Construction of thick, functional tissues in vitro is a major challenge in tissue engineering. There are several important factors to consider in the process, one of the biggest ones being the physiological requirement of cells which is to receive a sufficient amount of nutrients.
An efficient way of transporting these nutrients to each individual cell must be achieved in every living construct. In the case of most animals, particularly the vertebrae, nutrients are transported through their bodies with blood through a vascular system.
In native tissues, the exchange of chemicals (gases, nutrients, waste products, etc.) between the cell and it’s surroundings occurs through either active transport by fluid flow or diffusion. The main difference between the two is that with the former, external energy is involved in nutrient transportation process, which enables the substances to travel in both directions, independently from the concentration levels in and outside of the cell. In diffusion, the nutrients can only travel from the place with a higher concentration of a particular nutrient to the lower. More basics on nutrient transportation are explained here.
Regardless of the transportation mechanics, it is crucial that the receiving cell is very close to the point of exchange (wall of a blood vessel). This way, nutrient exchange is only possible in very short spatial domains (up to 200 μm). This yields the fact that for the proper functioning of the tissue, sufficient vascularisation is essential. In essence, this means that sufficient branching of blood vessels is required, from tubes several cm thick into fine, several μm thin capillaries. The reconstruction of sufficiently perfusable vascular systems is still one of the greatest challenges in tissue engineering.
We have already demonstrated the production of perfusable scaffolds [1-4] by embedded printing in biocompatible matrices. Here we describe a further approach for the fabrication of dense tubular scaffolds using a continuous perfusable channel applied in a single step by core-shell printing, which was developed in collaboration with the Medical Faculty of the University of Maribor. Core-shell 3D printing is based on the conventional micro-extrusion printing of two (or more) materials through a coaxial die, with the “shell” material enclosing the “core” material. By extruding the (bio-)ink through the shell chamber and a cross-linking agent through the core chamber of the nozzle, a tube is formed and stabilised at the interface between the two materials. By continuous deposition, a framework can be formed consisting of a single, flow-through tube.
Prototyping the nozzle
At IRNAS we are experienced in providing custom solutions for various bioprinting applications. For core-shell bioprinting, we have developed a custom nozzle that can be fed with material from two extruders simultaneously, allowing precise control of flow and deposition. The nozzle had to meet the following requirements:
- Enable Luer-Lock connectivity
- stable attachment to at least one extrusion head must be possible
- good axial alignment between core and shell compartments must be ensured
- it must be reusable
- must be fabricated with accessible manufacturing technique(s)
The prototype was designed as a housing for the core nozzle (a G27 needle with blunt end) and as a shell housing with Luer-Lock connection. A schematic diagram of the prototype is shown in Figure 1 and can be assembled in a simple 3-step process:
- A G27 needle (with Luer-Lock connector) is manually inserted into the prototype and attached. As the needle is inserted, its tip is automatically aligned with the central axis of the nozzle. This is achieved by anchoring the needle in two positions along the axis, one at each end, which compensates for the usual needle eccentricity.
- By mounting the needle on a 5 ml syringe and inserting the said syringe into one of the extruders (extruder A), the complete core-shell nozzle is attached to the extruder and can be positioned like any other conventional nozzle.
- Finally, the shell chamber is connected to a syringe in the secondary extruder (extruder B) via Luer-Lock and silicone tubing. After the procedure, the assembled assembly is ready for use.
Building perfusable scaffolds
After developing the nozzle prototype, we tested its functionality using an alginate-based ink as the shell material and calcium chloride solution as the extruded cross-linking agent in the core area. In order to produce a stable tubular filament and deposit it in a perfusable framework with a desired shape, the extrusion speeds of both materials and the spatial translation in XYZ directions must be adjusted accordingly. The exact protocol for optimising the extrusion parameters for custom materials will be described in detail in a future blog.
Essentially, it is necessary to find the appropriate flow and deposition rates that lead to a stable and continuous production of the hollow filament and mainly depend on the viscoelastic properties of the (bio-)ink, the concentration of the cross-linking agent and the resulting reaction rate. If the process involves cells, time and environmental parameters must also be taken into account in order to achieve high viability. With the described design and optimised manufacturing parameters, woodpile scaffolds were produced which showed good blood circulation and were colonised with endothelial cells for tissue engineering of vascular tissue engineering or other tubular structures, such as nephrons. Fluorescence microscopic images showing living human umbilical vein endothelial cells (HUVEC) are presented in Figure 2. A detailed description of the methods and results of this collaboration was published in the Journal of Visualised Experiments [5].
We were glad to be able to efficiently translate our future-proof innovation and technological development into delicate applications such as tissue engineering. Our VITAPRINT platform enables us to rapidly deliver a custom solution to the end environment for quick validation and testing, which in this case yet again played a crucial role in the development process. We hope to be able to form many more technological partnerships in the future and accelerate the road to success for scientific and industrial researchers, through adaptation of advanced custom technology.
References
- Stumberger, G. and B. Vihar, Freeform Perfusable Microfluidics Embedded in Hydrogel Matrices. Materials (Basel), 2018. 11(12).
- Vihar, B. Freeform gelatin microfluidics. 2017; Available from: http://archive.irnas.eu/bio-lab-symbiolab/2017/08/07/freeform-gelatin-microfluidics.
- Vihar, B. Freeform Gelatin Microfluidics2 – Making a Meniscus. 2017; Available from: http://archive.irnas.eu/bio-lab-symbiolab/2017/09/04/freeform-gelatin-microfluidics2-making-a-meniscus.
- Vihar, B. Making earlobe shaped channels using Vitaprint. 2017; Available from: http://archive.irnas.eu/bio-lab-symbiolab/2017/11/29/making-earlobe-shaped-channels-using-vitaprint.
- Milojevic, M., et al., Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures. J Vis Exp, 2019(151).
About the author
Boštjan Vihar
Biomimetics and tissue engineering enthusiast. He develops 3D bioprinting protocols for custom materials and applications. He manages science administration, but loves tinkering in the lab and finding new ways of measuring biological phenomena.
