3D Bioprinting of Vascularized Tissues for in vitro and in vivo Applications

Abstract

With a limited supply of organ donors and available organs for transplantation, the aim of tissue engineering with three-dimensional (3D) bioprinting technology is to construct fully functional and viable tissue and organ replacements for various clinical applications. 3D bioprinting allows for the customization of complex tissue architecture with numerous combinations of materials and printing methods to build different tissue types, and eventually fully functional replacement organs. The main challenge of maintaining 3D printed tissue viability is the inclusion of complex vascular networks for nutrient transport and waste disposal. Rapid development and discoveries in recent years have taken huge strides toward perfecting the incorporation of vascular networks in 3D printed tissue and organs. In this review, we will discuss the latest advancements in fabricating vascularized tissue and organs including novel strategies and materials, and their applications. Our discussion will begin with the exploration of printing vasculature, progress through the current statuses of bioprinting tissue/organoids from bone to muscles to organs, and conclude with relevant applications for in vitro models and drug testing. We will also explore and discuss the current limitations of vascularized tissue engineering and some of the promising future directions this technology may bring.

Keywords: 3D bioprinting; additive manufacturing; bioprinting; tissue engineering; vasculature.

Figure 1

Bioprinting process and techniques. (A) The typical workflow of bioprinting starts with the choosing the right type of cells, then culturing cells and preparing the bio-ink, printing the desired cell-laden scaffold, and finally used for transplantation, drug testing, or in vitro studies. (B) Inkjet bioprinters produce small droplets of hydrogel and cells in a sequential manner to construct tissues. (C) Laser-assisted bioprinters focuses a light source onto a donor layer (top) which propels the cells onto the print (arrow indicates direction of laser source). (D) Extrusion-based bioprinting produces a continuous supply of hydrogel and cells. (E) Stereolithography bioprinting uses digital light sources to selectively crosslink bio-inks layer by layer (arrows indicate direction of projected light). Reprinted with permission from Biotechnol. Adv. (Mandrycky et al., 2016), c 2021.

Figure 2

Blood vessel diameters, cell types, and compositions. Capillaries are typically made up of pericytes surrounding an extracellular lining. Arterioles and venules are surrounded by smooth muscle cells. Arteries and veins consist of fibroblasts surrounding a layer of smooth muscle cells. Reprinted with permission from Nat. Sci. Rep. (Schöneberg et al., 2018), c 2021.

Figure 3

Schematic diagram of the bioprinting process. First, a framework was fabricated with polydopamine-modified calcium silicate (PDACS)/poly-caprolactone (PCL) composite to support the entire mechanical stability. Second, the alginate/gelatin hydrogels encapsulated human umbilical vein endothelial cells (HUVEC) were dispensed into the pores. The Wharton’s jelly mesenchymal cells (WJMSC) were printed on the PDASC/PCL scaffold with the piezoelectric needle. The sequential dispensing of PDACS/PCL composite, hydrogel, and cells was repeated and stacked to build the three-dimensional (3D) scaffold (10 layers). Reprinted with permission from Mater. Sci. Eng. C Mater. Biol. Appl. (Chen et al., 2018), c 2021.

Figure 4

Schematic illustration for 3D hydrogel construct. (A) Schematic illustration of fabrication process for 3D hydrogel construct. (B) A photograph of 3D hydrogel constructs for vascular and bone formation. Reprinted with permission from Int. J. Mol. Sci. (Anada et al., 2019), c 2021.

Figure 5

Schematic of the bioplotting technique. (A) Schematic of the bioplotting technique used to print wood-pile scaffolds with vertical and lateral pores; a sacrificial support material provides a support to the biomaterial ink during the printing avoiding the collapse of the scaffold. (B) Wood-pile scaffold CAD model designed. (C) Schematic of the process used to add HUVECs to the bone construct, filling the whole interconnected pore network within the scaffolds. Reprinted with permission from Biofabrication (Chiesa et al., 2020), c 2021.

Figure 6

Process of making a 3D bioprinting (3DP) scaffold. (A) Schematic of a 3DP scaffold. Osteon-like fibrin hydrogels are co-printed with PCL for support. (B) Images of samples after 14 days implantation in vivo. (C) Micrographs of embedded, sectioned, and stained samples using Masson Trichrome at day 14 after implantation. Collagen is stained blue, cell nuclei are stained dark purple, and fibrin is stained pink. Black arrows indicate blood vessels. Reprinted with permission from Biofabrication (Piard et al., 2019), c 2021.

Figure 7

Histology of calvaria. (A) Histological examination of decalcified calvaria defects stained with HES staining. The left and right columns show histological images of regeneration area 1 and 2 months post-printing, respectively. NLB, native lamellar bone; nb, neoformed bone. White dash lines show the border of the calvaria defect. (B) Histological examination of decalcified calvaria defects stained with hematoxylin-eosin-saffron staining to assess vascularization. The left column shows histological images of regeneration areas 1 month post-printing. The middle and right columns, respectively, display histological images of regeneration areas 2 months post-printing and the magnification of the red squared areas. Black arrows indicate blood vessels. Reprinted with permission from Biofabrication (Kérourédan et al., 2019b), c 2021.

