Evaluation of Hydrogels Based on Oxidized Hyaluronic Acid for Bioprinting

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].

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[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

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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 and water loss showed modulus the whereas highest gels storage formed and in loss DMEM modulus had the whereas lowest gels values. formed Additionally, in DMEM the had gel the in waterlowest exhibited values. Additionally, the shortestgelation the gel in time, water while exhibited it did notthe gelshortest at all gelation in DMEM. time, Since while DMEM it did contains not gel severalat all in amino DMEM. acids Since with DMEM free amine contains functions, several the amino formation acids of with Schiff free Base amine to hydrazone functions, may the be formation massively disturbedof Schiff Base by trimmedto hydrazone aldehydes may be via massively imine formations disturbed with by thetrimmed amino aldehydes acids instead via ofimine the cross-linkerformations ADHwith the [15 ].amino acids instead of the cross-linker ADH [15].

Figure 2. ((aa)) Viscosities Viscosities of of LMW-HA LMW-HA and and LMW-AHA LMW-AHA with with lowest lowest and and second second highest highest equivalents equivalents of ofNaIO NaIO4, determined4, determined at atincreasing increasing shear shear stress; stress; (b ()b )Viscosities Viscosities of of HMW-HA HMW-HA and and HMW-AHA HMW-AHA with

lowest and highest equivalents of NaIONaIO4,, determined determined at at increasing increasing shear stress; (c) Determination of the gelation pointpoint ofof gelgel withwith 3.5%3.5% (w(w//w) HMW-AHA-0.5HMW-AHA-0.5 andand 0.10%0.10% ((ww//w)) adipic acid dihydrazidedihydrazide using time sweep measurements; (d) Determination of gelation kinetics (storage (continuous line) and loss (dashed lines) modulus) of identical gel compositions withwith 3.5% (w/ww)) HMW-AHA-0.5 HMW-AHA-0.5 and and 0.20% 0.20% (w/ww)) ADH ADH mixed mixed in in different different solvent solvent (distilled (distilled water, water, PBS and DMEM). 2.3. Mechanical Properties 2.3. Mechanical Properties Conﬁned compression tests were performed to compare cast gels with HMW-AHA-2.0 containing Confined compression tests were performed to compare cast gels with HMW-AHA-2.0 various concentrations of cross-linkers, since not all gels could be fully recovered from the containing various concentrations of cross-linkers, since not all gels could be fully recovered from the 96-well-plates without destruction. In Figure3, representative samples are shown and best compression 96-well-plates without destruction. In Figure 3, representative samples are shown and best stability was achieved with 0.25% (w/w) ADH. With increasing concentrations of the cross-linker, compression stability was achieved with 0.25% (w/w) ADH. With increasing concentrations of the gels lose mechanical stability, since the aldehyde functionalities of AHA are saturated with ADH and cross-linker, gels lose mechanical stability, since the aldehyde functionalities of AHA are saturated are not able to form further stable cross-links between polymer chains. with ADH and are not able to form further stable cross-links between polymer chains.

