Skip to main content
Log in

Chitosan-based composite bilayer scaffold as an in vitro osteochondral defect regeneration model

  • Published:
Biomedical Microdevices Aims and scope Submit manuscript

Abstract

Prolonged osteochondral tissue damage can result in osteoarthritis and decreased quality of life. Multiphasic scaffolds, where different layers model different microenvironments, are a promising treatment approach, yet stable joining between layers during fabrication remains challenging. Here, a bilayer scaffold for osteochondral tissue regeneration was fabricated using thermally-induced phase separation (TIPS). Two distinct polymer solutions were layered before TIPS, and the resulting porous, bilayer scaffold was characterized by seamless interfacial integration and a mechanical stiffness gradient reflecting the native osteochondral microenvironment. Chitosan is a critical component of both scaffold layers to facilitate cell attachment and the formation of polyelectrolyte complexes with other biologically relevant natural polymers. The articular cartilage region was optimized for hyaluronic acid content and stiffness, while the subchondral bone region was defined by higher stiffness and osteoconductive hydroxyapatite content. Following co-culture with chondrocyte-like (SW-1353 or mesenchymal stem cells) and osteoblast-like cells (MG63), cell proliferation and migration to the interface along with increased gene expression associated with relevant markers of osteogenesis and chondrogenesis indicates the potential of this bilayer scaffold for osteochondral tissue regeneration.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

References

  • H.M. Aydin, A three-layered osteochondral plug: Structural, mechanical, and in vitro biocompatibility analysis. Adv. Eng. Mater. 13(12), B511–B517 (2011)

    Article  Google Scholar 

  • D.L. Batchelar, M.T.M. Davidson, W. Dabrowski, I.A. Cunningham, Bone-composition imaging using coherent-scatter computed tomography: Assessing bone health beyond bone mineral density. Med. Phys. 33(4), 904–915 (2006)

    Article  Google Scholar 

  • J.E. Bekkers, T.S. de Windt, M. Brittberg, D.B. Saris, Cartilage repair in football (soccer) athletes: What evidence leads to which treatment? A critical review of the literature. Cartilage 3(1 Suppl), 43S–49S (2012)

    Article  Google Scholar 

  • R. Bexkens, P.T. Ogink, J.N. Doornberg, G. Kerkhoffs, D. Eygendaal, L.S. Oh, M.P.J. van den Bekerom, Donor-site morbidity after osteochondral autologous transplantation for osteochondritis dissecans of the capitellum: A systematic review and meta-analysis. Knee Surg. Sports Traumatol. Arthrosc. 25(7), 2237–2246 (2017)

    Article  Google Scholar 

  • B.E. Bobick, F.H. Chen, A.M. Le, R.S. Tuan, Regulation of the Chondrogenic phenotype in culture. Birth Defects Res. C. Embryo Today 87(4), 351–371 (2009)

  • B. Cecen, L.D. Kozaci, M. Yuksel, O. Ustun, B.U. Ergur, H. Havitcioglu, Biocompatibility and biomechanical characteristics of loofah based scaffolds combined with hydroxyapatite, cellulose, poly-l-lactic acid with chondrocyte-like cells. Mater. Sci. Eng. C 69, 437–446 (2016)

    Article  Google Scholar 

  • B.M. Chesnutt, A.M. Viano, Y. Yuan, Y. Yang, T. Guda, M.R. Appleford, et al., Design and characterization of a novel chitosan/nanocrystalline calcium phosphate composite scaffold for bone regeneration. J. Biomed. Mater. Res. A 88(2), 491–502 (2009)

    Article  Google Scholar 

  • C.-S. Chien, H.-O. Ho, Y.-C. Liang, P.-H. Ko, M.-T. Sheu, C.-H. Chen, Incorporation of exudates of human platelet-rich fibrin gel in biodegradable fibrin scaffolds for tissue engineering of cartilage. J. Biomed. Mater. Res. B Appl. Biomater. 100B(4), 948–955 (2012)

    Article  Google Scholar 

  • B.J. Cole, C. Pascual-Garrido, R.C. Grumet, Surgical management of articular cartilage defects in the knee. J. Bone Joint Surg. Am. 91(7), 1778–1790 (2009)

