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 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.
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.
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.
ObjectiveTo observe the effects of exosomes derived from rat mesenchymal stem cells (MSC-exosomes) on the rat experimental autoimmune uveitis (EAU) model.MethodsTwelve Lewis rats were randomly divided into experimental group and control group by random number table, with 6 rats in each group. Rats in the experimental group were established with EAU model, 100 μl of MSC-exosomes (50 μg) were periocular injected on the 9th day after modeling while the control rats were injected with the same volume of phosphate buffer. At different time points after modeling, the retinal structure was observed by hematoxylin and eosin (HE) staining, and the clinical and pathological manifestations were evaluated. T cells from the two groups were analyzed by flow cytometry. Immunohistochemical staining was used to observe the expression of macrophage surface marker CD68. The effect of MSC-exosomes on T cells was measured by lymphocyte proliferation assays. And flow cytometry was used to detect Th1, Th17 and regulatory T cells Variety. Electroretinogram (ERG) was used to evaluate the retinal function. Data were compared between the two groups using the t test.ResultsHE staining showed that the retina structure of the experimental group was more complete than that of the control group on the 15th day after modeling. Immunohistochemical staining showed that the positive expression of CD68 in the experimental group was significantly less than that in the control group. On the 15th day after modeling, the retinal pathological score of the experimental group was lower than that of the control group. On the 9th to 13th day after modeling, compared to the control group, the average clinical scores of the retina in the experimental group were lower, and the difference was statistically significant (t=3.665, 3.21, 3.181, 4.121, 3.227; P＜0.01). The results of T cell proliferation assay showed that exosomes (1.0, 10.0 μg/ml) inhibited the proliferation of T cells under different concentrations of R16 (1, 10, 30 μg/ml), and the difference was statistically significant (F=11.630, 4.188, 6.011; P＜0.05). The results of flow cytometry showed that the number of Th1, Th17 and Treg cell subsets in the experimental group was decreased compared with the control group, and the difference was statistically significant (t=7.374, 4.525, 6.910; P＜0.01). There was no difference in the proportion of cells in the T cells and lymph nodes (t=1.126, 0.493, 0.178; P=0.286, 0.632, 0.862). The results of ERG showed that, compared with the control group, the amplitudes of 0.01, 3.0 cd/m2 a wave and b wave of the experiment group were all increased on the 15th day after modeling, and the differences were statistically significant (t=3.604, 4.178, 4.551, 2.566, P＜0.05).ConclusionsMSC-exosomes can reduce the clinical and pathological manifestations of EAU, protect retinal function, reduce ocular macrophage infiltration, down-regulate the proportion of inflammatory cells in the eye, and inhibit T cell proliferation.
Objective To observe the therapeutic effect of mensenchymal stem cells (MSCs) for experimental autoimmune uveitis (EAU). Methods MSCs were obtained from Wistar rats and selected by plastic adherence. Lewis rats were divided into treatment group and control group, six rats in each group. EAU models were induced by immunization with an emulsion (0.2 ml) containing 30 mu;g interphotoreceptor retinoid-binding protein derived peptide R16 and complete Freundprime;s adjuvant. The clinical manifestations of two groups were observed. Nine to 11 day after modeling, 1 ml MSCs suspension, which contained 5times;106 MSCs, were injected into the rats in treatment group via tail vein, and the rats in control group were given equal volume of phosphate buffer solution. Fifteen day after modeling, the eyes were collected to test the proportion of interferon gamma;, interleukin-17 and Foxp3 positive cells by flow cytometry. The clinical scores were analyzed by mixed linear model and statistical analysis of variance of repeated measurement data. The results of flow cytometry were analyzed using independent-sample t test. Results Six days after immunization, mild dilatation and congestion of iris vascular was observed. Nine days after immunization, mild muddy anterior chamber, myosis and absent pupillary reaction to light were observed. Twelve days after immunization, muddy anterior chamber, occlusion of pupil and dimmed or disappeared red reflex were observed, and then inflammation was slowly reduced. From 11 to 15 days after immunization, the clinical score of treatment group was lower than that in control group, the difference was statistically significant (t=2.42, 2.21, 4.16, 5.24, 4.03; P＜0.05). The results of flow cytometry showed that MSCs treatment could decrease the proportion of CD4＋T cells, Th1 cells and Th17 cells, increase the proportion of Treg cells. Conclusion MSCs treatment can ameliorate EAU, up-regulate the expression of Treg cells and down-regulate the expression of CD4＋T cells, Th1 cells and Th17 cells.
