Scaffold contraction is a common but underestimated problem in the field of tissue engineering. It becomes particularly problematic when creating anatomically complex shapes such as the ear. The aim of this study was to develop a contraction-free biocompatible scaffold construct for ear cartilage tissue engineering. To address this aim, we used three constructs: (i) a fibrin/hyaluronic acid (FB/HA) hydrogel, (ii) a FB/HA hydrogel combined with a collagen I/III scaffold, and (iii) a cage construct containing (ii) surrounded by a 3D-printed poly-ɛ-caprolactone mold. A wide range of different cell types were tested within these constructs, including chondrocytes, perichondrocytes, adipose-derived mesenchymal stem cells, and their combinations. After in vitro culturing for 1, 14, and 28 days, all constructs were analyzed. Macroscopic observation showed severe contraction of the cell-seeded hydrogel (i). This could be prevented, in part, by combining the hydrogel with the collagen scaffold (ii) and prevented in total using the 3D-printed cage construct (iii). (Immuno)histological analysis, multiphoton laser scanning microscopy, and biomechanical analysis showed extracellular matrix deposition and increased Young's modulus and thereby the feasibility of ear cartilage engineering. These results demonstrated that the 3D-printed cage construct is an adequate model for contraction-free ear cartilage engineering using a range of cell combinations.
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References
1.
SivayohamE., and WoolfordT.J.Current opinion on auricular reconstruction. Curr Opin Otolaryngol Head Neck Surg, 20, 287, 2012.
2.
MobinS.S.N., RayE., WuT., ReinischJ., and UrataM.M.Review of options for burned ear reconstruction. J Craniofac Surg, 21, 1165, 2010.
3.
BicharaA.D., O'SullivanN.-A., PomerantsevaI., ZhaoX., SundbackA.C., VacantiJ.P., and RandolphA.M.The tissue-engineered auricle: past, present, and future. Tissue Eng Part B Rev, 18, 51, 2012.
4.
CaoY., VacantiJ.P., PaigeK.T., UptonJ., and VacantiC.A.Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear. Plast Reconstr Surg, 100, 297, 1997.
5.
HaischA., KläringS., GrögerA., GebertC., and SittingerM.A tissue-engineering model for the manufacture of auricular-shaped cartilage implants. Eur Arch Otorhinolaryngol, 259, 316, 2002.
6.
ShiehS.J., TeradaS., and VacantiJ.P.Tissue engineering auricular reconstruction: In vitro and in vivo studies. Biomaterials, 25, 1545, 2004.
7.
PleumeekersM.M., NimeskernL., KoevoetW.L., KopsN., PoublonR.M., StokK.S., and van OschG.J.The in vitro and in vivo capacity of culture-expanded human cells from several sources encapsulated in alginate to form cartilage. Eur Cell Mater, 27, 264, 2014.
8.
TingV., SimsD.C., BrechtL.E., McCarthyJ.G., KasabianA.K., ConnellyP.R., ElisseeffJ., GittesG.K., and LongakerM.T.In vitro prefabrication of human cartilage shapes using fibrin glue and human chondrocytes. Ann Plast Surg, 40, 413, 1998.
9.
ZhouL., PomerantsevaI., BassettE.K., BowleyC.M., ZhaoX., BicharaD.A., KulligK.M., VacantiJ.P., RandolphM.A., and SundbackC.A.Engineering ear constructs with a composite scaffold to maintain dimensions. Tissue Eng Part A, 17, 1573, 2011.
10.
YanagaH., ImaiK., FujimotoT., and YanagaK.Generating ears from cultured autologous auricular chondrocytes by using two-stage implantation in treatment of microtia. Plast Reconstr Surg, 124, 817, 2009.
11.
TogoT., UtaniA., NaitohM., OhtaM., TsujiY., MorikawaN., MotonubuN., and SuzukiS.Identification of cartilage progenitor cells in the adult ear perichondrium: utilization for cartilage reconstruction. Lab Invest, 86, 445, 2006.
12.
BahraniH., RamzkhahM., AshrafM.J., TanidehN., ChenariN., KhademiB., and GhaderiA.Differentiation of adipose-derived stem cells into ear auricle cartilage in rabbits. J Laryngol Otol, 126, 770, 2012.
13.
LeeF., and KurisawaM.Formation and stability of interpenetrating polymer network hydrogels consisting of fibrin and hyaluronic acid for tissue engineering. Acta Biomater, 9, 5143, 2013.
