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Bioprinting: 3D Printing Comes to Life

 

By Anthony Atala
Director

By James Yoo
Professor and Chief Scientific Officer,
Wake Forest Institute for Regenerative Medicine
Winston-Salem, NC

3D printing is driving innovation in many areas, from engineering and manufacturing to art and education. The technology is also being broadly applied in medicine—from printing prosthetic limbs to making patient-specific models of body parts that surgeons use as guides during reconstructive surgery. 3D printers have been used to make implants used in a small number of patients, including a titanium jawbone and a tailor-made, bioresorbable tracheal splint that saved a baby’s life. The next frontier in medical printing is bioprinting—using living cells to print replacement tissues and organs.

The idea of lab-built organs is not new and scientists have demonstrated the function of engineered airways, bladders, blood vessels and urine tubes in patients. These engineered structures have three key components: scaffolds, made of materials compatible with the body and fashioned in the shape of a tissue or organ; cells, ideally from the patient; and biomolecules, such as growth factors, to induce tissue formation.
A kidney prototype and ear and finger bone scaffolds made with Wake Forest Institute for Regenerative Medicine’s 3D printer.
This same “recipe” that was used to engineer human organs and tissue by hand is now used with 3D printers to produce structures such as bone, cartilage, blood vessels, cardiac tissue and heart valves that show promise for clinical use. The ultimate goal is to print complex organs such as livers and kidneys for transplant and to create composite tissues made up of skin, muscle, tendon, nerves, bone and blood vessels for reconstructive surgery.

The advantages of printing tissues, rather than engineering them by hand, are many. Printers allow the proper placement of multiple cell types, biomaterials and bioactive molecules in defined locations. They also offer the ability to control the size, microarchitecture and interconnectivity of pores in the scaffolds—essential to transporting oxygen and nutrients for cell survival. The technology also offers the option of using a patient’s medical images, such as MRI or CT scans, to tailor-make organs.

Of course, there are many challenges to overcome before printing organs for patients is a reality. The technologies that were designed to print molten plastics and metals must be adapted to print sensitive, living biological materials. And, the more central challenge is to reproduce the complex microarchitecture of living tissue to ensure that printed tissues have biological function.


Much to Accomplish Before Pressing “Print”

Regardless of whether an organ or tissue is engineered by hand or printed, there is much groundwork that must be accomplished. A thorough understanding of cell biology is vital to the process. Scientists must determine not only what types of cells to use, but how to expand them in the lab and how to keep them alive and viable throughout the engineering process. Do they need to be embedded in biocompatible material? If so, which biomaterial is most suitable? The bar for success is high—lab-engineered structures must function like native tissue.

Also important is the selection of printing strategy and printer type. There are currently three main approaches to 3D bioprinting. With the first, biomimicry, the goal is to manufacture structures identical to the cellular and extracellular components of a tissue or organ. This approach requires duplication not only of form and structure, but of organ microenvironment.

Research Fellow Young Joon Seol works on a project to print experimental muscle tissue for reconstructive surgery.

A second strategy, autonomous self-assembly, relies on the natural process of embryonic organ development. With this approach, the cell drives the process of tissue formation through cell signaling, the production of extracellular matrix and autonomous organization and patterning. The strategy requires detailed knowledge of the developmental mechanisms of embryonic tissue and organs.

A third approach, which can combine both biomimicry and self-assembly, is mini tissues. Because organs and tissues are made up of smaller, functional building blocks, these segments can be fabricated and assembled into larger structures. It is likely that combinations of all three strategies will be required to print complex 3D biological structures.


Bioprinter Options

As the field of bioprinting moves forward, new types of printers will likely be designed to meet the goal of printing functional replacement organs. Currently, there are three main printer types used to deposit and pattern biological materials—inkjet, microextrusion and laser-assisted. In addition, our institute has designed a system that is integrated—allowing for the printing of both solid and flexible materials.

Inkjet printers deliver controlled volumes of liquid to predefined locations and have been used to regenerate functional skin and cartilage in preclinical studies. They have also fabricated bone constructs, matured in vitro before implantation into mice.
A 3D printer at Wake Forest Institute for Regenerative Medicine at work on a kidney prototype.
The first inkjet bioprinters were commercially available desktop printers that were modified by scientists to have a third axis. The “ink” was replaced with a biologic material. These printers are characterized based on the mechanism used to generate the droplets: thermal, laser-induced, pneumatic, etc.

