Objective To introduce the research of mesenchymal stemcells(MSCs) transplantation for treating intervertebral disc degeneration. Methods The recent original articles about the MSCs transplantation for treating intervertebral disc degeneration were extensively reviewed. Results Transplanted MSCs in intervertebral disc can express chrondcyte-like phenotype in certain conditions, increase matrix synthesis and release intervertebral disc degeneration. Conclusion MSCs transplantation for treating intervertebral disc degeneration may be a future approach.
Objective To review the study progress of mesenchymal stem cells induced to differentiate intervertebral disc cells Methods The recent related literature was reviewed. The theorical and experimental studies were summarized. Results MSCs had the potential of multidirectional differentiation.International experimental studies indicated the potential of MSCs induced to differentiate intervertebral disc cells. Conclusion MSCs induced to differentiate intervertebral disc cells has the fine prospect.
Objective To explore the method that can inducethe mesenchymal stem cells (MSCs) to differentiate into the neuronlike cells in vitro．Methods The neuron-like cells were isolated froman SD rat (age, 3 months; weight, 200 g). They underwent a primary culture; theinduced liquid supernatant was collected, and was identified by the cell immunohistochemistry. The C3H1OT1/2 cells were cultured, as an MSCs model, and they were induced into differentiation by β-mercaptoethanol (Group A) and by the liquid supernatant of the neuron-like primary cells (Group B), respectively. The cells were cultured without any induction were used as a control (Group C). Immunohistochemistrywas used to identify the type of the cells． Results The result of the immunochemistry showed that the cells undergoing the primary culture expressed the neurofilament protein (NF) and the neuronspecific enolase (NSE), and they were neuron-like cells. β-mercaptoethanol could induce the C3H1OT1/2 cells toexpress NF and NSE at 2 h, and the expression intensity increased at 5 h. The liquid supernatant of the primarily-cultured neuron-like cells could induce theC3H1OT1/2 cells to express NF and NSE at 1 d, but the expression intensity induced by the liquid supernatant was weaker than that induced by β-mercaptoethanol. The positivity rate and the intensity expression of NSE were higher than those of NF. Conclusion MSCs can differentiate into the neuron-like cells by β-mercaptoethanol and the microenvironment humoral factor, which can pave the way for a further study of the differentiation of MSCs and the effectof the differentiation on the brain trauma repair.
OBJECTIVE: From the point of view of material science, the methods of tissue repair and defect reconstruct were discussed, including mesenchymal stem cells (MSCs), growth factors, gene therapy and tissue engineered tissue. METHODS: The advances in tissue engineering technologies were introduced based on the recent literature. RESULTS: Tissue engineering should solve the design and preparation of molecular scaffold, tissue vascularization and dynamic culture of cell on the scaffolds in vitro. CONCLUSION: Biomaterials play an important role in the tissue engineering. They can be used as the matrices of MSCs, the delivery carrier of growth factor, the culture scaffold of cell in bioreactors and delivery carrier of gene encoding growth factors.
Objective To establish a method of isolating and culturing adult human bloodderived mesenchymal stem cells(MSCs) and to investigate their osteogenic potential in vitro. Methods Thirty peripheral blood sampleswere collected from 30adult volunteers(15 ml per person).Adult human MSCs derived from peripheral blood were isolated from the lymphocyte separation fluid fraction of mononuclear cells, cultured in α-Modified Eagle’s Medium with low glucose containing 20% fetal bovine serum, and proliferated through a process of subculturing. The phenotype of MSCs was analyzed with flow cytometry. For in vitro osteogenic differentiation, MSCs from the second passage grew in the presence of osteogenic supplements (100 nmol/L dexamethasone,10 mmol/L β-glycerophosphate,50 μmol/L vitamin C, and 10 nmol/L 1,25-2-hydroxide vitamin D3). In the fifth passage cells, the activity of alkaline phosphatase, the expression level of collagen typeI, osteocalcin and osteonectin were determined. And the calcium tubercle formation would be examined after the continual one-month culture of the fifth passage. Results MSCs exsited in the pheripheral blood of adult human. And the clone forming efficiency of blood-derived MSCs was 0.27±0.22/106 mononuclear cells. The MSCs expressed CD44,CD54,CD105,and CD166，but did not CD14, CD34, CD45,and CD31.Under the function of osteogenic supplements, the MSCs were found to be higher activity of alkaline phosphatase and higher expression levels of collagen type Ⅰ, osteocalcin and osteonectin. And the calcium tubercle formation was examined throughtetracycline fluorescence labeling method. Conclusion The isolation and cultureconditions established for adult human MSCs may select a distinct population of peripheral blood-derived adherent cells. Adult human blood-derived MSCs possess osteogenic potential in vitro, and may be used as seed cells for bone tissue engineering.
