Singapore ARF Project [T206B1114]; National Natural Science Foundation of China [50928301, 51203108]; China MOE Chang Jiang Scholars ProgramSilk scaffolds having biomimetic hierarchical porous structures were achieved by carefully tuning liquid-liquid separation in regenerated silk fibroin solutions. Such scaffolds show greatly enhanced cellular responses

Chuah, Yon Jin

Goh, James C. H.

Li, Jing Liang

Liu, Xiang Yang

Wang, Hui

Xu, Hongyao

刘向阳

Xiamen University Institutional Repository

This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 1431--1433 1431Cite this: Chem. Commun.,2013,49, 1431Design and engineering of silk fibroin scaffolds withbiomimetic hierarchical structures†Hui Wang,ab Xiang Yang Liu,*ad Yon Jin Chuah,c James C. H. Goh,c Jing Liang Liaand Hongyao XudSilk scaffolds having biomimetic hierarchical porous structureswere achieved by carefully tuning liquid–liquid separation inregenerated silk fibroin solutions. Such scaffolds show greatlyenhanced cellular responses.In recent years, the fabrication of three dimensional (3D) polymericscaffolds has turned out to be one of the crucial steps in tissueengineering to provide the vital framework for the seeded cells toorganize into a functioning tissue.1 To date, Bombyx mori silk, a sortof fibril protein, has been attracting much attention in 3D polymericscaffold fabrication, due to its biocompatibility, minimal inflamma-tory reaction, robust biomechanical features, and relatively slowbiodegradation as well as sufficient supply based on the establishedmature sericulture industry.2 So far, the critical effect of porearchitecture in silk-based 3D scaffolds on the cell response hasbeen assessed to some extent.3 Although there is no generalconsensus as to what the optimal pore size for the cell growthand tissue formation is, it is commonly believed that the pores of100–200 mm will improve the cells and bone in-growth, and thepores of several micrometers support osteoblasts migration and giverise to a higher rate of mineralization.3,4 Furthermore, how todesign and fabricate the scaffolds that are both porous andmechanically strong is a challenge as the two go in the oppositedirection. In this regard, the scaffolds of a single dispersion of poresizes are of such a common dilemma. Hopefully, the scaffoldshaving multiple pore size distribution may balance the two oppositeeffects. Note that bone exhibits an assemblage of multi-level pores,macro to micro, for establishment of multiple functions. Thisincludes achieving a strong mechanical strength, accommodatingabundant blood vessels and marrow, and transporting nutrientsand oxygen.5 Based on the inspiration of bone, we expect to designand produce 3D silk scaffolds with multi-level porous structures soas to acquire the optimal biological and mechanical performance.The freeze drying technique (path i) ii) iii) shown in (Fig. 1)turns out to be one of the most popular fabrication techniques of 3Dsilk scaffolds due to its biofriendly nature and the ease of treatment.3Note that for the normal freezing drying process, the pores in thescaffolds were generated by ice crystallite templates, but the poresize is usually single-dispersed at a fixed temperature. In this study,we will combine the freeze drying technique with the followingFig. 1 Schematic illustration of processing stages in the formation of calciuminduced porous silk scaffolds. (i) ) (iii) Normal freeze drying process to producethe monopore sized scaffold. (i0) ) (iii 0) Ca2+ induced multiphase freeze dryingprocess to produce the multipore sized scaffold.a College of Materials, Xiamen University, Xiamen 361005, China and Departmentof Physics and Department of Chemistry, National University of Singapore,117542, Singapore. E-mail: phyliuxy@nus.edu.sg; Tel: +65 6516 2812b National Engineering Laboratory for Modern Silk, College ofTextile and Clothing Engineering, Soochow University, Suzhou 215123, Chinac Department of Orthopaedic Surgery, National University of Singapore,119074, Singapored College of Material Science and Engineering & State Key Laboratory forModification of Chemical Fibers and Polymer Materials, Donghua University,Shanghai, China† Electronic supplementary information (ESI) available: Experimental details.See DOI: 10.1039/c2cc38779dReceived 24th September 2012,Accepted 18th December 2012DOI: 