Enhanced Chondrogenic Differentiation of Dental Pulp Stem Cells Using Nanopatterned PEG-GelMA-HA Hydrogels

Nemeth Cameron L. (1), Janebodin Kajohnkiart (1), Yuan Alex E., Dennis James E., Reyes Morayma, and Kim Deok-Ho.

(1) University of Washington

Tissue Engineering Part A, 20(21-22): 2817-2829(2014)

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Abstract: We have examined the effects of surface nanotopography and hyaluronic acid (HA) on in vitro chondrogenesis of dental pulp stem cells (DPSCs). Ultraviolet-assisted capillary force lithography was employed to fabricate well-defined nanostructured scaffolds of composite PEG-GelMA-HA hydrogels that consist of poly(ethylene glycol) dimethacrylate (PEGDMA), methacrylated gelatin (GelMA), and HA. Using this microengineered platform, we first demonstrated that DPSCs formed three-dimensional spheroids, which provide an appropriate environment for in vitro chondrogenic differentiation. We also found that DPSCs cultured on nanopatterned PEG-GelMA-HA scaffolds showed a significant upregulation of the chondrogenic gene markers (Sox9, Alkaline phosphatase, Aggrecan, Procollagen type II, and Procollagen type X), while downregulating the pluripotent stem cell gene, Nanog, and epithelial–mesenchymal genes (Twist, Snail, Slug) compared with tissue culture polystyrene-cultured DPSCs. Immunocytochemistry showed more extensive deposition of collagen type II in DPSCs cultured on the nanopatterned PEG-GelMA-HA scaffolds. These findings suggest that nanotopography and HA provide important cues for promoting chondrogenic differentiation of DPSCs.

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Materials & Methods: Glass coverslips (BioScience Tools) were cleaned in a piranha solution consisting of a 3:1 ratio of 100% sulfuric acid (Sigma-Aldrich) and 30% aqueous hydrogen peroxide (SigmaAldrich) for 30min to remove organic material and provide additional hydroxyl groups before silane treatment. Then, coverslips were thoroughly cleaned using deionized water and dried under an air stream before being submerged in 2mM 3-(trimethoxysilyl) propyl methacrylate (Sigma-Aldrich) in anhydrous toluene (Sigma-Aldrich) for 60min. The glass coverslips were rinsed in toluene again and dried under an air stream. The cleaned and silane-treated coverslips were stored under vacuum inside a desiccator until used. UV curable nanopatterned polyurethane acrylate (PUA) (Minuta Tech) molds were prepared for fabrication. Characterization and synthesis were previously described.5 The PUA mold consisted of a pattern of ridge·groove·height dimensions of 800·800·500nm. Anisotropically nanopatterned PEG-GelMA-HA hydrogels were fabricated on the pretreated glass coverslips using UV-assisted CFL. A PUA mold was rinsed with 100% ethyl alcohol to remove organic contaminants and was carefully placed onto the surface. A small amount (*10mL) of PEG-GelMA-HA precursor solution was pipetted onto a single glass coverslip. The solution was drawn into the nanogrooves of the pattern through capillary action and cured by exposure to UV light (l=365nm) for 5min. After curing, the PUA mold was peeled off leaving a nanopatterned PEG-GelMA-HA hydrogel scaffold.

Microscopic Technique: Scanning Electron Microscope

Cell Type(s): DPSC