No doubt many of us have watched videos on social media of a “human ear” being 3D printed in a lab, indeed, many of us will be the ones posting such clips, where we use the complex structure of the ear to demonstrate the printability and performance of a bio-ink material. Besides the visually engaging results, it is worth to consider how such 3D bioprinting technology can offer real hope for patients with conditions such as microtia.
Microtia is a birth defect where the external ear is underdeveloped. Although there are several different options for auricular reconstruction, today the ‘Rib Cartilage Graft Reconstruction’ is the most effective. This treatment is based on sculpting the patient’s own rib cartilage into the form of an ear. Because the cartilage is the patient’s own living tissue, the reconstructed ear continues to grow as the child does. This is a very detailed and complicated surgical procedure with associated high-risk as well as chest-wall deformities.
3D bioprinting technology aims to create transplantable organs by applying cell-supporting bio-inks. One of the leading research areas of this technology focuses on the development of cartilage tissues for use in reconstructive surgery. This process aims to utilize the natural self-organizing properties of cells in order to produce a functional tissue. Although bioprinting technology is a promising method in itself, the properties of the bio-inks required are also crucial for the development of functional living tissues such as cartilage.
Synthetic biocompatible polymers and peptides have been utilized to develop hydrogels for cartilage tissue engineering. Their properties can be controlled and custom-designed to match the requirements of a given cell type. This is especially true for peptide-based hydrogels, in which the physical and the biomimetic properties can be tailored.
In the last few years, there has been a huge variety of research in cartilage bioprinting techniques, cell types, and bio-inks which have demonstrated the promising potential of such advanced bio-manufacturing process. Today this technology still has many challenges. However, it is hoped that in the future 3D bioprinting as a process to create transplantable cartilage can be translated into clinical practice and can provide a low-risk surgical solution for diseases such as Microtia.
Bioprinting is becoming widely used not only in regenerative medicine and tissue engineering applications but also in many other biomedical fields such as cancer research, as recently discussed on the Biogelx blog (see article). Thus, it is not surprising that scientists are now focusing on using this tool for novel approaches in stem cell research as well.
A recent article by Stephanie Willerth from University of Victoria in Canada describes advances in printing stem cells (particularly hiPSCs) by microfluidic extrusion and discusses the opportunities these advances open for high-throughput production of hiPSC-derived neural tissues. Willerth explains how these tissues can then be utilized for screening drug candidates for Alzheimer’s disease; which is yet another example of how bioprinting is becoming important in drug development.
Another innovative application of bioprinting as an enabling tool in stem cell research is the development of novel treatments for cardiac injury. Michael Davis from Emory University and Georgia Institute of Technology has worked extensively with stem cells and focuses his research in solving pediatric congenital heart defects using bioprinting technology combined with the reparative properties of stem cells. Scientists at Davis’ lab are working on a 3D printable patch which contains stem cells that will repair the surrounding damaged cardiac tissue when inserted into place. The group is also using bioprinting to create heart valves using skin cells from patients, thus minimizing the risk of organ rejection. Importantly for pediatric patients, this also allows the organ to grow with the patient, which means that a replacement will not be needed in the future.
In summary, the bioprinting of stem cells is being increasingly recognized as an innovative tool with extensive applications in biomedicine. Whilst the promise of such techniques is undeniable, it is important to remember that the bioinks utilized and several other factors in the printing process (as well as the culture period post printing) will be key to their success. Such factors can influence stem cells viability, differentiation, and function, so they should be carefully considered for future applications.
You can read a 2018 review of stem cell bioprinting technology here.
The future of drug screening, disease modeling, and precision medicine.
In the last few decades, both high-throughput screening and three-dimensional (3D) bioprinting technologies have emerged and have been proven to be instrumental for the advancement of drug screening, drug development, and tissue modelling.
High-throughput screening (HTS) is well established within the pharmaceutical industry for compound and drug discovery. It is, for the most part, the primary method of testing prior to compound validation and preclinical animal model studies prior to clinical trials. Today, the industry standard is still the use of two-dimensional (2D) cell culture for screening, as it has a long record of use and thus well-characterised cell lines and methods. This is despite the fact that the use of 2D culture is limiting due to its inability to capture in vivo-like cell-cell and cell-matrix interactions, and the fact many cell types display different phenotypes and varying genomic profiles in 2D versus 3D. Whilst the advantages of 3D cell culture are well understood, the reality is that many 3d culture systems can be challenging to reproduce to the levels required for HTS.
This is where 3D bioprintingis becoming recognised as a promising technology for HTS. It has the potential to produce more realistic, highly reproducible 3D cell cultures and support the bio-fabrication of 3D tissues and organoids.
