Opinion

Bioprinting scale-up and

constructs for transplantation

Ibrahim T. Ozbolat

Biomanufacturing Laboratory, The University of Iowa, Iowa City, IA, 52242, USA

Bioprinting is an emerging field that is having a revolu- biology of natural counterparts for pharmaceutical testing

tionary impact on the medical sciences. It offers great or cancer studies [8].

precision for the spatial placement of cells, proteins, Here I present recent approaches to the bioprinting

genes, drugs, and biologically active particles to better scale-up of functional tissue and organ constructs for

guide tissue generation and formation. This emerging transplantation, including bioprinting vascularized tissue

biotechnology appears to be promising for advancing and organ constructs in vitro and in situ bioprinting tech-

toward functional tissue and organ nology to build tissues directly in defect sites. I discuss

fabrication for transplantation, drug testing, research major roadblocks to this approach and provide potential

investigations, and cancer or disease modeling, and has solutions and future directions.

recently attracted growing interest worldwide among

researchers and the general public. In this Opinion, I Bioprinting of vascularized tissue and organ constructs

highlight possibilities for the bioprinting scale-up of func- in vitro

tional tissue and organ constructs for transplantation and Organ bioprinting holds great promise for the future, but

provide the reader with alternative approaches, their whole-organ bioprinting has remained elusive due to

limitations, and promising directions for new research several limitations associated with biology, bioprinting

prospects. technology, bioink material, and the post-bioprinting mat-

uration process [9]. The bioprinting of functional tissues is

Bioprinting: a promising technology to revolutionize an intermediate stage toward achieving organ-level com-

medicine plexity. In vitro fabrication of functional tissues is a

Bioprinting can be defined as the spatial patterning of sophisticated phenomenon comprising a hierarchical ar-

living cells and other biologics by stacking and assembling rangement of multiple types, including a multiscale

them using a computer-aided layer-by-layer deposition network of vasculature in stroma and parenchyma, along

approach to develop living tissue and organ analogs for with lymphatic vessels and, occasionally, neural and mus-

tissue engineering, , pharmacoki- cle tissue, depending on the tissue type. In vitro engineered

netic, and other biological studies [1]. It uses four tissue models that incorporate all of these components are

approaches to deposit living cells: inkjet [2], extrusion still far on the horizon. Bioprinting technology offers a

[3], acoustic [4] and laser [5] based. Given its great benefit great benefit in the hierarchical arrangement of cells or

in spatially arranging multiple cell types to recapitulate building tissue blocks in a 3D microenvironment, but the

tissue biology, bioprinting is a game-changer in the rapid bioink and the post-bioprinting maturation phase are as

development of tissue constructs and is receiving enor- important as the bioprinting process itself. The bioink

mous attention. Although bioprinting of functional 3D material is crucial because it should provide the spectrum

whole organs for transplantation remains in the realm of biochemical (i.e., chemokines, growth factors, adhesion

of science fiction, the field is moving forward, providing factors, or signaling proteins) and physical (i.e., interstitial

hope that shortages in tissue grafts and organ transplan- flow, mechanical and structural properties of extracellular

tation will be mitigated to some extent in the future ) cues to promote an environment for cell survival,

[6]. While current tissue-engineering strategies cannot motility, and differentiation [10]. In addition, the bioink

enable fabrication of fully functional tissues or organs should exhibit high mechanical integrity and structural

[7], bioprinting enables precise placement of biologics to stability without dissolving after bioprinting, enable dif-

recapitulate heterocellular tissue biology to some degree. ferentiation of autologous stem cells into tissue-specific cell

Current technology enables the development of organ or lineages, facilitate engraftment with the endogenous tis-

tissue constructs that do not require substantial vascu- sue without generating an immune response, demonstrate

larization, as well as mini-tissue models mimicking the bioprintability with ease of shear thinning, rapid solidifi-

cation and formability, and be affordable, abundant, and

commercially available with appropriate regulations for

Corresponding author: Ozbolat, I.T. ([email protected]).

