Additive manufacturing of tough GELS and elastomers and applications thereof

Abstract

Provided herein are methods of invention fabricating gels and elastomers into complex objects with properties of high toughness through additive manufacturing techniques that utilize vat photopolymerization. The method produces objects with long polymer chains that are highly entangled.

Description

[0001] TITLE OF THE INVENTION

[0002] Additive Manufacturing of Tough Gels and Elastomers and Applications Thereof

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 537,268 entitled "ADDITIVE MANUFACTURING OF TOUGH GELS AND ELASTOMERS AND APPLICATIONS THEREOF" filed September 8. 2023. and U.S. Provisional Patent Application No. 63 / 677,060 entitled "ADDITIVE MANUFACTURING OF TOUGH GELS AND ELASTOMERS AND APPLICATIONS THEREOF" filed July 30, 2024, the disclosures of which are incorporated herein by reference in their entireties.

[0005] BACKGROUND

[0006] Typical approaches to improve the mechanical properties of 3D printed objects include adding additional crosslinkers or post-curing the material after printing. However, such approaches result in materials with brittle properties and low molecular weight (short) polymer chains, which have low toughness compared to materials with very high molecular weight (long) chains and few crosslinks.

[0007] There is an unmet need to develop new 3D printing methods that provide stiffer materials without compromising the material toughness. The present disclosure provides a solution to this unmet need.

[0008] BRIEF DESCRIPTION OF THE FIGURES

[0009] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.

[0010] FIGs. 1 A-1C show a schematic overview of existing technology and invention innovation for 3D printing. (FIG. 1A) Highly tough and stiff single network gels and elastomers can be fabricated through molding / casting techniques; however, methods to 3D print these materials have not been described. These materials require low initiator concentrations and long curing times (3-12 hours) to achieve highly entangled polymer networks (far right) with desirable properties. (FIG. IB) Digital light processing (DLP) is a 3D printing technique that uses light to polymerize materials into defined shapes in a layer- by-layer process, where each layer is exposed to light for a short duration (typically 1-10 seconds) and thus high concentrations of initiator are required for rapid polymerization. Using a high concentration of initiator results in materials with low toughness and stiffness and decreased polymer entanglement (right). Thus, there is a clear mismatch between the current techniques to fabricate tough single network gels and DLP based 3D printing methods. (FIG. 1C) A system has been developed where polymerization is initiated within the liquid resin before printing using a redox initiator, which initially starts growing polymer chains and continues to grow polymer chains after light exposure during DLP printing. This results in 3D printed objects that have high toughness and stiffness due to their highly entangled polymer networks (right).

[0011] FIGs. 2A-2D show the impact of Redox initiator (+Redox) on the mechanical properties of DLP printed hydrogels, according to various embodiments. (FIG. 2A) Viscosity of polyacrylamide (PAm) chains formed with DLP only or DLP + Redox. Increased viscosity of the solution for DLP + Redox group indicates higher number of longer chains and higher degree of chain entanglements. (FIG. 2B) Representative compressive stressstrain curves and quantification of compressive modulus of water swollen acrylamide (Am) hydrogels 3D printed with DLP only or DLP + Redox. (FIG. 2C) Representative tensile stress-stretch curves for water swollen acrylamide (Am) hydrogels 3D printed DLP only or DLP + Redox. Quantification of tensile modulus, strain at failure, and work of fracture (Wf). (FIG. 2D) Representative oscillatory' shear rheology' plots and quantification of storage (G') and loss (G") modulus of water swollen acry lamide (Am) hydrogels 3D printed with DLP only or DLP + Redox. Overall, compressive, tensile, and shear mechanical properties of hydrogels are significantly improved through DLP + Redox as compared to DLP only.

[0012] FIGs. 3A-3F show 3D printing resolution and examples of DLP +Redox printed objects, according to various embodiments. (FIG. 3 A) XY and Z-resolution of 3D printed water-swollen acrylamide (Am) hydrogels with DLP only or DLP + Redox. The resolution of the 3D printed hydrogels is unaffected by DLP + Redox and micron sized features can be printed and washed with ease. (FIG. 3B) Examples of complex highly porous structures such as trabecular bone and hollow channels printed with DLP + Redox. (FIG. 3C) Multi-material structures (gray and yellow denote different materials) printed with DLP + Redox. (FIG. 3D) Stiff yet flexible structures like the porous Kanome lattice conforms to the shape of the underly ing object. (FIGs. 3E and 3F) Metamaterial auxetic structure printed with DLP + Redox can sustain large deformation (greater than 80%) and recover its original shape after deformation.

