This proposal is submitted by Italian Regenerative Medicine Infrastructure (IRMI), an infrastructure organised by 9 Italian Universities (Turin Politecnico and University, Milan, Trieste, Genova, Modena, Bologna, Pisa and La Sapienza Rome), 5 Research Organisations (IOR Bologna, CNR Rome, CNR Naples, ISMETT and RiMED Foundation Palermo) and 7 private societies (GVM, ABMedica, ABTremila, Genomnia, Igea, Chiesi Farmaceutici and UPMC Italy).The strategic goal of IRMI is to create an infrastructure that will facilitate the exchange of knowledge between the different disciplines that underlie regenerative medicine products. Thank to this strategy, will be encouraged the development of new products on the territory for the advanced therapies, consequently increasing the international competitiveness of Italian Biotechnology sector for products derived from human tissues and cells.

More specifically, the IRMI is supporting the growth in the scientific-industrial sector producing:

1. the development of existing biobanking structures, offering new opportunities for storage of both autologous and allogeneic cells and tissues;
2. the strengthening and rationalization of cell manipulation structures, working in a coordinated and collaborative organisation, following the example of the PACT created in the USA by National Lung, Heart and Blood Institute, also providing all the necessary services for the organization, management and analysis of clinical trials and the interface with authorities, both for clinical trials and for the marketing authorization of drugs.
3. the development of bioprinting through collaboration with Italian Digital Biomanufacturing Network (IDBN), a scientific network with European diffusion. 
4. the creation of support structures for research and industrial development of advanced therapy products, including translational clinical research;
5. research and development of new human tissues derivatives, 3D printed electro-spun biological membranes, biomatrices and biocompatible polymers, cellular products based on progenitors with high differentiation capabilities derived from iPS and active molecules able to regulate the proliferation and differentiation of stem cells, advanced therapies medicinal product based on autologous corneal epithelium
6. new professional figures for biobanking, the manipulation of cells and tissues, the research and development of biomaterials, the application of converging technologies, the bioprinting, the management of clinical trials and regulatory aspects of advanced therapies.

IRMI partners collaborate with Italian Digital Biomanufacturing Network (IDBN) a group of Italian academic institutions, FabLabs and industries involved in bioprinting research and application in medicine.

What is the challenge and the vision?

3D bioprinting, known as additive manufacturing, is driving major innovations in many areas, such as health, engineering, manufacturing, art, design, and education.

Available systems utilize one of following processes:

1) photopolymerization of liquid monomer,

2) sintering powdered materials,

3) thermal or chemical process and

4) printing materials such as chemical binder onto powder.

3D bioprinting techniques associated with imaging techniques are used to develop scaffolds customized in size and shape for personalised therapeutic applications, thus becoming an important enabling technology for tissue engineering. To bioprint scaffolds for human organs and tissues regeneration, many groups around the world have developed technologies to lay down matrices with live cells to form 3D organ/tissue structures.

3D bioprinting is actually developed to

1) create fully functional scaffolds for damaged tissues regeneration in patients and

2) manufacture small-sized human-based tissue models or organoids.

The use of 3D bioprinting in tissue engineering is diffusing and the research needs to solve present problems like type of biomaterials, cell types, growth factors and cell saving technology for a quick production.

Available bioprinting techniques

Currently, there are three major types of 3D bioprinting techniques: inkjet bioprinting, microextrusion bioprinting and laser-assisted bioprinting.

Inkjet 3D bioprinting

In inkjet-based bioprinting, droplets of cells or biomaterials are dispensed by thermal bubble, piezoelectric actuator or electromechanical valve-controlled pressure pulse. Even if in the inkjet printer the localized heating can be very high, it lasts for a very short duration of 2 μs and does not exert any significant impact on the viability of cells. Recently, acoustic field are used to generate droplets.

Microextrusion bioprinting

Microextrusion 3D bioprinters deposit layers of viscous material via a syringe under constant pressure and can be used also for multicellular cell spheroids, which possess the mechanical and functional properties of the tissue extracellular matrix (ECM).

Laser-assisted bioprinting

Uses the energy of pulsed laser to induce the transfer of materials with the advantage of being a nozzle-free technique and compatibility with a wide range of biomaterial viscosities.

Biomaterials for bioprinting

Biomaterials, supporting the cellular components and thus being important in the implementation of bioprinting fall into two categories:

1. curable polymers, forming mechanically resistant structures after solidification, require high temperatures or toxic solvents and thus cells are usually deposited on the scaffolds after fabrication, thus avoiding conditions harmful to the cells.
2. soft materials, with high water content, inside of which cells are capable of residing. Soft materials can comprise synthetic or natural polymers.

For live cells, the primary biomaterial used for printing is hydrogel. The most used biomaterials include polyethylene glycol (PEG)-based materials, collagen, hyaluronic acid, alginate and fibrin. Another group of materials that can be printed is cell-aggregate-based bioink, which can be tissue spheroids, cell pellets, or tissue strands.