clinical use [11]. A variety of bioinks, including naturally

Keywords: bioprinting; bioprinting for transplantation; vascularized-tissue printing;

in situ bioprinting. and synthetically derived materials, has been used for

0167-7799/ tissue , as detailed in the literature

ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2015.04.005 [12,13]. The post-bioprinting process is also crucial and

necessitates mechanical and chemical stimulation and

Trends in Biotechnology, July 2015, Vol. 33, No. 7 395

Opinion Trends in Biotechnology July 2015, Vol. 33, No. 7

signaling to regulate tissue remodeling and growth, the vascular tubes [19] using tissue spheroids as building

development of new bioreactor technologies enabling rapid blocks [26] that are printed inside a mold pattern; and

maturation of tissues, multiscale vascularization for sur- (ii) bioprinting of vasculature using direct extrusion of a

vivability of tissues, and mechanical integrity and inner- tubular network [16–18]. A recent study [3] demonstrated

vation for transplantation. hybrid biofabrication of vasculature in tandem with tissue

Although several researchers have studied bioprinting strands, where fibroblast tissue strands quickly fuse to

of tissue constructs, the fabrication of scale-up tissues with each other, maturate, and form the tissue around the

a high volumetric oxygen-consumption rate, such as cardi- vasculature (Figure 1C–E). Tissue strands were made

ac, , or tissue, is still a challenge. One major scaffold free and used as building blocks to construct the

roadblock is associated with the integration of the vascular scale-up tissue due to their quick fusion, folding, and

hierarchical network spanning arteries and veins down to maturation capabilities. This approach demonstrated the

capillaries. To bioprint vascularized thick tissues, highly proof of concept toward larger-scale perfusable tissues;

repeatable and straightforward technologies and protocols further work is needed to demonstrate a complex vascular

should be developed in logical steps, from simple to com- network within larger tissues with vascularization on

plex. Since it is difficult to print capillaries at the submi- multiple scales that can be envisioned using a Multi-

cron scale using current technology, an alternative could be Arm BioPrinter [27]. Although vascularization is impor-

to bioprint the macrovasculature and then leave nature to tant for larger-scale tissue constructs for transplantation,

create the capillaries. To this end, two alternative anastomosis to the circulatory system and functionality

approaches have been considered: (i) indirect bioprinting post-transplantation should also be considered. The vas-

by utilizing a fugitive ink that is removed by thermally cular network should be designed and bioprinted so that it

induced decrosslinking, leaving a vascular network behind can be sutured to a vascular network easily, and it should

[14,15]; and (ii) direct bioprinting of a vasculature network have certain properties, such as enough mechanical prop-

in a tubular shape [16–19] (Figure 1). erties to satisfy suture retention and burst pressure, suffi-

Several recent attempts have been made to bioprint a cient intactness of endothelium to prevent thrombosis, and

fugitive bioink to create vascular channels [15,20–22]. Cell a high patency rate to support occlusion-free circulation

laden were used to fabricate the tissue construct, [28]. Compared with indirect bioprinting of a vascular

and integration of the vascular network demonstrated network, the direct bioprinting of vasculature can be more

increased cell viability inside the construct; regions near convenient, suturing to the host at the time of implanta-

channels exhibited significant differences compared with tion.

regions away from channels. Although most researchers

have attempted to create a vascular network on a macro- From in vitro to in situ: regenerating tissues through

scale and generate an endothelium lining inside the lumen direct bioprinting into defect sites

via gluing endothelial cells through perfusion, Lee et al. Bioprinting living tissue constructs or cell laden scaffolds

[21] took one step forward and successfully achieved an- in vitro has been well studied, and thin tissues or tissues

giogenesis by sprouting endothelial cells within a fibrin that do not need vascularization, including skin, cartilage,

network loaded with other pericyte-like supporting cells and blood vessels, have been grown [12]. However, in situ

(Figure 1A,B). Their study demonstrated that endothelial bioprinting can enable the growth of thick tissues in critical

spouting generated a considerable increase in the perme- defects with the help of vascularization driven by nature in

ability of the tissue construct. More advanced angiogenesis lesions. Therefore, it is a promising direction for the bio-

and vasculogenesis have already been developed in micro- printing of porous tissue analogs that can engraft with

fluidic devices; several supporting cells have been endogenous tissue and regenerate new tissue along with

attempted and used in cancer metastasis studies [23]. De- vasculogenesis through the migration of progenitor cells

spite the flexibility in bioprinting channels and the ability into the tissue construct and sprouting of capillaries from

to create angiogenesis, this technology still faces major the endogenous tissue.

