Electrospun cross-linked gelatin fibers with controlled diameter: The effect of matrix stiffness on proliferative and biosynthetic activity of chondrocytes cultured in vitro Maciej Skotak, Sandra Noriega, Gustavo Larsen, Anuradha Subramanian Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Nebraska 68588-0643 Received 22 May 2009; revised 6 October 2009; accepted 24 March 2010 Published online 7 September 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.32850 Abstract: Nanofibrous scaffolds were prepared from gelatin solutions and were further cross-linked with glutaraldehyde (GA). The fiber diameter was varied from 100 to 1000 nm by controlling the applied voltage (4–15 kV) and the concentra- tion of the gelatin solution (4–15%). The tensile moduli and the tensile strength of the noncross-linked scaffolds varied from 20 to 120 MPa and 0.5 to 3.5 MPa, respectively. Cross- linking with GA led to an increase in both the tensile modu- lus and strength and correlated with cross-linker concentra- tion. Gelatin-based matrices were characterized by Fourier transform infrared spectroscopy and differential scanning cal- orimetry. High cellular viabilities and rounded morphology of chondrocytes was observed at the end of 7 days in culture with added matrix deposition and flattening of cells at 15 days. Matrix stiffness was noted to impact cell densities and the expression of chondrocytic markers, especially aggrecan. The ratios of collagen-II (C-II) to collagen-I (C-I) of 0.62 and 1.33 were noted on gelatin nanofibrous scaffolds cross-linked with 0.1% GA at the end of 7 and 15 days in culture, respec- tively. C-II/C-I ratios of 1.30 and 2.58 were noted on scaffolds cross-linked with 1.0% GA at the end of 7 and 15 days in cul- ture, respectively. V C 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 95A: 828–836, 2010. Key Words: gelatin, electrospinning, cross-linking, chondro- cyte phenotype INTRODUCTION The articular cartilage is the dense white tissue covering the articulating surfaces of bones. Chondrocytes, in the form of chondrons, 1 are the only cells present in the cartilage. They are sparsely dispersed within the extracellular matrix (ECM). Their major role is to maintain homeostatic balance and respond to catabolic processes. The regulation of chon- drocyte function (e.g., biosynthesis of type II collagen, pro- teoglycans synthesis and down-regulation of nitrous oxide production) by growth factors (transforming growth factor- b ), cytokines, and biomechanical forces (dynamic compres- sion) has been demonstrated. 2–5 An area that has not been studied as well, but is critical for the understanding of carti- lage function and maintenance during normal and disease states, is the regulation of chondrocyte function by the micromechanical environment or variation in substrate me- chanical property. For example, osteoarthritis is brought about by the degradation of the collagen fibrils in the ECM, 6 leading to a cartilage matrix with decreased tensile modulus and increased stiffness, which in turn perhaps leads to altered biosynthetic activity of chondrocytes. Our long-term objective is: (1) to develop a simplified model that lends itself to the analyses of the effects of micromechanical envi- ronment of the matrix on cellular functions and (2) to find thresholds and mechanisms leading to a positive versus neg- ative mechanotransduction. The ECM of the cartilage is mainly composed of collagen fibers, the orientation and percent composition of which varies from the tangential zone to the deep zone, with the chondrocytes embedded in the avascular matrix. The tech- nique of electrospinning (ES) has enabled the fabrication of nanofibrous matrices that mimic the scaffold topography of native tissues, including articular cartilage. ES, 7–10 a type of electrohydrodynamics (EHD) used in fiber production proc- esses, has been frequently used to prepare fibrous matrices with diameters in the range to that encountered in vivo. 11 Although the cellular response to nanofibrous matrices has been reported extensively, to the best of our knowledge, studies dealing with systematic evaluations of the effect of the parameters associated with electrospun matrices on the mitotic function of chondrocytes in vitro are limited. The in vitro approach offers the advantage of investigating the impact of one matrix parameter at a time; in this article, we have focused our efforts on elucidating the effect of matrix stiffness on the mitotic and biosynthetic activity of chondro- cytes seeded on electrospun matrices. We are cognizant that collagen, the main constituent of connective tissue and solutions of collagen in fluorinated alcohols such as 1,1,1,3,3,3-hexafluoropropanol or 2,2,2-tri- fluoroethanol (TFE), can be processed by EHD into fibrous structures with relative ease. However, the cytotoxicity of traces of fluorinated alcohols that may remain in the fibers Correspondence to: A. Subramanian; e-mail: asubramanian2@unl.edu Contract grant sponsor: NIH; contract grant number: 5R21EB006046-02 828 V C 2010 WILEY PERIODICALS, INC.