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Figure 3. Average standard force for the confined compression measurements of gels containing 3.5% Figure 3. Average standard force for the confined conﬁned compre compressionssion measurements of gels containing 3.5% (w/w) of HMW-AHA-2.0 and various concentrations of ADH. Graph represents average of 4 (w/ww)) ofof HMW-AHA-2.0HMW-AHA-2.0 and and various various concentrations concentrations of ADH. of ADH. Graph representsGraph represents average ofaverage 4 individual of 4 individual samples and their standard deviation. samplesindividual and samples their standard and their deviation. standard deviation. 2.4. Swelling Studies 2.4. Swelling Studies Depending onon the the aqueous aqueous solutions solutions the the gels gels were were incubated incubated in, swelling in, swelling ratios largerratios andlarger smaller and Depending on the aqueous solutions the gels were incubated in, swelling ratios larger and thansmaller 1 were than obtained1 were obtained (Figure 4(Figure). Besides 4). Besides the different the different swelling swelling studies studies shown shown in Figure in Figure4, gels 4, were gels smaller than 1 were obtained (Figure 4). Besides the different swelling studies shown in Figure 4, gels alsowere allowed also allowed to swell to inswell pure in distilled pure distilled water. water. These dataThese are data not are shown, not shown, since the since gels the were gels already were were also allowed to swell in pure distilled water. These data are not shown, since the gels were dissolvedalready dissolved overnight. overnight. Since the Since Schiff the Base Schiff chemistry Base chemistry is reversible is inreversible water and in awater constant and equilibrium a constant already dissolved overnight. Since the Schiff Base chemistry is reversible in water and a constant ofequilibrium free and bound of free polymer and bound chains polyme is present,r chains this is effect present, leads this to aeffect permanent leads to loss a permanent of polymer loss chains. of equilibrium of free and bound polymer chains is present, this effect leads to a permanent loss of Hyperosmolarpolymer chains. solutions Hyperosmolar with high solution concentrations with high of concentration sodium ions tendof sodium to result ions in tend gel shrinkageto result in and gel polymer chains. Hyperosmolar solutions with high concentration of sodium ions tend to result in gel decelerateshrinkage and the degradationdecelerate the of degradation the gels, since of sodiumthe gels, ionssince also sodium may ions have also some may shielding have some effects shielding on the shrinkage and decelerate the degradation of the gels, since sodium ions also may have some shielding ioniceffects repulsion on the betweenionic repulsion the negatively between charged the carboxylic negatively groups charged of AHA. carboxylic Accordingly, groups the gelof inAHA. 1 M effects on the ionic repulsion between the negatively charged carboxylic groups of AHA. NaClAccordingly, was the longestthe gel in lasting 1 M NaCl up to was 424 h,the until longest it was lasting not possible up to 424 to weighh, until anymore. it was not In possible DMEM to and weigh PBS, Accordingly, the gel in 1 M NaCl was the longest lasting up to 424 h, until it was not possible to weigh theanymore. hydrogels In DMEM dissolved and muchPBS, the faster, hydrogels after two dissolve daysd for much DMEM faster, and after six daystwo days for PBS. for DMEM Both solutions, and six anymore. In DMEM and PBS, the hydrogels dissolved much faster, after two days for DMEM and six PBSdays andfor PBS. DMEM Both contain solutions, a plurality PBS and of DMEM salts and contain amino a acids,plurality which of salts might and interfere amino acids, with thewhich gel days for PBS. Both solutions, PBS and DMEM contain a plurality of salts and amino acids, which network.might interfere Especially with with the DMEM, gel netw aminoork. acidsEspecially will diffuse with DMEM, into the networkamino acids of AHA will and diffuse ADH, into causing the might interfere with the gel network. Especially with DMEM, amino acids will diffuse into the divergencenetwork of of AHA the polymer and ADH, chains causing and subsequent divergence gel breakdown.of the polymer For PBS,chains which and contains subsequent no amino gel network of AHA and ADH, causing divergence of the polymer chains and subsequent gel acid,breakdown. the degradation For PBS, iswhich most contains likely governed no amino by acid, the adjusted the degradation neutral pH, is most which likely is very governed likely toby alter the breakdown. For PBS, which contains no amino acid, the degradation is most likely governed by the theadjusted reaction neutral balance pH, of which the formed is very Schiff likely base. to alter the reaction balance of the formed Schiff base. adjusted neutral pH, which is very likely to alter the reaction balance of the formed Schiff base.

Figure 4. Swelling tests with gels in different solutionsolution media,media, containingcontaining 3.5%3.5% ((ww//w) HMW-AHA-2.0 Figure 4. Swelling tests with gels in different solution media, containing 3.5% (w/w) HMW-AHA-2.0 and 0.5% (w/ww)) ADH. ADH. and 0.5% (w/w) ADH.

