Evaluation of Chondrogenesis of Bone Marrow and Adipose-derived Stem Cells Encapsulated in Agarose and Self-assembling Peptide Hydrogels

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Copyright Year	2005
Abstract	ENCAPSULATED IN AGAROSE AND SELF-ASSEMBLING PEPTIDE HYDROGELS *Kisiday, J D, †Kopesky, P W, †Szafranski ,J D, +Evans, C H, †Grodzinsky, A J; *McIlwraith, C W, *Frisbie, D D, *Colorado State University, Fort Collins, CO, †Massachusetts Institute of Technology, Cambridge, MA, +Harvard Medical School, Boston, MA, kisiday@lamar.colostate.edu INTRODUCTION: Bone marrow [1] and adipose tissue [2] contain pluripotent stem cells that are capable of differentiating into cells of a chondrogenic lineage. Therefore, each tissue may be considered as a cell source for cartilage resurfacing therapies [3]. In this study, we investigated the chondrogenic potential of bone marrow and adipose cells obtained from 2-5 year old horses, an animal source chosen for relevance to experimental models for human therapies [4]. Cultureexpanded cells were encapsulated in agarose and self-assembling peptide [5] hydrogels and analyzed for extracellular matrix synthesis and accumulation as indicators of chondrogenic differentiation. METHODS: Tissue harvest and, cell preparation – Bone marrow was harvested from the iliac crest and, resuspended in 0.8% ammonium chloride to lyse the majority of the red blood cells. The remaining cells were seeded in tissue culture flasks at a concentration of 0.66x10 nucleated cells/cm in low glucose DMEM supplemented with 10% FBS and 1 ng/ml FGF-2 [6] to allow for attachment of mesenchymal stem cells (MSCs) [7]. Adherent MSC colonies were allowed to grow for 1012 days, then reseeded at a concentration of 22x10 cells/cm. After reaching confluence, the cells were passaged once prior to seeding in agarose hydrogels. Preadipocytes were obtained from adipose tissue [2] collected subcutaneously from the tailhead fat pad. Washed tissue was digested in 0.1% collagenase for 3-4 hours with agitation. Preadipocytes were spun down and seeded into tissue culture flasks at a concentration of 3.5 x 10 cells/cm in low glucose DMEM supplemented with 10% FBS and 1 ng/ml FGF-2. After reaching confluence in 5-6 days, the cultures were passaged twice at a ratio of 1:3. Hydrogel encapsulation and culture in agarose and peptide scaffolds – Cells were seeded in either 2% agarose or 0.35% KLD12 peptide hydrogel at a concentration of 10x10 cells/ml. Cell-seeded hydrogels were cultured in high glucose DMEM supplemented with ITS+, 0.1 μM dexamethasone, and 37.5 μg/ml ascorbate-2-phosphate, with and without 10 ng/ml recombinant human TGF-β1 (R&D) [1]. Hydrogels were cultured for 21 days prior to analysis. Extracellular matrix analysis Total GAG accumulation in the scaffold were quantified using the DMMB dye binding assay. Over the final 24 hours of culture, protein and proteoglycan synthesis was quantified via H-proline and S-sulfate incorporation, respectively. Selected samples were radiolabeled with 50 μCi/ml S-sulfate followed by protein extraction in 4M guanidine, ethanol precipitation, and separation on a Superose 6 column to determine the size of newlysynthesized proteoglycans. Histological analyses (toluidine blue for proteoglycans, immunohistochemical staining for type II collagen) were used to spatially resolve extracellular matrix accumulation in the gel. RESULTS: Agarose hydrogel culture – Quantification of extracellular matrix synthesis: Adipose-derived cells from 3 animals (designated as #1, #2, and #3 in figs. 1, 2) were evaluated for extracellular matrix synthesis and GAG accumulation. For each animal, protein synthesis was 1.5-3-fold higher for TGF-β1 cultures relative to TGF-β1-free conditions (Fig. 1). A marrow culture (run in parallel with adipose cultures #1 and #2) showed similar increases in protein synthesis with TGF-β1 treatment, however the magnitude of synthesis was approximately 2-fold higher in marrow culture relative to adipose for matched TGF conditions. Proteoglycan synthesis in adipose cultures showed similar trends of approximately 2-fold increases with TGF-β1 treatment (Fig. 2). In marrow cultures, TGF-β1-free conditions showed comparable proteoglycan synthesis to TGF-β1-cultured adipose hydrogels. TGF treatment of marrow hydrogels stimulated nearly a 20fold increase in proteoglycan synthesis over TGF-free cultures. GAG accumulation (data not shown): All adipose cultures and TGF-β1-free marrow cultures contained 0.2-0.6 μg GAG/mg ww, while TGF-β1 marrow hydrogels contained 4.2 μg GAG/mg ww (s.e.m. values <10% of the mean for all conditions). Superose 6 analysis – Fractionation of newly-synthesized proteoglycans in marrow TGF-β1 cultures showed that the majority of S-sulfate incorporation was found in a single peak centered on the 7.5 ml elute fraction (Fig. 3), This profile was similar to superose 6 analysis of proteoglycans (aggrecan) extracted from adult equine cartilage (detected via DMMB binding, data not shown). All adipose cultures showed incorporation of S-sulfate over a wide range of proteoglycan sizes (Fig. 3, adipose #2 cultured in TGF). Histological analysis – No metachromatic staining was present for adipose cultures. Marrow TGF-β1 cultures showed pericellular staining around a subset of encapsulated cells (Fig. 5A) as well as intracellular immunohistochemical staining for type II collagen. Peptide hydrogel culture – Marrow and adipose-derived cells from the same animal were cultured in both agarose and peptide hydrogels +/TGF-β1. Agarose cultures showed similar trends of radiolabel incorporation (Fig. 4) and GAG accumulation (data not shown) as previously observed. In peptide cultures (Fig 4), TGF-β1 stimulated proline incorporation ~10 and 25fold for marrow and adipose cultures, respectively. Likewise, sulfate incorporation in TGF-β1 cultures increased ~25 and 50-fold over TGFfree cultures for marrow and adipose cells, respectively. Relative to agarose cultures, radiolabel incorporation in peptide hydrogels was ~4 to 9-fold (proline) and ~3-fold (sulfate) higher for matched TGF-β1 conditions. Adipose cell in peptide hydrogels cultured in TGF-β1-free conditions showed ~8 and ~3-fold increases over TGF-β1-free agarose cultures in proline and sulfate incorporation, respectively. Adipose/peptide cultures in TGF-β1 showed ~3550-fold increases in radiolabel incorporation relative to adipose/agarose hydrogels cultured in TGF-β1. DISCUSSION: The low metabolic activity and broad profile of both small and large proteoglycan synthesis demonstrated that adult equine adipose-derived cells showed poor TGF-β1-mediated chondrogenesis relative to marrow cultures when seeded in agarose. However, when seeded in peptide hydrogel, both marrow and adipose cells appear capable of chondrogenesis in the presence of TGF-β1. A possible explanation of the differential behavior between agarose and peptide hydrogels may be the physical properties of the gels. Agarose, an order of magnitude stiffer than peptide hydrogel [6], did not allow for cell migration or changes in morphology. In peptide hydrogels, both adipose and marrow-derived cells were able to elongate by displacing the soft peptide matrix, resulting in cell-cell contact and contraction of cell aggregates containing approximately hundreds of cells each (Fig. 5B). These data demonstrate that 3-D environment may have a significant influence on stem cell chondrogenesis and merit consideration in the design of cartilage resurfacing strategies.
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