Engineering biomaterials mimicking the biofunctionality of the extracellular matrix (ECM) is important in instructing and eliciting cell response. The native ECM is highly dynamic and has been shown to support cellular attachment, migration, and differentiation. The advantage of synthesizing an ECM-based biomaterial is that it mimics the native cellular environment. However, the ECM has tissue-specific composition and patterned arrangement. In this study, we have employed biomimetic strategies to develop a novel collagen/chitosan template that is embedded with the native ECM of differentiating human marrow stromal cells (HMSCs) to facilitate osteoblast differentiation. The scaffold was characterized for substrate stiffness by magnetic resonance imaging and nanoindentation and by immunohistochemical analysis for the presence of key ECM proteins. Gene expression analysis showed that the ECM scaffold supported osteogenic differentiation of undifferentiated HMSCs as significant changes were observed in the expression levels of growth factors, transcription factors, proteases, receptors, and ECM proteins. Finally, we demonstrate that the scaffold had the ability to nucleate calcium phosphate polymorphs to form a mineralized matrix. The results from this study suggest that the three-dimensional native ECM scaffold directly controls cell behavior and supports the osteogenic differentiation of mesenchymal stem cells.
LeeT.C., HoJ.T., HungK.S., ChenW.F., ChungY.H., YangY.L.Bone morphogenetic protein gene therapy using a fibrin scaffold for a rabbit spinal-fusion experiment. Neurosurgery, 58:373. 2006.
3.
HamiltonR., CampbellF.R.Immunochemical localizationof extracellular materials in bone marrow of rats. Anat Rec, 231:218. 2001.
4.
GordonM.Y.Extracellular matrix of the marrow microenvironment. Br J Haematol, 7:1. 1988.
5.
HockingA.M., ShinomuraT., McQuillanD.J.Leucine-rich repeat glycoproteins of the extracellular matrix. Matrix Biol, 17:1. 1998.
6.
ChenX.D., DusevichV., FengJ.Q., ManolagasS.C., JilkaR.L.Extracellular matrix made by bone marrow cells facilitates expansion of marrow-derived mesenchymal progenitor cells and prevents their differentiation into osteoblasts. J Bone Miner Res, 22:1943. 2007.
7.
ThibaultR.A., Scott BaggettL., MikosA.G., KasperF.K.Osteogenic differentiation of mesenchymal stem cells on pregenerated extracellular matrix scaffolds in the absence of osteogenic cell culture supplements. Tissue Eng Part A, 16:431. 2010.
8.
PhamQ.P., KasperF.K., Scott BaggettL., RaphaelR.M., JansenJ.A., MikosA.G.The influence of an in vitro generated bone-like extracellular matrix on osteoblastic gene expression of marrow stromal cells. Biomaterials, 29:2729. 2008.
9.
PittengerM.F., MackayA.M., BeckS.C., JaiswalR.K., DouglasR., MoscaJ.D.et al.Multilineage potential of adult human mesenchymal stem cells. Science, 284:143. 1999.
10.
SekiyaI., LarsonB.L., SmithJ.R., PochampallyR., CuiJ.G., ProckopD.J.Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells, 20:530. 2002.
11.
RavindranS., SongY., GeorgeA.Development of three-dimensional biomimetic scaffold to study epithelial–mesenchymal interactions. Tissue Eng Part A, 16:327. 2010.
12.
HeG., DahlT., VeisA., GeorgeA.Nucleation of apatite crystals in vitro by self-assembled dentin matrix protein 1. Nat Mater, 2:552. 2003.
13.
HennigJ., NauerthA., FriedburgH.RARE Imaging: a fast imaging method for Clinical, M.R. Mag Reson Med, 3:823. 1986.
14.
PaiA., LiX., MajumdarS.A comparative study at 3T of sequence dependence of T2 quantification in the knee. Mag Reson Imaging, 26:1215. 2008.
15.
StejskalE.O.Use of spin echoes in a pulsed magnetic-field gradient to study anisotropc, restricted diffusion and flow. J Chem Phys, 43:3597. 1965.
16.
TaylorD.G., BushellM.C.The spatial mapping of translational diffusion coefficients by the NMR imaging technique. Phys Med Biol, 30:345. 1985.
17.
