Biomacromolecules 2004, 5, 5-10
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Thermally Cross-Linked Oligo(poly(ethylene glycol) fumarate) Hydrogels Support Osteogenic Differentiation of Encapsulated Marrow Stromal Cells In Vitro Johnna S. Temenoff,† Hansoo Park,† Esmaiel Jabbari,†,‡ Daniel E. Conway,† Tiffany L. Sheffield,§ Catherine G. Ambrose,§ and Antonios G. Mikos*,† Department of Bioengineering, Rice University, 6100 Main, Houston, Texas 77251, and Department of Orthopaedics, University of TexassHouston, 6431 Fannin, Houston, Texas 77030 Received September 16, 2003; Revised Manuscript Received November 3, 2003
A novel polymer, oligo(poly(ethylene glycol) fumarate) (OPF), cross-linked with a thermal radical initiation system has recently been developed in our laboratory as an injectable, biodegradable cell carrier for regeneration of orthopaedic tissues. The cross-linking, swelling, and degradative properties of hydrogels prepared from OPF with poly(ethylene glycol) of two different chain lengths were assessed. The two OPF types had similar gelation onset times (∼3.6 min) but, when cross-linked for 8 min at 37 °C, exhibited significantly different swelling characteristics (fold swelling: 17.5 ( 0.2 vs 13.4 ( 0.4). Rat marrow stromal cells (MSCs) were then directly combined with the hydrogel precursors and encapsulated in a model OPF formulation at ∼14 million cells/mL, cultured in vitro in the presence of osteogenic supplements (dexamethasone), and monitored over 28 days via histology. MSC differentiation in these samples (6 mm diameter × 0.5 mm thick before swelling), as determined by Von Kossa staining for calcified matrix, was apparent by day 21. At day 28, mineralized matrix could be seen throughout the samples, many microns away from the cells. These exp eriments strongly support the usefulness of thermally cross-linked OPF hydrogels as injectable cell carriers for bone regeneration. Introduction In the past decade, research in the area of orthopaedic tissue engineering has endeavored to produce methods that restore defects in bone and cartilage. One such approach is to deliver cells to the site of injury and, thus, promote localized healing of the desired tissue. To reduce patient discomfort, an ideal cell carrier would be both injectable and biodegradable, so that cells could be introduced via minimally invasive means and a second surgery would not be required to remove the carrier material.1 A suitable injectable material should be biocompatible, both in regards to the resident cells and surrounding tissue, have biocompatible degradation products, demonstrate appropriate degradation times and mechanical properties to protect the defect site while promoting tissue growth, be easily sterilizable, and exhibit clinically relevant setting times and handling properties.1 Additionally, materials to be used as cell carriers (either with or without macroporosity) should have diffusional properties sufficient to allow nutrient exchange throughout the construct. Several synthetic injectable materials, including those based on acrylamides,2 poly(vinyl alcohol) (PVA),3 and poly(ethylene glycol) (PEG),4-6 have been examined for cell * To whom correspondence should be addressed. Dr. Antonios G. Mikos, Department of Bioengineering, Rice University, 6100 Main, MS 142, P.O. Box 1892, Houston, TX 77251-1892. Phone: (713) 348-5355. Fax: (713) 348-4244. E-mail:
[email protected]. † Rice University. ‡ Current address: Department of Orthopaedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. § University of TexassHouston.
encapsulation applications. The advantage of these hydrogel materials is that they have a high degree of swelling in aqueous environments and thus can promote viability of cells in constructs with a thickness of several millimeters.5 Our laboratory has recently developed a novel synthetic injectable hydrogel, oligo(poly(ethylene glycol) fumarate) (OPF), for orthopaedic tissue engineering applications. This material meets many of the requirements detailed above for an ideal injectable cell carrier. Hydrolysis of the ester bonds in the OPF backbone results in biodegradation of the resulting cross-linked hydrogels.7 The cytocompatibility of the linear OPF molecule and leachable fractions from the cross-linked hydrogels have been demonstrated in vitro using rat marrow stromal cells (MSCs).8 In addition, evidence of the biocompatibility of pre-cross-linked gels from various OPF formulations has been observed in vivo in rabbits.7 Depending on the selection of cross-linking molecules, the mechanical and degradative properties of these hydrogels may be altered as required for given applications.7,9,10 A cytocompatible, water-soluble radical initiation system, ammonium persulfate/N,N,N′,N′-tetramethylethylenediamine (APS/TEMED), has recently been identified for this oligomer, allowing for the encapsulation of rat MSCs at 37 °C within 10 min.11 Thus, cell-OPF hydrogel constructs can be formed on a clinically relevant time scale, and the spatial distribution of the encapsulated cells can be controlled. Additionally, the MSC-OPF solution is easily drawn into a syringe at room temperature, making the system relatively easy to manage in a clinical setting. In particular, while many previous PEG-based materials for cell encapsulation have
10.1021/bm030067p CCC: $27.50 © 2004 American Chemical Society Published on Web 11/26/2003
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Biomacromolecules, Vol. 5, No. 1, 2004
involved photo-cross-linking to form hydrogels,5,6,12 in this case, gelation is based only on changes in temperature, which may be especially advantageous in areas where light penetration is limited. As the next step in the investigation of OPF as an injectable cell carrier, this study was designed to characterize the gelation time, swelling, and degradative properties of OPF hydrogels using two different OPF formulations (OPF 10K and OPF 3K) cross-linked with PEG-diacrylate (PEG-DA). Subsequently, the differentiation of rat MSCs encapsulated in the OPF 10K hydrogels and cultured in the presence of osteogenic supplements (dexamethasone) was qualitatively evaluated via histology over 28 days in vitro. Materials and Methods pH Experiments. Solutions of 25 mM APS (EM Science, Gibbstown, NJ), 25 mM TEMED (Sigma-Aldrich, St. Louis, MO), as well as the combination APS/TEMED (25 mM each component) were prepared in triplicate in MSC culture media [DMEM high glucose (Gibco, Grand Island, NY) supplemented with 10% v/v fetal bovine serum (Gemini, Calabasas, CA), 10 mM β-glycerophosphate, 50 mg/L ascorbic acid, 250 µg/L fungizone, 100 mg/L ampicillin, and 50 mg/L gentamicin (all from Sigma-Aldrich)]. The pH of these solutions was recorded immediately after mixing with a digital pH meter (AP5, Fisher Scientific, PA). The pH change in reference to media containing no initiators was then calculated.13 OPF Synthesis and Characterization. Two formulations of OPF were synthesized from PEG of different nominal number average molecular weight: 10 000 g/mol (designated OPF 10K) or 3300 g/mol (OPF 3K), following established procedures.10,14 After synthesis, the resulting macromer was purified via recrystallization from ethyl acetate (Fisher Scientific, Pittsburgh, PA) and precipitation with ethyl ether (Fisher Scientific). The resulting powder was vacuum-dried at
