Rat Bone Marrow-derived Mesenchymal Stem Research Articles

The effect of rat bone marrow-derived mesenchymal stem cells on proliferation and angiogenesis of endothelial cells

Objective To investigate the effect of rat bone marrow-derived mesenchvmal stem cells (MSCs) on proliferation and angiogenesis of endothelial cells (ECs).Methods The rat bone marrow-derived mononuclear cells (BM-MNCs) were collected by density gradient centrifugation and cultured in different medium (DMEM and EGM-2) before identified by flow cytometry or immunofluorescenee.After direct and indirect co-culture,they were analysised by vascular network formation in vitro and immunofluorescence.Results Compared with DMEM,the BM-MNCs could formed typical colony-forming cells in EGM-2.After cuhured in DMEM and EGM-2 respectively,MNCs could develop into MSCs with greater than 95％ of cells expressing CD105,CD90 and CD73,and ECs with greater than 90％ of cells positive staining for CD31.Compared with indirect co-culture,direct co-culture of MSCs and ECs cells would result in significant reduction of tube quantity and tabe length (P ＜ 0.05).Furthermore,immunofluorescence staining demonstrated the number of ECs also significantly reduced (P ＜ 0.05).Conclusion Directly cocultured MSCs were able to inhibit ECs proliferation and vascular network formation. Key words: Rat bone marrow stem cells; Co-culture; Vascular network formation; Proliferation

An effective inducer of dopaminergic neuron-like differentiation.

Rat bone marrow-derived mesenchymal stem cells were cultured and passaged in vitro. After induction with basic fibroblast growth factor for 24 hours, passage 3 bone marrow-derived mesenchymal stem cells were additionally induced into dopaminergic neurons using three different combinations with basic fibroblast growth factor as follows: 20% Xiangdan injection; all-trans retinoic acid + glial-derived neurotrophic factor; or sonic hedgehog + fibroblast growth factor 8. Results suggest that the bone marrow-derived mesenchymal stem cells showed typical neuronal morphological characteristics after induction. In particular, after treatment with sonic hedgehog + fibroblast growth factor 8, the expressions of nestin, neuron-specific enolase, microtubuleassociated protein 2, tyrosine hydroxylase and vesicular monoamine transporter-2 in cells were significantly increased. Moreover, the levels of catecholamines in the culture supernatant were significantly increased. These findings indicate that Xiangdan injection, all-trans retinoic acid + glial-derived neurotrophic factor, and sonic hedgehog + fibroblast growth factor 8 can all induce dopaminergic neuronal differentiation from bone marrow-derived mesenchymal stem cells. In particular, the efficiency of sonic hedgehog + fibroblast growth factor 8 was highest.

Cultivation and spontaneous differentiation of rat bone marrow-derived mesenchymal stem cells on polymeric surfaces

Accumulating evidence demonstrated many physical and chemical cues from the local microenvironment could influence mesenchymal stem cells (MSCs) maintenance and differentiation. In this study, we systematically investigated the interaction of rat bone marrow-derived mesenchymal stem cells (rBMSCs) and polymeric substrates. Adhesion, proliferative capacity, cytoskeleton alteration, cytotoxicity, apoptosis, senescence, and adipogenesis potential of rBMSCs were determined on these polymeric inserts prepared from polyetherurethane (PEU) and poly(ether imide) (PEI). Inserts for culture plates were applied to ensure that the rBMSCs were solely in contact to the tested material. The explored inserts exhibited advancing contact angles of 84° (PEU) and 93° (PEI). Finally, the micromechanical properties determined by atomic force microscopy (AFM) indentation varied in the range from 6 GPa (PEU) to 13 GPa (PEI). We found that both PEU and PEI showed a good cell compatibility to rBMSCs. rBMSCs could adherent on both polymeric surfaces with the similar adhesion ratio and subsequent division rate. However, cells cultured on PEU exhibited higher apoptosis level and senescence ratio, which resulted in lower cell density (22061 ± 3000/cm(2)) compared to that on PEI (68395 ± 8000/cm(2)) after 20 days cultivation. Morphological differences of rBMSCs were detected after 5 days cultivation. Cells on PEU exhibited flat and enlarged shape with rearranged filamentous actin (F-actin) cytoskeleton, while cells on PEI and tissue culture plate (TCP) had similar spindle-shape morphology and oriented F-actin. After 20 days, lipid droplets were spontaneously formed in rBMSCs on PEU and PEI but not on TCP. Both PEU and PEI might trigger rBMSCs towards spontaneous adipogenic commitment, whereas PEI provided better cell compatibility on rBMSCs apoptosis, senescence and proliferation.

