Noninvasive in vivo 3D bioprinting

Abstract

Three-dimensional (3D) printing technology has great potential in advancing clinical medicine. Currently, the in vivo application strategies for 3D-printed macroscale products are limited to surgical implantation or in situ 3D printing at the exposed trauma, both requiring exposure of the application site. Here, we show a digital near-infrared (NIR) photopolymerization (DNP)-based 3D printing technology that enables the noninvasive in vivo 3D bioprinting of tissue constructs. In this technology, the NIR is modulated into customized pattern by a digital micromirror device, and dynamically projected for spatially inducing the polymerization of monomer solutions. By ex vivo irradiation with the patterned NIR, the subcutaneously injected bioink can be noninvasively printed into customized tissue constructs in situ. Without surgery implantation, a personalized ear-like tissue constructs with chondrification and a muscle tissue repairable cell-laden conformal scaffold were obtained in vivo. This work provides a proof of concept of noninvasive in vivo 3D bioprinting.

Fig. 1. Schematic diagram of DNP-based noninvasive 3D bioprinting.

The data of a customized CAD model were sent into the DMD chip through a control computer. The 980-nm NIR light with an optical pattern was casted across optical lens, and tissue onto the bioink, which was injected into the body to noninvasively fabricate a living tissue in vivo. The bioink contains UCNP@LAP nanoinitiators that can convert the NIR light to 365-nm light and then initiate the optical pattern–controlled polymerization of monomers.

Fig. 2. Characterization of UCNP@LAP nanoinitiators.

(A) Transmission electron microscopy images of aqueous UCNPs and UCNP@LAP nanoinitiators. UCNP@LAP nanoinitiators show an obvious shell structure. Scale bars, 20 (top) and 50 nm (bottom). (B) Elemental mapping of a single UCNP@LAP nanoinitiator, indicating the distribution of Y and P elements. Scale bar, 20 nm. (C) Thermogravimetric analysis (TGA) curves of UCNPs (black line) and UCNP@LAP nanoinitiators (red line) of the analyses performed under N₂ atmosphere. (D) Absorption spectrum of LAP (blue line) and up-conversion luminescence emission spectra of UCNPs (purple line) and UCNP@LAP nanoinitiators (black line) in aqueous solution upon 980-nm excitation. a.u., arbitrary units. (E) Degree of photocuring ratio of GelMA (15 wt %) versus time under 2-W NIR light for various concentrations of UCNP@LAP nanoinitiators: 0.5 (black), 1 (red), and 2 wt % (blue). (F) Degree of photocuring ratio versus time using UCNP@LAP (1 wt %) as the PIs for various powers of NIR light: 1.5 (black), 2.5 (red), and 3.5 W (blue). PL, photoluminescence.

Fig. 3. 3D bioprinting acellular constructs using DNP-based process in vitro.

(A) Scanning electron microscopy (SEM) images of fabricated constructs including three-ring microconstructs with decreasing widths, flower-like, Danboard-like, round cake-like, and a type of truss constructs. Scale bars, 200 μm. (B) Schematic diagram of printing setup used to estimate the tissue-penetration capacity. The bioink was deposited under the skin or muscle. (C) The images of ring constructs printed from bioink (control) or bioink covered over by skin or 0.5-mm-thick muscle by DNP process. Scale bars, 0.5 cm. Photo credit: Yuwen Chen, State Key Laboratory of Biotherapy and Cancer Center.

Fig. 4. 3D bioprinting acellular constructs by DNP-based process in vivo.

(A) H&E of the surrounding tissue of construct after DNP printing in vivo for 1 and 7 days. The arrow represents the printed construct. Scale bar, 100 μm. (B) CAD models and vitamin B12–stained triangle, cross, and two-layer cake-like constructs fabricated by noninvasive DNP-based process in vivo. Scale bar, 0.5 cm. Photo credit: Jiumeng Zhang, State Key Laboratory of Biotherapy and Cancer Center.

Fig. 5. Noninvasive 3D bioprinting ear-like tissue by the DNP-based process.

(A) Representative image of the normal ear. (B) Mirror image of (A). (C) Optimized ear-outline image of (B). (D) Image of printed ear-like construct from the bioink covered over by skin by DNP process. Scale bar, 2 mm. (E) The Live/Dead staining for ear constructs encapsulated with chondrocytes bioprinted from bioink covered by skin after culture for 7 days. Scale bar, 2 mm. (F) Noninvasive 3D bioprinting of ear-shaped construct in vivo by DNP-based process. The ear-shaped construct was printed subcutaneously in BALB/c nude mice. Scale bar, 5 mm. (G) Representative image of bioprinted ear-shaped construct at 1 month. Scale bar, 5 mm. (H) H&E and (I) collagen type II immunostaining of retrieved ear-shaped construct at 1 month. Scale bars, 50 μm. Photo credit: Yuwen Chen, State Key Laboratory of Biotherapy and Cancer Center.

Fig. 6. Noninvasive 3D bioprinting conformal ASC-laden scaffold for muscle defect repair by the DNP-based process.

(A) Schematic illustration of the conformal ASC-laden scaffold for muscle defect repair. (B) Representative images exhibit acceleration of the wound healing of the DNP group compared with control. Scale bar, 5 mm. (C) Percent closure of muscle wounds evaluated at day 10. **P < 0.01, n = 5. (D) H&E histological analysis of muscle wound healing at day 10 after treatments. Scale bar, 50 μm. Photo credit: Yuwen Chen, State Key Laboratory of Biotherapy and Cancer Center.