While there are many advantages to inkjet technology, a downside is the risk of exposing cells and materials to thermal and mechanical stress, and in the case of acoustic printers, to frequencies that may damage the cell membrane. Inkjet bioprinters are also limited in the viscosity of the bio-ink because of the excessive force required to eject drops.

While these drawbacks can be overcome, the methods may slow the process and expose cells to toxic materials. In addition, the cell concentration with inkjet technology may be lower than desired to prevent clogging the nozzle.

A second type of bioprinter, microextrusion, uses mechanical or pneumatic dispensing systems to extrude continuous beats of material, often consisting of cells mixed with hydrogel. Structures are printed in 2D with hydrogel and the material is then solidified either physically or chemically so the structures can be combined to create 3D shapes. This type of printer allows for a wider selection of biomaterials since more viscous materials can be printed through the needle. Another advantage is that the printer can deposit very high cell densities.

While cell viability is lower than with inkjet printers, viability is in the range of 40 to 86%, depending on nozzle gauge and extrusion pressure. With all printer types, it will be important for scientists to demonstrate not only viability of cells, but the ability of printed cells to perform essential functions in the construct.

A third printer type, laser-assisted bioprinting, is based on the principles of laser-induced forward transfer. These printers have been successfully applied to such biological materials as peptides, DNA and cells. This type of printer was used to make a noncellular, bioabsorbable trachea splint that saved a young child’s life.

These printers consist of a pulsed laser beam, a focusing system and a “ribbon” that has a donor transport support, a layer of biological material and a receiving substrate facing the ribbon. Focused laser pulses are used to generate a high-pressure bubble that propels cell-containing materials toward the collector substrate.

Because the technology is laser free, there is no problem with cell clogging. Other advantages are that the printer is compatible with a range of viscosities, can deposit cells at high density and can print mammalian cells with little effect on viability. Disadvantages include that the high resolution results in a low overall flow rate, it can be difficult to accurately target and position cells and that metallic residues are present in the final construct. While some of these challenges can be overcome, it is currently unclear whether this system can be scaled up for larger tissue sizes.

To overcome the disadvantages of relying solely on the cell-laden hydrogels used in most 3D printers, our institute has developed a hybrid system. Using hydrogels alone can make it difficult to fabricate structures with enough strength to form a clinically applicable size. Our hybrid system can concurrently print a synthetic biopolymer to provide physical strength and a cell-laden hydrogel to promote regeneration.

Instructor Hyun-Wook Kang oversees the 3D printer that will be used to print miniature organs for the Body on a Chip system.
What Makes the Ideal Biomaterial?

As scientists move away from hand-fashioning scaffolds to bioprinting them, additional biomaterials will need to be identified. The material must not only be printable, but also must be compatible with the body and support cellular attachment, proliferation and function. Also important is how quickly the material will degrade in the body. The degradation rates of the scaffold must match the cells’ activity in building a “home” from their own extracellular matrix, the molecules they secrete to provide structural and biochemical support.

In addition, material selection must be based on the mechanical properties needed for a particular structure. Different structural requirements will be needed for tissue types ranging from skin to liver and bone.


3D Bioprinting: The Future

While numerous biologic tissues have been printed and tested pre-clinically, challenges remain to further develop and harness 3D printing technologies for more complex tissues and organs. As scientists move away from modifying existing printers and begin to design new technologies, the range of materials can be extended and methods to deposit materials and cells with increasing precision and specificity can be developed.

Areas for future focus include:

  • Developing new biocompatible materials with sufficient mechanical strength to maintain their shape and withstand external stress after implantation.
  • Improving printer resolution. Complex organs such as kidney and liver have a detailed inner architecture that must be duplicated.
  • Developing methods to vascularize and innervate printed engineered tissue and organs, especially complex volumetric organs. While some groups have demonstrated generation of a branched vascular tree, a challenge is the time required for assembly and maturation.
  • Increase the speed of printing. Currently, conditions that increase printing speed, such as with the extrusion-based bioprinter, can lead to cell damage.
  • Develop in vivo bioprinting to regenerate tissues immediately after injury or during surgery. For example, 3D bioprinters can potentially be integrated into minimally invasive robotic surgical tools so that tissues can be removed and replaced during the same surgery.

 


Published Date : 2/10/2015

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