Cytocompatibility and osteogenic activity of a novel calcium phosphate silicate bioceramic: Silicocarnotite

In the present study, the effect of a novel bioceramic, silicon-containing calcium phosphate ceramic (silicocarnotite, Ca5 (PO4 )2 SiO4 , CPS) on attachment, proliferation, and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSC) has been investigated in comparison to hydroxyapatite (HA). The CPS showed a similar cell attachment behavior to HA, while the proliferation of rBMSC on CPS was significantly higher than that on HA, which indicated that CPS had a good cytocompatibility. Moreover, the expression of alkaline phosphatase activity and osteogenic-related genes, including Runx-2, osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), demonstrated that CPS enhanced the osteogenic differentiation of rBMSC and accelerated the differentiation process. The results suggest that CPS ceramic exhibits a good cytocompatibility and osteogenic activity, which might be used as a potential candidate material for bone tissue engineering.

Short-term, long-term and paracrine effect of human umbilical cord-derived stem cells in lung injury prevention and repair in experimental bronchopulmonary dysplasia

BackgroundBronchopulmonary dysplasia (BPD) remains a main complication of extreme prematurity and currently lacks efficient treatment. Rat bone marrow-derived mesenchymal stem cells (MSC) prevent lung injury in an oxygen-induced model of...

The Poor Osteoinductive Capability of Human Acellular Bone Matrix

Demineralized bone matrix (DBM) has extensive clinical use for bone regeneration because of its osteoinductive and osteoconductive aptitude. It is suggested that the demineralization process in bone matrix preparation is influential in maintaining osteoinductivity; however, relevant investigations, especially into the osteoinductivity of acellular bone matrix, are not often performed. This study addressed the osteoinductive capability of human acellular cancellous bone matrix (ACBM) after subcutaneous implantation in a rat model. The growth and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) seeded in this material were also studied. Without the demineralization process, the ACBM we obtained had an interconnected porous network and the micropores in the surface were clearly exposed. After the ACBM was subcutaneously implanted for 4 months, new osteoid formation was noted but not typical mature bone formation. rBM-MSCs grew well in the ACBM and kept a steady morphology after continuous culture for 28 days. However, no mineralized nodule formation was detected and the expression levels of genes encoding osteogenic markers were significantly decreased. These results demonstrated that human ACBM possess the structural features of native bone and poor osteoinductivity; nonetheless this material helped to preserve the undifferentiated phenotype of rBM-MSCs. Such insights may further broaden our understanding of the application of ACBM for bone regeneration and the creation of stem cell niches.

BMP-2, VEGF and bFGF synergistically promote the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) were treated with bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) dose-dependently and time-dependently. Together they caused a strong synergistic effect on the osteogenic differentiation of MSCs, with lower concentrations of each factor being enough to show the synergistic promotion (50ng BMP-2/ml, 1ng VEGF/ml and 10ng bFGF/ml). When both VEGF and bFGF were added in the early proliferating stage (the first 7days) and BMP-2 was added in the late differentiation stage (the last 7days), osteogenic differentiation of MSCs could be enhanced more effectively.

Osteopontin Promotes Mesenchymal Stem Cell Migration and Lessens Cell Stiffness via Integrin β1, FAK, and ERK Pathways

The use of mesenchymal stem cells (MSCs) for therapeutic applications has attracted great attention because MSCs home to and engraft to injured tissues after in vivo administration. The expression of osteopontin (OPN) is elevated in response to injury and inflammation, and its role on rat bone marrow-derived mesenchymal stem cells (rMSCs)-directed migration has been elucidated. However, the signaling pathways through the activation of which OPN promotes rMSCs migration and the involvement of cell mechanics during OPN-mediating rMSCs migration have not been well studied. In this study, we found that OPN activated focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways by the ligation of integrin β1 in rMSCs. Inhibitors of FAK and ERK pathways inhibited OPN-induced rMSCs migration, indicating the possible involvement of FAK and ERK activation in OPN-induced migration in rMSCs. In addition, atomic force microscopy analysis showed that OPN reduced cell stiffness in rMSCs via integrin β1, FAK, and ERK pathways, suggesting that the promotion of rMSCs migration might partially be contributing to the decrease in cell stiffness stimulated by OPN. To further examine the role of OPN on cell motility and stiffness, actin cytoskeleton of rMSCs was observed. The reduced well-defined F-actin filaments and the promoted formation of pseudopodia in rMSCs induced by OPN explained the reduction in cell stiffness and the increase in cell migration. The current study data have shown for the first time that OPN binding to integrin β1 promotes rMSCs migration through the activation of FAK and ERK pathways, which may be attributed to the change in cell stiffness caused by the reduction in the amount of organized actin cytoskeleton.

