Preparation of open porous polycaprolactone microspheres and their applications as effective cell carriers in hydrogel system Qingchun Zhang a , Ke Tan b , Zhaoyang Ye b , Yan Zhang a, ⁎, Wensong Tan b , Meidong Lang a, ⁎ a Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, China b State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237 China abstract article info Article history: Received 25 April 2012 Received in revised form 5 July 2012 Accepted 30 July 2012 Available online 4 August 2012 Keywords: Polycaprolactone Porous microspheres Paraffin Mesenchymal stem cells Microsphere/hydrogel composite Common hydrogel, composed of synthetic polymers or natural polysaccharides could not support the adhesion of anchorage-dependent cells due to the lack of cell affinitive interface and high cell constraint. The use of porous polyester microspheres as cell-carriers and introduction of cell-loaded microspheres into the hydrogel system might overcome the problem. However, the preparation of the open porous microsphere especially using polycaprolactone (PCL) has been rarely reported. Here, the open porous PCL microspheres were fabricated via the combined emulsion/solvent evaporation and particle leaching method. The microspheres exhibited porous surface and inter-connective pore structure. Additionally, the pore structure could be easily controlled by adjusting the processing parameters. The surface pore size could be altered from 20 μm to 80 μm and the in- ternal porosities were varied from 30% to 70%. The obtained microspheres were evaluated to delivery mesen- chymal stem cells (MSCs) and showed the improved cell adhesion and growth when compared with the non-porous microspheres. Then, the MSCs loaded microspheres were introduced into agarose hydrogel. MSCs remained alive and sustained proliferation in microsphere/agarose composite in 5-day incubation while a dec- rement of MSCs viabilities was found in agarose hydrogel without microspheres. The results indicated that the microsphere/hydrogel composite had a great potential in cell therapy and injectable system for tissue regeneration. © 2012 Elsevier B.V. All rights reserved. 1. Introduction In recent years, in situ forming hydrogels have been widely studied in tissue engineering due to the facile cell encapsulation and the injectable capacity [1,2]. From their strategies, three steps are generally involved: i. suspending cells in pre-gel solution, ii. injecting the mixture into damaged site, iii. crosslinking to form hydrogel in situ [1]. The use of in situ forming system ensures a spatial fit between implant and defect site, and allows cells to be evenly distributed in hydrogel system [3–5]. However, the low cell affinity and high cell constraint failed to achieve settlement of anchorage dependent cells such as fibroblasts, osteoblasts, endothelial and stem cells. Cells cannot spread out in common hydrogel, which results in loss of cell function and cell death [6,7]. Many approaches such as grafting of cell-adhesive moieties have been applied to solve this problem, but the results turned out to be unsatisfied probably due to the high cell constraint [8,9]. Recently, a physical hybridization of gelatin micro-carriers and agarose hydrogel has been used. The incorporated micro-carriers can provide a cell-affinitive interface and reduce the cell constraint to remain the cells alive and sustained proliferation in the agarose hydrogel [10]. However, to the best of our knowledge, the introduction of porous microspheres based on polyesters into hydrogel has not been reported. Porous microsphere systems have been recently developed as cell carriers in the field of tissue engineering [11–14]. Tissue cells or stem cells attach and subsequently proliferate upon microspherical sub- strate, and the construct is implanted into damaged site to induce neo-tissue formation [15,16]. Many studies have demonstrated that the microspheres with porous structure could achieve more efficient cell expansion than those with non-porous structure [13,17,18]. The porous structure can provide large specific surface areas and high interconnectivities for cell settlement, migration and growth. Polycaprolactone (PCL) has been generally used to fabricate po- rous scaffold to support cell growth due to its good biocompatibility and slow biodegradation [19–22]. Their microspheres have also been studied for the sustained cell delivery [23]. However, non- porous structure was an obstacle for PCL microspheres for cell culti- vation. The smooth surface and low specific surface area fail to obtain a sustained cell proliferation. Therefore, it is critical to create an open pore structure on PCL microspheres. Several methods have been ap- plied to prepare porous microspheres such as blended polymer ex- traction [24], self-assembly [17] and gas foaming [11], but few method is suitable for the preparation of porous PCL microspheres. Materials Science and Engineering C 32 (2012) 2589–2595 ⁎ Corresponding authors. Tel./fax: +86 21 64253916. E-mail addresses: zhang_yan@ecust.edu.cn (Y. Zhang), mdlang@ecust.edu.cn (M. Lang). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.07.045 Contents lists available at SciVerse ScienceDirect Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec