Highly Porous Electrospun Nanofibers Enhanced by Ultrasonication for Improved Cellular Infiltration Jung Bok Lee, M.S., 1, * Sung In Jeong, Ph.D., 1, * Min Soo Bae, M.S., 1 Dae Hyeok Yang, Ph.D., 1 Dong Nyoung Heo, M.S., 1 Chun Ho Kim, Ph.D., 2 Eben Alsberg, Ph.D., 3 and Il Keun Kwon, Ph.D. 1,4 A significant problem that affects tissue-engineered electrospun nanofibrous scaffolds is poor infiltration of cells into the three-dimensional (3D) structure. Physical manipulation can enhance cellular infiltration into electro- spun scaffolds. The porosity of electrospun nanofibers was highly enlarged by ultrasonication in an aqueous solution. The porosity and related property changes on a series of nanofibers were observed to be dependent on ultrasonication time and energy. To evaluate cell infiltration into the scaffold, fibroblasts were seeded onto these nanofibers and cultured for different lengths of time. The penetration levels of these cells into the scaffold were monitored using confocal lazer scanning microscopy. The cell infiltration potential was greatly increased with regard to an increase in pore size and porosity. These 3D nanofibrous scaffolds fabricated by an ultrasonication process allowed cells to infiltrate easily into the scaffold. This approach shows great promise for design of cell permeable nanofibrous scaffolds for tissue-engineering applications. Introduction T issue scaffolds are used in tissue engineering to repair damaged tissue and organs with a combination of cells and biomaterials. There are several methods for generating three-dimensional (3D) tissue-engineering scaffolds. These methods include solvent casting/particulate leaching, gas formation, emulsion lyophilization, electrospinning, and phase separation. Several polymers are used for this including biodegradable/biocompatible natural or synthetic polymers such as collagen, gelatin, chitosan, poly(l-lactic acid) (PLLA), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(e- caprolactone) (PCL), and poly (lactic acid-co-e-caprolactone). 1–5 To repair or replace damaged tissue, these scaffolds are often designed to mimic the structure and biological functions of natural extracellular matrix (ECM). The ECM guides cellular migration, provides mechanical support, and regulates cel- lular activities. 6,7 Electrospinning is a relatively simple and versatile method for forming nonwoven fibers by electrically charging a polymer solution as it is passed through a nee- dle. 8,9 Nanofibers fabricated by electrospinning provide nano-scaled fibers that mimic the structure of ECM. 10 These nanofibrous structures are widely used in tissue engineering due to their high porosity and surface area-to-volume ratio as well as their topographical features that can enhance cel- lular adhesion, migration, and proliferation. Polymer meshes fabricated by electrospinning are comprised of fibers with diameters as low as hundreds of nanometers, and exhibit porosities lower than 90% with pore sizes as low as several micrometers. 5,10–12 Although electrospun nano/micro-fibers have been studied as effective tissue-engineering scaffolds for regenerating tissue such as bone, cartilage, skin, blood vessels, 13–15 and nerves, 16 the small pore size and low overall thickness of these nanofibers may limit cellular infiltration into the scaffold. 5,11 Commonly, nanofibrous scaffolds form into extremely long, entangled, and densely packed 2-D structures rather than being shaped into porous 3D structures useful for tissue engineering. Recently, several studies have shown that nanofiber matrixes which allow cells to infiltrate into their porous structure are useful as tissue-engineering scaf- folds. 12,15,17–19 In a study by Pham et al., an alternating layered scaffold of PCL microfibers and nanofibers was fabricated to increase the pore size. 11 Increasing the pore size by adding microfibers allows cells to infiltrate the matrix; however, microfiber scaffolds are not on the same size scale as ECM components. Nam et al. and Ekaputra et al. tried to increase the pore size of electrospun scaffolds by using salt-leaching methods. 17,18 These studies have been able to fabricate the 3D nanofibrous architectures necessary for the seeded cells to penetrate inside of the scaffold. Despite this successful in- crease in pore size of the scaffold, there was a problem of structural collapse after leaching out the salt. 1 Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea. 2 Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea. 3 Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio. 4 Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea. *Co-first authorship. TISSUE ENGINEERING: Part A Volume 17, Numbers 21 and 22, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2010.0709 2695