Silk fibroin scaffolds enhance cell commitment ofadult rat cardiac progenitor cellsValentina Di Felice1*, Claudia Serradifalco1, Luigi Rizzuto1, Angela De Luca1, Francesca Rappa1,Rosario Barone1, Patrizia Di Marco2, Giovanni Cassata2, Roberto Puleio2, Lucia Verin3,4,Antonella Motta3,4, Claudio Migliaresi3,4, Annalisa Guercio2 and Giovanni Zummo11Department of Experimental Biomedicine and Clinical Neurosciences, University of Palermo, Italy2Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy3Department of Materials Engineering and Industrial Technologies and Biotech Research Centre, University of Trento, Italy4European Institute of Excellence in Tissue Engineering and Regenerative Medicine and INSTM Research Unit, Trento, Italy

Abstract

The use of three-dimensional (3D) cultures may induce cardiac progenitor cells to synthesize theirown extracellular matrix (ECM) and sarcomeric proteins to initiate cardiac differentiation. 3Dcultures grown on synthetic scaffolds may favour the implantation and survival of stem cells for celltherapy when pharmacological therapies are not efficient in curing cardiovascular diseases and whenorgan transplantation remains the only treatment able to rescue the patients life. Silk fibroin-basedscaffolds may be used to increase cell affinity to biomaterials and may be chemically modified toimprove cell adhesion. In the present study, porous, partially orientated and electrospun nanometricnets were used. Cardiac progenitor cells isolated from adult rats were seeded by capillarity in the 3Dstructures and cultured inside inserts for 21days. Under this condition, the cells expressed a highlevel of sarcomeric and cardiac proteins and synthesized a great quantity of ECM. In particular,partially orientated scaffolds induced the synthesis of titin, which is a fundamental protein insarcomere assembly. Copyright 2013 John Wiley & Sons, Ltd.

Received 8 September 2012; Revised 18 January 2013; Accepted 5 February 2013

Supporting information may be found in the online version of this article.

Keywords myocardial tissue; progenitor cells; Z-bodies; tissue engineering; natural polymers; silk fibroin

1. Introduction

Cardiovascular disease is the leading cause of deathworldwide in both low-income and middle-incomecountries (Nabel and Braunwald, 2012) and approxi-mately 1900 patients/million population are hospital-ized for acute myocardial infarction (AMI) in Europe(Widimsky et al., 2010). With larger infarcts, the non-infarcted myocardium remodels over time, becominghypertrophic and eventually progressing into failure.When the pharmacological approach no longer arrestsdisease evolution, organ transplantation remains the

only treatment able to rescue the patients life.However, recent studies have suggested the possibilityof replacing the injured cells with stem cells. Thisapproach would circumvent many of the limitations oforgan transplantation, such as the low availability oforgans, major surgical procedures, high costs andlong-term immunosuppression.

As discussed in a recent paper published in Nature(Laflamme and Murry, 2011), cardiac progenitor cells(CPCs) are among the cells that are closest to clinicaltrials of AMI. CPCs are partially differentiated cells locatedin the adult myocardium that can differentiate intofibroblasts, endothelial cells, cardiomyocytes of the conduc-tion system and working cardiomyocytes (Bernstein andSrivastava, 2012; Di Felice et al., 2009b). In 2003, PieroAnversas group isolated and characterized these cells forthe first time (Beltrami et al., 2003). We now know thatthere is most likely a unique population of c-Kit+ cells and

*Correspondence to: V. Di Felice, Dipartimento di BiomedicinaSperimentale e Neuroscienze Cliniche, Universit degli Studi diPalermo, Via del Vespro 129, 90127 Palermo, Italy. E-mail:vdfelice@inwind.it

Copyright 2013 John Wiley & Sons, Ltd.

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLEJ Tissue Eng Regen Med (2013)Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1739

that the most important surface markers of CPCs are c-Kit,MDR-1 and Sca-1 (Bernstein and Srivastava, 2012). The or-igin of these cells is unknown, although data suggest thatthey are present in themyocardiumduring human heart de-velopment and persist after birth (Di Felice and Zummo,2009; Serradifalco et al., 2011).

Currently, two clinical trials are in progress in the USA:one trial is implementing intracoronary injection of CPCsin coronary heart disease and congestive heart failure[phase I; NCT00474461 (Bolli et al., 2011)] and the otheris a dose escalation study of the safety and efficacy ofintracoronary delivery of cardiosphere-derived CPCs inpatients with ischaemic left ventricular dysfunction anda recent myocardial infarction [phase I; NCT00893360(Makkar et al., 2012)]. In these two clinical trials, stemcells are delivered by intracoronary injection. However,the delivery of stem cells alone to infarcted myocardiummay provide no structural support while the myocardiumheals, and the injected stem cells may not properly inte-grate into the myocardium because they may not besubjected to the mechanical forces that are known to drivemyocardial cellular physiology.

