)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/omcl/2016/9785890.pdf · Oxidative...

Research ArticleMetformin Decreases Reactive Oxygen SpeciesEnhances Osteogenic Properties of Adipose-DerivedMultipotent Mesenchymal Stem Cells In Vitroand Increases Bone Density In Vivo

Krzysztof Marycz12 Krzysztof A Tomaszewski3 Katarzyna Kornicka1

Brandon Michael Henry3 Sebastian WroNski4 Jacek Tarasiuk4 and Monika Maredziak25

1Electron Microscopy Laboratory Wroclaw University of Environmental and Life Sciences 5b Kozuchowska Street51-631 Wroclaw Poland2Wroclaw Research Centre EIT+ 147 Stablowicka Street 54-066 Wroclaw Poland3Department of Anatomy Jagiellonian University Medical College 12 Kopernika Street 31-034 Krakow Poland4Faculty of Physics and Applied Computer Science AGH University of Science and Technology 30 Mickiewicza Street30059 Krakow Poland5Department of Animal Physiology and Biostructure Faculty of Veterinary MedicineWroclaw University of Environmental and Life Sciences 31 Norwida Street 50-375 Wroclaw Poland

Correspondence should be addressed to Krzysztof Marycz krzysztofmaryczinteriapl

Received 28 December 2015 Revised 24 March 2016 Accepted 30 March 2016

Academic Editor Ange Mouithys-Mickalad

Copyright copy 2016 Krzysztof Marycz et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Due to its pleiotropic effects the commonly used drug metformin has gained renewed interest among medical researchers Whilemetformin is mainly used for the treatment of diabetes recent studies suggest that it may have further application in anticancerand antiaging therapies In this study we investigated the proliferative potential accumulation of oxidative stress factors andosteogenic and adipogenic differentiation potential of mouse adipose-derived stem cells (MuASCs) isolated frommice treated withmetformin for 8 weeks Moreover we investigated the influence of metformin supplementation on mice bone density and boneelement composition The ASCs isolated from mice who were treated with metformin for 8 weeks showed highest proliferativepotential generated a robust net of cytoskeletal projections had reduced expression of markers associated with cellular senescenceand decreased amount of reactive oxygen species in comparison to control group Furthermore we demonstrated that thesecells possessed greatest osteogenic differentiation potential while their adipogenic differentiation ability was reduced We alsodemonstrated that metformin supplementation increases bone density in vivo Our result stands as a valuable source of dataregarding the in vivo influence ofmetformin onASCs and bone density and supports a role formetformin in regenerativemedicine

1 Introduction

Over the past several years the pleiotropic effects of met-formin (N N1015840-dimethylbiguanide) a commonly used drugfor the treatment of type 2 diabetes mellitus (T2DB)have gained interest among both clinicians and medicalresearchers Metformin belongs to the group of biguanide

drugs that are mainly prescribed for insulin resistance (IR)for obesity and for patients suffering from polycystic ovariansyndrome (POS) [1] The general systemic effects of met-formin involve the reduction of glucose production in theliver and increased insulin sensitivity in peripheral tissuesMetformin has also been proposed as a calorie restrictionmimetic (CRM) that has been shown in combination with

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 9785890 19 pageshttpdxdoiorg10115520169785890

2 Oxidative Medicine and Cellular Longevity

calorie restriction to reduce morbidity and mortality inboth healthy and tumorigenic mice thus increasing overalllifespan [2]

In the field of regenerative medicine major scientificinterest is focused on the cytophysiological characteristicsand clinical application of mesenchymal stem cells (MSCs)Those cells are found inmany types of adult tissues includingbonemarrow and adipose tissue [3] Although adipose tissue-derived stem cells (ASCs) share similar characteristics withbone marrow mesenchymal stem cells (BMSCs) they haveseveral advantages over BMSCs They are easily accessiblewithminimal invasive harvesting procedure and the isolationyields a great number of cells crucial for effective stem celltherapies and bone engineering

Furthermore it has been shown that metformin mayinhibit the growth of tumor cells and thus may potentiallyfind wide application in anticancer therapies [4ndash8] Hirschet al [9] demonstrated that metformin inhibits cellulartransformation and selectively kills cancer stem cells ingenetically distinct breast cancer cells lines The inhibitoryeffect of metformin on tumor cells in vitro is associated notonly with its cytostatic properties but also with proapoptoticactions in the tumor cells [10] To date the signaling pathwaysof metforminrsquos mechanism of action have not been fullyunderstood and require further study However it seemsthat metformin affects insulin-like growth factor (IGF) path-ways by activation of 51015840-adenosinemonophosphate-activatedprotein kinase (AMPK) which plays a key role in insulinsignaling and energy sensing [11] Moreover it has beenshown that in the context of atherosclerosis metformininhibits NF-120581B activation decreases C-reactive protein levels[12] and inhibits the inflammatory response via a pathwayinvolving AMPK and the tumor suppressor PTEN [13]

Moreover recent studies have indicated antioxidativeproperties of metformin Increased production of the intra-cellular level of reactive oxygen species (ROS) results fromthe imbalance of the antioxidant scavenger enzymes thatmetabolize free radicals The antioxidant enzymes includemanganese superoxide dismutase (MnSOD) copper andzinc (CuZn) SOD catalase and glutathione peroxidaseWhen the level of ROS exceeds the defense mechanismsa cell is said to be in a state of ldquooxidative stressrdquo [14] Theenhanced production of ROS during environmental stressescan pose a threat to cells by causing peroxidation of lipidsoxidation of proteins and damage to nucleic acids ultimatelyleading to death of the cells Thus dealing with excessiveROS may improve cellular metabolism and in consequenceincrease the lifespan It was also shown that metforminexerted intracellular antioxidant properties by decreasingROS production through the inhibition of protein kinase Cactivity [15] Recent studies indicate that its primary effect isinmitochondria where it interferes with respiratory complexI and reduces ATP production leading to the activation ofAMP kinase [16]

However the potential role of metformin in reducingROS in ASC is still not well established

It has also been reported that metformin can induceMC3T3-E1 osteoblastic cell differentiation and bone matrixsynthesis via AMPK activation and subsequent induction of

endothelial nitric oxide synthase (eNOS) and bone morpho-genetic protein-2 (BMP-2) expression [17 18]Metforminwasalso found to enhance rat osteoblasts proliferation increasealkaline phosphatase activity and increase the number offormed mineralized nodules Our group recently showedthat metformin in a dose-dependent manner affects theviability morphology and ultrastructure of mouse bonemarrow-derived multipotent mesenchymal stromal cells andthe Balb3T3 embryonic fibroblast cell line [19] We have alsodemonstrated that the expression of tandem gene H19IGF2as well as Oct4 a pluripotent stem cell marker in partcorrelates with particular dosage application of metformin invitro Additionally we found a significant correlation betweenthe activity of the ASCs and osteopontin at the mRNA andprotein level in vitro and in vivo The mounting evidenceindicates that the range of metformin actions may be signif-icantly wider than once thought and thus the application ofmetforminmay open new avenues in the treatment of variousmedical conditions [20 21]

Bone development is a complex and dynamic processinvolving several different mechanisms that maintain thebalance between bone formation and bone resorption Thisbalance might be affected by different factors including phys-ical activity endogenous hormones or drugs [22] Essentialto this balance and to the formation of the skeletal systemis proosteoblastic differentiation of bone marrow stem cellsosteoclast activity and the osteoblast-adipocyte relationshipRecently it has been demonstrated that the complementsystem plays an important role in both bone developmentand homeostasis and specifically influences osteoblast andosteoclast activity [23] Moreover it has been shown thatcomplement plays a role in the bone repair process thusmak-ing a bridge between skeletal and immunological systems

Bearing in mind that previous research has indicated aproosteogenic effect ofmetformin onBMSCs or commitmentcell lines in vitro we became interested in the effect ofmetformin on the osteogenic potential of mice as well asthe effects of metformin on bone in vivo It has been shownthat metformin prevents bone loss induced by ovariectomyin rats [24] suggesting that metformin confers a protectiveeffect against bone loss Further research on rats showedthat 2 weeks of metformin supplementation led to increasedtrabecular volume osteocyte density and osteoblast numberin femoral metaphysis [25] Wang et al [26] using insulin-resistantmice also showed that 6weeks ofmetformin admin-istration protects femoral bone architecture [26] Howeverlittle is known about the effects of metformin on bonetrabecular architecture and bone density in healthy mice

The aim of our study was to investigate the effects ofmetformin on ASCs osteogenic differentiation potential invitro and on bone density andmineralization in healthymice

2 Materials and Methods

All reagents used in this experiment were purchased fromSigma-Aldrich (Poland) unless indicated otherwise

Oxidative Medicine and Cellular Longevity 3

21 Experimental Animals The experiment was performedon 4-week-old C57BL6 mice (119899 = 18) All mice were housedthree per cage in an ultraclean facility on ventilated racksand were provided with food and water ad libitum during thexperiment period Mice received a standard diet with 42fat (Morawski Labofeed H Poland) and were maintainedon a 12-h light-dark cycle at 22 plusmn 02∘C The animals werepurchased from the Animal Laboratory House WroclawMedical School and housed in the Animal ExperimentalLaboratory (Wroclaw Medical School Wroclaw Poland)

The animals used in the study were divided into twogroups (a) controlmice receiving drinkingwater only [119899 = 9]and (b) the group receiving metformin in drinking water atconcentration equal to 28mgday (Metformax 850 TevaPharmaceuticals Poland) for 8 weeks [119899 = 9] Waterwas changed every two days After the experiment themice were fasted for 24 h Body weight measurement wascarried out using electronic scale (RADWAG PSC1 seriesPoland) Bloodwas collected from the animals using a cardiacpuncture method Biochemical analysis of blood samplessuch as glucose and lipidsmeasurements was performedwithErba XL 300 platform (Erba Diagnostics Mannheim GmbHGermany) After euthanasia of the animals abdominal sub-cutaneous adipose tissue as well as both tibia bones wascollected from each animal

Cells or tissues obtained from animals treated with drugfor 8 weeks are designated as the 8-week metformin (Met 8)group and from the control group were designated as control

22 Isolation of Mice Adipose-Derived Mesenchymal StemCells (MuASCs) from Adipose Tissue The MuASCs wereisolated from subcutaneous adipose tissue of C57BL6 mice(119899 = 9 Met 8 mice 119899 = 9 control mice) Cells were isolatedusing a protocol previously described by Grzesiak et al [27]Tissue samples were placed in sterile Hanksrsquo balanced saltsolution (HBSS) containing 1 of antibioticsolution (PSAPenicillinStreptomycinAmphotericin B) and washed exten-sively to remove contaminating debris and erythrocytesAfter removing blood vessels the tissue was cut into smallpieces using surgical scissors Aspirates were then transferredinto sterile tubes and treated with collagenase type I enzyme(1mgmL) in PBS for 40min at 37∘C with gentle agitationThe obtained tissue homogenate was then centrifuged for10min at 1200timesgThe supernatant was then discarded whilethe cell pellet was resuspended in culture media and platedonto a conventional tissue culture flask

