Scaffold designs are critical for in vitro culture of tissue-engineered cartilage in three-dimensional environments to enhance cellular differentiation for tissue engineering and regenerative medicine. In the present study we demonstrated silk and fibrin/hyaluronic acid (HA) composite gels as scaffolds for nucleus pulposus (NP) cartilage formation, providing both biochemical support for NP outcomes as well as fostering the retention of size of the scaffold during culture due to the combined features of the two proteins. Passage two (P2) human chondrocytes cultured in 10% serum were encapsulated within silk-fibrin/HA gels. Five study groups with fibrin/HA gel culture (F/H) along with varying silk concentrations (2% silk gel only, fibrin/HA gel culture with 1% silk [F/H+1S], 1.5% silk [F/H+1.5S], and 2% silk [F/H+2S]) were cultured in serum-free chondrogenic defined media (CDM) for 4 weeks. Histological examination with alcian blue showed a defined chondrogenic area at 1 week in all groups that widened homogenously until 4 weeks. In particular, chondrogenic differentiation observed in the F/H+1.5S had no reduction in size throughout the culture period. The results of biochemical and molecular biological evaluations supported observations made during histological examination. Mechanical strength measurements showed that the silk mixed gels provided stronger mechanical properties for NP tissue than fibrin/HA composite gels in CDM. This effect could potentially be useful in the study of in vitro NP tissue engineering as well as for clinical implications for NP tissue regeneration.
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References
1.
EhrlichG.E.Low back pain. Bull World Health Organ, 81:671. 2003.
2.
KelseyJ.L., WhiteA.A.3rd. Epidemiology and impact of low-back pain. Spine (Phila Pa 1976), 5:133. 1980.
3.
GanJ.C., DucheyneP., VresilovicE.J., SwaimW., ShapiroI.M.Intervertebral disc tissue engineering I: characterization of the nucleus pulposus. Clin Orthop Relat Res, 411:305. 2003.
4.
KitaharaH.Histochemical study of the human intervertebral disc. Nippon Seikeigeka Gakkai Zasshi, 53:817. 1979.
5.
RisbudM.V., IzzoM.W., AdamsC.S., ArnoldW.W., HillibrandA.S., VresilovicE.J., VaccaroA.R., AlbertT.J., ShapiroI.M.An organ culture system for the study of the nucleus pulposus: description of the system and evaluation of the cells. Spine (Phila Pa 1976), 28:2652. 2003.
6.
GruberH.E., HanleyE.N.Jr.Biologic strategies for the therapy of intervertebral disc degeneration. Expert Opin Biol Ther, 3:1209. 2003.
7.
HubbellJ.A.Materials as morphogenetic guides in tissue engineering. Curr Opin Biotechnol, 14:551. 2003.
8.
DruryJ.L., MooneyD.J.Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials, 24:4337. 2003.
9.
BoydL.M., CarterA.J.Injectable biomaterials and vertebral endplate treatment for repair and regeneration of the intervertebral disc. Eur Spine J, 15,Suppl 3:S414. 2006.
10.
SakaiD., MochidaJ., YamamotoY., NomuraT., OkumaM., NishimuraK., NakaiT., AndoK., HottaT.Transplantation of mesenchymal stem cells embedded in Atelocollagen gel to the intervertebral disc: a potential therapeutic model for disc degeneration. Biomaterials, 24:3531. 2003.
11.
SakaiD., MochidaJ., IwashinaT., WatanabeT., SuyamaK., AndoK., HottaT.Atelocollagen for culture of human nucleus pulposus cells forming nucleus pulposus-like tissue in vitro: influence on the proliferation and proteoglycan production of HNPSV-1 cells. Biomaterials, 27:346. 2006.
12.
MizunoH., RoyA.K., ZaporojanV., VacantiC.A., UedaM., BonassarL.J.Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs. Biomaterials, 27:362. 2006.
13.
MwaleF., IordanovaM., DemersC.N., SteffenT., RoughleyP., AntoniouJ.Biological evaluation of chitosan salts cross-linked to genipin as a cell scaffold for disk tissue engineering. Tissue Eng, 11:130. 2005.
14.
Sha'banM., YoonS.J., KoY.K., HaH.J., KimS.H., SoJ.W., IdrusR.B., KhangG.Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold. J Biomater Sci Polym Ed, 19:1219. 2008.
15.