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Gels 2018, 4, x FOR PEER REVIEW 5 of 8 2.5. Bioprinting 2.5. Bioprinting Based on determined viscosities of LMW-AHA and HMW-AHA and degrees of oxidation HMW-AHA-2.0Based on determined was chosen viscosities for the bioprinting of LMW-AH experiments.A and HMW-AHA Since HMW-AHA-2.0 and degrees shows of oxidation similar viscosityHMW-AHA-2.0 as LMW-AHA was chosen after for extensive the bioprinting degradation experiments. but has Si muchnce HMW-AHA-2.0 higher degree shows of oxidation, similar manyviscosity more as aldehydeLMW-AHA functions after extensive will be available degradation for cross-linking.but has much Additionally,higher degree ADH of oxidation, concentrations many formore cross-linking aldehyde functions were varied will and be areavailable listed infor Table cross-linking.1. Additionally, ADH concentrations for cross-linking were varied and are listed in Table 1. The gel compositionTable used 1. Composition for formulation of gels F1 and was set printed ups used using for bioprinting. a needle with a diameter of 0.25 mm at 3.4 bar. It exhibited the lowest shape fidelity and the strands began to lose their forms during HMW-AHA-2.0 Concentration ADH Concentration Needle Diameter Specimen Pressure in Bar the printing process (Figurein % 5a). (w/ wAfter) printing eighin %t layers, (w/w) the strandsin were mm no longer visible because of the lowF1 stability. The gel with 3.5 highest stability was 0.05 used for F2. Because 0.25 of the immense 3.4 stiffness, caused byF2 higher concentration 3.5 of ADH and conseque 1.0nt higher cross-linking 0.25 density, 2.0it could only be printedF3 with a very high 3.5pressure of 4.5 bar and 0.075 a needle diameter 0.41 of 0.41 mm, producing 4.5 eight and twelve layers (Figure 5b) with a brittle appearance of the construct. The best printing results were achievedThe gelwith composition F3 (Figure 5c). used The for pressure formulation used fo F1r printing was printed could using be reduced a needle to 2.0 with bar a and diameter a needle of 0.25diameter mm atof 3.40.25 bar. mm It was exhibited applied. the After lowest eight shape layers, ﬁdelity the construct and the showed strands smooth beganto strands lose their and formsshape duringfidelity. the Additionally, printing process a smaller (Figure distance5a). After between printing the eightstrands layers, was theachieved strands compared were no longerto Wang visible et al. becauseprinting offour the layers low stability. with 3 mm The between gel with the highest strands stability [12]. was used for F2. Because of the immense stiffness, caused by higher concentration of ADH and consequent higher cross-linking density, it could only be printed with a veryTable high 1. Composition pressure of of 4.5 gels bar and and set a ups needle used diameter for bioprinting. of 0.41 mm, producing eight and twelve layers (FigureHMW-AHA-2.05b) with a brittle appearanceADH Concentration of the construct. in Needle The best Diameter printing resultsPressure were in Specimen achieved with F3Concentration (Figure5c). in The % pressure(w/w) used for% printing (w/w) could be reducedin mm to 2.0 bar and aBar needle diameterF1 of 0.25 mm was applied.3.5 After eight layers, the 0.05 construct showed smooth0.25 strands and3.4 shape ﬁdelity.F2 Additionally, a smaller3.5 distance between the 1.0 strands was achieved 0.25 compared to Wang2.0 et al. printingF3 four layers with 33.5 mm between the strands [ 0.07512]. 0.41 4.5

Figure 5. All gel compositions are listed in Table 1 1;;( (a) F1 was printed with 3 mm spacing between the strands; (b) F2 was printed with 3 mm spacing between the strands; (c) F3, printed with 2 mm spacing between the strands. 3. Conclusions 3. Conclusions Here we evaluated reversibly cross-linked hydrogels based on oxidized HA and ADH for 3D printing. Here we evaluated reversibly cross-linked hydrogels based on oxidized HA and ADH for 3D Mechanical and swelling properties can vary greatly,depending on the gel composition. Three different gel printing. Mechanical and swelling properties can vary greatly, depending on the gel composition. compositions based on HMW-AHA-2.0 could be used for printing, since HMW-AHA-2.0 offers moderate Three different gel compositions based on HMW-AHA-2.0 could be used for printing, since HMW- viscosity and the highest aldehyde functionalization for cross-linking with various amounts of ADH. AHA-2.0 offers moderate viscosity and the highest aldehyde functionalization for cross-linking with The short term stability of the formed hydrogels in PBS and DMEM is a beneficial feature for their use as various amounts of ADH. The short term stability of the formed hydrogels in PBS and DMEM is a sacrificial support materials, which can be easily washed out using cell compatible medium, e.g., DMEM. beneficial feature for their use as sacrificial support materials, which can be easily washed out using Still, the significant degradation of HA during the oxidation is a common issue, which must be solved for cell compatible medium, e.g., DMEM. Still, the significant degradation of HA during the oxidation is a common issue, which must be solved for future studies by introducing aldehyde functionalities via other more gentle modifications. Alternatively, biocompatible reducing reagents can be considered

