Effect And Mechanism Of Extracellular Matrix In The Treatment Of Osteoporosis

Part I Culturing on decellularized extracellular matrix enhances antioxidant properties of human mesenchymal stem cellsObjective:Our previous studies showed that decellularized cell-deposited extracellular matrix（ECM）provided an in vivo-mimicking microenvironment for mesenchymal stem cells（MSCs）and facilitated in vitro cell expansion.This study was conducted to analyze the cellular response of MSCs when culturing on the ECM,including reactive oxygen species（ROS）,intracellular antioxidative enzymes,and the resistance to exogenous oxidative stress.Methods:To prepare decellularized ECM deposited by MSCs,after MSCs reaching 90%confluence,100μM L-ascorbic acid was added for an additional culture period of eight days.Then cultured cells were incubated in extraction buffer[0.5%Triton X-100 and 20 mM NH₄OH in phosphate buffered saline（PBS）,pH=7.4].Decellularized ECM was stored under sterile conditionsat4°C.The morphology of decellularized ECM was analyzed by a scanning electron microscope.The biochemical composition of decellularized ECM was analyzed by Immunofluorescence staining.To investigate how culturing on ECM affected the proliferative potential of MSCs,we conducted DNA assays and FDA staining.We used flow cytometry to analyze the cell cycle distribution.Flow cytometry analysis of DCF fluorescence intensity showed intracellular ROS accumulation.Intracellular levels of H₂O₂ were detected with the Amplex?Red Hydrogen Peroxide Assay Kit（Invitrogen）according to the manufacturer’s instructions.Total SOD activity was analyzed by the SOD assay kit（Beyotime）according to the manufacturer’s instructions.Catalase activity was measured by a commercially available assay kit（Sigma-Aldrich）according to the manufacturer’s instructions.To investigate the protective effects of culturing on ECM on MSC osteogenesis,exogenous 100μM H₂O₂ was added to the osteogenic differentiation medium to create an oxidative microenvironment.Mineralization of the matrix was determined by Alizarin Red Staining.To quantify the calcified matrix,1%hydrochloric acid（Sigma-Aldrich）was added.RT-PCR and Western blot were conducted to assay gene and protein expression.Results:After decellularization,the architecture of cell-deposited ECM was characterized as nanofibrous,collagen fibrils and the matrix components were identified as type I and III collagens,fibronectin,and laminin.Compared to tissue culture polystyrene（TCPS）plates,culturing on ECM yielded a 2-fold increase of MSC proliferation and improved the percentage of cells in the S phase by2.4-fold.The levels of intracellular ROS and hydrogen peroxide（H₂O₂）in ECM-cultured cells were reduced by 41.7%and 82.9%,respectively.More importantly,ECM-cultured MSCs showed enhanced expression and activity of intracellular antioxidative enzymes such as superoxide dismutase and catalase,up-regulated expression of silent information regulator type 1,and suppressed phosphorylation of p38mitogen-activated protein kinase.Furthermore,a continuous treatment with exogenous 100μM H₂O₂ dramatically inhibited osteogenic differentiation of MSCs cultured on TCPS,but culturing on ECM retained the differentiation capacity formatrix mineralization and osteoblast-specific marker gene expression.Conclusion:We conclude that the cellular response of MSCs cultured on decellularized cell-deposited ECM showed attenuated intracellular ROS,enhanced antioxidative enzymes,and improved resistance to exogenous oxidative stress.Decellularized ECM can be an effective in vitro expansion system to produce sufficient amounts of MSCs and can potentially be an antioxidative treatment to prevent oxidative damage in transplanted MSCs.Future work will focus on identifying bioactive components,matrix mechanical properties of decellularized ECM,and investigating the underlying mechanism of the role of SIRT1 in ECM-cultured MSCPart II Spontaneous up-regulation of SIRT1 during osteogenic induction contributes to stem cells’ resistance to oxidative stress via antioxidant mechanismsObjective : Osteogenic differentiation of bone marrow-derived mesenchymal stem cells（BM-MSCs）is the central event in bone formation;however,accumulating data have suggested that oxidative stress has a deleterious impact on BM-MSC osteogenesis.In this study,we hypothesized that oxidative stress influenced BM-MSC osteogenesis differently in the early or late stages,in which silent information regulator type 1（SIRT1）played a critical role via antioxidant mechanisms.Methods:To evaluate the effects of H₂O₂ on cell viability of BM-MSCs,cells were exposed to different dosages of H₂O₂ ranging from 25 to 400 μM for 72 h.Cell viability was qualitatively observed by an FDA staining assay,quantitatively observed by a cell counting kit-8（CCK-8;Beyotime）.Flow cytometry analysis of DCF-DA fluorescence intensity showed intracellular ROS level.To evaluate the influence of H₂O₂ on the stages of osteogenic differentiation,BM-MSCs were treated with 25 μM,50 μM,and 100 μM H₂O₂ for 21 days.Untreated cells served as a control.To investigate the role of SIRT1 in the early stage of osteogenesis,BM-MSCs were treated with 5 μM Res V in the presence of 100 μM H₂O₂ for 7 days and subjected to the subsequent experiments.To investigate the role of SIRT1 in the late stage of osteogenesis,BM-MSCs were treated with 10 m M NAM in the presence of 50 μM H₂O₂ for the last 7 days