Biomacromolecules 2009, 10, 25–31
25
Articles Characterization of Gels Composed of Blends of Collagen I, Collagen III, and Chondroitin Sulfate Kate Stuart and Alyssa Panitch* Weldon School of Biomedical Engineering, Purdue University, 206 South Martin Jischke Drive, West Lafayette, Indiana 47907 Received August 7, 2008; Revised Manuscript Received October 30, 2008
Type I collagen is explored heavily for use in biomaterials, but the role of other extracellular matrix components in regulating collagen organization is gaining attention. We show that as the ratio of type III to type I collagen increases, fibril diameter decreases. A mixture of the two collagen types results in a more open structural network, corresponding to a more compliant material, as compared to a material composed of only one collagen type. Glycosaminoglycans also affect collagen organization and tissue properties. We show that chondroitin sulfate decreases the collagen fibril diameter. Additionally, chondroitin sulfate (CS) increases the void space of a collagen I or collagen III gel, resulting in a more compliant material, but the interactions between types I and III collagen negate the effects of CS. The simple combination of these components results in materials with unique structural, mechanical, and biological cues that can be useful in tailoring biomaterials for tissue engineering.
Introduction The organization of collagen, from fibril diameter and length, to the overall orientation of the fibrils in a network, provides unique morphologic and mechanical properties to individual tissues. Type I collagen is the most prevalent collagen, and its relative ease of isolation and low cost have led to numerous studies of this molecule for multiple tissue engineering applications.1 Type I collagen has the ability to self-associate to form fibrils in vitro that are similar to those seen in vivo,2 but the organization of collagen in vivo is often controlled by its interaction with other ECM components. Certain components, such as other types of collagens,3–6 noncollagenous proteins,4,7 proteoglycans,8–12 and glycosaminoglycans,13–18 have been studied in vitro. Of particular interest in compliant tissues, such as vasculature and skin, is type III collagen.19 Type III collagen is often found associated with type I collagen in vivo and is thought to aid in fibrillogenesis of type I collagen and regulate collagen fibril diameter.5,19–22 Type III collagen is present in reticular fibers which provide elasticity to tissues, while type I collagen fibrils often contribute to a tissue’s tensile strength.19 A defect in type III collagen synthesis, such as seen in type IV Ehlers-Danlos syndrome, results in patients with fragile skin and blood vessels, highlighting the importance of type III collagen in normal tissue development.23–25 The characteristics that type III collagen imparts to tissues in vivo make it a worthwhile molecule to study for tissue engineering applications. There has been little published regarding the use of type III collagen as a biomaterial. Given the prevalence of type III collagen in several tissues, including the arterial medial layer, there exists a need to better understand * To whom correspondence should be addressed. E-mail: apanitch@ purdue.edu.
the behavior of type III collagen alone and in the presence of type I collagen to develop a knowledge base for its use in tissue engineering. Type III collagen decreases type I collagen fibril diameter in vitro and affects the rate of fibrillogenesis.4,6,7,26,27 We wished to examine how these factors relate to the microstructural organization of the collagen network and mechanical properties of the resulting gels. Additionally, we have previously reported on the effects of chondroitin sulfate (CS) on collagen I scaffold properties.17 Physical interactions between protein and glycosaminoglycans (GAGs) such as CS are gaining wide attention and our previous work is extended here as the effect of CS on gels containing both types I and III collagen is examined.
Materials and Methods Materials. Type I collagen, rat tail, was purchased from BD Biosciences (Franklin Lakes, NJ). Type III collagen, bovine skin, was purchased from Millipore (Billerica, MA). Collagens were used as supplied: the type III collagen was provided with mild pepsin treatment, while the type I collagen was not. Both types of collagen were solubilized in 20 mM acetic acid at a concentration of 5 mg/mL. The type III collagen contained 10% type I collagen, so gels contained a maximum of 90% type III collagen. The type I collagen had some type III collagen contaminant, but this was always less than 10%, according to the manufacturer; we also verified the type III collagen content in the type I collagen solution via immunohistochemistry (see Supporting Information). Chondroitin-6-sulfate (shark cartilage) was purchased from Sigma-Aldrich (St. Louis, MO) and had an average molecular weight of 20 kDa. Gel Preparation. Stock collagens were kept at 5 mg/mL in 0.02 M acetic acid. Collagen was mixed on ice with 10× phosphate buffered saline (PBS), 1 M NaOH, and 1× PBS to give a final pH of 7.4 and salt concentration of 164 mM (1× PBS was composed of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.76 mM KH2PO4). Gelation occurred by heating the sample to 37 °C. CS was solubilized
10.1021/bm800888u CCC: $40.75 2009 American Chemical Society Published on Web 11/24/2008
26
Biomacromolecules, Vol. 10, No. 1, 2009
in 1× PBS at a concentration of 10 mg/mL and was added to the solution as part of the 1× PBS solution immediately prior to heating. Gels had a final total concentration of 4 mg/mL collagen with