10.1039/c2cc38779dwww.rsc.org/chemcommChemCommCOMMUNICATIONPublished on 19 December 2012. Downloaded by Xiamen University on 12/07/2015 10:40:31. View Article OnlineView Journal  | View Issue1432 Chem. Commun., 2013, 49, 1431--1433 This journal is c The Royal Society of Chemistry 2013materials engineering strategy: (1) to create multiscale freezingcompartments in a silk fibroin solution (i0 ) ii0 in Fig. 1), wepromote the phase separation of the silk fibroin solutions by addingan electrolyte. It follows from the binodal behavior that for the twocoexistence phases, one will be highly dispersed in the other phaseof protein solutions. According to the nucleation theories,6,7 thenumber and the size of the highly dispersed phase are determinedby the nucleation rate J (eqn (1) and (2), ESI†). Obviously, for a givensystem, a higher nucleation rate, which can be achieved at a highthermodynamic driving force, will give rise to smaller disperseddroplets. The change of the silk fibroin concentration can tune thenucleation rate. On the other hand, the addition of Ca2+ will greatlyalter the phase boundary, consequently the thermodynamic drivingforce effectively. Based on this principle, we should be able to tunethe size and size distribution of the dispersed silk fibroin dropletsby the concentrations of electrolytes and silk fibroin and otherstimuli.8 The freezing of the multiphase solutions will give rise tothe formation of multiscale ice crystallites (ii0 ) iii0 in Fig. 1). The icecrystallites formed in the ambient phase will be bigger and the sizecan be tuned by the freezing temperature and the freezing rate,while in the highly dispersed liquid droplets, the size of ice crystal-lites will be confined by the size of liquid droplets.7,9 The icetemplates created in this process should be of multilevel sizes. Ascan be seen, we fully take advantage of the liquid–liquid phaseseparation of protein solutions in the materials engineering.As is well known, Ca2+ ions play an important role in controllingthe interactions of proteins.10 In this study, Ca2+ ions were intro-duced into a regenerated silk fibroin (RSF) solution to create multi-phase solutions. Ca2+ ions can strongly bind to the opposite chargesor dipoles of silk fibroin molecules at the electrostatic double layer,to reduce the zeta potential (Fig. 2A). This leads to the aggregation offibroin molecules, giving rise to the liquid–liquid (L–L) phaseseparation: a highly dispersed dense proteinaceous phase and adilute phase (step (i0) in Fig. 1). These two liquid phases can beidentified by the change from a clear RSF solution to a translucentsolution immediately after adding Ca2+, causing the enhancement oflight scattering (Fig. 2A). The optical density (OD) increased signifi-cantly with increasing Ca2+, and levelled off when the concentrationof Ca2+ is higher than 10 mmol L1 (Fig. 2A). Fig. 2B shows that thesize of liquid droplets (or ‘‘particles’’) in the RSF solutions increasedwith the concentration of Ca2+ from 0 to 15 mmol L1 but decreasedat 20 mmol L1, and the size distribution changed from a narrowstate to a broader state. This implies that fibroin molecules aggrega-tion might be interrupted by strong electrostatic repelling interactiondue to high charge density of Ca2+. Note that such a level of Ca2+concentration is insufficient to cause the conformational transitionof silk fibroin immediately, and only induces the protein moleculesaggregation which is confirmed by circular dichroism (CD) spectraand fluorescence spectra (Fig. S1, ESI†). All the spectra show theunchanged characteristic feature of random coils with increasingCa2+ concentration.11 In the subsequent freezing process (ii0 ) iii0in Fig. 1), the bigger ice crystals form in the dilute silk fibroinphase, whereas the microice crystallites are created from the highlydispersed proteinaceous phase. The ice templates of multi-scale areremoved during the freeze-drying process, which gives rise to theformation of the multiscale pores structure (iii0 ) iv0 in Fig. 1).The morphology of calcium incorporated silk fibroin scaffolds(Ca/SFSs) is shown in Fig. 2C, together with those acquired from theconventional freeze-drying technique using pure silk fibroin (SFSs,as control). The pores in SFSs formed by slow cooling (20 1C) showthe smooth but not interconnected walls (Fig. 