23 Cells Culture All stages of the experiment were per-formed under aseptic standardized culture conditions (37∘C5 CO

2) Primary cultures were plated on T25 culture

flasks in culture media consisting of Dulbeccorsquos ModifiedEaglersquos Medium (DMEM) with nutrient F-12 Ham 10 ofFetal Bovine Serum (FBS) and 1 solution of PSA Themedium was changed every two days while passage of cellswas performed after reaching 80ndash90 confluence Prior tothe experiment cells were passed three times using trypsinsolution (TrypLE Life Technologies Carlsbad USA) inaccordance with manufacturerrsquos protocol

24 Immunophenotyping of MuASCs andMultipotency AssayIsolated cells were characterized by the expression of follow-ing markers CD29 CD44 CD45 CD73 and CD105 Prior tostaining cells were cultivated onto 24-well dishes designatedfor immunofluorescence preparations For each stainingcultures were run in triplicate The following antibodieswere used in the experiment rabbit anti-mouse integrin-b-1(CD29) dilution 1 100 rabbit anti-mouse CD44 (RampD Sys-tems Minneapolis USA) dilution 1 100 rabbit anti-mouseCD45 dilution 1 100 rabbit anti-mouse NT5E (CD73)dilution 1 200 rabbit anti-mouse endoglin (CD105) dilution1 200 Secondary antibody conjugated with atto-488 fluores-cence label goat anti-mouse IgG atto-488 was diluted 1 400Immunocytochemistry was performed in accordance withthe manufacturerrsquos instructions Briefly cells were incubatedwith certain primary antibodies overnight while incubationwith secondary antibodies lasted 1 hour and was performedin 37∘C in the dark To assess the binding specificity ofsecondary antibodies negative controls were included Tovisualize cells nuclei specimens were counterstained with410158406-diamidino-2-phenylindole (DAPI) The samples wereimaged using an inverted fluorescence microscope (AxioOb-serverA1 Carl Zeiss Jena Germany) and captured withCanon PowerShot Multipotency of MuASCs was confirmedby adipogenic and osteogenic differentiation of cells culturedin commercially available media (StemPro Life Technolo-gies) onto 24-well plates Adipogenic differentiation was con-firmed with Oil Red O to visualize lipid droplets Osteogenicdifferentiation was confirmed with Alizarin Red staining ofthe extracellular matrix mineralization Experiments werecarried out simultaneously and performed in triplicate

25 Cell Viability and Colony Forming Efficiency Assay Forthe assays cells were plated onto 24-well plates and inoculatedwith a 05mL volume of culturemedium at a concentration of2times10

4 cells per wellThe number of viable cells in the culturewas evaluated using resazurin-based assay kit (Alamar Blue)in accordance with the manufacturerrsquos instruction after 12 5 and 7 days of cell culture Briefly to perform theassay the culture medium was collected and replaced with amedium containing 10 of the dye Cells were then incubatedfor 2 hours at 37∘C followed by supernatant collectionAbsorbance levels of the supernatants were measured spec-trophotometrically using 96-well microplate reader (Spec-trostar Nano BMG Labtech Ortenberg Germany) The dyereduction was evaluated at a wavelength of 600 nm and areference wavelength of 690 nm Each test included a blankthat consisted of completemediumwith dyeThe cell numberwas obtained from the test data and allowed to calculatethe population doubling time (PDT) as well as proliferationfactor (PF)

The calculated values of PF reflect the activity of theexperimental group (Met 8) in relation to the control groupgiving an arbitrary value equal to 1 A PF value higher than 1indicated increased cellular activity while a PF lower than 1showeddecreased proliferation potential PDTwas calculatedusing online software (httpwwwdoubling-timecom)

To evaluate the ability of cells to form colonies a colonyforming efficiency (CFE) assay was performed Cells were


seeded onto 6-well plates at an initial density of 1times102 perwelland inoculated in culture media (DMEM with nutrient F-12Ham 10 FBS 1 PSA) After seven days of propagationcells were washed with 4 ice-cold paraformaldehyde andstained with pararosaniline Colonies of more than 50 cellswere counted and the efficiency of colony forming wascalculated using the formula presented as follows [28]

CFUfs [] = the number of coloniesinitial cell number

times 100 (1)

26 Evaluation of Cell Morphology Ultrastructure and Senes-cence Markers Cell morphology cellular composition andculture growth pattern were analyzed using an invertedfluorescence microscope (Axio ObserverA1 Zeiss Jena Ger-many) and a scanning electron microscope (SEM EVO LS15Zeiss Jena Germany)

The morphology of cells was evaluated on the 7th(standard culture) 12th (adipogenic stimulation) and 18th(osteogenic stimulation) days Prior to observation cells werecultured on 24-well plates After fixation in 4 ice-coldparaformaldehyde (40min) cells membranes were perme-abilized using 01 Triton X-100 for 15min After washingwithHBSS cells were stainedwith atto-565 labeled phalloidinfor 40min to visualize actin filaments Cells were thenwashed again with HBSS three times and the nuclei weresubsequently counterstained with diamidino-2-phenylindole(DAPI) for 5min During fluorescence staining cells wereincubated at room temperature in the dark

To confirm osteogenesis and adipogenesis Alizarin Redand Oil Red O staining were applied respectively Firstthe culture media were removed and the cells were thenwashed with HBSS followed by treatment with 4 ice-coldparaformaldehyde for 10min To visualize the extracellularmineralized matrix cells were incubated with 2 AlizarinRed solution for 10min After dye discarding cells werewashed a few times with distilled water For intracellularlipid droplets visualization paraformaldehyde was replacedwith 60 isopropanol and the cells were incubated with itfor 5min Then the cells were rinsed with distilled waterand Oil Red O solution was added for 15min followed byhematoxylin counterstaining for 1min Images were capturedusing a Canon PowerShot camera

Detailed cells morphology was evaluated using SEMCells were rinsed with distilled water and dehydrated in agraded ethanol series (concentrations from 50 to 100every 5min) Samples were then sprinkled with gold (Scan-Coat 6 Oxford) transferred to the microscope chamber andobserved using SE1 detector at 10 kV of filament tensionAdditionally the number of bone nodules the size of the nod-ules and the calcium and phosphorus concentration withinthe nodules were analyzed by SEM with energy dispersiveX-ray analysis (SEMEDX) A QUANTAX detector (BrukerBillerica USA) with 10 kV of filament tension was used toperform a line scan analysis of randomly selected cells Theobtained values of calcium and phosphorus deposition werepresented as weight percentage (wt)

To evaluate amounts of viable and dead cells a CellstainDouble Staining Kit was applied Live cells were stained with

Calcein-AMand emitted a green fluorescence signal whereasdead cell nuclei were stained with Propidium Iodide andemitted a redorange fluorescence Cells were then observedusing fluorescence microscopy All procedures were per-formed in accordance with the manufacturerrsquos instructionsImmunofluorescence staining for caspase-3 was performedusing anti-caspase 3 antibodies (anti-caspase-3 active anti-body produced in rabbit cat number C8487) Prior to investi-gation of senescence-associated 120573-galactosidase (120573-gal) cellswere stained using a commercially available kit (SenescenceCells Histochemical Staining Kit) following manufacturerrsquosprotocol

27 Analysis of Oxidative Stress and Superoxide DismutaseActivity (SOD) To evaluate stress levels the cells werecultured in medium without phenol red for 7 days andthe tests were performed on the 7th day Nitric oxide(NO) concentration was assessed with the Griess ReagentKit (Life Technologies) superoxide dismutase (SOD) wasassessed by SOD Assay Kit and the level of reactive oxygenspecies (ROS) was estimated with a 5-(and-6)-chloromethyl-2101584071015840-dichlorodihydrofluorescein diacetate (H2DCF-DA LifeTechnologies) solution All procedures were performed induplicate in accordance with the manufacturerrsquos protocols

28 Evaluation of Osteogenic and Adipogenic DifferentiationPotential of MuASCs For the in vitro osteogenic and adi-pogenic assays cells were planted onto 24-well plates atan approximate density of 2 times 104 cells per well Priorto osteogenic differentiation of MuASCs cells were cul-tured in STEMPRO Osteogenesis Differentiation Kit (LifeTechnologies Carlsbad USA) for a period of 18 days Foradipogenic differentiation cells were cultured in STEM-PRO Adipogenesis Differentiation Kit (Life TechnologiesCarlsbad USA) for 12 days The differentiation media weresupplemented with 1 of PSA and changed every two daysCultures expanded in standard culture media (DMEM withnutrient F-12 Ham 10 FBS 1 PSA) served as a control(nonstimulated) which allowed determining effectiveness ofthe differentiation process All procedures were performed inaccordance with the manufacturerrsquos instructions

29 Detection of Extracellular Form of Osteopontin (OPN)Osteocalcin (OCL) and Bone Morphogenetic Protein-2 (BMP-2) with Enzyme-Linked Immunosorbent Assay (ELISA) Theinvestigated markers were detected in the supernatants col-lected at the last day of the differentiation process the 18thday of osteogenic stimulation and the 12th day of adipogenicstimulation The presence of osteopontin (OPN) was deter-mined using MouseRat Osteopontin Quantikine ELISAKit (RampD Systems Minneapolis USA) whereas osteocalcin(OCN) was assessed with the Mouse Gla-Osteocalcin HighSensitive EIA Kit (Takara Otsu Japan) The level of bonemorphogenetic protein (BMP) was investigated with theMouse BMP-2 Quantikine ELISA Kit (RampD Systems Min-neapolis USA) All procedures were performed according tothemanufacturerrsquos protocolThe amount of detected proteinswas expressed as ngmL of supernatant


Table 1 Sequences of primers used in qPCR OCN Osteocalcin BMP-2 Bone morphogenetic protein-2 ADPQ Adiponectin B2M beta 2microglobulin

