The delivery of osteogenic factors is a proven therapeutic strategy to promote bone regeneration. Bone morphogenetic proteins (BMPs) constitute a family of cytokines with well-known osteogenic and bone regenerative abilities. However, clinical uses of BMPs require high doses that have been associated with complications such as osteolysis, ectopic bone formation, or hematoma formation. In the present work, we sought to improve bone tissue engineering through an approach that combines the use of bone marrow-derived mesenchymal stem cells (BMMSCs), composite scaffolds, and osteoinductive agents. We employed a composite gelatin/CaSO4 scaffold that allows for an early expansion of seeded BMMSCs, which is followed by an increased level of osteogenic differentiation after 10 days in culture. Furthermore, this scaffold enhanced bone formation by BMMSCs in a mouse model of critical-sized calvarial defect. More importantly, our results demonstrate that ex vivo pretreatment of BMMSCs with low amounts of BMP-2 (2 nM) and Wnt3a (50 ng/mL) for 24 h cooperatively increases the expression of osteogenic markers in vitro and bone regeneration in the critical-sized calvarial defect mouse model. These data provide a strong rationale for the development of an ex vivo cooperative use of BMP-2 and Wnt3a. Osteogenic factor cooperation might be applied to reduce the required amount of growth factors while obtaining higher therapeutic effects.
Get full access to this article
View all access options for this article.
References
1.
GiannoudisP.V., DinopoulosH., and TsiridisE.Bone substitutes: an update. Injury, 36Suppl 3, S20, 2005.
2.
GraysonW.L., BunnellB.A., MartinE., FrazierT., HungB.P., and GimbleJ.M.Stromal cells and stem cells in clinical bone regeneration. Nat Rev Endocrinol, 11, 140, 2015.
3.
TevlinR., McArdleA., AtashrooD., WalmsleyG.G., Senarath-YapaK., ZielinsE.R., PaikK.J., LongakerM.T., and WanD.C.Biomaterials for craniofacial bone engineering. J Dent Res, 93, 1187, 2014.
4.
ShiS., KirkM., and KahnA.J.The role of type I collagen in the regulation of the osteoblast phenotype. J Bone Miner Res, 11, 1139, 1996.
5.
JikkoA., HarrisS.E., ChenD., MendrickD.L., and DamskyC.H.Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2. J Bone Miner Res, 14, 1075, 1999.
6.
BarradasA.M., FernandesH.A., GroenN., ChaiY.C., SchrootenJ., van de PeppelJ., van LeeuwenJ.P., van BlitterswijkC.A., and de BoerJ.A calcium-induced signaling cascade leading to osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells. Biomaterials, 33, 3205, 2012.
7.
AxelradT.W., and EinhornT.A.Bone morphogenetic proteins in orthopaedic surgery. Cytokine Growth Factor Rev, 20, 481, 2009.
8.
DickinsonB.P., AshleyR.K., WassonK.L., O'HaraC., GabbayJ., HellerJ.B., and BradleyJ.P.Reduced morbidity and improved healing with bone morphogenic protein-2 in older patients with alveolar cleft defects. Plast Reconstr Surg, 121, 209, 2008.
9.
MinamideA., YoshidaM., KawakamiM., YamasakiS., KojimaH., HashizumeH., and BodenS.D.The use of cultured bone marrow cells in type I collagen gel and porous hydroxyapatite for posterolateral lumbar spine fusion. Spine (Phila Pa 1976), 30, 1134, 2005.
10.
YuanJ., CaoY., and LiuW.Biomimetic scaffolds: implications for craniofacial regeneration. J Craniofac Surg, 23, 294, 2012.
11.
ZaraJ.N., SiuR.K., ZhangX., ShenJ., NgoR., LeeM., LiW., ChiangM., ChungJ., KwakJ., WuB.M., TingK., and SooC.High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo. Tissue Eng Part A, 17, 1389, 2011.
12.
BoerckelJ.D., KolambkarY.M., DupontK.M., UhrigB.A., PhelpsE.A., StevensH.Y., GarciaA.J., and GuldbergR.E.Effects of protein dose and delivery system on BMP-mediated bone regeneration. Biomaterials, 32, 5241, 2011.
13.
LeviB., JamesA.W., NelsonE.R., LiS., PengM., CommonsG.W., LeeM., WuB., and LongakerM.T.Human adipose-derived stromal cells stimulate autogenous skeletal repair via paracrine Hedgehog signaling with calvarial osteoblasts. Stem Cells Dev, 20, 243, 2011.
14.
RipamontiU.Soluble and insoluble signals sculpt osteogenesis in angiogenesis. World J Biol Chem, 1, 109, 2010.
15.
WhiteA.P., VaccaroA.R., HallJ.A., WhangP.G., FrielB.C., and McKeeM.D.Clinical applications of BMP-7/OP-1 in fractures, nonunions and spinal fusion. Int Orthop, 31, 735, 2007.
16.
