| PartⅠTransforming growth factor-betal inhibits tissue engineering cartilage absorption via inducing the generation of regulatory T cellsObjective:The objective of the present study was to explore the mechanisms of transforming growth factor-betal (TGF-β1) inhibiting tissue engineering cartilage absorption.Methods:We transfected TGF-β1 gene into bone marrow mesenchymal stem cells (BMMSCs) and co-cultured with IFN-γ and TNF-α and CD4+T lymphocytes and then characterized the morphological changes, apoptosis and characterization of chondrogenic-committed cells from TGF-β1+ BMMSCs and explored the mechanisms.Results:Morphology Changes of BMMSCsUsing fluorescent microscopy, CM-DiI-positive cells showed orange-red, and intensive fluorescent particles was observed within the CM-DiI-positive cells. Nuclei showed rounded dark field. The morphology of cells in group A and control group moved gradually from slender spindle to roundness or polygon from day 3. On the 7th day, about half of the cells were in mitosis phase, showing larger sizes, polygon and larger nuclei. In group C, the morphology of cells showed a similar change as well. However, this was not so in group B cells to which IFN-y and TNF-a were added. Compared with other groups, group B showed sparse and small sizes cells, and hardly any cells was in mitosis.DNA Fragmentation in TGF-β1+ BMMSCsTUNEL assay with fluorescent microscopy was undertaken to determine the effect of proinflammatory cytokine and CD4+T lymphocytes on DNA fragmentation in BMMSCs. Compared with group A, IFN-y and TNF-a caused a significant increase in the number of TUNEL-positive cells in group B (p<0.01). The increase was concentration dependent and most obvious in 200 ng/ml (p<0.01). In group C, CD4+T lymphocytes (and TGF-β1) relieved the increase of TUNEL-positive cells induced by IFN-γ and TNF-α.Expression Levels of Treg Cells MarkersTo determine the function of CD4+T lymphocytes in group C, we analyzed Foxp3 by RT-PCR. Foxp3 is the intracellular molecular marker of Treg cells. Foxp3 was almost not expressed in group A or B. The Foxp3 expression of group C was significantly higher than the other two groups. CD25 and CD39 expressions of cells were examined by flow cytometry. As shown in group C,15.1±3.6% of cells were CD25+CD39+. We observed a significantly higher CD25+CD39- expression of group C cells relative to other groups. These expressions gradually increased starting from day 3 of adding CD4+T lymphocytes till day 7.Characterization of Chondrogenic-committed Cells from TGF-β1+ BMMSCsImmunostaining indicated that the chondrocyte-stimulated cells expressed type Ⅱ collagen. Compared with group A and C, group B had fewer type ⅡⅡⅠ collagen-positive cells. Western blot analysis indicated that the expression levels of type Ⅱ collagen in group B were significantly lower than those in group A and C, respectively. The difference was concentration dependent (200 ng/ml most obviously, p<0.01). There was no significant difference between group A and C.Conclusions:The data suggest that IFN-γand TNF-α induced BMMSCs apoptosis and tissue engineering cartilage absorption, but the newborn regulatory T (Treg) cells inhibited the function of IFN-γ and TNF-α and defended BMMSCs and tissue engineering cartilage finally. TGF-(31 has not only played an cartilage inductive role, but also inhibited the absorption of tissue engineering cartilage. In addition, the pathway put forward in our study may simulate the real reaction procedure after the implantation of BMMSCs and tissue engineering cartilage in vivo.Part ⅡTransforming growth factor-betal-induced Treg cells inhibit the absorption of tissue-engineered cartilage caused by endogenous IFN-γ and TNF-αObjective:In a previous study, we showed that interferon (IFN)-γ and tumor necrosis factor (TNF)-α induce BMMSCs apoptosis and absorption of tissue-engineered cartilage, but the induced regulatory T (iTreg) cells inhibit the function of IFN-y and TNF-α. In this study, we investigated the effect of iTreg cells on the absorption of tissue-engineered cartilage caused by endogenous IFN-γ and TNF-α.Methods:We transfected the TGF-(31 gene into BMMSCs co-cultured with CD4+T lymphocytes. Then, we assessed the expression of iTreg cell markers (Foxp3, CD25 and CD39) and IFN-γ and TNF-α and the level of