Double-network hydrogel particles, methods of making double-network hydrogel particles, and methods of making objects

Abstract

The present disclosure provides for double-network hydrogel particles, methods of making double-network hydrogel particles, methods of making objects (e.g., three-dimensional objects) using double-network hydrogel particle, and the like. In an aspect, double-network hydrogel particles are viscoelastic voxels (e.g., spherical or semi-spherical) that can be precisely manipulated in three-dimensional space and can be used in both soft matter science and biomanufacturing. The present disclosure provides for a voxelated bioprinting technology that enables the digital assembly of interpenetrating double-network hydrogel particles to form objects, in particular three-dimensional objects.

Description

[0001] DOUBLE-NETWORK HYDROGEL PARTICLES, METHODS OF MAKING DOUBLENETWORK HYDROGEL PARTICLES, AND METHODS OF MAKING OBJECTS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. provisional application entitled “Voxelated Bioprinting of Modular Double-Network Bio-Ink Droplets” having serial number 63 / 538,260 filed on September 13, 2023, which is entirely incorporated herein by reference.

[0004] STATEMENT ON FUNDING PROVIDED BY THE U.S. GOVERNMENT

[0005] This invention was made with Government support under Grant No. 2306012 awarded by the National Science Foundation. The Government has certain rights in the invention.

[0006] BACKGROUND

[0007] Additive manufacturing, also known as three-dimensional (3D) printing, can contribute to innovations in many areas, such as engineering, manufacturing, art, education, and medicine. One approach to 3D printing involves digital assembly of spherical particles (DASP). This technique facilitates placement of individual spherical particles at predetermined locations within a 3D space. A structure can be built up by adding more spherical particles to the predetermined locations to form a variety of structures of specified shapes, sizes, and densities.

[0008] SUMMARY

[0009] The present disclosure provides for double-network hydrogel particles, methods of making double-network hydrogel particles, methods of making objects (e.g., three- dimensional objects) using double-network hydrogel particle, and the like.

[0010] In an aspect, the present disclosure provides for a double-network hydrogel particle, comprising: a first polymer network-scaffold and a second polymer network-scaffold, wherein the first polymer network-scaffold and the second polymer network-scaffold are independent polymer network-scaffolds, wherein the first polymer network-scaffold and the second polymer network-scaffold form a double scaffold-network polymer composition, wherein the first polymer network-scaffold includes a reaction product of a A-polymer and a B-polymer, wherein the A-polymer includes a plurality of first click chemistry units and a polymer backbone, wherein the B-polymer includes a plurality of second click chemistry units and a polymer backbone, wherein the first click chemistry unit and the second click chemistry unit react with one another to form a click-chemistry reaction crosslink between the A-polymer and the B-polymer, wherein the second polymer network-scaffold is a reaction product of alginate polymers in the presence of a divalent cation. In an aspect, the A-polymer is selected from the group consisting of: a norbornene(NB)-polymer, an azide-polymer, a thiolpolymer, and a maleimide-polymer; and the B-polymer is selected from the group consisting of: a TZ-polymer, an alkyne-polymer, an alkene-polymer, a thiol-polymer. In an aspect, the A-polymer is a norbornene(NB)-polymer and the B-polymer is a tetrazine (TZ)-polymer , wherein the TZ-polymer includes a plurality of TZ units and a polymer backbone, wherein the NB-polymer includes a plurality of NB units and a polymer backbone, wherein the TZ unit and the NB units react with one another to form a click-chemistry reaction crosslink between the TZ-polymer and the NB-polymer.

[0011] The present disclosure provides for a three-dimensional object, comprising a plurality of double-network hydrogel particles as described above and herein.

[0012] The present disclosure provides for a method of making three-dimensional object, comprising: a) mixing an first alginate polymer with a B-polymer to form a first mixture; b) mixing an second alginate polymer with a A-polymer to form a second mixture, wherein the first alginate polymer and the second alginate polymer are identical; c) introducing, from a first inlet, the first mixture to a mixing area and introducing, from a second inlet, the second mixture to the mixing area, to form a third mixture, wherein a first polymer network-scaffold is formed by a reaction of the B-polymer and the A-polymer, wherein the B-polymer includes a plurality of second click chemistry units and a polymer backbone, wherein the A-polymer includes a plurality first click chemistry units and a polymer backbone, wherein the first click chemistry unit and the second click-chemistry unit react with one another to form a clickchemistry reaction crosslink between the A-polymer and the B-polymer; and d) flowing the third mixture out of a print nozzle to form a plurality of double-network hydrogel particle of any one of claims 1 to 17 to form the three-dimensional object, where each double-network hydrogel particle is adjacent at least one other double-network hydrogel particle.

[0013] The present disclosure provides for a method of making three-dimensional object, comprising: a) mixing an first alginate polymer with a tetrazine(TZ)-polymer to form a first mixture; b) mixing an second alginate polymer with a norbornene(NB)-polymer to form a second mixture, wherein the first alginate polymer and the second alginate polymer are identical; c) introducing, from a first inlet, the first mixture to a mixing area and introducing, from a second inlet, the second mixture to the mixing area, to form a third mixture, wherein a first polymer network-scaffold is formed by a reaction of the TZ-polymer and the NB-polymer, wherein the TZ-polymer includes a plurality of TZ units and a polymer backbone, wherein the NB-polymer includes a plurality of NB units and a polymer backbone, wherein the TZ unit and the NB units react with one another to form a click-chemistry reaction crosslink between the TZ-polymer and the NB-polymer; and d) flowing the third mixture out of a print nozzle to form a plurality of double-network hydrogel particle of any one of claims 1 to 17 to form the three-dimensional object, where each double-network hydrogel particle is adjacent at least one other double-network hydrogel particle.

[0014] BRIEF DESCRIPTION OF DRAWINGS

[0015] Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

[0016] Figs. 1A and 1 B illustrate the digital assembly of spherical particles (DASP) made of modular double-network bio-inks. Fig. 1 A illustrates that DASP uses spherical bio-ink droplets, not conventional one-dimensional (1D) bio-ink filaments, as the building blocks to create three-dimensional (3D) objects. In DASP printing, (i) droplets are deposited at precisely controlled locations and with prescribed volume in a sacrificial supporting matrix made of an aqueous yield-stress fluid, (ii) Under controlled swelling, the deposited droplets partially coalesce with their neighbors, (iii) Following a complete crosslinking of droplets and the removal of the sacrificial matrix, the printed particles form a free-standing 3D construct made of interconnected yet distinguishable hydrogel particles. Fig. 1 B illustrates that DASP 2.0 enables printing modular double-network (DN) bio-inks. The ink consists of alginate and poly(acrylamide) (PAM), which is functionalized with either norbornene (NB) or tetrazine (TZ) groups, (i) In an aqueous solution, the NB-PAM (blue) and TZ-PAM (red) form a network via click reaction between NB and TZ at RT without any external trigger, while alginate forms a network via ionic crosslinking, (ii) A custom-designed multi-channel print nozzle is developed to enable printing DN bio-inks. The nozzle mixes a pair of bio-inks, TZ-PAM (red arrow) / alginate (green arrow) + NB-PAM (blue arrow) / alginate (green arrow), homogeneously without damaging cells, (iii) In a DN hydrogel, the ionically crosslinked alginate network interpenetrates with the PAM network.

[0017] Figs. 2A-2F illustrates crosslinking kinetics and stiffness of single-network PAM hydrogels crosslinked through biofriendly click chemistry. Fig. 2A illustrates real-time characterization of storage (G', thick lines) and loss (G", thin lines) moduli for mixtures of NB- PAM and TZ-PAM at various molar ratios between NB and TZ groups. The concentration of TZ-PAM and NB-PAM solution are both fixed at 10% (w / v) before mixing. For TZ-PAM, the grafting ratio of TZ is fixed at 2.53%, defined as the molar ratio between the grafted tetrazine and the total number of chemical monomers on the PAM backbone. This value corresponds to on average of 52 TZ groups per polymer. In contrast, for NB-PAM, the grafting ratio of NB groups varies from 0.63 to 4.25%. The gelation time, tg, is defined as the point above which G' surpasses G". All measurements are conducted at RT, using a fixed strain 0.5% and an oscillatory shear frequency 1 Hz. Fig. 2B illustrates the dependencies of gelation time, tg, on the NB / TZ ratio at fixed polymer concentration of 10% (w / v) and on the polymer concentration, c, at fixed NB / TZ ratio of 1 . Fig. 2C illustrates the dependencies of storage modulus, G' , at gelation time, tg, on the NB / TZ ratio at fixed polymer concentration of 10% (w / v) and on the polymer concentration, c, at fixed NB / TZ ratio of 1. Fig. 2D illustrates the dependencies of storage (G', thick lines) and loss (G", thin lines) moduli of a completely crosslinked single-network PAM hydrogels on the oscillatory shear frequency. In all hydrogels, both the TZ-PAM and NB-PAM polymers have roughly the same grafting ratio of 2.5%, and they are mixed at the same concentrations ranging from 1.25 to 15% (w / v). Fig. 2E illustrates the dependencies of the experimentally measured (solid squares) and the theoretically predicted (dash line) shear modulus G for PAM hydrogels on the concentration, c, of PAM polymers, c’, overlap concentration of PAM solutions; c**, the crossover concentration at which the correlation length, is about the average distance between two neighboring functional groups on the same the polymer chain, dx. For c > c”, < dx. Fig. 2F illustrates a schematic of our theory explaining the relation between the concentration of PAM polymers and the network shear modulus. In a crosslinked network, the mesh size axexhibits two distinct regimes: ~ f ; (2) for c > c“, axbecomes saturated and ax® dx. The network modulus G ® kBT / ax, as indicated by the dashed line in Fig. 2E.

[0018] Figs. 3A-3E illustrates the stiffness and extensibility of DN polyacrylamide / alginate (PAM / AIg) hydrogels. The grafting ratio of TZ-PAM and NB-PAM is fixed at 2.5% unless otherwise specified. Fig. 3A illustrates dependencies of storage (G', solid lines) and loss (G”, dashed lines) moduli of completely crosslinked single-network PAM, single-network alginate (Alg), and double-network PAM / alginate hydrogels on oscillatory shear frequency. The composition of a crosslinked hydrogel is denoted as PAMxAlgy, where x is the concentration in (w / v)% for PAM consisting of equal amount of TZ-PAM and NB-PAM, and y is the concentration of alginate. Fig. 3B illustrates the equilibrium shear moduli of single-network PAM, single-network alginate, double-network PAM / alginate hydrogels. Fig. 3C illustrates photos of PAM10, Alg2, and PAMi0Alg2hydrogels under uniaxial tensile tests at a fixed strain rate of 0.02 / sec. For each formulation, the left, middle, and right panels are, respectively, captured at the beginning of the tensile test, right before breaking, and right after breaking. Samples are 13 mm in length and 2 mm in width of the center part. Fig. 3D illustrates stressstrain behavior of hydrogels. Dashed red line: a PAMwAlg2double-network hydrogel made from TZ-PAM with a grafting ratio of 2.53% and NB-PAM with a grafting ratio of 0.63%, which is denoted as PAMi01 / 4Alg2. Fig. 3E illustrates a two-parameter (G, e^) diagram-of- state that outlines the mechanical properties of all hydrogels formulations in (d). Empty circle corresponds to the hydrogel PAMioAlg2with mismatched TZ and NB grafting ratios in Fig. 3D, which is denoted as PAMio1 / 4Alg2. Fig. 4A-4F illustrates 3D printing of DN hydrogels. Fig. 4A illustrates the dependence of viscosity of the PAM (10%, w / v), Alg (2%, w / v), and the corresponding DN bio-ink, PAMioAlg2, on the shear rate, y, at RT. Fig. 4B illustrates a dual-inlet print nozzle with a static mixer chamber, (i) A 3D rendering of the static mixer chamber, (ii) Photos of the nozzle with a pair of DN PAM Alg2 bio-inks, flowing through the mixer chamber. The two bio-inks contain red and green microparticles, respectively, with a diameter of 50 pm. The left, middle, and right panels of (ii) are sequential photos when only the red channel flows, both channels flow, and only the green channel flows, respectively. Scale bar, 1 mm. Fig. 4C illustrates a DASP printed hollow sphere consisting of 42 interconnected yet distinguishable DN PAMi0Alg2hydrogel particles. Panels (i), (ii), and (iii) display the front view, computer- rendered model, and the hollow sphere that is cut into two pieces. Scale bar for panel (iii) is 1 mm. Fig. 4D illustrates a gyroid structure created by conventional printing that uses DN PAMioAlg2hydrogel filaments as building blocks. Panels (i), (ii), and (iii) respectively depict the 45° view, computer-rendered 3D model, and side view of the gyroid. Scale bar for panel (iii) is 5 mm. Fig. 4E illustrates photos of the gyroid structure from Fig. 4D undergoing cyclic compression testing with a compression strain rate of 0.005 / sec. Panels (i), (ii), (iii), and (iv) are captured at 0%, 30%, 60% compression strain, and full release in the first compression cycle. Scale bar, 5 mm. Fig. 4F illustrates mechanical behavior of the gyroid structures during cyclic compression. The gyroids are made of DN PA i0Alg2(solid lines) and singlenetwork Alg2(dashed lines) hydrogels. Insert: a zoom-in view of the compression curves.

[0019] Figs. 5A-5H illustrate cytocompatibility and in vivo stability of DASP printed DN polyacrylamide / alginate (PAM / AIg) scaffolds. The single-network and DN scaffolds are respectively made of Alg4and PAMi0Alg2hydrogels. Fig. 5A illustrates a strategy that utilizes PEG / dextran aqueous two-phase system to encapsulate cells during DASP printing, (i) A 3D rendering of the print nozzle for cell encapsulation. The print nozzle consists of three channels: one channel loaded with cell suspension in dextran (green flow) and two channels respectively loaded with the pair of bio-inks mixed with PEG (red and blue flow). During the extrusion, the bio-ink flows first encounter with the immiscible flow of the cell suspension and subsequently mix in the static mixer chamber, (ii) A schematic of the extruded bio-ink in which the cell suspension phase (green) is separated from the bio-ink phase (red), (iii) A representative microscopy image showing the distribution of cell-mimicking polystyrene microparticles in the extruded bio-ink. Scale bar, 50 pm. Fig. 5B illustrates a representative fluorescence confocal microscopy image from live / dead assay of DASP encapsulated Beta- TC-6 cells. Scale bar, 100 pm. Fig. 5C illustrates the viability of naked and DASP encapsulated Beta-TC-6 cells up to 3 days. Fig. 5D illustrates a glucose stimulation index of naked and DASP Beta-TC-6 cells in 3 days. Results in Fig. 5C and Fig. 5D are shown as mean ± standard deviation (S.D.) with sample size n=5 wells for naked Beta-TC-6 and n=5 scaffolds for DASP Beta-TC-6 in PAM / Alg. Fig. 5E illustrates an animal model for testing the mechanical robustness of DASP printed DN scaffolds in vivo, (i) A schematic of transplanting a DASP printed 5*5*4 lattice scaffold into the abdominal cavity of immunocompetent C57BL / 6 mice, (ii) A representative photograph of the surgery. Fig. 5F illustrates a representative photos of the scaffolds made of pure alginate hydrogel (i) before the transplantation and (ii) after being retrieved. Scale bars, 1 mm. Fig. 5G illustrates a representative photographs of the scaffolds made of DN hydrogel (i) before the transplantation, (ii) after being retrieved, (iii) located in the abdominal cavity (dashed circles), and (iv) under the digital microscopy after being retrieved. Scale bars, 1 mm. Fig. 5H illustrates the mechanical behavior of scaffolds retrieved at different time points under compression.