Gene Primer Sequence 51015840-31015840 Amplicon length (bp) Accesion no

OCN F GGTGCAGACCTAGCAGACACCA 100 NM 0010322982R CGCTGGGCTTGGCATCTGTAA

BMP-2 F CTACAGGGAGAACACCCGGA 280 NM 0075533R GGGGAAGCAGCAACACTAGAA

ADPQ F CCTGGAGAGAAGGGAGAGAAA 209 NM 0096054R CGAATGGGTACATTGGGAAC

B2M F CATACGCCTGCAGAGTTAAGCA 73 NM 0097353R GATCACATGTCTCGATCCCAGTAG

210 Analysis of Gene Expression Real-Time Reverse Tran-scription Polymerase Chain Reaction (qRT-PCR) of OPNBMP-2 and Adiponectin (ADPQ) After osteogenic stimula-tion on the 18th day of culture the cultures were rinsed withHBSS and lysed in a culture dishwith 05mLof TRIReagentThe total RNA was isolated using a phenol chloroformmethod The resulting samples were then diluted in DEPC-treated water The quantity and quality of the total RNA weredetermined using a nanospectrometer (VPA biowave II) Toremove any traces of genomic DNA samples were digestedusing DNase I RNase-free kit (Thermo Scientific WalthamUSA) Each reaction was performed with 100 ng of totalRNA which was then transcribed to complementary DNA(cDNA) with the Moloney murine leukemia virus reversetranscriptase (M-MLV RT) provided with the Tetro ReverseTranscriptase kit (Bioline London UK) Both RNA purifica-tion and cDNA synthesis were performed following theman-ufacturers protocol using a T100 Thermo Cycler (Bio-RadHercules USA) Quantitative RT-PCR was performed in atotal volume of 20120583L using the SensiFast SYBRampFluoresceinKit (Bioline London UK)The primer concentration in eachreaction equaled 500 nM and the primer sequences used foreach specific reaction are listed in Table 1 The analysis wasperformedusingCFXConnectTMReal-TimePCRDetectionSystem (Bio-Rad Hercules USA) To confirm the specificityof the reaction products an analysis of the dissociation curvewas performedThe value of the threshold cycle (Ct) was usedto calculate the fold change in relation to the expression of thehousekeeping gene B2M (beta-2 microglobulin)

211 Ex Vivo Analysis of Bone Mineral Concentration andDensity by Microtomography (120583CT) and SEM-EDX CalciumDeposition Analysis Prior to performing 120583CT analysis thebone samples were fixed in 4 formaldehyde in PBS for 48 hSpecimens were then dehydrated in an increasing ethanolgradient of 50 60 70 80 96 and 100 and thenanalyzed in 120583CT

The tomographic studies were carried out in the Labo-ratory of Micro and Nano Tomography (LMINT) Facultyof Physics and Applied Computer Science AGH Univer-sity of Science and Technology Krakow Poland The 120583CTmeasurements were performed using a Nanotom 180N (GESensing amp Inspection Technologies Phoenix X-ray GmbH)The machine is equipped with a nanofocus X-ray tube with

a maximum 180 kV voltage The tomograms were registeredusing a Hamamatsu 2300 times 2300 pixel 2D detector Thereconstruction of measured objects was performed usingdatosX ver 210 (GE) with the use of a Feldkamp algorithmfor cone beam X-ray CT [29] The postreconstruction datatreatment was performed using VGStudio Max 21 [30] andfree Fiji software [31] with the BoneJ plugin [32]

All examined specimens were scanned at 70 kV of sourcevoltage and 200120583A with a 360-degree rotation of the spec-imen in 1400 steps The exposure time was 500ms with aframe averaging of 3 and image skip of 1 applied resultingin a scanning time of 60 minutes The reconstructed imageshad a voxel size of 5 120583m3

For SEM-EDX analysis the femoral bones were fixed in25 glutaraldehyde for one hour washed in distilled waterdehydrated in a graded ethanol series sputtered with carbon(ScanCoat 6 Oxford) and imaged using an SEM at 10 kV offilamentrsquos tension Additionally specimens were prepared forEDX analysis according to the method described previouslyby our laboratory [33] Observations were performed at afilament tension of 10 kVThe detection ofmineralizedmatrixwas performed using SEM combined with EDX (BrukerCoventry UK) to analyze the surface distribution of calciumand phosphorus The values obtained were presented asweight percentage (wt)

212 Statistical Analysis All experiments were performedwith 119899 = 3 or more Statistical analysis was performedusingGraphPad Prism 5 software (La Jolla USA) Differencesbetween groups was determined using one-way analysis ofvariance (ANOVA) with post hoc Tukeyrsquos multiple compar-ison or Studentrsquos 119905-test Differences with a probability of 119901 lt005 were considered significant

3 Results

The clinical characteristics of the C57BL6 mice included inthe study assessed following the 24 h of fasting at the end ofthe experiment are presented in Table 2

31 Immunophenotyping Characterization of IsolatedMuASCsand Multipotency Assay In order to confirm multipotentcharacter of isolated ASCs immunohistochemical staining


CD29+

CD44+

CD73+

Staining results Negative controls

CD105+

CD45minus

Figure 1 Results of MuASCs immunophenotyping Obtained cells were positive for the mesenchymal markers CD29 CD44 CD73 andCD105 and show lack of expression for the hematopoietic marker CD45 Each marker was stained with a specific antibody and secondaryantibodies conjugated with atto-488 nuclei were counterstained with DAPI Magnification times100 scale bar 250 120583m

and multipotency assays were performed Immunohisto-chemical staining revealed that MuASCs population did notexpress CD45 hematopoietic marker (Figure 1) In contrastwe observed positive reactions for mesenchymal markerswhich are specific to multipotent stromal cells includingCD29 CD44 CD73 and CD105 (Figure 2) Moreover themultipotent character of isolated MuASCs was confirmed bypositive adipogenic and osteogenic differentiation (Figure 2)Formation of extracellular matrix mineralization was visu-alized by Alizarin Red staining (Figure 2(c)) In addition

Oil Red O staining was performed to reveal the formationof intracellular lipid droplets (Figure 2(b)) Cells cultured instandard medium served as a control (Figure 2(a))

32 Proliferation Factor (PF) Population Doubling Time(PDT) and Colony Forming Efficiency (CFE) of MuASCs dur-ing Seven-Day Viability Test The proliferation of MuASCswas evaluated after 1st 2nd 5th and 7th days of culture Theviability characteristics of MuASCs including PF PDT and


Standard mediumMu ASCs culture in

(a)

Adipogenic mediumMu ASCs culture in

(b)

Osteogenic mediumMu ASCs culture in

(c)

Figure 2 Results of multipotency assay Morphology of MuASCs cultured in standard (a) adipogenic (b) and osteogenic (c) conditionsCalcium deposits in mineralized extracellular matrix were visualized with Alizarin Red staining while lipid droplets formed in adipogenicstimulation with Oil Red O The images included in the figure were chosen to be representative Magnification times100 scale bar 250 120583m

Table 2 Clinical characteristics of C57BL6 mice

Parameters Control group(119899 = 9)

Metformin 8weeks (119899 = 9)

Body weight (g) 194 plusmn 055 2226 plusmn 11Fasting blood glucose(mmolL) 136 plusmn 01 16 plusmn 008

Fasting serumtriglyceride (mmolL) 0613 plusmn 0018 125 plusmn 001

Fasting serumcholesterol (mmolL) 134 plusmn 001 184 plusmn 002

CFE were assessed in comparison to the control group Theanalysis of cytophysiological activity of theMet 8 cells showedproliferation activity that was decreased after 24 hours ofpropagation However this was followed by a significantlyaccelerated proliferation on the 2nd 5th and 7th day ofculture The highest PF was recorded on 5th day (119901 lt 0001)of experimental culture (Figure 3(a))

The proliferation assay analysis showed that metformintaking contributed to the change in PDT The control groupreached PDT in 1604 plusmn 419 h Interestingly the time neededto double the population of MuASCs was considerablyreduced in Met 8 group (119901 lt 005) which was only 9258 plusmn58 h (Figure 3(b))

ASCs plated on low density tend to form colonies origi-nated from a single cell It was found that cells from the Met8 group displayed a significantly (119901 lt 005) higher CFE thanthe control group (Figure 3(c))

33 Evaluation of MuASCs Morphology During the initialstage of culture (from day one) we observed the formationof embryonic-like nodules (Figures 4(a) and 4(f)) whichdisplayed the largest morphology in the Met 8 group Thecontrol culture of MuASCs was characterized by large flatand irregularly shaped cells The cytoskeleton of those cellswas well developed and DAPI staining revealed centrallypositioned nuclei (Figures 4(b) and 4(c)) SEM picturesrevealed few actin projections filopodia and microvesicles

(MVs) that were located near to nucleus as well as on theedge of the cell body (Figures 4(d) and 4(e))

In the Met 8 group we observed more cells with afibroblast-like shape and the embryonic-like nodules wereof greatest size Moreover the size of the cells seemed to besmaller No apoptotic bodies were observed and the actincytoskeleton network was well organized (Figures 4(g) and4(h))The number of cells was visibly higher than in the othergroups The SEM imaging allowed visualization of multiplethin cellular projections (Figure 4(i)) and rich production ofMVs (Figure 4(j))

34 Evaluation of Senescence Markers To evaluate the per-centage of dead cells in each group we used a CellstainDouble Kit Calcein-AMPropidium Iodide staining revealedthat the number of dead cells was significantly reduced in theMet 8 group (Figure 5(a)(V VI)) in comparison to the control(Figure 5(a)(I II)) To determine if a decrease in cell growthwas connected to an increased apoptosis level we examinedthe activity of caspase-3 by immunofluorescence techniqueThe obtained pictures showed that the greatest fluorescencesignal was in the control group (Figure 5(a)(III)) with thesignal decreasing in theMet 8 group (Figure 5(a)(VII)) wherethe lowest activity of caspase-3 was observed

We also performed staining for senescence-associated 120573-galactosidase (120573-gal)The amount of blue 120573-gal positive cellswas higher in the control (Figure 5(a)(IV)) than in the Met 8group where we noticed a reduction in the number of 120573-galpositive cells (Figure 5(a)(VIII))

Moreover to provide quantitative data percentage ofcells positive for both caspase-3 (Figure 5(b)) and 120573-gal(Figure 5(c)) was calculated Both markers were decreased inMet 8 group

35 Oxidative Stress Accumulation and SOD Activity Toevaluate the effect ofmetformin on accumulation of oxidativestress inMuASCswe investigated the levels of reactive oxygenspecies (ROS) and nitric oxide (NO) in those cells Theamount of both ROS (Figure 6(a)) and NO (Figure 6(b))was significantly decreased in cells obtained from animals


1 2 5 700

05

15

20

23

1

Proliferation factor

Days of cultureMetformin 8 weeks

PF

lowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150

200

250

lowast

Population doubling time

(h)

Metformin 8 weeksControl

(b)

