Research Article Physical Activity Increases the Total

Research ArticlePhysical Activity Increases the Total Number ofBone-Marrow-Derived Mesenchymal Stem Cells Enhances TheirOsteogenic Potential and Inhibits Their Adipogenic Properties

Monika Marwdziak12 Agnieszka Umieszek23 Klaudia Chrzdstek3

Katarzyna Basinska3 and Krzysztof Marycz23

1Faculty of Veterinary Medicine University of Environmental and Life Sciences Norwida 31 Street 50-375 Wroclaw Poland2Wroclaw Research Centre EIT+ Stablowicka 147 Street 54-066 Wroclaw Poland3Faculty of Biology University of Environmental and Life Sciences Kozuchowska 5b Street 50-631 Wroclaw Poland

Correspondence should be addressed to Monika Marędziak monikamaredziakgmailcom

Received 5 March 2015 Revised 12 May 2015 Accepted 18 May 2015

Academic Editor Christian Dani

Copyright copy 2015 Monika Marędziak 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

Aging and sedentary lifestyle are common nowadays and are associated with the increasing number of chronic diseases Thusphysical activity is recommended as one of three healthy behavior factors that play a crucial role in health prophylaxis In the presentstudy we were interested whether physical activity influences the number and potential of bone-marrow-derived mesenchymalstem cells BMMSCs In this study four-week-oldmale C57Bl6mice were trained on a treadmill at progressive speeds over a 5-weekperiod Comparisons made between exercised (EX) and sedentary animal groups revealed (i) significantly higher number of MSCsin EX animals (ii) elevated alkaline phosphatase (ALP) activity (iii) increased level of osteopontin (OPN) and osteocalcin (OCL)and (iv) reduced marrow cavity fat The results obtained support the thesis that EX may play a substantial role in the regenerationof mesenchymal tissues Therefore EX may represent a novel nonpharmacological strategy of slowing down age-related decline ofthe musculoskeletal functions

1 Introduction

Bone marrow (BM) contains various populations of devel-opmentally early stemprogenitor cells including endothelialprogenitor cells (EPCs) very small embryonicepiblast-likestem cells (VSELs) hematopoietic stem cells (HSCs) andmesenchymal stem cells (MSCs) [1] MSCs are multipotentcells present in adult bone marrow umbilical vein andadipose tissue that possess capacity to proliferate and dif-ferentiate into various mesenchymal cell lineages includingadipocytes chondrocytes and osteocytes [2ndash4] These stemcell populations are defined by the expression of variousmolecules including CD90 CD105 and CD73 and theabsence of markers such as CD34 or CD45 [5] The combi-nation of MSC differentiation potential and their paracrineeffects makes them an attractive candidate for tissue repair

and regenerative medicine MSCs significantly influencethe regenerative process through various mechanisms forexample synthesis and secretion of membrane derived vesi-cles (MVs) [6 7] Increasing evidence has shown that theclinical application of bone-marrow-derived MSCs in somediseases leads to a successful regeneration of the damagedtissue [8] The mechanisms increasing the number of MSCsin marrow are still poorly understood Therefore searchingfor efficient nonpharmacological methods that would signifi-cantly increase the total number ofMSCs in the bonemarrowmay represent a helpful strategy for tissue regeneration

Active lifestyle provides many physiological as well aspsychological benefits Many diseases such as heart diseaseand type II diabetes have been reported to be associated withlimited physical activity Thus different types of exercises arerecommended as a prophylactic measure for example highly

Hindawi Publishing CorporationStem Cells InternationalVolume 2015 Article ID 379093 11 pageshttpdxdoiorg1011552015379093

2 Stem Cells International

aerobic endurance training program or moderate exercisesEach of them seems to act at different physiological levelsincluding improvement of immune andor endocrine systemAlthough there has been little research conducted concerningthe explanation of the mechanism of the positive effect ofexercises on health it has been demonstrated that exercisespositively correlate with increased content of red blood cells[9 10] and enhanced disease resistance via improved immunefunction [11] In turn moderate exercise has been reported asa factor that can boost immune function [12] while intenseexercise can reduce immune response causing a decreasein lymphocyte concentration natural killer cell activity andlymphocyte proliferation [13] In addition it was found thatthe exercise significantly reduces apoptosis and improvesviability of osteocytes as investigated using an osteopenicrat model [14] It was observed in adolescent individualsthat intensive exercises increased bone mass in the lumbarspine and femur [15] Further it appears that exercisesthrough biomechanical stimulation of the bone wall mayplay a prevention role in bone resorption and formation[16]

The collective data suggests that physical exercises rep-resent a nonpharmacological factor that positively affectslongevity prevention of metabolic diseases and stem cellmobilization [17ndash19]Therefore in the present study we havefocused on (i) quantitative analysis of the total number ofMSCs in the bone marrow (ii) quantitative analysis of earlyand late markers of osteogenesis in osteoblasts precursorsand (iii) evaluation of femur mineralization process in miceexposed to endurance exercise training

2 Materials and Methods

21 Animals and Exercise Training Protocol Sedentary (SED)and exercise-trained (EX) C57Bl6 mice (4 weeks old) werekept three per cage in an ultraclean facility on ventilated rackshoused in the Animal Experimental Laboratory (WroclawMedical School Norwida 34 Poland) Mice were maintainedon a 12 h light-dark cycle at 22 plusmn 02∘C The experiment wasconducted with the consent of the Local Ethics Committeefor Animal Experiments The animals were allocated toexperimental groups (6 animals per group) divided intosedentary animals which did not undergo physical activity(SE) and animals that exercised endurance (EX) Animalswere exercise-trained (119899 = 6) 3 days per week (dwk)(Monday Wednesday and Friday) for 5 weeks using anExer 36 Treadmill (Columbus Instruments Columbus OHUSA) Afterwards 5-wk progressive exercise protocol wasused The training started at 14 metersmin for 45min (wk 1)and was gradually increased to 24metersmin for 45min (wk5) The training portion of the protocol was always precededby a 10min warm-up at 10metersmin followed by a 5mincool-down at 10metersmin as described previously [20]Sedentary control mice (119899 = 6) were exposed to the treadmillbut not subjected to training Sampling was carried out afterthe last training period Bones (both femurs and tibiae)were collected dissected of muscle and fat and flushed with1mL of Iscoversquos modified Dulbecco medium (Sigma-Aldrich

Munich Germany) with 2 FBS (Invitrogen Carlsbad CAUSA) for bone marrow cell isolation