[0020] Fig. 6A-6G illustrates mechanical properties, printability, and biocompatibility of a DN hyaluronic acid / alginate (HA / AIg) bio-ink. Fig. 6A illustrates HA is functionalized with either norbornene (NB) or tetrazine (TZ) groups. Upon mixing, TZ-HA and NB-HA are crosslinked via the click reaction between TZ and NB groups, following the same crosslinking mechanism as that for creating a PAM network (Fig. 1 B). The grafting ratio of TZ-HA and NB-HA are both fixed at 27%. Fig. 6B illustrates dependencies of storage (G', solid lines) and loss (G", dashed lines) moduli of completely crosslinked single-network HA and DN HA / alginate hydrogels on oscillatory shear frequency. Single-network hydrogel (HA): TZ-HA (2%, w / v) + NB-HA (4%, w / v). The DN hydrogel (HA / alginate) is prepared by mixing a pair of bio-inks, TZ-HA (2%, w / v) I alginate (2%, w / v) + NB-HA (4%, w / v) / alginate (2%, w / v). Fig. 6C illustrates representative photos of the DN HA / alginate hydrogel under uniaxial tensile test at a fixed strain rate of 0.02 / sec. The left, middle, and right panels, respectively, are captured at the beginning of the tensile test, right before fracture, and right after fracture. Samples are 13 mm in length and 2 mm in width of the center part. Fig. 6D illustrates stressstrain behavior of the single-network HA and DN HA / alginate hydrogels in Fig. 6B. Fig. 6E illustrates a DASP printed 5*5*4 lattice scaffold consisting of 100 interconnected yet distinguishable DN HA / alginate hydrogel particles. Upper panel: top view; lower panel: side view. Scale bar, 1 mm. Fig. 6F illustrates cytocompatibility of the DN HA / alginate bio-ink. (i) A pair of HA / alginate bio-inks pre-loaded with cells are printed using a two-channel print nozzle, (ii) A representative fluorescence confocal microscopy image from live / dead assay of DASP encapsulated Beta-TC-6 cells within the DN HA / alginate hydrogel. Scale bar, 100 pm. (iii) Viability of naked and DASP encapsulated Beta-TC-6 cells within the DN HA / alginate hydrogel up to 3 days. Results are shown as mean ± standard deviation (S.D.) with sample size n=5 wells for naked Beta-TC-6 and n=5 scaffolds for DASP Beta-TC-6 in HA / Alginate. Fig. 6G illustrates representative photographs of the scaffolds made of DN HA / alginate hydrogel (i) before the transplantation, (ii) after being retrieved at 30 days. Scale bar, 1 mm. Fig. 7 illustrates a1H NMR spectrum of tert- butyl A / -(4-cyanobenzyl)carbamate.

[0021] Fig. 8 illustrates a1H NMR of tert-butyl A / -[4-(6-methyl-1 ,2,4,5-tetrazin-3- yl)benzyl]carbamate.

[0022] Fig. 9 illustrates a1H NMR of [4-(6-methyl-1 ,2,4,5-tetrazin-3-yl) phenyl]methanamine.

[0023] Fig. 10 illustrates a1H NMR of tetrazine modified poly(acrylamide-co-acrylic acid).

[0024] Fig. 11 illustrates a1H NMR of norbornene modified poly(acrylamide-co-acrylic acid).

[0025] Fig. 12 illustrates a1H NMR of tetrazine modified hyaluronic acid.

[0026] Fig. 13 illustrates a1H NMR of norbornene modified hyaluronic acid.

[0027] Fig. 14 illustrates swelling of a bulk DN hydrogel. Photos of the DN PAM Alg2hydrogel in DMEM media up to 24h. Scale bar, 10 mm.

[0028] Fig. 15 illustrates dynamic mechanical properties of a double-network hydrogel. Dependencies of storage (G', solid line) and loss (G", dashed line) moduli of completely crosslinked double-network PAM^4Alg2with a mismatched TZ and NB grafting ratio. PAM^4Alg2is made from TZ-PAM with a grafting ratio of 2.53% and NB-PAM with a grafting ratio of 0.63%. The composition of a crosslinked hydrogel is denoted as PAMJAlgy, where x is the concentration in (w / v)% for PAM consisting of equal amount of TZ-PAM and NB-PAM, y is the concentration in (w / v)% for alginate, and z is the molar ratio between NB and TZ groups.

[0029] Figs. 16A-16C illustrates setups of the DASP 2.0. Fig. 16A illustrates an assembly of the extrusion module, syringes, and extrusion nozzle. Fig. 16B illustrates (i) Schematic and (ii) photograph of the dual-inlet mixing module. Fig. 16C illustrates (i) Schematic and (ii) photograph of the triple-inlet mixing module.

[0030] Fig. 17 illustrates swelling of DASP printed DN bio-ink droplets. Photos of DASP printed DN bio-ink droplets in the supporting matrix until equilibrium. The droplets are made of made of DN PAMi0Alg2hydrogel. Scale bar, 500 m.

[0031] Fig. 18 illustrates snapshots of a DASP printed 1 D filament under uniaxial elongation. The bio-ink is PAMioAlg2and the tensile strain rate is 0.05 / sec. The left, middle, and right panels are, respectively, captured at the beginning of the tensile test, right before breaking, and right after breaking. Scale bar, 1 mm

[0032] Fig. 19 illustrates microstructure of hydrogels formed by aqueous-two phase system. Fluorescence confocal microscopy images depicting the PEG / dextran aqueous two-phase system mixed by the extrusion nozzle. Green phase, dextran with a concentration of 15% (w / v) and FITC labeled dextran with a concentration of 0.2% (w / v). Dark phase: PEG with a concentration of 15% (w / v) and alginate with a concentration of 2% (w / v), which is crosslinked by 20 mM calcium ion. The images are generated via 3D reconstruction from multiple 2D images. Figs. 20A-20B illustrate a DASP printed 3D lattice. The 5x5x4 lattice consists of 100 interconnected yet distinguishable DN PAMi0Alg2hydrogel particles. Fig. 20A and 20B illustrate display the top and lateral view, respectively. Scale bars, 1 mm.

[0033] Fig. 21 illustrates cytocompatibility of free polyacrylamide polymers. A representative fluorescence confocal microscopy image from live / dead assay of Beta-TC-6 cells. The cells with a density of 2xlO7 / mL are incubated in DMEM with 10% w / v polyacrylamide for 3h and subsequently suspended to cell culture media to reach a final density of 1 ,2xlO5 / mL for cell recovery. The image is taken 12 h after the recovery. Scale bar, 50 pm.

[0034] Fig. 22 illustrates in vivo stability of DASP printed scaffolds. Representative photographs of the retrieved DN PAMi0Alg2scaffolds. Fig. 22A a photograph of the scaffolds located near the liver in the abdominal cavity (dashed circles). Fig. 22B illustrate a photograph of all scaffolds retrieved at 30, 60 and 120 days after transplantation

[0035] DETAILED DESCRIPTION

[0036] In general, the present disclosure provides for double-network hydrogel particles, methods of making double-network hydrogel particles, methods of making objects (e.g., three-dimensional objects) using double-network hydrogel particle, and the like. Additional details are provided herein and in the Examples.

[0037] This disclosure is not limited to particular embodiments described, and as such may, of course, vary. The terminology used herein serves the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0038] Where a range of values is provided, each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0039] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, polymer chemistry, organic chemistry, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0040] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0041] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, dimensions, frequency ranges, applications, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence, where this is logically possible. It is also possible that the embodiments of the present disclosure can be applied to additional embodiments involving measurements beyond the examples described herein, which are not intended to be limiting. It is furthermore possible that the embodiments of the present disclosure can be combined or integrated with other measurement techniques beyond the examples described herein, which are not intended to be limiting.

[0042] It should be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0043] Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

[0044] Definitions

[0045] The term "composition" as used herein refers to a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0046] “Polymers” are understood to include, but are not limited to, homopolymers, copolymers, such as for example, block, graft, random and alternating copolymers, terpolymers, etc. and blends and modifications thereof.

[0047] The term “crosslinking” can generally refer to “partial crosslinking” or “fully crosslinking” based upon the way in which it is used. As used herein, the term "partial crosslinking" or "partially cross-linked" refers to reaction of a portion of the reactive sites of molecules of a polymer (e.g., alginate, A-polymer (e.g., NB-polymer), B-polymer (e.g., TZ- polymer)), to form cross-linking bonds between molecules of the polymer that cause the hydrogel to increase in viscosity, e.g., solidify. Full cross-linking refers to a degree of crosslinking between molecules of a polymer that is sufficient for the reaction rate of the cross- linking reaction to decrease to an insignificant rate. At full cross-linking, some reactive sites on the polymer molecules remain unreacted, but the degree of cross-linking between the molecules is sufficient to prevent reactive sites on adjacent molecules from coming close enough to undergo reaction to form a cross- linking bond. Partial cross-linking may also be considered in terms of the solidification of the double-network hydrogel particle produced therefrom. Partial cross-linking may refer to a degree of cross-linking sufficient to cause the hydrogel to solidify to the point that the storage modulus G' exceeds the loss modulus G". Full cross-linking refers to a degree of cross-linking at which the storage modulus G' and loss modulus G" become constant again after the cross-linking process. In an aspect, “crosslinking” refers to the formation of ionic or covalent bonds linking two molecules of a compound (e.g., alginate).

[0048] Discussion

[0049] The present disclosure provides for double-network hydrogel particles, methods of making double-network hydrogel particles, methods of making objects (e.g., three- dimensional objects) using double-network hydrogel particle, and the like. In an aspect, double-network hydrogel particles are viscoelastic voxels (e.g., spherical or semi-spherical) that can be precisely manipulated in three-dimensional space and can be used in both soft matter science and biomanufacturing. The present disclosure provides for a voxelated bioprinting technology that enables the digital assembly of interpenetrating double-network hydrogel particles to form objects, in particular three-dimensional objects.

[0050] In particular illustrative aspects, the double-network hydrogel particles can be made of independent polymer networks of polyacrylamide-based polymers, for example, and alginate or hyaluronic acid-based polymers, for example, and alginate. In an aspect, each of the polyacrylamide-based polymers or hyaluronic acid-based polymers can be independently crosslinked via additive-free and biofriendly a click chemistry between a pair of stoichiometrically matched polymers.

[0051] For example, the pair of matched polymers can be referred to as “A”-polymer and “B”-polymer, where the A-polymer includes a first click chemistry moiety and the B-polymer includes a second click chemistry moiety. The first and second click-reaction moieties are a matched pair that can react with one another using a click-reaction to form a click-reaction product (or crosslinking group). In a particular aspect, the pair of polymers can be two polyacrylamide-based polymers each including a paired click chemistry moiety such as norbornene (NB) and tetrazine (TZ) groups, respectively (e.g., NB-polyacrylamide and TZ- polyacrylamide). Multi-channel print nozzles can be used for on-demand mixing of the highly viscoelastic bio-inks (e.g., NB-polyacrylamide / alginate and TZ-polyacrylamide / alginate) with cells without significantly impairing cell viability. As illustrated in Example 1 , highly complex three-dimensional structures, such as a hollow sphere, can be made of a plurality of double-network hydrogel particles that are interconnected (e.g., partially cross-linked) yet distinguishable (e.g., using optical microscopy techniques). Example 1 also illustrates the cytocompatibility and in vivo stability of the scaffolds of double-network hydrogel particle through cell encapsulation and animal transplantation. b

[0052] The present disclosure provides for double-network hydrogel particles. The doublenetwork hydrogel particles include two independent and distinguishable polymer networkscaffolds (e.g., a first polymer network-scaffold and a second polymer network-scaffold). The two polymer network-scaffolds are formed independent of one another and can be formed in different time windows (while within the double-network hydrogel particle). The two polymer network-scaffolds are formed using different chemistries, which are described herein. In a simplistic non-chemical view (and non-limiting view), the two polymer networkscaffolds can be viewed as two independent fishing nets that overlay one another. In an aspect, the first polymer network-scaffold includes two polymers that are crosslinked using click chemistry. In short, each polymer used in the click reaction is prepared separately and isolated from one other until it is desired to form the first polymer network-scaffold using the click chemistry. In an aspect, the second polymer network-scaffold includes an alginate polymer (e.g, one or two alginate polymer compositions). In an aspect, an alginate polymer is mixed with each of the polymers above used in the click-chemistry reaction. The two alginate polymers can be identical (e.g., weight average molecular weight). In another aspect, the alginate polymer is only mixed with one of the polymers above used in the clickreaction reaction. The alginate polymers can be crosslinked together using a divalent cation (e.g., Ca2+).

[0053] In an aspect, a microfluidic printhead can be used to mix the precursor components (e.g., NB-polymer, TZ-polymer, alginate, cells, and the like) that are ultimately combined to form the double-network hydrogel particle (e.g., hydrogel voxels) using an apparatus (e.g., digital assembly of spherical particles (DASP)) as described in U.S. 2023 / 0271377, which is included herein by reference for the device and system to print. The compositions that can be mixed to from the double-network hydrogel particle can be adjusted (e.g., amount, concentration, etc.) to control the viscoelasticity and / or mesh size. A sacrificial support matrix can be used to support the double-network hydrogel particle when initially printed from the printhead. A plurality of double-network hydrogel particles can be assembled (e.g., printed) to form a structure, such as a one-dimensional, two-dimensional array, or a freestanding three-dimensional lattice structure. The method may include depositing a plurality of double-network hydrogel particle within the sacrificial support matrix and subsequently removing the sacrificial support matrix to form self-supporting three-dimensional lattice structures or objects, for example. The double-network hydrogel particles can be precisely positioned so that the double-network hydrogel particles are adjacent to one another and can interconnect (e.g., partially crosslink). Due to the chemical composition of the doublenetwork hydrogel particles, mild conditions that are amenable to living cells, tissue, and high- density organ tissues can be used to form the structures, which is unlike other processes that require the use of ultraviolet energy, high mechanical shearing, and the like, for example, that can be harmful to cells.

[0054] Now having described features of the present disclosure generally, additional features are provided herein and below. In an aspect, the first polymer network-scaffold includes: a polyacrylamide-based polymer (e.g., a polyacrylamide polymer), a polysaccharide-based polymer (e.g., a polysaccharide polymer such as a hyaluronic acidbased polymer), a polyethylene glycol-based polymer (e.g., a PEG polymer), or a graphed polymer (e.g., a bottlebrush polymer with polyethylene glycol or poly(dimethyl siloxane (PDMS as the side chains and methacrylate-based monomer, an acrylate-based monomer, an acrylamide-based monomers, or a vinyl-based monomer that form the backbone). The first polymer network-scaffold is formed upon mixing and cross-linking precursor components that are functionalized to achieve a click chemistry reaction. Click chemistry is used since the click chemistry cross-linking reaction will cross-link the polymer upon mixing without harsh reaction conditions and that that the cross-linking can happen as the doublenetwork hydrogel particle is printed and forms. The click chemistry can include one or more of the following moiety pairs: norbornene (NB) and tetrazine (TZ); azide and alkyne; thiol and alkene; or maleimide and thiol. The pairs of moieties are present on two separate polymers (e.g., A-polymer and B-polymer, where “A” and “B” correspond to the pair of click chemistry moieties (e.g., “A” = NB and “B” = TZ)) that are then mixed, upon which the click chemistry of the pairs occurs to form the first polymer network-scaffold. The first polymer networkscaffold can be the reaction product of the following precursor polymer components (A- polymer; B-polymer) (which are mixed to form the first polymer network-scaffold): NB- polymer and TZ-polymer; azide-polymer and alkyne-polymer; thiol-polymer and alkenepolymer; and maleimide-polymer and a thiol-polymer. In an aspect, the NB-polymer can be a NB-polyacrylamide, NB-polysaccharide, NB-polyethylene glycol, or NB-graphed polymer. In an aspect, the TZ-polymer can be a TZ-polyacrylamide, TZ-polysaccharide, TZ- polyethylene glycol, or TZ-graphed polymer. In an aspect, the azide-polymer can be an azide-polyacrylamide, azide-polysaccharide, azide-polyethylene glycol, or azide-graphed polymer. In an aspect, the alkyne-polymer can be an alkyne-polyacrylamide, alkynepolysaccharide, alkyne-polyethylene glycol, or alkyne-graphed polymer. In an aspect, the thiol-polymer can be a thiol-polyacrylamide, thiol-polysaccharide, thiol-polyethylene glycol, or thiol-graphed polymer. In an aspect, the alkene-polymer can be an alkene-polyacrylamide, alkene-polysaccharide, alkene-polyethylene glycol, or alkene-graphed polymer. In an aspect, the maleimide-polymer can be a maleimide-polyacrylamide, maleimide- polysaccharide, maleimide-polyethylene glycol, or maleimide-graphed polymer.

[0055] In an aspect, the pair of moieties (e.g., NB and TZ; azide and alkyne; thiol and alkene; or maleimide and thiol) used in the click chemistry reaction can be bonded to two different polymer (a pair of polymers (A-polymer and B-polymer) that form the first polymer network-scaffold), then the polymers (A-polymer and B-polymer) are mixed and click chemistry reaction can occur. One of the pair of the polymers (B-polymer) can include a plurality of units (e.g., TZ units, alkyne units, alkene units or thiol unites) and a polymer backbone, while the other of the pair (A-polymer) can include a plurality of units (e.g., NB unit, azide unit, thiol unit and maleimide unit, respectively) and a polymer backbone. The pair of unites (e.g., TZ units and the NB units, etc.) react with one another to form a clickchemistry reaction crosslink between the pair of polymers.