00

02

04

06

08

10

12

Colony forming efficiency

CFU

-E (

)

lowast


(c)

Figure 3 Seven-day viability test ofMuASCs Growth kinetics and proliferation potential ofMuASCs during seven-day culture Proliferationfactor calculated in comparison to control group (a) Time required for doubling the cell population expressed in hours (b) and the efficiencyto form single cell derived colonies (c) Results expressed as mean plusmn SD lowast119901 value lt 005 lowastlowastlowast119901 value lt 0001

treated with metformin (119901 lt 005 and 119901 lt 0001 resp) Toinvestigate the antioxidative defense of MuASC the activityof superoxide dismutase (SOD) was assessed We observedthatmetformin supplementation positively affects the activityof SOD as it was significantly increased in Met 8 group(Figure 6(c) 119901 lt 005)

36 Proliferation Factor (PF) and PDT during Osteogenic Dif-ferentiation The PF as well as the PDT was analyzed duringthe 18-day period of culturing MuASCs in osteogenesis-inducing medium Obtained data showed that metforminsupplementation influenced both the PF and PDT The pro-liferation of MuASCs from the Met 8 group was decreasingduring the first six days of propagation and reached thelowest value on the 6th day (119901 lt 0001) After that theproliferation activity was stabilized Significant differencesfrom controls were observed only later on the last two daysof the experiment the 16th (119901 lt 001) and 18th (119901 lt 005)days (Figure 7(a))

The estimation of PDT showed that the time needed todouble the population in the Met 8 group was significantlyincreased (119901 lt 005) in comparison to control culture(Figure 7(b))

37 The Morphology of Osteoblasts Originated from MuASCsAfter osteogenic induction cell morphology shifted fromtypical fibroblast-like cells to more flattened and cuboidalcells Moreover cytoskeletal projections became shorterAfter 18 days of propagation bone nodules became visibleand showed strong blackred signal after Alizarin Red stainwas applied (Figure 7(c)(II V)) Characteristic osteoblast-like cells were observed in each of the experimental groupshowever the cells in Met 8 group displayed the most effectiveosteogenesis (Figure 7(c)(IV V VI)) In this group bonenodules were the largest in size and Alizarin Red dyeabsorption was most abundant

The lowest efficiency of osteogenic induction wasobserved in control group where nodule diameters were


Control Metformin 8 weeks

DA

PID

API

Pha

lloid

in1s

t day

7th

day

lp

mvs

lp

fpfp

mvs

SEM

SEM

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

Figure 4 Morphology and cellular composition of MuASCs evaluated on 7th day of culture light microscope (a f) staining for DAPI (b g)DAPIphalloidin merged (c h) and SEM (d e i j) images Morphological features were indicated with proper abbreviations fp filopodialp lamellipodia and mvs microvesicles Magnification DAPI times50 scale bar 250 120583m DAPIphalloidin times100 scale bar 250 120583m SEM times5000scale bar 2 120583m



lc

(I) (V)

dc

(II)

dc

(VI)

Cas-3Cas-3

(III) (VII)

Calc

ein-

AM

P Io

dide

Casp

ase-

3

120573-gal

120573-gal

(IV) (VIII)

120573-g

alac

tosid

ase

lc

Cas-3

120573-gal

(a)

0

5

10

15

20

25 Cas-3 positive cells

()

lowastlowast

(b)0

5

10

15

20 120573-gal positive cells

() lowast

(c)

Figure 5 Evaluation of senescence markers in MuASCs after 7th day of culture Photographs showing different types of cellular senescence-associated markers Cells stained with Calcein-AM (I V) Propidium Iodide (II VI) antibodies for caspase-3 (III VII nuclei counterstainedwith DAPI) and senescence dye (IV VIII) showing cells with 120573-galactosidase accumulation Abbreviations used to describe different celltypes lc live cell dc dead cell cas-3 caspase-3 positive cell and120573-gal120573-galactosidase positive cellMagnifications Calcein-AMand P Iodidetimes50 scale bar 500 120583m caspase-3 times100 scale bar 250 120583m 120573-galactosidase times200 scale bar 125 120583m Data was also presented quantitatively bycalculating percentage of cells positive for caspase-3 (b) and 120573-gal (c) Results expressed as mean plusmn SD lowast119901 value lt 005 lowastlowast119901 value lt 001

much smaller and calcium deposits absorbed a smalleramount of Alizarin Red dye (Figure 7(c)(I II III))Interestingly in the Met 8 group we observed the lowestnumber of bone nodules (Figure 7(d)) however theyreached the greatest size (Figure 7(e)) Furthermore calcium

deposition was significantly higher in the Met 8 groupcompared to the control group (Figure 7(f) 119901 lt 0001)The obtained data suggest that metformin supplementationincreases MuASCs osteogenic differentiation potential andincreases its effectiveness


ROS

005

010

015

020ΔA


lowast

(a)

Nitric oxide (NO)

54

55

56

57

58

0

25

(120583M

mL)


lowastlowastlowast

(b)

SOD

0

5

10

15

Inhi

bitio

n (

)


lowast

(c)

Figure 6 Assessment of oxidative stress and SOD activity in MuASC To investigate whether treatment with metformin affects the oxidativestatus of MuASC we investigated the extracellular levels of ROS (a) and NO (b) Moreover the activity of antioxidant enzyme SOD wasevaluated (c) Obtained data indicates that metformin supplementation decreases the oxidative stress and simultaneously improves theantioxidative protection coming from SOD Results expressed as mean plusmn SD lowast119901 value lt 005 lowastlowastlowast119901 value lt 0001

38 Extracellular Identification of OPN OCN BMP-2and RT-PCR for OPN and BMP-2 On the 18th day ofosteogenic stimulation quantitative analysis of extracellularOPN showed increased levels in the Met 8 (Figure 8(a)119901 lt 0001) group in comparison to the control culture LikeOPNOCN concentrationwas also affected by themetforminsupplementation The OCN concentration was increased inthe Met 8 (119901 lt 001) group (Figure 8(b)) BMP-2 was alsoincreased in Met 8 group although without statistical sig-nificance (Figure 8(c)) Moreover RT-PCR analysis revealedincreased mRNA levels of both OPN (Figure 8(d) 119901 lt 005)and BMP-2 (Figure 8(e) 119901 lt 001) in Met 8 group

39 Proliferation Factor (PF) PDT Morphological Changesand ADPQ Expression during Adipogenic Differentiation

The PF as well as the PDT was estimated during a 12-dayperiod of culturing cells in adipogenic-inducing mediumMuASCs fromMet 8 group showed decrease in PF comparedto the control group (Figure 9(a)) The highest PF valuewas observed on the 2nd day and from that time point ageneral decreasing tendency was observed The PDT wasalso increased in metformin group and showed a significantdifference (119901 lt 005) in comparison to the control culture asit was reduced by approximately 35 (Figure 9(b))

Adipogenic differentiation was demonstrated by theaccumulation of intracellular lipid droplets indicated by OilRed O staining on 12th day of culture (Figure 9(c)(III VI))In the control group we observed the greatest amount oflipid droplets The ratio of red staining area was significantlydecreased in metformin group where adipocytes displayed


1 4 6 8 11 13 16 1806070809101112 Proliferation factor

Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150

200 Population doubling time

(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l

DAPI SEMAlizarin Red

bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15

20 Bone nodules number


(d)

0

20

40

60

80

100 Bone nodules size

(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15

20 Calcium and phosphorus

wt (

)

lowastlowastlowast


(f)

Figure 7 Evaluation of MuASCs osteogenic potential Growth kinetics and morphology of cells cultured under osteogenic conditionsProliferation factor (a) population doubling time (b) Morphology evaluation after 18 days of culture (c) DAPI staining (I IV) AlizarinRed (II V) and SEM pictures (III VI) bn bone nodule Mean bone nodules number (d) and size (e) Evaluation of calcium and phosphorusdepositions (f) Magnifications DAPI and Alizarin Red times100 scale bars 250120583m SEM times5000 scale bar 5 120583m Results expressed as mean plusmnSD lowast119901 value lt 005 lowastlowast119901 value lt 001 lowastlowastlowast119901 value lt 0001

a smaller size and were reduced in number (Figure 9(c)(VI))Furthermore DAPI (Figure 9(c)(I IV)) and phalloidin (Fig-ure 9(c)(II V)) staining revealed that cells from the controlgroup became enlarged and spread out and simultaneouslydecreased the number of cytoskeletal projections Moreoverwe calculate percentage of area stained with Oil Red O(Figure 9(d)) and it was significantly reduced in Met 8 group(119901 lt 001) Using RT-PCR we also assessed the expressionof adiponectin mRNA which was reduced in Met 8 group(Figure 9(e) 119901 lt 0001) The obtained data suggests thatmetformin inhibits adipogenic differentiation of MuASCs

310 Ex Vivo Analysis of Bone Mineral Concentration andDensity The morphology of murine tibia bones assessed byNano-CT is shown in Figure 10(a) control group (I II)and Met 8 (III IV) respectively Using Nano-CT we alsoevaluated bone density on certain parts of the bone (scanningbegan from the proximal epiphysis in the direction markedin Figure 9 by the white arrows) The greatest bone densitywas observed inMet 8 especially in the proximal 1ndash6mmareaof epiphysis in comparison to other groups (Figure 10(b))The smallest medullary cavity observed in the Met 8 groupwas at a distance between 2 and 7mm which indicates higher


012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)

Figure 8 Concentration of osteogenic-related proteins and reverse transcriptase-polymerase chain reaction (RT-PCR) results Extracellularprotein levels under osteogenic conditions Quantitative ELISA results for osteopontin (a) osteocalcin (b) and BMP-2 (c) Gene expressioninMuASCs cultured in osteogenesis-promoting conditions Osteopontin (d) and BMP-2 (e) Results expressed asmeanplusmn SD lowast119901 value lt 005lowastlowast119901 value lt 001 and lowastlowastlowast119901 value lt 0001


2 4 7 9 1206

08

10

12

14 Proliferation factor

Days of culture

PF

Metformin 8 weeks(a)

0

50

100

150

200


(h)

lowast


(b)

DAPI Phalloidin Oil Red O

(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)

