is available in different levels or columns of scaffold by fibrin glue seeding than by traditional seeding.
Objective To explore the feasibil ity of using PKH26 as a cell tracer to construct tissue engineered bone. Methods BMSCs isolated from the bone marrow of 1-week-old New Zealand white rabbit were cultured. The BMSCs at passage 3 were labeled with PKH26 and were observed under fluorescence microscope. The percentage of the labeled cells wasdetected by Flow cytometer. The labeled cells were induced to differentiate into osteoblasts in vitro and the morphology of the cells after induction was observed under inverted phase contrast microscope. The osteogenic induction was evaluated by ALP staining and Alizarin red staining. The cells labeled with PKH26 were seeded on the bio-derived bone to construct tissue engineered bone in vitro. Then the compound of cells and material were observed under fluorescence microscope. The compound of labeled cells and material were implanted into the rabbit thigh muscle, and the transformation of the labeled cells was observed by fluorescence microscope 14 and 28 days later. Results Fluorescence microscope observation: the BMSCs labeled by PKH26 were spherical and presented with red and uniform-distributed fluorescence, and the contour of the cells were clearly observed when they were adherent 24 hours after culture. Flow cytometric detection revealed that the percentage of labeled cells was 97.2%. After osteogenic induction, the morphology of the cells changed from long-fusiform to polygon-shape or cube-shape, more ECM was secreted, andthe ALP and the Alizarin red staining were positive. At 48 hours after culturing the PKH26 labeled BMSCs with bio-derived bone, the fluorescence microscope observation showed that there was red fluorescence on the surface and inside of the material. At 14 days after implantation, the labeled cells with red and l ight fluorescence were evident in the implantation area; while at 28 days, the cells with red fluorescence were still evident but less in quantity and weaker in fluorescence strength. Conclusion PKH26 can be used as BMSCs label for the construction of tissue engineered bone in vitro and the short-term tracing in vivo.
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.
Objective To study the effect of hypoxia on the prol iferation of hBMSCs and human placental decidua basal is-MSCs (hPDB-MSCs), and to provide the theoretical basis for discovering the new seed cells source for tissue engineering. Methods Density gradient centrifugation method was adopted to isolate and culture hBMSCs and hPDB-MSCs,flow cytometry (FCM) was appl ied to detect cell surface marker. After establ ishing the experimental model of CoC12 chemical hypoxia, MTT method was appl ied to evaluate the prol iferation of hBMSCs and hPDB-MSCs at different time points (6, 12, 24, 48, 72, 96 hours) with various CoC12 concentration (0, 50, 75, 100, 125, 150, 175, 200 μmol/L). Results FCM analysis revealed that hPDB-MSCs and hBMSCs expressed CD9, CD29, CD44, CD105, CD106 and human leucocyte antigen ABC (HLA-ABC), but both were absent for CD34, CD40L and HLA-DR. Compared with hBMSCs, hPDB-MSCs expressed stage-specific embryonic antigen 1 (SSEA-1), SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 better. The prol iferations of hPDB-MSCs and hBMSCs were inhibited within the first 12 hours under hypoxia condition, but promoted after 12 hours of hypoxia. Compared with the control group, the hBMSCs were remarkably prol iferated 24 hours after hypoxia with CoC12 concentration of 150 μmol/L (P lt; 0.05), while hPDB-MSCs were significantly prol iferated 12 hours after hypoxia with CoC12 concentration of 75 μmol/L (P lt; 0.05). Conclusion Compared with hBMSCs, hPDB-MSCs express more specific surface antigens of embryonic stem cells and are more sensitive to the prol iferation effects of chemical hypoxia, indicating it may be a new seed cells source for tissue engineering.
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 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.
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.