[0056] While particular embodiments are described below in relation to the click chemistry involving NB and TZ, the present disclosure is not limited to these and the other types of click chemistry described above and herein can applied as well to these particular embodiments. In particular aspects, the NB-polymer (A-polymer) can be a NB- polyacrylamide or a NB-hyaluronic acid, while the TZ-polymer (B-polymer) can be a TZ- polyacrylamide or a TZ-hyaluronic acid. In an aspect, the TZ-polymer (B-polymer) includes a plurality of TZ units and a polymer backbone, while the NB-polymer (A-polymer) includes a plurality of NB units and a polymer backbone. The TZ units and the NB units react with one another to form a click chemistry reaction crosslink between the TZ-polymer and the NB- polymer. In particular, the reaction crosslink is a click chemistry reaction product of TZ and NB. In an aspect, the click chemistry reaction crosslink includes: a

[0057] NB Polymer backbone

[0058] TZ Polymer backbone crosslinking group.

[0059] In an aspect, the ratio of pair of units (e.g., TZ units to NB units) can be about 0.9:1 .1 to 1.1 :0.9 or about 1 :1. In another aspect, the number of each unit (e.g., the TZ units and NB units) may not be the same, which can allow for some unreacted units and those units can react with units in adjacent double-network hydrogel particles in the object that is formed using a plurality of double-network hydrogel particles. In a particular aspect, the ratio of TZ units to NB units can be about 0.9: 1.1 to 1.1 :0.9 or about 1 :1. In another aspect, the number of the TZ units and NB units may not be the same, which can allow for some unreacted units and those units can react with units in adjacent double-network hydrogel particles in the object that is formed using a plurality of double-network hydrogel particles.

[0060] In an aspect, the weight average molecular weight of the polymers that form the polymer network-scaffold (e.g., a polyacrylamide-based polymer, a polysaccharide-based polymer (e.g., a hyaluronic acid-based polymer), a polyethylene glycol-based polymer, or a graphed polymer) can be about 10 kDa to 1000 kDa. In a particular aspect, the weight average molecular weight of the polyacrylamide polymer can be about 10 kDa to 1000 kDa. The weight average molecular weight of the NB-polyacrylamide is about 10 kDa to 1000 kDa and the weight average molecular weight TZ-polyacrylamide is about 10 kDa to 1000 kDa. In an aspect, the weight average molecular weight of TZ-polymer is about 80% to 120% or about 90% to 110% of a weight average molecular weight of the NB-polymer or the weight average molecular weight of NB-polymer is about 80% to 120% or about 90% to 110% of a weight average molecular weight of the TZ-polymer.

[0061] In an aspect, the alginate polymer may be a commercially available alginate. The alginate can be derived from alginic acid sodium salts obtained from brown algae. The double-network hydrogel particle (or one or two alginate polymer compositions) can include a concentration of alginate sufficient to produce a mesh size suitable for the particular application of the double-network hydrogel particle and the object formed from the doublenetwork hydrogel particles. In an embodiment, the double-network hydrogel particle can include greater than or equal to 1 w / v%, or greater than or equal to 1.5 w / v% alginate, where the w / v% is defined as the weight of the component (e.g., alginate polymer) divided by the volume of the double-network hydrogel particle and the quotient multiplied by 100. The double-network hydrogel particle can include less than or equal to 5 w / v% or less than or equal to 4 w / v% alginate. The double-network hydrogel particle can include about 1 w / v% to 5 w / v%, about 1 w / v% to 4 w / v%, about 1 .5 w / v% to 5 w / v%, or about 1 .5 w / v% to 4 w / v% alginate. The weight average molecular weight of the alginate polymer can be about 10 kDa to 1000 kDa.

[0062] As described herein, the alginate can be present in one or two alginate polymer compositions, where when present in two alginate polymer compositions, the two alginate polymer compositions are subsequently mixed together to form the double-network hydrogel particle. In an aspect, when the alginate is present in the two alginate polymer compositions, the alginate polymers are identical, while in another aspect, the alginate polymers can have different weight average molecular weights.

[0063] The second polymer network-scaffold is a reaction product of alginate polymers in the presence of the divalent cation. As described above, the alginate polymers can be from a single alginate polymer composition (e.g., mixed with either the NB-polymer or the TZ- polymer) or can be from two alginate polymer compositions (e.g., one with the NB-polymer and the other with the TZ-polymer). In an aspect, the second polymer network-scaffold includes divalent cationic bonding with carboxylate groups of the alginate polymers. In an aspect, the divalent cation can be Ca2+, Ba2+, Co2+, and Ni2+, where the divalent cation can be delivered as a salt of the divalent cation.

[0064] In an aspect, the first polymer network-scaffold and the second polymer networkscaffold are independent polymer network-scaffolds and have less than 5%, less than 10%, or less than 15% crosslinking bonds between the first polymer network-scaffold and the second polymer network-scaffold. In an aspect, the ratio of the first polymer networkscaffold to the second polymer network-scaffold is about 1 :1 to 1 :10 or about 1 :5 ratio.

[0065] In an aspect, the double polymer network-scaffold composition includes microislands. The micro-islands are volumetric domains between / among the first polymer network-scaffold and / or a second polymer network-scaffold. The micro-islands can include cells and / or fluid (e.g., dextran, polyethylene glycol). The micro-islands can have a volume of about 10'6to 10‘5pL.

[0066] In an aspect, the double-network hydrogel particle can have a spherical or semi- spherical shape. The double-network hydrogel particle can have a diameter of about 150 pm to 1500 pm, about 500 pm to 1500 pm, about 500 pm to 1200 pm, or about 1000 pm.

[0067] The present disclosure provides for objects or structures, such as three-dimensional objects, to be made of a plurality of double-network hydrogel polymer particles. A plurality of double-network hydrogel particles can be assembled to form the object structure (e.g., a one-dimensional object (e.g., a string of double-network hydrogel polymers), a two- dimensional array, or a free-standing three-dimensional lattice structure. In an aspect, the free-standing three-dimensional lattice structure can include one or more hollow volumetric domains within the free-standing three-dimensional lattice structure. One or more dimensions of the object can be on the scale of millimeters to centimeters or 10s of centimeters. Adjacent double-network hydrogel polymer particles can interpenetrate one another so that the double-network hydrogel polymer particles adhere or are bonded to one another, but are independent and distinguishable double-network hydrogel polymer particles when inspected using microscopy techniques, at least when the object is initially formed. In an aspect, crosslinks between the first polymer network-scaffold in adjacent double-network hydrogel polymer particles, crosslinks between the second polymer network-scaffold in adjacent double-network hydrogel polymer particles, or both can be present.

[0068] While the TZ and NB click chemistry moieties are described below, the other click chemistry moieties can be used in the same way. For purposes of illustration, alginate in a first double-network hydrogel polymer particle can crosslink with the alginate in an adjacent second double-network hydrogel polymer particle, or the TZ-polymer (B-polymer) in a first double-network hydrogel polymer particle can crosslink with the NB-polymer (A-polymer) in an adjacent second double-network hydrogel polymer particle, or both types of crosslinks can occur. As described in Example 1 , the free-standing three-dimensional lattice structure is robust and mechanically stable and can survive in a living animal for weeks, months, or longer, and substantially (e.g., about 80% or more, about 90% or more) maintain the three- dimensional structure and the double-network hydrogel polymer particles independent and distinguishable.

[0069] In an aspect, the double-network hydrogel particle and / or objects formed from the double-network hydrogel particles can include one or more types of cells of bacterial cells, animal tissue cells, human tissue cells, and combinations thereof. In other embodiments, the double-network hydrogel particle and / or objects formed from the double-network hydrogel particles do not include cells. When present, the cells can be present on the first polymer network-scaffold and / or a second polymer network-scaffold and / or within microislands (e.g., domains between / among the network-scaffolds) within the double-network hydrogel particles and / or in the domains between the double-network hydrogel particles of the object. The number or concentration of the cells can vary depending upon the purpose of the double-network hydrogel particle. The concentration and / or type of cells present can vary based on location within the object.

[0070] The present disclosure provides for methods of forming objects, such as freestanding three-dimensional objects. For purposes of illustration, only the TZ and NB moieties are described below, but each of the other pairs of click chemistry moieties can be used in the same way in the methods described herein. A first mixture can be made that includes a first alginate polymer with a TZ-polymer to form a first mixture. The first mixture can optionally include a solution including cells. A second mixture can be made that includes a second alginate polymer with a NB-polymer. The second mixture can optionally include a solution of cells. In another aspect, either the first mixture or the second mixture may not include the first or second alginate polymer, respectively. In an aspect, the first alginate polymer and the second alginate polymer are identical (e.g., same weight average molecular weight). In another aspect, the first alginate polymer and the second alginate polymer can be different (e.g., different weight average molecular weight).

[0071] Using a first inlet, the first mixture can be introduced into a mixing area. Simultaneously or sequentially, a second inlet can be used to introduce the second mixture to the mixing area. Upon mixing the first and second mixtures, a third mixture is formed. After mixing, a first polymer network-scaffold is formed by a reaction of the TZ-polymer (e.g., including a plurality of TZ units) and the NB-polymer (e.g., including a plurality of NB units). The TZ units and the NB units react with one another to form a click-chemistry reaction crosslink between the TZ-polymer and the NB-polymer.

[0072] The third mixture is flowed out of a print nozzle to form a plurality of double-network hydrogel particles. The third mixture is flowed out as the first polymer network-scaffold is forming. Each of the plurality of double-network hydrogel particles are sequentially formed (or two or more plurality of double-network hydrogel particles are formed is the hydrogel printer includes multiple print nozzles) to ultimately form the object (e.g., the three- dimensional object). Each double-network hydrogel particle is adjacent at least one other double-network hydrogel particle can interpenetrate one another. In an aspect, the interpenetrating can include crosslinking of TZ units and NB units in adjacent double-network hydrogel particles and / or crosslinking (e.g., when divalent cations are present) of alginate polymers in adjacent double-network hydrogel particles.

[0073] In an aspect, the method can include introducing the three-dimensional object (e.g., fully or partially constructed) to a solution including divalent cations. The presence of the divalent cations (e.g., Ca2+) can initiate formation of the second polymer network-scaffold by crosslinking the alginate polymers. In an aspect, the divalent cations can be present in the sacrificial support matrix, as described below.

[0074] Now having described the method in general, additional details are provided below. When the double-network hydrogel particles are formed using a DASP system, the doublenetwork hydrogel particles are formed with a sacrificial support matrix. The sacrificial support matrix is designed to have certain physical and chemical properties to enable printing of the double-network hydrogel particle using the DASP system. To serve as the supporting matrix for embedded printing, the sacrificial support matrix can possess five features: (1) the sacrificial support matrix must be aqueous and cytocompatible; (2) the sacrificial support matrix must be a yield stress fluid such that under stress it becomes fluidlike, allowing the movement of the printing nozzle; (3) the sacrificial support matrix must be able to self-heal to recover its original mechanical properties within a short time to localize a deposited droplet; (4) the sacrificial support matrix should provide an environment in which individual double-network hydrogel particle can swell and partially coalesce with each other; and (5) the sacrificial support matrix can be easily removed after printing and cross-linking of the hydrogel voxels. The sacrificial support matrix may be a yield stress fluid that becomes fluid-like at shear stresses greater than 10 pascals. The sacrificial support matrix of the present disclosure may comprise gelatin microparticles.

[0075] To maintain good fidelity of the double-network hydrogel particle during and immediately after printing, the hydrogel composition of the double-network hydrogel particle has a relatively high viscosity such that the hydrogel composition of a printed double-network hydrogel particle does not flow through the porous sacrificial support matrix in a short time before cross-linking can occur. Good fidelity of a double-network hydrogel particle refers to the double-network hydrogel particle having a generally spherical shape with a minimum of deformation caused by flow of the hydrogel composition into the sacrificial support matrix or dragging forces during separation of the microfluidic printhead from the double-network hydrogel particle. The click chemistry of the NB-polymer and TZ-polymer can be used to maintain good fidelity, but is should also be noted that the other click chemistry pairs of units can also maintain good fidelity.

[0076] In addition to viscoelasticity, the hydrogel composition of the double-network hydrogel particle should have a mesh size after crosslinking that is tunable to satisfy mass transport requirements of various applications. As used herein, the term “mesh size” refers to an average distance between molecules of the polymers-networks making up the of the double-network hydrogel particle after crosslinking and is related to the average size of the spaces or pores between polymer molecules through which bio-materials can diffuse into and out of the double-network hydrogel particle. The mesh size of the hydrogel composition of the of the double-network hydrogel particle after cross-linking may determine the size of molecules that can diffuse into or out of the double-network hydrogel particle and / or the rate at which molecules can diffuse into and out of the double-network hydrogel particle. For instance, a greater mesh size of the hydrogel composition may improve the efficiency of nutrient transport into the double-network hydrogel particle by providing greater diffusion rates of nutrients into the double-network hydrogel particle. In some applications, however, hydrogel compositions with a relatively small mesh size may be needed to reduce or prevent diffusion of certain species into the double-network hydrogel particle.

[0077] In embodiments, the hydrogel compositions of the of the double-network hydrogel particle have a viscosity sufficient to prevent the hydrogel composition from flowing into the pores in the sacrificial support matrix during and immediately after printing before any crosslinking can occur. The hydrogel compositions of the of the double-network hydrogel particle may have a viscosity of greater than or equal 100 pascal seconds (Pa-s), where the viscosity is determined using a stress-controlled rheometer (Anton Paar, MCR 302) with 25 mm plate-plate geometry at 20°C. The viscosity of the hydrogel composition is the value of the viscosity at the lowest shear rate 0.01 s1. The hydrogel composition of the doublenetwork hydrogel particle may have a viscosity of from 100 Pa-s to 150 Pa-s.

[0078] In an aspect, during cross-linking of the alginate in the double-network hydrogel particle, the divalent cation (e.g., Ca2+(as a salt of the divalent cation)) can be present in the sacrificial support matrix (e.g., present in the sacrificial support matrix while forming the double-network hydrogel particle or adding the divalent cation after formation of one or more of the double-network hydrogel particles) and can facilitate cross-linking between alginate molecules proximate the outer surface of one double-network hydrogel particle and alginate molecules proximate the outer surface of another adjacent double-network hydrogel particle. The cross-linking between alginate molecules of the two double-network hydrogel particles may cause the two double-network hydrogel particles to become attached or bonded to each other through a cross-linked alginate polymer network. Additionally, alginate molecules within each of the double-network hydrogel particle, in particular alginate molecules proximate the outer surfaces of each double-network hydrogel particle, may crosslink with each other to form a cross-linked alginate polymer matrix, which may cause the hydrogel voxel to solidify and become stiffen. This solidification may reduce or prevent flow of the hydrogel composition out of the double-network hydrogel particle and into the sacrificial support matrix and may allow the double-network hydrogel particle to develop the stiffness to become self-supporting. The crosslinking using the click chemistry and / or crosslinking of alginate can increase the viscosity of the materials to form the double-network hydrogel particle.

[0079] In an aspect, the alginate molecules of the double-network hydrogel particle may include a plurality of reactive sites capable of reacting to form the ionic or covalent bonds between alginate molecules that cross-link the alginate molecules. The components of the hydrogel, such as alginates in the hydrogel for example, may undergo crosslinking in the presence of the divalent cation (e.g., Ca2+) as part of the formation of the double-network hydrogel particle. The gelation time refers to the time that the hydrogel composition begins to solidify, which refers to the time when the storage modulus G' of the hydrogel exceeds the loss modulus G". As previously discussed, partial crosslinking of the alginates in the hydrogel composition may refer to a degree of crosslinking between the alginate molecules that is sufficient for the storage modulus and the loss modulus to change as a function of time. When the degree of cross-linking reaches full cross-linking, the storage modulus and loss modulus of the hydrogel voxels attain constant values again.