Figure 9 Evaluation of adipogenic potential Growth kinetics and morphology of cells cultured under adipogenic culture conditionsProliferation factor (a) population doubling time (b) Morphology evaluation after 12 days of culture (c) Nuclei staining with DAPI (IIV) phalloidin (II V) and Oil Red O (III VI) Magnifications DAPIphalloidin times100 scale bars 250120583m Oil Red O times50 scale bar 500 120583mOil Red O staining was quantified by calculation percentage of stained area (d) Moreover using RT-PCR mRNA levels of Adiponectin wereassessed (e) Results expressed as mean plusmn SD lowast119901 value lt 005 lowastlowast119901 lt 001

bone volume in this section (Figure 10(c)) Calcium concen-tration in the tibia was greater in Met 8 (Figure 10(d)) andsimilarly phosphorus deposition (Figure 10(e)) Moreoverwe observed increased tibia porosity in the control groupespecially at a distance between 4 and 5mm (Figure 10(f))

4 Discussion

Recently more emphasis is being paid on investigating themultidirectional action of metformin in vivo and in vitroWhile commonly known as an antidiabetic drug metformin


(I)

(III) (IV)

(II)

(a)

Cortical bone density

0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200

Distance from tibiarsquos head (mm)

Mea

n br

ight

ness

(b)

Medullary cavity volume in relation tocortical bone volume

2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35

8 10 12 14 16 18Distance from tibiarsquos head (mm)

(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity

ControlMetformin 8 weeks

(f)

Figure 10 Nano-CT and SEM-EDX analysis Nano-CT images of tibia bones and their transverse sections from control (I II) andMet 8 (IIIIV) mouse White arrows indicate direction of performed scanning Quantification of cortical bone density (b) and medullary cavity volume(c) in mice tibias by Nano-CT SEM-EDX assessment of calcium (d) and phosphorus (e) content in tibia from control andMet 8 group Meanbone porosity assessed by Nano-CT in the studied groups (f) Results expressed as mean 119899 = 5

is recognized not only as a hypoglycemic agent but also asa potential geroprotector a drug that protects against theaging process [34] It has been previously demonstrated thatmetformin increases the lifespan ofmice [2] and enhances theviability and proliferative activity of mesenchymal stromalstem cells [35] Thus its application as an antiaging drugin part follows the hypothesis that ldquowe are as old as our

adult stem cells arerdquo The antiaging effects of metforminare most likely explained by the upregulation of circulatinginsulin-like growth factor-1 (IGF-1) in peripheral blood thathas been recognized as a crucial factor in the context oflongevity [36] Bearing in mind the fact that mesenchymalstem cells are also partially linked with longevity by theirability to divide proliferate and eventually differentiate to


replace a damaged tissue [37] it is reasonable to investigatethe effects of metformin on the differentiation potential ofendogenousMSCs derived frommetformin-treated animals

The aim of the present study was to investigate how met-formin administration affects the osteogenic and adipogenicdifferentiation potential of ASCs isolated from healthy micethat previously received oral metformin supplementation for8 weeks with a dose that is recommended formice with DT2Additionally bearing in mind the divergent data concerningthe effects of metformin on bone mineral density and boneregenerative ability we also focused on the evaluation of bonedensity of healthy mice (without performing bone defects)treated with metformin We found that ASCs derived fromthe 8-week metformin- (Met 8-) treated animals present dif-ferent osteogenic and adipogenic differentiation potentialswith cells obtained from the Met 8 group presenting withmore osteogenic than adipogenic character

As observed in our study metformin administration hasan important influence on ASCs morphology proliferationrate and osteogenicadipogenic differentiation ability all ofwhich are essential features of tissue regeneration Recentlypublished data by our group revealed that metformin affectsstromal stem cellsrsquo proliferative activity in a dose-dependentmanner [19] We demonstrated that metformin increasesthe proliferative activity of BMSCs and mouse embryonicfibroblast cell line Balb3T3 after 48 h of culture when 1mMof metformin supplementation was used [19] Gao et al[38] have demonstrated that BMSCs treated in vitro with100 120583M metformin responded with an increase in prolifera-tive activity In turn Smieszek et al [19] demonstrated thatASCs compared to BMSCs are more sensitive to metforminadministration in vitro and thus even lower concentrationsof 1mM metformin resulted in decreased ASCs prolifera-tive activity Moreover it was previously demonstrated thatmetformin administered orally through 5 weeks at a dose of100mgday decreases expression of Ki-67 and osteopontin(OPN) in subcutaneous adipose tissue The inhibitory effectof metformin on stromal stem cells has been explainedby the activation of adenosine 5-monophosphate-activatedprotein kinase (AMPK) and inhibition of multiple molecularsignaling pathways which are important in protein synthesiscontrol [33 39]

In the current study we found that prolonged adminis-tration of metformin (8 weeks) at a concentration equal to28mgday in mice increases the viability and proliferativeactivity of ASCs and shortens their PDT Simultaneouslywe observed decreased activity of the senescence markerscaspase-3 and 120573-galactosidase in ASCs isolated from theMet 8 group in comparison to the control group cellsThus metformin supplementation may be a potential optionfor overcoming the detrimental effects of aging on ASCsand might be considered in the pretransplantation periodespecially in older patients to enhance the effects of ASCbased regenerative therapies

Furthermore we found that ASCs derived from Met 8groupwere characterized by elevated synthesis ofmembrane-derived vesicles (MVs) observed during electronmicroscopyAs previously demonstrated by Ratajczak [40] MVs are rich

in a broad range of growth factors that supports regenerativeprocesses MVs have also been demonstrated to be involvedin intercellular signaling and thus the elevated secretion ofMVs by the cells derived from the Met 8 group might lead toimprovement of regenerative abilities Besides elevated MVssecretary activity the ASCs derived from the Met 8 groupwere characterized by the development of extensive networkof filopodia that transferred the MVs between the cells aswell as a robust net of lamellipodia These structures play acrucial role in promoting both cell migration and extensionMoreover filopodia are involved in cellular processes likewound healing and guidance towards a chemoattractantwhichmakes them a valuable characteristic of cells to be usedfor regenerative medicine [41] Overall our obtained dataindicated elevated cytophysiological activity of ASCs derivedfrom the Met 8 group that from a regenerative point of viewseems to be fundamental

Moreover to investigate whether metformin administra-tion affects the oxidative status of MuASC we evaluate thelevels of ROS and NO in those cells Beyond its antidiabeticactivity justifying its use in the treatment of the type 2diabetes metformin has been shown to exhibit antioxidantproperties As it was shown by Mahrouf et al [15] metforminreduced intracellular ROS production in aortic endothelialcells stimulated by a short incubation with high levels ofglucoseMoreover data presented byAlgire et al [42] showedpreviously unrecognized inhibitory effects of metformin onROS production and somatic cell mutation providing anovel instrument for the reduction in cancer risk Recentlythe actual mechanism underlying the antioxidative effect ofmetformin was discovered As it was shown by Hou et al[43] metformin induced Trx expression through activationof AMPK-FOXO3 pathway which in consequence led toreduced intracellular ROS levels Our own data confirmedantioxidative effect of metformin as we observed decreasedROS and NO in MuASC from Met 8 group and simulta-neously improved antioxidant defense originated from SODactivity Obtained data provide a valuable information in thecontexts of diabetes treatment as increased ROS productionplays a key role in the onset and development of microvas-cular and macrovascular complications in patients withdiabetes Moreover targeting endogenous ROS productionmay improve ASC metabolism and proliferative potentialmaking them more efficient tool in cellular therapy [44 45]

Recently it has been thought that metformin mightimprove the osteogenic differentiation potential of stromalstem cells and thus find an application in pharmacotherapyin the course of the bone healing process Our results supportthis hypothesis and suggest several possible applications ofmetformin in regenerative medicine Firstly metformin canbe used orally before collection of adipose tissue for isolationof ASCs for clinical application or metformin might beused as an active agent that may be added to the cultureenvironment to increase activity of ASCs before patienttransplantation Such supplementation might be especiallyuseful in the elderly as it has been previously established thatosteogenic differentiation potential of ASCs declines with age[46ndash48] Furthermore the addition of proper concentrationsof metformin in various types of biomaterials used for bone


reconstruction may through release into the intercellularspace increase the bone healing process

The above applications of metformin are also supportedby previously published data It has been reported thatmetformin can induce MC3T3-E1 osteoblastic cell differen-tiation and bone matrix synthesis via AMPK activation andsubsequent induction of endothelial nitric oxide synthase(eNOS) and bone morphogenetic protein-2 (BMP-2) expres-sion [17 18] Metformin was also found to regulate SmallHeterodimer Partner (SHP) in MC3T3-E1 cells an orphannuclear receptor that stimulates osteoblast differentiation andaffects osteoblastic bone formation by interacting with thetranscription factor Runx2 [49]

In our study we found that 8 weeks of metformin admin-istration in healthy mice resulted in an elevated osteogenicdifferentiation potential of ASCs with simultaneous inhibi-tion of their adipogenic differentiation abilityThematurationof ASCs into osteoblasts is pivotal for fracture healing and theosseointegration of bone-anchored implants as well as thegeneral bone turnover processThe osteogenic differentiationis regulated by a number of key factors and signalingpathways Commonly used as markers at both gene andprotein levels are BMP-2 OPN and OCL Here we foundon both levels elevated expression of BMP-2 OPN andOCL in cells derived from the Met 8 group Interestinglyon the last day of experiment we observed that extracellularforms of those proteins were upregulated in the Met 8 whichindicates much robust osteogenic differentiation process inthat group As the mRNA levels are usually analyzed duringthe early phases of osteogenic differentiation we observedonly slight differences in BMP-2 OPN and OCL expressionbetween investigated groups In turn the development oflarge mineralized osteonodules was also observed in Met 8group As such our findings support the concept that stromalstem cells under the systemic influence of metformin mighthave wide application for use in bone healing procedures

Our results stand in good agreement with the work byShah et al [50] and Zhen et al [51] which showed thatthe proosteogenic action of metformin was possibly viastimulation of Runx2 and production of IGF-1 On the otherhand Wu et al [52] demonstrated no effect of metforminon osteogenic differentiation potential of BMSCs Kasai et al[53] usingMC3T3-E1 cells and primary osteoblasts observedno matrix mineralization after metformin supplementationHowever both of the above mentioned studies were per-formed on an in vitro model using different doses of met-formin Along with the elevated osteogenic differentiationpotential we observed inhibition of the adipogenic differenti-ation ability of the ASCs isolated from both of the metformingroups In both of these groups the cells were characterizedby a reduced proliferation activity Furthermore the cells didnot develop the typical morphological picture for adipogenicdifferentiation These results correlate with the findings ofNicpon et al [37] who also observed an inhibitory effect ofmetformin on adipogenic differentiation potential

