Objective To explore the feasibility of allogeneic marrow stromal stem cells(MSCs) as seed cells to construct tissue engineered bone bydetecting the expressions of interleukin 2(IL-2) and IL-2 receptor in rhesus monkeys after implanting these tissue engineered bones.Methods Engineered bones were constructed with osteoblasts which derived from allogeneic MSCs and bio-derived materials in vitro, and then were implanted to bridge 2.5 cm segmental bone defects of left radius in 15 rhesus monkeys as experimental group, bioderived materials only were implanted to bridge same size defects of right radius as control group. Every 3 monkeys were sacrificed in the 1st, the 2nd, the 3rd, the 6th andthe 12th weeks postoperatively and the expressions of IL-2 and IL-2 receptor in blood and graft samples were detected quantitatively by enzymelinked immuneosorbent assay (ELISA).Results There was no significant difference in the contents of IL-2 and its receptor between 2 groups（P＞0.05）. The contents ofIL-2 and its receptor increased from the 2nd week and maintained high level from the 2nd to the 6th week, but decreased after 6 weeks.ConclusionTissue engineered bones constructed with allogeneic MSCs and bio-derived materials show low immunogenicity. Allogeneic MSCs may be used as seed cells to construct tissue engineered bone.
Objective To explore the effect of the platelet-rich plasma (PRP) on proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) in China goat in vitro. Methods MSCs from the bone marrow of China goat were cultured. The third passage of MSCs were treated with PRP in the PRP group (the experimental group), but the cells were cultured with only the fetal calf serum (FCS) in the FCS group (the control group). The morphology and proliferation of the cells were observed by an inverted phase contrast microscope. The effect of PRP on proliferation of MSCs was examined by the MTT assay at 2,4,6 and 8 days. Furthermore, MSCs were cultured withdexamethasone（DEX）or PRP; alkaline phosphatase (ALP) and the calcium stainingwere used to evaluate the effect of DEX or PRP on osteogenic differatiation of MSCs at 18 days. The results from the PRP group were compared with those from the FCS group. Results The time for the MSCs confluence in the PRP group was earlier than that in the FCS group when observed under the inverted phase contrast microscope. The MTT assay showed that at 2, 4, 6 and 8 days the mean absorbance values were 0.252±0.026, 0.747±0.042, 1.173±0.067, and 1.242±0.056 in the PRP group, but 0.137±0.019, 0.436±0.052, 0.939±0.036, and 1.105±0.070 in the FCS group. The mean absorbance value was significantly higher in the PRP group than in the FCS group at each observation time (P＜0.01). Compared with the FCS group, the positive-ALP cells and the calcium deposition were decreased in the PRP group; however, DEX could increase boththe number of the positiveALP cells and the calcium deposition. Conclusion The PRP can promote proliferation of the MSCs of China goats in vitro but inhibit osteogenic differentiation.
Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2 (hBMP-2) gene and to study its expression in human umbil ical cord blood mesenchymal stem cells (HUMSCs). Methods hBMP-2 gene was ampl ified by PCR from a plasmid and was cloned into pDown by BP reaction. pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator (rtTA) were obtained with GATEWAY technology, and then were sequenced and analyzed by PCR. The recombinant vectors were transfected into 293FT cells respectively through l ipofectamine, and the lentiviral viruses were harvested from 293FT cells, then the titer was determined. Viruses were used to infect HUMSCs in tandem. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxycl ine concentrations (0, 10, 100 ng/mL, and 1, 10, 100 μg/mL) in the same induction time (48 hours), and the other by the same concentration (10 μg/mL) in different time points (12, 24, 48, and 72 hours). The expression of target gene hBMP-2 was indentified by ELISA method. After 2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Al izarin bordeaux staining method. Results Therecombinant inducible lentiviral vectors (pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA) were successfully constructed. The lentiviruses were also obtained and mediated by 293FT cells, and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively. HUMSCs could expression hBMP-2 by induction of doxycycl ine. The expression of hBMP-2 reached the peak at 10 μg/mL doxycl ine at 48 hours of induction. After 2-week osteogenic induction, a lot of mineral ization nodes were observed. Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.