Objective To investigate the effects of the recombinant plasmid pIRES-hBMP-2-hVEGF165 on differentiation and maturation of hBMSCs in vitro. Methods The co-expressing vector of hBMP-2 and hVEGF165 was constructed. The BMSCs were isolated and cultured from health adult human denoted marrow. By the l ipofection method, the reconstructed plasmids pIRES-hBMP-2-hVEGF165, pIRES-hBMP-2, pIRES-hVEGF165 and pIRES neo empty vector, weretransfected to hBMSCs (groups A, B, C and D). The untransfected cells were harvested as control group (group E). After4 weeks of culture, RT-PCR was employed to assay the hBMP-2, hVEGF165 and osteocalcin mRNA expression in hBMSCs. The expressions of hBMP-2 and hVEGF165 of BMSCs were assayed by Western blot. The level of ALP activities of BMSCs was determined. Col I was also determined by immunohistochemical staining. Results Compared to group E, the hBMSCs in group A secreted high level of hBMP-2, hVEGF165, Col I and osteocalcin; osteocalcin and Col I expressed at high level in group B, and hVEGF165 expressed at high level in group C. Otherwise, the expression of hVEGF165 in group B and the expressions of hBMP-2 and Col I in group C resemble to that of groups D and E, no expression or few expression was observed. The activities of ALP in groups A, B, C, D and E were 0.91 ± 0.03, 0.90 ± 0.02, 0.64 ± 0.03, 0.67 ± 0.01 and 0.66 ± 0.02, respectively. The activity of ALP of groups A and B were significantly increased compared with that of group E (P lt; 0.05); there was no significant difference among groups C, D and E (P gt; 0.05). Conclusion The recombinant plasmid pIRES-hBMP-2-hVEGF165 can be successfully transfected into BMSCs with cation l iposome-mediated transfection method, the exogenous hBMP-2 and hVEGF165 genes can be expressed constitutively in the transfected BMSCs, and it can enhance the differentiation abil ities of BMSCs.
Objective To evaluate the osteogenesis of bi phasic ceramic-l ike biologic bone (BCBB) with tissue engineering in repairing segmental bone defects. Methods BMSCs isolated from the femoral and tibial marrow of 2-weekold Japanese white rabbit were cultured to passage 3. Then 20 μL of the cell suspension at a concentration of 1 × 107 cells/mLwere seeded into 15 mm × 15 mm × 5 mm BCBB block; the construction of tissue engineered BCBB was completed after 8 days of compound culture. Forty-eight adult Japanese white rabbits were randomly divided into groups A, B, C and D, then BCBBs cultured with BMSCs in vitro for 8 days (group A) and only BCBBs without BMSCs (group B) were respectively implanted into the radius segmental bone defects of rabbits, autogenous il iac bone graft (group C) and empty defect (group D) were used as controls. The specimens were examined after 4, 8, 12 and 24 weeks, the osteogenesis was evaluated through X-ray radiograph and histology examination. Results X-ray examination: the border between the material and host’s bone was clear after 4 weeks, and blurred after 8 weeks in group A and group B; the density of some part of the edge of the material was similar to that of radius and there was high density imaging in the materials of group A after 12 weeks; there was much high density imaging in the materials of group B after 12 weeks. The medullary cavity of bone was formed and l ittle high density imaging in the materials of group A after 24 weeks. Some high density imaging still existed in the materials of group B after 24 weeks. The X-ray evaluated scores showed that the scores of group A was higher than that of group B, and there was significant difference between group A and group B after 12 and 24 weeks (P lt; 0.05). Histological examination: there was new bone formation in the materials and also new bone grew adhesively on the surface of BCBB in group A. While in group B only new bone grew and attached to the surface of BCBB. BCBB degraded more with the time and more new bone formed. The histological evaluation showed that the bone forming area in group A was more than that in group B, and there was significant difference between group A and group B (P lt; 0.05). Conclusion The osteogenesis of BCBB with tissue engineering was superior to only BCBB, BCBB could be used as a scaffold of bone tissue engineering.
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.