22 Preparation ofMSCs for FACS MurineMNCs (mononu-clear cells) were isolated from the BM of pathogen-free4-week-old C57BL6 mice Bone marrow cell suspensionsisolated by flushing femurs and tibiae were lysed in BDlysing buffer (BD Biosciences San Jose CA USA) for15min at room temperature and washed twice in phosphate-buffered saline (PBS) The following anti-mouse antibod-ies (BD Pharmingen) were used for staining anti-CD45(allophycocyanin-Cy7 clone 30F11) anti-CD45RB220 (PEclone RA-6B2) anti-Gr-1 (PE clone RB6-8 C5) anti-T-cellreceptor-120572120573 (PE clone H57-5970) anti-T-cell receptor-120574120575(PE clone GL3) anti-CD11b (PE clone M170) anti-Ter119(PE clone TER-119) and anti-Ly-6AE (also known as Sca-1 biotin and clone E13-1617 with streptavidin conjugated toPE-Cy5) MSCs (Sca-1+LinminusCD45minusCD31minusCD51+) wereisolated by the multiparameter live-cell sorting (INFLUXBD) as described previously [21] Monoclonal antibodies(mAbs) used in this study were added at saturating con-centrations The cells were incubated for 30 minutes on icewith different anti-mouse monoclonal antibodies washedtwice and resuspended in staining buffer at a concentrationof 5 times 106 cells per milliliter Samples were analyzed on aLSR II flow cytometer (BD Biosciences Mountain View CAUS) The following anti-mouse mAbs were purchased fromBD Pharmingen (San Diego CA) Ly-GAE (Sca-1 FITCclone D7) lineage marker (CD45R (also known as B220)PE clone RA3-6B2) Ly-6GLy-6C (PE clone RB6-8C5) T-cell receptor b (PE clone H57-597) T-cell receptor cd (PEclone GL3) CD11b (PE cloneM170) Ter119 (PE clone TER-119) CD51 (biotin cloneRMV-7with streptavidin conjugatedto PE-Cy5 as secondary Ab) CD31 (APC clone 390) andCD45 (APC-Cy7 clone 30-F11)The results are representativeof three independent experiments

23 Multipotency Assay Cells were seeded in 24-well platesat a concentration of 2 times 104 cells per well and subsequentlyadipogenic chondrogenic and osteogenic differentiationswere performed Stimulation of MSCs was performed usingStemPro Adipogenesis Chondrogenesis and OsteogenesisDifferentiation Kits (Life Technologies Poland) (StemProAdipogenesis Differentiation Kit StemPro ChondrogenesisDifferentiation Kit and StemPro Osteogenesis Differentia-tion Kit) according to the manufacturersrsquo instruction Dif-ferentiations began 24 h after inoculation and cells werecultured for 14 days Media were changed every three daysThe stage of adipogenic chondrogenic and osteogenic dif-ferentiation of MSCs was assessed using specific stainingOil Red O (Sigma-Aldrich Munich Germany) for detectingneutral lipid deposits Safranin O (Sigma-Aldrich MunichGermany) for glycosaminoglycans staining andAlizarin Red(Sigma-Aldrich Munich Germany) for calcium depositsPreparations were analyzed using Axio Observer A1 invertedmicroscope (Axio Observer A1 Carl Zeiss Jena Germany)while the documentationwasmade usingCannonPowerShotcamera

Stem Cells International 3

24 CFU-Fs Assay Assays for colony-forming unit fibrob-lasts (CFU-Fs) were prepared according to the protocoldescribed previously by Baker et al [20] MSCs derivedfrom bone marrow (200 cells) of exercising (119899 = 6) andsedentary animals (119899 = 6) were plated in collagen-coated35mm tissue culture plates The cultures were supplementedwith 120572-MEM with 15 FBS penicillin and streptomycinand the cells were maintained in culture for 14 d Next thecells were fixed with 4 paraformaldehyde for 10min andsubsequently stainedwith 05 crystal violet (Sigma-AldrichMunich Germany) in 20methanol for 5min Only coloniesconsisting of 20 aggregated cells or more were counted asCFU-Fs All experiments were performed in triplicate

25 Histology and Immunohistochemistry Both humeri of allanimals were fixed (4 formaldehyde in PBS 48 h Sigma-Aldrich) decalcified by EDTA (Osteosoft Merck Millipore)at 37∘C for 5 days and rinsed with PBS All samples weredehydrated in increasing ethanol gradient of 50 60 7080 96 and 100 Then the samples were embeddedin paraffin as previously described [22] The samples weresectioned transversely (HM 340E Microm) at a thicknessof 10 120583m dried rehydrated in alcohol series and stainedwith hematoxylin and eosin (Thermo Scientific LutterworthUK) Next the samples were dehydrated and mounted undera coverslip The slides were examined by light microscopy(Axio Zeiss) The thickness of the endosteum was measuredand normalized at photo area For immunohistochemistry6 120583m paraffin-embedded sections were placed on adhesiveplates and dried at 37∘C for 24 hours Samples were thendeparaffinized in xylene and rehydrated in alcohol serieswith decreasing concentrationsThe EnVision System (Dako)was used to visualize the antigen-antibody reaction Rabbitanti-CD105 (Abcam Cambridge UK) primary monoclonalantibodies were used All antibodies were diluted accordingto the manufacturerrsquos instructions Images were taken usingCannon PowerShot Camera

Quantification of CD105 positive staining was performedusing computer-assisted image analyses with a Java-basedopen source image processing software ImageJ v148c Imageanalysis was performed on six randomly chosen previouslycaptured images The analysis included color thresholdmethod binary conversion and measurement of the activeareas using measure plugin and expressed as percentage ofpositive staining per image

26 Osteogenic and Adipogenic Differentiation of MSCs Forosteogenesis half of the MSC plates were used After thefirst passage the cells were placed in 24-well culture platesat a concentration of 15 times 105 cells per well and culturedin osteogenicmedium (StemProOsteogenesisDifferentiationKit Life Technologies) Osteogenic differentiation began24 h after inoculation and was completed 21 days laterCulture media were changed every three days In additionto osteogenic differentiation adipogenesis assay was alsoperformed The cells were maintained in 120572-MEM with 15FBS penicillin and streptomycin for 14 days To quantify

adipogenesis cells were stained with Oil Red O (Sigma-Aldrich Munich Germany) eluted with isopropanol Rep-resentative images were captured using Axio Observer A1(Zeiss Oberkochen) For quantitative analysis absorbancewas measured at 490 nm following destaining with 100ethanol for 20min

27 Scanning Electron MicroscopyEnergy Dispersive X-RaySpectroscopy (SEM-EDX) Analysis Humeri were fixed in25 glutaraldehyde for one hour washed in distilled waterdehydrated in a graded ethanol series sputtered with gold(ScanCoat 6 Oxford) and imaged using a scanning electronmicroscope (EVO LS15 Zeiss) at 10 kV of filamentrsquos tensionAdditionally specimens were prepared for EDX analysisaccording to the method described previously by our labora-tory [23 24] Observations were performed at the filamentrsquostension of 10 kVThedetection ofmineralizedmatrix was per-formed using SEM combined with EDX (Bruker CoventryUK) by analyzing the surface distribution of calcium andphosphorus The values obtained were presented as weightpercentage (wt)

28 Alizarin Red S Staining Quantitative Alkaline Phos-phatase (ALP) Osteocalcin (OCL) and Osteopontin (OPN)Assay Alizarin Red S staining was performed to evaluate theformation of calcium deposits Briefly after a 21-day period ofosteogenic differentiation cells were fixed in 10 formalin for1 h at room temperature followed by washing with distilledwater and incubation for 5min in the working solutionof Alizarin Red S After staining cells were washed threetimes in distilled water and calcium deposits were observedunder an inverted microscope (Axio Observer A1 Zeiss)Photographs were acquired using Cannon PowerShot digitalcamera For quantitative analysis of Alizarin Red stainingplates were incubated with 10 acetic acid for 30min andthen the contents of the wells were collected into 15 mLsample tubes mixed heated to 85∘C for 10 minutes cooledon ice for 5 minutes and pH-neutralized with 10 ammo-nium hydroxide After the spin-down (1200timesg 15min) theabsorption of the supernatant at 405 nm wavelength wasmeasured using a microplate reader (SPECTROstar NanoBMG Labtech) Extracellular activity of ALP was determinedin the supernatants collected on the last day of cell incuba-tion The assay was performed using Alkaline PhosphataseColorimetric Assay Kit (Abcam Cambridge UK) accordingto the protocol provided by the manufacturer Experimentalsamples were prepared in duplicate and diluted twofold Acontrol sample of the background was included and thebackground was corrected by subtracting the values derivedfrom the zero standards from all standards samples and thecontrol sample p-nitrophenyl phosphate (pNPP) was usedas a substrate for phosphatase The substrate was hydrolyzedinto p-nitrophenol by alkaline phosphatase

