Providing fully mature and functional osteoblasts is challenging for bone tissue engineering and regenerative medicine. Such cells could be obtained from multipotent bone marrow mesenchymal stem cells (MSCs) after induction by different osteogenic factors. However, there are some discrepancies in results, notably due to the use of sera and to the type of osteogenic factor. In this study, we compared the osteogenic differentiation of bone marrow MSCs induced by dexamethasone (Dex) or bone morphogenetic proteins (BMPs) by assessing phenotypes in vitro and functional osteoblasts in vivo. Reducing the content of fetal calf serum from 10% to 2% significantly increased the mineral deposition and expression of osteoblastic markers during osteogenesis. In comparison to Dex condition, the addition of BMP4 greatly improved the differentiation of MSCs into fully mature osteoblasts as seen by high expression of Osterix. These results were confirmed in different supportive matrixes, plastic flasks, or biphasic calcium phosphate biomaterials. In contrast to Dex-derived osteoblasts, BMP4-derived osteoblasts from MSCs were significantly able to produce new bone in subcutis of nude mice in accordance with in vitro results. In conclusion, we describe a convenient ex vivo method to produce consistently mature functional osteoblasts from human MSCs with use of BMP4 and low serum.
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References
1.
DeschaseauxF., PontikoglouC., SensebeL.Bone regeneration: the stem/progenitor cells point of view. J Cell Mol Med, 14:103. 2010.
2.
DeschaseauxF., SensebeL., HeymannD.Mechanisms of bone repair and regeneration. Trends Mol Med, 15:417. 2009.
3.
FriedlaenderG.E., PerryC.R., ColeJ.D., CookS.D., CiernyG., MuschlerG.F., ZychG.A., CalhounJ.H., LaForteA.J., YinS.Osteogenic protein-1 (bone morphogenetic protein-7) in the treatment of tibial nonunions. J Bone Joint Surg Am, 83-A,Suppl 1:S151. 2001.
4.
GovenderS., CsimmaC., GenantH.K., Valentin-OpranA., AmitY., ArbelR., AroH., AtarD., BishayM., BornerM.G., ChironP., ChoongP., CinatsJ., CourtenayB., FeibelR., GeuletteB., GravelC., HaasN., RaschkeM., HammacherE., van der VeldeD., HardyP., HoltM., JostenC., KetterlR.L., LindequeB., LobG., MathevonH., McCoyG., MarshD., MillerR., MuntingE., OevreS., NordslettenL., PatelA., PohlA., RennieW., ReyndersP., RommensP.M., RondiaJ., RossouwW.C., DaneelP.J., RuffS., RuterA., SantavirtaS., SchildhauerT.A., GekleC., SchnettlerR., SegalD., SeilerH., SnowdowneR.B., StapertJ., TaglangG., VerdonkR., VogelsL., WeckbachA., WentzensenA., WisniewskiT.Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am, 84-A:2123. 2002.
5.
SilberJ.S., AndersonD.G., DaffnerS.D., BrislinB.T., LelandJ.M., HilibrandA.S., VaccaroA.R., AlbertT.J.Donor site morbidity after anterior iliac crest bone harvest for single-level anterior cervical discectomy and fusion. Spine, 28:134. 2003.
6.
LennonD., HaynesworthS., BruderS., JaiswalN., CaplanA.Human and animal mesenchymal progenitor cells from bone marrow: identification of serum for optimal selection and proliferation. In Vitro Cell Dev Biol Anim, 32:602. 1996.
7.
PittengerM.F., MackayA.M., BeckS.C., JaiswalR.K., DouglasR., MoscaJ.D., MoormanM.A., SimonettiD.W., CraigS., MarshakD.R.Multilineage potential of adult human mesenchymal stem cells. Science, 284:143. 1999.
8.
OsyczkaA.M., LeboyP.S.Bone morphogenetic protein regulation of early osteoblast genes in human marrow stromal cells is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling. Endocrinology, 146:3428. 2005.
AubinJ.E.Osteoprogenitor cell frequency in rat bone marrow stromal populations: role for heterotypic cell-cell interactions in osteoblast differentiation. J Cell Biochem, 72:396. 1999.
