Chen Zong

Different effects of intermittent and continuous fluid shear stresses on osteogenic differentiation of human mesenchymal stem cells

A reasonable mechanical microenvironment similar to the bone microenvironment in vivo is critical to the formation of engineering bone tissues. As fluid shear stress (FSS) produced by perfusion culture system can lead to the osteogenic differentiation of human mesenchymal stem cells (hMSCs), it is widely used in studies of bone tissue engineering. However, effects of FSS on the differentiation of hMSCs largely depend on the FSS application manner. It is interesting how different FSS application manners influence the differentiation of hMSCs. In this study, we examined the effects of intermittent FSS and continuous FSS on the osteogenic differentiation of hMSCs. The phosphorylation level of ERK1/2 and FAK is measured to investigate the effects of different FSS application manners on the activation of signaling molecules. The results showed that intermittent FSS could promote the osteogenic differentiation of hMSCs. The expression level of osteogenic genes and the alkaline phosphatase (ALP) activity in cells under intermittent FSS application were significantly higher than those in cells under continuous FSS application. Moreover, intermittent FSS up-regulated the activity of ERK1/2 and FAK. Our study demonstrated that intermittent FSS is more effective to induce the osteogenic differentiation of hMSCs than continuous FSS.

The interaction between β1 integrins and ERK1/2 in osteogenic differentiation of human mesenchymal stem cells under fluid shear stress modelled by a perfusion system

Fluid shear stress (FSS) is an important biomechanical factor regulating the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and is therefore widely used in bone tissue engineering. However, the mechanotransduction of FSS in hMSCs remains largely unknown. As β1 integrins are considered to be important mechanoreceptors in other cells, we suspect that β1 integrins should also be important for hMSCs to sense the stimulation of FSS. We used a perfusion culture system to produce FSS loading on hMSCs seeded in PLGA three‐dimensional (3D) scaffolds and investigated the roles of β1 integrins, FAK and ERK1/2 in FSS‐induced osteogenic differentiation of hMSCs. Our results showed that FSS not only markedly increased ALP activity and the expression of ALP, OCN, Runx2 and COLIα genes but also significantly enhanced the phosphorylation of ERK1/2, Runx2 and FAK. FSS‐induced activation of ERK1/2 and FAK was inhibited by blockade of the connection between β1 integrins and ECM with RGDS peptide and integrins β1 monoclonal antibody. Our study also found that FSS could upregulate the expression level of β1 integrins and that this upregulation could be abolished by PD98059. Further investigation indicated that FSS‐activated ERK1/2 led to the phosphorylation of IκBα and NFκB p65. The activation of NFκB p65 resulted in the upregulation of β1 integrin expression. Therefore, it could be inferred that β1 integrins should sense the stimulation of FSS and thus activate ERK1/2 through activating of FAK, and FSS‐activated ERK1/2 feedback to upregulate the expression of β1 integrins through activating NFκB. Copyright

Extracellular signal-regulated kinase1/2 activated by fluid shear stress promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells through novel signaling pathways.

It is a classical signaling pathway that the activation of extracellular signal-regulated kinase1/2 (ERK1/2) results in the phosphorylation of runt-related transcription factor 2 (Runx2) and thereby initiates the transcription of osteogenic genes. Recently, it is found that the activation of ERK1/2 resulted from fluid shear stress (FSS) also increased the expression of Runx2 and β1 integrins, and finally enhanced osteogenic differentiation. However, it has been remained largely unknown how ERK1/2 regulates the expression of Runx2 and β1 integrins. We use the perfusion culture system to produce FSS exerting on human bone marrow-derived mesenchymal stem cells (hMSCs) and thus activate ERK1/2. Our study demonstrated that FSS-activated ERK1/2 mediated the expression of osteogenic genes via two novel signaling pathways except for the classical signaling pathway: feedback up-regulation of β1 integrins expression via activating nuclear factor kappa B (NF-κB), and activation of bone morphogenesis proteins (BMPs)/mothers against decapentaplegic (Smad) pathway via activating NF-κB and thereby regulating Runx2 expression. These signaling pathways combined with the classical signaling pathway, with ERK1/2 as a hub node molecule, form a molecular signaling cross-talking network to induce the osteogenic differentiation of hMSCs. The understanding on the mechanism of FSS inducing the osteogenic differentiation of hMSCs will not only be helpful to develop the bone tissue engineering but also provide new targets for drug discovery for treatment of osteoporosis and other related bone-wasting diseases.

Biological, immunological and regenerative characteristics of placenta-derived mesenchymal stem cell isolated using a time-gradient attachment method.

It has been verified that placenta contains multi-lineage mesenchymal stem cells (MSCs). We have used a time-gradient attachment method to isolate placenta-derived MSCs (PMSCs). The morphology, differentiation potential, immunogenicity and xenogenic reconstruction potential of these PMSCs were examined. The results showed that PMSCs isolated using the time-gradient attachment method showed higher potential of in vitro proliferation and multi-lineage differentiation. PMSCs isolated using the time-gradient attachment method showed a low immunogenicity. HLA-A gene fragment and no HLA-DR gene fragment were detected in PMSCs isolated using the time-gradient attachment method, and the mixed lymphocyte reaction (MLR) assay identified that these cells inhibited the proliferation of the allogeneic T-lymphocytes induced by PHA. The transplantation in calvaria of rats showed that PMSCs had the higher xenogenic reconstruction potential. Finally, the significance of PMSCs isolated using the time-gradient attachment method in experimental and clinical applications is discussed.

