(B-74) Hybrid Bioprinting for the Fabrication of Heterogeneous Interfaces

Introduction:: While great strides have been made in the field of tissue engineering thanks to advancements in three-dimensional (3D) bioprinting, there are still limitations in constructing complex tissue engineering constructs (TECs) that may require multiple biomaterials and graded loadings of biofactors. Various instruments have been used to print biomaterials of varying stiffness and viscosity, but to create TECs that better mimic natural tissue, the printing platform should be able to extrude rigid biomaterials to support structural loads, print soft hydrogels to carry biofactors, and deliver patterned biofactors to spatially control biochemical cues for tissue regeneration. Previously we introduced a hybrid printing system, Hybprinter, which integrates molten material extrusion (MME), syringe microextrusion (SBM) and stereolithography (SLA) for the fabrication of a complex TEC. This study further introduces acoustic droplet ejection (ADE) to 2^nd-generation system called Hybprinter-Acoustic (Hybprinter-A). The introduction of ADE allows for precise deposition of biological cues, leading to more native-mimicking TECs. We have constructed two different TEC models that showcase the range of materials and geometries that can be combined using Hybprinter-A.


Materials and Methods:: Hybprinter-A system and printing procedure: The system was composed of three modules, including MME, SBM, and ADE. MME was used to print thermal plastics such as PCL as the rigid support; SBM was used to print soft hydrogel material for cell loading; ADE was used to print biologics such as growth factors in droplet forms. A customized software was developed for the coordination of the three modules in a layer-by-layer fashion. During printing of a single layer, a layer of rigid material was first printed, followed by the printing of hydrogel in the same layer, followed by the deposition of growth factors onto specific spots. The procedure was repeated for the next layers.

Preparation of GelMA-based hydrogel: The proposed work features a novel hydrogel system developed in our lab, composed of GelMA and xanthan gum for the improvement of printability. Briefly, 20% GelMA mixture was mixed with different concentrations of xanthan gum with 0.6% Igracure 2599 as photo initiator. After printing, the hydrogel was UV-crosslinked under a 3.4 mW/cm² UV lamp for 1 min.

Cell study: Human MSCs were cultured in DMEM media. Upon printing, cells (10⁶/mL) were mixed in 20% GelMA before mixing with xanthan gum, and printed using the previously described protocol. After printing, the complex structure was molded into disc shape and cultured in cell medium. Calcein AM and Ethidium homodimer-1 were used for live and dead cell staining respectively.


Results, Conclusions, and Discussions:: RESULTS: Fig. 1 shows the schematic, working principle and logistics of the Hybprinter-A system. Specifically, MME and SBM allows for the fabrication of materials with mechanical gradient, while the ADE module deposits growth factors forming a biological gradient. We have developed a customized software allowing for automated control of all three modules, and 3D printed adaptors for mechanical integration of the three modules.

Fig. 2 shows demonstration of 3D printed scaffolds using Hybprinter-A. Fig. 2a shows bridging two PCL scaffolds with hydrogel scaffold, mimicking soft-rigid interface. Fig. 2b shows the deposition of graded biological cues, demonstrated by color, onto the soft-rigid interface. Fig. 2c shows a complementary pattern of soft and rigid materials, where the soft material fills the gap between the rigid material. We were able to precisely control the resolution to enable pores in the complementary scaffold, allowing for nutrient distribution. Fig. 2d shows both vertical and horizontal patterns implemented in a hydrogel scaffold. Fig. 2e shows multiple colors representing different growth factors patterned in a single complimentary scaffold, which demonstrates one great advantage of the ADE module which is the capability of patterning unprecedented number of different growth factors and growth factor combinations.

Fig. 3 shows combination of all components including PCL scaffold, hydrogel, and growth factors (demonstrated with food dye). Cell viability study was also carried out in a 7-day live/dead staining experiment (Fig. 3c-e). Results showed excellent cell viability after 7 days of culturing. The fluorescent results also indicated a seamless integration of hydrogel and PCL scaffold, as the cells along the interface (indicated by the red dashed line) showed good viability and even started to spread.

DISCUSSION: The newly developed Hybprinter-A system demonstrates unprecedented capability of fabricating complex TECs, by integrating different bioprinting techniques. Using the Hybprinter-A system as a manufacturing platform, we are able to create both mechanical and biological gradient at the same time. The results have shown good integration of all three components, as well as excellent cytocompatibility. Results also show great potential in engineering bone-soft tissue interfaces, which has been found challenging by any existing fabrication technology alone.


Acknowledgements (Optional): : This research was partially funded through financial support from DoD grant W81XWH-20-1-0343 and W81XWH-22-1-0189, NIH grants U01AR069395, R01AR072613, and R01AR074458 from NIAMS, Stanford MCHRI postdoctoral fellowship, the Stanford Woods Institute for the Environment, Boswell Foundation, and Tad and Diane Taube Family Foundation.


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