The reaction productwasmeasured at 405 nmwavelengthusing a microplate reader (BMG Labtech) Sample readingswere applied to the standard curve to obtain the amountof pNP generated by the ALP sample Enzymatic activitywas determined using the following formula ALP activity


(UmL)=119860119881119879 where (i)119860 is the amount of pNPgeneratedby the samples (in nmol) (ii)119881 is the volume of sample addedto the assay well (in mL) and (iii) 119879 is the reaction time

The levels of osteocalcin (OCN) and osteopontin (OPN)were determined in the supernatants after 21 d of cell cul-ture The medium was collected in three separate repli-cates Prior to the protein level measurements samples werethoroughly mixed In order to determine the concentrationof extracellular proteins specific ELISA kits were used (i)Rat Gla-Osteocalcin High Sensitive ELISA Kit (Takara BioEurope Saint-Germain-en-Laye France) and (ii) MouseRatOsteopontin Quantikine ELISA Kit (RampD Systems UK)Quantitative determination ofOCNandOPNwas performedaccording to the manufacturersrsquo instructions The amount ofproteins detected was expressed as a ratio of proteinmass andsupernatant volume (wv)

29 Statistical Analysis Statistical analysis was performedusing Statistica 90 software (StatSoft Polska Sp z ooKrakow Poland) The significance of differences between theresults obtained was evaluated using the unpaired 119905-test orone-way ANOVAwith Fisherrsquos post hoc testThe results wereconsidered significant at 119901 le 005 All data were presented asmean plusmn SD

3 Results

31TheTotal Number ofMSCs Isolated fromBoneMarrow andClonogenic Potential In order to confirm cellsrsquo mesenchymalorigin the criteria for definition of MSCs were fulfilled(i) typical plastic adherent growth (ii) expression of MSCsmarkers and absence of surface hematopoietic markers and(iii) in vitro differentiation potential toward chondrogenicosteogenic and adipogenic lineage (Figure 1)

The number of colonies formed byMSC progenitors after21 days of culture was significantly higher in endurance-trained animals in comparison to the sedentary groupExercises resulted in the formation of 21 MSC colonies (21 plusmn2 119901 le 005) per well while in sedentary group 16 colonieswere observed (16 plusmn 3 119901 le 005) (Figure 2)

The number of MSCs isolated after endurance exerciseswas significantly elevated (4 plusmn 2 lowast 106 119901 le 005) in com-parison to the MSCs isolated from sedentary animals (18 plusmn2 lowast 10

6 119901 le 005) Moreover the immunohistochemicalstaining to detect CD105+ cells revealed more intensivereaction in endurance-trained animals (Figures 3(b) and 3(c))in comparison to the sedentary group (Figures 3(a) and 3(c))

32 The Concentration of Calcium and Phosphorus in theBoneWall SEM-EDXanalysiswas performed to estimate theeffect of EX on the bone mineralization Quantitative EDXanalysis demonstrated that the concentration of Ca and P inthe bone wall of sedentary animals was 42 higher (26wt plusmn6 119901 le 005) when compared to the exercised group (11 wt plusmn2 119901 le 005) The data are presented in Figure 4

33 MSC Osteogenic Potential A comparative analysisbetween both groups was performed to investigate whether

EX exerted an effect on the osteogenic differentiation poten-tial We analyzed early and late markers of osteogenesis aswell as Ca and P concentrations on the surface of osteoblastprecursors A significantly higher level of alkaline phos-phatase (ALP) was detected in endurance-trained animals(19 uUmL plusmn 1 119901 le 005) when compared to the sedentarygroup (16 uUmL plusmn 1 119901 le 005) (Figure 5(a)) A similarcorrelation was observed in the level of osteopontin (OPN)The highest OPN level equal to 48 plusmn 2 ngmL was recordedin endurance-trained animals while in sedentary group itamounted to 32 plusmn 2 ngmL (Figure 5(b)) This tendency wasalso retained with respect to osteocalcin (OCL) level in bothgroups investigated Higher OCL level (170 plusmn 13 ngmL 119901 le005) was detected in the exercised group compared with thesedentary group (130 plusmn 14 ngmL 119901 le 005) (Figure 5(c))Data obtained strongly correlated with quantitative analysisof Ca and P performed using SEM-EDX A higher concentra-tion of calcium (14 plusmn 1 wt 119901 le 005) and phosphorus (6 plusmn2wt119901 le 005) was observed in the exercised group in com-parison to the sedentary group where Ca and P were equal to9 plusmn 2wt and 3 plusmn 06wt respectively (119901 le 005) (Figures5(d) and 5(e)) Alizarin Red staining revealed significantdifferences (119901 le 005) in the MSC mineralization process inboth groups tested A considerably stronger reaction and thehighest percentage of absorbed Alizarin Red were observedin exercised animals (81 plusmn 9) (Figures 6(b) and 6(c)) whencompared to sedentary individuals (56 plusmn 3) (Figures 6(a)and 6(c))

34 ReducedNumber of Adipocytes in the BoneMarrowCavityand Inhibition of MSC Adipogenic Potential Picture of themarrow cavity was investigated using hematoxylin and eosin(H+E) staining Larger deposits of fat droplets were observedin sedentary animals in the marrow cavity (Figures 7(a)and 7(d)) when compared to the exercising animals (Figures7(b) and 7(e)) Quantitative evaluation of the total numberof adipocytes in the marrow cavity revealed that sedentaryanimals had 55 more of adipocytes (20 plusmn 3 119901 le 005)compared to the exercised animals (11 plusmn 2 119901 le 005)(Figure 7(f)) Moreover the thicker bone wall (328 120583m plusmn 36)was observed in exercising animals when compared to thecontrol individuals (223120583m plusmn 14 119901 le 005)

The adipogenic potential of MSCs isolated from seden-tary and endurance-trained animals was evaluated usingstandard MSC adipogenesis-inducing protocol Oil Red Ostaining revealed more extensive adipogenesis in sedentaryanimals in comparison to the exercised group (Figures 6(d)and 6(e)) Additionally it was found that the adipogenicdifferentiation potential of MSCs derived from endurance-trained animals was decreased based on the lower percentageof absorbed Oil Red O dye (17 plusmn 3 119901 le 005) compared tosedentary group (46 plusmn 3 119901 le 005) (Figure 6(f))

4 Discussion

In recent years much attention has been paid to the develop-ment of strategies aimed at slowing down aging process andits consequences Aging and sedentary lifestyle contribute tothe reduction of bone quantity and quality decreased muscle


00

200

200

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400

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600

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800

1000

1000

SSC-

A

FSC-A

100

100

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101

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102

103

103

PE-L

in

PE Cy5-Sca-1

0

200

400

600

800

1000

100

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102

103

SSC-

A

APC-Cy7 CD45

100

100

101

101

102

102

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103

APC

CD31

PE-Cy5 CD51

(a)

400120583m

Standard culture

400120583m

Chondrogenic stimulation

400120583m

Osteogenic stimulation

400120583m

Adipogenic stimulation

(b)

Figure 1 Analysis of MSC (a) phenotypes using flow cytometry The FACS profile of separated Sca-1 positive and Lin-negative cellsdemonstrating CD31negative CD45negative and CD51positive MSCs (a) (b) Morphology of MSCs cultured in DMEM medium supplementedwith 10 of FBS (standard culture) and the results of its chondrogenic osteogenic and adipogenic stimulation For visualization ofglycosaminoglycans and calcium deposits specific Safranin O and Alizarin Red staining were used respectively while lipid droplets werestained using Oil Red O

0

5

10

15

20

25

CFU-Fs

Col

ony

num

ber

Sedentary Exercise

lowastp lt 005

(a) (b) (c)

Figure 2 The number of MSC colony-forming units (CFU-Fs) (a) Representative images of stained cells collected from sedentary (b) andexercised (c) groups

mass and strength and weakened postural stability culmi-nating in an elevated risk of skeletal fractures Regenerativeability of an individual strongly correlates with the numberof stem cells that can serve as a backup population for therepairrejuvenation of damaged tissue [21 24] Therefore

seeking nonpharmacological methods that will increase thenumber of stem cells of different origin seems to be a majormedical challenge

Mechanical signals affect bone marrow-derived stem cellpopulations particularly mesenchymal stem cells (MSCs)


100120583m

(a)

100120583m

(b)

0

5

10

15

20

25

CD105 positive cells ()

Sedentary Exercise

lowastp lt 005

()

(c)

Figure 3 (a b) Immunohistochemical stainning the CD105+ cells stained immunohistchemically in the sedentary group (a) and exercised (b)group Positive reactions are indicated by red arrows Quantification of CD105 positive cells results representative of six randomly choosenpictures are expressed as mean plusmn SD (lowast119901 value lt 005)

0

10

20

30

Calcium and phosphorus in bone

Con

tent

()

Sedentary Ca Exercise Ca Sedentary P Exercise P

lowastp lt 005

lowastp lt 005

Figure 4The concentration of calcium and phosphorus in the boneafter endurance exercise training Statistical significance (119901 lt 005)

which has a significant impact on the bone and fat morphol-ogy [25] In this study we have shown that the endurance

exercise training (EX) increases the total number of mes-enchymal stemprogenitor cells (MSCs) in the bone marrowcavity Moreover in our previous study we have demon-strated that EX increased the total number of VSELs circulat-ing in PB and residing in BM Thus we could speculate thatEXmay have beneficial effects on the expansion of both earlydevelopmentally stem cells (VSELs) and stemprogenitorcells that might play an important role in tissue and organrejuvenation As previously reported circulating stem cellsare involved in repairing minor exercise-related tissue andorgan injuries [25] Moreover we have found that EXenhances the osteogenic potential of MSCs which is consis-tent with the results of Baker et al [20] The latter authorgroup reported that EX enhances hematopoiesis due tothe increased endocrine signaling from skeletal muscleandremodeling of the medullary hematopoietic niche

Given the fact that the boneniches are inhabited byMSCstheir number and differentiation potential appear to be acrucial factor in the context of body regenerative needsMSCspresent in bone marrow provide microenvironmental sup-port for hematopoietic stem cells (HSCs) and can additionallydifferentiate into various mesodermal lineages [21 26] Thepresent study demonstrates that the physical activity canincrease the total number of bone-marrow-derivedMSCsAnincreased number of MSCs in the bone marrow was stronglycorrelated with a higher number of fibroblast colony-forming


lowastp lt 005

0

5

10

15

20

Exercisecontrolosteogenesis

ExerciseSedentaryosteogenesis

Sedentarycontrol

ALP

ALP

(120583U

mL)

(a)

lowastp lt 005

0

5

1030

35

40

45

50

55



Sedentarycontrol

Osteopontin

Oste

opon

tin (n

gm

L)

(b)

lowastp lt 005

0

10

120

140

160

180



Sedentarycontrol

Osteocalcin

Oste

ocal

cin

(ng

mL)

(c)

lowastp lt 005

0

5

10

15



Sedentarycontrol

Calcium in culture

Con

tent

()

(d)

lowastp lt 005

0

1

2

3

4

5

6

7



Sedentarycontrol

Phosphorus in culture

Con

tent

()

(e)

Figure 5 The activity of ALP (a) and the concentration of OPN (b) OCL (c) calcium (d) and phosphorus (e) measured on the 21st day ofculture in osteogenic medium Statistical significance (119901 lt 005)


100120583m

(a)

100120583m

(b)

Alizarin Red absorption ()

Sedentary Exercise

lowastp lt 005

()

0

20

40

60

80

100

(c)

100120583m

(d)

100120583m

(e)

Oil Red O absorption ()

Sedentary Exercise

lowastp lt 005

()

0

20

40

60

(f)

Figure 6 Alizarin Red staining of osteoblasts derived fromMSCs collected from sedentary (a) and exercised (b) individuals and quantity ofabsorbed dye (c)(d e and f) Representative images of 21 d Oil RedO stained and quantified and differentiatedMSCs isolated from sedentary(d) and exercise-trained (e) animals Percentage of Oil Red O absorption (f) cells derived from sedentary and exercised animals Statisticalsignificance (119901 lt 005)

units (CFU-Fs) in exercised animals Similar results wereobtained by Baker at al [20] who reported a higher numberof CFU-Fs of MSCs in trained animals In the current workwe have found that EXdecreases the amount of fat in the bonemarrow cavityWehave noticed a lower number of adipocytesin the bone marrow cavity in endurance-trained individualswhen compared to sedentary animals Our data is consistentwith findings of other research groups that have also observeda decreased number of adipocytes in exercised animals andhumans [20] It is well known that the fatty bone marrow hasa negative effect on hematopoiesis This is due to not onlythe occupation of marrow space but also the protein factorsreleased by adipocytes such as neuropilin-1 [27] lipocalin2 [28] or adiponectin [29] which can significantly impairhematopoietic proliferation

In the perspective of stem cellsrsquo application in the clinicalpractice it is important not only to increase hematopoiesisbut also to achieve and maintain an adequate amount andquality of bone tissue Interestingly in our study osteoblastprecursors (OBs) exhibited a higher osteogenic potential in

endurance-trained animals when compared to the seden-tary group We have found a higher activity of alkalinephosphatase (ALP) an early marker of osteogenesis as wellas elevated levels of late markers of osteogenesis that isosteopontin (OPN) and osteocalcin (OCL) Furthermore itwas found that endurance training enhanced the processof mineralization in OBs Higher calcium (Ca) and phos-phorus (P) levels were detected in OBs derived from MSCsof exercised animals which highlighted the efficiency ofosteogenesis in vitro In contrast a lower level of Ca and Pwas observed in the humerus of trained animals This mightbe the result of Ca and P release to the peripheral bloodduring exercises which has been previously reported by otherauthors [30] Changes observed in differentiation potential aswell as the mineralization process of OBs derived from EXanimals might be the result of mechanical strain stimulus Itwas reported that the mechanical forces applied to the boneduring treadmill training program activated extracellularsignal-regulated kinase pathway (ERK12) which resultedin a more mature osteogenic phenotype of MSCs [20]


(a) (b)

The thickness of the endosteum

Sedentary Exercise

lowastp lt 005

0

100

200

300

400

Wid

th (120583

m)

(c)

100120583m

(d)

100120583m

(e)

Number of adipocytes

Num

ber o

f adi

pocy

tes

Sedentary Exercise

lowastp lt 005

0

5

10

15

20

25

(f)

Figure 7 The morphology of the bone marrow cavity and endosteum in sedentary (a) and exercised animals (b) Representative imagestaken in the central area of the marrow cavity of bones stained with hematoxylin and eosin from sedentary (d) or exercise-trained animals(e) The thickness of murine endosteum in exercise-trained animals and sedentary group (c) and the total number of adipocytes in the bonemarrow cavity in sedentary and endurance exercising animals (f) Number of adipocytes normalized on photo areas Statistical significance(119901 lt 005)

Additionally bone exposure for physical strain was shownto downregulate peroxisome proliferator-activated receptor(PPAR-120574) also known as the glitazone receptor (PPARG)that stimulates lipid uptake and inhibits adipogenesis [30 31]In our study we observed a significantly lower number oflipid droplets in adipocyte precursors derived from MSCsin EX animals This indicates the inhibitory effect of EX onthe adipogenic potential of MSCs Mechanical and physicalstress including exercises might influence changes in cellsactin organization Actin cytoskeleton dynamics have beenassociated with the regulation of adipogenic and osteogenicdifferentiation Signals forwarded from physical activity aretransduced by integrins to the actin cytoskeleton to switchmechanical signals into biochemical pathwaysmdashfrom adi-pogenic to osteogenic differentiation [32ndash34]

The study performed by Menuki at al [35] reported thatclimbing exercise enhanced MSC differentiation potential

and bone remodeling Another study demonstrated thatthe cyclic strain enhanced matrix mineralization in humanmesenchymal stem cells (hMSCs)

5 Conclusions

In conclusion we have shown that endurance trainingincreases the total number of mesenchymal stemprogenitorcells in the bone marrow Our results suggest that EXnot only supports the process of hematopoiesis butalso significantly enhances the osteogenic differentiationpotential of MSCs with simultaneous inhibition of theiradipogenic properties The results support the thesis thatendurance training can play a substantial role in theregeneration of mesenchymal tissues and thus represents anovel nonpharmacological manner of delaying age-relatedweakening of the musculoskeletal system


Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The research was supported by Wroclaw Research CentreEIT+ under the project ldquoBiotechnologies and AdvancedMedical TechnologiesrdquomdashBioMed (POIG010102-02-00308)financed from the European Regional Development Fund(Operational Programmed Innovative Economy 112) Thispaper was supported by Wroclaw Centre of Biotechnologyprogramme the Leading National Research Centre (KNOW)for years 2014ndash2018

References

[1] M Kucia W Wu and M Z Ratajczak ldquoBone marrow-derivedvery small embryonic-like stem cells their developmentalorigin and biological significancerdquo Developmental Dynamicsvol 236 no 12 pp 3309ndash3320 2007

[2] D G Phinney and D J Prockop ldquoConcise review mesenchy-mal stemmultipotent stromal cells the state of transdifferen-tiation and modes of tissue repairmdashcurrent viewsrdquo Stem Cellsvol 25 no 11 pp 2896ndash2902 2007

[3] A Malgieri E Kantzari M P Patrizi and S GambardellaldquoBone marrow and umbilical cord blood human mesenchymalstem cells state of the artrdquo International Journal of Clinical andExperimental Medicine vol 3 no 4 pp 248ndash269 2010

[4] V Sabapathy B Sundaram S Vm P Mankuzhy and S KumarldquoHuman whartonrsquos jelly mesenchymal stem cells plasticityaugments scar-free skin wound healing with hair growthrdquo PLoSONE vol 9 no 4 Article ID e93726 2014

[5] S A Jacobs V D Roobrouck C M Verfaillie and S W vanGool ldquoImmunological characteristics of human mesenchymalstem cells and multipotent adult progenitor cellsrdquo Immunologyand Cell Biology vol 91 no 1 pp 32ndash39 2013

[6] R J McCoy A Widaa K M Watters et al ldquoOrchestrat-ing osteogenic differentiation of mesenchymal stem cellsmdashidentification of placental growth factor as a mechanosensitivegene with a pro-osteogenic rolerdquo Stem Cells vol 31 no 11 pp2420ndash2431 2013

[7] L Biancone S Bruno M C Deregibus C Tetta and GCamussi ldquoTherapeutic potential of mesenchymal stem cell-derived microvesiclesrdquo Nephrology Dialysis Transplantationvol 27 no 8 pp 3037ndash3042 2012

[8] A Trounson R GThakar G Lomax andD Gibbons ldquoClinicaltrials for stem cell therapiesrdquo BMC Medicine vol 9 article 522011

[9] M A Konstam S Tursquomeh J Wynne J R Beck J Kozlowskiand B L Holman ldquoEffect of exercise on erythrocyte count andblood activity concentration after technetium-99m in vivo redblood cell labelingrdquoCirculation vol 66 no 3 pp 638ndash642 1982

[10] H Mairbaurl ldquoRed blood cells in sports Effects of exerciseand training on oxygen supply by red blood cellsrdquo Frontiers inPhysiology vol 4 article 332 2013

[11] N P Walsh M Gleeson R J Shephard et al ldquoPositionstatement Part one immune function and exerciserdquo ExerciseImmunology Review vol 17 pp 6ndash63 2011

[12] S L Nehlsen-Cannarella D C Nieman A J Balk-Lambertonet al ldquoThe effects of moderate exercise training on immuneresponserdquo Medicine and Science in Sports and Exercise vol 23no 1 pp 64ndash70 1991

[13] D C Nieman ldquoSpecial feature for the olympics effects ofexcercise on the immune systemrdquo Immunology and Cell Biologyvol 78 no 5 pp 496ndash501 2000

[14] V Mann C Huber G Kogianni D Jones and B Noble ldquoTheinfluence of mechanical stimulation on osteocyte apoptosisand bone viability in human trabecular bonerdquo Journal ofMusculoskeletal Neuronal Interactions vol 6 no 4 pp 408ndash4172006

[15] C Bagur-Calafat J Farrerons-Minguella M Girabent-Farresand J R Serra-Grima ldquoThe impact of high level basketballcompetition calcium intake menses and hormone levels inadolescent bone density a three-year follow-uprdquoThe Journal ofSports Medicine and Physical Fitness vol 55 no 1-2 pp 58ndash672015

[16] NMOcarinoUMarubayashi T G S Cardoso et al ldquoPhysicalactivity in osteopenia treatment improved the mass of bonesdirectly and indirectly submitted tomechanical impactrdquo Journalof Musculoskeletal Neuronal Interactions vol 7 no 1 pp 84ndash932007

[17] A E Hardman ldquoExercise in the prevention of atheroscleroticmetabolic and hypertensive diseases a reviewrdquo Journal of SportsSciences vol 14 no 3 pp 201ndash218 1996

[18] C H Lai T J HoWW Kuo et al ldquoExercise training enhancedSIRT1 longevity signaling replaces the IGF1 survival pathway toattenuate aging-induced rat heart apoptosisrdquo Age vol 36 no 5article 9706 2014

[19] I Keser E Suyani S Z Aki and A G T Sucak ldquoThe positiveimpact of regular exercise program on stem cell mobilizationprior to autologous stem cell transplantationrdquo Transfusion andApheresis Science vol 49 no 2 pp 302ndash306 2013

[20] J M Baker M De Lisio and G Parise ldquoEndurance exercisetraining promotes medullary hematopoiesisrdquo The FASEB Jour-nal vol 25 no 12 pp 4348ndash4357 2011