11.
PurpuraK.A., AubinJ.E., ZandstraP.W.Sustained in vitro expansion of bone progenitors is cell density dependent. Stem Cells, 22:39. 2004.
WeinsteinR.S., JilkaR.L., ParfittA.M., ManolagasS.C.Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. Potential mechanisms of their deleterious effects on bone. J Clin Invest, 102:274. 1998.
15.
KimH.J., ZhaoH., KitauraH., BhattacharyyaS., BrewerJ.A., MugliaL.J., Patrick RossF., TeitelbaumS.L.Glucocorticoids and the osteoclast. Ann NY Acad Sci, 1116:335. 2007.
16.
TarteK., GaillardJ., LatailladeJ.J., FouillardL., BeckerM., MossafaH., TchirkovA., RouardH., HenryC., SplingardM., DulongJ., MonnierD., GourmelonP., GorinN.C., SensebeL.Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation. Blood, 115. 1549.
17.
SelmaniZ., NajiA., ZidiI., FavierB., GaiffeE., ObertL., BorgC., SaasP., TiberghienP., Rouas-FreissN., CarosellaE.D., DeschaseauxF.Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lymphocyte and natural killer function and to induce CD4+CD25highFOXP3+ regulatory T cells. Stem Cells, 26:212. 2008.
18.
LivakK.J., SchmittgenT.D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25:402. 2001.
19.
ChangW., TuC., ChenT.H., BikleD., ShobackD.The extracellular calcium-sensing receptor (CaSR) is a critical modulator of skeletal development. Sci Signal, 1:ra1. 2008.
20.
da Silva MeirellesL., ChagastellesP.C., NardiN.B.Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci, 119:2204. 2006.
21.
ToriiY., HitomiK., YamagishiY., TsukagoshiN.Demonstration of alkaline phosphatase participation in the mineralization of osteoblasts by antisense RNA approach. Cell Biol Int, 20:459. 1996.
22.
FellahB.H., GauthierO., WeissP., ChappardD., LayrolleP.Osteogenicity of biphasic calcium phosphate ceramics and bone autograft in a goat model. Biomaterials, 29:1177. 2008.
23.
FischerE.M., LayrolleP., Van BlitterswijkC.A., De BruijnJ.D.Bone formation by mesenchymal progenitor cells cultured on dense and microporous hydroxyapatite particles. Tissue Eng, 9:1179. 2003.
24.
HabibovicP., KruytM.C., JuhlM.V., ClyensS., MartinettiR., DolciniL., TheilgaardN., van BlitterswijkC.A.Comparative in vivo study of six hydroxyapatite-based bone graft substitutes. J Orthop Res, 26:1363. 2008.
25.
Le NihouannenD., DaculsiG., SaffarzadehA., GauthierO., DelplaceS., PiletP., LayrolleP.Ectopic bone formation by microporous calcium phosphate ceramic particles in sheep muscles. Bone, 36:1086. 2005.
26.
YuanH., KurashinaK., de BruijnJ.D., LiY., de GrootK., ZhangX.A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics. Biomaterials, 20:1799. 1999.
27.
AubinJ.E., LiuF., MalavalL., GuptaA.K.Osteoblast and chondroblast differentiation. Bone, 17:77S. 1995.
28.
SteinG.S., LianJ.B.Regulation of cell cycle and growth control. BioelectromagneticsSuppl 1:247. 1992.
29.
SteinG.S., LianJ.B., OwenT.A.Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation. FASEB J, 4:3111. 1990.
30.
CanalisE., DeregowskiV., PereiraR.C., GazzerroE.Signals that determine the fate of osteoblastic cells. J Endocrinol Invest, 28:3. 2005.
OwenT.A., AronowM., ShalhoubV., BaroneL.M., WilmingL., TassinariM.S., KennedyM.B., PockwinseS., LianJ.B., SteinG.S.Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol, 143:420. 1990.
34.
