A biomimetic strategy for controllable degradation of chitosan scaffolds

Abstract

Controllable degradation of scaffolds plays an important role in tissue engineering applications. Here, we describe a biomimetic approach to control chitosan scaffold degradation by incorporating lysozyme-loaded poly(D,L-lactic-co-glycolic acid) microspheres in 3D chitosan scaffolds. In vitro degradation tests reveal that the degradation rate increased when the mass ratio of microspheres-to-chitosan increased whereas the contrast group showed a visible turning point at 28d. In vivo degradation rate was much faster than that in vitro, and the relationship between in vitro degradation and in vivo degradation was correlative. Finally, for determining the primary biocompatibility of the combined scaffolds, studies such as cytotoxicity assay, cell attachment study and histological evaluation were carried out. It is concluded that the combination method of enzyme and scaffold is suitable for chitosan scaffold degradation; it also demonstrates an alternative strategy for other biomaterials used in tissue engineering.

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References

  1. 1.

    V.F. Abdullin, A.B. Shipovskaya, V.I. Fomina, S.E. Artemenko, G.P. Ovchinnikova, and E.V. Pchelintseva: Physicochemical properties of chitosan from different raw material sources. Fibre Chem. 40, 40 (2008).

    CAS  Article  Google Scholar 

  2. 2.

    W.P. Wang, Y.M. Du, and X.Y. Wang: Physical properties of fungal chitosan. World J. Microbiol. Biotechnol. 24, 2717 (2008).

    CAS  Article  Google Scholar 

  3. 3.

    T. Tsujikawa, O. Kanauchi, A. Andoh, T. Saotome, M. Sasaki, Y. Fujiyama, and T. Bamba: Supplement of a chitosan and ascorbic acid mixture for Crohn’s disease: A pilot study. Nutrition 19, 137 (2003).

    CAS  Article  Google Scholar 

  4. 4.

    Y.J. Seol, J.Y. Lee, Y.J. Park, Y.M. Lee, I.C. Young-Ku, Rhyu, S.J. Lee, S.B. Han, and C.P. Chung: Chitosan sponges as tissue engineering scaffolds for bone formation. Biotechnol. Lett. 26, 1037 (2004).

    CAS  Article  Google Scholar 

  5. 5.

    D. Ren, H. Yi, W. Wang, and X. Ma: The enzymatic degradation and swelling properties of chitosan matrices with different degrees of N-acetylation. Carbohydr. Res. 340, 2403 (2005).

    CAS  Article  Google Scholar 

  6. 6.

    G.Y. Lu, L.J. Kong, B.Y. Sheng, G. Wang, Y.D. Gong, and X.F. Zhang: Degradation of covalently crosslinked carboxymethyl chitosan and its potential application for peripheral nerve regeneration. Eur. Polym. J. 43, 3807 (2007).

    CAS  Article  Google Scholar 

  7. 7.

    T. Freier, H.S. Koh, K. Kazazian, and M.S. Shoichet: Controlling cell adhesion and degradation of chitosan films by N-acetylation. Biomaterials 26, 5872 (2005).

    CAS  Article  Google Scholar 

  8. 8.

    Y. Yuan, B.M. Chesnutt, L. Wright, W.O. Haggard, and J.D. Bumgardner: Mechanical property, degradation rate, and bone cell growth of chitosan-coated titanium influenced by degree of deacetylation of chitosan. J. Biomed. Mater. Res. Part B 86, 245 (2007).

    Google Scholar 

  9. 9.

    J. Li, Y. Du, and H. Liang: Influence of molecular parameters on the degradation of chitosan by a commercial enzyme. Polym. Degrad. Stab. 92, 515 (2007).

    CAS  Article  Google Scholar 

  10. 10.

    Y. Wan, A.X. Yu, H. Wu, Z. Wang, and D. Wen: Porous-conductive chitosan scaffolds for tissue engineering II. In vitro and in vivo degradation. J. Mater. Sci. - Mater. Med. 16, 1017 (2005).

    CAS  Article  Google Scholar 

  11. 11.

    C. Cunha-Reis, K. TuzlaKoglu, E. Baas, Y. Yang, A. EI Haj, and R.L. Reis: Influence of porosity and fiber diameter on the degradation of chitosan fiber-mesh scaffolds and cell adhesion. J. Mater. Sci. - Mater. Med. 18, 195 (2007).

    CAS  Article  Google Scholar 

  12. 12.

    D. Ren, H. Yi, H. Zhang, W. Xie, W. Wang, and X. Ma: A preliminary study on fabrication of nanoscale fibrous chitosan membranes in situ by biospecific degradation. J. Membr. Sci. 280, 99 (2006).

    CAS  Article  Google Scholar 

  13. 13.

    Z. She, B. Zhang, C. Jin, Q. Feng, and Y. Xu: Preparation and in vitro degradation of porous three-dimensional silk fibroin/chitosan scaffold. Polym. Degrad. Stab. 98, 1316 (2008).

    Article  CAS  Google Scholar 

  14. 14.