[0080] While embodiments of the present disclosure are described in connection with the Examples and the corresponding text and figures, there is no intent to limit the disclosure to the embodiments in these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

[0081] EXAMPLE 1

[0082] Analogous of pixels to two-dimensional (2D) pictures, voxels — in the form of either small cubes or spherical particles — are the basic units of three-dimensional (3D) objects1. A prominent example is Minecraft®, one of the world’s best-selling video games. In the 3D- mosaic world of Minecraft®, everything — animals, houses, even the sun, and the moon — is made of small cubes. Players use these voxels with various functions and colors to construct their own artworks. The only limitation is the players’ imagination. A similar approach, if realized in 3D cell assembly, would provide a standardized method fortissue engineering, transforming basic and translational biomedicine. In principle, one can use cell-encapsulated hydrogel droplets as voxels, assemble the voxels to create hierarchical and organized 3D structures, and exploit biophysical2and biochemical3cues to program the assemblies to highly functional 3D tissues. However, this approach requires precisely manipulating highly viscoelastic bio-ink voxels in 3D space, which represents a grand challenge in both soft matter science and biomanufacturing.

[0083] Recently, building on the advancement of embedded 3D printing4~7, we proposed and showed the concept of a voxelated bioprinting technology that enables the digital assembly of spherical particles (DASP)89. DASP generates a highly viscoelastic aqueous droplet in an aqueous yield-stress fluid, deposits the droplet at a prescribed location, and assembles individual droplets into 3D structures by controlled polymer swelling (Fig. 1a). DASP is qualitatively different from existing embedded droplet printing technologies10 11, which rely on the classic Rayleigh-Plateau instability to generate droplets. As a result, these techniques can only handle low viscosity liquids and often involve biohazardous organic solvents; more importantly, they have no control over the absolute location of each droplet in 3D space. By contrast, DASP exploits nonlinear fluid dynamics to precisely manipulate bioink droplets in 3D space. The mechanism of DASP printing is somewhat reminiscent of the “tablecloth trick” that involves pulling a loaded tablecloth away from a table but leaving the plates behind. This trick relies on inertia. The key difference is that, in DASP, the “inertia” comes from the confinement force from the supporting matrix, so that the embedded droplet can be deposited at a prescribed location. Compared to the printing of spheroids12"14, in which solid-like cell aggregates are stacked with great care and subsequently fused through slow cell migration, in DASP the droplets are joined by relatively fast and more controllable polymer swelling. Thus, DASP provides a robust approach for the on-demand assembly of cell encapsulated hydrogel particles in a cytocompatible environment. Using DASP, we created multiscale porous scaffolds formed by interconnected yet distinguishable alginate hydrogel particles. In our proof-of-concept research, we encapsulated human islets in individual particles and demonstrated that the scaffolds allow for responsive insulin release8, highlighting the potential of DASP in biomedical applications. However, our prototype voxelated bioprinting technology can print pure alginate hydrogels only, which are brittle and have limited tunability in mechanical properties. These limit the applications of DASP not only in translational biomedicine, such as printing mechanically robust scaffolds as long-term transplants, but also in basic biomedical research, which requires modular biomaterials for controlling the microenvironment of voxels. Here, we develop a substantially improved voxelated bioprinting technology (DASP 2.0) that enables the digital assembly of interpenetrating alginate and polyacrylamide (PAM) double-network (DN) bio-ink droplets (Fig. 1b). We design and synthesize a pair of PAM polymers functionalized with tetrazine (TZ) and norbornene (NB) groups, respectively. Upon mixing, this pair of TZ-PAM and NB-PAM (A+B) polymers form a network via additive-free click reaction between TZ and NB groups (Fig. 1 b, i). For the single-network PAM hydrogels, we systematically investigate the dependence of crosslinking kinetics and stiffness on NB / TZ ratio and polymer concentration. The experimentally observed behavior can be well described by kinetic and scaling theories. A diagram-of-state is constructed to outline the stiffness and extensibility of DN PAM / alginate ((A+B) / C) hydrogels with various formulations. We engineer multi-channel print nozzles for on-demand mixing of highly viscoelastic bio-inks without significantly impairing cell viability (Fig. 1b, ii). Further, we demonstrate the capability of voxelated bioprinting by creating highly complex 3D structures such as a hollow sphere composed of interconnected yet distinguishable hydrogel particles. The voxel-printed DN scaffolds can repeatedly sustain a large extent of compression up to 60% without noticeable damage. Moreover, we transplant the DN scaffolds into abdominal cavity of immunocompetent mice to show that the scaffolds can be retrieved after 4 months with negligible impaired structural integrity and mechanical properties. Finally, we demonstrate the universality of using (A+B) / C type DN hydrogels as inks for DASP by replacing the polymer backbone of the A+B network from PAM to hyaluronic acid (HA) without compromising mechanical robustness. The developed cytocompatible DN biomaterials (Fig. 1 b, Hi) and printing systems pave the way for voxelated bioprinting highly complex yet organized tissue constructs for basic and translational biomedicine.

[0084] Results

[0085] Crosslinking kinetics of single-network PAM hydrogels:

[0086] To be suitable for DASP printing, the bio-ink should possess a gelation time of sufficient duration. This allows the printed droplets to undergo partial swelling, enabling them to partially coalesce with adjacent droplets before complete crosslinking occurs (Fig. 1a). However, the crosslinking time should not be too long, otherwise the bio-ink may diffuse through the porous supporting matrix, leading to uncontrollable droplet shape8. Thus, it is essential to quantify the crosslinking kinetics of single-network PAM hydrogels.

[0087] Unlike typical hydrogels formed by crosslinking precursor polymer chains using small molecules as crosslinking agents, our single-network PAM gels are formed by crosslinking a pair of linear PAM polymers. The two polymers are of the same molecular weight (MW) but are functionalized by tetrazine (TZ) (Supplementary Fig. 3) and norbornene (NB) groups, respectively. Upon reaction, the TZ and NB groups form a crosslink via additive-free click reaction at room temperature (RT) (Fig. 1 b, i). Therefore, the crosslinking kinetics of our single-network PAM hydrogels is expected to depend on both the concentration, c, of the polymers and the grafting ratio, fx, of the functional groups, which is defined as the molar ratio between the grafted functional groups and the total number of chemical monomers on the PAM backbone.

[0088] To investigate the crosslinking kinetics of PAM hydrogels, we maintain the concentrations of TZ-PAM and NB-PAM solutions at 10% (w / v) before mixing while varying the molar ratio between NB and TZ groups. To do so, we keep the grafting ratio of TZ-PAM, fxTZ, at 2.53% (Supplementary Fig. 4); on average, this value corresponds to approximately 50 TZ groups per polymer. We vary the grafting ratio of NB groups, fx NB, from 0.63% to 4.25% (Supplementary Fig. 5). These formulations cover NB / TZ ratios spanning approximately one order of magnitude, ranging from 0.25 to 1 .68. For each mixture with equal amount of TZ-PAM and NB-PAM polymers, we employ a stress-controlled rheometer to continuously monitor its viscoelasticity in real time for 12 hours (see Methods). We determine the gelation time, tg, above which the storage modulus, G exceeds the loss modulus, G", as denoted by the arrow in Fig. 2a. As the NB / TZ ratio increases from 0.25 to 1.68, tqdecreases by nearly 40 times from 616 sec to 16 sec (solid squares in Fig. 2b).

[0089] The decrease in the gelation time with the increase of NB / TZ ratio can be explained by a simple kinetic theory. The rate of forming crosslinks, d.Cxl<lt, is proportional to the probability of a NB group to meet with a TZ group, which is the product of the concentrations, (CWB 0- Cx) and (CTZ 0- Cx), of the two in the mixture:

[0090] Here, k is a constant determined by the reaction rate between a NB and a TZ, and CVB 0and CTZ,t, respectively, are the concentrations of unreacted NB and TZ groups at reaction time t = 0. Integrating eq. (1), one obtains the relation between the concentration of crosslinks and reaction time:

[0091] At the gelation time tg, each polymer has on average one crosslink, forming a giant molecule that percolates the whole volume of the solution15. As each polymer carries about

[0092] 50 functional groups, the concentration of crosslinks relative to the initial concentration of the functional groups, / ? = Cx g / CTZ 0« 1 / 50. Substituting this relation to eq. (2), one obtains the dependence of the gelation time on the ratio between NB and TZ groups, CNB 0 / CTZ 0

[0093] Equation (3) predicts that, for CNB 0A CTZ 0, tgdecreases rapidly with the increase of CNBfi / CTZ,o- This theory explains well the experimentally observed gelation time with the fitting parameter / c ~ 1.8 x io-2L ■ mol~1sec~1, as shown by the solid line on the left panel of Fig. 2b.

[0094] For CNB 0= CTZ 0= Co, eq. (3) predicts that the gelation time is inversely proportional to the initial concentration of functional groups. To test this prediction, we synthesize TZ- PAM and NB-PAM polymers with grafting ratios of 2.53% and 2.57%, respectively; this ensures that CNBfi / CTZfi® 1. We prepare a series of solution mixtures consisting of equal amount of TZ-PAM and NB-PAM polymers but increase the polymer concentration c from 1.25 to 15% (w / v). The experimentally measured gelation time decreases from 1352 to 40 sec (solid circles in Fig. 2b). This behavior can be well explained by our theory, as shown by the solid line on the right panel of Fig. 2b. Moreover, the fitting parameter, k « 1.5 x 10-2L ■ mol~lsec~l, agrees reasonably well with that for CNB 0A CTZ 0.

[0095] The gelation theory is based on the physical picture that at the gelation point there is on average one crosslink per polymer. This implies two consequences for the hydrogel stiffness at the gelation point: (i) The stiffness should be constant if the polymer concentration is the same; (ii) the stiffness increases with the polymer concentration. Indeed, these predictions are verified by our experiments. For the hydrogel formulations with the same polymer concentration but various NB / TZ ratios, the shear storage modulus at the gelation time, G'(c^), is nearly a constant of 13 Pa (left panel in Fig. 2c). In contrast, for the hydrogel formulations of the same NB / TZ ratio but various polymer concentrations, G’(tg) increases with the polymer concentration (right panel in Fig. 2c). Taken together, our results provide the scientific foundation for prescribed gelation time of the single-network PAM hydrogels in a wide range.

[0096] Stiffness of single-network PAM hydrogels:

[0097] To explore the stiffness of single-network PAM hydrogels, we fix TZ-PAM and NB- PAM polymers at a stoichiometrically matched grafting ratio of 2.5% and mix them at equal concentrations from 1 .25% to 15% (w / v). We wait for sufficiently long enough time to allow the hydrogel to be completely crosslinked, as exemplified by the saturated G' in Fig. 2a. For all hydrogel formulations, G' is nearly independent of the frequency within the range of 0.1- 20 Hz (thick lines, Fig. 2d). Moreover, G' is more than 10 times greater than the loss modulus G" (thin lines, Fig. 2d). These results indicate that the PAM hydrogels are elastic networks; therefore, one can take G' at the lowest frequency as the equilibrium network shear modulus G.

[0098] Interestingly, the dependence of hydrogel stiffness on concentration exhibits two distinct regimes. As the polymer concentration increases from 1 .25 to 10% (w / v), G increases rapidly from 385 Pa to 2.1 *104Pa by a power law with an exponent of 2.1 ±0.2 (dashed orange line, Fig. 2e). Further increasing the polymer concentration results in a negligible increase (Figs. 2d, e). This two-regime behavior contradicts conventional understanding that the hydrogel stiffness increases with polymer and crosslinker concentrations.

[0099] We develop a scaling theory to describe the dependence of hydrogel stiffness on the functionality fxand concentration c of PAM polymers. To form a crosslink, a TZ must meet a NB. Thus, the crosslink concentration is determined by the average distance between a TZ and a NB group. This distance also equals the correlation length, which is defined as the nearest distance of a monomer from another monomer on the neighboring polymer chains, as indicated by the dashed circles in Fig. 2f. Hence, the size of a network strand, ax, is approximately the correlation length: ax« f. The shear modulus of the network is about kBT per volume pervaded by a network strand:

[0100] G « kBT / ax3« kBT / (4)

[0101] Because water is a good solvent for PAM, decreases by ~ f?F(c / c*)-3 / 4, where c’is the polymer overlap concentration, and RF« W3 / sis the end-to-end distance of a single PAM chain in a dilute solution with N being the number of Kuhn monomers per polymer15. This gives the dependence of the network shear modulus on polymer concentration:

[0102] However, this power law behavior will stop at concentrations greater than c”, at which the correlation length f (c”) is about the average distance, dx, between two neighboring functional groups (TZ or NB) on the same polymer as marked in Fig. 2f. In this case, all functional groups are reacted, and the network shear modulus saturates at:

[0103] In a good solvent, dx~ b(fx1 / nK')3 / 5(7) Here, b = 1.6 nm is the Kuhn length of PAM, nK= Cra / cos2Q) « 6.5 is the number of chemical monomers per Kuhn monomer with the Flory’s characteristic ratio Cx= 8.5 and the main C-C chain bond angle 0 = 68°16, and fx= 40 is the average number of chemical repeating units within the polymer section of size dx« 4.8 nm. Substituting eq. (7) into (6), one obtains

[0104] Thus, for a fixed fx, we expect two regimes for stiffness:

[0105] At relatively low concentrations, c* < c < c“, the network stiffness increases with the polymer concentration by a power of 9 / 4. In contrast, at high concentration, c > c”, the network stiffness saturates and becomes a constant. The theoretical predictions [see eq. (9)] explain the experiments remarkably well (Fig. 2e). Note that scaling theory does not include prefactors, which typically have values on the order of unit. For instance, our theory predicts that the maximum hydrogel stiffness is Gmax« 37 kPa [eq. (8)]. Comparing the prediction against the experimentally measured value 22 kPa, one obtains a prefactor of 0.6. Nevertheless, the combination of experiments and theory shows that the TZ / NB-PAM singlenetwork hydrogels allow for prescribed stiffness from ~400 Pa to 22 kPa, sufficient for cell encapsulation and most biomedical applications16~18.

[0106] Stiffness and extensibility of DN hydrogels:

[0107] To prepare a DN hydrogel, we use TZ-PAM and NB-PAM, respectively, to prepare two solutions, dissolve the same amount of alginate (Alg) in each of the two solutions, mix the pair of the solutions to initiate the crosslinking of PAM network, and then transfer the partially crosslinked hydrogel to a bath containing 20 mM Ca2+to incubate for 10 min at RT to completely crosslink both the alginate and PAM networks. We denote a DN hydrogel formulation as PAMxAlgy, where x is the concentration in (w / v)% for TZ or NB-PAM before mixing, and y is the concentration of alginate. In all formulations, we use TZ-PAM and NB- PAM with stoichiometrically matched grafting ratio of 2.5% at a concentration of 10% (w / v); this formulation results in a single-network PAM hydrogel PAM of shear modulus approximately 20 kPa (Fig. 2d), comparable to that of most organs in abdominal space19. Because the alginate network contains carboxylic acid groups and the PAM network contains carboxylic acid and amide groups, and that both the functional groups are sensitive to ions and pH, we equilibrate and completely crosslink hydrogel in cell culture media (DMEM) with 2 mM CaCI2for 24 hours before characterizing its mechanical properties. Indeed, all the hydrogel samples swell in DMEM, as exemplified by a swelling ratio of 7% in size for PAMioAlg2(Supplementary Fig. 8). Consequently, the shear modulus of PAM10 hydrogel decreases dramatically from 21 kPa in water to 5.3 kPa in cell culture media. Yet, the hydrogel remains an elastic solid, as evidenced by the nearly frequency-independent G’ as well as small loss factors, tan 5 = G" / G’ ® 0.02, within the frequency ranging from 0.1 to 20 Hz (solid grey line, Fig. 3a). Similarly, frequency-independent dynamic moduli are observed for 2% (w / v) alginate hydrogel, Alg2, and DN hydrogels (blue and red lines, Fig. 3a). These results show that all hydrogels are non-dissipative, elastic solids.

[0108] The shear modulus of a DN hydrogel is approximately the sum of the moduli of its constituent singlet-network hydrogels. For instance, Alg2and PAMw exhibit shear moduli of 4.5 kPa and 5.3 kPa, respectively, resulting in a combined modulus of 9.8 kPa, which closely matches the value of 11 kPa for PAMi0Alg2. This observation holds true for other DN hydrogel formulations as well. When the concentration of alginate increases from 1% to 3% (w / v), the stiffness of single-network alginate hydrogels increases linearly from 1.2 kPa to 6.6 kPa (dashed bars, Fig. 3b). The corresponding DN hydrogels show a similar increase in stiffness from 8.2 kPa to 14 kPa with a nearly the same magnitude (solid bars, Fig. 3b).