Gels 2018, 4, 82 6 of 8 future studies by introducing aldehyde functionalities via other more gentle modifications. Alternatively, biocompatible reducing reagents can be considered for reducing the reversible hydrazone bond to a stable hydrazide species, which will increase stability under cell culture conditions and eventually allow long term applications as bioink. Moreover, the use of specially designed multifunctional macromolecular cross-linkers bearing several hydrazide functions might further enhance stability of the hydrogels. All in all, the hyaluronic acid based hydrogels presented here are a promising platform for future development of new bioinks based on modified polysaccharides.

4. Materials and Methods

4.1. Materials and Reagents High (1.3 MDa) and low (50 kDa) molecular weight HA, abbreviated as HMW-HA and LMW-HA were purchased from Dagmar Kohler-BaccaraRose, Alpen, Germany. Adipic acid dihydrazide and 3-Methyl-2-benzothiazolinone hydrazone were acquired from Sigma-Aldrich, St. Louis, MO, USA. The following reagents were purchased from Merck KGaA, Darmstadt, Germany: Acetaldehyde, adipic acid dihydrazide (ADH), disodium phosphate dodecahydrate, potassium chloride, monopotassium phosphate, sodium chloride, sodium hydroxide, hydrochloride acid 32%. Calcium chloride dihydrate was acquired from Acros Organics, Geel, Belgium; Dulbecco’s Modiﬁed Eagle Medium (DMEM) from Invitrogen Life Technologies GmbH, Karlsruhe, Germany; iron(III) chloride from Alfa Aeser GmbH & Co. KG, Karlsruhe, Germany; ethylene glycol from Carl Roth GmbH & Co. KG, Karlsruhe, Germany; sodium periodate (NaIO4) from Thermo Fisher Scientiﬁc, Waltham, MA, USA; sulfamic acid from Grussing GmbH Analytica, Filsum, Germany.

4.2. Synthesis of AHA The oxidation of HMW-HA and LMW-HA was performed according to Su et al., with some modiﬁcations. Throughout this manuscript, the products are termed according to the molecular weight of HA used and the equivalents of oxidizing reagent used for the oxidation step of the disaccharide unit. AHA is used as an abbreviation of aldehyde containing HA. For example, LMW-AHA-0.5 was obtained from the oxidation of low molecular weight HA and 0.5 eq. of NaIO4. Various amounts (0.1, 0.2, 0.35, 0.5 eq. for LMW-HA, 0.5 and 2.0 for HMW-HA) of NaIO4 referred to one dimer unit of HA were used to generate different degrees of oxidation. The reaction process of LMW-AHA-0.5 is described as a representative example. In short, 2 g of LMW-HA was dissolved in 100 mL distilled water. A solution of 0.586 g NaIO4 in 15 mL distilled water was added slowly and the mixture was stirred at room temperature for 2 h. To stop the reaction, 2 mL ethylene glycol was added and the reaction stirred for 1 h. For puriﬁcation, the reaction mixture was dialyzed against distilled water for at least 3 days and lyophilized to obtain a white solid foam (Alpha 1–2 LD, Martin Christ Gefriertrocknungsanlage GmbH, Osterode, Germany). For synthesis with HMW-HA, the polysaccharide was dissolved in 160 mL instead of 100 mL to guarantee homogeneous solutions.