    K.V.H. Prashanth, K. Lakshman, T.R. Shamala, and R.N. Tharanathan: Biodegradation of chitosan-graft-polymethylmethacrylate films. Int. Biodeterior. Biodegrad. 56, 115 (2005).

    CAS  Article  Google Scholar 

  15. 15.

    C. Picart, A. Schneider, O. Etienne, J. Mutterer, P. Schaaf, C. Egles, N. Jessel, and J.C. Voegel: Controlled degradability of polysaccharide multilayer films in vitro and in vivo. Adv. Funct. Mater. 15, 1771 (2005).

    CAS  Article  Google Scholar 

  16. 16.

    Y. Hong, H. Song, Y. Gong, Z. Mao, C. Gao, and J. Shen: Covalently crosslinked chitosan hydrogel: Properties of in vitro degradation and chondrocyte encapsulation. Acta Biomater. 3, 23 (2007).

    CAS  Article  Google Scholar 

  17. 17.

    A.J. Kuijpers, P.B. Wachem, M.J.A. Luyn, G.H.M. Engbers, J. Krijgsveld, S.A.J. Zaat, J. Dankert, and J. Feijen: In vivo and in vitro release of lysozyme from crosslinked gelatin hydrogels: A model system for the delivery of antibacterial proteins from prosthetic heart valves. J. Controlled Release 67, 323 (2000).

    CAS  Article  Google Scholar 

  18. 18.

    F. Kang, G. Jiang, A. Hinderliter, P.P. DeLuca, and J. Singh: Lysozyme stability in primary emulsion for PLGA microsphere preparation: Effect of recovery methods and stabilizing excipients. Pharm. Res. 19, 629 (2002).

    CAS  Article  Google Scholar 

  19. 19.

    L.A. Morozova-Roche: Equine lysozyme: The molecular basis of folding, self-assembly and innate amyloid toxicity. FEBS Lett. 581, 2587 (2007).

    CAS  Article  Google Scholar 

  20. 20.

    M.F. Mossuto, A. Dhulesia, G. Devlin, E. Frare, J.R. Kumita, P.P. Laureto, M. Dumoulin, A. Fontana, C.M. Dobson, and X. Salvatella: The non-core regions of human lysozyme amyloid fibrils influence cytotoxicity. J. Mol. Biol. 402, 783 (2010).

    CAS  Article  Google Scholar 

  21. 21.

    R. Mishra, K. Sörgjerd, S. Nyström, A. Nordigården, Y.C. Yu, and P. Hammarström: Lysozyme amyloidogenesis is accelerated by specific nicking and fragmentation but decelerated by intact-protein binding and conversion. J. Mol. Biol. 366, 1029 (2007).

    CAS  Article  Google Scholar 

  22. 22.

    D. Cerven, G. DeGeorge, and D. Bethell: 28-Day repeated dose oral toxicity of recombinant human apo-lactoferrin or recombinant human lysozyme in rats. Regul. Toxicol. Pharm. 51, 162 (2008).

    CAS  Article  Google Scholar 

  23. 23.

    T. Jiang, S.P. Nukavarapu, M. Deng, E. Jabbarzadeh, M.D. Kofron, S.B. Doty, W.I. Abdel-Fattah, and C.T. Laurencin: Chitosan-poly(lactide-co-glycolide) microsphere-based scaffolds for bone tissue engineering: In vitro degradation and in vivo bone regeneration studies. Acta Biomater. 6, 3457 (2010).

    CAS  Article  Google Scholar 

  24. 24.

    M. Wang, Q. Feng, X. Guo, Z. She, and R. Tan: A dual microsphere based on PLGA and chitosan for delivering the oligopeptide derived from BMP-2. Polym. Degrad. Stab. 96, 107 (2011).

    CAS  Article  Google Scholar 

  25. 25.

    F. Ganji and M.J. Abdekhodaie: Chitosan-g-PLGA copolymer as a thermosensitive membrane. Carbohydr. Polym. 80, 740 (2010).

    CAS  Article  Google Scholar 

  26. 26.

    H. Tan, J. Wu, L. Lao, and C. Gao: Gelatin/chitosan/hyaluronan scaffold integrated with PLGA microspheres for cartilage tissue engineering. Acta Biomater. 5, 328 (2009).

    CAS  Article  Google Scholar 

  27. 27.

    V.K. Nandagiri, P. Gentile, V. Chiono, C. Tonda-Turo, A. Matsiko, Z. Ramtoola, F.M. Montevecchi, and G. Ciardelli: Incorporation of PLGA nanoparticles into porous chitosan-gelatin scaffolds: Influence on the physical properties and cell behavior. J. Mech. Behav. Biomed. Mater. 4, 1318 (2011).

    CAS  Article  Google Scholar 

  28. 28.

    A.M. Martins, R.C. Pereira, I.B. Leonor, H.S. Azevedo, and R.L. Reis: Chitosan scaffolds incorporating lysozyme into CaP coatings produced by a biomimetic route: A novel concept for tissue engineering combining a self-regulated degradation system with in situ pore formation. Acta Biomater. 5, 3328 (2009).

    CAS  Article  Google Scholar 

  29. 29.