[0109] Next, we perform uniaxial tensile tests to quantify the extensibility of DN hydrogels. To do so, we take a fully crosslinked hydrogel film measuring approximately 1 mm in thickness and cut it into a dog-bone shape sample. We then subject the sample to a fixed strain rate of 0.02 / sec while capturing the process using a camera, as visualized by the photos in Fig. 3c and by Supplementary Movie 1 . For all hydrogels, we find that the stress increases almost linearly with tensile strain until the hydrogels reach their breaking point. However, we note that the single-network Alg2hydrogel is often too brittle for reliable measurements. These findings further confirm that the hydrogels are nearly pure elastic networks with little energy dissipation.

[0110] The DN PAMi0Alg2hydrogel exhibits significantly improved extensibility compared to the single-network PAM10 hydrogel. While PAM10 has a tensile breaking strain efof 0.36, the PAMwAlg2hydrogel demonstrates approximately three times greater extensibility with efof 1.0, as shown in Figs. 3c, d and by Supplementary Movie 1. Remarkably, although increasing the alginate concentration stiffens the corresponding DN hydrogels, e remains nearly the same at 1 (solid lines in Fig. 3d and filled circles in Fig. 3e). We attribute the observed enhancement in network extensibility to the characteristics of DN hydrogels. Upon deformation, the weak alginate network fractures first; this process effectively prevents localized and amplified stress along PAM network strands, thereby improving network extensibility. However, the network extensibility cannot exceed that afforded by the network strand of the PAM network. In theory, the maximum elongation at break, X „x, for a network strand equals the ratio between its initial size, ax, and contour length, Lm(lx.

[0111] However, typical single-network hydrogels, which often have a wide distribution in network strand size, cannot reach this maximum extensibility. When subjected to deformation, the relatively short network strands would break first, resulting in extensibility lower than the prediction based on average network size ax. By contrast, in a DN hydrogel, the weak network can undergo fracture to prevent localized, amplified stress near network defects or along network strands, and therefore, avoid premature failure of the strong network. Consequently, a DN hydrogel may reach the theoretical extensibility ~Tmax.2021

[0112] We develop a scaling theory to describe the dependence of network theoretical extensibility 'Tmaxon network stiffness G. In a DN PAM / alginate hydrogel, the PAM network has on average NxKuhn monomers per network strand. In a good solvent, the end-to-end distance, or the size of the network strand, is a self-avoiding random walk of Kuhn monomers with size of b: ax^ bNx3 / 5(11)

[0113] Recall eq. (10), the theoretical extensibility of the network is: Tmax~ Nx2 / S(12)

[0114] For an unentangled network, its shear modulus G is about kBT per volume pervaded by the network strand,

[0115] Thus, one can correlate to the network stiffness G,

[0116] &ax « (kBT / G)2 / 9b~2 / 3oc G~2 / 9(14)

[0117] Equation (14) predicts that extensibility of the DN hydrogel increases with the decrease of PAM hydrogel stiffness.

[0118] To test this prediction, we synthesize another DN hydrogel, PAMw174Alg2, in which the TZ-PAM has a grafting ratio of 2.53%, while the NB-PAM has a mismatched grafting ratio of 0.63%. This formulation results in a lower network shear modulus of DN hydrogel G « 7.4 kPa (Supplementary Fig. 9) and higher maximum extensibility,max ;1 / 4« 2.3 (dashed line in Fig. 3d and empty circle in Fig. 3e). Considering that the ionic crosslinked alginate network has negligible contribution to and the equilibrium shear modulus G1 / 4, G1 / t, and Gaigare respectively 7.4, 11.5, and 4.5 kPa (Figs. 3b, e), the ratio of the extensibility of the softer DN hydrogel with NB / TZ ratio 1 / 4 to that of the stiffer one with NB / TZ ratio 1 / 1 is predicted t 1.2. Remarkably, this value agrees well with the experimentally measured ratio 1 .2. These results collectively show that the extensibility of the DN hydrogel is determined by the PAM network.

[0119] Notably, compared to the classical DN PAM / alginate hydrogel of the same composition, which exhibits a remarkable extensibility with ef® 2022, our DN hydrogel is much less stretchable. This significant difference in absolute extensibility is likely due to the difference in network topology. In the classical DN hydrogel, the PAM network is formed through in situ free radical polymerization of acrylamide monomers in an alginate solution. This polymerization is an uncontrolled reaction, resulting in a wide distribution in network strand size and leading to relatively large extensibility of a single-network PAM hydrogel with ef« 6.5. In contrast, in our DN hydrogels, the PAM network is formed through the click reaction between TZ-PAM and NB-PAM precursor polymers, which have a relatively low and fixed molecular weight. Furthermore, the reactive groups further segment the linear PAM polymers into small sections, thereby further reducing the network’s extensibility.

[0120] DASP printing of DN hydrogels:

[0121] DASP printing requires highly viscous and shear-thinning bio-inks, such that during printing, an extruded droplet can grow uniformly in the yield-stress fluid supporting matrix9. We conduct shear tests to measure the viscosity of the DN PAMi0Alg2bio-ink and its constituent PAMw and Alg2solutions. The PAM-io solution is nearly a Newtonian fluid with a low viscosity of 0.2 Pa-s, which is nearly independent of shear rate ranging from 0.1 to 1000 S'1, as shown by the squares in Fig. 4a. In contrast, the Alg2solution is a shear-thinning fluid, with the apparent viscosity decreasing with the shear rate by a power of -0.63, as shown by the triangles in Fig. 4a. However, the magnitude of the viscosity, 2.6 Pa-s, remains much lower than ~20 Pa-s required for DASP printing. Consequently, both constituent bio-inks are unsuitable for DASP printing. Remarkably, the viscosity of the DN bio-ink significantly increases to 17.1 Pa-s while maintaining shear-thinning behavior, as shown by the solid circles in Fig. 4a. These properties make the DN bio-ink PAMi0Alg2suitable for DASP printing9. Moreover, the crosslinked DN hydrogel has a significantly improved extensibility compared to its single-network constituents and a stiffness comparable to that of the organs in the abnormal cavity. Thus, we use PAMi0Alg2as the DN bio-ink for the subsequent DASP printing.

[0122] For DASP printing, the bio-inks are required to be extruded through the nozzle in a liquid state and rapidly crosslink once being deposited. Due to the rapid crosslinking of DN bio-inks upon mixing (Fig. 2a), premixing and loading the DN bio-inks into a single syringe, as performed in DASP 1.0, would clog the print nozzle and syringe. To avoid the clogging, we separately load the TZ-PAM and the NB-PAM in two syringes and then mix them on- demand during the printing. To do so, we engineer a dual-inlet print nozzle with an inner static mixer chamber (Supplementary Fig. 10). The design of the mixer chamber utilizes a structure called Quadro™ Square, as depicted by a computer-rendered 3D model in Fig. 4b, i. We use stereolithography printing to fabricate this nozzle, which has an outer diameter of 2.7 mm. The inner channel of the nozzle has a narrowest dimension of 425 pm. These dimensions are much larger than the size of typical cells, ~30 pm23, and even some large cell aggregates such as islets, ~200 pm24. This design avoids mechanical shear-induced damage to the cells during extrusion.

[0123] To evaluate the mixing capability of the dual-inlet print nozzle, we use a camera to monitor the mixing process in real-time. To visualize the mixing, we incorporate red and green mica microparticles with the diameter of 50 pm into the respective bio-inks. The pair of bio-inks is extruded through the two inlets of the nozzle at a flow rate of 0.15 pL / s, which is the same as that used in DASP printing. Initially, we turn on only the red channel, allowing the mixer chamber to be filled with red particles (left, Fig. 4b, ii). Subsequently, we switch on the green channel, and within 120 sec, the mixer chamber transitions to a greyish purple color because of thorough mixing (middle, Fig. 4b, ii). During this mixing process, approximately 36 pL of bio-ink passes through the chamber, which is around the volume of the chamber itself. Finally, we turn off the red channel, and within another 240 sec, the chamber completely changes to a green color (right, Fig. 4b, iii). The smooth transition of colors in the mixer chamber demonstrates the nozzle’s capability to homogeneously mix the viscoelastic bio-inks (Supplementary Movie 2).

[0124] To showcase the capabilities of DASP 2.0, we seek to fabricate a structure reminiscent of a raspberry: A hollow sphere with a shell composed of a single layer of hydrogel particles. Achieving this goal perhaps represents one of the most formidable tasks in voxelated bioprinting, as it demands precise generation, deposition, and assembly of individual particles in 3D space. Furthermore, it requires a robust connection between neighboring hydrogel particles to ensure mechanical integrity of the structure.

[0125] Building on our previously established knowledge and leveraging the newly developed extrusion module, the printed droplets in the supporting matrix undergo isotropic swelling by 14% in diameter (Supplementary Fig. 11), enabling them to coalesce into a hollow sphere consisting of only one layer of interconnected yet distinguishable DN hydrogel particles. This sphere has a diameter of 7 mm and comprises 42 hydrogel particles, each approximately 1 mm in diameter, as shown by a representative photo in Fig. 4c, i and illustrated by the 3D rendering in Fig. 4c, ii. All the particles are interconnected yet distinguishable, resulting in a mechanically robust free-standing hollow sphere that can be easily manipulated without additional protection (Supplementary Movie 3). Furthermore, we confirm the hollow nature of the sphere by cutting it into two pieces, revealing a single layer of hydrogel particles (Fig. 4c, Hi; Supplementary Movie 4). Compared with the simple cubic lattice structure printed in our previous work8, fabricating such a hollow sphere with high surface curvature represents a greater challenge. This is primarily due to the risk of the structure collapsing if the hydrogel itself is mechanically brittle. Therefore, the successful fabrication of this hollow sphere using DASP 2.0 represents a significant advancement in voxelated bioprinting.

[0126] In a DASP printed structure, the bridges between neighboring droplets may be weaker than the bulk hydrogel, which would impair the mechanical robustness of the structure. To this end, we quantify the extensibility of a DASP printed 1D filament that comprises a sequence of interconnected droplets (Supplementary Fig. 12). As expected, the 1 D filament has a tensile breaking strain efof 0.7, which is slightly lower than that of bulk hydrogel of 1.0 (Figs. 3c, d). Nevertheless, the breaking strain of the 3D filament remains much larger than the constituent single-network hydrogels (e^ « 0.36). These results further support that DASP enables the fabrication of integrated structures consisting of interconnected droplets.

[0127] DASP 2.0 can be easily converted to conventional one-dimensional (1 D) filamentbased bioprinting, as demonstrated by the creation of a gyroid cubic structure (Fig. 4d). Unlike other mesh-like structures such as waffles or honeycombs in which the channels are separated by walls, the gyroid is a highly porous structure consisting of an interconnected network of channels and voids (Fig. 4d, ii). To 3D print such a complex geometric pattern, each layer must slightly protrude beyond the previous layer, forming the so-called hangovers. This poses a challenge for 3D bioprinting, as bio-inks often lack the mechanical strength to support the subsequent hangover layer. However, this caveat can be circumvented using our DN bio-ink, which not only is mechanically strong but also possesses relatively fast crosslinking kinetics. Indeed, with DASP 2.0, we successfully print our DN bio-ink into a gyroid with high fidelity, as confirmed by the 3x4 array of tunnel-like holes with a diameter of 1 .5 mm on the side view (Figs. 4d, i & iii), precisely capturing the intended design (Fig. 4d, ii; Supplementary Movie 5). To assess the mechanical properties of the DN gyroid, we perform a cyclic compression test at a strain rate of 0.005 / sec. The DN gyroid can sustain a large compression strain of 60% without fracturing (Figs. 4e, i-iii). Upon stress release, it completely recovers its original height, as visually depicted in Fig. 4e, iv and Supplementary Movie 6. The stress-strain profiles during loading and unloading almost overlap (solid lines, Fig. 4f). Quantitatively, the energy dissipation efficiency, defined as the ratio between the integrated area in the hysteresis loop and that under the compression curve, is relatively small with a value of 27.5%. This elastic, non-dissipative behavior persists over eight cycles of cyclic compression, as evidenced by the nearly perfectly overlaying of stress-strain profiles (solid lines, Fig. 4f). In contrast, the gyroid made of pure alginate exhibits plastic, dissipative behavior with the energy dissipation efficiency remarkably high of 80.2% at the first cycle. After eight compression cycles, the height of the gyroid decreases to 74% of its original value, as indicated by the increase of onset strain above which the stress becomes positive (dashed line, inset of Fig. 4f). Moreover, as the number of compression cycles increase to eight, the stress at 60% strain decreases by approximately 30% from 8.6 kPa to 6.0 kPa. These results demonstrate that DASP printing DN bio-inks enables the fabrication of highly deformable, elastic, non-dissipative, and multiscale porous scaffolds.

[0128] Cytocompatibility and in vivo stability of DASP printed DN scaffolds:

[0129] Compared to traditional scaffolds based on bulky hydrogels, the DASP-printed DN scaffolds offer a promising platform for cell-based therapy, which often involves encapsulating therapeutic cells for transplantation. The multiscale porosity has previously been shown to facilitate efficient nutrient transport8, while the mechanical robustness is expected to allow the scaffolds to withstand constant mechanical perturbation resulting from the movement of the transplant recipient. To demonstrate this potential, we begin by investigating the cytocompatibility of both the printing process and the bio-ink by encapsulating cells into each hydrogel droplet. To do so, we modify the print nozzle by adding an extra channel, creating a three-channel nozzle (Supplementary Figs. 10b, c). Similar to the original print nozzle, two channels are used to load the DN ink. However, the third channel is dedicated to loading cells, as illustrated in Fig. 5a, i. This design ensures that the cells are not loaded directly to DN ink, as this could change the rheological properties of the ink. Such alterations can be significant and unpredictable at high cell density, rendering the ink unsuitable for DASP printing. Additionally, following our previously developed method25, we incorporate 15% (w / v) dextran and 17% (w / v) polyethylene glycol) (PEG) polymers into the cell suspension and the DN ink, respectively. Compared to the DN ink, these polymer solutions are of relatively low viscosity and do not impact the printing process. This method results in an aqueous two-phase system, in which both the dextran and PEG solutions are aqueous and cytocompatible yet have a small interfacial tension that is sufficient to lead to phase separation26. During the extrusion process, the DN ink encounters the dextran phase in the static mixer chamber (Fig. 5a, i). After extrusion, the dextran phase is expected to phase separate from DN ink, forming an island-sea-like morphology, as illustrated in Fig. 5a, ii. Indeed, this microstructure is confirmed by visualizing the distribution of dextran phase and cell-mimicking polystyrene microspheres with a diameter of 20 pm, respectively shown by a representative fluorescence confocal microscopy 3D image in Supplementary Fig. 13 and a bright field microscopy image in Fig. 5a, iii (see Methods). These results demonstrate that, together with the aqueous two-phase system, the three- channel nozzle allows for transforming the DN ink into a sponge-like structure with a solid hydrogel filled with cell-laden liquid voids.

[0130] By replacing the cell-mimicking microspheres with Beta-TC-6 cells, we print a 5*5*4 DN lattice (Supplementary Fig. 14). Beta-TC-6 cells are a pancreatic beta cell line that retains the function of glucose-stimulated insulin secretion, allowing us to test both the cell viability and the transport properties of the multiscale porous scaffold. The live / dead assay reveals that the viability of Beta-TC-6 cells immediately after printing is 61 ,3± 8.0%, and it remains above 60% for the subsequent three days (Figs. 5b, c). This viability is consistent with the values 40-80% reported for extrusion-based bioprinting27, but it is considerably lower compared to DASP 1 .0, which uses pure alginate as the hydrogel and exhibits a viability of 90%8. The lower viability can be attributed to the cytotoxicity of free polyacrylamide polymers present in the DN ink before complete crosslinking. Although polyacrylamide-based hydrogels are generally considered as non-toxic and extensively used in biomedical research, our study reveals that free polyacrylamide polymers exhibit some degree of cytotoxicity. Indeed, live / dead assay conducted on cells directly mixed with pure polyacrylamide polymers with the same concentration as that used for the DN bio-ink shows a cell viability less than 1% (Supplementary Fig. 15). Nevertheless, in DASP 2.0 more than half of the cells survive and maintain their viability for three days, which is a typical waiting time before transplantation in clinical settings.

