title: Ionic osmolytes and intracellular calcium regulate tissue production in chondrocytes cultured in a 3D charged hydrogel.
abstract: The goal of this study was to investigate the role of fixed negative charges in regulating cartilage-like tissue production by chondrocytes under static and dynamic three-dimensional culture, and to determine whether intracellular calcium ([Ca(2+)]i) is involved in mediating this response. Initial experiments using the 3D neutral hydrogel were conducted in static isotonic culture with ionic and non-ionic osmolytes added to the culture medium. Tissue production by bovine chondrocytes with non-ionic osmolytes was 1.9-fold greater than with ionic osmolytes, suggesting that the ionic nature of the osmolyte is an important regulator of tissue production. To investigate fixed negative charges, a 3D culture system containing encapsulated chondrocytes was employed based on a synthetic and neutral hydrogel platform within which negatively charged chondroitin sulfate was incorporated in a controlled manner. Incorporation of negative charges did not affect the mechanical properties of the hydrogel; however, intracellular ion concentration was elevated from the culture medium (330 mOsm) and estimated to be similar to that in ~400 mOsm culture medium. With dynamic loading, GAG synthesis decreased by 26% in neutral hydrogels cultured in 400mOsm medium, and increased by 26% in charged gels cultured in 330 mOsm. Treatment of chondrocyte-seeded hydrogels with the Ca(2+) chelator BAPTA-AM decreased GAG synthesis by 32-46% and was similar among all conditions, suggesting multiple roles for Ca(2+) mediated tissue production including with ionic osmolytes. In conclusion, findings from this study suggest that a dynamic ionic environment regulates tissue synthesis and points to [Ca(2+)]i signaling as a potential mediator.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, United States.
authors.forename: Nikki L
authors.initials: NL
authors.lastname: Farnsworth
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, United States.
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Mead
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, United States.
authors.forename: Lorena R
authors.initials: LR
authors.lastname: Antunez
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, CO, United States; BioFrontiers Institute, University of Colorado Boulder, Boulder, CO, United States.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Palmer
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, United States; BioFrontiers Institute, University of Colorado Boulder, Boulder, CO, United States; Material Science and Engineering Program, University of Colorado Boulder, Boulder, CO, United States. Electronic address: stephanie.bryant@coloradol.edu.
authors.forename: Stephanie J
authors.initials: SJ
authors.lastname: Bryant
mesh_terms: D001696:Biomechanical Phenomena; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D019902:Chondrocytes / Q000378:metabolism / Q000502:physiology*; D002809:Chondroitin Sulfates; D003094:Collagen / Q000378:metabolism; D005453:Fluorescence; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000378:metabolism*; D007150:Immunohistochemistry; D007424:Intracellular Fluid / Q000378:metabolism*; D007477:Ions / Q000378:metabolism*; D008689:Methacrylates; D009994:Osmolar Concentration; D011092:Polyethylene Glycols; D011509:Proteoglycans / Q000096:biosynthesis*
chemical_list: D007477:Ions; D008689:Methacrylates; D011509:Proteoglycans; C421283:poly(ethylene glycol)-dimethacrylate; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D011092:Polyethylene Glycols; D002809:Chondroitin Sulfates; D003094:Collagen; D002118:Calcium
keywords: Anabolic activity; Chondroitin-sulfate; Dynamic load; Osmolarity; PEG