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3D-bioprinting technique produces blood vessel networks for tissue engineering applications

By Joseph Bennington-Castro April 17, 2017
Researchers produced blood vessel networks using a new three-dimensional printing technique, which involves using a digital micromirror array device to project digital masks onto a specially formulated cell-encapsulating hydrogel, which polymerizes in response to UV light. Credit: Biomaterials/Elsevier.

Three-dimensional (3D) printing technology has found widespread application, ranging from electronics to biomedicine. Researchers have also sought, with limited success, to use 3D printing technologies for tissue engineering purposes, in hopes of creating life-like tissues with embedded vasculature (blood vessel network) capable of transporting biological materials when implanted into the body. In a big step forward for the field, scientists have developed a new 3D-printing technique called microscale continuous optical bioprinting (μCOB), which can rapidly print tissue with complex, cell-laden vasculature networks, complete with blood vessels that branch out into many smaller blood vessels. As reported recently in Biomaterials, the bioprinted prevascularized tissues, when grafted onto skin wounds of mice, can grow and integrate with the host vasculature network within two weeks.

“For any tissue implant, it won’t survive if there’s no connection to the host blood vessel system, so it’s important to have vasculature networks in the tissue before implanting it,” says study lead author Shaochen Chen, a biomaterials researcher at the University of California, San Diego. “We can now create these blood vessels in the 3D-printed tissue or model, and in the future, you can use them for repairing or regenerating tissues.”

A major challenge in tissue engineering is creating tissues with functional blood vessels that transport blood, nutrition, oxygen, and waste within the engineered tissue. One approach to achieving this goal is incorporating growth factors that promote the recruitment of the host vasculature into the engineered tissue, but this technique is not always effective. “If the tissue isn’t prevascularized, then the host vasculature doesn’t know where to go,” Chen says. Naturally, a potentially better approach is to encapsulate endothelial cells—those that form the inner lining of blood vessels—and supportive cells into the artificial tissue (that is, prevascularize the engineered tissue) before implantation. Furthermore, combining this approach with 3D printers is ideal because the technology would allow for the large-scale printing of highly customizable tissues.

Traditional 3D-printing technologies are not suited for biologically active materials because they require heat and physical extrusion forces, both of which can damage cells. In recent years, researchers have modified nozzle-based technologies to incorporate cell-encapsulating bioinks and stereolithography systems to write 3D structures into hydrogels containing cells, making the technologies more suited for the printing of vascularized tissues. However, these nozzle- and laser-based bioprinting techniques may still be too time-consuming for large-scale tissue printing (a major concern being the survival of the integrated cells) and/or may lack adequate mechanical stability of tissues. A more apt approach is to use a next-generation 3D-printing technology called digital light processing (DLP), which uses a highly controllable digital micromirror array device to project designs onto a hydrogel solution, which polymerizes in response to the UV light. In recent work, Chen and his colleagues have used the technology to produce liver tissue and artificial microfish (microscopic fish-shaped robots that can “swim” in liquids) to detect and remove toxins.

In the new work, Chen and his colleagues developed μCOB, a DLP-based technology for bioprinting prevascularized artificial tissue. The technique utilizes a digital micromirror array device that is made up of about 2 million micromirrors; these mirrors can be individually controlled to project a precise image onto a photo-polymerizable monomer solution on the fabrication stage. A motorized syringe pump system below the stage adds and removes the prepolymer solution. Digitized and controlled by a computer, the printing process involves feeding a series of digital photo masks (which can be sliced from 3D computer models of native organs) to the micromirror device, which shines the patterned UV light onto the solution to polymerize it, while simultaneously moving the stage.

For the matrix material needed to 3D-print the tissue constructs, the team used two biocompatible and photo-polymerizable hydrogels: Glycidal methacrylate-hyaluronic acid (GM-HA) and gelatin methacrylate (GelMa). Haluronic acid is involved in numerous cellular responses, including cell signaling, wound healing, and the formation of new blood vessels (the inclusion of the methacrylate groups makes it photo-polymerizable). GelMa has previously been used in engineering microvascular networks. For the vasculature, the researchers also created three masks of hexagonal patterns with gradient widths.

Within just one minute, the researchers printed a prevascularized tissue construct measuring just 4 mm by 5 mm by 600 mm, which they then cultured for a day. To test the new technology, they grafted the 3D-printed tissue under the skin of mice. After two weeks, they found significant amounts of endothelial vessels with red blood cells within the prevascularized tissues, suggesting the artificial tissues grew and merged with the mice’s blood vessel network. In comparison, when the researchers implanted non-prevascularized tissue (that is, 3D-printed tissue without cells), they only found very limited endothelial networks after two weeks, and these networks were only at the periphery of the implants.

Producing hollow vascular networks within hydrogel blocks using stereolithographic bioprinting is challenging, says Yu Shrike Zhang, an associate bioengineer and instructor of medicine at Brigham and Women’s Hospital, Harvard Medical School. “This paper is among the first to report the generation of perfusable vascular patterns within hydrogels, using a sacrificial, photo-crosslinkable material that can be subsequently removed using an enzyme,” says Zhang, who was not involved in the research. “It is novel and of great interest to the community, especially for those who are using stereolithographic bioprinting.” But, he adds, the degradation process for the sacrificial material is relatively lengthy, potentially limiting the cell type and viability and perhaps the complexity of the bioprinted pattern.

Chen and his colleagues are working toward optimizing the system to eventually conduct clinical trials. In particular, they are working on creating patient-specific tissues that use human-induced pluripotent stem cells (stem cells generated from adult cells), as the current technology uses human umbilical vein endothelial cells. In the more immediate future, Chen foresees the technology being useful for testing new drugs, an expensive process that currently takes many years. “Now we can print human cells in these gels and form human tissue, such as liver and heart tissue,” he says. “And then we can use that tissue to test whether a drug compound is effective or toxic. It could assist in the research of pharmaceutical companies.”

Read the abstract in Biomaterials