[0131] Next, we test the insulin release of the lattice scaffolds using static glucose- stimulated insulin secretion (GSIS) measurement. Specifically, we expose the Beta-TC-6 cell-encapsulated scaffolds to low and high glucose media and measure the amount of released insulin. The release index is defined as the ratio between insulin release amounts in high glucose media over that in low glucose media. On the first day, the lattice scaffolds exhibit a relatively low release index with a value of 1.39±0.56. Yet, this value is comparable to that of the naked counterpart, which exhibits a release index of 1 ,31±0.42 (Fig. 5d). This is likely because that the cells are in a recovery phase after detachment from the culture flask. In the subsequent days, the release index of the naked cells increases significantly, reaching a value of 6.78±1 .03 on day 3, which is about twice the value of 2.98±0.83 observed for the lattice scaffolds (Fig. 5d). This difference is consistent with our previous studies, in which we found that the release index of islets encapsulated 3*3*3 lattice scaffolds created using DASP 1.0 is about half that of the naked counterpart. Nonetheless, the release index of DASP Beta-TC-6 cells on day 3 is much higher than one, which exceeds the standard line for clinical transplantation. Taken together, our results show that DASP 2.0 enables encapsulation of living cells while maintaining reasonable cell viability and function.

[0132] To further confirm the suitability of the DN scaffolds for clinical transplantation, we perform in vivo studies to evaluate their mechanical robustness. We transplant the 5*5*4 lattice scaffolds into the abdominal cavity of immunocompetent C57BL / 6 mice, as respectively illustrated and photographed in Figs. 5e, i and ii. This in vivo environment is characterized by vigorous mechanical perturbation resulting from intestinal peristalsis and body movement. Two bio-ink formulations are used to prepare the scaffolds: (i) 4% (w / v) alginate, our previous prototype formulation for DASP 1.0, denoted as Alg4, and (ii) DN bioink for DASP 2.0, a formulation explored for cell encapsulation, denoted as PAMioAlg2(Fig. 5a). Both Alg4and PAMi0Alg2scaffolds exhibit similar stiffness, with respective apparent compression moduli of 5.6 and 5.8 kPa (dashed red line and dark line, Fig. 5h). However, a significant difference in in vivo stability is observed for these two scaffolds. Alg4scaffolds fragment only 7 days after the transplantation and can be hardly retrieved, as shown in Fig. 5f. This phenomenon can be attributed to the fragility of the alginate scaffold, which fractures under a relatively small strain of 37% during compression test (dashed red line, Fig. 5h), and limited stability of ionic crosslinks in physiological conditions28. In contrast, the PAMi0Alg2scaffolds retain their integrity without observable deformation for 120 days after the transplantation, as shown by Figs. 5g, i and ii. As observed in different mice, the PAMi0Alg2scaffolds are widely distributed in the abdominal cavity, in contact with the stomach, intestine (Fig. 5g, iii), bladder, liver (Supplementary Fig. 16a), and sometimes slightly adhering to fat tissue, yet all scaffolds could be successfully retrieved (Supplementary Fig. 16b). Moreover, the retrieved scaffolds maintain the pre-transplant lattice structure in which individual particles are interconnected yet distinguishable (Fig. 5g, iv).

[0133] The integrity of the retrieved DN scaffolds is further confirmed by their mechanical robustness. For scaffolds retrieved 30 days after transplantation, their yield compression stress decreases from 39.3 kPa to 23.8 kPa compared to its pre-transplant value; however, the yield compression strain remains remarkably high of approximately 60% (solid blue line, Fig. 5h). Moreover, the stress-strain curve nearly perfectly overlaps with that of the pretransplant one, and this behavior persists for scaffolds retrieved after longer periods of transplantation for 60 and 120 days (purple and green lines, Fig. 5h). Taken together, our results show that the DASP 2.0 printed DN scaffolds possess sufficient mechanically robustness to maintain long-term in vivo stability.

[0134] Universality of DN (A+B) / C hydrogels as bio-inks for DASP 2.0:

[0135] We note that the cytocompatibility of the PAM / alginate DN bio-ink is relatively low, with cell viability ~60% (Fig. 5c), largely because free PAM-based polymers are not completely cytocompatible (Supplementary Fig. 15). Yet, the concept of (A+B) / C DN hydrogels based on TZ-NB click chemistry is expected to be applicable to other biopolymers, where A, B, and C respectively represent TZ-polymer, NB-polymer, and alginate. To test this, we replace the PAM polymer by hyaluronic acid (HA), a natural polysaccharide widely used in biomaterials development. Following the same chemistry as for the PAM (Fig. 1 b), we functionalize a pair of HA polymers, respectively, with TZ and NB at nearly the same grafting ratio of 27%, corresponding to approximately 250 TZ or NB groups per polymer (Supplementary Figs. 6, 7). Upon mixing, the pair of TZ-HA and NB-HA crosslink to form a network via additive-free click reaction, as illustrated in Fig. 6a.

[0136] Compared to the single-network hydrogels, the improvement in the mechanical properties of the HA / alginate DN hydrogel is comparable to that observed for the PAM / alginate formulation. For instance, the shear modulus of a single-network HA is about 500 Pa (solid grey line, Fig. 6b). By contrast, for the DN HA / alginate hydrogel, G is about 3050 Pa, approximately the sum of HA and alginate single-network hydrogels (red solid line, Fig. 6b). Moreover, unlike the single-network HA hydrogel that is brittle with a tensile breaking strain of 0.6, the DN HA / alginate hydrogel is significantly more stretchable with ef= 1.02, as shown in Figs. 6c, d and Supplementary Movie 7.

[0137] As expected, the HA / alginate is suitable for DASP printing, as demonstrated by the success of creating a 5*5x4 lattice scaffold (Fig. 6e). To test the cytocompatibility of the HA / alginate hydrogel, we directly mix Beta-TC-6 cells with the bio-ink at a cell density of 5 million / mL. Live / dead assay reveals that the viability of HA / alginate encapsulated Beta-TC-6 cells remains above 80% for three days after the printing (Fig. 6f). This survival rate is significantly higher than 60% observed in PAMi0Alg2, and importantly, meets the standards for most applications27. Finally, we transplant the DASP printed 5x5x4 lattice scaffolds made of HA / alginate into the mouse abdominal cavity. The integrity of the retrieved scaffolds is confirmed 30 days after transplantation (Fig. 6g), indicating that HA / alginate DN scaffolds are sufficiently robust to maintain long-term in vivo stability. Taken together, these results demonstrate that the concept of (A+B) / C hydrogels provides a universal strategy for the development of modular DN bio-inks for DASP printing. Discussion:

[0138] The advancement of voxelated bioprinting requires three indispensable efforts: (i) biomaterial voxels, (ii) technology development, and (iii) demonstration of biomedical applications. Through a combination of biomaterials development and hardware engineering, we have significantly advanced our voxelated bioprinting technology from DASP 1.0 to DASP 2.0. Compared to DASP 1 .0 that can print pure alginate voxels only, DASP 2.0 allows for printing DN hydrogel voxels consisting of interpenetrating alginate and PAM or HA polymers. Unlike classical DN PAM / alginate hydrogels crosslinked by cytotoxic radical polymerization of acrylamide monomers22, our DN hydrogel is formed by crosslinking PAM or HA polymers through a biofriendly click chemistry. This is achieved by controlled synthesis of a pair of precursor PAM or HA polymers, which are functionalized with prescribed fractions of tetrazine and norbornene groups, respectively. Upon mixing, a pair of tetrazine and norbornene groups forms a covalent bond through click reaction without any external trigger. In the meantime, the alginate network is formed by ionic crosslinking. These two kinds of crosslinking mechanisms are orthogonal, allowing for modular control over hydrogel mechanical properties through its constituent polymer networks. Further, we engineer multi-channel print nozzles to enable on-demand mixing of highly viscoelastic bioinks without significantly impairing cell viability. Exploiting the print nozzle allows for the fabrication of highly complex 3D structures such as a hollow sphere made of one layer of interconnected yet distinguishable hydrogel particles. Finally, we conduct in vitro studies to show that voxel-printed DN scaffolds are cytocompatible, as well as in vivo studies to demonstrate that the scaffolds are mechanically robust to be easily retrieved after being transplanted into immunocompetent mice.

[0139] The concept of using a pair of stoichiometrically matched functionalized polymers for hydrogel synthesis offers precise control over crosslinking kinetics and stiffness. In classical HA29and PEG30based hydrogels, multiple-functional polymers are pre-mixed with small molecule crosslinkers, which upon external triggers such as light crosslink the polymers to form a network. As a result, the gelation kinetics is often sensitive to the type and conditions of chemical reactions and can be difficult to be precisely controlled. By contrast, our hydrogels are crosslinked by click reaction, a process that is relatively insensitive to reaction conditions. The gelation kinetics can be well-described by a one-parameter theory accounting the concentration of crosslinkers. Moreover, the hydrogel stiffness increases with the polymer concentration by a power of 2.1 ; this relation is well-described by a simple polymer physics model that the network stiffness is kBT per correlation volume of the polymer solution. Compared to the linear dependence of stiffness on crosslinker concentration in conventional hydrogels, this power law behavior offers a wider range of tunable hydrogel stiffness. Unlike single-network hydrogels whose stiffness and extensibility are intrinsically negatively correlated, the developed DN bio-ink offers modular control over mechanical properties. For example, the stiffness of the DN hydrogel is about the sum of the moduli of its constituent single-network hydrogels. In contrast, the elongation at break is determined by the size of PAM network strand. Consequently, one can control the extensibility of a DN hydrogel by varying the crosslinking density of the PAM network and control the stiffness by varying the concentration of alginate. Such a modular control over the mechanical properties may offer an approach to designing tough biomaterials for applications such as engineering blood vessels31and cartilage repair32.

[0140] In the context of hardware engineering, the capability of integrating multiple flows into a single print nozzle offers versatility in processing bio-inks. For instance, the developed dual-inlet print nozzle allows for mixing a pair of highly viscoelastic biomaterials on demand. The design of the nozzle is inspired by the commercially available static mixer Quadro™ Square. Yet, leveraging the capability of stereolithography printing, the dimension of the print nozzle is dramatically reduced to ensure a minimal dead volume; this allows us to use a relatively small amount of bio-ink less than 1 mL during processing including but not limited to bioprinting. An example is using this nozzle to rapidly extrude mixtures of A+B hydrogels onto the rheometer for the real-time characterization of crosslinking kinetics and dynamic mechanical properties.

[0141] The developed triple-inlet print nozzle allows for leveraging the existing aqueous two- phase system26to encapsulate cells at high density without significantly impairing cell viability. Moreover, the printed hydrogel voxels possess a multiscale porous structure, in which cell containing microscale liquid domains are randomly dispersed into a solid DN hydrogel. This structure may offer advantages in efficient nutrient transport essential to cell survival33. We note that free PAM polymers are not suitable for long-term cell culture because of limited cytocompatibility (Supplementary Fig. 15). Although this caveat can be mitigated using the triplet-inlet print nozzle to avoid directly mixing the cells with the PAM polymers for a long time, the cytocompatibility of the bio-ink remains relatively low with a cell viability of 60%. However, this issue can be solved by replacing the PAM backbone with a HA polymer, which is cytocompatible and can be directly pre-mixed with cells during the printing without significantly impairing cell viability.

[0142] The success of voxelated printing DN bio-inks relies on on-demand mixing, extruding, and crosslinking A+B type biomaterials without external trigger. Such a capability is enabled by integrating additive-free click chemistry and a multi-channel print nozzle. This design strategy has important implications in embedded bioprinting, in which the deposited bio-inks are trapped inside a thick supporting matrix. This matrix prevents easy access to external triggers such as UV or heat, which are often required to solidify the printed structures34. In our printing process, the additive-free click chemistry allows the bio-inks to be solidified before removing the supporting matrix, enabling the fabrication of highly complex 3D objects with high fidelity (Figs. 4c, d). This capability may be appealing for administering biomaterials in vivo, which is an environment with limited access to external triggers. Finally, the developed multi-channel can be easily adapted to medical syringes and needles for administering the DN bio-inks as a class of injectable biomaterials35.

[0143] We note a few limitations and potential challenges associated with applying DASP 2.0 for basic and translational biomedicine. For instance, to exploit DASP 2.0 to understand and control cell-matrix interactions, it is necessary to render the DN hydrogels cell- instructive. To this end, when preparing the DN HA / alginate hydrogel, the TZ-HA and NB-HA are mixed with a mismatched NB / TZ ratio of 2 / 1 (Fig. 6a), resulting in considerable unreacted free NB groups in the hydrogel network. These free NB groups may be used to conjugate extracellular matrix derived peptides for cell engagement3637. Moreover, degradable crosslinkers3839and growth factors40may be conjugated into the hydrogel network to promote cell migration and vascularization.

[0144] In the context of translational biomedicine, our results highlight the potential of voxelated bioprinting in manufacturing cell-encapsulated multiscale porous and mechanically robust scaffolds as therapeutic transplants41 42. For instance, a potential application is to encapsulate human islets in a voxelated 3D scaffold, which may be used as a transplant to reverse type 1 diabetes. Since a voxelated 3D scaffold consists of interconnected yet distinguishable hydrogel particles, it may combine the advantages of classical microencapsulation technology in high surface-to-volume ratio4344and macro-devices in easy retrievability4546. However, it remains to be determined the biomaterial biocompatibility and the ability of voxelated 3D scaffolds to allow long-term cell survival and function. Nevertheless, DASP 2.0 represents a significantly advanced voxelated bioprinting technology, paving the way for engineering highly complex yet organized functional 3D tissue constructs.

[0145] Methods

[0146] Materials and reagents for chemical synthesis, bioprinting, and cell culture:

[0147] Chemical synthesis. 4-(Aminomethyl)benzonitrile hydrochloride (Cat. No. 631396), triethylamine (Cat. No. 471283), di-tert-butyl decarbonate (Boc20, Cat. No. 205249), hydrazine (anhydrous, Cat. No.215155), trifluoroacetic acid (Cat. No. T6508), poly(acrylamide-co-acrylic acid) partial sodium salt (MW 150 kDa, acrylamide ~80 wt. %, Cat. No. 511471) are purchased from Sigma-Aldrich, USA. Nickel(ll) trifluoromethanesulfonate (Ni(OTf)2, Cat. No. N08615G), 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)- 4-methylmorpholinium chloride (DMTMM, Cat. No. D29195G), and 5-norbornene-2- methylamine (Cat. No. N09075G) are purchased from Tokyo Chemical Industry (TCI), Fisher Scientific, USA.

[0148] Voxelated bioprintinq. Gelatin from porcine skin (gel strength 300, Type A, Cat. No. G2500), alginic acid sodium salt from brown algae (medium viscosity, Cat. No. A2033), dextran from Leuconostoc spp. with MW approximately 40,000 Da (Cat. No. 31389), polystyrene microparticles with a diameter of 20 pm (Cat. No. 74491), and fluorescein isothiocyanate-dextran (2000 kDa, Cat. No. FD2000S) are purchased from Sigma-Aldrich, USA. Polyethylene glycol) (PEG) with MW of 8 kDa (Cat. No. 43443) is purchased from Fisher Scientific, USA. Red and green mica powders for visualization of bio-ink flows are purchased from the SEISSO, China.

[0149] Cell culture and characterization. Beta-TC-6 cells are received as a gift from Dr. Yong Wang, which was purchased from the American Type Culture Collection (Cat. No. CRL-11506). Dulbecco’s Modified Eagle Medium (DMEM, high glucose, GlutaMax, Cat. No. 10566-016), DMEM (high glucose, without calcium, Cat. No. 21068-028), FBS (Cat. No. 10438-018), Pen / Strep (Cat. No. 15140-122), 2-mercaptoethanol (Cat. No. 21985- 023) are purchased from Gibco, Fisher Scientific, USA. Chemical dye for live / dead assay including fluorescein diacetate (Cat. No. F7378), propidium iodide (Cat. No. P4170) are purchased from Sigma-Aldrich, USA.

[0150] Chemical and polymer synthesis:

[0151] Compound I (Cl): tert-butyl / V-(4-cyanobenzyl) carbamate. Cl is prepared following a previously reported procedure47with minor modifications. To a solution of 4- (aminomethyl)benzonitrile hydrochloride (10.00 g, 59.3 mmol) in dichloromethane (250 mL), triethylamine (20.7 mL, 148.5 mmol) and Boc2G (14.23 g, 65.2 mmol) are gently added. The reaction mixture is stirred at RT for 12 h. The resulting solution is washed with 10% HCI (200 mL) to remove the excessive triethylamine and unreacted 4-(aminomethyl)benzonitrile hydrochloride if any. Finally, DCM is removed in vacuo. The obtained product is a white solid with 98% yield. The product is verified by1H-NMR (Supplementary Fig. 1) and used for the synthesis of CH without further purification.

