Three-dimensional plotted scaffolds with controlled pore size gradients: Effect of scaffold geometry on mechanical performance and cell seeding efﬁciency Jorge M. Sobral, Soﬁa G. Caridade, Rui A. Sousa, João F. Mano ⇑ , Rui L. Reis 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, 4806-909 Taipas, Guimarães, Portugal Institute for Biotechnology and Bioengineering, PT Associated Laboratory, Guimarães, Portugal article info Article history: Received 16 June 2010 Received in revised form 26 October 2010 Accepted 1 November 2010 Available online 4 November 2010 Keywords: Tissue engineering Regenerative medicine Three-dimensional plotting Porosity gradient Seeding efﬁciency abstract Scaffolds produced by rapid prototyping (RP) techniques have proved their value for tissue engineering applications, due to their ability to produce predetermined forms and structures featuring fully intercon- nected pore architectures. Nevertheless, low cell seeding efﬁciency and non-uniform distribution of cells remain major limitations when using such types of scaffold. This can be mainly attributed to the inade- quate pore architecture of scaffolds produced by RP and the limited efﬁciency of cell seeding techniques normally adopted. In this study we aimed at producing scaffolds with pore size gradients to enhance cell seeding efﬁciency and control the spatial organization of cells within the scaffold. Scaffolds based on blends of starch with poly(e-caprolactone) featuring both homogeneously spaced pores (based on pore sizes of 0.75 and 0.1 mm) and pore size gradients (based on pore sizes of 0.1–0.75–0.1 and 0.75–0.1– 0.75 mm) were designed and produced by three-dimensional plotting. The mechanical performance of the scaffolds was characterized using dynamic mechanical analysis (DMA) and conventional compression testing under wet conditions and subsequently characterized using scanning electron microscopy and micro-computed tomography. Osteoblast-like cells were seeded onto such scaffolds to investigate cell seeding efﬁciency and the ability to control the zonal distribution of cells upon seeding. Scaffolds featur- ing continuous pore size gradients were originally produced. These scaffolds were shown to have inter- mediate mechanical and morphological properties compared with homogenous pore size scaffolds. The pore size gradient scaffolds improved seeding efﬁciency from 35% in homogeneous scaffolds to 70% under static culture conditions. Fluorescence images of cross-sections of the scaffolds revealed that scaf- folds with pore size gradients induce a more homogeneous distribution of cells within the scaffold. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Tissue engineering aims at restoring or regenerating a damaged tissue by combining cells with three-dimensional (3D) porous scaf- folds. After isolation and eventual in vitro expansion, cells are seeded on 3D scaffolds and implanted directly or at a later stage in the patient [1]. Control of the cellular micro-architecture inside the scaffolds is of great importance when developing tissue engi- neering constructs. Moreover, early studies suggest that the mi- cro-architecture of scaffolds might inﬂuence cell attachment and orientation and induce different biological behaviors [2–4]. Ulti- mately, optimizing and controlling these characteristics could lead to better implants when attempting to restore damaged tissues. Rapid prototyping (RP) is one of the most promising techniques for designing and producing scaffolds for tissue engineering appli- cations [5–11]. Many studies on the optimization of RP techniques and scaffolds fabricated by these techniques have been reported in the past few years [12–17]. The scaffolds are usually characterized by their 100% interconnected pores, fully computer controlled architecture and high porosities, which facilitate nutrient perfu- sion, essential to ensure cell viability. However, these techniques also present some drawbacks, including low resolution, which only allows fabrication of scaffolds with large pore sizes compared with the dimensions of a cell. This often leads to low cell seeding efﬁ- ciencies (25–40%) and to a non-uniform distribution of cells along the scaffolds [18]. From a tissue engineering point of view it is known that high cell densities are closely related to improved tis- sue formation in 3D scaffolds [19–23]. However, achieving a high cell seeding efﬁciency is very difﬁcult, mainly due to the intrinsic scaffold characteristics (large pore size, poor cell–material adhe- sion and open pore architecture, among others) and limited cell seeding techniques [24]. The shortage of cells upon seeding re- quires longer periods of cell culture in order to obtain viable con- structs. There is also some evidence that the growth of the cells 1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.11.003 ⇑ Corresponding author. Address: 3B’s Research Group – Biomaterials, Biode- gradables and Biomimetics, AvePark, Zona Industrial da Gandra, S. Cláudio do Barco, 4806-909 Caldas das Taipas, Guimarães, Portugal. Tel.: +351 253510904; fax: +351 253510909. E-mail address: jmano@dep.uminho.pt (J.F. Mano). Acta Biomaterialia 7 (2011) 1009–1018 Contents lists available at ScienceDirect Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat