Associate Professor New Jersey Institute of Technology Newark, New Jersey, United States
Introduction: Three-dimensional (3D) bioprinting offers a promising solution to address tissue and organ shortages for transplantation by fabricating functional tissues. However, successful tissue fabrication requires bioprinting strategies that replicate the biological and structural features of native tissue. While embedded printing approaches can create architecturally and biologically complex structures, traditional support baths lack relevant bioactivity and heterogeneity. In this study, we present a method to create dense cellular structures by depositing cell-only bioinks into photocurable support matrices. This approach can utilize multiple cell types and functional hydrogels, enabling the creation of highly heterogeneous and complex structures. In this study, our focus is on creating dense cellular structures within cell-instructive hydrogels with osteoinductive cues to spatially control stem cell osteogenesis, offering potential for bone tissue model and osteochondral tissue interface fabrication with relevant complexities.
Materials and
Methods: Bioprinting: Support matrix inks were prepared with 5 or 10 w/v% methacrylated hyaluronic acid (MeHA, functional support) in PBS containing 0.05% (w/v) photoinitiator, LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate). Cell-only bioinks were prepared by collecting respective cell type (NIH 3T3s), human umbilical vein endothelial cells (HUVECs) or human mesenchymal stem cells (hMSCs) into a 50-mL centrifuge tube and loading into 3 mL syringe.
Osteogenic differentiation studies: hMSCs (passage 4, Lonza) embedded within MeHA (5 or 10 w/v%) with and without tricalcium phosphate (TCP, 1 w/v%) or bone morphogenetic protein-2 (BMP-2, 100 ng/mL) functionalization were used for differentiation studies. Samples were cultured for 28 days in growth media and then 14 days in osteogenic induction media. Osteogenic differentiation of hMSCs were characterized through Alkaline Phosphatase (ALP) activity and Alizarin Red staining (ARS).
Results, Conclusions, and Discussions:
Results: In his study, a MeHA support matrix was printed layer-by-layer instead of using a bulk support bath. Each layer was partially crosslinked by a brief blue light exposure to provide sufficient mechanical strength to support the following layers. At the desired layer, a secondary cell-only ink (or a hydrogel or a cell-laden hydrogel ink) was printed within the viscous, uncured support layer prior to partial crosslinking. After deposition of the secondary ink, additional layers could be printed to form a large scale construct and the whole construct was fully cured via blue light exposure for 75 s. It was possible to fabricate structures at clinically relevant sizes with multiple cell aggregate layers (Figure 1Ai), which can be composed of different cell lines, and also embedded dense cell aggregates into functional supports printed as a strut or a matrix (Figures 1Aii&1Aiii). These results clearly show that, using this approach, dense cellular structures with high material and biological heterogeneity can be fabricated.
To show the potential of the developed method and importance of matrix functionalization, hMSCs embedded in biphasic MeHA matrix (Figure 1B) (with 5-10% MeHA, MeHA w/ and w/o TCP, and MeHA w/ and w/o BMP-2 regions) used for in vitro bone regeneration. hMSCs embedded in 5% MeHA (Figures 1Ci&Di), w/ TCP (Figures 1Cii&Dii), and w/ BMP-2 (Figures 1Ciii&Diii) regions showed significantly higher amount of calcium deposition, indicating spatial enhancement of osteogenesis in continues dense cell strands.
Conclusions and Ongoing Work: The developed layer-by-layer printing method with photocurable MeHA support matrix enables the fabrication of dense cellular structures with high heterogeneity. Matrix functionalization plays a crucial role in directing stem cell behavior, as evidenced by the spatial control of osteogenesis achieved within biphasic MeHA matrices. This approach holds promise for creating tissue-engineered constructs with tailored material properties and spatially controlled cellular responses, advancing applications in tissue interface engineering and regenerative medicine. Ongoing studies focus on spatial control of stem cell osteogenesis and chondrogenesis to create osteochondral tissue.
Acknowledgements (Optional): We acknowledge the funding from NSF/DMR - CAREER 2044479.