challenges. First, loading cells in hydrogels does not sup- The idea of in situ bioprinting was first proposed by

port cell–cell interactions, because those take place in vivo, Weiss using inkjet technology [29]; however, translating

and limited phenotypic stability and activity of cells are bioprinters into operating rooms was considered to be

observed during prolonged in vitro incubation. Second, challenging due to the perception that surgeons can be

while fibrin is a suitable environment for angiogenesis considered artists and prefer off-the-shelf solutions, such

because it has a crucial role in blood clotting [24], it is as using prefabricated tissue constructs and cutting or

not a convenient environment for tissue-specific cells, such carving them into a form to be implanted into the defect

as islets in a pancreas or follicles in a lymph node; a site. Limited research has been performed on in situ bio-

scaffold-free environment should be considered for these. printing since Weiss proposed this technology. Inkjet-

A recent article [25] demonstrated contiguous vasculariza- based bioprinting of skin cells has been tested for burn

tion of cell aggregates in tumor spheroid models and robust wounds [30], and laser-assisted bioprinting has been per-

angiogenesis into the fibrin matrix where spheroids were formed to test the feasibility of printing nanohydroxyapa-

encapsulated, showing the possibility of generating anas- tite particles on a mouse model [31]. The idea of bioprinting

tomosis of vascular networks of stromal and parenchymal skin cells (fibroblasts and keratinocytes zonally) has been

tissues in vitro. considered feasible for transitioning the technology to

The other approach is direct bioprinting of a vascular clinical settings, with the hope of repairing major wound

network via: (i) bioprinting of scaffold-free branched defects of soldiers on the battlefield.

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Opinion Trends in Biotechnology July 2015, Vol. 33, No. 7

(A) Vascular channels (C) Stage 1

Detachable Fibrin nozzle Co-axial Embed endothelial nozzle and supporng cells

Stage 2

Capillary network is formed (D) within fibrin scaffold Tissue

strands

Stage 1 Stage 2 Stage 3 Stage 3

Angiogenic sproung is formed Vasculature from vascular channels to the Key: capillary network Endothelial cells Assembly through Paral fusion of ssue Complete fusion of ssue Supporng cells hybrid bioprinng strands around the strands ghtly adhering vasculature to the vasculature (B) (E)

Capillary network Sproung

Endothelium

100µm

TRENDS in Biotechnology

Figure 1. Vascularized tissue construct bioprinting in vitro. In the first approach, (A) a fugitive ink is bioprinted to create vascular channels inside a collagen support

structure along with deposition of endothelial and fibroblast cells in a fibrin scaffold, resulting in capillary network formation in fibrin scaffold followed by endothelial

sprouting from vascular channel to capillary network in 9 days, where (B) a immunohistochemistry image shows integration of large sprouts from the parental vascular

channel (red) to the capillary network (green). In the second approach, (C) a hybrid bioprinting approach can be applied using a Multi-Arm BioPrinter, where (D) the tissue

assembly is created by placing the vasculature in the middle, resulting in fusion and maturation of scaffold-free tissue strands around the vasculature, further resulting in

(E) the tight fusion of the fibroblast tissue to the vasculature in 1 week [3]. The co-axial nozzle used in that study can enable continuous printing of a single lumen

vasculature throughout the fabrication of larger-scale hybrid tissue constructs. Reproduced and adapted, with permission, from [21] (B).

In situ bioprinting is challenging, and further system- and regulatory issues related to animals or humans neces-

atic research is required to take the technology into a sitating safe delivery of the tissue construct under anes-

robust state. There are major limitations associated with thesia. In addition, in situ bioprinting can sometimes

its biological, , and engineering aspects, such as increase the duration and cost of surgery.

printing difficulties on nonhorizontal surfaces, the need for Bioprinting ex vivo on explants can be considered a

highly advanced robotics bioprinters coupled with comput- transitional stage (Figure 2A) in which tissue constructs

er-aided design technologies (i.e., scanning the defect site can be built and engineered inside explants [32]. In situ

and providing an interactive user interface for surgeons), bioprinting is promising for developing tissue analogs

the requirement for a highly effective extrudable bioink directly on the defect model in operating rooms

enabling instant solidification in a living body (without the (Figure 2B), which paves the way to developing associated

need for an external solidifier, such as a ultraviolet light or enabling technologies for humans in the future. When the

a chemical crosslinker), the need for a biologically appeal- technology is translated into clinics, it will have several

ing ink for enhanced tissue formation, the requirement for benefits: (i) direct bioprinting of tissue constructs into

technologies that do not interfere with in vivo conditions defects can eliminate the need for preshaping or reshaping