The future of bioprinting in pharmaceutical applications
Bioprinting technology has advanced significantly in recent years, although it is still limited by the speed of printing and resolution. Therefore depending on the complexity of the printed construct, it may currently be a better candidate for use in lower throughput, larger scale models in 96-well or fewer plates. Nonetheless, in the future, with the advances being made in the precision, accuracy, and scale of the bioprinters, this technology might offer solutions for many current challenges of the drug development such as disease modelling, drug optimisation, toxicity screening and precision medicine.
Disease modelling and drug optimization
Better disease models are crucial for better understanding the behaviour, development, treatment, prevention, and cure of disease. Bioprinting of 3D cell-hydrogel constructs, when combine with microfluidic systems, has allowed the creation of complex, reproducible organ-on-a-chip models which can replicate body wide diseases by incorporating many external features such as fluid or air flow and a combination of tissue types into a single 3d model. Such technology enables researchers to conduct disease progression studies for better understanding and ultimately treating the disease.
Drug toxicity screening
When developing new drug treatments, toxic effects must be well characterised and monitored in vivo in animal models. The opportunity to more effectively model such effects prior to animal studies by utilising 3D cell culture models generated using bioprinting technology may allow for the drug to be modified or withdrawn prior to costly to in-vivo modelling.
One of the most heavily investigated side effects in drug development is the liver toxicity since it can result from over-exposure to toxins, leading to toxic hepatitis, inflammation of the liver, and eventual cirrhosis if exposed over a long period of time. These side effects further lead to what is known as drug-induced liver injury which is life-threatening. The complexity of the liver combined with the substantial role it plays in drug metabolism has created a demand for physiologically improved liver models which can be addressed by novel bioprinting approaches and improved liver models.
Precision medicine is the tailoring of therapy based on a patient’s genetic information. The use of a patient’s own cells allows for an amount of data to be collected for long term studies of disease progression and drug treatment response.
The use of 3D cell culture models in precision medicine allows for cells from the patient to be cultured and expanded to study the disease ex vivo while still maintaining in vivo genotype. Using the patient’s own cells, 3D bioprinting can be leveraged to create a large quantity of patient-specific disease models for use in parallel with clinical trials or independently through precision medicine initiatives for targeting patient or disease-specific genetic, proteomic, and phenotypic characteristics.
In summary, the potential of 3d bioprinting applications in high-throughput screening is vast, with drug screening and toxicity studies, disease modelling, and most recently precision medicine applications having a greater potential in the future due to the increased complexity, physiological relevance and reproducibility bioprinting can offer. By using bioprinting as a tool in high-throughput screening, physiologically relevant models can be realized for the improvement of healthcare.
Printing techniques and materials which have the potential in this research area
Chondral and osteochondral lesions represent one of the most challenging scenarios for the orthopaedic surgeon and for the patient, as the current therapeutic strategies are not providing satisfactory results in restoring function or slowing the progression of osteoarthritis. Based on research outcomes in the recent years, tissue engineering seems to have the potential to address the issue of osteoarticular loss and provide a viable alternative to current treatments by applying 3d bioprinting techniques in the clinical environment.
It is known that cartilage injuries cannot heal spontaneously, and that any type of repair will be characterised by fibrocartilage i.e. scar tissue. This tissue lacks properties such as resistance to shear and compression which make hyaline cartilage so distinctive, and in turn leads to degenerative changes and arthritis.
Which bioprinting techniques work for osteoarticular tissues?
With the boom of 3D bioprinting, there has been widespread development of printers and additive manufacturing techniques, which allow the biofabrication of complex structures. These technologies include fused deposition modelling (FDM), stereolithography, extrusion printing, inkjet printing and laser-based techniques. With regards to cartilage regeneration, the hydrogel-based scaffolds are the main materials used due to their inherent compatibility with chondral tissue. Therefore, inkjet and extrusion printers are the most commonly used machines in cartilage tissue engineering. Having said this, it has been extrusion-based methods that have been most widely used in recent years. The popularity of this printing technique is most likely due to its simplicity, diversity, and predictability. Whilst in comparison to inkjet, for the moment extrusion-based bioprinting has lower speed and resolution, it offsets this by being able to offer advantages such as higher cell densities, a wider range of printable biomaterials (viscosity in the range of 30-6 x 107 mPa/s) and relatively inexpensive equipment.
Featured materials for cartilage bioprinting
Properties of bio-inks are also crucial for the development of functional living tissues such as cartilage by 3D bioprinting. Bio-inks based on the combination of scaffold and cells should satisfy biological features, biodegradability, and printability. Generally, available scaffold options for bio-inks are hydrogels, decellularized ECM (dECM) and microcarriers.