[0013] FIGs. 4A-4F shows the versatility of the DLP + Redox method to acrylamide hydrogels formed with (FIG. 4A, FIG. 4B) varying amounts of crosslinker, (FIG. 4C, FIG. 4D) varying the type or chemistry of crosslinker, as well as (FIG. 4E, FIG. 4F) vary ing the type of co-monomer. according to various embodiments. Overall, the mechanical properties (stiffness and toughness) can be readily tuned yielding a formulation that is soft and highly stretchable to one that is stiff yet tough. Degradation (enzymatic, hydrolytic, pH) of the entangled hydrogels can be engineered through incorporation of crosslinkers like GelMA, MeHA, PEGDA. Functionality such as thermo-responsiveness, tissue-adhesion, zwitterionic anti-fouling can be endowed to these stiff yet tough hydrogels through co-polymerization of monomers with acrylamide using DLP + redox. (FIGs. 4A, 4C, and 4E) Representative tensile stress-stretch curves for acrylamide (Am) hydrogels. (FIGs. 4B, 4D, and 4F) Quantification of tensile modulus, strain at failure, and work of fracture (Wf). All hydrogels used for mechanical testing were 3D printed with DLP + Redox method and swollen in water.

[0014] FIGs. 5A-5G show wet adhesion of DLP + Redox 3D printed hydrogels to various tissues, according to various embodiments. Adhesive co-polymer 3D printed objects can be fabricated by adding anionic monomers (e.g. acrylic acid to obtain poly(acrylamide-co- acrylic acid) to the 3D printing resin, which can undergo hydrogen bonding with tissue (FIG. 5 A). Adhesion can be stabilized by coating the surface of the 3D printed object with a cationic polymer (e.g. chitosan) before adhering the object to tissues, where the cationic polymer diffuse into the tissue and object and undergoes hydrogen bonding to create an entangled bridges between the 3D printed object and the tissue. This process was carried out in the subsequent figures. (FIG. 5B) Images before (left) and during (right) of 180-degree peel (top) and lap shear (bottom) adhesion tests with various tissues, and respective (FIG. 5C) interfacial toughness and (FIG. 5D) shear strength values. These tests confirm that DLP + Redox 3D printed hydrogels fabricated with adhesive co-polymers such as poly(acrylamide- co-acrylic acid) adhere to a wide range of tissues with useful adhesion levels that are on par with some of the highest values published. (FIG. 5E) Wet adhesion to heart tissue is not negatively affected by the presence of blood. (FIGs. 5F, 5G) Examples of large 3D printed scaffolds adhered to heart tissue and intestinal tissue. (FIG. 5F) 3D printed scaffolds conform to the curvature of heart tissue and small intestine and remain adhered when tissues are expanded / stretched.

[0015] FIGs. 6A-6C show7that patterned adhesion can be programmed into 3D printed structures. (FIG. 6A) 3D printed metamaterials have directional adhesive properties. Peeling force is highest when peeled from the front, while it is lowest when peeled from the back or when unpattemed gels are peeled. These metamaterial structures are enabled by the 3D printing process. (FIG. 6B) Perpendicular and parallel adhesive ridges can be created with 3D printing to provide spatial patterns of adhesive and non-adhesive materials. This is shown with the manual peeling of the gel and the corresponding tensile testing data showing peaks and valleys of force measurements (top). Ridges that are parallel to the peeling direction show a constant force value during peeling (bottom). (FIG. 6C) Gels can be printed with void spaces inside them that can act as regions where depots of drugs, gels and cells can be injected after adhesion on tissues.

[0016] FIGs. 7A-7H show the effect of photoinitiator concentration (I) on mechanical performance and processability of highly entangled hydrogels. (FIG. 7A) When I is high, too many polymer chains are initiated, resulting in a lower molecular weight. Precursor solution (W = 2.63, C = 0) when exposed to light results in lower molecular weight (shorter) chains with increasing I concentration as indirectly evidenced by the reduction in viscosity of solution (h) at a fixed final concentration of 15 mg mL'1. The number of monomers per chain can be estimated as (2I)'1. Hence, the molecular weight of the polymer chain can be estimated to be -950 kDa (I = 0.01 wt.%), -95 kDa (I = 0. 1 wt.%), and -19 kDa (I = 0.5 wt.%). (FIG. 7B) Representative photorheology (storage modulus G', closed circles and loss modulus G", open circles) over time and (FIG. 7C) Time to reach gel point (tgei, measured at the crossover between G" and G') for highly entangled hydrogels (W = 2.63, C = 3.5 x 1 O'4). Shaded area indicates visible light exposure. (FIG. 7D) Schematic representation of hydrogel fabrication between glass slides with light irradiation. (FIG. 7E) Representative stress (o) - stretch (A) curves of samples stretched uniaxially to rupture, (FIG. 7F) tensile modulus (ET), and (FIG. 7G) work of fracture (Wf) for highly entangled hydrogels (W = 2.63, C = 3.5 x 1 O'4). (FIG. 7H) Digital light processing (DLP) of precursor resin with varying I concentration at a constant energy (120 mJ cm'2per layer). Decreasing 1 concentration (less than 0.1 wt.%) requires a long exposure time to reach gel point, limiting the 3D printing of structures (e.g., pyramid) within reasonable time and energy dose. Data are reported as mean ± SD; n > 3; ns = not significant; *p < 0.05; ****p < 0.0001.

