Pre-osteoblastic cell response on three-dimensional, organic-inorganic hybrid material scaffolds for bone tissue engineering Konstantina Terzaki,1,2 Maria Kissamitaki,1,2 Amalia Skarmoutsou,3 Costas Fotakis,4,2 Costas A. Charitidis,3 Maria Farsari,2 Maria Vamvakaki,1,2 Maria Chatzinikolaidou1,2 1
Department of Materials Science and Technology, University of Crete, P.O. Box 2208, GR-71303 Heraklio, Greece Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology Hellas (FORTH), Greece 3 School of Chemical Engineering, National Technical University of Athens, Greece 4 Department of Physics, University of Crete, Greece 2
Received 15 June 2012; revised 28 October 2012; accepted 29 October 2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34516 Abstract: Engineering artificial scaffolds that enhance cell adhesion and growth in three dimensions is essential to successful bone tissue engineering. However, the fabrication of three-dimensional (3D) tissue scaffolds exhibiting complex micro- and nano-features still remains a challenge. Few materials can be structured in three dimensions, and even those have not been characterized for their mechanical and biological properties. In this study, we investigate the suitability of three novel materials of different chemical compositions in bone tissue regeneration: a hybrid material consisting of methacryloxypropyl trimethoxysilane and zirconium propoxide, a hybrid organic–inorganic material of the above containing 50 mole% 2-(dimethylamino)ethyl methacrylate (DMAEMA) and a pure organic material based on polyDMAEMA. More specifically, we
study the mechanical properties of the aforementioned materials and evaluate the biological response of pre-osteoblastic cells on them. We also highlight the use of a 3D scaffolding technology, Direct femtosecond Laser Writing (DLW), to fabricate complex structures. Our results show that, while all three investigated materials could potentially be used as biomaterials in tissue engineering, the 50% DMAEMA composite exhibits the best mechanical properties for structure fabrication with C 2013 Wiley Periodicals, Inc. DLW and strong biological response. V J Biomed Mater Res Part A: 00A:000–000, 2013.
Key Words: MC3T3-E1 pre-osteoblasts, three-dimensional scaffold fabrication, hybrid material, nanomechanical characterization, cell adhesion
How to cite this article: Terzaki K, Kissamitaki M, Skarmoutsou A, Fotakis C, Charitidis CA, Farsari M, Vamvakaki M, Chatzinikolaidou M. 2013. Pre-osteoblastic cell response on three-dimensional, organic-inorganic hybrid material scaffolds for bone tissue engineering . J Biomed Mater Res Part A 2013:00A:000–000.
INTRODUCTION
In tissue engineering, a number of parameters significantly influence the cellular response on a functional cell-scaffold construct, such as the material chemistry, the material porosity and pore size, interconnectivity, mechanical properties, cell seeding density, and various exogenous growth factors.1 Scaffold characteristics related to their mechanical and chemical properties are considered crucial. It has been acknowledged that cells seeded on scaffolds can recognize differences related to the materials’ physical and mechanical properties and subsequently change their response and functions. Earlier studies have reported the influence of the scaffold stiffness on cell adhesion,2 morphology,3,4 proliferation,5,6 and differentiation.5 Considering in situ bone repair, current strategies for surgical intervention include the use of autografts and allografts.
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Examples of commercially available allografts are InfuseV Bone Graft (http://www.medtronic.com/patients/lumbar-degenerative-disc-disease/surgery/index.htm), a collagen carrier sponge with bone morphogenetic protein 2 designed to treat degenerative disc disease, and TrinityV EvolutionTM (http://www.orthofix.com/common_products.asp?pid¼90& cid¼45), a cancellous bone containing viable adult stem cells, osteoprogenitor cells and a demineralized bone component. Each approach has limitations, such as cost, variability in osteogenic capacity, and lot-to-lot variability. Previous work has described7 donor-site morbidity in the use of autografts and the risk of immunogenic rejection and disease transmission in the use of allografts. To overcome these inherent limitations of autografts and allografts, synthetic bone-graft substitutes have been developed as an alternative. There is a major clinical need for versatile biomaterial systems for bone R
Correspondence to: Maria Chatzinikolaidou; e-mail:
[email protected] Contract grant sponsor: European Union (European Social Fund—ESF) Contract grant sponsor: Greek national funds (Operational Program ‘‘Education and Lifelong Learning’’ of the National Strategic Reference Framework (NSRF) - Research Funding Program: Heracleitus II-Investing in knowledge society through the European Social Fund) Contract grant sponsor: ITN TOPBIO; contract grant number: PITN-GA-2010-264362 Contract grant sponsor: Special Account for Research Fund of the University of Crete
C 2013 WILEY PERIODICALS, INC. V
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FIGURE 1. DLW allows the fabrication of readily assembled, fully 3D structures. (a) a typical porous structure (b) a micro ballerina.