Hydrogels are water-swellable, yet water-insoluble, cross-linked networks. Often these are naturally-derived polymers, in which the 3D environment provided is able to maintain a high-water content, which resembles biological tissues and facilitates cell proliferation. The only limitation of such hydrogels for tissue engineering might be their inability to maintain a uniform 3D structure. However, researchers have been able to overcome this problem by applying synthetic hydrogels or natural-synthetic hydrogels hybrids.
Synthetic biocompatible polymers and peptides have been utilised to develop hydrogels for cartilage tissue engineering. Their properties can be controlled and custom-designed to match the requirements of a given cell type. This is especially true for peptide-based hydrogels., in which the physical and the biomimetic properties can be tailored.
It has been proven that chondrocytes change their function and morphology based on the ECM. Therefore, being able to provide the appropriate ECM structure is considered paramount in cartilage tissue engineering.
Peptide-based Hydrogel Inks
Synthetic ECM mimics are increasingly being used as they can provide solutions to some of the issues that naturally-derived materials pose. The main benefit of synthetic hydrogels is consistency. There should be no batch to batch variations as they are produced using known quantities, optimised procedure, and generally the chemistry of the polymer does not change unless required. As they are synthetic, there are no components from animal-derived sources which is an important factor for cell culture and tissue engineering research with the ultimate aim of clinical applicability.
Printing an organ for transplant seems like something from a futuristic movie but the reality of this is a lot closer than expected. The concept of 3D printers has been around for decades, particularly in engineering fields, however, the extent of their potential in the medical field, is now being realised and the opportunities they can provide are vast. The manufacture of orthopaedic devices matched specifically to an individual patient, 3D printed bones for reconstructive surgery planning, and custom 3D printing for hearing aid and dental industries are just a few applications of 3D printing which have been used to improve the lives of patients. The next step is not only to improve the standard of life of patients but to save lives. Printing cells in a bioink scaffold which will ultimately lead to production of an organ which can be transplanted into a human body without having to wait for a donor could save precious time which some may not have.
The theory, and the goal that many 3D bioprinting researchers are working towards, is that from a simple cell biopsy (e.g. from the patients themselves), these cells can be proliferated and printed into the appropriate shape within a bioink, and allowing this bioprinted tissue to grow will produce a perfectly matched organ. Whilst researchers continue to develop bioprinting technology and address the challenges of how to print complicated and intricate architecture required to ensure appropriate blood supply and oxygen levels through printed tissues, it is important to consider but what are the regulatory implications, when this theory becomes in reality?
The U.S Food and Drug Administration (FDA) would appear to be the right agency to regulate 3D printed organs as its mission “to promote and protect the public health.” explicitly applies to regulation of “cellular and tissue-based products. However, it is yet to decide on appropriate regulations which cover a manufactured organ rather than a human organ. There are many complex points to be considered as a printed organ used for transplant is not entirely comparable to one single procedure or device currently regulated such as drugs, biological products, and medical devices. A human organ according to US Dictionary Act is “born alive at any stage of development”, this does not accurately describe a manufactured organ which would be grown from manipulated human cells to produce a completely new organ. Furthermore, the printed organ transplant process would also be much less invasive with no significant detrimental effect on the donor (indeed the cell donor could be the patient themselves). This difference would also suggest human and 3D printed organs are treated differently. Taking a different perspective, a 3D printed organ could be regulated in comparison to a drug. Whilst it does seem strange to parallel an organ with a drug, the printed organ’s intended use matches that of a drug; a drug is taken to allow a person with an illness to live a better standard of life or cure the illness completely, this would be the purpose of a 3D printed organ to potentially cure a critical illness. It has also been proposed that 3D printed organs should be regulated as a “biological product,” with these products being defined as “a virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood component or derivative, allergenic product, protein or analogous product, . . . applicable to the prevention, treatment, or cure of a disease or condition of human beings.” From this list, it is the term protein that must be considered, In order to print human cells and enable these to develop into a transplantable printed organ, matrices, culture media, and bioinks will be involved which will contain proteins. Thus it seems logical that such printed organs would fall within the scope of biological products. On the other hand, 3D printed organs could not be regulated as a medical device since the organ would have a chemical action within the human body with the intention of replacing a malfunctioning organ. Medical devices are generally not made of biological material, they could be metal or plastic implants or devices that aid in the recovery or standard of life of a person.
It seems that 3D printed organs could be covered by multiple regulations due to the complexity of the applications it could be used for. And whilst the existing regulatory framework could not predict such a disruptive technology, it seems that the basis for a framework to support 3D bioprinting does exist. Like organ printing technology itself in which very little is instinctive, its regulation will be equally complex. Could the FDA compile a new regulation to cover the use of printed organs for transplant if it became a widely routine procedure, or will it be covered by one or more of the regulations already in place? As the industry looks to address to the scientific challenges associated with progressing organ printing in the lab, it must also start to address these regulatory questions for when the theory becomes a reality.