CarrageeE.J., HurwitzE.L., and WeinerB.K.A critical review of recombinant human bone morphogenetic protein-2 trials in spinal surgery: emerging safety concerns and lessons learned. Spine J, 11, 471, 2011.
17.
TannouryC.A., and AnH.S.Complications with the use of bone morphogenetic protein 2 (BMP-2) in spine surgery. Spine J, 14, 552, 2014.
18.
KoharaH., and TabataY.Enhancement of ectopic osteoid formation following the dual release of bone morphogenetic protein 2 and Wnt1 inducible signaling pathway protein 1 from gelatin sponges. Biomaterials, 32, 5726, 2011.
19.
LeeS.S., HuangB.J., KaltzS.R., SurS., NewcombC.J., StockS.R., ShahR.N., and StuppS.I.Bone regeneration with low dose BMP-2 amplified by biomimetic supramolecular nanofibers within collagen scaffolds. Biomaterials, 34, 452, 2013.
20.
MiyazonoK., KamiyaY., and MorikawaM.Bone morphogenetic protein receptors and signal transduction. J Biochem, 147, 35, 2010.
21.
UlsamerA., OrtunoM.J., RuizS., SusperreguiA.R., OssesN., RosaJ.L., and VenturaF.BMP-2 induces Osterix expression through up-regulation of Dlx5 and its phosphorylation by p38. J Biol Chem, 283, 3816, 2008.
22.
OrtunoM.J., Ruiz-GaspaS., Rodriguez-CarballoE., SusperreguiA.R., BartronsR., RosaJ.L., and VenturaF.p38 regulates expression of osteoblast-specific genes by phosphorylation of osterix. J Biol Chem, 285, 31985, 2010.
FuentealbaL.C., EiversE., IkedaA., HurtadoC., KurodaH., PeraE.M., and De RobertisE.M.Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal. Cell, 131, 980, 2007.
25.
Rodriguez-CarballoE., UlsamerA., SusperreguiA.R., Manzanares-CespedesC., Sanchez-GarciaE., BartronsR., RosaJ.L., and VenturaF.Conserved regulatory motifs in osteogenic gene promoters integrate cooperative effects of canonical Wnt and BMP pathways. J Bone Miner Res, 26, 718, 2011.
26.
SoleimaniM., and NadriS.A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat Protoc, 4, 102, 2009.
27.
SawyerA.A., SongS.J., SusantoE., ChuanP., LamC.X., WoodruffM.A., HutmacherD.W., and CoolS.M.The stimulation of healing within a rat calvarial defect by mPCL-TCP/collagen scaffolds loaded with rhBMP-2. Biomaterials, 30, 2479, 2009.
28.
GlowackiJ., and MizunoS.Collagen scaffolds for tissue engineering. Biopolymers, 89, 338, 2008.
29.
TakahashiY., YamamotoM., and TabataY.Enhanced osteoinduction by controlled release of bone morphogenetic protein-2 from biodegradable sponge composed of gelatin and beta-tricalcium phosphate. Biomaterials, 26, 4856, 2005.
30.
GrabowskiG., and CornettC.A.Bone graft and bone graft substitutes in spine surgery: current concepts and controversies. J Am Acad Orthop Surg, 21, 51, 2013.
31.
KuoT.F., LeeS.Y., WuH.D., PomaM., WuY.W., and YangJ.C.An in vivo swine study for xeno-grafts of calcium sulfate-based bone grafts with human dental pulp stem cells (hDPSCs). Mater Sci Eng C Mater Biol Appl, 50, 19, 2015.
32.
TsaiC.C., SuP.F., HuangY.F., YewT.L., and HungS.C.Oct4 and Nanog directly regulate Dnmt1 to maintain self-renewal and undifferentiated state in mesenchymal stem cells. Mol Cell, 47, 169, 2012.
33.
TsumakiN., and YoshikawaH.The role of bone morphogenetic proteins in endochondral bone formation. Cytokine Growth Factor Rev, 16, 279, 2005.
34.
RegardJ.B., ZhongZ., WilliamsB.O., and YangY.Wnt signaling in bone development and disease: making stronger bone with Wnts. Cold Spring Harb Perspect Biol, 4, 2012.
35.
HoeppnerL.H., SecretoF.J., and WestendorfJ.J.Wnt signaling as a therapeutic target for bone diseases. Expert Opin Ther Targets, 13, 485, 2009.
36.
SunX., SuJ., BaoJ., PengT., ZhangL., ZhangY., YangY., and ZhouX.Cytokine combination therapy prediction for bone remodeling in tissue engineering based on the intracellular signaling pathway. Biomaterials, 33, 8265, 2012.
CooperG.M., MooneyM.P., GosainA.K., CampbellP.G., LoseeJ.E., and HuardJ.Testing the critical size in calvarial bone defects: revisiting the concept of a critical-size defect. Plast Reconstr Surg, 125, 1685, 2010.
39.
GomesP.S., and FernandesM.H.Rodent models in bone-related research: the relevance of calvarial defects in the assessment of bone regeneration strategies. Lab Anim, 45, 14, 2011.
40.