Objective To determine if mesenchymal stem cells ( MSCs) could be reconstructed as a vehicle for angiopoietin-1 ( Ang1) gene therapy in lung injury. Methods MSCs were obtained from adult male inbred mice and cultured to passage four. The cells were identified by fluorescence-activated cell sorting ( FACS) analysis and cell differentiation detection. Lentiviral vectors contained GFP and Ang1 gene were conducted in 293T cells through three plasmids co-transfection method. Then MSCs were transduced with Ang1 gene efficiently through lentiviral vectors. The mRNA expression of Ang1 in MSCs was detected by RT-PCR before and after transfection. Also fluorescence from MSCs was detected by fluorescence microscope every day after transfection. Two hours after LPS inhalation, mice were infused via jugular veinwith normal saline ( NS group) , lentiviral vector carrying Ang1 ( Ang1 group) , lentiviral vector carrying GFP ( MSCs group) , and lentiviral vector carrying Ang1 /GFP ( MSCs-Ang1 group) , respectively. Kaplan-Meier survival analysis was performed to compare the effects of MSCs-Ang1 on survival. And ectogenic MSCs origined lung cells were investigated in receipt mice. Results After passaged and purification,MSCs were confirmed to have the potential of differentiation. The lentiviral vectors carrying Ang1 and GFP were also identified. After transfection, the mRNA expression of Ang1 in MSCs was enhanced. Through the fluorescence microscope,MSCs get the most green fluorescence expression five days after the transfection when MOI was 20. Kaplan-Meier survival analysis showed that MSCs-Ang1 infusion had improved survival rates of lung injury rats compared with the control, but it did not reach statistical significance ( P = 0. 066) . Cells expressing GFP in lung tissues can be observed after MSCs were transplanted in vivo. Conclusions MSCs expressing Ang1 high can be constructed through lentiviral vector transfer, and MSCs-origined cells can be detected in receipt lungs after transplantation. So MSCs may serve as a vehicle for gene therapy in lung injury.
Objective To investigate the curative effects of homograft of the mesenchymal stem cells(MSCs) compbined with the medical collagen membrane of the guided tissue regeneration(MCMG) on the full thickness defects of the articular cartilage. Methods MSCs derived from New Zealand rabbits aged 3-4 months weighing 2.1-3.4 kg were cultured in vitro with a density of 5.5×108/ml and seeded onto MCMG. The MSC/MCMG complex was cultured for 48 h and transplanted into the fullthickness defects on the inboardcondyle and trochlea. Twenty-seven healthy New Zealand rabbits were randomly divided into 3 groups of 9rabbits in each. The cartilage defects in the inboard condyle and trochlea werefilled with the auto bone marrow MSCs and MCMG complex (MSCs/ MCMG) in Group A (Management A), with only MCMG in Group B (Management B)and with nothing in Group C (Management C). Three rabbits were killed at 4, 8 and 12 weeks after operation in each group, and the reparative tissue samples evaluated grossly,histologically and immunohistochemically were graded according tothe gross and histological scale. Results Four weeks after transplantation, the cartilage and subchondralbone were regenerated in Group A;for 12 weeks, the regenerated cartilage gradually thicked; 12 week after transplantation, the defect was repaired and the structures of the carticular surface and subchondral bone was in integrity.The defects in Group A were repaired by the hylinelike tissue and the defects in Groups B and C were repaired by the fibrous tissues. Glycosaminoglycan and type Ⅱcollagen in Groups A,B and C were reduced gradually.The statistical analysis on the gross at 12 weeks and the histologicalgradings at 4 weeks,8 weeks and 12 weeks showed that the inboardcondylar repairhad no significant difference compared with the rochlearepair(Pgt;0.05).Management A was significantly better than Managements B and C (Plt;0.05), and Management B was better than Management C(Plt;0.05). Conclusion Transplantation of the MSCs combined with MCMG on the full thickness defects of the articular cartilage is a promising approach to the the treatment of cartilage defects. MCMG can satisfy the demands of the scaffold for the tissue-engineered cartilage.