    Google Scholar 

  • A. Di Luca, B. Ostrowska, I. Lorenzo-Moldero, A. Lepedda, W. Swieszkowski, C. Van Blitterswijk, L. Moroni, Gradients in pore size enhance the osteogenic differentiation of human mesenchymal stromal cells in three-dimensional scaffolds. Sci. Rep. 6, 22898 (2016)

    Article  Google Scholar 

  • D.E. Discher, P. Janmey, Y.L. Wang, Tissue cells feel and respond to the stiffness of their substrate. Science 310(5751), 1139–1143 (2005)

    Article  Google Scholar 

  • R. Dorati, C. Colonna, C. Tomasi, I. Genta, G. Bruni, B. Conti, Design of 3D scaffolds for tissue engineering testing a tough polylactide-based graft copolymer. Mater. Sci. Eng. C Mater Biol Appl. 34, 130–139 (2014)

    Article  Google Scholar 

  • A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Matrix elasticity directs stem cell lineage specification. Cell 126(4), 677–689 (2006)

    Article  Google Scholar 

  • S.J. Florczyk, D.J. Kim, D.L. Wood, M. Zhang, Influence of processing parameters on pore structure of 3D porous chitosan-alginate polyelectrolyte complex scaffolds. J. Biomed. Mater. Res. A 98(4), 614–620 (2011)

    Article  Google Scholar 

  • S.J. Florczyk, M. Leung, S. Jana, Z.S. Li, N. Bhattarai, J.I. Huang, et al., Enhanced bone tissue formation by alginate gel-assisted cell seeding in porous ceramic scaffolds and sustained release of growth factor. J. Biomed. Mater. Res. A 100A(12), 3408–3415 (2012)

    Article  Google Scholar 

  • S.J. Florczyk, K. Wang, S. Jana, D.L. Wood, S.K. Sytsma, J.G. Sham, et al., Porous chitosan-hyaluronic acid scaffolds as a mimic of glioblastoma microenvironment ECM. Biomaterials 34(38), 10143–10150 (2013)

    Article  Google Scholar 

  • S.J. Florczyk, F.M. Kievit, K. Wang, A.E. Erickson, R.G. Ellenbogen, M.Q. Zhang, 3D porous chitosan-alginate scaffolds promote proliferation and enrichment of cancer stem-like cells. J. Mater. Chem. B 4(38), 6326–6334 (2016)

    Article  Google Scholar 

  • M. Frydrych, C.Y. Wan, R. Stengler, K.U. O'Kelly, B.Q. Chen, Structure and mechanical properties of gelatin/sepiolite nanocomposite foams. J. Mater. Chem. 21(25), 9103–9111 (2011)

    Article  Google Scholar 

  • A. Galperin, R.A. Oldinski, S.J. Florczyk, J.D. Bryers, M.Q. Zhang, B.D. Ratner, Integrated bi-layered scaffold for osteochondral tissue engineering. Adv. Healthc. Mater. 2(6), 872–883 (2013)

    Article  Google Scholar 

  • P. Gupta, M. Adhikary, J.C. M, M. Kumar, N. Bhardwaj, B.B. Mandal, Biomimetic, Osteoconductive non-mulberry silk Fiber reinforced Tricomposite scaffolds for bone tissue engineering. ACS Appl. Mater. Interfaces 8(45), 30797–30810 (2016)

    Article  Google Scholar 

  • A. He, L. Liu, X. Luo, Y. Liu, F. Liu, X. Wang, et al., Repair of osteochondral defects with in vitro engineered cartilage based on autologous bone marrow stromal cells in a swine model. Sci. Rep. 7, 40489 (2017)

    Article  Google Scholar 

  • Y.Y. Hsu, J.D. Gresser, D.J. Trantolo, C.M. Lyons, P.R.J. Gangadharam, D.L. Wise, Effect of polymer foam morphology and density on kinetics of in vitro controlled release of isoniazid from compressed foam matrices. J. Biomed. Mater. Res. 35(1), 107–116 (1997)

    Article  Google Scholar 

  • E.B. Hunziker, Articular cartilage repair: Basic science and clinical progress. A review of the current status and prospects. Osteoarthr. Cartil. 10(6), 432–463 (2002)

