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.
Similar content being viewed by others
References
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).
W.P. Wang, Y.M. Du, and X.Y. Wang: Physical properties of fungal chitosan. World J. Microbiol. Biotechnol. 24, 2717 (2008).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
L.A. Morozova-Roche: Equine lysozyme: The molecular basis of folding, self-assembly and innate amyloid toxicity. FEBS Lett. 581, 2587 (2007).
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).
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).
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).
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).
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).
F. Ganji and M.J. Abdekhodaie: Chitosan-g-PLGA copolymer as a thermosensitive membrane. Carbohydr. Polym. 80, 740 (2010).
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).
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).
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).
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).
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).
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).
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).
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).
R. Krishnamurthy, J.A. Lumpkin, and R. Sridhar: Inactivation of lysozyme by sonication under conditions relevant to microencapsulation. Int. J. Pharm. 205, 23 (2000).
Q. Deng, C. Zhou, and B. Luo: Preparation and characterization of chitosan nanoparticles containing lysozyme. Pharm. Biol. 44, 336 (2006).
A. Jaklenec, E. Wan, M.E. Murray, and E. Mathiowitz: Novel scaffolds fabricated from protein-loaded microspheres for tissue engineering. Biomaterials 29, 185 (2008).
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).
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).
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).
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).
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).
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).
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).
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1557/jmr.2012.176