Volume-by-volume bioprinting of chondrocytes-alginate bioinks in high temperature thermoplastic scaffolds for cartilage regeneration

Abstract

Biofabrication technologies with layer-by-layer simultaneous deposition of a polymeric matrix and cell-laden bioinks (also known as bioprinting) offer an alternative to conventional treatments to regenerate cartilage tissue. Thermoplastic polymers, like poly-lactic acid, are easy to print using fused deposition modeling, and the shape, mesh structure, biodegradation time, and stiffness can be easily controlled. Besides some of them being clinically approved, the high manufacturing temperatures used in bioprinting applications with these clinically available thermoplastics decrease cell viability. Geometric restriction prevents cell contact with the heated printed fibers, increasing cell viability but comprising the mechanical performance and biodegradation time of the printed parts. The objective of this study was to develop a novel volume-by-volume 3D-biofabrication process that divides the printed part into different volumes and injects the cells after each volume has been printed, once the temperature of the printed thermoplastic fibers has decreased. In order to show the suitability of this process, chondrocytes were isolated from osteoarthritic patient samples and after characterization were used to test the feasibility of the process. Human chondrocytes were bioprinted together with poly-lactic acid and apoptosis, proliferation and metabolic activity were analyzed. This novel volume-by-volume 3D-biofabrication procedure prints a mesh structure layer-by-layer with a high adhesion surface/volume ratio, driving a rapid decrease in the temperature, avoiding contact with cells in high temperature zones. In our study, chondrocytes survived the manufacturing process, with 90% of viability, 2 h after printing, and, after seven days in culture, chondrocytes proliferated and totally colonized the scaffold. The use of the volume-by-volume-based biofabrication process presented in this study shows valuable potential in the short-term development of bioprint-based clinical therapies for cartilage injuries. Impact statement: 3D bioprinting represents a novel advance in the area of regenerative biomedicine and tissue engineering for the treatment of different pathologies, among which are those related to cartilage. Currently, the use of different thermoplastic polymers, such as PLA or PCL, for bioprinting processes presents an important limitation: the high temperatures that are required for extrusion affect the cell viability and the final characteristics of the construct. In this work, we present a novel bioprinting process called volume-by-volume (VbV) that allows us to preserve cell viability after bioprinting. This procedure allows cell injection at a safe thermoplastic temperature, and also allows the cells to be deposited in the desired areas of the construct, without the limitations caused by high temperatures. The VbV process could make it easier to bring 3D bioprinting into the clinic, allowing the generation of tissue constructs with polymers that are currently approved for clinical use.