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.
【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.
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 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.
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 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 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.
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 investigate the adhesiveness of osteoblasts and vascular endothel ial cells from rat BMSCs co-cultured on allogeneic freeze-dried partially bone in vitro. Methods The BMSCs were isolated from 4-week-old SD rats (weighing 100-110 g) and cultured in vitro. The third generation of BMSCs were induced into osteoblasts and vascular endothel ial cells. The osteoblasts and vascular endothel ial cells after being induced for 7 days in a ratio of 1 to 1 were directlyco-cultured (experimental group), while the second generation of uninduced BMSCs was used as a control (control group). The growth and prol iferation abil ity were analyzed by MTT examination and the growth curve was drawn at 1-8 days. The osteoblasts and vascular endothel ial cells after being induced for 14 days were implanted in the allogeneic freeze-dried partially bone coated by 20% Col I or not at different densities (0.25 × 106/mL、0.50 × 106/mL、1.00 × 106/mL、2.00 × 106/mL、4.00 × 106/mL), as modified group and unmodified group, the cell adherence rate was calculated after 24 hours. These two kinds of cells were implanted in the pre-disposal treated allogeneic freeze-dried partially bone and observed by scanning electron microscope. Results ALP staining of osteoblasts showed that there were blue grains in cytoplasm at 7 days. CD31 and CD34 immunocytochemical staining of vascular endothelial cell showed that there were positive signals in the cytoplasm at 14 days. The MTT test showed that the prol iferation level of the experimental group was lower than those of the control group. There were significant differences in absorbance value between two group from 3 days to 8 days (P lt; 0.05). The cell adherence rate increased with increasing seeding density when the seeding density was (0.25-1.00) × 106/mL. The cell adherence rate reached the peak when the seeding density was 1.00 × 106/mL. The cell adherence rate decreased when the seeding density was more than 2.00 × 106/mL. There were significant differences in cell adherence rate between modified group and unmodified group at different seeding densities (P lt; 0.05). The prol iferation of the osteoblasts and endothel ial cells presented better growth and histocompatibil ity under scanning electron microscope. Conclusion The growing behavior of two kinds of cells is good in the allogeneic freezedried partially bone coated by 20% Col I , which can be used in reconstrction of vascularized tissue engineered bone.
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.