    Article  Google Scholar 

  • E.B. Hunziker, I.M. Driesang, Functional barrier principle for growth-factor-based articular cartilage repair. Osteoarthr. Cartil. 11(5), 320–327 (2003)

    Article  Google Scholar 

  • S. Jana, S.J. Florczyk, M. Leung, M.Q. Zhang, High-strength pristine porous chitosan scaffolds for tissue engineering. J. Mater. Chem. 22(13), 6291–6299 (2012)

    Article  Google Scholar 

  • X. Jin, J. Zhuang, Z. Zhang, H. Guo, J. Tan, Hydrothermal synthesis of hydroxyapatite nanorods in the presence of sodium citrate and its aqueous colloidal stability evaluation in neutral pH. J. Colloid Interface Sci. 443, 125–130 (2015)

    Article  Google Scholar 

  • F.M. Kievit, S.J. Florczyk, M.C. Leung, O. Veiseh, J.O. Park, M.L. Disis, M. Zhang, Chitosan-alginate 3D scaffolds as a mimic of the glioma tumor microenvironment. Biomaterials 31(22), 5903–5910 (2010)

    Article  Google Scholar 

  • G. Kim, M. Okumura, T. Ishiguro, T. Kadosawa, T. Fujinaga, Preventive effect of hyaluronic acid on the suppression of attachment and migration abilities of bovine chondrocytes by IL-1 alpha in vitro. J. Vet. Med. Sci. 65(3), 427–430 (2003)

    Article  Google Scholar 

  • I.Y. Kim, S.J. Seo, H.S. Moon, M.K. Yoo, I.Y. Park, B.C. Kim, C.S. Cho, Chitosan and its derivatives for tissue engineering applications. Biotechnol. Adv. 26(1), 1–21 (2008)

    Article  Google Scholar 

  • H.L. Kim, G.Y. Jung, J.H. Yoon, J.S. Han, Y.J. Park, D.G. Kim, et al., Preparation and characterization of nano-sized hydroxyapatite/alginate/chitosan composite scaffolds for bone tissue engineering. Mater. Sci. Eng. C Mater. Biol. Appl. 54, 20–25 (2015)

    Article  Google Scholar 

  • C.B. Knudson, W. Knudson, Hyaluronan and CD44: Modulators of chondrocyte metabolism. Clin. Orthop. Relat. Res. (427 Suppl), S152–S162 (2004)

  • E. Kon, M. Delcogliano, G. Filardo, M. Fini, G. Giavaresi, S. Francioli, et al., Orderly osteochondral regeneration in a sheep model using a novel nano-composite multilayered biomaterial. J. Orthop. Res. 28(1), 116–124 (2010)

    Google Scholar 

  • B. Kreklau, M. Sittinger, M.B. Mensing, C. Voigt, G. Berger, G.R. Burmester, et al., Tissue engineering of biphasic joint cartilage transplants. Biomaterials 20(18), 1743–1749 (1999)

    Article  Google Scholar 

  • S.L. Levengood, M. Zhang, Chitosan-based scaffolds for bone tissue engineering. J. Mater. Chem. B 2(21), 3161–3184 (2014)

    Article  Google Scholar 

  • T.J. Levingstone, A.C. Ramesh, R.T. Brady, P. Brama, J.P. Gleeson, F.J. O'Brien, Collagen-based multilayered scaffold shows potential for osteochondral defect repair. J. Tissue Eng. Regen. Med. 8, 82–83 (2014)

    Google Scholar 

  • T.J. Levingstone, E. Thompson, A. Matsiko, A. Schepens, J.P. Gleeson, F.J. O'Brien, Multi-layered collagen-based scaffolds for osteochondral defect repair in rabbits. Acta Biomater. 32, 149–160 (2016)

    Article  Google Scholar 

  • Z.S. Li, M.Q. Zhang, Chitosan-alginate as scaffolding material for cartilage tissue engineering. J. Biomed. Mater. Res. A 75A(2), 485–493 (2005)

    Article  Google Scholar 

  • Z.S. Li, H.R. Ramay, K.D. Hauch, D.M. Xiao, M.Q. Zhang, Chitosan-alginate hybrid scaffolds for bone tissue engineering. Biomaterials 26(18), 3919–3928 (2005)

