Achieving cell spreading and proliferation inside hydrogels that are compatible with microencapsulation technology represents a major challenge for tissue engineering scaffolding and for the development of three-dimensional cell culture models. In this study, microcapsules of 650–900 μm in diameter were fabricated from oxidized alginate covalently cross-linked with gelatine (AlGel). Schiff's base bond formed in AlGel, detected by Fourier transform infrared spectroscopy, which confirmed the cross-linking of oxidized alginate with gelatine. Biological properties of alginate based hydrogels were studied by comparing the viability and morphology of MG-63 osteosarcoma cells encapsulated in gelatine and RGD-modified alginate. We hypothesized that the presence of gelatine and RGD will support cell adhesion and spreading inside the microcapsules and finally, also vascular endothelial growth factor (VEGF) secretion. After 4 days of incubation, cells formed extensive cortical protrusions and after 2 weeks they proliferated, migrated, and formed cellular networks through the AlGel material. In contrast, cells encapsulated in pure alginate and in RGD-modified alginate formed spherical aggregates with limited cell mobility and VEGF secretion. Metabolic activity was doubled after 5 days of incubation, making AlGel a promising material for cell encapsulation.
Get full access to this article
View all access options for this article.
References
1.
RowleyJ.A., MadlambayanG., and MooneyD.J.Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials, 20, 45, 1999.
2.
MuruaA., PorteroA., OriveG., HernándezR.M., de CastroM., and PedrazJ.L.Cell microencapsulation technology: towards clinical application. J Control Release, 132, 76, 2008.
3.
AwadH.A., WickhamM.Q., LeddyH.A., GimbleJ.M., and GuilakF.Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials, 25, 3211, 2004.
4.
JenA.C., WakeM.C., and MikosA.G.Review: hydrogels for cell immobilization. Biotechnol Bioeng, 50, 357, 1996.
5.
LeeJ., CuddihyM.J., and KotovN.A.Three-dimensional cell culture matrices: state of the art. Tissue Eng Part B Rev, 14, 61, 2008.
6.
MeliL., JordanE.T., ClarkD.S., LinhardtR.J., and DordickJ.S.Influence of a three-dimensional, microarray environment on human Cell culture in drug screening systems. Biomaterials, 33, 9087, 2012.
7.
GeckilH., XuF., ZhangX., MoonS., and DemirciU.Engineering hydrogels as extracellular matrix mimics. Nanomedicine, 5, 469, 2010.
8.
TibbittM.W., and AnsethK.S.Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng, 103, 655, 2009.
9.
MauneyJ.R., VollochV., and KaplanD.L.Role of adult mesenchymal stem cells in bone tissue-engineering applications: current status and future prospects. Tissue Eng, 11, 787, 2005.
10.
KneserU., SchaeferD.J., PolykandriotisE., and HorchR.E.Tissue engineering of bone: the reconstructive surgeon's point of view. J Cell Mol Med, 10, 7, 2006.
11.
StreetJ., BaoM., DeGuzmanL., BuntingS., PealeF.V.Jr., FerraraN., et al.Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci U S A, 99, 9656, 2002.
12.
SlaughterB.V., KhurshidS.S., FisherO.Z., KhademhosseiniA., and PeppasN.A.Hydrogels in regenerative medicine. Adv Mater, 21, 3307, 2009.
13.
AlbrechtD.R., UnderhillG.H., WassermannT.B., SahR.L., and BhatiaS.N.Probing the role of multicellular organization in three-dimensional microenvironments. Nat Methods, 3, 369, 2006.
14.
NicodemusG.D., and BryantS.J.Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Eng Part B Rev, 14, 149, 2008.
15.
LeeK.Y., and MooneyD.J.Alginate: properties and biomedical applications. Prog Polym Sci (Oxford), 37, 106, 2012.
16.
KongH.J., SmithM.K., and MooneyD.J.Designing alginate hydrogels to maintain viability of immobilized cells. Biomaterials, 24, 4023, 2003.
17.
RiceJ.J., MartinoM.M., De LaporteL., TortelliF., BriquezP.S., and HubbellJ.A.Engineering the regenerative microenvironment with biomaterials. Adv Healthc Mater, 2, 57, 2013.
18.
KangS.W., ChaB.H., ParkH., ParkK.S., LeeK.Y., and LeeS.H.The Effect of conjugating RGD into 3D alginate hydrogels on adipogenic differentiation of human adipose-derived stromal cells. Macromol Biosci, 11, 673, 2011.
19.
FonsecaK.B., BidarraS.J., OliveiraM.J., GranjaP.L., and BarriasC.C.Molecularly designed alginate hydrogels susceptible to local proteolysis as three-dimensional cellular microenvironments. Acta Biomater, 7, 1674, 2011.