[21] M Kucia M Masternak R Liu et al ldquoThe negative effect ofprolonged somatotrophicinsulin signaling on an adult bonemarrow-residing population of pluripotent very small embry-onic-like stem cells (VSELs)rdquo Age vol 35 no 2 pp 315ndash3302013

[22] K Marycz J Krzak W Urbanski and C Pezowicz ldquoIn vitroand in vivo evaluation of sol-gel derived TiO

2

coatings basedon a variety of precursors and synthesis conditionsrdquo Journal ofNanomaterials vol 2014 Article ID 350579 14 pages 2014

[23] K Marycz A Smieszek J Grzesiak A Donesz-Sikorska and JKrzak-Ros ldquoApplication of bone marrow and adipose-derivedmesenchymal stem cells for testing the biocompatibility ofmetal-based biomaterials functionalized with ascorbic acidrdquoBiomedical Materials vol 8 no 6 Article ID 065004 2013

[24] R Starosta A Brzuszkiewicz A Bykowska et alldquoA novel copper(I) complex [CuI(221015840-biquino-line)P(CH

2

N(CH2

CH2

)2

O)3

]ndashSynthesis characterisation andcomparative studies on biological activityrdquo Polyhedron vol 50no 1 pp 481ndash489 2013

[25] M Kucia R Reca F R Campbell et al ldquoA population of verysmall embryonic-like (VSEL) CXCR4+SSEA-1+Oct-4+ stemcells identified in adult bone marrowrdquo Leukemia vol 20 no 5pp 857ndash869 2006


[26] K Marycz K Mierzejewska A Smieszek et al ldquoEnduranceexercise mobilizes developmentally early stem cells into periph-eral blood and increases their number in bonemarrow implica-tions for tissue regenerationrdquo Stem Cells International In press

[27] C J Rosen and M L Bouxsein ldquoMechanisms of disease isosteoporosis the obesity of bonerdquo Nature Clinical PracticeRheumatology vol 2 no 1 pp 35ndash43 2006

[28] Z Belaid-Choucair Y Lepelletier G Poncin et al ldquoHumanbone marrow adipocytes block granulopoiesis through neuro-pilin-1-induced granulocyte colony-stimulating factor inhibi-tionrdquo Stem Cells vol 26 no 6 pp 1556ndash1564 2008

[29] B Maitra E Szekely K Gjini et al ldquoHuman mesenchymalstem cells support unrelated donor hematopoietic stem cellsand suppress T-cell activationrdquo Bone Marrow Transplantationvol 33 no 6 pp 597ndash604 2004

[30] V David A Martin M-H Lafage-Proust et al ldquoMechani-cal loading down-regulates peroxisome proliferator-activatedreceptor 120574 in bonemarrow stromal cells and favors osteoblasto-genesis at the expense of adipogenesisrdquo Endocrinology vol 148no 5 pp 2553ndash2562 2007

[31] T Yokota K Oritani I Takahashi et al ldquoAdiponectin a newmember of the family of soluble defense collagens negativelyregulates the growth of myelomonocytic progenitors and thefunctions of macrophagesrdquo Blood vol 96 no 5 pp 1723ndash17322000

[32] P Muller A Langenbach A Kaminski and J Rychly ldquoModu-lating the actin cytoskeleton affectsmechanically induced signaltransduction and differentiation in mesenchymal stem cellsrdquoPLoS ONE vol 8 no 7 Article ID e71283 2013

[33] P S Mathieu and E G Loboa ldquoCytoskeletal and focal adhesioninfluences on mesenchymal stem cell shape mechanical prop-erties and differentiation down osteogenic adipogenic andchondrogenic pathwaysrdquo Tissue EngineeringmdashPart B Reviewsvol 18 no 6 pp 436ndash444 2012

[34] V Zablotskii O Lunov B Novotna et al ldquoDown-regulationof adipogenesis of mesenchymal stem cells by oscillating high-gradient magnetic fields and mechanical vibrationrdquo AppliedPhysics Letters vol 105 no 10 Article ID 103702 2014

[35] KMenuki T Mori A Sakai et al ldquoClimbing exercise enhancesosteoblast differentiation and inhibits adipogenic differentia-tion with high expression of PTHPTHrP receptor in bonemarrow cellsrdquo Bone vol 43 no 3 pp 613ndash620 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


aerobic endurance training program or moderate exercisesEach of them seems to act at different physiological levelsincluding improvement of immune andor endocrine systemAlthough there has been little research conducted concerningthe explanation of the mechanism of the positive effect ofexercises on health it has been demonstrated that exercisespositively correlate with increased content of red blood cells[9 10] and enhanced disease resistance via improved immunefunction [11] In turn moderate exercise has been reported asa factor that can boost immune function [12] while intenseexercise can reduce immune response causing a decreasein lymphocyte concentration natural killer cell activity andlymphocyte proliferation [13] In addition it was found thatthe exercise significantly reduces apoptosis and improvesviability of osteocytes as investigated using an osteopenicrat model [14] It was observed in adolescent individualsthat intensive exercises increased bone mass in the lumbarspine and femur [15] Further it appears that exercisesthrough biomechanical stimulation of the bone wall mayplay a prevention role in bone resorption and formation[16]

The collective data suggests that physical exercises rep-resent a nonpharmacological factor that positively affectslongevity prevention of metabolic diseases and stem cellmobilization [17ndash19]Therefore in the present study we havefocused on (i) quantitative analysis of the total number ofMSCs in the bone marrow (ii) quantitative analysis of earlyand late markers of osteogenesis in osteoblasts precursorsand (iii) evaluation of femur mineralization process in miceexposed to endurance exercise training

2 Materials and Methods

21 Animals and Exercise Training Protocol Sedentary (SED)and exercise-trained (EX) C57Bl6 mice (4 weeks old) werekept three per cage in an ultraclean facility on ventilated rackshoused in the Animal Experimental Laboratory (WroclawMedical School Norwida 34 Poland) Mice were maintainedon a 12 h light-dark cycle at 22 plusmn 02∘C The experiment wasconducted with the consent of the Local Ethics Committeefor Animal Experiments The animals were allocated toexperimental groups (6 animals per group) divided intosedentary animals which did not undergo physical activity(SE) and animals that exercised endurance (EX) Animalswere exercise-trained (119899 = 6) 3 days per week (dwk)(Monday Wednesday and Friday) for 5 weeks using anExer 36 Treadmill (Columbus Instruments Columbus OHUSA) Afterwards 5-wk progressive exercise protocol wasused The training started at 14 metersmin for 45min (wk 1)and was gradually increased to 24metersmin for 45min (wk5) The training portion of the protocol was always precededby a 10min warm-up at 10metersmin followed by a 5mincool-down at 10metersmin as described previously [20]Sedentary control mice (119899 = 6) were exposed to the treadmillbut not subjected to training Sampling was carried out afterthe last training period Bones (both femurs and tibiae)were collected dissected of muscle and fat and flushed with1mL of Iscoversquos modified Dulbecco medium (Sigma-Aldrich

Munich Germany) with 2 FBS (Invitrogen Carlsbad CAUSA) for bone marrow cell isolation