RaucciA., BellostaP., GrassiR., BasilicoC., MansukhaniA.Osteoblast proliferation or differentiation is regulated by relative strengths of opposing signaling pathways. J Cell Physiol, 215:442. 2008.
35.
van EsJ.H., van GijnM.E., RiccioO., van den BornM., VooijsM., BegthelH., CozijnsenM., RobineS., WintonD.J., RadtkeF., CleversH.Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature, 435:959. 2005.
36.
KatagiriT., YamaguchiA., KomakiM., AbeE., TakahashiN., IkedaT., RosenV., WozneyJ.M., Fujisawa-SeharaA., SudaT.Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J Cell Biol, 127:1755. 1994.
37.
DrissiH., HushkaD., AslamF., NguyenQ., BuffoneE., KoffA., van WijnenA., LianJ.B., SteinJ.L., SteinG.S.The cell cycle regulator p27kip1 contributes to growth and differentiation of osteoblasts. Cancer Res, 59:3705. 1999.
38.
NishimoriS., TanakaY., ChibaT., FujiiM., ImamuraT., MiyazonoK., OgasawaraT., KawaguchiH., IgarashiT., FujitaT., TanakaK., ToyoshimaH.Smad-mediated transcription is required for transforming growth factor-beta 1-induced p57(Kip2) proteolysis in osteoblastic cells. J Biol Chem, 276:10700. 2001.
39.
UranoT., YashirodaH., MuraokaM., TanakaK., HosoiT., InoueS., OuchiY., ToyoshimaH.p57(Kip2) is degraded through the proteasome in osteoblasts stimulated to proliferation by transforming growth factor beta1. J Biol Chem, 274:12197. 1999.
40.
HaynesworthS.E., GoshimaJ., GoldbergV.M., CaplanA.I.Characterization of cells with osteogenic potential from human marrow. Bone, 13:81. 1992.
41.
JaiswalN., HaynesworthS.E., CaplanA.I., BruderS.P.Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem, 64:295. 1997.
42.
ShaoJ., XuX., LiG., ZhangW., QiJ., WangJ., CaiM., CuiL., ZhangL., ZhaoQ., DengL.Inhibitory effects of pharmacological doses of dexamethasone on mineralization of mesenchymal progenitor cells in vitro. Pharmazie, 64:674. 2009.
43.
DerfoulA., PerkinsG.L., HallD.J., TuanR.S.Glucocorticoids promote chondrogenic differentiation of adult human mesenchymal stem cells by enhancing expression of cartilage extracellular matrix genes. Stem Cells, 24:1487. 2006.
44.
IndrawattanaN., ChenG., TadokoroM., ShannL.H., OhgushiH., TateishiT., TanakaJ., BunyaratvejA.Growth factor combination for chondrogenic induction from human mesenchymal stem cell. Biochem Biophys Res Commun, 320:914. 2004.
45.
OshinaH., SotomeS., YoshiiT., TorigoeI., SugataY., MaeharaH., MarukawaE., OmuraK., ShinomiyaK.Effects of continuous dexamethasone treatment on differentiation capabilities of bone marrow-derived mesenchymal cells. Bone, 41:575. 2007.
46.
LaneN.E., YaoW.Glucocorticoid-induced bone fragility. Ann NY Acad Sci, 1192:81.
47.
QiX., LiT.G., HaoJ., HuJ., WangJ., SimmonsH., MiuraS., MishinaY., ZhaoG.Q.BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc Natl Acad Sci USA, 101:6027. 2004.
48.
UristM.R.Bone: formation by autoinduction. Science, 150:893. 1965.
YingQ.L., NicholsJ., ChambersI., SmithA.BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell, 115:281. 2003.
51.
FujiiM., TakedaK., ImamuraT., AokiH., SampathT.K., EnomotoS., KawabataM., KatoM., IchijoH., MiyazonoK.Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation. Mol Biol Cell, 10:3801. 1999.
52.
KawabataM., ImamuraT., MiyazonoK.Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev, 9:49. 1998.
53.
NishimuraR., KatoY., ChenD., HarrisS.E., MundyG.R., YonedaT.Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12. J Biol Chem, 273:1872. 1998.