In vitro chondrogenesis by BMP6 gene therapy

In this study, the promotion of in vitro chondrogenesis was investigated by using chitosan scaffolds and rat bone marrow-derived mesenchymal stem cells (rBMSCs) which are transfected by BMP6 (bone morphogenetic protein-6) encoding gene. For this purpose, plasmid DNA (pShuttle-rBMP6), the expression vector consisting of the coding sequence of the BMP6 was obtained, and then, it was entrapped in chitosan scaffolds to obtain a gene-activated matrix (GAM). The chitosan scaffolds performed the controlled and sustained release of plasmid DNA, thus they continuously provided the modification of rBMSCs to induce chondrogenic differentiation. In addition, the cells were transfected by lipid-based agent (Lipofectamine) and then, these modified cells were inoculated into the chitosan scaffolds. Furthermore, a group of chitosan scaffolds with nontransfected rBMSCs with recombinant BMP6 free in culture medium was used as control. Comparative results showed that, mitochondrial activities of modified rBMSCs by Lipofectamine and chitosan GAM were significantly higher than those of nontransfected rBMSCs. The observations from scanning electron microscopy analysis confirmed that BMP6 gene-modified rBMSCs differentiated to the chondrogenic phenotype. Highest amount of glycosaminoglycan contents of rBMSCs on GAM concluded that BMP6 gene-activated chitosan scaffold has a potential in the application of cartilage regeneration.

Osteogenesis and angiogenesis induced by porous β-CaSiO3/PDLGA composite scaffold via activation of AMPK/ERK1/2 and PI3K/Akt pathways

As a potential bioactive material, β-calcium silicate (β-CS) has attracted particular attention in the field of bone regeneration. In this study, porous β-CS/Poly-d,l-Lactide-Glycolide (PDLGA) composite scaffolds were developed with the goals of controlling the degradation rate and improving the mechanical and biological properties. The compressive strength and toughness were significantly enhanced by PDLGA modification of porous β-CS ceramic scaffolds. The effects of the ionic extract from β-CS/PDLGA composite scaffolds on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs), proliferation of human umbilical vein endothelial cells (HUVECs) and the related mechanisms were investigated. It was shown that bioactive ions from β-CS/PDLGA scaffolds could enhance cell viability, alkaline phosphatase (ALP) activity, calcium mineral deposition, and mRNA expression levels of osteoblast-related genes of rBMSCs without addition of extra osteogenic reagents. The activation in AMP-activated protein kinase (AMPK), extracellular signal-related kinases (ERK) 1/2 and RUNX-2 were observed in rBMSCs cultured in the extract of β-CS/PDLGA, and these effects could be blocked by AMPK inhibitor Compound C. The extracts of β-CS/PDLGA composites stimulated HUVECs proliferation that was associated with phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) as well as an increase in nitric oxide (NO) production and secretion of vascular endothelial growth factor (VEGF). The inductions were abolished by the addition of phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. The composite scaffolds were implanted in critical sized rabbit femur defects (6 × 10 mm) for 4, 12 and 20 weeks with β-tricalcium phosphate (β-TCP) as controls. Sequential histological evaluations and radiographs revealed that β-CS/PDLGA dramatically stimulated new bone formation and angiogenesis. The biodegradation rate of the β-CS/PDLGA scaffolds was lower than that of β-TCP at each time point examined, and matched the new bone formation rates. These data suggest that β-CS/PDLGA could promote bone regeneration in vivo, which might be ascribed to the enhanced osteogenic differentiation of mesenchymal stem cells (MSCs) and increased angiogenic activity of endothelial cells (ECs).

MIR-499 INDUCES CARDIAC DIFFERENTIATION OF RAT BONE MARROW-DERIVED MESENCHYMAL STEM CELLS

ObjectivesTo test the hypothesis that over-expressing miR-499 in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) induces them to differentiate into cardiomyocyte-like cells.MethodsThe fourth passage of rat BM-MSCs were infected with...

