The second part of this article detailed how today, vets use 3D printing (3DP) for various applications – including surgery rehearsal, the creation of endoprostheses and exoprostheses, the design of patient-specific surgical guides, and, finally, student and client education.
All of these applications stem directly from their counterparts in human medicine and will not be covered again in this article.
However, some of the most recent developments associated with 3DP have yet to infiltrate veterinary medicine and are still confined to the human field. One of these main applications is bioprinting.
This article – the third and final of this series – defines the concept of bioprinting and explores the latest developments of this technology in the field of human medicine.
Definition and industrial status
Bioprinting is an umbrella term representing any of the following processes:
- the printing of bioactive scaffolds impregnated with growth factors designed to stimulate tissue regeneration
- the printing of bioactive scaffolds impregnated with drugs that concentrate their activity in the microenvironment
- the printing of customised bioactive implants
- the printing of living tissue (Ballard et al, 2018)
In less than two decades, bioprinting-related research has increased substantially, with peer-reviewed publications pertaining to this field growing from 24 to 792 from 2000-15 (Rodriguez-Savador et al, 2017).
Over the past 27 years, the total market for additive manufacturing (3DP) has grown by 26.2% and was estimated to be worth US$5.1 billon (£4.1 billion) in 2015 (Liaw and Guvendiren, 2017). Within this market, the medical and health care sectors are estimated to account for nearly 16% of the total revenue (Liaw and Guvendiren, 2017).
Over the past decade, the increasing demand for – and accessibility to – 3DP-customised, patient-specific surgical guides, prostheses or other medical products has been the driving force behind this surge in the market. More recently, as the technology of 3DP continues to grow in ease of use and accessibility, 3D bioprinting has progressively become a more tangible reality.
The market potential of 3D bioprinting is expected to grow from US$411 million (£336 million) in 2016 to US$1,332.6 million (£1,091.2 million) by 2021 (MarketsandMarkets, 2017). This significant growth expectation is driven by several factors, including the potential for bioprinting to satisfy the demand for organ transplantation and tissue regeneration, the use of bioprinted tissues in drug testing and cosmetics testing, and a growing research and development landscape that increases public and private investments (MarketsandMarkets, 2017; Vijayavenkataraman et al, 2018).
Main impetus behind bioprinting: organ donor shortage and drug testing
Organ shortage is a major problem across the world. In the US alone, due to the opt-in system in place, more than 120,000 patients were waiting for an organ in 2016, with 48% of them waiting more than 24 months (Wu et al, 2017). What if printing entire host‑specific functional organs became possible?
Take a moment to also consider the possibility of using bioprinted tissues as in vitro models on which drugs are tested – eventually obliterating the use of animal testing and the inherent risks associated with trying drugs on human subjects for the first time.
While a considerable way to go still exists to reach this end result, significant progress has been made towards achieving this goal over the past five years.
Bioprinting could also offer a valid alternative when it comes to drug testing. It takes on average 12 years and US$2.6 billion (£2.1 billion) to develop a new drug (Sullivan, 2018). But beyond time and money, ethical concerns of animal testing also exist – not to mention some drugs successfully work on animals, but fail to work on humans for unforeseen reasons.
In 2006, eight volunteers took part in what has become known as the “Norwich Park” clinical trial. Six were given an IV immunomodulating drug that has successfully passed the animal testing phase, while two men received a placebo. All six men who received the tested compound developed multiorgan failure within hours of IV injection and required weeks of hospitalisation; all men survived (Caffrey, 2016).
Scaffolds versus ‘bioprinters’: same goal, different approach
Tissues can be engineered via 3DP in two main ways – tissue engineering (also known as indirect cell assembly) and direct cell assembly.
Tissue engineering/indirect cell assembly
Tissue engineering involves first forming a 3D scaffold out of a biocompatible material. These scaffolds are then modified during something called a “fabrication process” to meet specific needs depending on the target tissue/organ (that is, biodegradability, porosity, size, shape and so on). The scaffold is then either seeded with living cells and cultured in vitro in an incubator, or placed within the body, where living cells can infiltrate and populate it.
In both approaches, growth factors, hormones and chemical signals are key in triggering differentiation and functionality. Several scaffold fabrication methods exist, including electrospinning, phase separation, freeze‑drying, self-assembly, solvent casting, material injections and 3DP (commonly referred to as additive manufacturing; Ravichandran et al, 2012; Pedde et al, 2017).
3DP represents several advantages over more conventional fabrication methods, including the possibility of creating a layered scaffold made of multiple biomaterials, as well as creating complex geometries and graded macroscale architecture (Khatiwala et al, 2012; Do et al, 2015; Bajaj et al, 2014). The end result is the creation of a 3D milieu that best mimics a natural extracellular matrix and that will eventually be able to display signals critical for the determination of cellular fate (proliferation, differentiation, migration and so on).
Direct cell assembly
In the direct cell assembly method, both cells and materials are embedded together at the same time. Instead of printing a scaffold that will be seeded with cells, a mixture of cells and gel (extracellular matrix components) are printed directly together to control the spatial distribution of cells, and even realise in situ repairs (Yan et al, 2018).
