EuropeanJT Connelly Cells et andal. Materials Vol. 22 2011 (pages 168-177) DOI: 10.22203/eCM.v022a13Ligands regulate chondrogenesis in ISSN 3D hydrogels1473-2262 FIBRONECTIN- AND COLLAGEN-MIMETIC LIGANDS REGULATE BONE MARROW STROMAL CELL CHONDROGENESIS IN THREE-DIMENSIONAL HYDROGELS J.T. Connelly1,§, T.A. Petrie2, A.J. García1,2 and M.E. Levenston3,* 1George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA 2Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA 3Department of Mechanical Engineering, Stanford University, Stanford, CA, USA §Current address: Centre for Cutaneous Research, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, London, UK Abstract Introduction Modifi cation of tissue engineering scaffolds with bioactive Mesenchymal stem cells (MSCs) are an attractive cell molecules is a potential strategy for modulating cell behavior source for tissue engineering and cell-based therapies given and guiding tissue regeneration. While adhesion to RGD their multilineage differentiation potential and ability to be peptides has been shown to inhibit in vitro chondrogenesis, expanded in culture (Caplan, 1991; Pittenger et al., 1999). the effects of extracellular matrix (ECM)-mimetic The repair of damaged articular cartilage is one particular ligands with complex secondary and tertiary structures application that would be well suited for MSCs as a result are unknown. This study aimed to determine whether of the limited capacity for cartilage regeneration and the collagen- and fi bronectin-mimetic ligands would retain rapid dedifferentiation of chondrocytes during monolayer biologic functionality in three-dimensional (3D) hydrogels, expansion (Benya and Shaffer, 1982). Many populations whether different ECM-mimetic ligands differentially of mesenchymal progenitors such as bone marrow stromal infl uence in vitro chondrogenesis, and if effects of ligands cells (BMSCs) display chondrogenic-like differentiation on differentiation depend on soluble biochemical stimuli. in three-dimensional (3D) culture environments and in the A linear RGD peptide, a recombinant fi bronectin fragment presence of transforming growth factor-β (TGF-β) family containing the seven to ten Type III repeats (FnIII7-10) and growth factors (Johnstone et al., 1998; Caterson et al., a triple helical, collagen mimetic peptide with the GFOGER 2001; Bosnakovski et al., 2004). However, recent studies motif were covalently coupled to agarose gels using the have shown that even over extended culture periods, sulfo-SANPAH crosslinker, and bone marrow stromal engineered cartilage constructs derived from BMSCs cells (BMSCs) were cultured within the 3D hydrogels. have a distinct composition and inferior mechanical The ligands retained biologic functionality within the properties compared with those derived from native agarose gels and promoted density-dependent BMSC articular chondrocytes (Mauck et al., 2006; Connelly et spreading. Interactions with all adhesive ligands inhibited al., 2008b). It is therefore likely that additional signals stimulation by chondrogenic factors of collagen Type II are required for complete chondrogenic differentiation and aggrecan mRNA levels and deposition of sulfated and the development of a functional tissue replacement. glycosaminoglycans. In medium containing fetal bovine Interactions between cells and the extracellular matrix serum, interactions with the GFOGER peptide enhanced (ECM) provide important cues for the differentiation mRNA expression of the osteogenic gene osteocalcin and development of many tissues, including cartilage. whereas FnIII7-10 inhibited osteocalcin expression. In For example, integrin-mediated adhesion to fi bronectin conclusion, modifi cation of agarose hydrogels with ECM- is required for precartilage condensation of limb bud mimetic ligands can infl uence the differentiation of BMSCs cells (Bang et al., 2000), and the presence of specifi c in a manner that depends strongly on the presence and nature fi bronectin isoforms infl uences the extent of condensation of soluble biochemical stimuli. (White et al., 2003). Interactions with Type II collagen can also enhance the in vitro chondrogenesis of BMSCs Keywords: Chondrogenesis, mesenchymal progenitors, (Bosnakovski et al., 2006). Although much work is still extracellular matrix, fibronectin, collagen, tissue required to fully understand the complex roles of cell- engineering, microenvironment. matrix interactions in chondrogenesis, it is clear that the ECM provides key signals for guiding this process. A potential strategy for controlling cell-matrix interactions in vitro is to engineer synthetic matrices to present specific ligands in a precise manner. The incorporation of peptides