Vascular endothelial growth factor participates in modulating the C6 glioma-induced migration of rat bone marrow-derived mesenchymal stem cells and upregulates their vascular cell adhesion molecule-1 expression

Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to be able to migrate towards glioma, but the molecular mechanisms responsible for this migratory behavior still require further elucidation. This study aimed to test the role of vascular endothelial growth factor (VEGF) in the C6 glioma-induced migration of BMSCs, evaluate the effect of VEGF on the migratory capacity and vascular cell adhesion molecule-1 (VCAM-1) expression of BMSCs and explore the role of VCAM-1 in the VEGF-induced migration of BMSCs. The results showed that C6 glioma cells significantly increased the migration of BMSCs in vitro, which was partially blocked by a VEGF neutralizing antibody, and 20 ng/ml recombinant rat VEGF164 incubation enhanced the migration of BMSCs. Moreover, 12 h of 20 ng/ml VEGF164 incubation upregulated the VCAM-1 expression of BMSCs and the blocking of VCAM-1 reduced the VEGF164-induced migration of BMSCs. The data also revealed that LY294002, an inhibitor of phosphoinositide-3-kinase (PI3K), decreased the VEGF-induced migration and VCAM-1 expression of BMSCs. These findings indicate that VEGF participates in mediating the C6 glioma-induced migration of BMSCs by upregulating their VCAM-1 expression, and that PI3K is involved in the signal transduction of VEGF164-induced migration and VCAM-1 expression of BMSCs.

Therapeutic gene expression in transduced mesenchymal stem cells can be monitored using a reporter gene

We constructed a recombinant adenovirus construct Ad5-sr39tk-IRES-VEGF(165) (Ad5-SIV) that contained a mutant herpes viral thymidine kinase reporter gene (HSV1-sr39tk) and the human vascular endothelial growth factor 165 (VEGF(165)) gene for noninvasive imaging of gene expression. The recombinant adenovirus Ad5-SIV was transfected into rat bone marrow-derived mesenchymal stem cells (MSCs), and we measured the expression of HSV1-sr39tk and VEGF(165) to evaluate the feasibility of monitoring VEGF(165) expression using reporter gene expression. The MSCs were infected with Ad5-SIV at various levels of infection (MOI), ranging from 0 to 100 infectious units per cell (IU/cell). The mRNA and protein expression levels of the reporter and therapeutic genes were determined using real-time RT-PCR, Western blot, ELISA and immunofluorescence. The HSV1-sr39tk expression in the MSCs was also detected in vitro using a cellular uptake study of the reporter probe (131)I-FIAU. Gene expression was also evaluated in vivo by micro-Positron Emission Tomography/Computed Tomography (micro-PET/CT) imaging 1day after injecting Ad5-SIV-tranfected MSCs into the left foreleg of the rat. The right foreleg was injected with non-transfected MSCs and served as an internal control. The real-time RT-PCR results demonstrated a good correlation between the expression levels of HSV1-sr39tk mRNA and VEGF(165) mRNA (R(2)=0.93, P<0.05). The cellular uptake of (131)I-FIAU increased with increasing viral titers (R(2)=0.89; P<0.05), and in the group that received an MOI of 100, a peak value of 30.15%±1.11% was found at 3 hours of incubation. The uptake rates increased rapidly between 30 and 150 minutes and reached a plateau after 150 minutes. The uptake rates of (131)I-FIAU by the Ad5-SIV-infected cells were significantly higher than by the Ad5-EGFP-infected cells for all time points (t=18.43-54.83, P<0.05). Moreover, the rate of VEGF(165) protein secretion was highly correlated with the uptake rate of (131)I-FIAU (R(2)=0.84, P<0.05). The radioactivity on the micro-PET/CT images was significantly higher in the left foreleg (which received the transfected MSCs) compared with the control foreleg. These results suggest that radionuclide reporter gene imaging may be used to monitor gene expression in vivo.

