title: Wide-range stiffness gradient PVA/HA hydrogel to investigate stem cell differentiation behavior.
abstract: Although stiffness-controllable substrates have been developed to investigate the effect of stiffness on cell behavior and function, the use of separate substrates with different degrees of stiffness, substrates with a narrow range stiffness gradient, toxicity of residues, different surface composition, complex fabrication procedures/devices, and low cell adhesion are still considered as hurdles of conventional techniques. In this study, a cylindrical polyvinyl alcohol (PVA)/hyaluronic acid (HA) hydrogel with a wide-range stiffness gradient (between ∼20kPa and ∼200kPa) and cell adhesiveness was prepared by a liquid nitrogen (LN2)-contacting gradual freezing-thawing method that does not use any additives or specific devices to produce the stiffness gradient hydrogel. From an in vitro cell culture using the stiffness gradient PVA/HA hydrogel, it was observed that human bone marrow mesenchymal stem cells have favorable stiffness ranges for induction of differentiation into specific cell types (∼20kPa for nerve cell, ∼40kPa for muscle cell, ∼80kPa for chondrocyte, and ∼190kPa for osteoblast). The PVA/HA hydrogel with a wide range of stiffness spectrum can be a useful tool for basic studies related with the stem cell differentiation, cell reprogramming, cell migration, and tissue regeneration in terms of substrate stiffness.
  
  
  CONCLUSIONS
  It is postulated that the stiffness of the extracellular matrix influences cell behavior. To prove this concept, various techniques to prepare substrates with a stiffness gradient have been developed. However, the narrow ranges of stiffness gradient and complex fabrication procedures/devices are still remained as limitations. Herein, we develop a substrate (hydrogel) with a wide-range stiffness gradient using a gradual freezing-thawing method which does not need specific devices to produce a stiffness gradient hydrogel. From cell culture experiments using the hydrogel, it is observed that human bone marrow mesenchymal stem cells have favorable stiffness ranges for induction of differentiation into specific cell types (∼20kPa for nerve, ∼40kPa for muscle, ∼80kPa for cartilage, and ∼190kPa for bone in our hydrogel system).
authors.affiliation: Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 330-714, Republic of Korea.
authors.forename: Se Heang
authors.initials: SH
authors.lastname: Oh
authors.affiliation: Department of Advanced Materials, Hannam University, Daejeon 305-811, Republic of Korea.
authors.forename: Dan Bi
authors.initials: DB
authors.lastname: An
authors.affiliation: Department of Advanced Materials, Hannam University, Daejeon 305-811, Republic of Korea.
authors.forename: Tae Ho
authors.initials: TH
authors.lastname: Kim
authors.affiliation: Department of Advanced Materials, Hannam University, Daejeon 305-811, Republic of Korea. Electronic address: jhlee@hnu.kr.
authors.forename: Jin Ho
authors.initials: JH
authors.lastname: Lee
mesh_terms: D000328:Adult; D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003460:Crystallization; D004247:DNA / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008875:Middle Aged; D011142:Polyvinyl Alcohol / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: C062364:polyvinyl alcohol hydrogel; D011142:Polyvinyl Alcohol; D006820:Hyaluronic Acid; D004247:DNA
keywords: Differentiation; Hyaluronic acid; Polyvinyl alcohol; Stem cell; Stiffness gradient