22 Preparation ofMSCs for FACS MurineMNCs (mononu-clear cells) were isolated from the BM of pathogen-free4-week-old C57BL6 mice Bone marrow cell suspensionsisolated by flushing femurs and tibiae were lysed in BDlysing buffer (BD Biosciences San Jose CA USA) for15min at room temperature and washed twice in phosphate-buffered saline (PBS) The following anti-mouse antibod-ies (BD Pharmingen) were used for staining anti-CD45(allophycocyanin-Cy7 clone 30F11) anti-CD45RB220 (PEclone RA-6B2) anti-Gr-1 (PE clone RB6-8 C5) anti-T-cellreceptor-120572120573 (PE clone H57-5970) anti-T-cell receptor-120574120575(PE clone GL3) anti-CD11b (PE clone M170) anti-Ter119(PE clone TER-119) and anti-Ly-6AE (also known as Sca-1 biotin and clone E13-1617 with streptavidin conjugated toPE-Cy5) MSCs (Sca-1+LinminusCD45minusCD31minusCD51+) wereisolated by the multiparameter live-cell sorting (INFLUXBD) as described previously [21] Monoclonal antibodies(mAbs) used in this study were added at saturating con-centrations The cells were incubated for 30 minutes on icewith different anti-mouse monoclonal antibodies washedtwice and resuspended in staining buffer at a concentrationof 5 times 106 cells per milliliter Samples were analyzed on aLSR II flow cytometer (BD Biosciences Mountain View CAUS) The following anti-mouse mAbs were purchased fromBD Pharmingen (San Diego CA) Ly-GAE (Sca-1 FITCclone D7) lineage marker (CD45R (also known as B220)PE clone RA3-6B2) Ly-6GLy-6C (PE clone RB6-8C5) T-cell receptor b (PE clone H57-597) T-cell receptor cd (PEclone GL3) CD11b (PE cloneM170) Ter119 (PE clone TER-119) CD51 (biotin cloneRMV-7with streptavidin conjugatedto PE-Cy5 as secondary Ab) CD31 (APC clone 390) andCD45 (APC-Cy7 clone 30-F11)The results are representativeof three independent experiments

23 Multipotency Assay Cells were seeded in 24-well platesat a concentration of 2 times 104 cells per well and subsequentlyadipogenic chondrogenic and osteogenic differentiationswere performed Stimulation of MSCs was performed usingStemPro Adipogenesis Chondrogenesis and OsteogenesisDifferentiation Kits (Life Technologies Poland) (StemProAdipogenesis Differentiation Kit StemPro ChondrogenesisDifferentiation Kit and StemPro Osteogenesis Differentia-tion Kit) according to the manufacturersrsquo instruction Dif-ferentiations began 24 h after inoculation and cells werecultured for 14 days Media were changed every three daysThe stage of adipogenic chondrogenic and osteogenic dif-ferentiation of MSCs was assessed using specific stainingOil Red O (Sigma-Aldrich Munich Germany) for detectingneutral lipid deposits Safranin O (Sigma-Aldrich MunichGermany) for glycosaminoglycans staining andAlizarin Red(Sigma-Aldrich Munich Germany) for calcium depositsPreparations were analyzed using Axio Observer A1 invertedmicroscope (Axio Observer A1 Carl Zeiss Jena Germany)while the documentationwasmade usingCannonPowerShotcamera














3 Results










4 Discussion



00

200

200

400

400

600

600

800

800

1000

1000

SSC-

A

FSC-A

100

100

101

101

102

102

103

103

PE-L

in

PE Cy5-Sca-1

0

200

400

600

800

1000

100

101

102

103

SSC-

A

APC-Cy7 CD45

100

100

101

101

102

102

103

103

APC

CD31

PE-Cy5 CD51

(a)

400120583m

Standard culture

400120583m


400120583m


400120583m


(b)


0

5

10

15

20

25

CFU-Fs

Col

ony

num

ber

Sedentary Exercise

lowastp lt 005

(a) (b) (c)






100120583m

(a)

100120583m

(b)

0

5

10

15

20

25


Sedentary Exercise

lowastp lt 005

()

(c)


0

10

20

30


Con

tent

()


lowastp lt 005

lowastp lt 005






lowastp lt 005

0

5

10

15

20



Sedentarycontrol

ALP

ALP

(120583U

mL)

(a)

lowastp lt 005

0

5

1030

35

40

45

50

55



Sedentarycontrol

Osteopontin

Oste

opon

tin (n

gm

L)

(b)

lowastp lt 005

0

10

120

140

160

180



Sedentarycontrol

Osteocalcin

Oste

ocal

cin

(ng

mL)

(c)

lowastp lt 005

0

5

10

15



Sedentarycontrol

Calcium in culture

Con

tent

()

(d)

lowastp lt 005

0

1

2

3

4

5

6

7



Sedentarycontrol


Con

tent

()

(e)



100120583m

(a)

100120583m

(b)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

80

100

(c)

100120583m

(d)

100120583m

(e)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

(f)






(a) (b)


Sedentary Exercise

lowastp lt 005

0

100

200

300

400

Wid

th (120583

m)

(c)

100120583m

(d)

100120583m

(e)


Num

ber o

f adi

pocy

tes

Sedentary Exercise

lowastp lt 005

0

5

10

15

20

25

(f)





5 Conclusions





Acknowledgments


References























2




2

N(CH2

CH2

)2

O)3





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














3 Results










4 Discussion



00

200

200

400

400

600

600

800

800

1000

1000

SSC-

A

FSC-A

100

100

101

101

102

102

103

103

PE-L

in

PE Cy5-Sca-1

0

200

400

600

800

1000

100

101

102

103

SSC-

A

APC-Cy7 CD45

100

100

101

101

102

102

103

103

APC

CD31

PE-Cy5 CD51

(a)

400120583m

Standard culture

400120583m


400120583m


400120583m


(b)


0

5

10

15

20

25

CFU-Fs

Col

ony

num

ber

Sedentary Exercise

lowastp lt 005

(a) (b) (c)






100120583m

(a)

100120583m

(b)

0

5

10

15

20

25


Sedentary Exercise

lowastp lt 005

()

(c)


0

10

20

30


Con

tent

()


lowastp lt 005

lowastp lt 005






lowastp lt 005

0

5

10

15

20



Sedentarycontrol

ALP

ALP

(120583U

mL)

(a)

lowastp lt 005

0

5

1030

35

40

45

50

55



Sedentarycontrol

Osteopontin

Oste

opon

tin (n

gm

L)

(b)

lowastp lt 005

0

10

120

140

160

180



Sedentarycontrol

Osteocalcin

Oste

ocal

cin

(ng

mL)

(c)

lowastp lt 005

0

5

10

15



Sedentarycontrol

Calcium in culture

Con

tent

()

(d)

lowastp lt 005

0

1

2

3

4

5

6

7



Sedentarycontrol


Con

tent

()

(e)



100120583m

(a)

100120583m

(b)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

80

100

(c)

100120583m

(d)

100120583m

(e)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

(f)






(a) (b)


Sedentary Exercise

lowastp lt 005

0

100

200

300

400

Wid

th (120583

m)

(c)

100120583m

(d)

100120583m

(e)


Num

ber o

f adi

pocy

tes

Sedentary Exercise

lowastp lt 005

0

5

10

15

20

25

(f)





5 Conclusions





Acknowledgments


References























2




2

N(CH2

CH2

)2

O)3





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results










4 Discussion



00

200

200

400

400

600

600

800

800

1000

1000

SSC-

A

FSC-A

100

100

101

101

102

102

103

103

PE-L

in

PE Cy5-Sca-1

0

200

400

600

800

1000

100

101

102

103

SSC-

A

APC-Cy7 CD45

100

100

101

101

102

102

103

103

APC

CD31

PE-Cy5 CD51

(a)