    Article  Google Scholar 

  • E. Lopez-Ruiz, G. Jimenez, M.A. Garcia, C. Antich, H. Boulaiz, J.A. Marchal, M. Peran, Polymers, scaffolds and bioactive molecules with therapeutic properties in osteochondral pathologies: what’s new? Expert Opin. Ther. Pat. 26(8), 877–890 (2016)

    Article  Google Scholar 

  • H.-T. Lu, M.-S. Hsieh, C.-W. Cheng, L.-F. Yao, T.-Y. Hsu, J. Lan, et al., Alterative effects of an oral alginate extract on experimental rabbit osteoarthritis. J. Biomed. Sci. 22(1), 64 (2015)

    Article  Google Scholar 

  • H. Madry, C.N. van Dijk, M. Mueller-Gerbl, The basic science of the subchondral bone. Knee Surg. Sports Traumatol. Arthrosc. 18(4), 419–433 (2010)

    Article  Google Scholar 

  • P. Malmberg, H. Nygren, Methods for the analysis of the composition of bone tissue, with a focus on imaging mass spectrometry (TOF-SIMS). Proteomics 8(18), 3755–3762 (2008)

    Article  Google Scholar 

  • R. Marom, I. Shur, R. Solomon, D. Benayahu, Characterization of adhesion and differentiation markers of osteogenic marrow stromal cells. J. Cell. Physiol. 202(1), 41–48 (2005)

    Article  Google Scholar 

  • T. Nie, L. Xue, M. Ge, H. Ma, J. Zhang, Fabrication of poly(L-lactic acid) tissue engineering scaffolds with precisely controlled gradient structure. Mater. Lett. 176, 25–28 (2016)

    Article  Google Scholar 

  • G.G. Niederauer, M.A. Slivka, N.C. Leatherbury, D.L. Korvick, H.H. Harroff, W.C. Ehler, et al., Evaluation of multiphase implants for repair of focal osteochondral defects in goats. Biomaterials 21(24), 2561–2574 (2000)

    Article  Google Scholar 

  • S.H. Oh, I.K. Park, J.M. Kim, J.H. Lee, In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method. Biomaterials 28(9), 1664–1671 (2007)

    Article  Google Scholar 

  • S.H. Oh, T.H. Kim, J.H. Lee, Creating growth factor gradients in three dimensional porous matrix by centrifugation and surface immobilization. Biomaterials 32(32), 8254–8260 (2011)

    Article  Google Scholar 

  • R. Olivares-Navarrete, E.M. Lee, K. Smith, S.L. Hyzy, M. Doroudi, J.K. Williams, et al., Substrate stiffness controls osteoblastic and Chondrocytic differentiation of mesenchymal stem cells without exogenous stimuli. PLoS One 12(1), e0170312 (2017)

    Article  Google Scholar 

  • D. Schaefer, I. Martin, G. Jundt, J. Seidel, M. Heberer, A. Grodzinsky, et al., Tissue-engineered composites for the repair of large osteochondral defects. Arthritis Rheum. 46(9), 2524–2534 (2002)

    Article  Google Scholar 

  • M. Schinhan, M. Gruber, P. Vavken, R. Dorotka, L. Samouh, C. Chiari, et al., Critical-size defect induces unicompartmental osteoarthritis in a stable ovine knee. J. Orthop. Res. 30(2), 214–220 (2012)

    Article  Google Scholar 

  • K. Schlichting, H. Schell, R.U. Kleemann, A. Schill, A. Weiler, G.N. Duda, D.R. Epari, Influence of scaffold stiffness on subchondral bone and subsequent cartilage regeneration in an ovine model of osteochondral defect healing. Am. J. Sports Med. 36(12), 2379–2391 (2008)

    Article  Google Scholar 

  • S.-J. Seo, C. Mahapatra, R.K. Singh, J.C. Knowles, H.-W. Kim, Strategies for osteochondral repair: Focus on scaffolds. J. Tissue Eng. 5, 2041731414541850–2041731414541850 (2014)