and subjected to the subsequent experiments.ALP staining and activity measurement were conducted to evaluate early osteogenesis.Alizarin Red S staining and quantitation of matrix mineralization were conducted to evaluate late osteogenesis.RT-PCR and Western blot were conducted to assay gene and protein expression.Results:A continuous exposureto sublethal concentrations of hydrogen peroxide（H₂O₂）,ranging from 25μM to 100 μM for 21 days,resulted in the complete inhibition of BM-MSC osteogenesis.We found that a 7-day treatment with H₂O₂ retarded lineage commitment of BM-MSCs towards osteoblasts,as evidenced by a significant reduction of alkaline phosphatase activity（a typical marker for early osteogenesis）.However,moderate oxidative stress did not affect latedifferentiated BM-MSCs,as there were comparable levels of matrix mineralization（a typical marker for late osteogenesis）and of calcification-related gene expression.In addition,we observed a spontaneous up-regulation of SIRT1 and intracellular antioxidant enzymes such as superoxide dismutase 2,catalase,and glutathione peroxidase 1,which accounted for the enhanced resistance to oxidative stress upon osteogenic differentiation.Activation of SIRT1 by resveratrol ameliorated the effect of H₂O₂ on early-differentiated BM-MSCs and inhibition of SIRT1 by nicotinamide aggravated the effect of H₂O₂ on late-differentiated BM-MSCs,indicating that the SIRT1-mediated pathway was actively involved in MSCs’ osteogenic differentiation and antioxidant mechanisms.Conclusion : we demonstrated that exogenous H₂O₂-induced oxidative stress inhibited the osteogenic lineage commitment of BM-MSCs,but late-differentiated BM-MSCs showed an enhanced resistance to oxidative stress.Our findings uncovered a critical role of SIRT1 in regulating the spontaneous enhancement of the antioxidant defense system and resistance to oxidative stress in BM-MSCs upon osteogenic induction.Understanding the relationship between SIRT1 and antioxidant enzymes during osteogenic differentiation will provide a new strategy that could potentially protect MSCs from extracellular oxidative stress and facilitate the clinical applications of MSCs in bone tissue engineering.Part III Culturing on decellularized extracellular matrix reduce reactive oxygen species in monocyte and inhibit osteoclastic differentiationObjective:This study was conducted to analyze the cellular response of monocyte when culturing on the ECM,during osteoclasts induction and the potential mechanism.Methods : Modified adherence filtration was use to obtain C57BL/6 mouse bone marrow monocytes.The cell surface antigens were marked by CD11 b fluorescent-antibody and detected by flow cytometry.To evaluate how culturing on ECM affected the proliferative potential of monocytes,microscope observing and DNA assays were conducted.The morphology of monocytes cultured on ECM was analyzed by a scanning electron microscope.To investigate the effects of ECM on osteoclast differentiation,monocytes were seeded on TCPS and ECM.Osteoclasts induction were conducted by M-CSF and RANKL.Osteoclast differentiation was evaluated by TRAP staining,F-actin ring staining and RT-PCR.To investigate if ECM inhibit osteoclast differentiation through reduce ROS in monocyte,flow cytometry analysis of DCF fluorescence intensity was used to show intracellular ROS accumulation,TRAP staining and RT-PCR were used to show osteoclast differentiation.To investigate if ECM inhibit osteoclast differentiation through blocking NF-κB pathway,Immunofluorescence staining was conducted to trace p65,Western blot was conducted to show p65 and inhibitory κB protein（IκB）protein levels.To investigate the effects of protein composition of ECM on osteoclast differentiation,COLI and FN were coated on TCPS,then Osteoclasts induction were conducted on them.Osteoclast differentiation was evaluated by TRAP staining,F-actin ring staining and RT-PCR.Results: Flow cytometry showed CD11 b positive rates was 96%.Compared to TCPS,culturing on ECM yielded increasing of monocyte proliferation on day 5 and 7.After 5 days Osteoclasts induction,culturing on TCPS showed a lager TRAP positive cell number and F-actin ring area.Flow cytometry analysis of DCF-DA fluorescence intensity showed less intracellular ROS level when culturing on ECM.After add exogenous H₂O₂,ROS level did not show significant different between monocytes culturing on ECM and TCPS.More TRAP positive cells were observed when culturing on ECM with H₂O₂ compared to that without H₂O₂,but still less that culturing on TCPS.Immunofluorescence staining showed p65 transferred from cytoplasm to nucleus in cells culturing on TCPS.Cells culturing on ECM showed less p65 and IκB protein.After add exogenous H₂O₂,more p65 transferred from cytoplasm to nucleus in cells culturing on ECM,but less than cells culturing on TCPS.Exogenous H₂O₂ enhanced p65 and IκB protein level n cells culturing on ECM.After Osteoclasts induction on COLI,FN and ECM,cells culturing on FN and ECM showed less TRAP positive level,smaller F-actin ring area and less osteoclast gene expression.ECM showed more osteoclast inhibition than FN.Conclusion : Culturing on ECM reduce TRAP positive cell number,decrease F-actin ring formation,down-regulate osteoclast gene expression through reducing intracellular ROS level and blocking NF-κB pathway.FN,the main protein composition of ECM,also has a inhibit function in osteoclast differentiation.