54.
BlokhuisT.J., LindnerT.Allograft and bone morphogenetic proteins: an overview. Injury, 39,Suppl 2:S33. 2008.
55.
CheungA.Bone morphogenetic proteins in orthopaedic surgery. Curr Orthop, 20:424. 2006.
56.
KawaguchiJ., MeeP.J., SmithA.G.Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors. Bone, 36:758. 2005.
57.
LeeM.H., KwonT.G., ParkH.S., WozneyJ.M., RyooH.M.BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun, 309:689. 2003.
58.
NakashimaK., ZhouX., KunkelG., ZhangZ., DengJ.M., BehringerR.R., de CrombruggheB.The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell, 108:17. 2002.
59.
WinnierG., BlessingM., LaboskyP.A., HoganB.L.Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev, 9:2105. 1995.
60.
IshidouY., KitajimaI., ObamaH., MaruyamaI., MurataF., ImamuraT., YamadaN., ten DijkeP., MiyazonoK., SakouT.Enhanced expression of type I receptors for bone morphogenetic proteins during bone formation. J Bone Miner Res, 10:1651. 1995.
61.
NakaseT., NomuraS., YoshikawaH., HashimotoJ., HirotaS., KitamuraY., OikawaS., OnoK., TakaokaK.Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing. J Bone Miner Res, 9:651. 1994.
62.
SatoM., OchiT., NakaseT., HirotaS., KitamuraY., NomuraS., YasuiN.Mechanical tension-stress induces expression of bone morphogenetic protein (BMP)-2 and BMP-4, but not BMP-6, BMP-7, and GDF-5 mRNA, during distraction osteogenesis. J Bone Miner Res, 14:1084. 1999.
63.
YoshimuraY., NomuraS., KawasakiS., TsutsumimotoT., ShimizuT., TakaokaK. Colocalization of noggin and bone morphogenetic protein-4 during fracture healing. J Bone Miner Res, 16:876. 2001.
64.
BandyopadhyayA., TsujiK., CoxK., HarfeB.D., RosenV., TabinC.J.Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet, 2:e216. 2006.
65.
TsujiK., CoxK., BandyopadhyayA., HarfeB.D., TabinC.J., RosenV.BMP4 is dispensable for skeletogenesis and fracture-healing in the limb. J Bone Joint Surg Am, 90,Suppl 1:14. 2008.
66.
SiddappaR., MartensA., DoornJ., LeusinkA., OlivoC., LichtR., van RijnL., GasparC., FoddeR., JanssenF., van BlitterswijkC., de BoerJ.cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo. Proc Natl Acad Sci USA, 105:7281. 2008.
67.
WojtowiczA.M., TemplemanK.L., HutmacherD.W., GuldbergR.E., GarciaA.J.Runx2 overexpression in bone marrow stromal cells accelerates bone formation in critical-sized femoral defects. Tissue Eng Part A, 16:2795. 2010.
68.
ZhouG., LiuW., CuiL., WangX., LiuT., CaoY.Repair of porcine articular osteochondral defects in non-weightbearing areas with autologous bone marrow stromal cells. Tissue Eng, 12:3209. 2006.
69.
GronthosS., ChenS., WangC.Y., RobeyP.G., ShiS.Telomerase accelerates osteogenesis of bone marrow stromal stem cells by upregulation of CBFA1, osterix, and osteocalcin. J Bone Miner Res, 18:716. 2003.
70.
ChaseL.G., LakshmipathyU., SolchagaL.A., RaoM.S., VemuriM.C.A novel serum-free medium for the expansion of human mesenchymal stem cells. Stem Cell Res Ther, 1:8. 2010.
71.
FelkaT., SchaferR., De ZwartP., AicherW.K.Animal serum-free expansion and differentiation of human mesenchymal stromal cells. Cytotherapy, 12:143. 2010.
72.
SchallmoserK., RohdeE., ReinischA., BartmannC., ThalerD., DrexlerC., ObenaufA.C., LanzerG., LinkeschW., StrunkD.Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum. Tissue Eng Part C Methods, 14:185. 2008.
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