Figure 8

3D cell printing of skeletal muscle construct. (A) Schematic illustration of the decellularized extracellular matrix (dECM) bioink preparation, muscle construct fabrication, and volumetric muscle loss (VML) treatment. (B) Design of muscle construct. (C) 3D cell printing of muscle construct using a granule-based reservoir system. (D) 3D cell printed muscle construct. Reprinted with permission from Biomaterials (Choi et al., 2019), c 2021.

Figure 9

Bioprinting using organ building blocks. (A) Step-by-step illustration of bioprinting using organ building blocks (OBBs). (B) An image sequence showing the embedded 3D printing of a branched, hierarchical vascular network within a compacted EB-based tissue matrix connected to inlet and outlet tubes, seen entering the tissue from the left and right. Reprinted with permission from Sci. Adv. (Skylar-Scott et al., 2019), c 2021.

Figure 10

Skin bioprinter prototype and in situ bioprinting concept. (A) Schematic demonstrating scale, design, and components of the skin bioprinter. (B) The main components of the system consist of 260 μm diameter nozzles, driven by up to eight independently dispensing systems connected to a print-head with an XYZ movement system, in addition to the 3D wound scanner. All components are mounted on a frame small enough to be mobile in the operating room. (C) Skin bioprinting concept. Wounds are first scanned to obtain precise information on wound topography, which then guides the print-heads to deposit specified materials and cell types in appropriate locations (Images courtesy of LabTV – National Defense Education Program, Washington, DC, United States). (D) Example of skin bioprinting process, where markers that are placed around the wound area used as reference points (a) prior to scanning with a hand-held ZScanner™ Z700 scanner (b). Geometric information obtained via scanning is then input in the form of an STL file to orient the scanned images to standard coordinate system (c). The scanned data with its coordinate system is used to generate the fill volume and the path points for nozzle head to travel to print the fill volume (d). Output code is then provided to the custom bioprinter control interface for generation of nozzle path needed to print fill volume (e,f). Reprinted with permission from Sci. Rep. (Albanna et al., 2019), c 2021. (E) This system facilitates the depositing of multiple cell types with high precision and control. Layering of fibroblasts (green) and keratinocytes (red) is shown.

Figure 11

The 3D intestinal model. (A) the designed 3D intestinal model. (B) Optical (transparent, epithelium; and pink, capillary) images of the crypt and villus regions for the fabricated intestinal model. Reprinted with permission from ACS Appl. Mater. Interfaces (Han et al., 2020), c 2021.

Figure 12

Angiogenesis of tumor spheroids. Schematic describing the morphological changes and angiogenesis of tumor spheroids of glioblastoma cells seeded onto the vascularized tissue. Reprinted with permission from Int. J. Mol. Sci. (Han et al. 2020), c 2021.

Figure 13

Vascular network formation on the 3D cell printed vascular platform (VP). (A) Design of VP for induction of natural 3D vascular network. (B) Confocal microscope images of z-stacked 3D vascular network formed on VP (top panel, left, scale bar: 100 μm) and EC junctions formed on the surface of vascular network (top panel, right, scale bar: 50 μm). Confocal microscopy images of vascular tube formation by z-stacked (bottom left) and transverse cross-sectional (bottom right, scale bar: 50 μm) views. Reprinted with permission from Biofabrication (Park et al., 2018), c 2021.

Figure 14

Chip designs used for the biofabrication of printed tissue frameworks with on-chip vascular-like channels. Chips designs with (A) ready-made channels and (B) sacrificial gel-made channels were evaluated. Sequence of events (1–5) indicates the necessary working steps to fabricate both types of tissue models. Sacrificial gel-made channels can be used to incorporate endothelial cells that will form monolayers on the channel surface after removing the sacrificial gel. The inset photograph in (A) shows the channel formation at step 5. The inset photograph in (B) shows the presence of endothelial cells inside the sacrificial gel at step 3. Reprinted with permission from Front. Bioeng. Biotechnol. (Duarte Campos et al., 2020a), c 2021.

Figure 15

Flowchart for building tissue-chip-models for glioblastoma. Step 1, GBM cells are isolated from a specimen obtained through removal surgery. Step 2, off-the-shelf porcine BdECM bioink is obtained. Step 3, patient-derived cancer cells are printed with the BdECM bioink to produce a patient-specific GBM-on-a-chip. To mimic the heterogeneous GBM ecology, several other inks are used in the printing process, including a vascular cell-laden BdECM bioink and a silicone ink. Step 4, the chip is cultured for 1–2weeks to recapitulate the pathological features. Step 5, various candidate drug combinations are tested using the chip. Step 6, the drug combinations are prioritized according to their efficiencies and the best combination is identified. Step 7, the physician uses the test results to design a treatment plan for the patient. Reprinted with permission from Nat. Biomed. Eng. (Yi et al., 2019), c 2021.