GruberH.E., LeslieK., IngramJ., NortonH.J., HanleyE.N.Cell-based tissue engineering for the intervertebral disc: in vitro studies of human disc cell gene expression and matrix production within selected cell carriers. Spine J, 4:44. 2004.
16.
CloydJ.M., MalhotraN.R., WengL., ChenW., MauckR.L., ElliottD.M.Material properties in unconfined compression of human nucleus pulposus, injectable hyaluronic acid-based hydrogels and tissue engineering scaffolds. Eur Spine J, 16:1892. 2007.
17.
KoganG., SoltesL., SternR., GemeinerP.Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnol Lett, 29:17. 2007.
18.
BrobergK.B.On the mechanical behaviour of intervertebral discs. Spine (Phila Pa 1976), 8:151. 1983.
19.
ParkS.H., CuiJ.H., ParkS.R., MinB.H.Potential of fortified fibrin/hyaluronic acid composite gel as a cell delivery vehicle for chondrocytes. Artificial organs, 33:439. 2009.
20.
ParkS.H., ParkS.R., ChungS.I., PaiK.S., MinB.H.Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation. Artif Organs, 29:838. 2005.
21.
ChouC.H., ChengW.T., KuoT.F., SunJ.S., LinF.H., TsaiJ.C.Fibrin glue mixed with gelatin/hyaluronic acid/chondroitin-6-sulfate tri-copolymer for articular cartilage tissue engineering: the results of real-time polymerase chain reaction. J Biomed Mater Res A, 82:757. 2007.
22.
PerettiG.M., RandolphM.A., ZaporojanV., BonassarL.J., XuJ.W., FellersJ.C., YaremchukM.J.A biomechanical analysis of an engineered cell-scaffold implant for cartilage repair. Ann Plast Surg, 46:533. 2001.
23.
Seda TigliR., GhoshS., LahaM.M., ShevdeN.K., DaheronL., GimbleJ., GumusdereliogluM., KaplanD.L.Comparative chondrogenesis of human cell sources in 3D scaffolds. J Tissue Eng Regen Med, 3:348. 2009.
KimH.J., KimU.J., Vunjak-NovakovicG., MinB.H., KaplanD.L.Influence of macroporous protein scaffolds on bone tissue engineering from bone marrow stem cells. Biomaterials, 26:4442. 2005.
ChoiK.H., YooB.S., ParkS.R., ChoiB.H., MinB.H.Novel imaging analysis system to measure the spatial dimension of engineered tissue construct. Artif Organs, 34:158. 2009.
32.
WhitleyC.B., RidnourM.D., DraperK.A., DuttonC.M., NegliaJ.P.Diagnostic test for mucopolysaccharidosis. I. Direct method for quantifying excessive urinary glycosaminoglycan excretion. Clin Chem, 35:374. 1989.
33.
RossoF., MarinoG., GiordanoA., BarbarisiM., ParmeggianiD., BarbarisiA.Smart materials as scaffolds for tissue engineering. J Cell Physiol, 203:465. 2005.
34.
O'HalloranD.M., PanditA.S.Tissue-engineering approach to regenerating the intervertebral disc. Tissue Eng, 13:1927. 2007.
35.
PaesoldG., NerlichA.G., BoosN.Biological treatment strategies for disc degeneration: potentials and shortcomings. Eur Spine J, 16:447. 2007.
36.
ChenW.H., LiuH.Y., LoW.C., WuS.C., ChiC.H., ChangH.Y., HsiaoS.H., WuC.H., ChiuW.T., ChenB.J., DengW.P.Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma. Biomaterials, 30:5523. 2009.
37.
GruberH.E., StaskyA.A., HanleyE.N.Jr.Characterization and phenotypic stability of human disc cells in vitro. Matrix Biol, 16:285. 1997.
38.
PodskubkaA., PovysilC., KubesR., SprindrichJ., SedlacekR.[Treatment of deep cartilage defects of the knee with autologous chondrocyte transplantation on a hyaluronic Acid ester scaffolds (Hyalograft C)]Acta Chir Orthop Traumatol Cech, 73:251. 2006.
39.
RobertH., BahuaudJ.Autologous chondrocyte implantation. A review of techniques and preliminary results. Rev Rhum Engl Ed, 66:724. 1999.
ZhangY., LiZ., ThonarE.J., AnH.S., HeT.C., PietrylaD., PhillipsF.M.Transduced bovine articular chondrocytes affect the metabolism of cocultured nucleus pulposus cells in vitro: implications for chondrocyte transplantation into the intervertebral disc. Spine (Phila Pa 1976), 30:2601. 2005.
