gels Communication Evaluation of Hydrogels Based on Oxidized Hyaluronic Acid for Bioprinting Matthias Weis, Junwen Shan, Matthias Kuhlmann, Tomasz Jungst, Jörg Tessmar and Jürgen Groll * Department for Functional Materials in Medicine and Dentistry, University of Würzburg, Pleicherwall 2, 97070 Würzburg, Germany; [email protected] (M.W.); [email protected] (J.S.); [email protected] (M.K.); [email protected] (T.J.); [email protected] (J.T.) * Correspondence: [email protected]; Tel.: +49-931-20173610 Received: 15 August 2018; Accepted: 27 September 2018; Published: 9 October 2018 Abstract: In this study, we evaluate hydrogels based on oxidized hyaluronic acid, cross-linked with adipic acid dihydrazide, for their suitability as bioinks for 3D bioprinting. Aldehyde containing hyaluronic acid (AHA) is synthesized and cross-linked via Schiff Base chemistry with bifunctional adipic acid dihydrazide (ADH) to form a mechanically stable hydrogel with good printability. Mechanical and rheological properties of the printed and casted hydrogels are tunable depending on the concentrations of AHA and ADH cross-linkers. Keywords: biofabrication; bioprinting; hyaluronic acid 1. Introduction Hydrogels based on polysaccharides have attracted a high level of attention in recent decades, especially in the fields of Tissue Engineering and Regenerative Medicine [1–4]. The polysaccharides used for these applications are biopolymers, which are chosen in order to mimic the structural support of the native extracellular matrix and allow the three dimensional growth and proliferation of incorporated cells [5,6]. Hyaluronic acid, as one prominent example, is a polysaccharide which is an exisiting component of different extracellular matrices and therefore quite promising as cell carrier for several in-vitro approaches [7]. However, in many cases the natural polysaccharides need to be specially functionalized using chemically active groups for cross-linking reactions to improve the mechanical stability and ensure the shape fidelity of the newly formed hydrogel constructs [8]. In our group, thiol-ene chemistry has been used successfully to develop a hydrogel system based on thiol-functionalized hyaluronic acid together with allyl-functionalized poly(glycidol)s, which can be cast as a cell carrier hydrogel and bioprinted, allowing a generation of better defined 3D hydrogel constructs [9]. The group of Boccaccini introduced an oxidized alginate/gelatin-based hydrogel system for bioprinting. The aldehyde functionalities of the oxidized alginate were used for secondary cross-linking with the amino functions of gelatin using Schiff Base chemistry [10]. Without subsequent reduction, the Schiff Base chemistry provides a reversible reaction of an aldehyde with an amine, followed by elimination of water to form the final imine, which is however still labile to hydrolysis. Special amine derivatives, such as hydrazide or aminooxy groups, lead to much greater hydrolytic stability via formation of hydrazones or oximes [11], which might be interesting for the long term application of the hydrogels. The hydrazone system, as one example, has been published recently by the group of Burdick, which used an excess of adipic acid dihydrazide (ADH) to covalently functionalize acid functions of hyaluronic acid (HA) using carbodiimide chemistry as multiple hydrazide species and combined this polymer with oxidized HA (AHA) as aldehyde species [12]. Gels 2018, 4, 82; doi:10.3390/gels4040082 www.mdpi.com/journal/gels Gels 2018, 4, 82 2 of 8 Gels 2018, 4, x FOR PEER REVIEW 2 of 8 [12].With With this strategy,this strategy, they createdthey created two multifunctionaltwo multifunctional and macromolecularand macromolecular cross-linking cross-linking partners partners for a forstable a stable but still but reversible still reversible hydrogel. hydrogel. Su et al. Su also et cross-linkedal. also cross-linked AHA with AHA ADH, with forming ADH, anforming injectable an injectablehydrogel forhydrogel nucleus for pulposus nucleus regenerationpulposus regeneration [13]. [13]. InIn this study,study, wewe investigated investigated oxidized oxidized hyaluronic hyaluronic acid acid (AHA), (AHA), synthesized synthesized from from low low (LMW) (LMW) and andhigh high molecular molecular weight weight (HMW) (HMW) HA with HA differentwith different oxidation oxidation degrees, degrees, for its hydrogelfor its hydrogel formation formation as well as suitabilitywell as suitability for bioprinting for bioprinting with ADH with as ADH low molecular as low molecular weight bifunctional weight bifunctional cross-linker. cross-linker. Since the SinceSchiff the Base Schiff system Base using system both using hydrazide both hydrazide functions of functions the small of ADH the small leads ADH to a chemically leads to a cross-linked chemically cross-linkednetwork in dynamicnetwork equilibrium,in dynamic weequilibrium, expected tunablewe expected printability tunable of theprintability obtained of polysaccharide the obtained polysaccharidehydrogels by varying hydrogels the cross-linkerby varying the concentration. cross-linker concentration. 