Bioprinted tissues

Up to date, no functional human-sized organ has been printed but bioprinting can regenerate simple tissues such as bone, cartilage, skin and nerve and complex organs such as teeth, nose, ears, heart, and liver.

Bone

Bone can heal and regenerate when orthopedic surgeons use porous scaffold materials with or without growth factors and cells. 3D-bioprinted bone scaffolds, were used to reach successfully the object. Using 3D bioprinting, an anatomically shaped scaffold can be created to match the actual defect of patients based on the medical imaging data.

Cartilage

Cartilage is an aneural and avascular tissue containing few cells. Articular cartilage is composed of different regions with differing properties, sizes and morphology. 3D bioprinting technology is the right technique for cartilage regeneration. 3D-bioprinted constructs have also demonstrated some in vivo success in repairing cartilage defects.

Skin

In skin injuries, autograft, allograft, wound dressing, and tissue-engineered substitutes are the current treatment choices. A skin inkjet bioprinting method using Collagen precursor, keratinocytes and fibroblasts, printed in a layer-by-layer manner, has been developed and the 3D-bioprinted skin showed similar morphology and histology to native skin layers prior to in vivo implantation.

Vascular

Several 3D bioprinting techniques are used to form vascular channels from sacrificial channels to vessel-like constructs using hyaluronan hydrogels, from Pluronic F127 printing to agarose template. All these channels were lined with endothelial cells and perfused with blood under high-pressure pulsatile flow.

Nerve

To facilitate nerve regeneration neural stem cells, collagen hydrogel and vascular endothelial growth factor (VEGF)-releasing fibrin gel were printed to construct an artificial neural tissue. The 3D bioprinting technology can be used to create nerve graft with multi-lumen channels.Bioprinting is considered a promising approach to nerve graft fabrication and to nerve regeneration.

Trachea

A 3D bioprinted scaffold in PCL coated with MSCs in fibrin was used for tracheal reconstruction in an in vivo tracheal defect model. The bioprinted trachea was remodelled and covered with regenerated respiratory mucosa, allowing basic respiratory functions. 3D bioprinting and medical imaging used together can create patient-specific tissues for implantation.

Cardiac tissue

Application of 3D bioprinting in cardiac tissue regeneration is still in very early stages.

Heart valve

Heart valve is particularly suitable for 3D bioprinting technology and micro-extrusion bioprinters were used for manufacturing cellularized heart valves for clinical use.

Organs

Liver, kidney and thyroid gland are the first organs for which 3D bioprinting is more than a hope and new biprinting technologies allow to forecast possible application in teh next few years.

Future perspective

Engineering issues

All the current bioprinting technologies (inkjet, micro-extrusion and laser bioprinting) present advantages and disadvantages. Research needs to be done to

1. improve resolution, speed, reproducibility, viability and biocompatibility of the bioprinting process;
2. invent new dispensing technologies;
3. identify new biomaterials with increasing speed, precision, and specificity.
4. for clinical usage, it is also necessary to increase the speed of fabrication for creating larger structures.  
5. develop real-time automatic controls during the printing process.

Biomaterial issues

The available printable biomaterials are very limited and the native ECM is still difficult to double. Thus research is needed to develop new biomaterials that can be easily manipulated by the bioprinting technology to be dispensed in complex 3D structures and to maintain cellular viability and function, including new cytocompatible cross-link/gelation mechanisms.

Biological issues

3D bioprinting opens very important biological research issues from the cell source and quantity. To cell survival and vascularization within the printed structures, from angiogenesis to pore size, porosity and distribution, from mechanical properties and functions to tissue maturation in bioreactors. Research is needed to translate 3D printing techniques from bench to bedside.

Drug testing

The need for alternative drug testing methods are becoming increasingly urgent beyond the desire to eliminate the need to harm the animals. There are many diseases and conditions that animal testing will produce inconsistent or false positive reactions and find themselves unable to simulate in the human trial. Being able to bioprint humanlike tissue for drug testing purposes would remove the inherent unreliability of animal testing, and save money on drug development. Testing drugs on bioprinted human tissue would drastically reduce – if not entirely eliminate – the need for animal testing. Valuable research time and money would no longer need to be wasted on extended trials that may be effective in animal models but will be found to not work in human models. Additionally it could find treatments that work on human physiology that would have never made it past the initial animal test phase due to poor results.

Cosmetic testing

3D bioprinting’s allure has attracted blooming interest from the skincare industry, with three leading firms each launching skin printing initiatives in mid-2015 that they hope will revolutionise cosmetic testing. The initial challenge is making skin slivers for new product experiments conducted in multiwell analytical plates, but success could lead to much grander schemes. The expertise gained could feed into pharmaceutical research, and even help enable patients’ own cells to be made into almost perfectly compatible skin grafts and eventually replacement organs. 