SiveJ.I., BairdP., JeziorskM., WatkinsA., HoylandJ.A., FreemontA.J.Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol, 55:91. 2002.
44.
RobertsS., MenageJ., DuanceV., WottonS., AyadS.Volvo Award in basic sciences. Collagen types around the cells of the intervertebral disc and cartilage end plate: an immunolocalization study. Spine (Phila Pa 1976), 16:1030. 1991.
45.
TappH., HanleyE.N.Jr., PattJ.C., GruberH.E.Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair. Exp Biol Med (Maywood), 234:1. 2009.
GruberH.E., FisherE.C.Jr., DesaiB., StaskyA.A., HoelscherG., HanleyE.N.Jr.Human intervertebral disc cells from the annulus: three-dimensional culture in agarose or alginate and responsiveness to TGF-beta1. Exp Cell Res, 235:13. 1997.
48.
AliniM., LiW., MarkovicP., AebiM., SpiroR.C., RoughleyP.J.The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix. Spine (Phila Pa 1976), 28:446. 2003.
49.
ThompsonJ.P., OegemaT.R.Jr., BradfordD.S.Stimulation of mature canine intervertebral disc by growth factors. Spine (Phila Pa 1976), 16:253. 1991.
50.
LeungV.Y., ChanD., CheungK.M.Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine J, 15,Suppl 3:S406. 2006.
51.
ChibaK., AnderssonG.B., MasudaK., ThonarE.J.Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate. Spine (Phila Pa 1976), 22:2885. 1997.
52.
RaghunathJ., RolloJ., SalesK.M., ButlerP.E., SeifalianA.M.Biomaterials and scaffold design: key to tissue-engineering cartilage. Biotechnol Appl Biochem, 46:73. 2007.
53.
SakaiD., MochidaJ., IwashinaT., HiyamaA., OmiH., ImaiM., NakaiT., AndoK., HottaT.Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc. Biomaterials, 27:335. 2006.
54.
PerkaC., ArnoldU., SpitzerR.S., LindenhaynK.The use of fibrin beads for tissue engineering and subsequential transplantation. Tissue Eng, 7:359. 2001.
55.
YoonD.M., CurtisS., ReddiA.H., FisherJ.P.Addition of hyaluronic acid to alginate embedded chondrocytes interferes with IGF-1 signaling in vitro and in vivo. Tissue Eng Part A, 15:3449. 2009.
56.
PerkaC., SchultzO., LindenhaynK., SpitzerR.S., MuschikM., SittingerM., BurmesterG.R.Joint cartilage repair with transplantation of embryonic chondrocytes embedded in collagen-fibrin matrices. Clin Exp Rheumatol, 18:13. 2000.
57.
BuckwalterJ.A., SmithK.C., KazarienL.E., RosenbergL.C., UngarR.Articular cartilage and intervertebral disc proteoglycans differ in structure: an electron microscopic study. J Orthop Res, 7:146. 1989.
58.
ChungC., MesaJ., MillerG.J., RandolphM.A., GillT.J., BurdickJ.A.Effects of auricular chondrocyte expansion on neocartilage formation in photocrosslinked hyaluronic acid networks. Tissue Eng, 12:2665. 2006.
59.
KawasakiK., OchiM., UchioY., AdachiN., MatsusakiM.Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels. J Cell Physiol, 179:142. 1999.
60.
CrevenstenG., WalshA.J., AnanthakrishnanD., PageP., WahbaG.M., LotzJ.C., BervenS.Intervertebral disc cell therapy for regeneration: mesenchymal stem cell implantation in rat intervertebral discs. Ann Biomed Eng, 32:430. 2004.
61.
SternS., LindenhaynK., SchultzO., PerkaC.Cultivation of porcine cells from the nucleus pulposus in a fibrin/hyaluronic acid matrix. Acta Orthop Scand, 71:496. 2000.
62.
ParkS.H., ParkS.R., MinB.H.Reinforcement fibrin-hyaluronic acid composite gel for tissue engineering cartilage genesis. Key Eng Mat, 342–343:253. 2007.
NumataK., CebeP., KaplanD.L.Mechanism of enzymatic degradation of beta-sheet crystals. Biomaterials, 31:2926. 2010.
65.
WangY., KimU.J., BlasioliD.J., KimH.J., KaplanD.L.In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells. Biomaterials, 26:7082. 2005.
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