Tissue-engineering strategies are a promising therapeuticapproach, in which new matrices are being developed tosupport cellular activity. Tissue engineering for cardiacmuscle is possible, even though the construction of thishighly organized tissue is very difficult to achieve. The maingoals are to select the proper cell source and to reach a highdegree of differentiation through a bioactive 3D scaffold(Pelacho et al., 2007; Singh and Williams, 2008).

Scaffoldsmay reduce the number of cells needed for eachimplantation via driving cell fate by mimicking the special-ized micro-environment (niche) where CPCs are locatedin vivo and resembling the elasto-mechanical forces of theheart wall. The chemistry, geometry and mechanical prop-erties of scaffolds for tissue engineering should be designedso that the scaffolds act as templates for cell adhesion, acti-vation and extracellular matrix (ECM) production, whichwill lead to the regeneration of damaged tissues (Stoppatoet al., 2011). Considering natural ECM as a model, biopoly-mer matrices and particularly proteins are attracting the in-terest of many researchers. Likemany natural polymers, silkis a promising candidate for various medical applications.The uniqueness of silk is derived from its multi-functionalnature; it possesses adaptable mechanical performancecoupled with controllable degradation and excellent bio-compatibility. Fibroin-based materials can be obtained bydirectly treating degummed fibres or after fibroin dissolu-tion and dialysis. Active efforts have been made to developsilk for the production of scaffolds, such as films, hydrogels,nano- and micro-nets and sponges, that can be tailored anddesigned to specific applications (Fini et al., 2005; Wanget al., 2006). The potential of applying silk-based materialsto induce the regeneration of various mammalian tissues,including bone, cartilage, tendon and skin, is increasinglyreported (Bondar et al., 2008; Unger et al., 2007). Silkfibroin can be used as a polymer on its own or withother molecules to increase cell affinity to biomaterials(Cai et al., 2002; Ghanaati et al., 2010) and may be

chemically modified to improve cell adhesion (Morgan etal., 2008). We have recently demonstrated that porous fi-broin scaffolds may be used for 3D chondrocyte culture,and these cells adhere to the structure using the a5, b1and b3 integrin subunits (Wang et al., 2010).

Integrins are the main constituent of costameres,which are peripheral Z-disk and subsarcolemmalproteins that transduce force from the sarcomere tothe sarcolemma and anchor cardiomyocytes to theECM (Danowski et al., 1992).

Cardiac stem cells must organize costameres, titin an-chors and sarcomeres to be considered fully differentiated.Titin is a giant protein that connects each costamere to aperipheral Z-disk and is one of the proteins that transducesbiomechanical stress signals from the Z-disk to the nucleus(Frank and Frey, 2011).

Whether a polymer can induce the correct expression ofsarcomeric proteins and promote the Z-disk signalling net-work is not known, but for its particular interactions withintegrin subunits, silk fibroin may be a good candidate. Inthe present study, we evaluated whether the properties ofsilk fibroin scaffolds influence CPC differentiation andintegrin, cardiac and sarcomeric protein expressionin vitro. Specifically, three different geometries weredesigned for the scaffolds, which were obtained using afibroinwater solution, two sponges with different poresizes and distributions and an electrospun nanometric net.

2. Materials and methods

2.1. Material preparation

2.1.1. Fibroinwater solution

Bombyx mori cocoons (kindly provided by Socio Lario,Cassina Rizzardi, Como, Italy) were boiled for 1.5 hin an aqueous solution containing 1.1 g/l Na2CO3(10 g silk/l solution) and then for another 1.5 h in bathof water containing 0.4 g/l Na2CO3. The cocoons wererinsed thoroughly with distilled warm water to extractthe glue-like sericin proteins and finally air-dried.

The fibroinwater solution was prepared by dissolvingfibroin in an aqueous solution containing 9.3M LiBr (10%w/v; Fluka Chemical) at 65 C for 2h, followed by dialysis(for 3 days) against distilled water with a 3500Da MWCOmembrane (Slyde-A-Lyzer, Pierce) to eliminate the salt.The resulting solution was concentrated by dialysingagainst a PEGwater solution (25%w/v) for 5 h and filteredthrough a 160250mm filter (Duran Group). The finalconcentration of silk fibroin in the aqueous solution wasapproximately 15% w/v, as determined using a NanoDropND-1000 Spectrophotome