20.
ZhouH., and XuH.H.K.The fast release of stem cells from alginate-fibrin microbeads in injectable scaffolds for bone tissue engineering. Biomaterials, 32, 7503, 2011.
21.
SunJ., XiaoW., TangY., LiK., and FanH.Biomimetic interpenetrating polymer network hydrogels based on methacrylated alginate and collagen for 3D pre-osteoblast spreading and osteogenic differentiation. Soft Matter, 8, 2398, 2012.
22.
HaqueT., ChenH., OuyangW., MartoniC., LawuyiB., UrbanskaA.M., et al.In vitro study of alginate-chitosan microcapsules: an alternative to liver cell transplants for the treatment of liver failure. Biotechnol Lett, 27, 317, 2005.
23.
DahlmannJ., KrauseA., MöllerL., KensahG., MöwesM., DiekmannA., et al.Fully defined in situ cross-linkable alginate and hyaluronic acid hydrogels for myocardial tissue engineering. Biomaterials, 34, 940, 2013.
24.
GelseK., PöschlE., and AignerT.Collagens—structure, function, and biosynthesis. Adv Drug Deliv Rev, 55, 1531, 2003.
25.
TanH., ChuC.R., PayneK.A., and MarraK.G.Injectable in situ forming biodegradable chitosan-hyaluronic acid based hydrogels for cartilage tissue engineering. Biomaterials, 30, 2499, 2009.
26.
HuangY., OnyeriS., SieweM., MoshfeghianA., and MadihallyS.V.In vitro characterization of chitosan-gelatin scaffolds for tissue engineering. Biomaterials, 26, 7616, 2005.
27.
BernhardtA., DespangF., LodeA., DemmlerA., HankeT., and GelinskyM.Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate—gelatine—hydroxyapatite scaffolds with anisotropic pore structure. J Tissue Eng Regen Med, 3, 54, 2009.
28.
AlmeidaP.F., and AlmeidaA.J.Cross-linked alginate-gelatine beads: a new matrix for controlled release of pindolol. J Control Release, 97, 431, 2004.
29.
LiaoH., ZhangH., and ChenW.Differential physical, rheological, and biological properties of rapid in situ gelable hydrogels composed of oxidized alginate and gelatin derived from marine or porcine sources. J Mater Sci Mater Med, 20, 1263, 2009.
30.
BalakrishnanB., and JayakrishnanA.Self-cross-linking biopolymers as injectable in situ forming biodegradable scaffolds. Biomaterials, 26, 3941, 2005.
31.
BalakrishnanB., MohantyM., UmashankarP.R., and JayakrishnanA.Evaluation of an in situ forming hydrogel wound dressing based on oxidized alginate and gelatin. Biomaterials, 26, 6335, 2005.
32.
NguyenT.P., and LeeB.T.Fabrication of oxidized alginate-gelatin-BCP hydrogels and evaluation of the microstructure, material properties and biocompatibility for bone tissue regeneration. J Biomater Appl, 27, 311, 2012.
33.
AugstA.D., KongH.J., and MooneyD.J.Alginate hydrogels as biomaterials. Macromol Biosci, 6, 623, 2006.
34.
DruryJ.L., and MooneyD.J.Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials, 24, 4337, 2003.
35.
BoaniniE., RubiniK., PanzavoltaS., and BigiA.Chemico-physical characterization of gelatin films modified with oxidized alginate. Acta Biomater, 6, 383, 2010.
36.
ManjuS., MuraleedharanC.V., RajeevA., JayakrishnanA., and JosephR.Evaluation of alginate dialdehyde cross-linked gelatin hydrogel as a biodegradable sealant for polyester vascular graft. J Biomed Mater Res Part B Appl Biomater, 98B, 139, 2011.
37.
SakaiS., YamaguchiS., TakeiT., and KawakamiK.Oxidized alginate-cross-linked alginate/gelatin hydrogel fibers for fabricating tubular constructs with layered smooth muscle cells and endothelial cells in collagen gels. Biomacromolecules, 9, 2036, 2008.
38.
BouhadirK.H., LeeK.Y., AlsbergE., DammK.L., AndersonK.W., and MooneyD.J.Degradation of partially oxidized alginate and its potential application for tissue engineering. Biotechnol Prog, 17, 945, 2001.
39.
SarkarB., PapageorgiouD.G., SilvaR., ZehnderT., Gul-E-NoorF., BertmerM., et al.Fabrication of alginate gelatin crosslinked hydrogel microcapsules and evaluation of the microstructure and physio-chemical properties. J Mater Chem B, 2, 1470, 2014.
