生物技术与生物材料

抽象的

Biomaterials 2019: Volume-by-volume bioprinting of chondrocytes-alginate bioinks in high temperature thermoplastic scaffolds for cartilage regeneration- Adri�n Rodriguez- Universit� degli Studi di Cagliari

Adrián Rodriguez

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.

Bioprinting technologies have emerged as a powerful tool for tissue engineering (TE) due to the ability to mimic the 3D structure of any tissue. The use of biomaterials, cells, and biomolecules combined with this manufacturing technique is gaining increasing interest within the scientific community.1–3 There is a wide range of different bioprinting technologies available and the selection of an appropriate technology must be based on the characteristics of the tissue you want to regenerate.4 Joint cartilage is a non-vascular and non-innervated special connective tissue, composed of a specific extracellular matrix (ECM).5 The healing process of cartilage tissue is slow and results in a fibrous scar-like tissue that lacks the functional properties of the hyaline cartilage leading to further tissue degeneration.6 Current surgical treatments are not very effective, and this is motivating the development of new approaches. In this sense, TE and, in particular, bioprinting have emerged as a potential alternative.7 Additive manufacturing (AM) techniques are based on the principle of adding material layer-by-layer, allowing the manufacture of complex external and internal shapes with a mesh structure.8–10 The use of biopolymers for AM technologies has emerged during the last years.11,12 Parameters of the biopolymer deposition process, and the structure of the mesh, play an important role and can affect the structural, mechanical, biodegradation time, and cellular properties of AM scaffolds.13 Among different options, thermoplastic polymers can be easily printed using the 3D printing technology known as fused deposition modeling (FDM). This technology consists of a nozzle with a heater that melts a thermoplastic filament and deposits it in a controlled and organized manner, layer-by-layer, on a surface. After one layer has been printed, the z-axis moves and a new layer is printed.14 In this way, scaffolds are manufactured as a mesh, being rigid enough to ensure the maintenance of the required structure, and at the same time being flexible. The use of already clinically approved thermoplastic polymers presents a promising approach in treating cartilage defects.15,16 However, the high manufacturing temperatures of thermoplastic polymers decrease cell viability and also limit the scaffold geometry due to the need to avoid direct cell contact with the printed filaments. In order to solve these problems, low temperature biomaterials and novel printing procedures have been explored,17 but new biomaterials need a lot of time and many procedures before they can be approved for in-patient use. Therefore, new procedures based on currently approved biomaterials have to be investigated. The aim of this work was to implement a novel bioprinting method for high melting temperature thermoplastics, such as PLA, that allows high cell viability without shape and mesh restrictions. This method consists of a volume-by-volume (VbV) 3D-biofabrication process with an algorithm 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. This 3D-bioprinting method has the main objective of avoiding thermal damage to the cells. Moreover, it allows us to manufacture scaffolds with the desired biodegradation time and stiffness to enhance the performance and outcomes of the treatment. The biological feasibility of the printing process was assessed by printing bioinks of human chondrocytes together with PLA fibers and testing cell viability, distribution, and proliferatio.