Objective To study the culture and purification of the fetal mouse liver mesenchymal stem cells(MSCs) in vitro and to investigate their differentiation potential and the composite ability with true bone ceramic(TBC). Methods The single cell suspension of MSCs was primarily cultured and passaged, which was prepared from the fetal mouse liver; the flow cytometry was applied to detectCD29, CD34, CD44 and CD45. The osteogenic differentiation was induced in chemical inducing system; the osteogenic induction potency was tested. The purified fetal mouse liver MSCs were compounded with TBC covered with collagen type Ⅰ in vitro and the cell attachment and proliferation to the TBC were observed. Results The primary MSCs of fetal mouse liver were easy to culture in vitro. They proliferated well and were easy to subcultured. The proliferation ability of primary and passaged MSCs was similar. Flow cytometric analysis showed the positive results for CD29, CD44 and the negative results for CD34, CD45. After 7 days of induction, the MSCs expressed collagen type I and alkaline phosphatase(ALP) highly. After 14 days of induction, the fixed quantity of ALP increased significantly. After 28 days of induction, calcium accumulation was observed by Von Kossa’s staining. Many liver MSCs attached to the surface of TBC. Conclusion The MSCs of the fetalmouse liver can be obtained, subcultured and purified easily. After culturing in chemical inducing system, the MSCs of fetal mouse liver can be successfully induced to osteoblast-like cells, attach to the surface of TBC and proliferate well.
Objective To study the effect of adenovirus bone morphogenetic protein 2 gene(Ad-BMP-2) transfer inducing mesenchymal stem cells (MSCs) compounded with fibrin gel on repair of rabbit cartilage defect. Methods ①BMP-2 and collagen type Ⅱ in MSCs transferred by Ad-BMP-2 were examined by RT-PCR, aniline dyeing and immunohistochemical analysis in vitro. ②MSCs were cultured in fibrin gel for 9 days, and were examined with electron microscope. ③Fortytwo rabbits suffering from cartilage defect were divided into 3 groups:the defects were treated with Ad-BMP-2 transfer inducing MSCs compounded with fibrin in group A, with MSCs compounded with fibringel in group B and with no implants in group C as control. HE and aniline dyeing, immunohistochemical analysis and biomechanics study were carried out in the 4th, 8thand 12th weeks. Results ①The positive results were observed for BMP-2 and collagen type Ⅱ with RT-PCR on the 3rd day and 5th day respectively, being statisticallysignificant difference when compared with control group(P＜0.05). ②Ad-BMP-2 transfer inducing MSCs cultured in fibrin gel were positively stained by aniline dyeing and immunohistochemstry. ③The therapy effect of group A was better than that of the other two groups in histology, biochemistry and biomechanics, and the biomechanic and histological features of repaired cartilage were similar to those of the natural cartilage. Conclusion Ad-BMP-2 can induce the expressionof collagen type Ⅱ and mucopolysaccharide in MSCs by secreting BMP-2, and can reconstruct articular cartilage defects better when compounded with fibrin gel.
Objective To investigate the possibility of repairing articular cartilage defects with the mesenchymal stem cells(MSCs) seeded type Ⅰ collagen-glycosaminoglycan(CG) matrices after being cultured with the chondrogenic differentiation medium. Methods The adherent population of MSCs from bone marrow of10 adult dogs were expanded in number to the 3rd passage. MSCs were seeded intothe dehydrothermal treatment (DHT) crosslinked CG matrices; 2×106 cells per 9mm diameter samples were taken. Chondrogenic differentiation was achieved by the induction media for 3 weeks. Cell contractility was evaluated by the measuement of the cell-mediated contraction of the CG matrices with time inculture.The in vitro formation of the cartilage was assessed by an assayemploying immunohistochemical identification of type Ⅱ collagen and by immunohistochemistry to demonstrate smooth muscle actin （SMA）. The cells seededingCGs wereimplanted into cartilage defectsof canine knee joints. Twelve weeks after surgery, the dogs were sacrificed and results were observed. Results There was significant contraction of the MSCsseeded DHT crosslinked CG scaffolds cultured in the cartilage induction medium. After 21 days, the MSCseeded DHT crosslinked matrices were contracted to 64.4%±0.3%; histologically, the pores were found to be compressedandthe contraction coupled with the newly synthesized matrix, transforming the MSCsseeded CG matrix into a solid tissue in most areas. The type Ⅱ collagen staining was positive. The SMA staining was positive when these MSCs were seeded and the contracted CGs were implanted into the cartilage defects of the canine knee joints to repair the cartilage defects. The function of the knee joints recovered and the solid cartilaginous tissue filled the cartilage defects. Conclusion The results demonstrates that MSCs grown in the CG matrices can produce a solid cartilaginous tissuecontaining type Ⅱ collagen after being cultured with the chondrogenic differentiation medium and implanted into cartilage defects. We hypothesize that the following steps can be performed in the chondrogenic process: ①MSCs express SMA, resulting in matrix contraction, thus achieving a required cell density (allowing the cells to operate in a necessary society)； ②Cells interact to form a type Ⅱ collagencontaining extracellular matrix (and cartilaginous tissue)； ③Other factors, suchas an applied mechanical stress, may be required to form a mature cartilage with the normal architecture.