Inhibition of hypoxia and serum deprivation-induced apoptosis by salvianolic acid in rat mesenchymal stem cells

ObjectiveTo investigate the influence and mechanism of salvianolic acid B (SalB) on apoptosis inhibition in rat bone marrow-derived mesenchymal stem cells (BMSCs) induced by hypoxia and serum deprivation (hypoxia/SD). MethodsSalB concentration of 0.1, 1, 10 or 100 mg/L (drug groups) were investigated for their ability to inhibit apoptosis in rat BMSCs. BMSCs in both the apoptosis model and drug groups were cultured under hypoxic conditions for 6 h, after which cell apoptosis and change in mitochondrial membrane potential (MMP) were detected using flow cytometry. Activation of caspase-3 was detected using western blot analysis. ResultsHypoxia/SD induced apoptosis in rat BMSCs. The early apoptosis rate was lower in the drug groups compared to the apoptosis model group (P<0.05). SalB was found to inhibit the reduction in MMP and decrease the activation of caspase-3. Conclusion0.1, 1 and 10 mg/L of SalB inhibits activation of caspase-3 and early apoptosis of rat BMSCs induced by hypoxia/SD and could therefore enhance the survival rate of grafted stem cells.

Macrochanneled bioactive ceramic scaffolds in combination with collagen hydrogel: A new tool for bone tissue engineering

New tissue-engineering tool for bone regeneration is described to facilitate homogeneous cell seeding and effective osteogenic development. Calcium phosphate (CaP) scaffolds with macrochanneled and well-defined pore structure was developed, however, a large portion of the cells seeded directly within the scaffold easily penetrates without good adhesion to the scaffold surface. To overcome this, a method was exploited to dispense cells evenly throughout the CaP scaffold using collagen hydrogel. Rat bone marrow-derived mesenchymal stem cells (MSCs) were mixed within a neutralized collagen solution, which was then infiltrated into the macrochanneled pore space and gelled to result in macrochanneled bioceramic scaffold combined with MSCs-hydrogel. MSCs contained within the hydrogel-CaP scaffolds were highly viable, with similar growth pattern to those in the collagen hydrogel. Cells seeded by this approach were initially almost double in number compared with those seeded directly onto the CaP scaffold and had an active proliferation more than 14 days. Assessments of the MSCs showed significantly higher alkaline phosphatase levels in the combined scaffold, which was accompanied by enhanced osteogenesis including the expression of genes [collagen type I, bone sialoprotein, and osteopontin (OPN)] and proteins (OPN and osteocalcin). Extracellular calcium was also elevated significantly in the combined scaffold compared to the CaP scaffold. In addition, mechanical strength of the constructs was improved significantly in the combined scaffold compared to the CaP scaffold. Based on these, the cell culturing and tissue engineering strategy within the macrochanneled bioactive ceramic scaffolds could be improved greatly by the combinatory approach of using collagen hydrogel.

Effects of laminar shear stress on expression of MT1‐MMP in rat bone marrow‐derived mesenchymal stem cells

ObjectiveTo investigate the effect of laminar shear stress on the expression of membrane type‐1 matrix metalloproteinase (MT1‐MMP) in rat bone marrow‐derived mesenchymal stem cells (rMSCs).MethodsrMSCs were isolated from SD rat lower extremities and loaded by laminar shear stress with the parallel‐plate flow chamber system, and then the expression of MT1‐MMP mRNA of rMSCs was examined by real‐time PCR.ResultsShear stress with different magnitude of 5, 15, 30 dynes/cm2 all induced MT1‐MMP mRNA expression, especially high shear stress, 30 dynes/cm2, which was time‐dependent. The expression of MT1‐MMP mRNA was inhibited by SB202190, an inhibitor of p38, but enhanced by wortmannin, an inhibitor of PI3K/Akt.ConclusionShear stress can activate the expression of MT1‐MMP. The amount of MT1‐MMP expression is closely related to stimulating time and the strengths of shear stress, and p38MAPK and PI3K/Akt signal pathway may play a critical role during the process.

MiR-499 induces cardiac differentiation of rat mesenchymal stem cells through wnt/β-catenin signaling pathway

ObjectiveTo test the hypothesis that over-expressing miR-499 in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) induces them to differentiate into cardiomyocyte-like cells through the wnt/β-catenin signaling pathway. MethodsRat BM-MSCs were infected with lentiviral vectors bearing miR-499. The expression of cardiac-specific markers, NKx2.5, GATA4, MEF2C, and cTnI in these cells were examined by rtPCR or Western blot analysis and the activity of the wnt/β-catenin signaling pathway was evaluated by measuring the phosphorylation status of β-catenin. ResultsOver-expression of miR-499 in rat BM-MSCs increased the expression of cardiac-specific genes, such as NKx2.5, GATA4, MEF2C, and cTnI and decreased the ratio of phosphorylated/dephosphorylated β-catenin in the wnt/β-catenin signaling pathway, thus activating the pathway. Knocking down the expression of Dvl, an adaptor molecule in the wnt/β-catenin signaling, partially blocked the role of the miR-499 and decreased those cardiac-specific genes. ConclusionOver-expression of miR-499 in rat BM-MSCs induces them toward cardiac differentiation through the activating the wnt/β-catenin signal pathway.

