Dissertation Mid-session report:
Section Page Detail
Introduction An introduction to current science that is used in regenerative medicine. The need for specific personal medicines.- no need for transplants from others. Many die on the waiting list. No rejection, therefore no need to take immunosuppressants for years after transplantation.
Allows the production of highly heterogeneous tissue structures that is simply not possible with conventional tissue engineering (Campbell and Weiss, 2007).
A brief history of 3d bioprinting. Comparison with conventional 3D tissue generation using the support of scaffolds that are solid and porous (Hollister, 2005).
A paragraph explaining the paper.
Use of 3D printing not only to print the cells but to create scaffolds from which the cells can grow and can then be safely implanted into patients.
Bio-inks and modifications to the printer Introduction to biomaterials used in printing.
How do the printers work? There are a few main forms of actuation that allow the ink, cells, to be ejected from the cartilage (printer not physical). One is thermal, where an element is heated to around 300 degrees Celsius for around 2 microseconds to generate pulse pressure building mini bubbles of air that allow the ejection of ink drops of around 10-150 picolitres from the nozzle. (Canfield et al 1997). There are advantages and disadvantages to this form.
Adv- fast. The heat doesn’t kill the cells (Cui et al 2010). Cheap and almost ubiquitous amongst different cell types (Murphy and Atala, 2014).
Disadvantages- irregular sizing. Frequent clogging of the nozzle. Although they do not kill the cells, they do apply thermal stress which can affect the cell survival following printing.
Another form of ink ejection is through a piezoelectric system. Where voltage pulsation causes a constriction which generates pressure on the ink leading to its deposition (de Jong et al. 2006).
Adv- no heat exposure. Less clogging. Uniform droplets.
Disadvantages- voltage may cause damage to the cell membrane and cell lysis (Seetharam and Sharma, 1991).
As both mechanisms are not ideal, a further look into the components of the inks may contribute to overcoming these technical difficulties.
Moving away from inkjet there is the form of laser printing. As the name suggests a laser-transparent plate is placed within the machine which is lightly coated in a metal film and the biological material in liquid form. The focussed laser that is exposed which evaporates the film, allowing the liquid jet to deposit on the substrate in a liquid form. (Serra et al. 2009)
Advantages- high resolution with thin rows, less focussed when thickening the biological material.
Avoids clogging (Shaw-Stewart et al 2012). The maximum cell density of 10^8 cells per ml.
Disadvantage- for each cell type you want to print you need a different coated metallic film (Murphy et al 2013). This will make printing multi/ heterogenous materials more difficult.
It is slow, 100 drops per second, and will be even slower with a gelatin-based bio-ink (Guillotin and Guillemot, 2011).
There is also a chance of metallic contamination from the film and is expensive due to the metals being titanium or gold (Duocastella et al, 2010).
Stereolithography- the least popular form of 3D printing (S. Jana and A. Lerman 2015). This technique uses a liquid polymer which can be solidified upon illumination (Fisher et al. 2002)
Why are they needed: to provide an external support environment similar to the extracellular matrix. To allow smooth printing and not clogging up the nozzle.
Choice of viscosity.
There are three commonly used materials added into bio-inks, examples of collagen, gelatin, and alginate.
Collagen- abundant and easy to purify from animal and human tissue.
Collagen has cell adhesion domain sequences required for cell interactions.
Disadvantage- temperature sensitive. Easily degrades. 40/41
Solution- crosslinking- Yeo et al. 42
Gelatin- similar to collagen in that it is produced by the partial hydrolysis of collagen. Has high water absorption and low immunogenicity.
Problem- doesn’t retain shape. Collapses after a while.
Solution- can be used in the centre of scaffolds or printing products and will dissolve following incubation in medium creating a channel within which nutrients from the medium (mimicking the extracellular fluid) and oxygen can reach the inner cells of the graft allowing further proliferation of the cells and general cellular processes to take place as usual. 50. Further crosslinking may be possible to increase the strength of the ink
Alginate- from seaweed. Can become a gel in presence of divalent ions.
Problem- in the presence of monovalent ions such as sodium, which is commonly found in the extracellular fluid of cells, the crosslinking of the alginate molecules by the divalent ions are replaces which leads to a lack of the mechanical properties and therefore a lack of survival rate of the cells.
There are of course other biomaterials that can be added to the cell mixtures to provide appropriate bio-ink properties that are required for printing and contribute to the cell survival and development post printing. For example the use of recombinant spider silk proteins allowing adherence and proliferation of systems a week after printing.. 65
3D Bioprinted cartilage The collaboration of multiple attempts to print vascular materials.
What went well?
What could be improved?
Blood vessels and heart valves The collaboration of multiple attempts to print vascular materials.
What went well?
What could be improved?
1. Heart valve. By Hockaday et al. use of alginate based bio links. Use in porcine models showed nearly 100% viability over 21 days.
Future of bioprinting Discussion of the possible outcomes of science with this ability to print biomaterials for function in the body.
Also understanding the limitations of science. How we first need an understanding of the complex processes in order to replicate them.
Conclusion Summary of previous research and potential future ventures of this science, touching on the ethical perspectives as a thought-provoking ending.
Research into how much of the organ is required for function.
Skin grafting on 3D printed cartilage constructs in vivo.
The printed cartilage must be covered in skin that can grow on the surface of the construct.
Can integrated cartilage serve as a bed for a full-thickness skin graft?
Conclusions: A 3D bioprinted cartilage that has been allowed to integrate in-vivo is a sufficient base for a full-thickness skin graft. This finding accentuates the clinical potential of 3D bioprinting for reconstructive purposes.