© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4736 www.advmat.de www.MaterialsViews.com COMMUNICATION Adv. Mater. 2010, 22, 4736–4740 By Aleksander Skardal, Jianxing Zhang, Lindsi McCoard, Siam Oottamasathien, and Glenn D. Prestwich* Dynamically Crosslinked Gold Nanoparticle – Hyaluronan Hydrogels [∗] Dr. G. D. Prestwich Departments of Medicinal Chemistry and Bioengineering Center for Therapeutic Biomaterials The University of Utah 419 Wakara Way, Suite 205, Salt Lake City, UT 84108 (USA) E-mail: gprestwich@pharm.utah.edu A. Skardal Department of Bioengineering Center for Therapeutic Biomaterials The University of Utah 419 Wakara Way, Suite 205, Salt Lake City, UT 84108 (USA) Dr. J. Zhang, L. McCoard Center for Therapeutic Biomaterials The University of Utah 419 Wakara Way, Suite 205, Salt Lake City, UT 84108 (USA) Dr. S. Oottamasathien Department of Surgery Division of Pediatric Urology Center for Therapeutic Biomaterials The University of Utah 419 Wakara Way, Suite 205, Salt Lake City, UT 84108 (USA) DOI: 10.1002/adma.201001436 Bioprinting employs three-dimensional (3D) deposition of cells and biomaterials to create organized structures with organ- appropriate architecture. Such engineered organs could offer alternatives to inadequate donor organ supplies, [1,2] and bio- printed human tissues could improve predictability during pre- clinical evaluation of therapeutic agents. [3] However, scalability of bioprinting is limited by lack of extrudable, biocompatible mate- rials that can retain form, be remodeled by cells, be removed to create lumens, and offer layer-to-layer connectivity following assembly. To address these needs, we developed dynamically crosslinkable materials using gold nanoparticles (AuNPs) as multivalent crosslinkers. Specifically, 24 nm AuNPs and thiol- modified biomacromonomers derived from hyaluronic acid (HA) and gelatin were used to form printable semi-synthetic extracellular matrix (sECM) hydrogels. AuNP-sECMs are unique in having dynamic crosslinks; that is, both intra-gel and inter-gel covalent interactions can form and reform during and after printing. Moreover, AuNP-thiol crosslinking is reversible in the presence of benign thiols such as cysteine. In a proof-of- concept experiment, AuNP-sECMs were used to print tubular tissue constructs using an automated bioprinting system. In bioprinting, cells (the “bio-ink”) and hydrogels (the “bio- paper”) are deposited into precise 3D geometries by a 3-axis printer in a fashion enabling maturation into functional tis- sues. [4,5] Recently, cell aggregates and cell rods were printed into tubular assemblies that fused into seamless structures. [6,7] However, diffusion cannot readily occur in densely-packed cell aggregates lacking ECM, and tissue constructs exceeding 0.2–0.3 mm in diameter show reduced viability. One solution is to employ hybrid bio-inks, consisting of cells suspended in printable biomaterials. Biomaterials can be separated into two categories: monolithic, i.e., processible materials of fixed chemical composition, or living, i.e., materials that may be crosslinked into new forms in the presence of cells. [8] We now divide living biomaterials into statically crosslinked and dynam- ically crosslinked materials, and we illustrate the use of dynam- ically crosslinked AuNP-sECM biomaterials for bioprinting. Injectable sECMs suitable for regenerative medicine [8] and drug evaluation [9] employ in situ crosslinking of thiol-modified HA (CMHA-S) and thiol-modified gelatin (Gtn-DTPH) with polyethylene glycol diacrylate (PEGDA). The sECMs are com- mercially available as Extracel and HyStem for research use, and are in clinical use for veterinary medicine as the Sentrx line. Clinical products for human use are undergoing FDA review. However, these statically crosslinked sECMs were not suitable for bioprinting, as fully crosslinked gels frac- tured during printing and partially crosslinked gels clogged the printer. Despite the well-known thiophilicity of gold sur- faces, particularly in preparing self-assembled monolayers, [10] AuNPs have not been employed as structural components in hydrogels. By employing the unique size, multivalency, and thiophilicity of AuNPs to crosslink thiolated biomacromono- mers, printable, extrudable, cytocompatible and biodegrad- able hydrogels were produced. The reversible and dynamic crosslinking provided the increased experimental flexibility required for bioprinting. The AuNP hydrogels were prepared by first dissolving CMHA-S and Gtn-DTPH in sterile degassed water (2% w/v) at pH 7.4, followed by addition of a suspension of 24 nm AuNP (15:1 v:v). Gels formed within 24 h ( Figure 1A and Figure 1B). Using CMHA-S as the only macromolecular thiol afforded non-cell-adherent hydrogels, while inclusion of Gtn-DTPH (3:1 CMHA-S: Gtn-DTPH by volume) produced cell-adherent hydro- gels. In the absence of AuNPs, soft disulfide-crosslinked gels with a G ′, or storage modulus, of 10–20 Pa form. [11] First, to confirm biocompatibility, a LIVE/DEAD viability/ cytotoxicity assay and an MTS mitochondrial activity assay was performed on cells cultured on the surface of AuNP-sECM hydrogels. NIH 3T3, HepG2 C3A, and Int-407 cells showed >95% viability at day 3 and at day 7 (Supporting Informa- tion, Figure S1). Cell viability was not statistically different on PEGDA-crosslinked CMHA-S-Gtn-DTPH hydrogels. MTS absorbance data demonstrated significant increases in cell number (p < 0.01) from day 0 to 3 and day 3 to 7 within each sample set (Supporting Information, Figure S2).