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
]. 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
]. 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
]. With this strategy, they created two multifunctional and macromolecular cross-linking partners for a stable but still reversible hydrogel. Su et al. also cross-linked AHA with ADH, forming an injectable hydrogel for nucleus pulposus regeneration [13
In this study, we investigated oxidized hyaluronic acid (AHA), synthesized from low (LMW) and high molecular weight (HMW) HA with different oxidation degrees, for its hydrogel formation as well as suitability for bioprinting with ADH as low molecular weight bifunctional cross-linker. Since the Schiff Base system using both hydrazide functions of the small ADH leads to a chemically cross-linked network in dynamic equilibrium, we expected tunable printability of the obtained polysaccharide hydrogels by varying the cross-linker concentration.
Here we evaluated reversibly cross-linked hydrogels based on oxidized HA and ADH for 3D printing. Mechanical and swelling properties can vary greatly, depending on the gel composition. Three different gel compositions based on HMW-AHA-2.0 could be used for printing, since HMW-AHA-2.0 offers moderate viscosity and the highest aldehyde functionalization for cross-linking with various amounts of ADH. The short term stability of the formed hydrogels in PBS and DMEM is a beneficial feature for their use as sacrificial support materials, which can be easily washed out using 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 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.
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.
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
) of HMW-AHA-2.0 and 0.5% (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:
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.