Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

 

In recent years, three-dimensional (3D) bioprinting technology has become more accessible to researchers, allowing the technique to become more widely utilized for fabrication of tissue analogs. Bioprinting promises to revolutionize biomedical research by facilitating the rapid and repeatable fabrication of multifaceted tissue constructs. The crux of the bioprinting technology lays in the ability to precisely control the deposition of biomaterials (known as bioinks) in three dimensions. This allows the generation of complex scaffolds with distinct regions of matrix compositions, bioactive factors, and cells that can more accurately recapitulate native tissue structure.

Bioprinting has been utilized for the fabrication of constructs for many tissue applications including cartilage1, skin2, muscle3, and bone4. These tissues are attractive for bioprinting due to their intrinsic striated micro-architectures that are suitable for recapitulation via layer-by-layer deposition. In particular, skin possesses a well-defined multilayered structure5, which is suitable for fabrication through layer-by-layer deposition techniques such as bioprinting. Additionally, bioprinting can be utilized to generate constructs that possess the necessary anatomical dimensions and shapes to repair the tissue defect. The ability to generate biomaterials with patient-specific size and shape6 can begin to address the demand for partial repairs of many tissues including but not limited to bone defects, cartilage damage, and skin lesions whose extent varies from patient-to-patient.

In this study, two tissue analogs (articular cartilage and skin) were fabricated through the bioprinting of pre-cellularized bioinks. Ensuring adequate blending of a bioink with cell suspension that can ensure uniform cell distribution while preserving cell viability can be a challenge. Bioinks suitable for bioprinting via extrusion are often highly viscous and therefore require extensive mixing to ensure a homogenous blend. Mechanical damage to cells can occur under harsh mixing conditions and negatively affect viability. Studies have shown that most cell death during the inkjet printing process occurs during preparation such as mixing7,8. While traditional mixing with agitation9 may be sufficient for low viscosity bioinks suitable for inkjet printing10, mixing of cells into a high viscosity bioink more suitable for extrusion bioprinting is more difficult. Addressing this need, the use of mixing nozzles has become more popular for the blending of bioinks during the printing process11. These mixers have also been widely utilized in microfluidics research where the mixing of fluids with low Reynolds number is important12. The utilization of a continuous mixing process to blend a cell suspension into a bioink would allow for uniformity during the printing process. However, since cell suspensions possess low viscosity compared to a bioink, difficulties will arise in preventing sedimentation of the cells during the printing process9,13,14. Alternatively, the mixing of cells into a bioink prior to printing may address this issue.

To minimize cell death during blending into a bioink, we developed a technique based on a passive mixing unit to blend cells into a bioink in the minimal number of steps. The chaotic mixing generated through the flow of the materials through the mixing unit is sufficient to reproducibly blend two components together15,16. This method was primarily developed to simplify the blending of any cell suspension with any bioink that suitable for extrusion bioprinting. The number of steps in the mixing process was minimized to eliminate user-to-user variation in mixing. Excessive mixing steps can be time consuming and not applicable to all bioinks, particularly when cells are involved. Secondary, we aimed to develop a mixing process that was self-contained to both preserve sterility and minimize sample loss.

In this manuscript, we demonstrate the blending of a cell suspension with a bioink using a passive mixing unit technique that minimizes handling and results in high cell viability and uniform distribution. These pre-cellularized bioinks are then utilized to bioprint either a cartilage or skin construct with one or two cell types, respectively that are cultured for up to 6 weeks. The bioink utilized is an alginate-nanocellulose blend, which previously has shown suitability for bioprinting1.