containing the integrin adhesive *Address for correspondence: sequence Arginine-Glycine-Aspartic acid (RGD) into Marc E. Levenston non-adhesive hydrogels was reported to enhance MSC 233 Durand Building survival and osteogenic differentiation (Shin et al., Stanford University 2005). However, studies in our laboratory suggest that Stanford, CA 94305-4038, USA alginate and agarose hydrogels functionalized with RGD E-mail: [email protected] peptides inhibit BMSC chondrogenesis (Connelly et al., www.ecmjournal.org 168 JT Connelly et al. Ligands regulate chondrogenesis in 3D hydrogels 2007; Connelly et al., 2008a). In addition to modulating USA), and the AMV reverse transcriptase kit was from integrin-mediated adhesion, engineered matrices also Promega (Madison, WI, USA). The Sybr Green master provide a means for introducing small molecules (Benoit mix was from Applied Biosystems (Foster City, CA, USA), et al., 2008) or larger moieties such as chondroitin and the primers were from Invitrogen. sulfate (Varghese et al., 2008) that can enhance in vitro chondrogenesis. Although interactions with the RGD motif Preparation of ligand-modifi ed agarose inhibit BMSC chondrogenesis, interactions with adhesive A monobiotinylated fi bronectin fragment containing the sequences that mimic other ECM proteins and target a seventh to tenth Type III repeats (FnIII7-10) of human distinct set of integrin receptors may induce different fi bronectin was expressed in JM109 cells and purifi ed by responses. For example, α5β1 binding to fibronectin affi nity chromatography as previously described (Petrie requires a synergy site containing the “PHSRN” sequence et al., 2006). A biotin tag was coupled to the cysteines in in addition to RGD (Aota et al., 1994), and α2β1 binds to the GFOGER peptide using the EZ-link Maleimide-PEG- the “GFOGER” motif within fi brillar collagens (Reyes Biotin kit according to the manufacturer’s instructions. and Garcia, 2003). However, the effects of ECM-mimetic The synthetic peptides and fi bronectin fragment were then ligands with complex secondary and tertiary structures on conjugated to agarose hydrogels with the heterobifunctional chondrogenesis are unknown. The goals of this study were sulfo-SANPAH crosslinker (Dodla and Bellamkonda, to determine whether fi bronectin- and collagen-mimetic 2006). Briefl y, the primary amines on the ligands were ligands, targeting the α5β1 integrins and α2β1 integrins, reacted with the NHS-ester group of the sulfo-SANPAH respectively, retain biologic functionality when conjugated in PBS at room temperature for 4 h in the dark with a to 3D agarose hydrogels, whether interactions with 10-fold molar excess of the crosslinker. Seaprep agarose different ECM-mimetic ligands differentially infl uence solutions were prepared in Ca2+/Mg2+-free PBS, autoclaved, BMSC chondrogenesis in 3D hydrogels, and whether and cooled to 37 ºC. One part peptide/sulfo-SANPAH the responses to such engineered matrices depend on the solution was combined with three parts 4 % agarose and nature of soluble biochemical stimuli presented through mixed thoroughly to yield a 3 % agarose solution. The the culture medium. mixture was exposed to 365 nm ultraviolet light for 3 min to activate the photoreactive groups of the sulfo-SANPAH and conjugate the peptide to CH groups in the agarose. Materials and Methods The agarose was allowed to gel at 4 ºC for 20 min and washed four times with fi vefold excess PBS over 3 days Materials to remove the unbound peptide and crosslinker. Controls The synthetic peptides GRGDSP (RGD) and the non-adhesive were prepared as stated previously but without addition of control GRGESP (RGE) were obtained from Bachem (King the sulfo-SANPAH. Final ligand densities in the agarose of Prussia, PA, USA), and the collagen mimetic peptide gels were determined by dot blot detection of biotinylated GGYGGGPC[GPP]5GFOGER[GPP]5GPC (GFOGER) ligands (Fig. 1). Gels were digested with agarase (4 U/ was prepared by the Emory University Microchemical gel) at 45 ºC for 4 h and blotted onto nitrocellulose Facility. The sulfo-SANPAH crosslinker, EZ-link (n = 3/condition). Membranes were probed with alkaline Maleimide-PEG-Biotin kit, and Ultralink Immobilized phosphatase-conjugated anti-biotin antibodies and Avidin were from Pierce (Rockford, IL, USA). Seaprep developed with the ECF substrate and Fuji Image Analyzer agarose was from Cambrex (Frederick, MD, USA). (Fuji, Tokyo, Japan). For an input concentration of 8 μM Immature bovine hind limbs were from Research 87 (400 μg/mL) FnIII7-10, the conjugation effi ciency was (Marlborough, MA, USA). Recombinant human TGF-β1 approximately 30 %, resulting in a fi nal density of 2.4 was from R&D Systems (Minneapolis,