Large Scale Manufacturing of Precisely Patterned Flexible Soft Tissue Grafts with High Porosity

Introduction: : The nipple areolar complex (NAC) is an aesthetic landmark on the chest that is often neglected during breast reconstruction. Current clinical nipple reconstruction involves skin flap suturing, a technique in which skin is pulled and sutured to create a raised protrusion in the shape of a nipple that is limited by the loss of elevated projection within 12 months of surgery. Patients experience psychological, emotional, and physical stress coupled with dissatisfaction with current nipple reconstruction techniques due to the lack of clinically viable solutions. [1] Stereolithography 3D printing has the potential to create personalized NAC constructs with precisely positioned features down to 25 μm and physiologically relevant mechanical properties. [2] Printed constructs intended for implantation within the body or interaction with living materials must meet the conditions required for the growth and support of tissue within the matrix. In human tissues, cells must be within 200 μm of a blood vessel or nutrient supply to survive. [3] This minimum distance allows for gas exchange, waste removal, and nutrient delivery to support tissue survival and growth. When this minimum distance is not achieved for large scale 3D printed constructs, a necrotic core forms in the center of the tissue where cells begin to die due to a lack of nutrient supply. The design of porous networks within 3D printed scaffolds aids in delivering nutrients and removing waste to prevent the formation of a necrotic core.

Materials and

Methods: : The quantitative and qualitative analysis of the 3D printed constructs was performed through micro-CT imaging. A micro-CT scanner (SkyScan 1072, Bruker) was used to scan all samples. The images were acquired with a pixel size of 40.7 μm, voltage of 40 kV, and current of 200 μA. The obtained acquisition data was reconstructed (NRecon, Bruker) to provide axial cross sections for the total height of the samples. Strut integrity was measured through brightfield imaging capturing the bottom layer of 3D printed NACs with 0.4 and 0.5 mm strut diameter and 10 and 15% infill density.
Constructs were printed with Formlabs 4B printer and post-processed according to manufacturer’s recommendations. For cytotoxicity testing, 3D-printed disks were created using three different materials: Formlabs Biomed Durable, Flexible 80A, and Elastic 50A commercial resins. Prints were sterilized through serial dilutions of ethanol and phosphate buffered saline solution under UV for 30 mins. Consistent with ISO 10993-5, a monolayer of human mesenchymal stem cells were seeded into a culture dish at 30,000 cells/well and allowed to equilibrate to the culture vessel under incubation at 37 °C. After 24 hours, cell culture medium was removed and replaced with calcein AM and ethidium homidimer-1 for live/dead staining.
Compression testing was performed at room temperature on an Instron 5565 mechanical tester at a constant displacement rate of 1.3 mm/min, with a preload of 0.1 N (strain=0). Elastic modulus for each sample was calculated by determining the slope of the linear region of the stress-strain curve (strain region 3-5%).

Results, Conclusions, and Discussions:: Robustness of NAC internal geometry was assessed through MicroCT imaging to visualize pore area through the entire height of the construct. Physiologically relevant dimensions of 20 mm areola diameter, 10 mm nipple dimeter, and 12 mm nipple height were incorporated in print designs. Individual layers throughout the 12 mm nipple height were reproducibly printed and maintained integrity with identical layers in the z direction. Pore structures were also confirmed with brightfield imaging to assess the integrity of the struts on the inferior layer of the implants. Strut diameters were measured across 0.4 mm and 0.5 mm strut diameters printed with 10% and 15% infill density. Consistency in strut diameter was measured across each geometry to verify reproducibility.
Biocompatibility was also assessed through ISO 10993-5 testing for biological evaluation of medical devices through in vitro cytotoxicity. Results showed positive cellular response to biomaterials demonstrated by >90% viability across samples incubated with Formlabs Biomed Durable, Biomed Elastic 50A, Biomed Flexible 80A resins.
Mechanical properties were also assessed through compressive testing of samples. Capturing the mechanical properties of the tissue is critical for the NAC, as one of the primary concerns of patients that undergo nipple reconstruction focus on the desire for a natural looking appearance. [4] Soft tissues have a stiffness range between 0.1 and 500 kilopascals depending on the tissue of origin. [5] Compressive modulus results demonstrate that the 0.4 mm 10% infill NAC geometry exhibited a compressive modulus of 500 kilopascals, satisfying the requirement for soft tissue mechanics. By altering the strut diameter, infill density, infill pattern, and material type, we can reliably produce tissue mimics with a wide range of mechanical stiffness for soft tissue fabrication. Using a combination of additive manufacturing design software and prototyping methods, we introduce an innovative approach to enable the design and rapid prototyping of SLA printed flexible NACs and soft tissue implants with interconnected and structurally stable large pores.

Acknowledgements and/or References (Optional):: 1. Lovelace, D. L., McDaniel, L. R. & Golden, D. Long-Term Effects of Breast Cancer Surgery, Treatment, and Survivor Care. J. Midwifery Womens Health 64, 713–724 (2019).
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