XuH.H., OthmanS.F., MaginR.L.Monitoring tissue engineering using magnetic resonance imaging. J Biosci Bioeng, 106:515. 2008.
18.
L1W., HongL., HuL., MaginR.L.Magnetization transfer imaging provides a quantitative measure of chondrogenic differentiation and tissue development. Tissue Eng Part C Methods, 16:1407. 2010.
19.
DietrichO., BiffarA., Baur-Melnyk, ReiserM.F.Technical aspects of MR diffusion imaging of the body. Eur J Radiol, 76:314. 2010.
20.
JuneR.K., FyhrieD.P., MolecularN.M.R T2 values can predict cartilage stress relaxation. Biochem Biophys Res Commun, 377:57. 2008.
21.
WollN.L., HeaneyJ.D., BronsonS.K.Osteogenic nodule formation from single embryonic stem cell-derived progenitors. Stem Cells Dev, 15:865. 2006.
22.
WangY., VollochV., PindrusM.A., BlasioliD.J., ChenJ., KaplanD.L.Murine osteoblasts regulate mesenchymal stem cells via WNT and cadherin pathways: mechanism depends on cell-cell contact mode. J Tissue Eng Regen Med, 1:39. 2007.
23.
CoburnC.T., HajriT., IbrahimiA., AbumradN.A.Role of CD36 in membrane transport and utilization of long-chain fatty acids by different tissues. J Mol Neurosci, 16:117. 2001.
24.
HinoJ., MiyazawaT., MiyazatoM., KangawaK.Bone morphogenetic protein-3b (BMP-3b) is expressed in adipocytes and inhibits adipogenesis as a unique complex. Int J Obes 10.1038/ijo.2011.124.
25.
WoodR.J.Vitamin D and adipogenesis: new molecular insights. Nutr Rev, 66:40. 2008.
26.
PlaceE.S., EvansN.D., StevensM.M.Complexity in biomaterials for tissue engineering. Nat Mater, 8:457. 2009.
27.
GeorgeA., RavindranS.Protein templates in hard tissue engineering. Nano Today, 5:255. 2010.
28.
PekY.S., GaoS., ArshadM.S., LeckK.J., YingJ.Y.Porous collagen-apatite nanocomposite foams as bone regeneration scaffolds. Biomaterials, 29:4300. 2008.
29.
GeigerM., LiR.H., FriessW.Collagen sponges for bone regeneration with rhBMP-2. Adv Drug Deliv Rev, 55:1613. 2003.
30.
ZhangY., Jayarama ReddyV., WongS.Y., LiX., SuB., RamakrishnaS.et al.Enhanced biomineralization in osteoblasts on a novel electrospun biocomposite nanofibrous substrate of hydroxyapatite/collagen/chitosan. Tissue Eng Part A, 16:1949. 2010.
31.
ChandyT., SharmaC.Chitosan as a biomaterial. Biomater Art Cells Art Org, 18:1. 1990.
32.
XiaW., LiuW., CuiL., LiuY., ZhongW., LiuD.et al.Tissue engineering of cartilage with the use of chitosan-gelatin complex scaffolds. J Biomed Mater Res B Appl Biomater, 71:373. 2004.
33.
WuX., BlackL., Santacana-LaffitteG., PatrickC.W.Preparation and assessment of glutaraldehyde-crosslinked collagen-chitosan hydrogels for adipose tissue engineering. J Biomed Mater Res A, 81:59. 2007.
34.
KimK.W., ThomasR.L., LeeC., ParkH.J.Antimicrobial activity of native chitosan, degraded chitosan, and O-carboxymethylated chitosan. J Food Prot, 66:1495. 2003.
35.
TanW., KrishnarajR., DesaiT.A.Evaluation of nanostructured composite collagen—chitosan matrices for tissue engineering. Tissue Eng, 7:203. 2001.
36.
HeG., GeorgeA.Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro. J Biol Chem, 279:11649. 2004.
37.
ChenY., BalB.S., GorskiJ.P.Calcium and collagen binding properties of osteopontin, bone sialoprotein, and bone acidic glycoprotein-75 from bone. J Biol Chem, 267:24871. 1992.
38.