397

Opinion Trends in Biotechnology July 2015, Vol. 33, No. 7 (A) Cells migrang (B) from defect

Printed cells In situ bioprinng Bioprinter nozzle

Defect

Bioprinter Explant

Bioprinter Fixture Bioprinter nozzle nozzle Stem cells Plasmid DNA Calvarial defects Rat

Bioink Printed ssue inside a carlage defect Biodegradable microparcle

(C)

In situ hybrid bioprinng in operang rooms Large defects

Dura substute Printed Printed frame(biological or made of synethc) composite biodegradable loaded with hard stem cells and genes

TRENDS in Biotechnology

Figure 2. In situ bioprinting. (A) Bioprinting cells into explants in situ can be considered an intermediate step toward (B) bioprinting tissue constructs directly into animal

models, where DNA can also be printed for bioprinting mediated to transfect and differentiate printed stem cells into multiple lineages. This will have a great

impact on (C) transitioning in situ bioprinting technology into operating rooms for humans in the near future; for example, large, deep calvarial defects can be regenerated

on a synthetic or biological Dura substitute, where a hard polymeric biodegradable frame can be printed along with stem cells and genes loaded in hydrogels to generate

the vascularized tissues. Image in (C) reproduced courtesy of Christopher Barnatt.

the scaffold based on the defect geometry. This can avoid When bioprinting stem or progenitor cells in situ, cells are

the laborious nature of scaffold preparation and the risks exposed to the natural environment with growth factors that

associated with contamination and limited activity of cells can induce their differentiation into the desired lineages;

in vitro; (ii) for bioprinting of cell-laden tissue constructs (iii) in situ bioprinting can enable the precise deposition of

for critical- or large-size defects, in situ bioprinting can cells, genes, or cytokines inside the defect, unlike manual

eliminate the need for differentiation of stem or progenitor interventions, such as prefabricated scaffolds, in which the

cells in vitro, which might be expensive and time consuming. shape can alter due to swelling, contraction, or deformations.

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Opinion Trends in Biotechnology July 2015, Vol. 33, No. 7

Localized control via bioprinting, such as printing different Two approaches are discussed: (i) in vitro bioprinting of

cytokines at different layers, is an asset for future bioprint- tissue and organ constructs with a focus on alternative

ing research; (iv) standard defects made by surgery tools are technologies in bioprinting a multiscale vascular network

easily addressed by manipulation and bioprinting of tissue in tandem with the rest of the tissue construct, and (ii) in situ

analogs. However, naturally occurring defects resulting bioprinting of tissue construct for regeneration of large

from trauma, surgical excision, or other issues are random defects that will one day enable the repair of body parts

in morphology and geometry and need to be captured pre- directly in patients in operating rooms. Although the tech-

cisely; laser- or image-based scanning systems can overcome nology shows a great deal of promise for bioprinting at

this challenge. In situ bioprinting can eliminate the need for organ-level complexity, there is still a long way to go to

multiple operations because the tissue constructs can be realize this ambitious vision. There is a need for advance-

bioprinted into the defect immediately; and (v) twisting ment in several areas, including new cell sources and stem

multi-axis robotic arms can enable angled deposition and cell technology, novel bioinks, and advanced fully automat-

printing of the bioink into nonhorizontally oriented defects. ed bioprinter technologies and bioprinting processes. Over-

Defects on the live model can be at random locations, and it is coming current impediments in these technologies along

not convenient for the surgeon to change the position of the with advancements in in vivo integration is essential for

anesthetized model during bioprinting. developing seamlessly automated technology from autolo-

As a result of these benefits, in situ bioprinting of tissue gous stem cell isolation to tissue or organ construct trans-

analogs can be applied to various sites on the body, such as plantation.

deep dermal or extremity injuries and calvarial or cranio-

facial defects during maxillofacial or neurosurgeries. In the Acknowledgments

future, in situ bioprinting technology could be considered This work has been supported by National Science Foundation Awards

CMMI 1462232, CAREER 1349716, Diabetes in Action Research and

for humans (Figure 2C). For large calvarial defects that

Education Foundation, and the Grow Iowa Values Funds. The author

need a great deal of microvascularization along with struc-

would like to express his gratitude to G. Dai, Christopher Barnatt, Yin

tural support, one can envision bioprinting a frame (using

Yu, and Kerim Moncal for providing some of the high-quality images in

hard osteoconductive , such as a blend of poly- the figures.

caprolactone, , and ) in tandem

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