Besides finding that prolonged administration of met-formin has a proosteogenic action we also found that ithas an impact on cortical bone density as well as onmedullary cavity volumeThe combined investigation of 120583CT

and energy dispersive X-ray spectroscopy (EDX) revealeda strong correlation between calciumphosphorus level andtrabecular bone density Our data is in agreement with thefindings of Sedlinsky et al [25] who observed that oralmetformin application improves bone lesion regeneration inboth control and diabetic rats Moreover the authors demon-strated that metformin administration enhanced the expres-sion of osteoblast-specific transcription factor Runx2Cbfa1and activated AMPK in a time-dependent manner [25]However Jeyabalan et al [54] demonstrated no significantdifferences in cortical and trabecular bone architecture inmetformin-treated rodents (rat and mice after ovariectomy)compared to controls Furthermore the authors showed thatmetformin had no effect on bone resorption but reduced thebone formation rate in trabecular bone Future studies shouldfurther assess the in vivo effects of metformin on bone

5 Conclusions

Our results demonstrate that orally administratedmetforminin healthy mice enhances the osteogenic differentiationpotential of ASCs which results in an elevated expressionand secretion of BMP-2 OCL and OPN Additionallywe observed that metformin supplementation significantlyreduced oxidative stress in MuASC and simultaneouslyincreased SOD activity Moreover the development of highlymineralized osteonodules in vitro is enhanced by systemicmetformin administration Finally we found that 8-weekmetformin supplementation leads to increased tibial bonedensity in mice Our results shine a promising light on thepotential application of metformin as an auxiliary strategyin healthy individuals to enhance ASCs obtained for clinicalapplication

Ethical Approval

This study was conducted with the approval of the SecondLocal Bioethics Committee at the Department of Biologyand Animal Breeding Wroclaw University of Environmentaland Life SciencesWroclaw Poland (Dec number 1772010 of11152010)

Competing Interests

The authors declare that there are no competing interestsassociated with the publication of this paper

Acknowledgments

The research was supported by Wroclaw Research CentreEIT+ under the project ldquoBiotechnologies and AdvancedMedical Technologiesrdquo BioMed (POIG010102-02-00308)financed from the European Regional Development Fund(Operational Programmed Innovative Economy 112)Krzysztof A Tomaszewski was supported by the Foundationfor Polish Science (FNP)


References

[1] M Pugeat and P H Ducluzeau ldquoInsulin resistance polycysticovary syndrome and metforminrdquo Drugs vol 58 supplement 1pp 41ndash46 1999

[2] V N Anisimov LM Berstein P A Egormin et al ldquoMetforminslows down aging and extends life span of female SHR micerdquoCell Cycle vol 7 no 17 pp 2769ndash2773 2008

[3] P C Baer ldquoAdipose-derived mesenchymal stromalstem cellsan update on their phenotype in vivo and in vitrordquo WorldJournal of Stem Cells vol 6 no 3 pp 256ndash265 2014

[4] I N Alimova B Liu Z Fan et al ldquoMetformin inhibits breastcancer cell growth colony formation and induces cell cyclearrest in vitrordquo Cell Cycle vol 8 no 6 pp 909ndash915 2009

[5] E Karnevi K Said R Andersson and A H RosendahlldquoMetformin-mediated growth inhibition involves suppressionof the IGF-I receptor signalling pathway in human pancreaticcancer cellsrdquo BMC Cancer vol 13 article 235 2013

[6] E Giovannucci D M Harlan M C Archer et al ldquoDiabetesand cancer a consensus reportrdquoDiabetes Care vol 33 no 7 pp1674ndash1685 2010

[7] Q Luo D Hu S Hu M Yan Z Sun and F Chen ldquoIn vitro andin vivo anti-tumor effect of metformin as a novel therapeuticagent in human oral squamous cell carcinomardquo BMC Cancervol 12 article 517 2012

[8] B Liu Z Fan S M Edgerton et al ldquoMetformin induces uniquebiological and molecular responses in triple negative breastcancer cellsrdquo Cell Cycle vol 8 no 13 pp 2031ndash2040 2009

[9] H A Hirsch D Iliopoulos and K Struhl ldquoMetformin inhibitsthe inflammatory response associated with cellular transforma-tion and cancer stem cell growthrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 110 no3 pp 972ndash977 2013

[10] H-P Chen J-J Shieh C-C Chang et al ldquoMetformin decreaseshepatocellular carcinoma risk in a dose-dependent mannerpopulation-based and in vitro studiesrdquo Gut vol 62 no 4 pp606ndash615 2013

[11] D G Hardie ldquoRole of AMP-activated protein kinase in themetabolic syndrome and in heart diseaserdquo FEBS Letters vol582 no 1 pp 81ndash89 2008

[12] S-N Li XWang Q-T Zeng et al ldquoMetformin inhibits nuclearfactor 120581b activation and decreases serum high-sensitivity C-reactive protein level in experimental atherogenesis of rabbitsrdquoHeart and Vessels vol 24 no 6 pp 446ndash453 2009

[13] S A Kim and H C Choi ldquoMetformin inhibits inflammatoryresponse via AMPK-PTEN pathway in vascular smoothmusclecellsrdquo Biochemical and Biophysical Research Communicationsvol 425 no 4 pp 866ndash872 2012

[14] V J Thannickal and B L Fanburg ldquoReactive oxygen speciesin cell signalingrdquo The American Journal of PhysiologymdashLungCellular and Molecular Physiology vol 279 no 6 pp L1005ndashL1028 2000

[15] M Mahrouf N Ouslimani J Peynet et al ldquoMetformin reducesangiotensin-mediated intracellular production of reactive oxy-gen species in endothelial cells through the inhibition of proteinkinase Crdquo Biochemical Pharmacology vol 72 no 2 pp 176ndash1832006

[16] M R Owen E Doran and A P Halestrap ldquoEvidence thatmetformin exerts its anti-diabetic effects through inhibition ofcomplex 1 of the mitochondrial respiratory chainrdquo BiochemicalJournal vol 348 no 3 pp 607ndash614 2000

[17] A M Cortizo C Sedlinsky A D McCarthy A Blanco andL Schurman ldquoOsteogenic actions of the anti-diabetic drugmetformin on osteoblasts in culturerdquo European Journal ofPharmacology vol 536 no 1-2 pp 38ndash46 2006

[18] I Kanazawa T Yamaguchi S Yano M Yamauchi and TSugimoto ldquoMetformin enhances the differentiation and min-eralization of osteoblastic MC3T3-E1 cells via AMP kinaseactivation as well as eNOS and BMP-2 expressionrdquo Biochemicaland Biophysical Research Communications vol 375 no 3 pp414ndash419 2008

[19] A Smieszek A Czyrek K Basinska et al ldquoEffect of metforminon viability morphology and ultrastructure of mouse bonemarrow-derived multipotent mesenchymal stromal cells andBalb3T3 embryonic fibroblast cell linerdquo BioMed ResearchInternational vol 2015 Article ID 769402 14 pages 2015

[20] T V Kourelis and R D Siegel ldquoMetformin and cancer newapplications for an old drugrdquo Medical Oncology vol 29 no 2pp 1314ndash1327 2012

[21] B Viollet B Guigas N Sanz Garcia J Leclerc M Foretz and FAndreelli ldquoCellular and molecular mechanisms of metforminan overviewrdquo Clinical Science vol 122 no 6 pp 253ndash270 2012

[22] A Teti ldquoBone development overview of bone cells and signal-ingrdquo Current Osteoporosis Reports vol 9 no 4 pp 264ndash2732011

[23] P Schoengraf J D Lambris S Recknagel et al ldquoDoes com-plement play a role in bone development and regenerationrdquoImmunobiology vol 218 no 1 pp 1ndash9 2013

[24] Y Gao Y Li J Xue Y Jia and J Hu ldquoEffect of the anti-diabetic drug metformin on bone mass in ovariectomized ratsrdquoEuropean Journal of Pharmacology vol 635 no 1ndash3 pp 231ndash2362010

[25] C Sedlinsky M S Molinuevo A M Cortizo et al ldquoMetforminprevents anti-osteogenic in vivo and ex vivo effects of rosiglita-zone in ratsrdquo European Journal of Pharmacology vol 668 no 3pp 477ndash485 2011

[26] C Wang H Li S-G Chen et al ldquoThe skeletal effects ofthiazolidinedione and metformin on insulin-resistant micerdquoJournal of Bone andMineral Metabolism vol 30 no 6 pp 630ndash637 2012

[27] J Grzesiak K Marycz J Czogala K Wrzeszcz and J NicponldquoComparison of behavior morphology and morphometry ofequine and canine adipose derived mesenchymal stem cells inculturerdquo International Journal of Morphology vol 29 no 3 pp1012ndash1017 2011

[28] T Chen Y Zhou and W-S Tan ldquoInfluence of lactic acidon the proliferation metabolism and differentiation of rabbitmesenchymal stem cellsrdquo Cell Biology and Toxicology vol 25no 6 pp 573ndash586 2009

[29] L A Feldkamp L C Davis and J W Kress ldquoPractical cone-beam algorithmrdquo Journal of the Optical Society of America AOptics Image Science and Vision vol 1 no 6 pp 612ndash619 1984

[30] Volume Graphics GmbH Reference Manual VGStudio MaxRelease 20 Volume Graphics GmbH 2013 httpwwwvolumegraphicscomenproductsvgstudio-max

[31] httpfijiscFiji[32] M DoubeMM Kłosowski I Arganda-Carreras et al ldquoBoneJ

free and extensible bone image analysis in ImageJrdquo Bone vol 47no 6 pp 1076ndash1079 2010

[33] M Zakikhani R Dowling I G Fantus N Sonenberg andM Pollak ldquoMetformin is an AMP kinase-dependent growthinhibitor for breast cancer cellsrdquo Cancer Research vol 66Article ID 10269 2006


[34] S Kalra R Sahay and A Unnikrishnan ldquoMetformin and thepromise of geroprotectionrdquo Indian Journal of Endocrinology andMetabolism vol 16 no 4 pp 496ndash498 2012

[35] A Smieszek K Basinska K Chrząstek and K Marycz ldquoInvitro and in vivo effects ofmetformin on osteopontin expressionin mice adipose-derived multipotent stromal cells and adiposetissuerdquo Journal ofDiabetes Research vol 2015 Article ID 81489616 pages 2015

[36] D van Heemst ldquoInsulin IGF-1 and longevityrdquo Aging andDisease vol 1 no 2 pp 147ndash157 2010

[37] J Nicpon K Marycz and J Grzesiak ldquoTherapeutic effect ofadipose-derived mesenchymal stem cell injection in horses suf-fering from bone spavinrdquo Polish Journal of Veterinary Sciencesvol 16 no 4 pp 753ndash754 2013

