Photocrosslinkable hyaluronan-gelatin hydrogels for two-step bioprinting

Abstract

Bioprinting by the codeposition of cells and biomaterials is constrained by the availability of printable materials. Herein we describe a novel macromonomer, a new two-step photocrosslinking strategy, and the use of a simple rapid prototyping system to print a proof-of-concept tubular construct. First, we synthesized the methacrylated ethanolamide derivative of gelatin (GE-MA). Second, partial photochemical cocrosslinking of GE-MA with methacrylated hyaluronic acid (HA-MA) gave an extrudable gel-like fluid. Third, the new HA-MA:GE-MA hydrogels were biocompatible, supporting cell attachment and proliferation of HepG2 C3A, Int-407, and NIH 3T3 cells in vitro. Moreover, hydrogels injected subcutaneously in nude mice produced no inflammatory response. Fourth, using the Fab@Home printing system, we printed a tubular tissue construct. The partially crosslinked hydrogels were extruded from a syringe into a designed base layer, and irradiated again to create a firmer structure. The computer-driven protocol was iterated to complete a cellularized tubular construct with a cell-free core and a cell-free structural halo. Cells encapsulated within this printed construct were viable in culture, and gradually remodeled the synthetic extracellular matrix environment to a naturally secreted extracellular matrix. This two-step photocrosslinkable biomaterial addresses an unmet need for printable hydrogels useful in tissue engineering.

FIG. 1.

(A) Chemical synthesis of hyaluronic acid (HA) (methacrylated HA [HA-MA]). (B) Chemical synthesis of gelatin ethanolamide methacrylate (GE-MA).

FIG. 2.

New hydrogels (HA-MA:GE-MA [4:1]) support cell growth and proliferation. HepG2 C3A, NIH 3T3, and Int-407 cells were encapsulated in either Extracel™ or the new hydrogels. Biocompatibility was determined using an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (see text for details); increased absorbance from day 0 to 3 and day 3 to 7 indicate proliferation of cells on both materials. *p < 0.05.

FIG. 3.

Cell-free hydrogels are biocompatible in vivo. Hydrogels were injected subcutaneously in nude mice and evaluated at 2 or 4 weeks. (A, B) Hematoxylin and eosin–stained images of HA-MA:GE-MA (4:1) gels. (C, D) Hematoxylin and eosin–stained images of Extracel. At 2 and 4 weeks, surrounding tissues show no signs of inflammation or other immune responses. In addition, after 4 weeks, some integration between the tissue and hydrogels is evident. D, dermis; H, hydrogel. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Stiffness increases with time of irradiation. The moduli G′ and G″ were determined as a function of the time of irradiation at 365 nm. On the basis of these data, 120 s was selected for the first irradiation period to obtain a gel-like fluid.

FIG. 5.

Bioprinting of the new hydrogel. (A) A stacked ring bioprinting procedure was used to build a tubular construct. (B) The dual syringe deposition tool of the Fab@Home Model 1 printer. (C) Bioprinted tissue constructs. The cell-containing ring contains fluorescing HA-BODIPY, whereas the clear cell-free hydrogel forms the core and an outer supporting halo. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Printed tissue construct after 3 weeks of culture. (A) Gross image of construct. (B) Masson Trichrome (dark blue, positive for collagen)–stained neotissue adjacent to cell-free lumen (light blue). Cell nuclei appear black. (C) Masson Trichrome–stained image of a HA-MA:GE-MA hydrogel, indicating that the blue stain in (B) is not due to gelatin, but to cell-secreted collagen. (D) Positive immunohistochemistry staining of procollagen (brown). (E) Positive immunohistochemistry staining of procollagen in murine dermal tissue sample as a positive control. Color images available online at www.liebertonline.com/ten.