repair that mimic the architecture and mechanical and biostimulating functions of native bone.8 Zirconium propoxide (ZPO) is a particularly suitable biomaterial due to its advantageous mechanical properties such as high strength, toughness, and stability. Although our work considers ZPO as a component in a hybrid biomaterial for a tissue engineering application, most prior work using it derives from the area of prosthetic substitution.9–12 Organic materials possess certain functionalities that affect their interactions with cells. Numerous organic coatings on solid substrates have been employed for the development of cell culture scaffolds as well as antibacterial surfaces, whereas organic nanoparticles have been used in drug and gene delivery agents. Among them, poly(2-(dimethylamino ethyl)methacrylate) (PDMAEMA)-based materials have been extensively used on surfaces and coatings and were shown to exhibit antibioadherent properties against bacteria, macrophages, and fibroblasts similarly to other cationic biocides.13–16 A recent study on PDMAEMA17 proposed that polymers with a branched architecture and an intermediate molecular weight are promising candidates for efficient gene delivery, since they combine low cytotoxicity with acceptable cell transfection. Hybrid organic–inorganic materials combining the above mentioned mechanical, chemical, and biological material properties have emerged recently as a new class of materials in tissue engineering. These hybrid materials possess different properties compared to their component materials and constitute high-performance and multifunctional materials with an excellent balance between strength, toughness, and tunable chemical and mechanical characteristics.18 Composite scaffolds based on hydroxyapatite, the most widely studied hybrids so far, were shown to possess improved mechanical properties and osteoconductivity.19,20 ZrO2 incorporated within poly(e-caprolactone) matrices was shown to develop advanced composite substrates with improved mechanical and biological performance.21 In tissue engineering, the ability to control tissue formation in three dimensions is essential.22 Although several fab-
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rication methods have been used to produce scaffolds, those methods are unable to produce three-dimensional (3D) submicron and nanoscale scaffolds with precise control of the geometry, a crucial factor for the recent developments in the field of tissue engineering. Femtosecond laser-induced two-photon polymerization is a promising technique that fulfills these requirements. In Direct fs Laser Writing (DLW), the beam of an ultrafast laser is tightly focused into the volume of a photosensitive material, initiating multiphoton polymerization within the material.23 By moving the beam focus three-dimensionally, arbitrary 3D, high-resolution structures can be written into the volume of the material (Fig. 1). A variety of materials have been structured using DLW including purely organic polymers,24 organic-inorganic hybrids,25 biodegradable materials,26 and proteins.27,28 In this article, we present our investigations into the suitability of materials that can be structured in complex, 3D geometries using DLW for bone tissue scaffolds. First, we describe the synthesis and characterization of chemical and mechanical properties of three different materials comprising methacryloxypropyl trimethoxysilane (MAPTMS), ZPO, and/or 2-(dimethylamino)ethyl methacrylate (DMAEMA). Next, we use these materials to fabricate 2D films, and investigate the cell viability and proliferation of pre-osteoblastic cells on them. Additionally, we explore the influence of the materials’ chemical composition on cell proliferation. Finally, we report on the pre-osteoblastic cell adhesion on 3D scaffolds fabricated from the hybrid organic–inorganic material composition containing 50% DMAEMA, within the first hour and up to 3 days in culture. MATERIALS AND METHODS
Material synthesis All the chemicals used in this work were obtained from Sigma-Aldrich (Germany) and were used as received unless otherwise stated. The material used for the fabrication of 2D films and 3D structures is an organic–inorganic composite, produced by the addition of MAPTMS (99%) to ZPO (70% in propanol). DMAEMA (>99%) was also added
PRE-OSTEOBLASTIC CELL RESPONSE
ORIGINAL ARTICLE
which was copolymerized with MAPTMS upon photopolymerization. ZPO and the alkoxysilane groups of MAPTMS served as the inorganic network forming moieties. 4,4-bis(diethylamino)benzophenone was used as the photoinitiator. MAPTMS was first hydrolyzed using HCl solution (0.1 M) at a 1:0.1 ratio. After 5 min, the ZPO was slowly added to the hydrolyzed MAPTMS at a 3:7 ZPO:MAPTMS molar ratio. After stirring for 15 min, DMAEMA was added at a DMAEMA/MAPTMS molar ratio 5:5. Following another 30 min, water was added to the mixture at a 2.5:5 MAPTMS:H2O molar ratio. Finally, the photoinitiator, at a 1% (w/w) concentration with respect to photopolymerizable methacrylate moieties was added to the mixture. After stirring for a further 15 min, the composite was filtered using a 0.22 lm syringe filter. A hybrid material comprising only MAPTMS and ZPO in the absence of DMAEMA was also prepared following a procedure similar to that described above. This material was used as a control material to assess the effect of DMAEMA on the chemical, mechanical and biological properties of the films and 3D cell scaffolds. Sample preparation Three types of specimens, all prepared on glass substrates, were employed in this study: (i) thin films for the quantification of cell proliferation (ii) 3D square blocks with dimensions 200 200 10 lm3 (l w h) for the investigation of cell adhesion and cell morphology by immunocytochemical staining and (iii) 3D scaffolds with bar distances of 1, 2, and 5 lm for the investigation of cell adhesion by SEM. Materials specimens used for the adhesion, viability and proliferation experiments were incubated for 1 h in ethanol, airdried under sterile conditions in a laminar flow and rinsed briefly with alpha-minimal essential medium (MEM) cell culture medium without fetal bovine serum (FBS) prior to cell seeding. Hybrid thin film preparation. Thin films were prepared by drop-casting or spin-coating onto 100-lm thick silanized glass substrates, and they were dried in the oven at 50 C for 5 min before the photopolymerization. The heating process led to the condensation of the alkoxide groups and the formation of the inorganic matrix. Next, the methacrylate moieties were polymerized using a KrF excimer laser, operating at 248 nm, resulting in the formation of irreversible and fully saturated aliphatic CAC covalent bonds that further increase the connectivity of the material. Finally, the samples were developed for 30 min in a 50:50 solution of 1-propanol:isopropanol, and were further rinsed with isopropanol. Fabrication of hybrid blocks and 3D scaffolds by DLW. The experimental setup employed for 3D structure fabrication has been previously described extensively.29 A Ti:Sapphire femtosecond laser (Femtolasers Fusion, 800 nm, 75 MHz,