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Bioprinting: Science or Fiction?

 

The liveliest—literally—field of 3D printing may sound like something from a sci-fi movie, but (spoilers) it’s real and happening now.

 

By Arif Sirinterlikci and Lauren Walk
Department of Engineering
Robert Morris University
Moon Twp, PA

Cornell University’s Lawrence Bonassar calls bioprinting the intersection of three technologies: tissue engineering, regenerative medicine, and 3D printing. It’s the 3D printing of biological media for replacement of human tissue or biofriendly engineered materials such as scaffolds and drug release mechanisms for helping the healing process of human tissue.

Bioprinting, along with tissue engineering and regenerative medicine, is trying to fill a needed niche between organ and tissue donation—there are only so many donors—and implants and replacements based on engineered materials that have limited service lives due to wear and fatigue failures and that may also be prone to product recalls.

As early as 1988, Robert J. Klebe from University of Texas Health Sciences Center presented a vision of the bioprinting process in his publication, Cytoscribing: A Method for Micropositioning Cells and the Construction of Two- and Three-Dimensional Synthetic Tissues. Since then, hundreds of studies have been conducted including successful printing of bones, menisci, vasculature, heart valves, and ears as well as bioresorbable tracheal splints. Many experimental 3D bioprinters have been developed, and some have become commercialized, including the high-end Envisiontec Bioplotter as well as the lower-cost Seraph Robotics’ Bioprinter, which was developed at Cornell University. Most of these machines allow automated, rapid, scalable, and personalized printing as they utilize living cells. They also achieve uniformity while reducing printing time, costs, and expertise needed for tissue engineering. The Envisiontec Bioplotter is able to print with a wide range of materials, including hydroxypapatite, titanium, and tricalcium phosphate (TCP) for bone regeneration; polycaprolactone (PCL), polylactic-co-glycolic acid (PLGA), and poly-l-lactide (PLLA) for drug delivery; agar, alginate, collagen, chitosan, fibrin, and gelatin for soft tissue biofabrication and organ printing; and polyurethane and silicone for concept modeling.

Envisiontec Bioplotter printing a silicone mesh model.

The bioprinting process is currently realized through two different techniques: biological laser printing—the technology used in the Envisiontec bioplotter—and biological ink-jet printing—the basis of the Seraph Robotics Bioprinter.


Biological Laser Printing

Biological laser printing (BioLP) is an automated CAD based transfer process where a laser beam moves cells covered by a medium, usually within microbeads or microcapsules, onto the receiving substrate. It is capable of rapidly depositing living cells onto a variety of surfaces. Unlike other techniques such as ink-jetting printing, the process delivers a small volume of a variety of biomaterials without using an orifice, and eliminates potential clogging issues and damage to the cells. Today’s laser-assisted bioprinting technology applications include laser-based micro patterning of cells in gelatin, cell assembly, bioprinting of skin, and laser-engineered microenvironments for cell culture.

After intensive efforts in late 1990s for using biological media in sensor development and coupling it with lasers in fabrication, BioLP was developed in 2004 by a group of researchers from the Naval Research Laboratory and the Hebrew University of Jerusalem. They used a laser-based printing method to deposit bacteria, with the ability to respond to various chemical stressors, onto agar-coated surfaces and into microtiter plates. Initial work yielded smaller printing spots, increased resolution, and better repeatability compared to other related techniques. Deposition rates up to 100 pixels of biological material per second were achieved. The original cell printing experiments not only demonstrated close to 100% viability, they also were the first steps toward using BioLP to create heterogeneous 3D tissue constructs. More recently, in 2012 Vienna University of Technology (VUT) researchers developed a new variant, called 3D Photografting, to grow biological tissue or to fabricate microsensors by using two-photon lithography. The VUT scientists start with a hydrogel scaffold which is made from macromolecules arranged in a loose meshwork. The 3D Photografting method is then used to introduce selected biomolecules into the hydrogel meshwork where the laser beam is focused by breaking photochemically labile bonding. The laser produces intermediates which are very reactive and consequently attached to the hydrogel very quickly. Based on the laser’s lens system, resolutions as fine as 4 µm can be obtained.

Two views of a Cornell University bioprinter printing cartilage cells encapsulated in collagen hydrogel to help develop a new earlobe.Two views of a Cornell University bioprinter printing cartilage cells encapsulated in collagen hydrogel to help develop a new earlobe.

Laser-assisted bioprinting process has proven to be suitable for depositing multiple cell-types adjacently, producing larger-scale cell arrays and multilayer cell constructs. Recent work in the field includes biofabrication using laser in patterning stem cells, microbeads, and cell-loaded microcapsules at Rensselaer Polytechnic Institute (Troy, NY); in-situ and in-vivo bioprinting of cells and biomaterials at the University of Bordeaux (Bordeaux, France); and skin tissue generation by laser cell printing at the Laser Zentrum Hannover E.V. (Hannover, Germany).


Biological Ink-jet Printing

For many years, ink-jet technology has been used as a helpful tool in providing a noncontact technique to print inks in a rapid manner. Recently, this technology has been applied in the medical field by using encapsulated cells as the ink (bio-ink) in order to print tissues and organs, including heterogeneous tissue and microvascular cell assembly as well as biomaterials. With help from a pressurized air-supply controlled by solenoid valves, these bioprinters deposit encapsulated cells onto the substrate to generate 3D constructs such as an earlobe.

According to Thomas Boland of the University of Texas at El Paso, bio ink-jet printing can produce 100 million drops per second. That makes this method very rapid as well as convenient since the printer can be transported with ease and can use many different types of ink. The drops that are produced by the printer are very small; usually in range of 20–60 µm. When using different types of ink, it is important to determine the printability of the material. The printability can be determined by using the surface tension, density, viscosity, and the critical dimension of the printing material.

There are many types of inks that can be used with biological ink-jet printing, including alginate, chitosan, and collagen as well as reactive inks such as cross-linkers and proteins, and polypeptides. When printing and manipulating live cells, it is important to be aware of the apoptosis—the natural death rate of the cells. According to Boland, when cells are printed the apoptosis rate is 3.5 ± 1.3% while cells that are pipetted have a rate of 3.2 ± 1.6%. Thus, the inkjet printing of cells does not have any significant effect on the death rate of the cells, opening doors to endless opportunities and advantages in the medical field. Recent work with this method included printing of cartilage-based constructs including artificial ears at Cornell University (Ithaca, NY) and Princeton (Princeton, NJ) as well as other tissue and organ constructs at Wake Forest Institute for Regenerative Medicine (Winston-Salem, NC).


Handling Scaffolds for Tissue Growth

Wei Su from Drexel University (Philadelphia) recently presented his findings on fabricating scaffolds for tissue growth. Tissue scaffolds are used in tissue engineering to provide a temporary 3D structure on which cells can be manually or automatically attached (through 3D printing) and then grow and create new tissue. Tissue scaffolds are not easily constructed; there are many challenges. For example, the biomaterials being used must be carefully considered to see if they are biocompatible, biodegradable, or bioresorbable. Creating the right porosity and pore size of the scaffold is also a challenge. A large surface area gives the cell a place to attach and grow while a large pore volume adequately houses the cell. A high porosity is important because it makes it easier for the diffusion of nutrients and vascularization.

Once these challenges are overcome, a complete set of design constraints for the scaffold needs to be considered. Biophysical constraints deal with the scaffold’s structural integrity, strength, stability, and degradation, as well as the cell’s specific size, shape, porosity, and inter-architecture. Biological requirements deal with where the cell will attach, grow, and how the new tissue forms. Some other constraints include how the scaffold will perform anatomically and the manufacturability of the scaffold. After all of the challenges and constraints of the tissue scaffolds are handled, these scaffolds can be used to create 3D prints of organs, blood vessels, and even to create 3D models for drug release mechanisms.


The Future

Today’s successes in printing tissue for skin, heart valves, or earlobes can be complemented by successful printing and implantation of complex tissue assemblies with multi-material presence as well as complete bioficial organs such as a heart or a kidney in the future. However, successful bioficial organ printing relies on future developments in hardware and associated processes as well as successful printing of blood vessels and, especially, tissue with embedded vasculature. Recent research is showing promise in getting complex printed tissue to attach itself to the vascular network of an existing environment after implantation. Current studies are focusing on advancing printable cellular microfluidic channels, living lithography, fabrication of cellular materials with embedded vascular networks, printable Human Umbilical Vein Endothelial Cells (HUVEC) networks and direct printing of blood vessels. Hydrogel biopapers and BiOLP are showing promise in fabricating tissue with embedded vasculature. Additional attempts are also being made in bone and skin repair by directly printing on the existing tissue and in-vitro diagnostics for understanding the dynamics of the biochemistry of the organs and tissues.

 

This article was first published in the 2014 edition of the Medical Manufacturing Yearbook. Click here for PDF.   


Published Date : 4/7/2014

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