Objective To investigate the results of human amniotic membrane(HAM) which are loaded with marrow mesenchymal stem cells(MSCs) and epidermis cells in treating fullthickness skin defect combined with radiation injury. Methods Eight minipigs were used in this study. Three round fullthickness wounds（Ф3.67cm）, which combined with radiation injury, were created on the dorsum of each side close to the vertebral column in each animal. Among 48 wounds, 24 left side wounds were treated with HAM loaded with MSCs and epidermis cells as experimental group (group A), 16 right side wounds with simple HAM (HAM group, group B) and 8 right side wounds with oil gauze as control (group C). The granulation tissue, reepithelization and wound area were observed after 1,2 and 3 weeks. Immunohistochemistry was performed using vWF as a marker for blood vessels.Image analysis was employed to test new area of wound at different interval time and healing rate of wound.Results The healing time of group A was 6 to 7 days faster than that of group C and 5 to 6 days faster than that of group B. After 15-17 days of graft, there were significant differences in new area of wound and healing rate between group A and groups B,C(Plt;001). New epidermis fully covered whole wound surface in group A, and their granulation tissue, which contained a lot of vWF, fibroblasts, capillaries and collagen, grew well. Many inflammatory cells still were seen in groups B and C, and their contents of vWF, fibroblasts, capillaries and collagen in granulation tissue were smaller than that in group A.Conclusion The graft of HAM loaded with MSCs and epidermis cells played an effective role in promoting healing of wound combined radiation injury with high quality.
ObjectiveTo comprehensively analyze the recent advancements in the field of mesenchymal stem cells (MSCs) aging,and summary its achievements and its difficulty at the present. MethodsThe literature about MSCs aging was reviewed and analyzed. ResultsInducible telomerase reactivation of MSCs is successful to extend the life span of senescent cells,but it also has potential safety hazard.The age range presented in the research of age-related cell senescence is inconsistent,resulting in different outcomes.Many ways to improve cell in vitro culture conditions will help delay aging.Recent research indicates that oxidative stress theory is seemed to not completely explain cell aging. ConclusionFurther research of MSCs aging mechanism will help the tissue engineering transform to clinical application.
Mesenchymal stem cells (MSCs) are considered as an ideal treatment for multiple diseases including ocular disease. Recent studies have demonstrated that MSCs-derived exosomes have similar functions with MSCs. Exosomes are nanovesicles surrounded by a phospholipid layer that shuttle active cargo between different cells. They are capable of passing the biological barrier and have potentials to be utilized as natural carrier for the ocular drug delivery.
Objective To observe the epidermal width, the amount variation and distribution character of epidermal stem cells(ESCs) and the wound healing rate at different periods of diabetes mellitus(DM) rats after trauma, thento study the correlation of them. Methods Forty-eight Wistarrats were divided into DM group and normal control group randomly(n=24).TheDM rats were induced by streptozocin (STZ) and then made chronic healing wound by special perforex.At the 3rd, 4th, 7th,14th and 21st days after trauma, the healing rate was calculated, the wound edge and granulation tissue were obtained for haematoxylin-eosin (HE) staining and immunohistochemical staining of keratin 19(K19) and β1 integrin. Then the epidermal width, the area and the gradation value of positive unit(PU) were measured. Results At the 3rd, 7th,14th and 21st days after trauma, the wound healing rates of normal rats were 24.48%±3.37%, 50.46%±1.26%, 92.82%±2.12% and 99.41%±0.66% respectively, while those of DM rats were 2.43%±1.02%, 40.59%±1.65%, 80.77%±3.57% and 85.40%±0.94% respectively, showing significant differences (Plt;0.01). Before trauma, there was no significant difference in the epidermal width between normal rats and DM rats (Pgt;0.05). However, at the 3rd, 7th, 14th and 21st days after trauma, the epidermal widths of normal rats were 26.43±3.21, 33.29±3.52, 31.53±3.35 and 26.01±3.19 μm respectively, while those of DM rats were23.58±2.33, 31.02±3.38, 33.72±5.49 and 21.80±4.02 μm respectively,the epidermal widths in DM rats were significantly lower than those in normal rats(Plt;0.01). The average PU value of K19 in normal rats were 91.68%, 93.14%, 72.27% and 70.31% respectively, while those in DM rats were 40.29%, 40.79%, 29.94% and 10.37% respectively. The average PU value of β1 integrin in normal rats were 49.6%, 91.16%, 77.13% and 57.17% respectively, while those inDM rats were 38.94%, 24.16%, 61.36% and 38.83%. The results indicated that the average PU values of K19 and β1 integrin in DM rats were significantly lower than those in normal rats(Plt;0.05). Conclusion The amountand activity decrease of ESCs may be one of the important mechanisms of difficult recovering wounds of DM rats.