2. Results Results and and Discussion Discussion 2.1. AHA AHA Synthesis Synthesis and and Characterization The oxidation of HA was performed using NaIONaIO4 introducing dialdehyde functionalities in in several HA dimer units, units, resulting resulting in in a a simultaneous simultaneous ring ring opening opening of of the the glucuronic glucuronic acid acid [14]. [14]. Aldehyde formationformation was was proven proven by Fourier-transformby Fourier-transform infrared infrared (FTIR) (FTIR) spectroscopy spectroscopy with the with apparent the − apparentpresence ofpresence a shoulder of ata 1730shoulder cm 1atbeside 1730 thecm fingerprint−1 beside the area fingerprint (Figure1a) [area13]. (Figure Quantification 1a) [13]. of Quantificationthe aldehyde groups of the was aldehyde performed groups using awas 3-methyl-2-benzothiazolinone performed using a 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrazoneassay (MBTH-assay). hydrochloride As expected, assay (MBTH-assay). the degree of As oxidation expected, (DO) the increased degree of with oxidation increasing (DO) amounts increased of withoxidation increasing reagent amounts used for reaction.of oxidation By varying reagent the us amountsed for reaction. of the oxidation By varying reagent, the we amounts achieved of a DOthe oxidationbetween 1.4% reagent, and 8.0%we achieved for LMW-AHA a DO between and 4.6% 1.4% and and 15.6% 8.0% for for HMW-AHA LMW-AHA (Figure and 4.6%1b), whichand 15.6% proves for HMW-AHAgood control (Figure over the 1b), amount which of proves available good aldehyde control ov functions.er the amount of available aldehyde functions. Figure 1. ((aa)) IR-spectra IR-spectra of of native hyaluronic acid (HA) (black) and low molecular weight oxidized −1 hyaluronic acid (LMW-AHA-0.5) (orange) with a zoom to thethe shouldershoulder atat 17301730 cmcm−1; ;((bb)) Average Average of degree of oxidation (DO) according to equivalents of NaIO4 used, referred to one dimer unit of HA. 2.2. Rheology 2.2. Rheology The oxidation of HA, however, was accompanied with a significant decrease in viscosity of the The oxidation of HA, however, was accompanied with a significant decrease in viscosity of the pure AHA solutions (5% w/w), indicating a progressing degradation of HA chains during oxidation. pure AHA solutions (5% w/w), indicating a progressing degradation of HA chains during oxidation. Depending on the amount of NaIO4 used, viscosity of LMW-AHA varied between 0.01 to 0.06 Pa·s, Depending on the amount of NaIO4 used, viscosity of LMW-AHA varied between 0.01 to 0.06 Pa∙s, and higher amounts of oxidizing reagent tended to result in lower viscosity and consequently stronger and higher amounts of oxidizing reagent tended to result in lower viscosity and consequently degradation (Figure2a). This was observable for both LMW-AHA and HMW-AHA (Figure2b), stronger degradation (Figure 2a). This was observable for both LMW-AHA and HMW-AHA (Figure whereas the highest oxidized HMW-AHA showed viscosity in a similar range as LMW-AHA. 2b), whereas the highest oxidized HMW-AHA showed viscosity in a similar range as LMW-AHA. The gelation and hydrogel formation of a representative sample of 3.5% (w/w) HMW-AHA-0.5 The gelation and hydrogel formation of a representative sample of 3.5% (w/w) HMW-AHA-0.5 together with 0.10% (w/w) ADH cross-linker is shown in Figure2c. For this combination, we determined a together with 0.10% (w/w) ADH cross-linker is shown in Figure 2c. For this combination, we determined a very short gelation time of less than 1 min, but it was still long enough to allow mixing Gels 2018, 4, 82 3 of 8 Gels 2018, 4, x FOR PEER REVIEW 3 of 8 very short gelation time of less than 1 min, but it was still long enough to allow mixing of cells. In addition toof cells. the polymers In addition in pureto the water, polymers we also in pure measured water, the we gelationalso measured time of the gels gelation in other time aqueous of gels solutions. in other Toaqueous improve solutions. cell compatibility, To improve we comparedcell compatibilit demineralizedy, we compared water, phosphate demineralized buffered water, saline phosphate (PBS) and Dulbecco’sbuffered saline Modified (PBS) Eagle and MediumDulbecco’s (DMEM) Modified (Figure Ea2gled). TheMedium gels in (DMEM) distilled water(Figure showed 2d). The the highestgels in storagedistilled