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 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 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.
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.
【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 evaluate sex determining region of the Y (Sry) as a engrafting track of the transplanted BMSCs survival and new bone formation in the osteonecrosis of the femoral head (ONFH) of rabbit. Methods Fortynine 4-5-month-old New Zealand White rabbits were included, weighing 2.0-2.5 kg, 48 females and 1 male. BMSCs of the rabbits were isolated by density gradient separation method, the third passage cells were marked by 1, 1’-dioctadecyl-3, 3, 3’, 3’-tetramethyl indocarbocyanine perchlorate (DiI) and the concentration of cell suspension was 2.5 × 108/ mL. The animal model of ONFH were establ ished with 48 female rabbits by injecting l iquid nitrogen, and femoral head was not dislocated.The animal model were divided into 3 groups, 16 rabbits in each group. Group A only establ ished animal model as control. Autologous BMSCs (4 μL) marked by DiI was transplanted in the ONFH models of the group B. Allogenic BMSCs (4 μL) marked by DiI was transplanted in ONFH models of the group C. The femoral head were observed by X-ray, HE staining and Masson staining, and the regenerating trabecular volume percentages was determined at 2, 4, 6 and 8 weeks after operation respectively. The examples of the heart, lung, l iver, spleen and kidney were obtained. The transplanted BMSCs were traced by fluorescence microscope, the Sry gene expression was detected by PCR for cells survival. Results All rabbits survived till the end of experiment. The X-ray showed gradual necrosis in the femoral head of group A. HE and Masson staining results indicated that compared with the group A, the recovery condition of the necrotic femoral head in the groups B and C was better. At each time of groups B and C, the regenerating trabecular volume percentages were higher than that of the group A significantly (P lt; 0.01). There was no significant difference between groups B and C (P gt; 0.05). The cells marked by DiI were not founded in the tissues of the heart, lung, l iver, spleen and kidney in groups B and C at each time. PCR showed that the expression of Sry gene were not observed at the heart, lung, l iver, spleen and kidney of three groups at each time. The expression of Sry gene was clearly identified in the femoral head of all 16 rabbits in the group C at each time point. Conclusion Allografting of BMSCs transplanted into the femoral head can survive and induce new bone formation without redistribution.
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 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.
Objective To explorer the survival time of autogeneic BMSCs labeled by superparamagnetic iron oxide (SPIO) in rabbit intervertebral discs and the rule of migration so as to prove bases of gene therapy preventing intervertebral disc degeneration. Methods Twelve rabbits were used in this experiment, aged 8-10 weeks, weighing 1.5-2.0 kg and neglecting their gender. BMSCs were separated from rabbits bone marrow by density gradient centrifugation and cultivated, and the 3rd generation of BMSCs were harvested and labeled with SPIO, which was mixed with poly-l-lysine. The label ing efficiency was evaluated by Prussian blue staining and transmission electron microscope. Trypanblau stain and MTT were performed to calculate the cell’ s activity. Rabbits were randomly divided into experimental group (n=8) and control group (n=4), the labeled BMSCs and non-labeled BMSCs (5 × 105/mL) were injected into their own intervertebral discs (L1,2, L2,3, L3,4 and L4,5), respectively. At 2, 4, 6 and 8 weeks, the discs were treated with Perl’s fluid to observe cell survival and distribution. Results The label ing efficiency of BMSCs with SPIO was 95.65% ± 1.06%, the cell activity was 98.28% ± 0.85%. There was no statistically significant difference in cell prol iferation within 7 days between non-labeled and labeled cells (P gt; 0.05). After 8 weeks of operation, the injected cells was al ive. ConclusionLabeled BMSCs with SPIO is feasible in vitro and in vivo, and the cells can survive more than 8 weeks in rabbit discs.