[38] Y Gao J Xue X Li Y Jia and J Hu ldquoMetformin regulatesosteoblast and adipocyte differentiation of rat mesenchymalstem cellsrdquo Journal of Pharmacy and Pharmacology vol 60 no12 pp 1695ndash1700 2008

[39] C-F Sum J M Webster A B Johnson C Catalano B GCooper and R Taylor ldquoThe effect of intravenous metformin onglucose metabolism during hyperglycaemia in Type 2 diabetesrdquoDiabetic Medicine vol 9 no 1 pp 61ndash65 1992

[40] M Z Ratajczak ldquoThe emerging role of microvesicles in cellulartherapies for organtissue regenerationrdquo Nephrology DialysisTransplantation vol 26 no 5 pp 1453ndash1456 2011

[41] P K Mattila and P Lappalainen ldquoFilopodia molecular archi-tecture and cellular functionsrdquo Nature Reviews Molecular CellBiology vol 9 no 6 pp 446ndash454 2008

[42] C Algire OMoiseeva X Deschenes-Simard et al ldquoMetforminreduces endogenous reactive oxygen species and associatedDNA damagerdquo Cancer Prevention Research vol 5 no 4 pp536ndash543 2012

[43] X Hou J Song X-N Li et al ldquoMetformin reduces intracellularreactive oxygen species levels by upregulating expression ofthe antioxidant thioredoxin via the AMPK-FOXO3 pathwayrdquoBiochemical andBiophysical ResearchCommunications vol 396no 2 pp 199ndash205 2010

[44] K Kornicka B Babiarczuk J Krzak and K Marycz ldquoTheeffect of a solndashgel derived silica coating doped with vitamin Eon oxidative stress and senescence of human adipose-derivedmesenchymal stem cells (AMSCs)rdquo RSC Advances vol 6 no35 pp 29524ndash29537 2016

[45] K Marycz K Kornicka K Basinska and A Czyrek ldquoEquinemetabolic syndrome affects viability senescence and stressfactors of equine adipose-derived mesenchymal stromal stemcells new insight into EqASCs isolated from ems horses inthe context of their agingrdquo Oxidative Medicine and CellularLongevity vol 2016 Article ID 4710326 17 pages 2016

[46] M S Choudhery M Badowski A Muise J Pierce and D THarris ldquoDonor age negatively impacts adipose tissue-derivedmesenchymal stem cell expansion and differentiationrdquo Journalof Translational Medicine vol 12 article 8 2014

[47] M Marędziak K Marycz K A Tomaszewski K Kornickaand B M Henry ldquoThe influence of aging on the regenerativepotential of human adipose derived mesenchymal stem cellsrdquoStem Cells International vol 2016 Article ID 2152435 15 pages2016

[48] K Kornicka KMarycz K A TomaszewskiMMarędziak andA Smieszek ldquoThe effect of age on osteogenic and adipogenicdifferentiation potential of human adipose derived stromal stemcells (hASCs) and the impact of stress factors in the course

of the differentiation processrdquo Oxidative Medicine and CellularLongevity vol 2015 Article ID 309169 20 pages 2015

[49] W G Jang E J Kim I-H Bae et al ldquoMetformin inducesosteoblast differentiation via orphan nuclear receptor SHP-mediated transactivation of Runx2rdquo Bone vol 48 no 4 pp885ndash893 2011

[50] M Shah B Kola A Bataveljic et al ldquoAMP-activated proteinkinase (AMPK) activation regulates in vitro bone formation andbone massrdquo Bone vol 47 no 2 pp 309ndash319 2010

[51] D Zhen Y Chen and X Tang ldquoMetformin reverses thedeleterious effects of high glucose on osteoblast functionrdquoJournal of Diabetes and Its Complications vol 24 no 5 pp 334ndash344 2010

[52] W Wu Z Ye Y Zhou and W-S Tan ldquoAICAR a smallchemical molecule primes osteogenic differentiation of adultmesenchymal stem cellsrdquo International Journal of ArtificialOrgans vol 34 no 12 pp 1128ndash1136 2011

[53] T Kasai K Bandow H Suzuki et al ldquoOsteoblast differentiationis functionally associated with decreased AMP kinase activityrdquoJournal of Cellular Physiology vol 221 no 3 pp 740ndash749 2009

[54] J Jeyabalan B Viollet P Smitham et al ldquoThe anti-diabeticdrug metformin does not affect bone mass in vivo or fracturehealingrdquo Osteoporosis International vol 24 no 10 pp 2659ndash2670 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


calorie restriction to reduce morbidity and mortality inboth healthy and tumorigenic mice thus increasing overalllifespan [2]

In the field of regenerative medicine major scientificinterest is focused on the cytophysiological characteristicsand clinical application of mesenchymal stem cells (MSCs)Those cells are found inmany types of adult tissues includingbonemarrow and adipose tissue [3] Although adipose tissue-derived stem cells (ASCs) share similar characteristics withbone marrow mesenchymal stem cells (BMSCs) they haveseveral advantages over BMSCs They are easily accessiblewithminimal invasive harvesting procedure and the isolationyields a great number of cells crucial for effective stem celltherapies and bone engineering

Furthermore it has been shown that metformin mayinhibit the growth of tumor cells and thus may potentiallyfind wide application in anticancer therapies [4ndash8] Hirschet al [9] demonstrated that metformin inhibits cellulartransformation and selectively kills cancer stem cells ingenetically distinct breast cancer cells lines The inhibitoryeffect of metformin on tumor cells in vitro is associated notonly with its cytostatic properties but also with proapoptoticactions in the tumor cells [10] To date the signaling pathwaysof metforminrsquos mechanism of action have not been fullyunderstood and require further study However it seemsthat metformin affects insulin-like growth factor (IGF) path-ways by activation of 51015840-adenosinemonophosphate-activatedprotein kinase (AMPK) which plays a key role in insulinsignaling and energy sensing [11] Moreover it has beenshown that in the context of atherosclerosis metformininhibits NF-120581B activation decreases C-reactive protein levels[12] and inhibits the inflammatory response via a pathwayinvolving AMPK and the tumor suppressor PTEN [13]

Moreover recent studies have indicated antioxidativeproperties of metformin Increased production of the intra-cellular level of reactive oxygen species (ROS) results fromthe imbalance of the antioxidant scavenger enzymes thatmetabolize free radicals The antioxidant enzymes includemanganese superoxide dismutase (MnSOD) copper andzinc (CuZn) SOD catalase and glutathione peroxidaseWhen the level of ROS exceeds the defense mechanismsa cell is said to be in a state of ldquooxidative stressrdquo [14] Theenhanced production of ROS during environmental stressescan pose a threat to cells by causing peroxidation of lipidsoxidation of proteins and damage to nucleic acids ultimatelyleading to death of the cells Thus dealing with excessiveROS may improve cellular metabolism and in consequenceincrease the lifespan It was also shown that metforminexerted intracellular antioxidant properties by decreasingROS production through the inhibition of protein kinase Cactivity [15] Recent studies indicate that its primary effect isinmitochondria where it interferes with respiratory complexI and reduces ATP production leading to the activation ofAMP kinase [16]

However the potential role of metformin in reducingROS in ASC is still not well established

It has also been reported that metformin can induceMC3T3-E1 osteoblastic cell differentiation and bone matrixsynthesis via AMPK activation and subsequent induction of

endothelial nitric oxide synthase (eNOS) and bone morpho-genetic protein-2 (BMP-2) expression [17 18]Metforminwasalso found to enhance rat osteoblasts proliferation increasealkaline phosphatase activity and increase the number offormed mineralized nodules Our group recently showedthat metformin in a dose-dependent manner affects theviability morphology and ultrastructure of mouse bonemarrow-derived multipotent mesenchymal stromal cells andthe Balb3T3 embryonic fibroblast cell line [19] We have alsodemonstrated that the expression of tandem gene H19IGF2as well as Oct4 a pluripotent stem cell marker in partcorrelates with particular dosage application of metformin invitro Additionally we found a significant correlation betweenthe activity of the ASCs and osteopontin at the mRNA andprotein level in vitro and in vivo The mounting evidenceindicates that the range of metformin actions may be signif-icantly wider than once thought and thus the application ofmetforminmay open new avenues in the treatment of variousmedical conditions [20 21]

Bone development is a complex and dynamic processinvolving several different mechanisms that maintain thebalance between bone formation and bone resorption Thisbalance might be affected by different factors including phys-ical activity endogenous hormones or drugs [22] Essentialto this balance and to the formation of the skeletal systemis proosteoblastic differentiation of bone marrow stem cellsosteoclast activity and the osteoblast-adipocyte relationshipRecently it has been demonstrated that the complementsystem plays an important role in both bone developmentand homeostasis and specifically influences osteoblast andosteoclast activity [23] Moreover it has been shown thatcomplement plays a role in the bone repair process thusmak-ing a bridge between skeletal and immunological systems

Bearing in mind that previous research has indicated aproosteogenic effect ofmetformin onBMSCs or commitmentcell lines in vitro we became interested in the effect ofmetformin on the osteogenic potential of mice as well asthe effects of metformin on bone in vivo It has been shownthat metformin prevents bone loss induced by ovariectomyin rats [24] suggesting that metformin confers a protectiveeffect against bone loss Further research on rats showedthat 2 weeks of metformin supplementation led to increasedtrabecular volume osteocyte density and osteoblast numberin femoral metaphysis [25] Wang et al [26] using insulin-resistantmice also showed that 6weeks ofmetformin admin-istration protects femoral bone architecture [26] Howeverlittle is known about the effects of metformin on bonetrabecular architecture and bone density in healthy mice

The aim of our study was to investigate the effects ofmetformin on ASCs osteogenic differentiation potential invitro and on bone density andmineralization in healthymice

2 Materials and Methods

All reagents used in this experiment were purchased fromSigma-Aldrich (Poland) unless indicated otherwise

















times 100 (1)
























3 Results




CD29+

CD44+

CD73+


CD105+

CD45minus







(a)


(b)


(c)



















1 2 5 700

05

15

20

23

1



PF

lowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150

200

250

lowast


(h)


(b)

00

02

04

06

08

10

12


CFU

-E (

)

lowast


(c)









DA

PID

API

Pha

lloid

in1s

t day

7th

day

lp

mvs

lp

fpfp

mvs

SEM

SEM

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)




lc

(I) (V)

dc

(II)

dc

(VI)

Cas-3Cas-3

(III) (VII)

Calc

ein-

AM

P Io

dide

Casp

ase-

3

120573-gal

120573-gal

(IV) (VIII)

120573-g

alac

tosid

ase

lc

Cas-3

120573-gal

(a)

0

5

10

15

20


()

lowastlowast

(b)0

5

10

15


() lowast

(c)





