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.
ObjectiveTo explore the effects of cryopreservation on the cell survival rate, cell viability, early apoptosis, migration ability, and tendon-related marker expression of tendon-derived stem cells (TDSCs) in rat patellar tendons.MethodsThe patellar tendon tissues were harvested from 12 4-month-old male Sprague Dawley rats; 12 patellar tendon tissues from 6 rats were cryopreserved (the experimental group), and the other 12 patellar tendon tissues were not treated (the control group). The patellar tendons were digested with 0.3% type I collagenase to obtain nucleated cells. The survival rate of nucleated cells was detected by trypan blue exclusion assay, and colony-forming ability by crystal violet staining. TDSCs were isolated and cultured to passage 3 (P3). The cell viability of TDSCs was detected by Alamar Blue method, the early apoptosis by Annexin V-FITC/PI assay, the cell migration ability by Transwell method, and the mRNA expressions of tendon-related markers [collagen type I (Col1α1), scleraxis (Scx), and tenomodulin (Tnmd)] by real-time quantitative PCR.ResultsThe survival rate of nucleated cells was 91.00%±3.63% in the control group, and was 61.65%±4.76% in the experimental group, showing significant difference (t=12.010, P=0.000). The formation of the primary nucleated cell clones was observed in 2 groups. At 12 days, the number of colonies forming of the experimental group [(8.41±0.33)/1 000 nucleated cells] was significantly lower than that of the control group [(15.19±0.47)/1 000 nucleated cells] (t=28.910, P=0.000). The percentage of TDSCs in the active nucleated cells in the experimental group (1.37%±0.09%) was significantly lower than that in the control group (1.67%±0.10%) (t=5.508, P=0.003). The growth trend of TDSCs (P3) in the 2 groups was consistent within 14 days. There was no significant difference in absorbance (A) value between 2 groups at each time point (P>0.05). The early apoptotic rate of TDSCs was 1.67%±0.06% in the experimental group and was 1.63%±0.06% in the control group, showing no significant difference (t=0.707, P=0.519). Under microscope, TDSCs adhered to the lower chamber of the Transwell chamber; the number of cells was 445.00±9.70 in the experimental group and was 451.50±12.66 in the control group, showing no significant difference (t=0.998, P=0.342). The relative mRNA expressions of Col1α1, Scx, and Tnmd were 3.498±0.065, 0.062±0.002, and (4.211±0.211)×10–5 in the experimental group and were 3.499±0.113, 0.062±0.001, and (4.341±0.274)×10–5 in the con-trol group, showing no significant difference (t=0.013, P=0.991; t=0.042, P=0.969; t=0.653, P=0.549).ConclusionThe survival rate of nucleated cells in cryopreserved rat tendon tissues is lower, but a large number of active TDSCs, and its cell viability, early apoptosis rate, migration ability in vitro, and cell tenogenic differentiation ability are remained.
Objective To study the vascularization of the compositeof bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. Methods Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly(n=15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), AdLacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvacular density counting(MVD) were made 4, 8, and 12 weeks after operation. Results In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (Plt;0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defectedarea was filled with fibrous tissue. Conclusion BMP-2 gene therapy, by -upregulating VEGF expression, indirectly induces vascularization ofgrafts,promotes the living of seed cells, and thus accelerates new bone formation.
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 study the differenation of adult marrow mesenchymal stem cells(MSCs) into vascular endothelial cells in vitro and to explore inducing conditions. Methods MSCs were isolated from adult marrow mononuclear cells by attaching growth. MSCs were divided into 4 groups to induce: the cells seeded at a density of 5×103/cm2 in 2% and 15% FCS LDMEM respectively (group1 and group 2)， at a density of 5×104/cm2 in 2% and 15% FCS LDMEM respectively (group 3 and group 4); vascular endothelial growth factor(VEGF) supplemented with Bovine pituitary extract was used to induce the cell differentiation. The differentiated cells were identified by measuring surfacemarks (CD34, VEGFR2, CD31 and vWF ) on the 14th day and 21st day and performed angiogenesis in vitroon the 21st day.The cell proliferation index（PI）of different inducing conditions were measured. Results After induced in VEGF supplemented with Bovine pituitary extract, the cells of group 3 expressed the surface marks CD34, VEGFR-2, CD31 and vWF on the 14th day, the positive rates were 8.5%, 12.0%, 40.0% and 30.0% respectively, and on the 21st day the positive ratesof CD34 and VEGFR2 increased to 15.5％ and 20.0％, while the other groups did not express these marks; the induced cells of group 3 showed low proliferating state(PI was 10.4%) and formed capillary-like structure in semisolid medium. Conclusion Adult MSCs can differentiate into vascular endothelial cellsafter induced by VEGF and Bovine pituitary extract at high cell densities and low proliferatingconditions，suggesting that adult MSCs will be ideal seed cells forthe therapeutic neovascularization and tissue engineering.
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.
ARDS 是引起重症患者呼吸衰竭的主要原因, 尽管医疗技术有了很大的进步, 但对ARDS 的治疗只局限在器官支持层面, 其病死率仍高达40% [ 1] 。ARDS的主要病理改变为肺泡上皮细胞和毛细血管内皮细胞受损, 通透性增加, 富含蛋白质的液体渗出积聚于肺间质和肺泡。因此促进损伤肺毛细血管内皮细胞和肺泡上皮细胞的有效修复可能是ARDS治疗的关键所在。随着干细胞工程学的发展, 间充质干细胞( MSC) 作为一种理想的组织修复来源, 在ARDS 治疗中的应用受到越来越多的关注, 这可能为ARDS 的治疗开辟一条新的途径。
Objective To investigate the possibility of establishing the human bone marrow mesenchymal stem cells (hMSCs) bank as to provide an alternative source for the seed cells of tissue engineering. Methods The cell surface antigensof the purified, expanded hMSCs and the ones following cryopreservation were detected by flow cytometry, cultured in a special medium to induce the ostoegenic and chondrocytic- differentiation. Morphology was studied by light and electronic microscopes. The detection of alkaline phosphatase, collagen type Ⅰ, osteocalcin, and collagen type Ⅱ were also performed by immunochemistry and molecular biology.Results The phenotype and expansion possibility of hMSCs after cryopreservation were remained. It could expand for 10 generations. The doubling time was 40 h.Conclusion The bank of hMSCs is inipiently established and can provide eligible seed cells for tissue engineering.
Objective To study the integration of rat marrow stromal stem cells (MSCs) after transplantation into acellular extracellular matrix (AECM). Methods We got 16 femurs from 8 Kunming rats, the femurs were treated by Triton X100 toget AECM, MSCs were collected from femoral marrow of 20 Kunming rats about a mouth old by PBS 4ml, centrifugalized and primary cultured in bottles,then therat MSCs were transplanted into AECM at a concentration of 5×106/ml and culturedfor 7 days. The integration of the donor cells was observed using one phase contrast microscope, a light microscope and a scanning electron microscope (SEM).Results In AECM bone lacunas there were MSCs nucleuses stained blue. The nucleuses were unevenly distributed in AECM with more in the peripheral AECM than in the central AECM and with more in the layer anear culture medium than in the layer far away from culture medium.AECM possessed a good spatial scaffold structure, the marrow stromal stem cells were well integrated into AECM.Conclusion AECM can be usedas a good scaffold material for tissue engineered bone construction.