Faculteit Dierengeneeskunde
 
In the upcoming series of interviews with researchers of Utrecht University, Utrecht Medical Center or other ULS-partners, we want to give more insight into current developments in the replacement, reduction and refinement of laboratory animal experimentation within these research facilities.

September 2015

Does 3D-bioprinting pave the way for animal-free testing methods?


Nowadays, 3D-printing is a popular industrial method to produce geometric structures in rapid prototyping and manufacturing, as well as in consumer households to produce desired 3D-structures of jewelry and so on1. Researchers of the University Medical Center Utrecht and Utrecht University also apply 3D-printers to produce cartilage tissue constructs in order to repair damaged cartilage tissue in (eventually) the human body. “Be aware of the fact that this is far more complex than the “normal” 3D-printing process, because we use living cells”, says dr. Jos Malda, Associate Professor at the Department of Orthopedics of UMC Utrecht and the Faculty of Veterinary Medicine of Utrecht University. “When making these anatomically shaped constructs we have to deal with a complex interaction of chondrocytes, growth factors, matrix and/or biomaterials’’. Recently, Malda and colleagues developed reinforced hydrogels with a special 3D-printing technique, by printing multiple layers of microfibers and cells to improve the biological and mechanical properties of the construct2 (watch the YouTube video below). These microfibers function as support material, to improve the stiffness of the construct, and it approaches the characteristics of the natural articular cartilage tissue. In the mechanically demanding environment of articular tissue, it is important that the construct can respond to these mechanical loads. 

Replacement and reduction
What about the implementation of the 3R’s in Malda’s research? Malda and colleagues unfortunately still have to use animal models in the initial stage of their research, for several reasons. Rat experiments have shown the safety of the biomaterials in the past, as well as the efficacy of these biomaterials in repairing and stimulating cartilage tissue. But currently they are also using 3D-printed cell models and donor tissue to test the stability and mechanical properties of the materials. Finally, the customized cartilage tissue construct is being implanted in horses to evaluate the effectiveness of the customized cartilage tissue implant, which has been done recently in the research group of Malda. Dr. Malda: ‘’This is not only useful for the eventual implantation of the constructs in the human body, but gives also insights into articular tissue problems in horses. So we’re helping horses as well.”

And what about reducing the number of experimental animals? Dr. Malda: “We used 8 horses in our latest research to test the reinforced hydrogels with (or without) microfibers and we used several joints per animal to reduce the number of animals”.

Printed joint-in-a-dish
Although the research of Malda will not replace animal experiments completely, in the future it may be possible to apply the 3D-bioprinting technique to answer specific research questions, as a replacement method. The fabrication of 3D-constructs can lead to a printed “joint-in-a-dish” model, for example as an in-vitro method to test medicines for articular diseases like arthrosis. Above this, these 3D-constructs contribute to the reduction of animal experiments. At the moment Malda is also working together with the Department of Clinical Sciences of Companion Animals (with dr. Bart Spee) to produce 3D-printed liver constructs. In the future this can lead for example to an predictive in-vitro liver model for preclinical drug testing, that will be more efficient than an animal model.

Biofabrication facility
To help researchers in applying the 3D-bioprinting technique, Malda offers his 3D-printers to other researchers interested in developing 3D-tissue constructs within the Biofabrication Facility. This facility is a leading European knowledge center in the area of biofabrication, bringing together engineers, materials scientists, cell biologists, clinicians and commercial partners to create a fostering environment for the development, evaluation and clinical translation of 3D- tissue constructs.
The Biofabrication Facility could be the first step towards the development of complete organs in 3D. However, we wonder if it would be possible to print complete organs in 3D in the nearby future. “We are still very far away in achieving this. We are optimizing the production of liver cell patches or patches of cardiac muscles, for direct use in-vivo. We’re not using complete organs because we want to help the human body to stimulate the surrounding cells to repair damaged tissue itself’’.

In conclusion: Dr. Malda and colleagues take the 3R’s into account and are conducting important research towards the regeneration of articulating joints. Above this, their research could also lead to new advancements in the area of preclinical drug testing.

3D-bioprinting developments
Other developments in the area of 3D-bioprinting are based on the screening of drugs and vaccines or can lead to new in-vitro models of disease3. Companies are eager to find alternative testing methods because of the complete ban on animal testing for cosmetics. For example, L’oreal USA has announced a partnership with 3D-Bioprinting company Organovo Holdings, to develop 3D- printed skin tissue for product evaluation4. This could lead to new advancements in in-vitro methods for evaluating product safety and performance.
Organovo Holdings recently launched its initial product, a 3D-human liver tissue construct for use in Toxicology testing (exVive3D™ Liver), which retains liver function for at least 40 days. Other products are still in the developmental stage and planned for release at the end of 2016, like human kidney tissue. For more information, go to the website of Organovo.

1. Malone, E. & Lipson, H. Fab@ Home, the personal desktop fabricator kit. Rapid Prototyping J. 13, 245–255 (2007).
2. Visser J. et al., Reinforcement of hydrogels using three-dimensionally printed microfibers. Nature communications 6, article nr. 6933 (april 2015)
3. S.V. Murphy and A. Atala, 3D-bioprinting of tissues and organs, Nature Biotechnology 32, pp 773-785
4. Press release of partnership L’oreal USA with Organovo