apoptosis of BMMSCs. In addition, we characterized chondrogenic-committed cells from TGF-β1+ BMMSCs and explored the role of iTreg cells.Results:Expression of iTreg cell markers, IFN-y and TNF-aTo determine whether the CD4+T lymphocytes convert into iTreg cells, we analyzed Treg cell markers Foxp3 and CD25 and CD39 (tested by RT-PCR and flow cytometry, respectively). In group B, in which TGF-β1+ BMMSCs were co-cultured with CD4+T cells (1×106 CD4+T lymphocytes), we detected gene expression of Foxp3 and CD25+CD39+ cells (17.58±0.45%). Foxp3 gene, and CD25+CD39+ cells were nearly undetectable in the groups.To determine the function of the CD4+T lymphocytes, we analyzed gene expression and protein production of IFN-y and TNF-a by RT-PCR and Western blot. We detected gene expression and protein production of IFN-y and TNF-a in group B and control B, in which co-cultured with CD4+T cells. There were no significant differences in IFN-y and TNF-a expression between group B and control B. IFN-y and TNF-a were nearly undetectable in the groups without CD4+T lymphocytes (group A and control A).DNA Fragmentation in TGF-β1+ BMMSCsA TUNEL assay together with fluorescence microscopy was undertaken to determine the effect of proinflammatory cytokines on DNA fragmentation in BMMSCs. In group B and control B, which included CD4+T lymphocytes (1×104, 1×105 or 1×106), the amount of BMMSCs apoptosis observed was greater than in group A and control A, without CD4+T lymphocytes (p<0.01), and the highest level of apoptosis was detected in control B (p<0.05, compared with group B). However, BMMSCs apoptosis did not increase with the number of different CD4+T lymphocytes. In TGF-β1+ BMMSCs and TGF-β1- BMMSCs, the levels of apoptosis were approximately 1.5 and 3 times higher, respectively, than in groups without CD4+T lymphocytes.Characterization of Chondrogenic-committed Cells from TGF-β1+ BMMSCsImmunostaining indicated that the chondrocyte-stimulated cells expressed type Ⅱ collagen. During the experiment, TGF-β1- BMMSCs (group control A and control B) were not induced to differentiate intochondrocytes. Compared with group A, group B presented fewer type Ⅱ collagen-positive cells (p<0.05) on day 7. However, in group B, in which 1×104, 1×105 or 1×106 CD4+T lymphocytes were added, chondrocytes were still detectable. Western blot analysis indicated that the expression levels of type Ⅱ collagen in group B were significantly lower than in group A (p<0.05). Type II collagen was not detected at any time in controls A and B.Conclusions:CD4+T cells led to the absorption of tissue-engineered cartilage through the secretion of endogenous IFN-y and TNF-a, whose inflammatory functions were concomitantly suppressed by iTreg cells converted from CD4+T cells. This study is clinically relevant and adds to our understanding of the mechanism of tissue-engineered cartilage absorption.Part ⅢApplication of TGF-β1 promoting Bio-Oss osteogenesisObjective:To explore the methods of inhibiting of inflammation around the Bio-Oss, promoting osteogenesis in vivo, providing references for improving the clinical success rate of dental implants.Methods:BMMSCs were cultured in medium with Bio-Oss, and biocompatibility was determined by distribution of cells and the material and the effection of proliferation and differentiation of BMMSCs caused by Bio-Oss. Rabbit alveolar bone defect was modeled and grouping repaired, changing the microenvironment around Bio-Oss and exploring Bio-Oss osteogenesis in vivo.Results:The cells was ordered along the long axis of the body and attached to the surface of Bio-Oss uniformly. There were not morphological differences comparing with the control group. Bio-Oss played no impact on proliferation and ALP activity of TGF-β1+ BMMSCs.30,60,90 days after alveolar bone defect repairing, Bio-Oss scattering fused with the surrounding bone tissue gradually. Bio-Oss osteogenesis was lagged when the pro-inflammatory cytokines IFN-y and TNF-a were involved. Osteogenesis was not dramatically different from the group involved in TGF-β1+ MMSCs or not.Conclusions:TGF-β1+ BMMSCs have good biocompatibility with Bio-Oss. The dual role of guided bone regeneration and controled local inflammation by TGF-β1+ BMMSCs in vivo is not clear. |
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