    Article  Google Scholar 

  • D.E. Shepherd, B.B. Seedhom, The 'instantaneous' compressive modulus of human articular cartilage in joints of the lower limb. Rheumatology (Oxford) 38(2), 124–132 (1999)

    Article  Google Scholar 

  • R. Shu, R. McMullen, M.J. Baumann, L.R. McCabe, Hydroxyapatite accelerates differentiation and suppresses growth of MC3T3-E1 osteoblasts. J. Biomed. Mater. Res. A 67A(4), 1196–1204 (2003)

    Article  Google Scholar 

  • Y.P. Singh, J.C. Moses, B.K. Bhunia, S.K. Nandi, B.B. Mandal, Hierarchically structured seamless silk scaffolds for osteochondral interface tissue engineering. J. Mater. Chem. B 6(36), 5671–5688 (2018)

    Article  Google Scholar 

  • G.D. Smith, G. Knutsen, J.B. Richardson, A clinical review of cartilage repair techniques. J. Bone Joint Surg. Br. Vol. 87B(4), 445–449 (2005)

    Article  Google Scholar 

  • R. Trombetta, J.A. Inzana, E.M. Schwarz, S.L. Kates, H.A. Awad, 3D printing of calcium phosphate ceramics for bone tissue engineering and drug delivery. Ann. Biomed. Eng. 45(1), 23–44 (2017)

    Article  Google Scholar 

  • J. Venkatesan, S.-K. Kim, Chitosan composites for bone tissue engineering—An overview. Mar. Drugs. 8(8), 2252–2266 (2010)

  • K. Wang, F.M. Kievit, A.E. Erickson, J.R. Silber, R.G. Ellenbogen, M.Q. Zhang, Culture on 3D chitosan-hyaluronic acid scaffolds enhances stem cell marker expression and drug resistance in human glioblastoma Cancer stem cells. Adv. Healthc. Mater. 5(24), 3173–3181 (2016)

    Article  Google Scholar 

  • S. Weiner, H.D. Wagner, THE MATERIAL BONE: Structure-mechanical function relations. Annu. Rev. Mater. Sci. 28(1), 271–298 (1998)

    Article  Google Scholar 

  • S. Yang, K.F. Leong, Z. Du, C.K. Chua, The design of scaffolds for use in tissue engineering. Part I. Traditional factors. Tissue Eng. 7(6), 679–689 (2001)

    Article  Google Scholar 

  • H.S. Yoo, E.A. Lee, J.J. Yoon, T.G. Park, Hyaluronic acid modified biodegradable scaffolds for cartilage tissue engineering. Biomaterials 26(14), 1925–1933 (2005)

    Article  Google Scholar 

  • P. Yusong, S. Qianqian, P. Chengling, W. Jing, Prediction of mechanical properties of multilayer gradient hydroxyapatite reinforced poly(vinyl alcohol) gel biomaterial. J. Biomed. Mater. Res. B Appl. Biomater. 101B(5), 729–735 (2013)

    Article  Google Scholar 

  • R.Y. Zhang, P.X. Ma, Poly(alpha-hydroxyl acids) hydroxyapatite porous composites for bone-tissue engineering. I. Preparation and morphology. J. Biomed. Mater. Res. 44(4), 446–455 (1999)

    Article  Google Scholar 

Download references

Acknowledgements

The authors acknowledge support for this work by the Kyocera Professor Endowment and NIH grant (R01CA172455) to Miqin Zhang. Ariane E. Erickson acknowledges support from the National Science Foundation Graduate Research Fellowship Program (DGE–1256082]. Part of this work was conducted at the Molecular Analysis Facility, a National Nanotechnology Coordinated Infrastructure site at the University of Washington which is supported in part by the National Science Foundation (ECC-1542101), the University of Washington, the Molecular Engineering & Sciences Institute, and the Clean Energy Institute.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Miqin Zhang.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(DOCX 112 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Erickson, A.E., Sun, J., Lan Levengood, S.K. et al. Chitosan-based composite bilayer scaffold as an in vitro osteochondral defect regeneration model. Biomed Microdevices 21, 34 (2019). https://doi.org/10.1007/s10544-019-0373-1

Download citation

  • Published:

  • DOI: https://doi.org/10.1007/s10544-019-0373-1

Keywords

Navigation