[0017] FIGs. 8A-8B show the effect of photoabsorber (Tartrazine, T) on photo-crosslinking of highly entangled hydrogels (W = 2.63, C = 3.5 x 1 O'4). (FIG. 8A) Representative photorheology (storage modulus G', closed circles and loss modulus G", open circles) of precursor over time with vary ing concentration of T. Photorheology' over the duration of light exposure (shaded region) demonstrates that addition of T slows the induction of gel point by minimizing light penetration. (FIG. 8B) Viscosity (h) as a function of shear rate (g) indicates low viscosity (< 1 Pa s) of the precursor resin, making it suitable for DLP.

[0018] FIGs. 9A-9D show the comparison of hydrogels (W = 2.63. C = 3.5 x 10'4) fabricated with different methods of free-radical polymerization. (FIG. 9A) Schematic representation of hydrogel fabrication between glass slides with redox reaction, light, or thermal initiation. (FIG. 9B) Representative stress (a) - stretch (X) curves of samples stretched uniaxially to rupture, (FIG. 9C) tensile modulus (ET). and (FIG. 9D) work of fracture (Wf) for highly entangled hydrogels. Data are reported as mean ± SD; n > 3; ns = not significant; *p < 0.05; **p < 0.01. Redox: APS (0.05 wt.%) and TEMED (0.1 wt.%); Light: LAP (0.1 wt.%); and Thermal: VA-044 (5 mM) at 45 °C.

[0019] FIGs. 10A-10B show the mechanics of CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 1 O’4) match those obtained via traditional casting with low photoinitiator concentration. (FIG. 10A) Representative tensile stress (c) - stretch (A) curves of cast and CLEAR printed hydrogels. (FIG. 10B) Hydrodynamic diameter (dh) measured for a photopolymer solution in the absence of crosslinker obtained by dissolving (W = 2.63. C = 0) hydrogel formed via casting or CLEAR. Hydrogels are cast using low photoinitiator concentration (0.01 wt.%, i.e., photoinitiator to monomer molar ratio: / = 3.7 x 10'5) to ensure low' I / C (= 0.1) and therefore low' initiation points compared to crosslinker. Data are reported as mean ± SD; n > 3: ns = not significant.

[0020] FIGs. 1 1A-11C show the mechanics of CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x IO"4) are stretch rate insensitive. (FIG. 11A) Representative tensile stress (G) - stretch (X) curves, (FIG. 1 IB) tensile modulus (ET), (FIG. 11C) w ork of fracture (Wf) for CLEAR printed hydrogels. Data are reported as mean ± SD; n = 3; ns = not significant.

[0021] FIGs. 12A-12F show- CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x IO"4) exhibit superior mechanical properties under compression and shear than those with DLP alone. (FIG. 12 A) Polymer content (<p), (FIG. 12B) representative compressive stress (G) - strain (y) curves, (FIG. 12C) compressive modulus (Ec), (FIG. 12D) representative rheology' (storage modulus G1, closed and loss modulus G", open as a function of angular frequency, co), (FIG. 12E) storage modulus (G1), and (FIG. 12F) loss modulus (G") measured under constant oscillation angular frequency (1 rad s'1) for hydrogels fabricated with DLP and CLEAR. Data are reported as mean ± SD; n > 3; *p < 0.05; **p < 0.01; ****p < 0.0001.

[0022] FIGs. 13A-13B show' dye diffusion through DLP and CLEAR printed hydrogels (W = 2.63, C = 3.5 x IO’4). (FIG. 13A) Time-series fluorescence micrographs depicting diffusion of calcein (green, molecular weight = 622 Da) Scale bar: 500 mm. (FIG. 13B) Quantification of diffusive permeability (Pd) of calcein through DLP and CLEAR printed hydrogels. Slower diffusion of the dye in the CLEAR printed hydrogels can be attributed to the higher polymer content and increased degree of crosslinks (both physical entanglements and covalent bonds). Data are reported as mean ± SD; n > 8; **p < 0.01.