In vitro reprogramming of rat bone marrow-derived mesenchymal stem cells into insulin-producing cells by genetically manipulating negative and positive regulators

Islet cell replacement therapy represents the most promising approach for the cure of type 1 diabetes if autoimmunity to β cells is under control. However, this potential is limited by a shortage of pancreas donors. To address the donor shortage problem, we determined whether bone marrow-derived mesenchymal stem cells (bmMSCs) can be directly reprogrammed to islet lineages by simultaneously forced suppression and over-expression of key regulator genes that play critical roles during pancreas development. Here, we report that rat bmMSCs were converted in vitro into insulin-producing cells by suppressing two-repressor genes repressor element-1 silencing transcription factor/neuronal restrictive silencing factor (Rest/Nrsf) and sonic hedgehog (Shh) and by over-expressing pancreas and duodenal transcription factor 1 (Pdx1). The reprogrammed bmMSCs expressed both genes and proteins specific for islet cells. These converted cells were capable of releasing insulin in a glucose-responsive manner. Our study suggests that bmMSCs may ultimately be reprogrammed to functional insulin-secreting cells.

Effect of the calcium sensing receptor on rat bone marrow-derived mesenchymal stem cell proliferation through the ERK1/2 pathway

Migration and proliferation of bone marrowderived mesenchymal stem cells (BMSCs) is critical to treatment of ischemic injury. The calcium sensing receptor (CaSR) has an important role in maintaining systemic calcium homeostasis, which is related to cell proliferation, apoptosis and paracrine signaling. We hypothesize that CaSR may enhance BMSC proliferation. Rat BMSCs were incubated with various calcium concentrations for 48 h in vitro to activate CaSR. To investigate potential mechanisms responsible for growth enhancement by calcium, the rat BMSC cell cycle progression was analyzed by fluorescence-activated cell sorting (FACS), and induction of apoptosis confirmed by cytofluorimetric analysis using propidium iodide and Annexin V-FITC double staining. Since the mitogen-activated protein kinase (MAPK) signaling pathway was one of the most significantly affected by CaSR, MAPK activation was measured by western blotting. Calcium exposure significantly enhanced rat BMSCs proliferation, as well as the proportion of the population in S phase, in a dose-dependent manner, effects which were abolished by NPS2390 (a CaSR antagonist) and U0126 (a MEK1/2 inhibitor). These results demonstrate that CaSR is involved in rat BMSC proliferation, as seen by an increased proliferation index, decreased apoptosis, and ERK1/2 activation, and provide important insight into the cellular and molecular mechanisms by which CaSR affects cell proliferation. A CaSR agonist may prove useful to enhance BMSC survival during transplantation.

Collagen Three-Dimensional Hydrogel Matrix Carrying Basic Fibroblast Growth Factor for the Cultivation of Mesenchymal Stem Cells and Osteogenic Differentiation

Three-dimensional (3D) collagen hydrogels have been extensively used for cell culture experiments and are more closely representative of in vivo conditions than monolayer (2D) culture. Here we cultured rat bone marrow-derived mesenchymal stem cells (MSCs) in collagen hydrogels containing varying concentrations of basic fibroblast growth factor (bFGF) to examine the effect of bFGF on MSC proliferation and osteogenic differentiation in 3D culture. The optimal bFGF concentration that promoted the greatest degree of cell proliferation and expression of the early osteogenic induction marker alkaline phosphatase was also determined. Subsequent quantitative real-time polymerase chain reaction analysis of gene expression demonstrated that bFGF promoted significant upregulation of the bone-related genes: collagen type I, osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN) for periods of up to 21 days. Immunofluorescence staining and fluorescence-activated cell sorting analysis further supported the enhanced osteogenic differentiation of cells as a greater proportion of cells were found to express OPN. Matrix mineralization within the collagen hydrogels was enhanced in the presence of bFGF, as assessed by calcium detection using von Kossa staining. These results clearly demonstrate a positive effect of bFGF on proliferation and osteogenic induction of MSCs in 3D collagen hydrogels when applied at the appropriate concentration. Moreover, collagen hydrogel constructs containing MSCs and appropriate growth factor stimulus might be a potentially useful biological tool for 3D bone tissue engineering.