4.3. Characterization of AHA FTIR-spectrometry (Nicolet iS10 FT-IR, Thermo Fisher Scientiﬁc, Waltham, MA, USA) was used to prove the formed aldehyde functions. To determine the quantity of aldehyde functionalization, a MBTH-assay was performed. For this assay, a solution containing 1% (w/w) MBTH, a solution containing 1% (w/w) FeCl3, 1.6% (w/w) sulfamic acid and a solution with 0.01% (w/w) AHA were prepared. The MBTH solution was mixed with AHA solution in a ratio of 1:1 in a total volume of 400 µL. After 30 min 200 µL of the solution containing FeCl3 and sulfamic acid were added and chilled for another 10 min. The mixture was diluted to 1 mL and the absorption measured via UV/VIS-spectrometer (GENESYS 10 S Bio Spectrophotometer, Thermo Fisher Scientiﬁc, Waltham, MA, USA). Various concentrations (0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 10 µg/µL) of an acetaldehyde solution were treated using the same procedure for calibration. Gels 2018, 4, 82 7 of 8

4.4. Gelation with ADH For cross-linking reactions, stock solutions of ADH (50 mg/mL), LMW-AHA (150 mg/mL) and HMW-AHA (50 mg/mL) in distilled water were mixed to form gels with ﬁnal concentrations of 8.0, 9.0, 10.0, 11.0 and 12.0% (w/w) for LMW-AHA, 3.0, 3.5, 4.0 and 4.5% (w/w) for HMW-AHA and 0.050, 0.10, 0.25, 0.50, 0.75% (w/w) of ADH.

4.5. Rheology Rheological measurements were carried out with a rheometer Physica MCR 301 from Anton Paar GmbH (Graz, Austria). A cone-plate setting with 60 mm cone diameter and an opening-angle of 0.5◦ was used for rotation measurements with shear rates between 0.1–100.0 s−1. Also, the gelation point of various hydrogel formations was investigated through taking oscillation measurements at 4 ◦C with a frequency of 15.9 s−1 and a deformation of 1.0%.

4.6. Mechanical Tests Mechanical conﬁned compression tests were performed with a universal testing machine (Zwick/Roell Z010 from Zwick GmbH & Co. KG, Ulm, Germany), using a 100-N load cell and maximum penetration depth of 2 mm. Gels were formed in 96-well-plates with a diameter of 6.4 mm and height of 6.2 mm.

4.7. Swelling Properties For swelling properties, gels with a ﬁnal concentration of 3.5% (w/w) of HMW-AHA-2.0 and 0.5% (w/w) ADH were tested in distilled water, phosphate buffered saline (PBS), 1.0 M NaCl solution and DMEM. The gels were incubated at 37 ◦C with the solutions for 16, 41, 64, 88, 112, 136, 160, 208, 256, 328, 376 and 424 h, and weighed after those times. The swelling ratio q was calculated with m0 (weight of gels before swelling) and mx (weight of gels at time x) via the following equation: m q = 0 mx

4.8. Bioprinting Printing tests were performed with a bioplotter (3D Discovery Gen 1, RegenHU Ltd., Villaz-St-Pierre, Switzerland). Cannulas with a diameter of 0.25 and 0.41 mm and a pressure up to 4.5 bar were used for printing. For better adhesion of the printed constructs, the covers of microtiter plates were coated with a HA solution. The number of printed layers were 8 and 12. Gels with a final composition of 3.5% (w/w) HMW-AHA-2.0 with 0.05, 0.075 and 1.0% (w/w) ADH were used for printing.

Author Contributions: Conceptualization, J.T. and J.G.; Methodology, M.W., M.K. and T.J.; Validation, M.W.; Formal Analysis, M.W.; Investigation, M.W.; Resources, J.G.; Data Curation, M.W.; Writing—Original Draft Preparation, J.S.; Writing—Review & Editing, J.T. and J.G.; Visualization, M.W. and J.S.; Supervision, M.K., T.J.; Project Administration, M.W. and M.K.; Funding Acquisition, J.T. and J.G. Funding: This work was supported by the German Research Foundation (DFG) within the collaborative research center TRR225 (subproject A02). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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