14.
MöllerL., KrauseA., DahlmannJ., GruhI., KirschningA., and DrägerG.Preparation and evaluation of hydrogel-composites from methacrylated hyaluronic acid, alginate, and gelatin for tissue engineering. Int J Artif Organs, 34, 93, 2011.
15.
NoguchiT., YamamuroT., OkaM., KumarP., HyonK.S., and IkadaY.Poly(vinyl alcohol) hydrogel as an artificial articular cartilage: evaluation of biocompatibility. J Appl Biomater, 2, 101, 1991.
16.
MolinaroG., LerouxJ.C., DamasJ., and AdamA.Biocompatibility of thermosensitive chitosan-based hydrogels: an in vivo experimental approach to injectable biomaterials. Biomaterials, 23, 2717, 2002.
17.
BottK., UptonZ., SchrobbackK., EhrbarM., HubbellJ.A., LutolfM.P.and Rizzi, S.C. The effect of matrix characteristics on fibroblast proliferation in 3D gels. Biomaterials, 31, 8454, 2010.
18.
LeeK.Y., and MooneyD.J.Hydrogels for tissue engineering applications. Chem Rev, 101, 1869, 2001.
19.
LeeC.H., SinglaA., and LeeY.Biomedical applications of collagen. Int J Pharm, 221, 1, 2001.
20.
SewingJ., KlingerM., and NotbohmH.Jellyfish collagen matrices conserve the chondrogenic phenotype in two- and three-dimensional collagen matrices. J Tissue Eng Regen Med (2015); [Epub ahead of print]; DOI: 10.1002/term.1993.
21.
HuiT.Y., CheungK.M.C., CheungW.L., ChanD., and ChanB.P.In vitro chondrogenic differentiation of human mesenchymal stem cells in collagen microspheres: influence of cell seeding density and collagen concentration. Biomaterials, 29, 3201, 2008.
22.
JurgensW.J., KroezeR.J., Zandieh-DoulabiB., Dijk vanA., RendersG.A., van MilligenF.J., RittM.J., and HelderM.N.One-step surgical procedure for the treatment of osteochondral defects with adipose-derived stem cells in a caprine knee defect: a pilot study. Biores Open Access, 2, 315, 2013.
LeeJ.-S., HongJ.M., JungJ.W., ShimJ.-H., OhJ.-H., and ChoD.-W.3D printing of composite tissue with complex shape applied to ear regeneration. Biofabrication, 6, 024103, 2014.
27.
WoodruffM.A., and HutmacherD.W.The return of a forgotten polymer—polycaprolactone in the 21st century. Prog Polym Sci, 35, 1217, 2010.
28.
ZukP, ZhuM., AshjianP., De UgarteD.A., HuangJ.I., MizunoH., AlfonsoZ.C., FraserJ.K., BenhaimP., HedrickM.H.Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 13, 4279, 2002.
29.
StollerP., CelliersP.M., ReiserK.M., and RubenchikA.M.Quantitative second-harmonic generation microscopy in collagen. Appl Opt, 42, 5209, 2003.
30.
CoxG., KableE., JonesA., FraserI., ManconiF., and GorrellM.D.3-Dimensional imaging of collagen using second harmonic generation. J Struct Biol, 141, 53, 2003.
31.
CampagnolaP.J., MillardA., TerasakiM., HoppeP.E., MaloneC.J., and MohlerW.A.Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys J, 82, 493, 2002.
32.
StruplerM., PenaA.-M., HernestM., TharauxP.-L., MartinJ.-L., BeaurepaireE., and Schanne-KleinM.C.Second harmonic imaging and scoring of collagen in fibrotic tissues. Opt Express, 15, 4054, 2007.
33.
MansfieldJ.C., WinloveC.P., MogerJ., and MatcherS.J.Collagen fiber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy. J Biomed Opt, 13, 044020, 2013.
34.
SuP.J., ChenT., LiT.H., ChouC.K., ChenC.H., HoY.Y., HuangC.H., ChuangS.J., HuangY.Y., LeeH.S., and DongC.Y.The discrimination of type I and type II collagen and the label-free imaging of engineered cartilage tissue. Biomaterials, 31, 9415, 2010.
35.
BrockbankK.G.M., MacLellanW.R., XieJ., Hamm-AlvarezS.F., ChenZ.Z., and Schenke-LaylandK.Quantitative second harmonic generation imaging of cartilage damage. Cell Tissue Bank, 9, 299, 2008.