40.
YeJ., XiongJ., and SunR.The fluorescence property of Schiff's bases of carboxymethyl cellulose. Carbohydr Polym, 88, 1420, 2012.
41.
IssaR.M., KhedrA.M., and RizkH.1H NMR, IR and UV/VIS spectroscopic studies of some schiff bases derived from 2-aminobenzothiazole and 2-amino-3-hydroxypyridine. J Chin Chem Soc (Taipei), 55, 875, 2008.
42.
BajpaiS.K., and SharmaS.Investigation of swelling/degradation behaviour of alginate beads crosslinked with Ca2+ and Ba2+ ions. React Funct Polym, 59, 129, 2004.
43.
GomezC.G., RinaudoM., and VillarM.A.Oxidation of sodium alginate and characterization of the oxidized derivatives. Carbohydr Polym, 67, 296, 2007.
SobralJ.M., CaridadeS.G., SousaR.A., ManoJ.F., and ReisR.L.Three-dimensional plotted scaffolds with controlled pore size gradients: effect of scaffold geometry on mechanical performance and cell seeding efficiency. Acta Biomater, 7, 1009, 2011.
46.
Leal-EgañaA., Dietrich-BraumannU., Díaz-CuencaA., NowickiM., and BaderA.Determination of pore size distribution at the cell-hydrogel interface. J Nanobiotechnol, 9, 24, 2011.
47.
KhattakS.F., SpataraM., RobertsL., and RobertsS.C.Application of colorimetric assays to assess viability, growth and metabolism of hydrogel-encapsulated cells. Biotechnol Lett, 28, 1361, 2006.
48.
EvangelistaM.B., HsiongS.X., FernandesR., SampaioP., KongH.J., BarriasC.C., et al.Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix. Biomaterials, 28, 3644, 2007.
49.
KongH.J., BoontheekulT., and MooneyD.J.Quantifying the relation between adhesion ligand-receptor bond formation and cell phenotype. Proc Natl Acad Sci U S A, 103, 18534, 2006.
50.
LeeJ.W., ParkY.J., LeeS.J., LeeS.K., and LeeK.Y.The effect of spacer arm length of an adhesion ligand coupled to an alginate gel on the control of fibroblast phenotype. Biomaterials, 31, 5545, 2010.
51.
HuntN.C., SheltonR.M., HendersonD.J., and GroverL.M.Calcium-alginate hydrogel-encapsulated fibroblasts provide sustained release of vascular endothelial growth factor. Tissue Eng Part A, 19, 905, 2013.
52.
BidarraS.J., BarriasC.C., BarbosaM.A., SoaresR., and GranjaP.L.Immobilization of human mesenchymal stem cells within RGD-grafted alginate microspheres and assessment of their angiogenic potential. Biomacromolecules, 11, 1956, 2010.
53.
KeshawH., ForbesA., and DayR.M.Release of angiogenic growth factors from cells encapsulated in alginate beads with bioactive glass. Biomaterials, 26, 4171, 2005.
54.
HuntN.C., SmithA.M., GbureckU., SheltonR.M., and GroverL.M.Encapsulation of fibroblasts causes accelerated alginate hydrogel degradation. Acta Biomater, 6, 3649, 2010.
55.
YuanY., TseK.T., SinF.W.Y., XueB., FanH.H., and XieY.Ex vivo amplification of human hematopoietic stem and progenitor cells in an alginate three-dimensional culture system. Int J Lab Hematol, 33, 516, 2011.
56.
ChayosumritM., TuchB., and SidhuK.Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm. Biomaterials, 31, 505, 2010.
57.
HuebschN., AranyP.R., MaoA.S., ShvartsmanD., AliO.A., BencherifS.A., et al.Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat Mater, 9, 518, 2010.
58.
BidarraS.J., BarriasC.C., FonsecaK.B., BarbosaM.A., SoaresR.A., and GranjaP.L.Injectable in situ crosslinkable RGD-modified alginate matrix for endothelial cells delivery. Biomaterials, 32, 7897, 2011.
59.
CaiK., ZhangJ., DengL.H., YangL., HuY., ChenC., et al.Physical and biological properties of a novel hydrogel composite based on oxidized alginate, gelatin and tricalcium phosphate for bone tissue engineering. Adv Eng Mater, 9, 1082, 2007.
60.
DetschR., SarkerB., GrigoreA., and BoccacciniA.R.Alginate and gelatine blending for bone cell printing and biofabrication. IASTED International Conference Biomedical Engineering Innsbruck. Austria: ACTA Press, 2013, pp. 451–455.
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.
