(J-376) 3D Printing Lipid Laden Tissue for Adipose Tissue Engineering and Reconstructive Medicine

Introduction:: Subcutaneous adipose tissue is frequently damaged as the result of disease, trauma, or congenital defects. In 2020, more than 5.2 million tumor removals, 47,000 dog bites, and 68,000 birth defects resulted in reconstructive procedures [1]. Adipose tissue reconstruction is achieved by transplantation of either engineered acellular biomaterials or autologous fat grafting. However, these treatments have poor graft retention rates and fail to conformally fill the damaged region or recreate the desired adipose tissue microstructure. 3D printing adipose tissue is a potential therapeutic solution which can address these shortcomings by fabricating patient specific models with controlled microstructure from isolated adipocytes with high precision, reproducibility, and repeatability [2]. Patient specific anatomical models can be generated by converting standard medical imaging files from CT, MRI, or 3D ultrasound datasets into solid objects [3]. Tissue microstructure can be engineered by printing parameter optimization to create the optimal tissue porosity to promote nutrient diffusion and enhance the interface between the host and implanted tissue [4]. To date, 3D printing mature adipocytes has not been achieved due to their large size, high buoyancy, and fragility under shear stress. Here we demonstrate a novel approach to create a lipid laden tissue by first creating an adipocyte laden alginate bioink then fabricating engineered tissues via freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting.


Materials and Methods:: Mature adipocytes were isolated from primary human adipose tissue obtained from panniculectomies through blunt manual dissection followed by an enzymatic digestion using collagenase type I. The adipocytes were passed through a 350 µm sieve to remove ECM components that would clog the syringe. Isolated adipocytes were suspended in a 6% alginate hydrogel then centrifuged to create a high cell density bioink. FRESH 3D printing was performed on a LulzBot Mini fitted with a Replistruder v4 syringe pump extruder [5] using a standard CaCl₂ gelatin support bath [6]. A 10x10x5mm CAD model was generated on Fusion 360 and printed with a rectilinear infill pattern at 50% infill density. To evaluate adipocyte morphology, the constructs were stained with BODIPY (1:4000) and DAPI (1:500) and imaged on a Zeiss Confocal Microscope.


Results, Conclusions, and Discussions:: Due to the buoyancy of adipocytes, FRESH 3D bioprinting is an ideal fabrication method as the alginate bioink is crosslinked directly upon extrusion into the CaCl₂ gelatin support bath. The color of the construct (Fig 1A) is a combination of the clear alginate and orange adipocytes. The uniform color of the construct suggests a homogenous lipid distribution. The 10x10x5mm tissue printed with a 50% infill density demonstrates the precision of FRESH 3D printing with dimensional errors < 0.6%. Though adipocytes are susceptible to shear forces, BODIPY staining revealed that printed cells maintained their unilocular lipid-filled morphology during the fabrication process with a total yield of a 66.32% lipid density (Fig 1B). It is likely that increasing the infill density will increase the lipid density.

FRESH bioprinting was used to print adipocytes in an alginate bioink and maintained mature unilocular cells throughout the process. The novel 3D printing approach establishes a platform to create patient specific lipid laden scaffolds, making this methodology of great value for adipose tissue reconstructive medicine. Future studies will further optimize the FRESH bioprinting parameters, evaluate the viability and function of 3D printed adipocytes in culture using MTT assays, and transition to fabrication of patient specific models.


Acknowledgements (Optional): : This work was supported by the Robert Downey Jr Footprint Coalition’s Science Engine and crowd-sourced funding on the Experiment platform.


References (Optional): : 1) American Society of Plastic Surgeons. 2020. 2) Cui, H. Advanced Healthcare Materials. 2016. 3) Tan, B. Royal Society of Chemistry. 2021. 4) Behre, A. Advanced Healthcare Materials. 2022. 5) Tashman, J. HardwareX. 2021. 6) Lee, A. Science. 2019.