400120583m

Standard culture

400120583m


400120583m


400120583m


(b)


0

5

10

15

20

25

CFU-Fs

Col

ony

num

ber

Sedentary Exercise

lowastp lt 005

(a) (b) (c)






100120583m

(a)

100120583m

(b)

0

5

10

15

20

25


Sedentary Exercise

lowastp lt 005

()

(c)


0

10

20

30


Con

tent

()


lowastp lt 005

lowastp lt 005






lowastp lt 005

0

5

10

15

20



Sedentarycontrol

ALP

ALP

(120583U

mL)

(a)

lowastp lt 005

0

5

1030

35

40

45

50

55



Sedentarycontrol

Osteopontin

Oste

opon

tin (n

gm

L)

(b)

lowastp lt 005

0

10

120

140

160

180



Sedentarycontrol

Osteocalcin

Oste

ocal

cin

(ng

mL)

(c)

lowastp lt 005

0

5

10

15



Sedentarycontrol

Calcium in culture

Con

tent

()

(d)

lowastp lt 005

0

1

2

3

4

5

6

7



Sedentarycontrol


Con

tent

()

(e)



100120583m

(a)

100120583m

(b)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

80

100

(c)

100120583m

(d)

100120583m

(e)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

(f)






(a) (b)


Sedentary Exercise

lowastp lt 005

0

100

200

300

400

Wid

th (120583

m)

(c)

100120583m

(d)

100120583m

(e)


Num

ber o

f adi

pocy

tes

Sedentary Exercise

lowastp lt 005

0

5

10

15

20

25

(f)





5 Conclusions





Acknowledgments


References























2




2

N(CH2

CH2

)2

O)3





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


00

200

200

400

400

600

600

800

800

1000

1000

SSC-

A

FSC-A

100

100

101

101

102

102

103

103

PE-L

in

PE Cy5-Sca-1

0

200

400

600

800

1000

100

101

102

103

SSC-

A

APC-Cy7 CD45

100

100

101

101

102

102

103

103

APC

CD31

PE-Cy5 CD51

(a)

400120583m

Standard culture

400120583m


400120583m


400120583m


(b)


0

5

10

15

20

25

CFU-Fs

Col

ony

num

ber

Sedentary Exercise

lowastp lt 005

(a) (b) (c)






100120583m

(a)

100120583m

(b)

0

5

10

15

20

25


Sedentary Exercise

lowastp lt 005

()

(c)


0

10

20

30


Con

tent

()


lowastp lt 005

lowastp lt 005






lowastp lt 005

0

5

10

15

20



Sedentarycontrol

ALP

ALP

(120583U

mL)

(a)

lowastp lt 005

0

5

1030

35

40

45

50

55



Sedentarycontrol

Osteopontin

Oste

opon

tin (n

gm

L)

(b)

lowastp lt 005

0

10

120

140

160

180



Sedentarycontrol

Osteocalcin

Oste

ocal

cin

(ng

mL)

(c)

lowastp lt 005

0

5

10

15



Sedentarycontrol

Calcium in culture

Con

tent

()

(d)

lowastp lt 005

0

1

2

3

4

5

6

7



Sedentarycontrol


Con

tent

()

(e)



100120583m

(a)

100120583m

(b)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

80

100

(c)

100120583m

(d)

100120583m

(e)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

(f)






(a) (b)


Sedentary Exercise

lowastp lt 005

0

100

200

300

400

Wid

th (120583

m)

(c)

100120583m

(d)

100120583m

(e)


Num

ber o

f adi

pocy

tes

Sedentary Exercise

lowastp lt 005

0

5

10

15

20

25

(f)





5 Conclusions





Acknowledgments


References























2




2

N(CH2

CH2

)2

O)3





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100120583m

(a)

100120583m

(b)

0

5

10

15

20

25


Sedentary Exercise

lowastp lt 005

()

(c)


0

10

20

30


Con

tent

()


lowastp lt 005

lowastp lt 005






lowastp lt 005

0

5

10

15

20



Sedentarycontrol

ALP

ALP

(120583U

mL)

(a)

lowastp lt 005

0

5

1030

35

40

45

50

55



Sedentarycontrol

Osteopontin

Oste

opon

tin (n

gm

L)

(b)

lowastp lt 005

0

10

120

140

160

180



Sedentarycontrol

Osteocalcin

Oste

ocal

cin

(ng

mL)

(c)

lowastp lt 005

0

5

10

15



Sedentarycontrol

Calcium in culture

Con

tent

()

(d)

lowastp lt 005

0

1

2

3

4

5

6

7



Sedentarycontrol


Con

tent

()

(e)



100120583m

(a)

100120583m

(b)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

80

100

(c)

100120583m

(d)

100120583m

(e)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

(f)






(a) (b)


Sedentary Exercise

lowastp lt 005

0

100

200

300

400

Wid

th (120583

m)

(c)

100120583m

(d)

100120583m

(e)


Num

ber o

f adi

pocy

tes

Sedentary Exercise

lowastp lt 005

0

5

10

15

20

25

(f)





5 Conclusions





Acknowledgments


References























2




2

N(CH2

CH2

)2

O)3





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


lowastp lt 005

0

5

10

15

20



Sedentarycontrol

ALP

ALP

(120583U

mL)

(a)

lowastp lt 005

0

5

1030

35

40

45

50

55



Sedentarycontrol

Osteopontin

Oste

opon

tin (n

gm

L)

(b)

lowastp lt 005

0

10

120

140

160

180



Sedentarycontrol

Osteocalcin

Oste

ocal

cin

(ng

mL)

(c)

lowastp lt 005

0

5

10

15



Sedentarycontrol

Calcium in culture

Con

tent

()

(d)

lowastp lt 005

0

1

2

3

4

5

6

7



Sedentarycontrol


Con

tent

()

(e)



100120583m

(a)

100120583m

(b)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

80

100

(c)

100120583m

(d)

100120583m

(e)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

(f)






(a) (b)


Sedentary Exercise

lowastp lt 005

0

100

200

300

400

Wid

th (120583

m)

(c)

100120583m

(d)

100120583m

(e)


Num

ber o

f adi

pocy

tes

Sedentary Exercise

lowastp lt 005

0

5

10

15

20

25

(f)





5 Conclusions





Acknowledgments


References























2




2

N(CH2

CH2

)2

O)3





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100120583m

(a)

100120583m

(b)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

80

100

(c)

100120583m

(d)

100120583m

(e)


Sedentary Exercise

lowastp lt 005

()

0

20

40

60

(f)






(a) (b)


Sedentary Exercise

lowastp lt 005

0

100

200

300

400

Wid

th (120583

m)

(c)

100120583m

(d)

100120583m

(e)


Num

ber o

f adi

pocy

tes

Sedentary Exercise

lowastp lt 005

0

5

10

15

20

25

(f)





5 Conclusions





Acknowledgments


References























2




2

N(CH2

CH2

)2

O)3





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)


Sedentary Exercise

lowastp lt 005

0

100

200

300

400

Wid

th (120583

m)

(c)

100120583m

(d)

100120583m

(e)


Num

ber o

f adi

pocy

tes

Sedentary Exercise

lowastp lt 005

0

5

10

15

20

25

(f)





5 Conclusions





Acknowledgments


References























2




2

N(CH2

CH2

)2

O)3





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgments


References























2




2

N(CH2

CH2

)2

O)3





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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