Tissue engineering holds the promise for treating disease, trauma, and congenital abnormalities. Researchers hope to create revolutionary new therapies that will re-establish damaged tissue and organ function. A part of this vision is to be able to use genetic engineering to control the activation of primitive regenerative genes, which are inactive in humans. This grand vision is still far from being a reality. In the meantime, most tissue engineering research deals with the therapeutic delivery of one or more types of tissue building blocks, including stem cells, growth factors and other signaling molecules, and scaffolds for delivering the cells or signaling molecules.

While maintaining the larger vision for tissue engineering, ICES Research Professor Phil Campbell and Robotics Institute Research Professor Lee Weiss and their collaborators, have focused on three areas: 1) directing stem cell fates using inkjet printed patterns of growth factors; 2) computer vision-based cell tracking for studying and controlling stem cultures to create the vast number of cells needed for regeneration; and 3) creating superior, cost effective scaffolds manufactured from donated blood plasma. Campbell and Weiss describe their focus as "delivering directly into the body minimal sets of biological building blocks as cues to induce and guide the body to repair itself."

This body of work requires a collaborative team effort. The team includes Professor Takeo Kanade of the Robotics Institute; graduate students Kang Li from Electrical and Computer Engineering and Eric Miller from Biomedical Engineering; former post-doctoral research fellow Julie Jadlowiec Phillippi (now at UPMC); Professor Alan Waggoner of Biological Sciences and the Molecular Biosensor and Imaging Center (MBIC); and Chemical Engineering Professor Lynn Walker. The team also includes Johnny Huard, professor of orthopedic surgery at the University of Pittsburgh School of Medicine and director of the Stem Cell Research Center at Children's Hospital of UPMC, and biologists working with the Stem Cell Research Center. 

Innovative Inkjet System for Bioprinting
Campbell and Weiss have been developing a methodology using printed "bioinks" to direct how and where stem cells grow to establish the biological cues required for regeneration. Their bioprinting research, which is currently funded by a four-year, approximately $1.5 million National Institutes of Health (NIH) grant, was recently featured in the media for their creation and use of an innovative inkjet system to print concentration-modulated patterns of growth factors, or "bio-ink" patterns.

These bio-ink patterns were used to direct stem cells derived from mouse muscle and to differentiate them into either muscle cells or bone cells. The results of this research have the potential to revolutionize the design of replacement body tissues. The team uses a custom-built inkjet printer for their experiments. Julie Jadlowiec Philippi describes the way in which they use inkjet technology commonly found in printers, but "instead of ink, we load the printer with proteins." 

The printer deposits patterns of growth factor proteins (the "bio-inks") onto a glass slide coated with a matrix of fibrin, which is the material produced by the body to stop bleeding when it receives a cut or fracture. Fibrin has the ability to naturally bind and immobilize growth factors. The slide is then placed in a culture dish with mouse muscle-derived stem cells. The printing system allows for a precise placement of the bio-inks on the matrix. The experiments show that stem cells that are exposed to bone morphogenetic protein (a growth factor) generate bone cells, while those cells that are not exposed generate muscle cells. Another feature of the procedure is that overprinting allows for a varied concentration of bio-inks that allow the researchers to see the impact of these changes on the stem cells and to determine appropriate patterns.

Such experimental approaches represent potentially efficient methods for: screening growth factors; determining dosages and combinations for subsequent in vivo investigations and therapy development; and discovery for stem cell culture conditions for both expansion and differentiation. Campbell and Weiss believe that bioprinted patterns will, at minimum, prove to have important applications as in vitro toolsets for basic biological discovery like cell screening assays. They also believe that these capabilities will lead to improved therapy designs, even if they are only simple designs.

Bioprinting is an emerging field representing diverse deposition processes, and inkjet bioprinters will become more widely available to a broach range of investigators over the next several years. Campbell and Weiss believe that it is likely that new unexpected applications will emerge as more investigators gain access to this technology.

Funding for the Future
The translational application of stem cell biology, or stem cell engineering, promises to revolutionize medicine. A basic challenge to realizing this goal is to provide in vitro production capabilities that can regulate adult stem cell expansion, to allow for self-renewal, without differentiation, in order to create sufficient numbers of cells for clinical applications. Therefore, in the development of the production process, cell culture conditions for expansion should be tailored to maintain "stemness," which is a measure of the cell population's ability to replicate without differentiation. Once it differentiates, it will not create more replications of itself. Characterizing and quantifying metrics of stemness ideally requires measuring the spatiotemporal histories of cell fates of each cell from within populations. Measuring the spatiotemporal histories means tracking individual cell locations over time, and it allows the researcher to measure the lineage of cells. Weiss explains that, "you want to know the 'mother-daughter' relationships of the cells."

The capability to track such metrics in vitro in real-time would offer unique opportunities for stem cell engineering applications, including efficient discovery and on-line process monitoring and control. Toward this goal, Campbell, Weiss, and Kanade were just awarded a four-year, $1.5 million NIH grant to develop a computer vision system that tracks and quantifies in real-time the spatiotemporal histories of stem cell divisions in vitro from phase-contrast imagery, and to apply this system to develop strategies that maximize the yield of adult stem cell proliferation ex vivo while maintaining the potential for differentiation.

In other developments, Campbell and Weiss, along with their partner James Burgess, a neurosurgeon at Allegheny General Hospital, have created a start-up company, CarMell, LLC, to develop scaffold and substitute graft materials made out of blood plasma using a proprietary manufacturing process. These novel plasma-based plastics (PBPs) address a multi-billion dollar and growing annual market. Campbell, Weiss, and Burgess believe that PBPs will provide better outcomes at lower costs than currently available alternatives.

All of this new work, including the original bioprinting research, was made possible with the help of initial seed funding by the Pennsylvania Infrastructure Technology Alliance (PITA) and by the Philip and Marsha Dowd Engineering Seed Fund. In 2003, graduate student Eric Miller received a one-year Dowd Fellowship on the "Development of Ink-Jet Based Processes for Tissue Engineering and Cellular Interaction Studies." Campbell notes that, "both PITA and the Dowd fund were crucial in initiating the bioprinting project." Additional funding for the bioprinting project has come from the Navy, the Army, and the National Science Foundation.

Much of the funding has also been used in educational endeavors, including classroom instruction; training undergraduates, graduate students, and post-doctoral researchers; and ongoing outreach programs. 


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