[0152] Compound II (Cll): tert-butyl / V-(4-(6-methyl-1 ,2,4,5-tetrazin-3-yl)benzyl)carbamate Cll is prepared following a previously reported procedure48. Cl (2.00 g, 8.6 mmol), acetonitrile (4.5 mL, 86.2 mmol), Ni(OTf)2(1.53 g, 4.3 mmol), and hydrazine (13.8 mL, 430.6 mmol) are added to a 60 mL high pressure tube (ACE GLASS, Sigma-Aldrich, Cat. No. Z568872). The tube is sealed, and the reaction mixture is stirred at 60 °C for 24 h. The resulting mixture is diluted with EtOAc (200 mL), transferred to a separatory funnel, and washed with brine (100 mL) for three times to remove the excessive hydrazine and acetonitrile. The intermediate is further oxidized into Cll following a previously reported method48. Finally, EtOAc is removed in vacuo and the crude product is obtained as dark purple solid. The crude product is further purified by silica gel column chromatography using gradient elution with EtOAc / Hexane from 8:92 to 20:80. The yield for the synthesis of CH is 59%. The product is verified by1H-NMR (Supplementary Fig. 2).

[0153] Compound III (CHI'): 4-(6-Methyl-1 ,2,4,5-tetrazin-3-yl)benzenemethanamine

[0154] To a solution of Cll (5 g, 16.6 mmol) in DCM (25.4 mL), trifluoroacetic acid (25.4 ml_, 332 mmol) is added and the reaction mixture is stirred at RT for 2 h. Then, DCM is removed in vacuo and saturated NaHCO3solution is added dropwise to neutralize the residual trifluoroacetic acid. At increased pH caused by NaHCO3, most of Cl 11 precipitates due to the transformation from HCI salt state to original state, yet there is a slight amount of Cl 11 dissolved in the aqueous phase. Finally, the precipitated product and the minor portion in the aqueous phase are extracted with DCM. After the removal of DCM in vacuo, Cl 11 is obtained as a pink solid with 99% yield. The product is verified by1H-NMR (Supplementary Fig. 3) and used for modifying polymers without further purification.

[0155] Tetrazine-modified polv(acrylamide-co-acrylic acid)

[0156] DMTMM (4.66 g, 16.8 mmol) is added to a solution of poly(acrylamide-co-acrylic acid) (PAmAc) (10 g, 140 mmol chemical monomer) in 300 mL mixed solvent of water / MeOH (2:1 v / v). The reaction mixture is stirred at RT for 30 min, then Cl II (0.84g, 4.2 mmol) suspended in 10 ml MeOH is added. The reaction mixture is further stirred at RT for 48 h. The resulting polymer solution is precipitated in 1 .5 L acetone twice. Then, the precipitated polymer is collected and redissolved in 200 mL water and dialyzed (Spectrum™, 3.5 kDa molecular weight cut-off (MWCO), Cat. No. 132112T) against DI water for further purification. During dialysis, the outer phase is refreshed 3 times / day for 72 h. Finally, the polymer is dried through lyophilization. The grafting density measured by1H-NMR (Supplementary Fig. 4) is 2.53%, which is defined as the molar ratio between the grafted tetrazine and total number of chemical monomers on the PAmAc backbone.

[0157] Norbornene-modified poly(acrylamide-co-acrylic acid)

[0158] DMTMM is added to the solution of PAmAc (10 g, 140 mmol chemical monomer) in 300 mL mixed solvent of water / MeOH (2:1 v / v). The reaction mixture is stirred at RT for 30 min, then 5-norbornene-2-methylamine dissolved in 10 ml MeOH is added and the mixture is further stirred at RT for 48 h.

[0159] To synthesize NB-PAmAc with various grafting density, we change the molar ratio of the feeding norbornene to the total number of chemical monomers on the polymer backbone. Specifically, 5-norbornene-2-methylamine is added as 0.35 g (2.8 mmol), 0.52 g (4.2 mmol), 0.69 g (5.6 mmol), and 1.04 g (8.5 mmol); whereas DMTMM is respectively added as 3.09 g (11.2 mmol), 4.64 g (16.8 mmol), 6.18 g (22.4 mmol), and 9.38 g (34.0 mmol). The resulting polymer solution is precipitated in 1 .5 L acetone twice. Then, the precipitated polymer is collected and redissolved in 200 mL water and dialyzed against DI water (Spectrum™, 3.5k MWCO, Cat. No. 132112T) for further purification. During dialysis, the outter phase is refreshed 3 times / day for 3 days. Finally, the product is dried through lyophilization. The grafting ratio measured by1H-NMR (Supplementary Fig. 5) is 0.63%, 1.51%, 2.57%, and 4.25%, respectively. Tetrazine-modified sodium hyaluronate

[0160] DMTMM (2.00 g, 7.2 mmol) is added to a solution of sodium hyaluronate (HA) (2 g, 5 mmol chemical monomer) in 200 mL mixed solvent of water / MeOH (2:1 v / v). The reaction mixture is stirred at RT for 30 min, then CUI (0.36 g, 1.8 mmol) suspended in 10 ml MeOH is added. The reaction mixture is further stirred at RT for 48 h. The resulting polymer solution is precipitated in 1 L ethanol for twice. Then, the precipitated polymer is collected and redissolved in 200 mL water and dialyzed (Spectrum™, 3.5 kDa molecular weight cut-off (MWCO), Cat. No. 132112T) against DI water for further purification. During dialysis, the outer phase is refreshed 3 times / day for 72 h. Finally, the polymer is dried through lyophilization. The grafting density measured by1H-NMR (Supplementary Fig. 6) is 27 %, which is defined as the molar ratio between the grafted tetrazine and total number of chemical monomers on the HA backbone. Norbornene-modified sodium hyaluronate

[0161] DMTMM (2.66 g, 9.6 mmol) is added to a solution of sodium hyaluronate (HA) (2 g, 5 mmol chemical monomer) in 200 mL mixed solvent of water / MeOH (2:1 v / v). The reaction mixture is stirred at RT for 30 min, then 5-norbornene-2-methylamine (0.3 g, 2.4 mmol) suspended in 10 ml MeOH is added. The reaction mixture is further stirred at RT for 48 h. The resulting polymer solution is precipitated in 1 L ethanol twice. Then, the precipitated polymer is collected and redissolved in 200 mL water and dialyzed (Spectrum™, 3.5 kDa molecular weight cut-off (MWCO), Cat. No. 132112T) against DI water for further purification. During dialysis, the outer phase is refreshed 3 times / day for 72 h. Finally, the polymer is dried through lyophilization. The grafting density measured by1H-NMR (Supplementary Fig. 7) is 27 %, which is defined as the molar ratio between the grafted norbornene and total number of chemical monomers on the HA backbone.

[0162] Instrument setup of DASP 2.0:

[0163] Building on DASP 1 .08, we develop the instrument of DASP 2.0 the by improving the extrusion module and the print nozzle. First, we develop an extrusion module for simultaneously loading and extruding up to three syringes (Supplementary Fig. 10a). Second, the print nozzle of DASP 2.0 is made of a dispensing needle (26G, McMaster-Carr) and an upstream mixing module with an inner static mixer chamber, such that bio-inks can be mixed on-demand during extrusion. The mixing module is manufactured through stereolithography 3D printing (Formlabs 3+). To demonstrate printing DN bio-inks and cell encapsulated DN scaffolds, we respectively design a dual-inlet and a triple-inlet print nozzle, as visualized in Supplementary Figs. 10b, c. The dual-inlet print nozzle allows a pair of bioinks to be mixed in the mixer chamber. The triple-inlet print nozzle ensures that a pair of bioinks meet the cell suspension before being mixed in the static mixer chamber. During printing, the print nozzle is attached to the extrusion module and connected with syringes (1 ml_, Shanghai Bolige Industrial & Trade Co., Ltd) through tubing (Cat. No. BB31695-PE / 5, Scientific Commodities Inc).

[0164] Bio-inks:

[0165] To explore the crosslinking kinetics and stiffness of single-network PAM hydrogels (Fig. 2), we prepare the bio-inks by respectively dissolving TZ-PAM and NB-PAM in DI water. This approach avoids possible effects from salt ions on the conformation of polymer chains, simplifying the corresponding theoretical calculations. In the subsequent study on the stiffness and extensibility of DN hydrogels (Fig. 3), we use 150 mM NaCI solution to respectively prepare PAM, alginate, and PAM / alginate DN bio-inks. Specifically, to prepare a DN bio-ink, we respectively dissolve TZ-PAM and NB-PAM in 150 mM NaCI solution, and then add the same amount of alginate to each of the two solutions. These bio-inks are isotonic and suitable for the following mechanical test in which the hydrogels are required to equilibrate in the cell culture media. For cell encapsulation and animal studies, the concentrations of the TZ-PAM, NB-PAM, and alginate are respectively fixed at 10%, 10%, and 2% (w / v). Additionally, the NaCI solution is replaced by DI water with 15% (w / v) PEG and 40 Mm HEPES.

[0166] To prepare the HA, alginate, and DN HA / alginate bio-inks in Fig. 6, we use 150 mM NaCI solution to prepare TZ-HA, NB-HA, and alginate bio-ink. Following the same protocol described in PAM / alginate bio-ink, we prepare HA / Alginate bio-ink with the concentration of TZ-HA, NB-HA, and alginate respectively fixed at 2%, 4%, and 2% (w / v). For cell encapsulation and animal studies, the NaCI solution is replaced by DMEM (without calcium) media supplemented with 30 Mm HEPES. Rheometry:

[0167] Rheological measurements are performed using a stress-controlled rheometer (Anton Paar MCR 301) equipped with a parallel-plate geometry of diameter 25 mm at 20°C. To prevent the evaporation of water from hydrogels, we apply mineral oil to seal the edge of the geometry for all samples. The correction to the measured moduli from the mineral oil is negligible according to our previous studies8.

[0168] To prepare the sample for single-network PAM or HA hydrogels, we use dual-inlet print nozzle to manually extrude the bio-ink on the bottom geometry of the rheometer, lower the geometry to a 1 mm gap, and trim the edge of the liquid. We characterize the crosslinking kinetics by monitoring in real time the storage modulus G' and loss modulus G" at an oscillatory shear frequency of 1 Hz and a shear strain of 0.5% for 12 h. This duration is sufficient for complete crosslinking of the hydrogel. The time taken for mixing the bio-inks is included when determining crosslinking kinetics. Following the measurements on kinetics, we apply an oscillatory frequency shear on the crosslinked hydrogel to characterize its shear moduli. The frequency is changed from 0.1 to 100 Hz and the strain is fixed at 0.5 %. The resulting data, shown in Fig. 2d, encompasses a frequency range from 0.1 to 20 Hz due to sample slippage at higher frequencies. In parallel, we measure the shear moduli of singlenetwork hydrogels in DMEM (Figs. 3a, b, e). Unlike DI water, DMEM contains salt ions that are known to slightly affect hydrogel mechanical properties. To this end, we incubate a hydrogel in DMEM for 24 hours to reach equilibrium before measurement.

[0169] To prepare the sample of a single-network alginate hydrogel, we prepare a film of alginate solution with an approximate thickness of 1 mm. To crosslink the film, we immerse it in a bath consisting of 20 mM CaCI2and 120 mM NaCI in deionized water (DI water) for 10 min. We then transfer the solidified film to cell culture media (DMEM) supplemented with 2 mM CaCI2and incubate for 24 hours to equilibrate the film. We cut the film into a disk with a diameter of 25 mm, matching the dimension of the rheometer geometry. The hydrogel disk is loaded onto the bottom geometry of the rheometer, and the upper geometry is lowered until it contacts the sample, which is indicated by a positive normal force of approximately 0.1 N. To characterize the shear moduli, an oscillatory shear is applied with a fixed strain of 0.5% and various frequency ranging from 0.1 to 100 Hz. Similar procedure is used to prepare and measure the dynamic mechanical properties of DN hydrogel samples.

[0170] We measure the dependence of bio-ink viscosity on shear rate y in the range of 0.1 to 1000 1 / sec. For all bio-inks, the viscosity is nearly constant at shear rates < 1 s1and dramatically decreases at shear rates >10 s1. We take the value at the lowest shear rate 0.1 S'1as the viscosity of a bio-ink. Uniaxial tensile test:

[0171] Uniaxial tensile tests are performed using a motorized tension test stand (MARK- 10 ESM303) equipped with a digital force gauge (MARK-10 M5-05) and two clamps (Mark-10 G1062). We use the same equilibrated hydrogel film in DMEM for rheological measurements to prepare the sample for tensile tests. Specifically, after rheological measurements, we detach the film from the geometry of the rheometer and use a die to cut the film into a dog bone-shape with the center part 2 mm in width and 13 mm in length. The sample is stretched at a fixed strain rate of 0.02 / sec until it fractures. Throughout this process, stress is recorded at a rate of 3 data points per second.

[0172] Evaluation of the mixing capability of the multi-channel print nozzle: We use a digital camera (Hayear, 16 MP) equipped with a long working distance lens to monitor the mixing process in real-time. To visualize the mixing, we incorporate red and green mica microparticles (50 pm) with a concentration of 0.5% (w / v), respectively, into a pair of bio-inks made of PAMioAlg2. The two bio-inks are simultaneously extruded through the dual-inlet nozzle’s respective inlets, each at a consistent flow rate of 0.15 pL / s, matching the flow rate used in DASP printing. The progression of the mixing process is visually demonstrated by initially activating the red channel, followed by activation of the green channel, and culminating with the deactivation of the red channel.

[0173] 3D printing of the double-network and alginate hydrogel:

[0174] We use our developed extrusion nozzle equipped with a dual-inlet print nozzle to demonstrate the printability of DN bio-inks (Figs. 4c, d; Fig. 6e). The method for DASP printing is the same as that used in our previous publication8. For filament-based printing, the movement of the nozzle is programmed by the slicer software (Ultimaker Cura 4.6).

[0175] Once the printing process is completed, the printed structure is left in the supporting matrix for 2 minutes for PAMi0ALg2bio-ink and 10 min for HA / alginate bio-ink. This period facilitates the solidification of the PAM and HA networks. Then, we immerse the supporting matrix in a bath containing 20 mM calcium and 120 mM NaCI at 37 °C for 10 min. This process not only dissociates the gelatin supporting matrix but also completely crosslinks the alginate hydrogel. Following the removal of the supporting matrix, the printed 3D structures are kept in saline (150 mM NaCI in DI water) supplemented with 2 mM CaCI2for 24 hours and imaged using a camera (Canon EOS 7D) equipped with a long working distance objective.

[0176] DASP printing scaffolds encapsulated with Beta-TC-6 cells:

[0177] For cell encapsulation within PAMi0Alg2, we use the extrusion module equipped with a triple-inlet print nozzle, in which one channel is loaded with cell suspension and the other two are loaded with a pair of bio-inks. To prepare the cell suspension, we culture Beta-TC-6 cells (ATCC) in multiple T75 flasks to obtain a relatively large number of cells required by the fabrication. The culture medium is DMEM (4.5 g / L D-Glucose) supplemented with 10% FBS, 1% Pen / Strep, 0.0005% 2-Mercaptoethanol and 20 mM HEPES. To harvest the cells, we rinse each flask using 1x PBS, apply 3 mL Trypsin-EDTA for 2 min to detach the cells from the substrate, and subsequently add 6 mL DMEM culture media to neutralize the Trypsin- EDTA. The cell pellet is collected by centrifuging the cell suspension at 600 g for 5 min. The pellet is then resuspended in DMEM (high glucose, without calcium) containing 15% (w / v) dextran. The final concentration of the cell suspension is 20 million / mL.

[0178] To prepare the bio-inks, we respectively dissolve TZ-PAM and NB-PAM in DI water with a concentration of 10% (w / v). Subsequently, PEG is introduced into the solution with a concentration of 15% (w / v). The osmotic pressure from the PEG polymers is approximately 440 kPa49, which is comparable to the osmotic pressure generated by 90 mM NaCI. To achieve a neutral pH of the bio-inks, we add 1 M ( / V-2-hydroxyethylpiperazine- / V-2- ethanesulfonic acid) (HEPES) stock buffer to reach a final HEPES concentration of 40 mM. Importantly, this pH adjustment is accomplished with minimal impact on the overall volume of the bio-ink. Taken PEG and HEPES together, the osmotic pressure resulting from polymers in the bio-ink is about 540 kPa, which is close to the physiological osmotic pressure of 730 kPa caused by ions. Finally, we dissolve the same amount of alginate in each of the two solutions with a concentration of 2% (w / v).