Through computer-aided design software, the shape of the final construct is determined. A multinozzle printer then lays down in a specific order the different components of the tissue to be – that is, the different cell types and various components of the gel that represent the extracellular matrix.
The use of 3D printing enables the creation of a very controlled and specific architecture within the printed tissue, such that cell types can be printed in specifically repeatable patterns. This enables different cell types to share precise spatial relationships that mimic human tissues better than other models.
As such, bioprinting also offers greater reproducibility in experiments, for once the path and order in which the tissue is to be printed is established, several tissue samples can be printed following the exact same path.
Once the print is finished, it is placed into an incubator, which mimics the condition of the body, and provides the nutrients and oxygen necessary for the cells to survive, grow and begin forming a network. Using this specific workflow, the company Aspect Biosystems has managed to reproduce a segment of a bronchi that constricts under the presence of histamine in the same fashion it does in a living body – showing the possibility that, one day, drugs could be tested on tissue generated from human cells, and that actually “printing human tissues on demand” may not be that far off (Mohamed, 2017).
More recently, the same company has teamed up with the National Research Center of Canada in trying to reproduce the blood-brain barrier (BBB) via 3D printing. This collaboration stems from a research effort aiming at better understanding BBB dysfunction in patients with Alzheimer’s disease (Iftikhar, 2019).
Bioprinted tissues: a new market
Aspect Biosystems is not the only company aiming at providing 3D-printed tissues for drug research (and, eventually, organ replacement).
In 2015, the company Organovo announced its first 3D-printed human kidney tissue. In 2016, a team of researchers from Harvard University, in partnership with Roche Pharma, reported the bioprinting of human proximal tubules and embedded them in an extracellular matrix where they were maintained for more than two months.
The engineered 3D tubules exhibited significantly enhanced epithelial morphology and functional properties relative to the same cells grown on 2D controls (Homan et al, 2016). On introducing the nephrotoxin, ciclosporin A, the epithelial barrier was disrupted in a dose-dependent manner – once again showing promise in terms of the validity of 3D-printed tissues for future drug testing.
Similarly, the start-up Nano3D Biosciences enables the 3D printing of spheroids that mimic native tissue environments. Following a partnership with the Houston Methodist Research Institute, Nano3D Biosciences published a study showing it could construct 3D tissue models of breast tumours. Data from the study suggested this bioprinted in vitro breast tumour model was advantageous due to its ability to rapidly form “large” tumours (the tumour cell composition and density of which can be controlled) and accurately mimic the in vivo tumour microenvironment.
Last, but certainly not least, preliminary tests using chemotherapy drugs such as doxorubicin suggested these 3D models may better model actual tumour response to therapeutics than 2D cell cultures (Jaganathan et al, 2014).
Potential for localised therapeutics
Bioprinting can also be used for local drug delivery – the advantages of which are:
- limiting or even obliterating systemic toxicity
- superior extended release compared to currently available compounds with a superficial drug-eluting coating (Ballard et al, 2018)
In the US, the Food and Drug Administration approved a 3D-printed form of levetiracetam (Ballard et al, 2018). In 2017, a team from the Washington University School of Medicine published a proof of concept in vitro study that showed the feasibility of printing patient‑specific hernia meshes made of gentamicin polylactic filament (Ballard et al, 2017).
Such 3D-printed meshes impregnated with antibiotics theoretically present several advantages over conventional ones – mainly:
- being patient tailored with predetermined sizes (eliminating material waste and reducing time in the operating room)
- potentially eliminating complications associated with IV antibiosis by enabling the focal delivery of antibiotics (Ballard et al, 2017).
Future directions: hormone therapy and chemotherapy
If most attention is currently focused on the potential use of 3DP in enhancing drug delivery (mainly antibiotics), the same principles can be applied to the delivery of hormones and other agents.
A 2017 study showed the feasibility of generating functional hormonal tissue based on a 3D scaffold. Laronda et al (2017) created a 3D-printed scaffold that mimicked ovarian tissue and seeded these with ovarian follicles at different stages of maturity before implementing them in mice with bilateral oophorectomy.
The 3D printed constructs demonstrated folliculogenesis and tissue growth, showing promises that this technology may one day be able to help the female population with diminished ovarian function, including the inability to go through puberty, early menopause and infertility (Laronda et al, 2017).
In another study, Tappa et al (2017) demonstrated the feasibility of 3D printing customisable and biodegradable oestrogen and progesterone-eluting constructs. As with the work of Laronda et al, the findings of this latter study hold promise in potentially generating patients’ specific intrauterine devices, pessaries or hormone therapy devices that would be able to release a tailored hormone dosage over a tailored period of time (Tappa et al, 2017).
Finally, building on all the recent developments that took place in the field of bioprinting, applications to the field of oncology in the future do seem possible. In time, patient tailored targeted chemotherapeutic implants could well become a possibility.
Conclusion
In conclusion, bioprinting is a relatively new, but rapidly progressing, field that holds considerable promises for the future. While most of the research presented in this article is still at its very early stages, it is not unreasonable to imagine a future where some of the bioprinting applications described here become tangible to the world of veterinary medicine.
from Vet Times
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