ChaiY.C., RobertsS.J., DesmetE., KerckhofsG., van GastelN., GerisL., CarmelietG., SchrootenJ., and LuytenF.P.Mechanisms of ectopic bone formation by human osteoprogenitor cells on CaP biomaterial carriers. Biomaterials, 33, 3127, 2012.
41.
AtariM., Caballe-SerranoJ., Gil-RecioC., Giner-DelgadoC., Martinez-SarraE., Garcia-FernandezD.A., BarajasM., Hernandez-AlfaroF., Ferres-PadroE., and Giner-TarridaL.The enhancement of osteogenesis through the use of dental pulp pluripotent stem cells in 3D. Bone, 50, 930, 2012.
42.
HanS.M., HanS.H., CohY.R., JangG., Chan RaJ., KangS.K., LeeH.W., and YounH.Y.Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells. Exp Mol Med, 46, e101, 2014.
43.
DupontK.M., SharmaK., StevensH.Y., BoerckelJ.D., GarciaA.J., and GuldbergR.E.Human stem cell delivery for treatment of large segmental bone defects. Proc Natl Acad Sci U S A, 107, 3305, 2010.
44.
LiuY., WangL., KikuiriT., AkiyamaK., ChenC., XuX., YangR., ChenW., WangS., and ShiS.Mesenchymal stem cell-based tissue regeneration is governed by recipient T lymphocytes via IFN-gamma and TNF-alpha. Nat Med, 17, 1594, 2011.
45.
CaplanA.I., and DennisJ.E.Mesenchymal stem cells as trophic mediators. J Cell Biochem, 98, 1076, 2006.
46.
GaoX., UsasA., ProtoJ.D., LuA., CumminsJ.H., ProctorA., ChenC.W., and HuardJ.Role of donor and host cells in muscle-derived stem cell-mediated bone repair: differentiation vs. paracrine effects. FASEB J, 28, 3792, 2014.
KimR.Y., OhJ.H., LeeB.S., SeoY.K., HwangS.J., and KimI.S.The effect of dose on rhBMP-2 signaling, delivered via collagen sponge, on osteoclast activation and in vivo bone resorption. Biomaterials, 35, 1869, 2014.
49.
ShuiW., ZhangW., YinL., NanG., LiaoZ., ZhangH., WangN., WuN., ChenX., WenS., HeY., DengF., ZhangJ., LuuH.H., ShiL.L., HuZ., HaydonR.C., MokJ.M., and HeT.C.Characterization of scaffold carriers for BMP9-transduced osteoblastic progenitor cells in bone regeneration. J Biomed Mater Res A, 102, 3429, 2014.
50.
ZhangH., WangJ., DengF., HuangE., YanZ., WangZ., DengY., ZhangQ., ZhangZ., YeJ., QiaoM., LiR., WeiQ., ZhouG., LuuH.H., HaydonR.C., and HeT.C.Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla (SCAPs). Biomaterials, 39, 145, 2015.
51.
PoyntonA.R., and LaneJ.M.Safety profile for the clinical use of bone morphogenetic proteins in the spine. Spine (Phila Pa 1976), 27, S40, 2002.
52.
TsujiK., BandyopadhyayA., HarfeB.D., CoxK., KakarS., GerstenfeldL., EinhornT., TabinC.J., and RosenV.BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing. Nat Genet, 38, 1424, 2006.
53.
JingW., SmithA.A., LiuB., LiJ., HunterD.J., DhamdhereG., SalmonB., JiangJ., ChengD., JohnsonC.A., ChenS., LeeK., SinghG., and HelmsJ.A.Reengineering autologous bone grafts with the stem cell activator WNT3A. Biomaterials, 47, 29, 2015.
54.
LinG.L., and HankensonK.D.Integration of BMP, Wnt, and notch signaling pathways in osteoblast differentiation. J Cell Biochem, 112, 3491, 2011.
55.
MinearS., LeuchtP., JiangJ., LiuB., ZengA., FuererC., NusseR., and HelmsJ.A.Wnt proteins promote bone regeneration. Sci Transl Med, 2, 29ra30, 2010.
56.
LysdahlH., BaatrupA., FoldagerC.B., and BungerC.Preconditioning human mesenchymal stem cells with a low concentration of BMP2 stimulates proliferation and osteogenic differentiation in vitro. Biores Open Access, 3, 278, 2014.
57.
ChenY., WhetstoneH.C., YounA., NadesanP., ChowE.C., LinA.C., and AlmanB.A.Beta-catenin signaling pathway is crucial for bone morphogenetic protein 2 to induce new bone formation. J Biol Chem, 282, 526, 2007.
58.
SapkotaG., AlarconC., SpagnoliF.M., BrivanlouA.H., and MassagueJ.Balancing BMP signaling through integrated inputs into the Smad1 linker. Mol Cell, 25, 441, 2007.
59.
KrauseU., HarrisS., GreenA., YlostaloJ., ZeitouniS., LeeN., and GregoryC.A.Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy. Proc Natl Acad Sci U S A, 107, 4147, 2010.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