[0179] We load the cell suspension into a 250-pL syringe and load the pair bio-inks respectively into two 1-mL syringes. During printing, these three syringes are driven by the extrusion module at the same speed such that the flow ratio between cell suspension and the two bio-ink channels are 1 :4:4. We DASP print 5*5x4 lattice scaffolds (Supplementary Fig. 14) encapsulated with Beta-TC-6 cells. The printed scaffolds are transferred to a 24-well plate with 1 scaffold in 1 ml_ Beta-TC-6 culture medium per well. To study the distribution of cell-mimicking microparticles in the extruded bio-ink (Fig. 5a, iii), we replace the Beta-TC-6 cells with polystyrene microparticles at the same concentration of 20 million / mL in suspension.

[0180] To characterize the porosity of the aqueous two-phase system, we label the dextran phase with fluorescence and capture the distribution of the dextran phase in 3D under the fluorescence confocal microscopy. Specifically, we add a trace amount of FITC-labeled dextran (2000 KDa) to the dextran phase with a concentration of 0.2% (w / v). Subsequently, we use the extrusion nozzle to mix the dextran phase with the counterpart phase containing 15% (w / v) PEG and 2% (w / v) alginate. Such mixture is crosslinked by 20 mM calcium ion right before the imaging.

[0181] For cell encapsulation within HA / alginate, we first prepare the bio-inks by respectively dissolving TZ-HA and NB-HA in DMEM cell culture media (without calcium) supplemented with 30 Mm HEPES with a concentration of 2 and 4% (w / v). Additionally, we dissolve the same amount of alginate in each of the two solutions with a concentration of 2% (w / v). Next, we directly mix the Beta-TC-6 cell suspension with the TZ-HA phase and load the pair of bioinks in two syringes; the final cell density in the TZ-HA phase is 5 million / mL. Finally, we use the dual-inlet nozzle (Supplementary Fig. 10b) to mix the pair of solutions on-demand, extrude the bio-ink, and print the 5x5x4 lattice scaffolds encapsulated with Beta-TC-6 cells, following the same printing process described in Fig. 4. The printed scaffolds are transferred to a 24-well plate with 1 scaffold in 1 mL Beta-TC-6 culture medium per well.

[0182] Compression tests:

[0183] To perform compression tests on the printed scaffolds, we use a rheometer (Anton Paar MCR 301) with a normal force resolution of 0.5 mN, sufficient for capturing the small deformation of the scaffolds. To ensure that the samples remain fully hydrated throughout the tests, we conduct the experiments within an aqueous environment. Specifically, the sample is situated in an open container filled with saline containing 2 mM Ca2+that mimics the DMEM composition. We place the sample, along with the container, onto the bottom geometry of the rheometer. Subsequently, the upper geometry is lowered into the saline to establish contact with the sample. Throughout the compression measurements, the upper plate is programmed with a predefined moving profile, which exerts cyclic and / or large oneway compression at a constant strain rate of 0.005 / sec. The parameters monitored during the test include the normal force, gap size, and time. Utilizing the pre-measured dimensions of the samples, we calculate stress and strain values.

[0184] To quantify the hysteresis of gyroid structures made of DN bio-ink (Fig. 4f), we introduce energy dissipation efficiency (DE), which is defined as the ratio between the integrated area in the hysteresis loop and that under the compression curve:

[0185] Here, e is the compression strain and a is the stress.

[0186] Cytocompatibility of DASP 2.0:

[0187] We use Beta-TC-6 cells to perform live / dead assay to assess the cytocompatibility of DASP 2.0. To prepare the working solution, stock solutions of propidium iodide (750 pM in Dulbecco’s phosphate buffered saline (DPBS)) and fluorescein diacetate (1 mM in dimethyl sulfoxide (DMSO)) are added to Hanks’ balanced salt solution (HBSS), yielding final concentrations of 3.75 pM and 0.2 pM, respectively. To stain both the naked Beta-TC-6 cells and the encapsulated cells, we replace the culture media with the working solution at a volume of 1 mL per well for the 24-well plate. Fluorescence images are captured using a confocal microscope (Leica SP8) equipped with a 10x dry objective with a numerical aperture of 0.3. The image acquisition is performed under multitrack mode, where red fluorescence from propidium iodide is excited by a HeNe laser (552 nm) and collected through a bandpass filter of 600 / 700 nm. Green fluorescence from fluorescein diacetate is excited by an Argon laser (488 nm) and collected through a bandpass filter of 500 / 540 nm.

[0188] To visualize cells encapsulated in hydrogel particles, we acquire a stack of images along z-axis to capture almost all the cells. Subsequently, these images are combined to generate a z-projection image, as shown in Fig. 5b and Fig. 6f, ii; this method enables a comprehensive assessment of the fraction of living cells. The survival rate is calculated as the ratio between the number of green dots and the total number of green and red dots. Glucose-stimulated insulin secretion (GSIS) test: We follow our previous method8to perform GSIS test on naked Beta-TC-6 cells and 5x5x4 DASP scaffolds encapsulated with Beta-TC-6 cells. During GSIS test, DASP printed scaffolds are placed in the 24-well plate with one scaffold per well and naked Beta-TC-6 cells are seeded in the 24-well plate with 1 .2x105cells per well, such that the number of encapsulated and naked Beta-TC-6 cells in each well are equivalent. Before the test, the naked and encapsulated Beta-TC-6 cells undergo a pre-incubation stage in a basal-glucose solution consisting of Krebs-Ringer bicarbonate buffer (KRB) supplemented with 2 mM D- glucose. The pre-incubation involves two cycles, with each cycle separated by a 1-hour interval. This pre-incubation process not only completely suppresses the cell pathway for insulin release, but also removes the residual insulin that may be trapped within the scaffold. Following the pre-incubation cycles, the washing solution is substituted with 1 mL of fresh basal-glucose solution, which is then incubated for an additional hour. Subsequently, the entire media is collected and transferred to 1 ,5-mL centrifuge tubes. Then, we add 1 mL high-glucose solution (KRB supplemented with 25 mM D-glucose) to the well with scaffold inside and further incubate for 1 hour, at which the media is once again collected. All the collected solutions are centrifuged at 600 g for 3 min and an aliquot of 600 L is extracted from the supernatant. This approach ensures effective exclusion of any potential cell debris. The insulin concentration in the collected supernatant is measured through mice insulin ELISA kits (Mercodia, Fisher Scientific, Cat. No. 10124710) and the release index is calculated by normalizing the insulin concentration upon stimulation to the basal value. Animal studies:

[0189] The animal studies are performed following a protocol approved by the University of Virginia Animal Care and Use Committee (Protocol No. 4360-08-21). Animals are housed in cages supplied with food pile, water, and corncob bedding. The cages are located in an animal facility with a 12-h light / 12-h dark cycle, a temperature of 22 °c, and a relative humidity of 50%. In our animal studies based on PAMi0Alg2(Figs. 5e-h), we use two types of DASP printed 5x5x4 lattice as scaffolds for transplantation. The first type is DN scaffolds that are the same as those tested for cytocompatibility and GSIS but without cells encapsulated. The second type is pure alginate single-network scaffolds printed by DASP 1.0. For each type, we transplant two scaffolds to the abdominal cavity of one immunocompetent C57BL / 6 mouse (male, 12 weeks old, Jackson Laboratory) and use 6 mice per group. For the group transplanted with pure alginate single-network scaffolds, we sacrifice all 6 mice 7 days after the transplantation. The scaffolds fracture into pieces can barely be retrieved. For the group transplanted with DN scaffolds, we sacrifice 2 mice and retrieve the scaffolds, respectively, at 30, 60, and 120 days after the transplantation. The retrieved scaffolds are preserved in saline supplemented with 0.02% sodium azide. Subsequently, the scaffolds are imaged using a camera (Canon EOS 7D) and a digital microscope (Hayear, 16 MP), followed by compression tests.

[0190] In animal studies based on HA / alginate (Fig. 6g), we transplant two scaffolds made of DN HA / alginate hydrogel to the abdominal cavity of one immunocompetent C57BL / 6 mouse (male, 12 weeks old, Jackson Laboratory) and use 3 mice in total. The scaffolds are retrieved 30 days after the transplantation.

[0191] Figs. 7-22 illustrate various features of the present disclosure as described in Example 1 .

[0192] References

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[0210] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1 percent to about 5 percent” should be interpreted to include not only the explicitly recited concentration of about 0.1 weight percent to about 5 weight percent but also include individual concentrations (e.g., 1 percent, 2 percent, 3 percent, and 4 percent) and the sub-ranges (e.g., 0.5 percent, 1.1 percent, 2.2 percent, 3.3 percent, and 4.4 percent) within the indicated range. The term “about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase “about ‘x’ to ‘y’” includes “about x’ to about ‘y

[0211] Many variations and modifications may be made to the above-described aspects. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

Claims

CLAIMSWhat is claimed:

1. A double-network hydrogel particle, comprising a first polymer network-scaffold and a second polymer network-scaffold, wherein the first polymer network-scaffold and the second polymer network-scaffold are independent polymer network-scaffolds, wherein the first polymer network-scaffold and the second polymer network-scaffold form a double scaffold-network polymer composition, wherein the first polymer network-scaffold includes a reaction product of a A-polymer and a B-polymer, wherein the A-polymer includes a plurality of first click chemistry units and a polymer backbone, wherein the B-polymer includes a plurality of second click chemistry units and a polymer backbone, wherein the first click chemistry unit and the second click chemistry unit react with one another to form a click-chemistry reaction crosslink between the A-polymer and the B-polymer, wherein the second polymer network-scaffold is a reaction product of alginate polymers in the presence of a divalent cation.

2. The double-network hydrogel particle of claim 1 , wherein the A-polymer is selected from the group consisting of: a norbornene(NB)-polymer, an azide-polymer, a thiol-polymer, and a maleimide-polymer; and the B-polymer is selected from the group consisting of: a TZ- polymer, an alkyne-polymer, an alkene-polymer, a thiol-polymer.

3. The double-network hydrogel particle of claim 1 , wherein A-polymer is a norbornene(NB)-polymer and the B-polymer is a tetrazine (TZ)-polymer, wherein the TZ- polymer includes a plurality of TZ units and a polymer backbone, wherein the NB-polymer includes a plurality of NB units and a polymer backbone, wherein the TZ unit and the NB units react with one another to form a click-chemistry reaction crosslink between the TZ- polymer and the NB-polymer..

4. The double-network hydrogel polymer of claim 3, wherein the reaction crosslink is a click-chemistry reaction product of TZ and NB.

5. The double-network hydrogel polymer of claim 3, wherein the click-chemistry reactionNB Polymer backboneTZ Polymer crosslink includes: a backbone crosslinking group.

6. The double-network hydrogel polymer of claim 3, wherein the polymer of TZ-polymer is hyaluronic acid (HA) or polyacrylamide (PAM), wherein the polymer of NB-polymer is HA or PAM, wherein TZ-polymer and NB-polymer have the same polymer.

7. The double-network hydrogel polymer of claims 1 to 6, wherein the second polymer network-scaffold includes divalent cationic bonding with carboxylate groups of the alginate polymers.

8. The double-network hydrogel polymer of claim 7, wherein divalent cation is selected from Ca2+, Ba2+, Co2+, and Ni2+.

9. The double-network hydrogel polymer of claim 1 , wherein the first polymer networkscaffold and the second polymer network-scaffold are independent polymer networkscaffolds and have less than 10 % crosslinking bonds between the first polymer networkscaffold and the second polymer network-scaffold.

10. The double-network hydrogel polymer of claims 2 to 6, wherein a weight average molecular weight of TZ-polymer is about 90% to 110% of a weight average molecular weight of the NB-polymer.11 . The double-network hydrogel polymer of claims 2 to 6, wherein the ratio of TZ units to NB units is 0.9:1.1 to 1.1 :0.9.

12. The double-network hydrogel polymer of claim 1 to 6, wherein the ratio the first polymer network-scaffold to the second polymer network-scaffold is about 1 :1 to 1 :10.

13. The double-network hydrogel polymer of claims 1 to 6, further comprising cells within the double polymer network-scaffold particle.

14. The double-network hydrogel polymer of claims 1 to 6, wherein the double polymer network-scaffold particle includes micro-islands.

15. The double-network hydrogel polymer of claims 1 to 6, wherein cells are present within the micro-islands.

16. The double-network hydrogel polymer of claims 1 to 6, wherein dextrin is present within the micro-islands.

17. The double-network hydrogel polymer of claims 1 to 6, wherein the micro-islands have a volume of about 106to 105pL.

18. The double-network hydrogel polymer of claims 1 to 6, wherein the double-network hydrogel particle has a diameter of about 0.5 to 1.5 mm.

19. The double-network hydrogel particle of claim 1 , wherein the first polymer network is selected from the group consisting of: a polyacrylamide-based polymer, a polysaccharide- based polymer, a polyethylene glycol-based polymer, and a graphed polymer.

20. A three-dimensional object, comprising a plurality of double-network hydrogel particles of claim 1 to 19.21 . The three-dimensional object of claim 20, wherein the adjacent double-network hydrogel particles interpenetrate one another.

22. The three-dimensional object of claim 20, wherein there are crosslinks between the first polymer network-scaffold in adjacent double-network hydrogel particles, wherein there are crosslinks between the second polymer network-scaffold in adjacent double-network hydrogel particles, or both.

23. The three-dimensional object of claim 20, wherein the three-dimensional object is a two-dimensional arrays of distinguishable double-network hydrogel particles or a freestanding three-dimensional lattice structure of distinguishable double-network hydrogel particles.

24. The three-dimensional object of claim 20, wherein the free-standing three-dimensional lattice structure has a hollow region without double-network hydrogel particles.

25. A method of making a three-dimensional object, comprising: a) mixing a first alginate polymer with a B-polymer to form a first mixture; b) mixing a second alginate polymer with a A-polymer to form a second mixture, optionally wherein the first alginate polymer and the second alginate polymer are identical; c) introducing, from a first inlet, the first mixture to a mixing area and introducing, from a second inlet, the second mixture to the mixing area, to form a third mixture, wherein a first polymer network-scaffold is formed by a reaction of the B-polymer and the A-polymer, wherein the B-polymer includes a plurality of second click chemistry units and a polymer backbone, wherein the A-polymer includes a plurality first click chemistry units and a polymer backbone, wherein the first click chemistry unit and the second click-chemistry unit react with one another to form a click-chemistry reaction crosslink between the A-polymer and the B-polymer; and d) flowing the third mixture out of a print nozzle to form a plurality of doublenetwork hydrogel particle of any one of claims 1 to 19 to form the three-dimensional object, where each double-network hydrogel particle is adjacent at least one other double-network hydrogel particle.

26. The method of making three-dimensional object of claim 25, further comprising introducing the three-dimensional object to a solution including divalent cations, wherein a second polymer network-scaffold is formed by a reaction of the alginate polymers and the divalent cations.

27. The method of making three-dimensional object of claim 25, wherein a first cell solution is mixed with the first alginate polymer and the B-polymer to form the first mixture, wherein a second cell solution is mixed with the second alginate polymer and the A-polymer to form the second mixture, or both, optionally wherein the first cell solution and the second cell solution are identical.

28. A method of making a three-dimensional object, comprising: a) mixing an first alginate polymer with a tetrazine(TZ)-polymer to form a first mixture; b) mixing an second alginate polymer with a norbornene(NB)-polymer to form a second mixture, wherein the first alginate polymer and the second alginate polymer are identical; c) introducing, from a first inlet, the first mixture to a mixing area and introducing,from a second inlet, the second mixture to the mixing area, to form a third mixture, wherein a first polymer network-scaffold is formed by a reaction of the TZ-polymer and the NB-polymer, wherein the TZ-polymer includes a plurality of TZ units and a polymer backbone, wherein the NB-polymer includes a plurality of NB units and a polymer backbone, wherein the TZ unit and the NB units react with one another to form a click-chemistry reaction crosslink between the TZ-polymer and the NB-polymer; and d) flowing the third mixture out of a print nozzle to form a plurality of doublenetwork hydrogel particle to form the three-dimensional object, where each double-network hydrogel particle is adjacent at least one other double-network hydrogel particle.

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