36.
LilledahlM.B., PierceD.M., RickenT., HolzapfelG.A., and DaviesC.D.L.Structural analysis of articular cartilage using multiphoton microscopy: Input for biomechanical modeling. IEEE Trans Med Imaging, 30, 1635, 2011.
37.
YehA.T., Hammer-WilsonM., Sickle vanD.C., BentonH.P., ZoumiA., TrombergB.J., and PeavyG.M.Nonlinear optical microscopy of articular cartilage. Osteoarthritis Cartilage, 13, 345, 2005.
38.
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.
39.
NehrerS., BreinanH., RamappaA., YoungG., ShortkroffS., LouieL.K., SledgeC.B., YannasI.V., and SpectorM.Matrix collagen type and pore size influence behaviour of seeded canine chondrocytes. Biomaterials, 18, 769, 1997.
40.
LeeC.R., GrodzinskyA.J., and SpectorM.The effects of cross-linking of collagen-glycosaminoglycan scaffolds on compressive stiffness, chondrocyte-mediated contraction, proliferation and biosynthesis. Biomaterials, 22, 3145, 2001.
41.
KobayashiS., TakebeT., ZhengY.-W., MizunoM., YabukiY., MaegawaJ., and TaniguchiH.Presence of cartilage stem/progenitor cells in adult mice auricular perichondrium. PLoS One, 6, e26393, 2011.
42.
AhearneM., LiuK., El HajA.J., ThenK.Y., RauzS., and YangY.Online monitoring of the mechanical behavior of collagen hydrogels: influence of corneal fibroblasts on elastic modulus. Tissue Eng Part C Methods, 16, 319, 2010.
43.
WudebweU.N.G., BannermanA., Goldberg-OppenheimP., PaxtonJ.Z., WilliamsR.L., and GroverL.M.Exploiting cell-mediated contraction and adhesion to structure tissues in vitro. Philos Trans R Soc Lond B Biol Sci, 370, 1, 2015.
44.
KlumpersD.D., ZhaoX., MooneyD.J., and SmitT.H.Cell mediated contraction in 3D cell-matrix constructs leads to spatially regulated osteogenic differentiation. Integr Biol, 5, 1174, 2013.
45.
MaL., GaoC., MaoZ., ZhouJ., and ShenJ.Biodegradability and cell-mediated contraction of porous collagen scaffolds: the effect of lysine as a novel crosslinking bridge. J Biomed Mater Res A, 71, 334, 2004.
46.
ChenC.-H., ShyuV.B.-H., ChenJ.-P., and LeeM.-Y.Selective laser sintered poly-ɛ-caprolactone scaffold hybridized with collagen hydrogel for cartilage tissue engineering. Biofabrication, 6, 015004, 2014.
47.
TempleJ.P., HuttonD., HungB.P., HuriP.Y., CookC.A., KondraguntaR., JiaX., and GraysonW.L.Engineering anatomically shaped vascularized bone grafts with hASCs and 3D-printed PCL scaffolds. J Biomed Mater Res A, 102, 4317, 2014.
48.
ParkS., KimG., JeonY.C., KohY., and KimW.3D polycaprolactone scaffolds with controlled pore structure using a rapid prototyping system. J Mater Sci Mater Med, 20, 229, 2009.
49.
XuT., BinderK.W., AlbannaM., DiceD., ZhaoW., YooJ.J., and AtalaA.Hybrid printing of mechanically and biologically improved constructs for cartilage tissue engineering applications. Biofabrication, 5, 015001, 2013.
50.
JurgensW.J., KroezeR.J., BankR.A., RittM.J., and HelderM.N.Rapid attachment of adipose stromal cells on resorbable polymeric scaffolds facilitates the one-step surgical procedure for cartilage and bone tissue engineering purposes. J Orthop Res, 29, 853, 2011.
51.
HoogendoornR.J.W., LuZ., KroezeR.J., BankR.A., WuismanP.I., and HelderM.N.Adipose stem cells for intervertebral disc regeneration: current status and concepts for the future. J Cell Mol Med, 12, 2205, 2008.
52.
ParkS.H., KimT.G., KimH.C., YangD.-Y., and ParkT.G.Development of dual scale scaffolds via direct polymer melt deposition and electrospinning for applications in tissue regeneration. Acta Biomater, 4, 1198, 2008.