Hepatogenic engineering from human bone marrow mesenchymal stem cells in porous polylactic glycolic acid scaffolds under perfusion culture

Bone marrow mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering. We used mesenchymal stem cells from human bone marrow (hMSCs) as the seeding cells to investigate the potential of hepatocytic differentiation of hMSCs in porous polylactic glycolic acid (PLGA) scaffolds under perfusion induction. hMSCs were seeded and proliferated in PLGA scaffolds, and then induced into hepatocyte‐like cells with hepatogenic medium in perfusion and static cultures. The results showed that hMSCs could be induced into hepatocyte‐like cells in PLGA scaffolds with hepatogenic medium in both static and perfusion induction systems. However, perfusion induction was more effective for cellularity in PLGA scaffolds than in static induction. Cells in the scaffold induced by the hepatogenic medium expressed hepatocyte‐specific genes cytokeratin 19 (CK19), α‐fetoprotein (αFP), cytokeratin 18 (CK18), albumin and cytochrome P4503A4 (CYP3A4) in a time‐dependent manner. Induced cells stained positive for αFP and albumin. Induced cells also acquired the functional characteristics of hepatocytes, i.e. secretion of urea and albumin. In a comparison of survival and hepatogenic differentiation of hMSCs between perfusion and static induction, perfusion induction increased the survival and the uniform distribution of induced cells in scaffolds, which resulted in a higher efficiency of hepatogenesis in the PLGA construct with hMSCs. The oscillatory perfusion induction system combined with the hepatogenic medium should be a valuable and convenient tool for in vitro hepatic tissue engineering using hMSCs. Copyright

A novel therapy strategy for bile duct repair using tissue engineering technique: PCL/PLGA bilayered scaffold with hMSCs

The current clinical treatments for complications caused by hepatobiliary surgery still have some inevitable weakness. The aim of the study was to fabricate a tissue‐engineered bile duct that utilized a novel bilayered polymer scaffold combined with human bone marrow‐derived mesenchymal stem cells (hMSCs) for new treatment of biliary disease. The biocompatibility of polycaprolactone (PCL) (PCL)/poly(lactide‐co‐glycolide) (PLGA) scaffold with hMSCs was first examined, and the hMSC–PCL/PLGA constructs (MPPCs) prepared. The MPPCs and blank scaffolds were then transplanted into 18 pigs for evaluation its efficacy on bile duct repairing, respectively. In vitro, the PCL/PLGA scaffold was verified to support the adhesion, proliferation and matrix deposition of hMSCs. There was no sign of bile duct narrowing and cholestasis in all experimental animals. At 6 months, the MPPCs had a superior repairing effect on the bile duct injury, compared with the blank PCL/PLGA scaffolds. Therefore, the implanted scaffolds could not only support the biliary tract and allow free bile flow but also had direct or indirect positive effects on repair of injured bile duct. Copyright

Studies on Culture and Osteogenic Induction of Human Mesenchymal Stem Cells under CO2-Independent Conditions

Human mesenchymal stem cells (hMSCs) are one of the important factors that regulate bone anabolism. Osteoporosis resulting from microgravity during spaceflight may possibly be due to a decrease in osteogenesis mediated by hMSCs. This speculation should be verified through culture and osteogenic induction of hMSCs in a microgravity environment during spaceflight. Control of CO2 is a key component in current experimental protocols for growth, survival, and proliferation of in vitro cultured cells. However, carrying CO2 tanks on a spaceflight and devoting space/mass allowances for classical CO2 control protocols make experimentation on culture and osteogenesis difficult during most missions. Therefore, an experimental culture and osteogenic medium was developed through modifying the components of buffer salts in conventional culture medium. This experimental medium was used to culture and induce hMSCs under CO2-independent conditions. The results showed that culture and induction of hMSCs with conventional culture medium and conventional osteogenic medium under CO2-independent conditions resulted in an increase of pH in medium. The proliferation of hMSCs was also inhibited. hMSCs cultured with experimental culture medium under CO2-independent conditions showed a proliferation potential that was the same as those cultured with conventional culture medium under CO2-dependent conditions. The experimental osteogenic medium could promote hMSCs to differentiate into osteoblast-like cells under CO2-independent conditions. Cells induced by this induction system showed high alkaline phosphatase activity. The expression levels of osteogenic genes in cells induced with experimental osteogenic medium under CO2-independent conditions were not significantly different from those cells induced with conventional osteogenic medium under CO2-dependent conditions. These results suggest that the experimental culture and induction system could be used to culture hMSCs and induce the osteogenesis of hMSCs in the atmospheric conditions common to spaceflights without additional CO2.