2C1 and C2). Suchpores may prevent cells from moving freely in and/or out. The poresize distribution displays a broadly dispersed peak around 100 mm(Fig. 2D1). In contrast, the morphology of Ca/SFSs exhibits on onehand the pores similar to those in SFSs (Fig. 2C3), which are mainlyto accommodate cells in tissue engineering. On the other hand, thenumerous tiny micropores (1–20 mm) are found on the walls aroundthe bigger pores. These tiny pores can improve the interconnectivityacross the big pores (Fig. 2C4). These tiny pores will facilitate greatlythe materials exchange between the bigger pores. The pore sizedistribution of Ca/SFSs displays double peaks with two magnitudedifferences, one peak at B90 mm, while another peak at B10 mm(Fig. 2D2). This strongly indicates the formation of two distinct levelsof pore structures in Ca/SFSs. The same structures of Ca/SFSs andSFSs are found everywhere in the scaffolds, expect for a thin layerformed on the bottom surface of scaffolds (Fig. S2, ESI†).The Fourier transform infrared (FTIR) spectra of both SFSs andCa/SFSs show fibroin peaks at 1620 and 1516 cm1 (Fig. S3A, ESI†),assigned to b-sheets.12 They confirm that the Ca2+ does not changeobviously the dominant secondary structure in the silk scaffolds beforefreezing, indicating that the incorporation of Ca2+ modifies mainly themorphological structure of Ca/SFSs. The compression testing revealsthat the Ca2+ ions incorporation does not jeopardize obviously themechanical performance of the silk scaffolds (Fig. S3B, ESI†), althoughCa/SFSs are a bit weaker than SFSs, which can be attributed to theoccurrence of the smaller pores in the walls of Ca/SFSs. Noticeably, theCa/SFSs modulus (4.89  1.16 MPa) is greater than some reportedsilk-based scaffolds (0.07–3 MPa),13 even though they were preparedby using a lower concentration of RSF solution (3.5 wt%).Fig. 2 (A) The influence of Ca2+ ions on physical states, optical density and zetapotential of the RSF solution. (B) High Performance Particle Sizer (HPPS) measure-ment (size distribution by volume) of RSF solution with 0, 5, 10, 15, 20 mmol L1Ca2+ ions. (C) SEM micrographs of SFSs (C1, C2) and Ca/SFSs (C3, C4). (D) The poresize distribution of pure SFSs (D1) has a broad peak centered at B100 mm, and Ca/SFSs (D2) has two distinct levels of pore structure peaks, tiny micropores (1–20 mm)appearing on the walls around the B90 mm bigger pores.Communication ChemCommPublished on 19 December 2012. Downloaded by Xiamen University on 12/07/2015 10:40:31. View Article OnlineThis journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 1431--1433 1433Finally, we aimed to assess whether the multi-level porearchitecture in silk scaffolds could improve human bone marrow-derived mesenchymal stem cells (HMSCs) responses for itspotential tissue engineering and biomedical applications. Incomparison with SFSs, HMSCs on Ca/SFSs after 1 day of cellculture show a much higher degree of spreading (Fig. 3A), andformed the cellular extension to bridge the adjacent micropores. After 7 days, the morphologies of HMSCs grown on theCa/SFSs show a much thicker attachment. After 14 days,HMSCs formed the cell sheets and extracellular matrices(ECM), concealing the porous structures of both SFSs andCa/SFSs. MTT assay was employed to evaluate the viability ofHMSCs cultured on different scaffolds (Fig. 3B). Ca/SFSs show asignificant enhancement compared with SFSs (p o 0.05). Thisresult clearly indicates that the tiny pores on the walls of thebigger pores within the Ca/SFSs may help the cells proliferationoutwards, allowing more and more cells to grow in a congenialenvironment. This leads to fewer dead cells in the initial growthphase due to overcrowding. Osteoblastic differentiation wasfurther assessed by measuring mRNA expression for alkalinephosphatase (ALP), type I collagen (Col I) and osteocalcin (OCN)via the reverse transcription-polymerase chain reaction(RT-PCR) (Fig. 3C). After 14 days of osteogenic culture, all threeosteogenic markers were significantly up-regulated in Ca/SFSscompared with SFSs (p o 0.05). These results suggest thatdifferentiation and maturation of HMSCs to the osteoblasticphenotype has indeed been improved by Ca/SFSs. To excludethe possibility that cell differentiation was induced by Ca2+stimulation from scaffolds, we monitored the Ca2+ releasingprocess