In Vitro Cytotoxicity of Redox Radical Initiators for Cross-Linking of Oligo(poly(ethylene glycol) fumarate) Macromers Johnna S. Temenoff, ²,# Heungsoo Shin, ²,# Daniel E. Conway, ² Paul S. Engel, ‡ and Antonios G. Mikos* ,² Department of Bioengineering and Department of Chemistry, Rice University, 6100 Main, Houston, Texas 77251 Received July 9, 2003; Revised Manuscript Received September 3, 2003 A novel hydrogel system based on oligo(poly(ethylene glycol) fumarate) (OPF) is currently being investigated as an injectable carrier for marrow stromal cells (MSCs) for orthopedic tissue engineering applications. This hydrogel is cross-linked using the redox radical initiators ammonium persulfate (APS) and ascorbic acid (AA). In this study, two different persulfate oxidizing agents (APS and sodium persulfate (NaPS)) with three reducing agents derived from ascorbic acid (AA, sodium ascorbate (Asc), and magnesium ascorbate- 2-phosphate (Asc-2)) and their combinations were examined to determine the relationship between pH, exposure time, and cytotoxicity for rat MSCs. In addition, gelation times for specific combinations were determined using rheometry. pH and cell viability data after 2 h for combinations ranging from 10 to 500 mM in each reagent showed that there was a smaller pH change and a corresponding higher viability at lower concentrations, regardless of the reagents used. At 10 mM, there was less than a 1.5 unit drop in pH and greater than 90% viability for all initiator combinations examined. However, MSC viability was significantly reduced with concentrations of 100 mM and higher of the initiator combinations. At 100 mM, exposure to NaPS/Asc-2 resulted in significantly more live cells than exposure to APS/AA or NaPS/Asc, but at this concentration, NaPS/Asc-2 exhibited significantly longer OPF gelation onset times than APS/ AA. At all combination concentrations, exposure time (10 min vs 2 h) did not significantly affect MSC viability. These data indicate that final pH and/or radical formation have a large impact on MSC viability and that multiple, intertwined testing procedures are required for identification of appropriate initiators for cell encapsulation applications. Introduction In recent years, a variety of hydrogel materials have been explored for use as cell carriers in orthopedic tissue engineering. Such carrier materials play an important role in promotion of tissue regeneration through localized reten- tion of cells at the defect site. Both natural- and synthetic- based materials have been studied as a means of cell transplantation to improve healing of cartilage and non-load- bearing bone defects. 1-17 Natural materials previously em- ployed to encapsulate cells include collagen, 1-3 fibrin, 4-7 alginate 8 and derivatives, 9 chitosan, 10 and agarose. 11 Ad- ditionally, synthetic materials based on acrylamides, 12 poly- (vinyl alcohol) (PVA), 13 poly(ethylene oxide) (PEG) 14-16 and copolymers of PEG, and poly(propylene oxide) 17 have been studied for this application. These synthetic hydrogels possess key advantages such as ease of mass production, assurance of pathogen removal, and tailorability for specific applica- tions. 18 In addition, PEG- and PVA-based hydrogels have been photopolymerized using ultraviolet light to form a three- dimensional structure around cells within seconds to minutes, allowing control of cellular distribution within the gel. 13,16 Our laboratory is currently investigating a novel, biode- gradable, in-situ cross-linkable, synthetic hydrogel material based on oligo(poly(ethylene glycol) fumarate) (OPF) for use as an injectable carrier for marrow stromal cells (MSCs) to aid regeneration of orthopedic injuries. This oligomer can be cross-linked in the presence of the water-soluble redox initiators ammonium persulfate (APS) and ascorbic acid (AA) to form a solid network. 19 Because initiation is a bimolecular reaction, gelation is dependent on temperature and concen- tration, rather than the presence of light, to commence curing. While this may be advantageous for certain clinical applica- tions in which light penetration is limited, the cytotoxicity of this initiator system must be evaluated before it can be used as part of a cell transplantation paradigm. Previous studies in our laboratory have demonstrated that the linear OPF molecule is noncytotoxic 20 and the resulting cross-linked hydrogel is biocompatible. 20,21 However, because MSCs are present during the cross-linking reaction, the cytotoxicity of all components that may interact with the cells must be determined. Therefore, this study was undertaken * To whom correspondence should be addressed. Mailing address: Department of Bioengineering, Rice University, 6100 Main, MS 142, PO Box 1892, Houston, TX 77251-1892. Tel: (713) 348-5355. Fax: (713) 348-4244. E-mail: mikos@rice.edu. ² Department of Bioengineering. # These authors contributed equally to this work. ‡ Department of Chemistry. 1605 Biomacromolecules 2003, 4, 1605-1613 10.1021/bm030056w CCC: $25.00 © 2003 American Chemical Society Published on Web 10/15/2003