53.
KimJ.-J., BaeW., KimJ., KimJ., LeeE., KimH., and KimE.Mineralized polycaprolactone nanofibrous matrix for odontogenesis of human dental pulp cells. J Biomater Appl, 28, 1069, 2014.
54.
LeeJ.W., KimY., ParkK., JeeK.S., ShinJ.W., and HahnS.B.Importance of integrin β1-mediated cell adhesion on biodegradable polymers under serum depletion in mesenchymal stem cells and chondrocytes. Biomaterials, 25, 1901, 2004.
55.
LamC.X.F., HutmacherD.W., SchantzJ.-T., WoodruffM.A., and TeohS.H.Evaluation of polycaprolactone scaffold degradation for 6 months in vitro and in vivo. J Biomed Mater Res A, 90, 906, 2009.
56.
XieX., WangY., ZhaoC., GuoS., LiuS., JiaW., TuanR.S., and ZhangC.Comparative evaluation of MSCs from bone marrow and adipose tissue seeded in PRP-derived scaffold for cartilage regeneration. Biomaterials, 33, 7008, 2012.
57.
LiQ., TangJ., WangR., BeiC., XinL., ZengY., and TangX.Comparing the chondrogenic potential in vivo of autogeneic mesenchymal stem cells derived from different tissues. Artif Cells Blood Substit Immobil Biotechnol, 39, 31, 2011.
58.
JungS.-N., RhieJ., KwonH., JunY.J., SeoJ.W., YooG., OhD.Y., AhnS.T., WooJ., and OhJ.In vivo cartilage formation using chondrogenic-differentiated human adipose-derived mesenchymal stem cells mixed with fibrin glue. J Craniofac Surg, 21, 468, 2010.
59.
WuL., PrinsH.J., HelderM.N., van BlitterswijkC.A., and KarperienM.Trophic effects of mesenchymal stem cells in chondrocyte co-cultures are independent of culture conditions and cell sources. Tissue Eng Part A, 18, 1542, 2012.
60.
WuL., PrinsHJ., LeijtenJ.C., HelderM., EvseenkoD., MoroniL., van BlitterswijkC., LinY., and KarperienM.Chondrocytes co-cultured with stromal vascular fraction of adipose tissue presents more intense chondrogenic cues characteristics than with adipose stem cells. Tissue Eng Part A, 22, 336, 2016.
61.
HellingmanC.A., VerwielE.T.P., SlagtI., KoevoetW., PoublonR.M.L., Nolst-TrenitéG.L., and Baatenburg de JongR.J.Differences in cartilage-forming capacity of expanded human chondrocytes from ear and nose and their gene expression profiles. Cell Transplant, 20, 925, 2011.
62.
DuynsteeM.L., Verwoerd-VerhoefH.L., and VerwoerdC.D., and Van OschG.J.The dual role of perichondrium in cartilage wound healing. Plast Reconstr Surg, 110, 1073, 2002.
63.
SterodimasA., and de FariaJ.Human auricular tissue engineering in an immunocompetent animal model. Aesthet Surg J, 33, 283, 2013.
64.
KobayashiS., TakabeT., InuiM., IwaiS., KanH., ZhengY.-W., MaegawaJ., and TaniguchiH.Reconstruction of human elastic cartilage by a CD44+ CD90+ stem cell in the ear perichondrium. Proc Natl Acad Sci U S A, 108, 14479, 2011.
NaumannA., DennisJ., AwadallahA., CarinoD.A., MansourJ.M., KastenbauerE., and CaplanA.I.Immunochemical and mechanical characterization of cartilage subtypes in rabbit. J Histochem Cytochem, 50, 1049, 2002.
67.
ParkS.S., JinH.-R., ChiD.H., and TaylorR.S.Characteristics of tissue-engineered cartilage from human auricular chondrocytes. Biomaterials, 25, 2363, 2004.
68.
Bastiaansen-JenniskensY.M., KoevoetW., De BartA., ZuurmondA-M., BankR.A., VerhaarJ.A., De GrootJ., and van OschG.J.TGFbeta affects collagen cross-linking independent of chondrocyte phenotype but strongly depending on physical environment. Tissue Eng. Part A, 14, 1059, 2008.
69.
HanE., ChenS.S., KlischS.M., and SahR.L.Contribution of proteoglycan osmotic swelling pressure to the compressive properties of articular cartilage. Biophys J, 101, 916, 2011.
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