DelanyA.M., HankensonK.D.Thrombospondin-2 and SPARC/osteonectin are critical regulators of bone remodeling. J Cell Commun Signal, 3:227. 2009.
39.
GeorgeA., VeisA.Phosphorylated proteins and control over apatite nucleation, crystal growth, and inhibition. Chem Rev, 108:4670. 2008.
40.
EbisawaT., TadaK., KitajimaI., TojoK., SampathT.K., KawabataM., MiyazonoK., ImamuraT.Characterization of bone morphogenetic protein-6 signaling pathways in osteoblast differentiation. J Cell Sci, 112:3519. 1999.
41.
ChoT.J., GerstenfeldL.C., EinhornT.A.Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J Bone Miner Res, 17:513. 2002.
42.
ChenL., JiangW., HuangJ., HeB.C., ZuoG.W., ZhangW.et al.Insulin-like growth factor 2 (IGF2) potentiates BMP9-induced osteogenic differentiation and bone formation. J Bone Miner Res, 25:2447. 2010.
43.
LuH.H., SongW.X., LuoX., ManningD., LuoJ., DengZ.L.et al.Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. J Orthop Res, 25:665. 2007.
44.
ParisuthimanD., MochidaY., DuarteW.R., YamauchiM.Biglycan modulates osteoblast differentiation and matrix mineralization. J Bone Miner Res, 20:1878. 2005.
45.
BoskeyA.L., SpevakL., DotyS.B., RosenbergL.Effects of bone CS-proteoglycans, DS-decorin, and DS-biglycan on hydroxyapatite formation in a gelatin gel. Calcif Tissue Int, 61:298. 1997.
46.
Di CesareP.E., FangC., LeslieM.P., TulliH., PerrisR., CarlsonC.S.Expression of cartilage oligomeric matrix protein (COMP) by embryonic and adult osteoblasts. J Orthop Res, 18:713. 2000.
47.
MosigR.A., DowlingO., DiFeoA., RamirezM.C., ParkerI.C., AbeE.et al. Loss of MMP-2 disrupts skeletal and craniofacial development and results in decreased bone mineralization, joint erosion and defects in osteoblast and osteoclast growth. Hum Mol Genet, 16:1113. 2007.
48.
JinE.J., ChoiY.A., Kyun ParkE., BangO.S., KangS.S.MMP-2 functions as a negative regulator of chondrogenic cell condensation via down-regulation of the FAK-integrin beta1 interaction. Dev Biol, 308:474. 2007.
49.
BanerjeeC., McCabeL.R., ChoiJ.Y., HiebertS.W., SteinJ.L., SteinG.S.et al.Runt homology domain proteins in osteoblast differentiation: AML-3/CBFA1 is a major component of a bone specific complex. J Cell Biochem, 66:1. 1997.
50.
DucyP., ZhangR., GeoffroyV., RidallA.L., KarsentyG.Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell, 89:747. 1997.
51.
ChoiJ.Y., PratapJ., JavedA., ZaidiS.K., XingL., BalintE.et al.Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development. Proc Natl Acad Sci U S A, 98:8650. 2001.
52.
KomoriT., YagiH., NomuraS., YamaguchiA., SasakiK., DeguchiK.et al.Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell, 89:755. 1997.
53.
Macías-SilvaM., AbdollahS., HoodlessP.A., PironeR., AttisanoL., WranaJ.L.MADR2 is a substrate of the TGFbeta receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell, 87:1215. 1996.
54.
MassaguéJ.TGF-beta signal transduction. Annu Rev Biochem, 67:753. 1998.
55.
ZhangZ., SongY., ZhangX., TangJ., ChenJ., ChenY.Msx1/Bmp4 genetic pathway regulates mammalian alveolar bone formation via induction of Dlx5 and Cbfa1. Mech Dev, 120:1469. 2003.
56.
Dziedzic-GoclawskaA., ToverudS.U., KaminskiA., BoassA., YamauchiM.Decreased heterotopic osteogenesis in vitamin-D-deficient, but normocalcemic guinea pigs. Bone Miner, 19:127. 1992.
57.
BaeH., ZhaoL., ZhuD., KanimL.E., WangJ.C., DelamarterR.B.Variability across ten production lots of a single demineralized bone matrix product. J Bone Joint Surg Am, 92:427. 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.