Two projects are partnerships with startups: L’Oréal's US-based global technology incubator has joined forces with Organovo in the US; and German-headquartered BASF with French firm Poietis. Meanwhile, US consumer products giant Procter & Gamble has invited research proposals from Singaporean academics within a five-year S$60 million (£27.4 million) programme with the country’s Agency for Science, Technology and Research (A*Star).

Why is it good for Europe?

A recent report by Grand View Research Inc. shows that 3D Bioprinting is a legitimate global market, that is set to be worth a staggering 1,82 Bilion USD within less than 10 years.

A study, entitled Open innovation in industry, including 3D printing, has been provided by Policy Department A of the European Parliament at the request of the ITRE committee, describes the mutual reinforcement of open innovation and additive manufacturing and addresses recommendations for different policy levels.

The EU has identified 3D printing as one of the technologies that will drive forward the development of future products and services, starting funding research into 3D printing during the first ever research funding round, which ran from 1984 to 1987. Under the latest round of research funding, which ran from 2007 to 2013, it spent over EUR 160 million on over 60 research projects in 3D printing. Under Horizon 2020, the funding round that runs from 2014 to 2020, EU plans to continue funding 3D printing, and it is asking researchers to submit their proposals.

3D printing is an innovation key for intelligent manufacturing and digitalized processes, but also for personalized healthcare. Bioprinting will completely innovate

1. organs and tissues regeneration,
2. medical devices production and their personal customisation,
3. drug pharma pill individualisation.

Bioprinting is maturing and expanding as a sector. There are now approximately 80 institutions worldwide conducting research, developing the printer technologies and applying the capabilities in ever more complex areas. Despite its growing potential, and two recent reports explore its market growth, research funding remains relatively low. In the USA, at present, bioprinting receives approximately $500 million, in comparison with $5 billion for cancer research and $2.8 billion for HIV/AIDS research.

Other tangible indicators of the ‘maturing’ of bio-printing are the announcement of the first ever Master’s degree and the first global conference. The master’s degree is the result of an international collaboration between universities in Australia, Germany and the Netherlands – which should begin to develop the range of critical skills – a mix of IT, CAD and biology.

“Body on a chip technology” may be one of the first major areas of application for bioprinting, putting the functionality of organs onto a chip, so that it can be used for drug and toxicity testing. Liver and other functions have been achieved and a ‘full body’ on a chip is the aim of US research. The benefits will include faster and more effective drug development; about 30% of drugs fail at the human trial stage because of differences in functionality between animal and human tests. With the cost of drug development ranging from $3.7 to $12 billion, and an average of about $1 billion, and a time to market of 12 years and drug companies facing ever greater challenges in developing effective drugs, the potential of body on a chip technology is huge.

Longer term bioprinting could address the shortage of transplantable organs. Organ transplant waiting lists are growing: about 120,000 people in the US and about 64,000 in Europe are waiting for organs at any one time. Organ donations, primarily from deceased donors totalled only 9534 in the EU, enabling just over 30,000 transplants and a similar number of operations in the US too. Many die while waiting. Bioprinting organs using an individual’s own stem cells could not only increase the availability of organs but also improve survival rates with earlier intervention and lower rejection rates.

Bioprinting is in its early days, but has emerged from its rather speculative ‘science fiction’ image and is maturing as a technology. Investment is needed at national and international levels, by government, academic and corporate organisations, if its potential is to be achieved. The opportunity is there, but skills in many areas are also needed.

3D bioprinting has the potential to impact on vast addressable markets, ultimately estimated to be in excess of $150bn. With a reasonable estimate of market size forecast at $6bn by the next decade, 3D bioprinting is currently the most promising application of 3D printing. 

What would it take to do it?

North America is leading in 3D bioprinting, as Europe slowly gains ground following in suit. 

In Europe many National initiatives are running and here below some are presented:

• the Fraunhofer Additive Manufacturing Alliance integrates fifteen Fraunhofer Institutes throughout , which addresses the issue of additive manufacturing and thus covers the entire process chain. This includes the development, application and implementation of additive manufacturing methods and processes as well as related materials;
• Layerwise from Belgium and Xilloc from Netherlands are the major companies dealing with 3D printed medical and dental implants. Xilloc was in the news recently for creating the first customized 3D-printed lower jaw for an 83-year old patient with a serious jaw infection;
• In Belgium Sirris, in collaboration with FLK (a research centre located in Germany), has decided to launch a project called “Bi4Life” which stands for BioPrinting of Biactive Bicomponent Biomaterial for LIFE science industrial products. Sirris is willing to involve companies active in cell therapy, in vitro diagnostics, drug discovery and medical device, all of which are technological areas for which Belgium (and more particularly Wallonia) has fully integrated value chains;
• In Holland MERLN Institute for Technology-Inspired Regenerative Medicine is focused on bioprinting research and clinical application.

The aim of the project is to fund with FET projects collaborative panEuropean collaboration to create an effective European network on Bioprinting.