Diabetic retinopathy is a serious complication of diabetes and is the leading cause of blindness in people with diabetes. At present, there are many views on the pathogenesis of diabetic retinopathy, including the changes of retinal microenvironment caused by high glucose, the formation of advanced glycation end products, oxidative stress injury, inflammatory reaction and angiogenesis factor. These mechanisms produce a common pathway that leads to retinal degeneration and microvascular injury in the retina. In recent years, cell regeneration therapy plays an increasingly important role in the process of repairing diseases. Different types of stem cells have neurological and vascular protection for the retina, but the focus of the target is different. It has been reported that stem cells can regulate the retinal microenvironment and protect the retinal nerve cells by paracrine production, and can also reduce immune damage through potential immunoregulation, and can also differentiate into damaged cells by regenerative function. Combined with the above characteristics, stem cells show the potential for the repair of diabetic retinopathy, this stem cell-based regenerative therapy for clinical application provides a pre-based evident. However, in the process of stem cell transplantation, homogeneity of stem cells, cell delivery, effective homing and transplantation to damaged tissue is still a problem of cell therapy.
Objective To study the effect of transforming growth factor β1(TGF-β1) and insulin-like growth factor 1(IGF-1) during the induction course from marrow mesenchymal stem cells (MSCs) to chondrocytes and to observe the effect of cell density on cell induction. Methods Differential time adherent methods were used to purify MSCs obtained from the bone marrow of Kunming mice. MSCs were cultured under special conditionsto induce themto differentiate into chondrocytes. Toluidine blue staining and immunofluoresence were used to identify those induced chondrocytes.TGF-β1 and IGF-1 were used individually or in combination under two different culture patterns: pellet culture and monolayer culture. According to different growth factors, experiment included 3 experimental groups(TGF-β1+IGF-1 group,10 ng/mland 50 ng/ml respectively;TGF-β1 group, 10 ng/ml; and IGF-1 group, 50 ng/ml) and control group(without growth factor). In TGF-β1+IGF-1 group, toluidine blue staining and immunofluoresence staining were carried out at 14 days and 21 days. The effect ofTGF-β1 and IGF-1 on the expression of collagen Ⅱgene was detected by RT-PCR at 7, 14 and 21 days of induction; the expressionsof collagen Ⅱ were compared between two culture patterns. Results In TGF-β1+IGF-1 group, the histological examination and immunofluoresence showed that those inducted chondyocytes could express collagen Ⅱ at 14 days. The gel electrophoresis results showed that the fragment of collagen Ⅱ gene was seen in TGF-β1+IGF-1 group andTGF-β1 group and that no fragment ofcollagen Ⅱ gene was seen in IGF-1 group and control group. The expression of collagen Ⅱ gene was ber in TGF-β1+ IGF-1 group than inTGF-β1 group, showing significant difference(Plt;0.05). Cells expressed more collagen Ⅱ under pellet culture than under monolayer culture. Conclusion IGF-1 could enhance the effect ofTGF-β1 during the induction course from MSCs to chondrocytes. A certain extent of high cell density is more effective for MSCs to differentiate into chondrocytes.
Objective To review the research appl ication and advance of synovium-derived mesenchymal stem cells (SMSCs) in tissue engineering. Methods The recent related l iterature was reviewed, concerning isolation method, characteristics of SMSCs, and its appl ication in tissue engineering. Results SMSCs are multi potent cell population with characteristics of easy isolation and high prol iferation, which have been appl icated in the cartilage, tendon, l igament, and bone tissue engineering. Conclusion SMSCs is a new member of mesenchymal stem cells family. It appears to be promising seedcells for tissue engineering, but further research is needed.