Objective To study the influence of three different ways of myogenic induction on Ca2+ regulation of mesenchymal stem cells (MSCs) derived from umbilical cord blood. Methods From January 2007 to April 2010, three different ways of myogenic induction including the adoptions of 5azacytidine, extraction of myocardium, and myocardial differentiation medium were used to induce MSCs derived from the umbilical cord blood of dogs in Xinhua Hospital of Shanghai Jiaotong University. Confocal laser scanning microscope was used to detect cells induced by the three abovementioned methods, cardiomyocytes and Ca2+ combined with Fluo3/AM inside the MSCs. For each group of cells, 2 to 5 visual fields were chosen, and 30 visual fields were recorded for each kind of cells. The mean fluorescence intensity of ten images shot in one minute was used to reflect the concentration of free intracellular Ca2+. Furthermore, the change of the concentration was continuously monitored by optical density(OD) value. Results After induction, the Ca2+ concentration inside the MSCs was significantly higher than that inside the cardiomyocytes (F=59.400, P=0.000). There was a statistical difference among the intracellular Ca2+ concentration induced respectively by 5azacytidine, extraction of myocardium, and myocardial differentiation medium (F=18.988, P=0.000). No significant difference existed between the intracellular Ca2+ concentration induced by 5-azacytidine and extraction of myocardium (OD value: 1 076.88±44.65 vs. 1 040.90±37.48, P=0.186), while the intracellular Ca2+ concentration induced by 5azacytidine was significantly higher than that induced by myocardial differentiation medium (OD value: 1 076.88±44.65 vs. 973.91±46.49, P=0.001), and the intracellular Ca2+ concentration induced by extraction of myocardium was significantly higher than that induced by myocardial differentiation medium (OD value: 1 040.90±37.48 vs. 973.91±46.49, P=0.001). The concentration of intracellular Ca2+ induced by all the three different methods fluctuated spontaneously, which was quite similar with the cardiomyocytes, but the frequency and the scope of the fluctuation were quite different. Ca2+ was released instantly by KCl stimulation in the two groups of MSCs pretreated by 5-aza and extraction of myocardium. Though MSCs pretreated by myocardial differentiation medium had response to KCl stimulation, Ca2+ could not be released in this group. On the contrary, the duration of Ca2+ release was prolonged. Conclusion Ca2+ regulation system of MSCs derived from umbilical cord blood can be influenced by these myogenic inductions. However, the reason and effect of the differences need to be elucidated by further investigation.
Objective To review the recent progress of the researches in construction of tissue engineered osteochondral composites, and to discuss the challenges in construction of tissue engineered osteochondral composites. Methods The recent literature on the construction of tissue engineered osteochondral composites was extensively reviewed and analyzed. Results The studies on the construction of tissue engineered osteochondral composites are relatively more in vivo, the current focus is that different tissues derived mesenchymal stem cells are widely used to be seed cells; single-phase scaffold has been limited, studies on biphase scaffold and triphase scaffold are new trends; the design and performance of bioreactor need to be further optimized in the future. Conclusion The construction of tissue engineered osteochondral composites will be a promising method for the treatment of cartilage defects.
ObjectiveTo review the research progress of the co-culture system for constructing vascularized tissue engineered bone. MethodsThe recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. ResultsThe construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. ConclusionThe strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.
Objective To review the research progress of the seed cells, scaffolds, growth factors, and the prospects for clinical application of the intervertebral disc regeneration. Methods The recent literature concerning the regeneration strategies and tissue engineering for treatment of degenerative intervertebral disc disease was extensively reviewed and summarized. Results Seed cells based on mesenchymal stem cells (MSCs) and multiple-designed biomimetic scaffolds are the hot topic in the field of intervertebral disc regeneration. It needs to be further investigated how to effectively combine the interactions of seed cells, scaffolds, and growth factors and to play their regulation function. Conclusion The biological regeneration of intervertebral disc would have a very broad prospects for clinical application in future.