ROS

005

010

015

020ΔA


lowast

(a)

Nitric oxide (NO)

54

55

56

57

58

0

25

(120583M

mL)


lowastlowastlowast

(b)

SOD

0

5

10

15

Inhi

bitio

n (

)


lowast

(c)








Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150


(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l


bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15



(d)

0

20

40

60

80


(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15


wt (

)

lowastlowastlowast


(f)





012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































times 100 (1)
























3 Results




CD29+

CD44+

CD73+


CD105+

CD45minus







(a)


(b)


(c)



















1 2 5 700

05

15

20

23

1



PF

lowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150

200

250

lowast


(h)


(b)

00

02

04

06

08

10

12


CFU

-E (

)

lowast


(c)









DA

PID

API

Pha

lloid

in1s

t day

7th

day

lp

mvs

lp

fpfp

mvs

SEM

SEM

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)




lc

(I) (V)

dc

(II)

dc

(VI)

Cas-3Cas-3

(III) (VII)

Calc

ein-

AM

P Io

dide

Casp

ase-

3

120573-gal

120573-gal

(IV) (VIII)

120573-g

alac

tosid

ase

lc

Cas-3

120573-gal

(a)

0

5

10

15

20


()

lowastlowast

(b)0

5

10

15


() lowast

(c)





ROS

005

010

015

020ΔA


lowast

(a)

Nitric oxide (NO)

54

55

56

57

58

0

25

(120583M

mL)


lowastlowastlowast

(b)

SOD

0

5

10

15

Inhi

bitio

n (

)


lowast

(c)








Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150


(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l


bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15



(d)

0

20

40

60

80


(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15


wt (

)

lowastlowastlowast


(f)





012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























3 Results




CD29+

CD44+

CD73+


CD105+

CD45minus







(a)


(b)


(c)



















1 2 5 700

05

15

20

23

1



PF

lowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150

200

250

lowast


(h)


(b)

00

02

04

06

08

10

12


CFU

-E (

)

lowast


(c)









DA

PID

API

Pha

lloid

in1s

t day

7th

day

lp

mvs

lp

fpfp

mvs

SEM

SEM

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)




lc

(I) (V)

dc

(II)

dc

(VI)

Cas-3Cas-3

(III) (VII)

Calc

ein-

AM

P Io

dide

Casp

ase-

3

120573-gal

120573-gal

(IV) (VIII)

120573-g

alac

tosid

ase

lc

Cas-3

120573-gal

(a)

0

5

10

15

20


()

lowastlowast

(b)0

5

10

15


() lowast

(c)





ROS

005

010

015

020ΔA


lowast

(a)

Nitric oxide (NO)

54

55

56

57

58

0

25

(120583M

mL)


lowastlowastlowast

(b)

SOD

0

5

10

15

Inhi

bitio

n (

)


lowast

(c)








Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150


(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l


bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15



(d)

0

20

40

60

80


(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15


wt (

)

lowastlowastlowast


(f)





012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CD29+

CD44+

CD73+


CD105+

CD45minus







(a)


(b)


(c)



















1 2 5 700

05

15

20

23

1



PF

lowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150

200

250

lowast


(h)


(b)

00

02

04

06

08

10

12


CFU

-E (

)

lowast


(c)









DA

PID

API

Pha

lloid

in1s

t day

7th

day

lp

mvs

lp

fpfp

mvs

SEM

SEM

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)




lc

(I) (V)

dc

(II)

dc

(VI)

Cas-3Cas-3

(III) (VII)

Calc

ein-

AM

P Io

dide

Casp

ase-

3

120573-gal

120573-gal

(IV) (VIII)

120573-g

alac

tosid

ase

lc

Cas-3

120573-gal

(a)

0

5

10

15

20


()

lowastlowast

(b)0

5

10

15


() lowast

(c)





ROS

005

010

015

020ΔA


lowast

(a)

Nitric oxide (NO)

54

55

56

57

58

0

25

(120583M

mL)


lowastlowastlowast

(b)

SOD

0

5

10

15

Inhi

bitio

n (

)


lowast

(c)








Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150


(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l


bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15



(d)

0

20

40

60

80


(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15


wt (

)

lowastlowastlowast


(f)





012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


(b)


(c)



















1 2 5 700

05

15

20

23

1



PF

lowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150

200

250

lowast


(h)


(b)

00

02

04

06

08

10

12


CFU

-E (

)

lowast


(c)









DA

PID

API

Pha

lloid

in1s

t day

7th

day

lp

mvs

lp

fpfp

mvs

SEM

SEM

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)




lc

(I) (V)

dc

(II)

dc

(VI)

Cas-3Cas-3

(III) (VII)

Calc

ein-

AM

P Io

dide

Casp

ase-

3

120573-gal

120573-gal

(IV) (VIII)

120573-g

alac

tosid

ase

lc

Cas-3

120573-gal

(a)

0

5

10

15

20


()

lowastlowast

(b)0

5

10

15


() lowast

(c)





ROS

005

010

015

020ΔA


lowast

(a)

Nitric oxide (NO)

54

55

56

57

58

0

25

(120583M

mL)


lowastlowastlowast

(b)

SOD

0

5

10

15

Inhi

bitio

n (

)


lowast

(c)








Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150


(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l


bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15



(d)

0

20

40

60

80


(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15


wt (

)

lowastlowastlowast


(f)





012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 2 5 700

05

15

20

23

1



PF

lowast

lowast

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150

200

250

lowast


(h)


(b)

00

02

04

06

08

10

12


CFU

-E (

)

lowast


(c)









DA

PID

API

Pha

lloid

in1s

t day

7th

day

lp

mvs

lp

fpfp

mvs

SEM

SEM

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)




lc

(I) (V)

dc

(II)

dc

(VI)

Cas-3Cas-3

(III) (VII)

Calc

ein-

AM

P Io

dide

Casp

ase-

3

120573-gal

120573-gal

(IV) (VIII)

120573-g

alac

tosid

ase

lc

Cas-3

120573-gal

(a)

0

5

10

15

20


()

lowastlowast

(b)0

5

10

15


() lowast

(c)





ROS

005

010

015

020ΔA


lowast

(a)

Nitric oxide (NO)

54

55

56

57

58

0

25

(120583M

mL)


lowastlowastlowast

(b)

SOD

0

5

10

15

Inhi

bitio

n (

)


lowast

(c)








Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150


(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l


bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15



(d)

0

20

40

60

80


(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15


wt (

)

lowastlowastlowast


(f)





012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















DA

PID

API

Pha

lloid

in1s

t day

7th

day

lp

mvs

lp

fpfp

mvs

SEM

SEM

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)




lc

(I) (V)

dc

(II)

dc

(VI)

Cas-3Cas-3

(III) (VII)

Calc

ein-

AM

P Io

dide

Casp

ase-

3

120573-gal

120573-gal

(IV) (VIII)

120573-g

alac

tosid

ase

lc

Cas-3

120573-gal

(a)

0

5

10

15

20


()

lowastlowast

(b)0

5

10

15


() lowast

(c)





ROS

005

010

015

020ΔA


lowast

(a)

Nitric oxide (NO)

54

55

56

57

58

0

25

(120583M

mL)


lowastlowastlowast

(b)

SOD

0

5

10

15

Inhi

bitio

n (

)


lowast

(c)








Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150


(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l


bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15



(d)

0

20

40

60

80


(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15


wt (

)

lowastlowastlowast


(f)





012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















lc

(I) (V)

dc

(II)

dc

(VI)

Cas-3Cas-3

(III) (VII)

Calc

ein-

AM

P Io

dide

Casp

ase-

3

120573-gal

120573-gal

(IV) (VIII)

120573-g

alac

tosid

ase

lc

Cas-3

120573-gal

(a)

0

5

10

15

20


()

lowastlowast

(b)0

5

10

15


() lowast

(c)





ROS

005

010

015

020ΔA


lowast

(a)

Nitric oxide (NO)

54

55

56

57

58

0

25

(120583M

mL)


lowastlowastlowast

(b)

SOD

0

5

10

15

Inhi

bitio

n (

)


lowast

(c)








Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150


(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l


bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15



(d)

0

20

40

60

80


(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15


wt (

)

lowastlowastlowast


(f)





012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ROS

005

010

015

020ΔA


lowast

(a)

Nitric oxide (NO)

54

55

56

57

58

0

25

(120583M

mL)


lowastlowastlowast

(b)

SOD

0

5

10

15

Inhi

bitio

n (

)


lowast

(c)








Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150


(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l


bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15



(d)

0

20

40

60

80


(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15


wt (

)

lowastlowastlowast


(f)





012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Days of culture

Metformin 8 weeks

lowastlowastlowast

lowastlowastlowast

(a)

0

50

100

150


(h)

lowast


(b)

Met

form

in 8

wee

ksC

ontro

l


bn

bn

(I) (II) (III)

(IV) (V) (VI)

(c)

0

5

10

15



(d)

0

20

40

60

80


(120583m

)

lowastlowastlowast


(e)

Ca P Ca P0

5

10

15


wt (

)

lowastlowastlowast


(f)





012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















012345

2526272829

Osteopontin

OPN

(ng

mL)

lowastlowastlowast


(a)

0

20

40

60

80 Osteocalcin

OCL

(ng

mL)

lowastlowast


(b)

0

10

20

30

40

50 BMP2

BMP2

(ng

mL)


(c)

10

11

12Osteopontin

0

02Qn

(oste

opon

tinB

2M)

lowast


(d)

058

060

062

BMP-2

0

02

063

Qn

(BM

P-2

B2M

)

lowastlowast


(e)



2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















2 4 7 9 1206

08

10

12


Days of culture

PF


0

50

100

150

200


(h)

lowast


(b)


(I) (II) (III)

(IV) (V) (VI)

Met

form

in 8

wee

ksC

ontro

l

(c)

0

10

20

30 Oil Red O

Stai

ned

area

()

lowastlowast


(d)

07

08

09 Adiponectin

Qn

(AD

PQG

APD

H)

lowastlowast


(e)



4 Discussion



(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(I)

(III) (IV)

(II)

(a)


0 2 4 6 8 10 12 1460

80

100

120

140

160

180

200


Mea

n br

ight

ness

(b)


2 4 6 8 10 12 14 16 18000002004006008010012014


Con

trib

utio

n

(c)

8 10 12 14 16 18

Phosphorus content

8

10

12

14

16


wt (

)

(d)

wt (

)

Calcium content

15

20

25

30

35


(e)


0002004006008

01012014

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Poro

sity

Mean bone porosity


(f)




















5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































5 Conclusions


Ethical Approval


Competing Interests


Acknowledgments



References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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