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Biomanufacturing

Biomanufacturing - Poster Session C

Poster B2 - Integrating Photopatterning with 3D Bioprinting for Spatially Localizing Biomolecules

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Native tissues are spatially complex and heterogeneous, and are often characterized by the transition of biological cues such as growth factors and peptides in a gradient manner. 3D bioprinting techniques such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH) have enabled the fabrication of hierarchical tissue constructs, however, it is challenging to generate gradients with extrusion-based printing. Photopatterning is a promising tool for locally tailoring the biomolecular environment within the 3D bioprinted constructs. In photopatterning, light is used to initiate chemical reactions that cause selective binding of biomolecules in defined areas (Figure 1A). There have been numerous examples of growth factor and peptide patterning within scaffolds in the literature. However, the majority of these approaches are unable to replicate the multi-layered macro-scale complex 3D geometry of FRESH bioprinted constructs. For this reason, we propose to incorporate photopatterning with 3D bioprinting through a novel dual-head 3D bioprinting platform which would generate structurally intricate and spatial heterogeneous scaffolds. To demonstrate this capability, thiolated heparin was conjugated onto collagen-based scaffolds in order to enhance growth factor binding efficacy in specific locations. This was achieved via a thiol-norbornene click reaction initiated by UV light between thiol-heparin and norbornene-functionalized collagen (NorCol). NorCol was chosen as the main hydrogel of the bioink as collagen is the most abundant protein within the body and cells can be encapsulated within the NorCol unlike acidified collagen. Heparin was chosen as a model biomolecule due to its ability to spontaneously immobilize and prolong the half-life of growth factors without denaturing.

Materials and

Methods:  NorCol was synthesized by modification of neutralized collagen with carbic anhydride. Briefly, collagen was dissolved within acetic acid, and carbic anhydride was added to the dissolved collagen at pH 9. After two hours of reaction, the solution was dialyzed for one week and then freeze-dried. The degree of functionalization was determined using a 2,4,6-Trinitrobenzene sulfonic acid (TNBS) assay. Following the NorCol synthesis, the bioink was prepared by mixing 15 mg/ml NorCol with 1% sodium alginate and 0.5% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The FRESH support bath was generated through washing gelatin microspheres with thiolated-FITC-heparin. Our open-source 3D bioprinter was modified by assembling a fiber-optic light-pipe as a secondary printhead (Figure 1B). Light from an Omnicure S2000 UV light source was collimated, filtered, and then delivered from the tip of the fiber-optic cannula. After the printing process, scaffolds were washed with phosphate-buffered saline and imaged under a confocal microscope. Immobilization of heparin within each bioprinted layer was quantified using the FITC groups on the functionalized heparin. Scaffolds were also imaged by optical coherence tomography (OCT) to assess shape fidelity.

Results, Conclusions, and Discussions: In this study, a collagen-based scaffold was successfully photopatterned with heparin through a novel photoconjugation approach. In order to assess the functionality of the synthesized NorCol, we performed a degree of functionalization assay using 2,4,6-Trinitrobenzene sulfonic acid (TNBS) and assessed its photocrosslinkability using photorheology. TNBS assay results demonstrated that collagen was highly modified with norbornene with 54% degree of functionalization. Photorheology data further confirmed that the NorCol produced was photocrosslinkable (Figure 1C). OCT images of the scaffolds after the printing process indicated that structures were printed without any printing defects (Figure 1D). Confocal images demonstrated selective patterning of heparin within the scaffold only where the light was irradiated. In summary, the results reported herein highlight the potential of the proposed technology as a versatile bioconjugation strategy that combines the capability of FRESH printing technique with photopatterning. Future work will focus on the inclusion of stem cells into the bioink formulation and investigating their differentiation depending on the growth factors or other biomolecules that are patterned onto the scaffold through this approach.

Acknowledgements (Optional):