from scaffolds. At the first day, the dissolution of Ca2+from the Ca/SFSs reaches a saturated state, and Ca2+ ions willnot be released further in the remaining period of cell culture(Fig. S3C, ESI†). Based on the fact that the culture medium waschanged every three days, the improved HMSCs response ismainly attributed to the multi-level pore structures in Ca/SFSs,rather than the dissolution of Ca2+ into the culture medium.It is capable of providing an environment suitable for cellattachment, growth and osteogenesis of HMSCs. Thesefindings clearly demonstrate the crucial role of the microporeson the walls around the bigger pores and highlight an importantasset of silk fibroin scaffolds with a multi-level porous architecturepresented in this communication.In summary, we have designed and fabricated 3D silk fibroinscaffolds with multi-level porous structures by taking advantage ofthe L–L phase separation of RSF solutions by introducing Ca2+.Furthermore, it was found that tiny pores on the bigger pore wallsplay a significant role in HMSCs attachment, proliferation, anddifferentiation, without weakening the mechanical performance ofthe scaffolds. The relative simple methodology and the outstandingperformance of the multi-level porous silk scaffolds suggest thatCa/SFSs may provide excellent 3D scaffolds for bone and hardtissue engineering applications.This work was supported by the Singapore ARF Project(Grant T206B1114), the National Natural Science Foundationof China (50928301, 51203108) and the China MOE ChangJiang Scholars Program.Notes and references1 (a) L. L. Hench and J. M. Polak, Science, 2002, 295, 1014;(b) V. Olivier, N. Faucheux and P. Hardouin, Drug Discovery Today,2004, 9, 803.2 (a) C. Veparia and D. L. Kaplan, Prog. Polym. Sci., 2007, 32, 991;(b) F. G. Omenetto and D. L. Kaplan, Science, 2010, 329, 528.3 (a) R. Nazarov, H. J. Jin and D. L. Kaplan, Biomacromolecules, 2004,5, 718; (b) Y. F. Zhang, W. Fan, Z. C. Ma, C. T. Wu, W. Fang, G. Liuand Y. Xiao, Acta Biomater., 2010, 6, 3021; (c) L. P. Yan, J. M. Oliveira,A. L. Oliveira, S. G. Caridade, J. F. Mano and R. L. Reis, ActaBiomater., 2012, 8, 289; (d) B. B. Mandal and S. C. Kundu, Biomaterials,2009, 30, 2956.4 W. Sun, J. E. Puzas, T. J. Sheu, X. Liu and P. M. Fauchet, Adv. Mater.,2007, 19, 921.5 (a) M. M. Stevens, Mater. Today, 2008, 11, 18; (b) R. Murugan andS. Ramakrishna, Compos. Sci. Technol., 2005, 65, 2385.6 (a) A. P. H. J. Schenning, F. B. G. Benneker, H. P. M. Geurts andX. Y. Liu, J. Am. Chem. Soc., 1996, 118, 8549; (b) Y. Y. Diao andX. Y. Liu, Adv. Funct. Mater., 2012, 22, 1354.7 K. Q. Zhang and X. Y. Liu, Nature, 2004, 429, 739.8 L. J. De Cock, O. De Wever, H. Hammad, B. N. Lambrecht,E. Vanderleyden, P. Dubruel, F. De Vos, C. Vervaet, J. P. Remonand B. G. De Geest, Chem. Commun., 2012, 48, 3512.9 (a) N. Du and X. Y. Liu, Appl. Phys. Lett., 2002, 81, 445; (b) X. Y. Liuand N. Du, J. Biol. Chem., 2004, 279, 6124; (c) T. H. Zhang andX. Y. Liu, Angew. Chem., Int. Ed., 2009, 48, 1308.10 P. Zhou, X. Xie, D. P. Knight, X. H. Zong, F. Deng and W. H. Yao,Biochemistry, 2004, 43, 11302.11 Y. H. Yang, Z. Z. Shao, X. Chen and P. Zhou, Biomacromolecules,2004, 5, 773.12 X. Hu, D. Kaplan and P. Cebe, Macromolecules, 2006, 39, 6161.13 (a) V. Karageorgiou and D. Kaplan, Biomaterials, 2005, 26, 5474;(b) Y. F. Zhang, C. T. Wu, T. Friis and Y. Xiao, Biomaterials, 2010,31, 2848; (c) U. J. Kim, J. Parka, H. J. Kima, M. Wadac andD. L. Kaplan, Biomaterials, 2005, 26, 2775.Fig. 3 (A) SEM micrographs showing adherence, growth, and cell sheetformation by HMSCs cultured on SFSs and Ca/SFSs for 1, 7 and 14 days. (B) Cellproliferation on SFSs and Ca/SFSs for 1, 7 and 14 days. (C) The bone-related geneexpression of ALP, COLI and OCN by HMSCs cultured on the SFSs and Ca/SFSs for14 days. Levels, quantified using RT-PCR, are normalized to the housekeepinggene, GAPDH. *Significant difference between two groups at p o 0.05.ChemComm CommunicationPublished on 19 December 2012. Downloaded by Xiamen University on 12/07/2015 10:40:31. View Article Online

Design and engineering of silk fibroin scaffolds with biomimetic hierarchical structures

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Design and engineering of silk fibroin scaffolds with biomimetic hierarchical structures

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