    H.M. Wong, J.J. Wang, and C.H. Wang: In vitro sustained release of human immunoglobulin G from biodegradable microspheres. Ind. Eng. Chem. Res. 40, 933 (2001).

    CAS  Article  Google Scholar 

  30. 30.

    G. Jiang, B.H. Woo, F. Kang, J. Singh, and P.P. DeLuca: Assessment of protein release kinetics, stability and protein polymer interaction of lysozyme encapsulated poly(D, L-lactide- co-glycolide) microspheres. J. Controlled Release 79, 137 (2002).

    CAS  Article  Google Scholar 

  31. 31.

    S. Sharif and D.T. O’Hagan: A comparison of alternative methods for the determination of the levels of proteins entrapped in poly(lactide-co-glycolide) microparticles. Int. J. Pharm. 115, 259 (1995).

    CAS  Article  Google Scholar 

  32. 32.

    D. Blanco and M.J. Alonso: Protein encapsulation and release from poly(lactide-co-glycolide) microspheres: Effect of the protein and polymer properties and of thecoencapsulation of surfactants. Eur. J. Pharm. Biopharm. 45, 285 (1998).

    CAS  Article  Google Scholar 

  33. 33.

    J.M. Bezemer, R. Radersma, D.W. Grijpma, P.J. Dijkstra, J. Feijen, and C.A. Blitterswijk: Zero-order release of lysozyme from poly(ethylene glycol)/poly(butylenes terephthalate) matrices. J. Controlled Release 64, 179 (2000).

    CAS  Article  Google Scholar 

  34. 34.

    R. Krishnamurthy, J.A. Lumpkin, and R. Sridhar: Inactivation of lysozyme by sonication under conditions relevant to microencapsulation. Int. J. Pharm. 205, 23 (2000).

    CAS  Article  Google Scholar 

  35. 35.

    Q. Deng, C. Zhou, and B. Luo: Preparation and characterization of chitosan nanoparticles containing lysozyme. Pharm. Biol. 44, 336 (2006).

    CAS  Article  Google Scholar 

  36. 36.

    A. Jaklenec, E. Wan, M.E. Murray, and E. Mathiowitz: Novel scaffolds fabricated from protein-loaded microspheres for tissue engineering. Biomaterials 29, 185 (2008).

    CAS  Article  Google Scholar 

  37. 37.

    P.B. Malafaya, T.C. Santos, M. Griensven, and R.L. Reis: Morphology, mechanical characterization and in vivo neovascularization of chitosan particle aggregated scaffolds architectures. Biomaterials 29, 3914 (2008).

    CAS  Article  Google Scholar 

  38. 38.

    Y.S. Nam, S.H. Song, J.Y. Choi, and T.G. Park: Lysozyme microencapsulation within biodegradable PLGA microspheres: Urea effect on protein release and stability. Biotechnol. Bioeng. 70, 270 (2000).

    CAS  Article  Google Scholar 

  39. 39.

    C. Pérez, P. Jesús, and K. Griebenow: Preservation of lysozyme structure and function upon encapsulation and release from poly(lactide-co-glycolic) acid microspheres prepared by the water-in-oil-in-water method. Int. J. Pharm. 248, 193 (2002).

    Article  Google Scholar 

  40. 40.

    A. Aubert-Pouëssel, D.C. Bibby, M.C. Venier-Julienne, F. Hindré, and J.P. Benoît: A novel in vitro delivery system for assessing the biological integrity of protein upon release from PLGA microspheres. Pharm. Res. 19, 1046 (2002).

    Article  Google Scholar 

  41. 41.

    Y. Hiraoka, H. Yamashiro, K. Yasuda, Y. Kimura, T. Inamoto, and Y. Tabata: In situ regeneration of adipose tissue in rat fat pad by combining a collagen scaffold with gelatin microspheres containing basic fibroblast growth factor. Tissue Eng. 12, 1475 (2006).

    CAS  Article  Google Scholar 

  42. 42.

    F. Ungaro, M. Biondi, I. d’Angelo, L. Indolfi, F. Quaglia, P.A. Netti, and M.I.L. Rotonda: Microsphere-integrated collagen scaffolds for tissue engineering: Effect of microsphere formulation and scaffold properties on protein release kinetics. J. Controlled Release 113, 128 (2006).

    CAS  Article  Google Scholar 

  43. 43.

    X. Niu, Q. Feng, M. Wang, X. Guo, and Q. Zheng: In vitro degradation and release behavior of porous poly(lactic acid) scaffolds containing chitosan microspheres as a carrier for BMP-2-derived synthetic peptide. Polym. Degrad. Stab. 94, 176 (2009).

    CAS  Article  Google Scholar 

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Acknowledgments

Financial supports from the Natural Science Foundation of China (31000441, 81171459) are gratefully acknowledged. The authors also thank Dr. Sun Y for the collaboration in the in vivo studies, as well as Dr. Liao W for the initial assistance with the AFM equipment.

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Correspondence to Yuangang Liu.

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Liu, Y., Zhou, C. & Sun, Y. A biomimetic strategy for controllable degradation of chitosan scaffolds. Journal of Materials Research 27, 1859–1868 (2012). https://doi.org/10.1557/jmr.2012.176

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