[0023] FIGs. 14A-14D show mechanical properties of as-printed, non-swollen hydrogels. (FIG. 14A) Representative tensile stress (o) - stretch ( ) curves, and (FIG. 14B) tensile modulus (ET) for DLP and CLEAR printed hydrogels (W = 2.63, C = 3.5 x 10'4). The benefits of CLEAR are realized in hydrogels in an as-prepared state without swelling. (FIG. 14C) Representative tensile stress (o) - stretch (X) curves, and (FIG. 14D) tensile modulus (ET) for CLEAR printed highly entangled hydrogels (W = 2.63) with varying BisAm (crosslinker) concentration such that C = 0; 7.1 x 10'5; 3.5 x 10’4for BisAm concentration 0; 0.01; 0.05 wt.%, respectively. It is interesting to note that in the non-swollen state, there are no significant differences in the tensile modulus of CLEAR printed highly entangled hydrogels with or without crosslinker, highlighting that the contribution of entanglements to the mechanics supersedes that of covalent crosslinks and that at low crosslinker concentrations the entanglements outnumber covalent crosslinks. Data are reported as mean ± SD; n > 3; ns = not significant; **p < 0.01.

[0024] FIG. 15A-15D show highly entangled hydrogels (W = 2.63. C = 0) in the absence of crosslinker are held together solely by physical entanglements. (FIG. 15 A) Time-lapse photographs of DLP and CLEAR printed pyramids submerged in water. Physical entanglements act as slip (non-permanent) crosslinks and as a result, polymer chains can dissolve in water over time; however, the rate of dissolution of the CLEAR printed entangled network is slower than that for DLP alone, highlighting the possibility of increased chain entanglement. Scale bar: 10 mm. (FIG. 15B) Viscosity (h) of solution containing polymer chains formed through DLP or CLEAR dissolved at a fixed concentration of 15 mg mL'1. The higher viscosity of solutions obtained through CLEAR can be an indirect indicator of higher molecular weight polymer chains than those with DLP. Data are reported as mean ± SD; n > 3; *p < 0.05. (FIG. 15C) Representative rheology (storage modulus G', closed circles and loss modulus G", open circles as a function of angular frequency, co) of a diluted polymer solution (15 mg mL1) fabricated with DLP or CLEAR. (FIG. 15D) Representative rheology (storage modulus G' and loss modulus G" as a function of angular frequency, co) of a polymer solution (at different concentrations) obtained with the CLEAR approach. There is a strong frequency dependence of storage modulus across all polymer solution concentrations, indicating slippage of polymer chains and dynamics of chain entanglements. Increasing the polymer solution concentration from 15 to 90 mg mL'1, shows a strong shift in the crossover frequency, indicating the presence of higher degrees of entanglements and thus a longer time scale is required for chains to completely slide past one another.

[0025] FIGs. 16A-16B show assessment of the size distribution of photopolymers printed with DLP or CLEAR. Printed hydrogel (W = 2.63, C = 0) in the absence of crosslinker is dissolved in water at a fixed concentration to create a solution containing polymer chains. (FIG. 16 A) Representative GPC chromatogram for polymers fabricated with DLP or CLEAR, tabulated data in Table 2. (FIG. 16B) Hydrodynamic diameter (dh) measured for a photopolymer solution for polymers fabricated with DLP or CLEAR. There is no significant difference between the measured hydrodynamic diameters of DLP and CLEAR samples. Data are reported as mean ± SD; n > 3; ns = not significant.

[0026] FIGs. 17A-17B show' assessment of degradation of CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 10’4) with (FIG. 17A) non-degradable, covalent [N,N0- (methylene)bisacrylamide; BisAm] or (FIG. 17B) degradable, disulfide [N,N0- bis(acryloyl)cystamine; BAC] crosslinker. Photograph of CLEAR printed hydrogels after submersion in water or accelerated degradation (i.e., reducing agent, 0.1 M b- mercaptoethanol, MCE) for seven days at 65 °C. In the presence of MCE, the disulfide crosslinks break via thiol-disulfide exchange; how ever, covalent crosslinks remain unaffected by the increased temperature or presence of a reducing agent. For CLEAR printed entangled hydrogels with degradable crosslinks, the hydrogels can be completely dissolved, indicating the absence of netw ork defects (e.g., permanent chemical crosslinks or network defects such as back-biting). Once the disulfide crosslinks reduce, the hydrogels are only held together byslip crosslinks (i.e., entanglements) and can therefore dissolve away over time. This provides qualitative evidence that CLEAR printing does not introduce network defects that can significantly influence the network topology.

[0027] FIGs. 18A-18H show differences in properties of hydrogels (W = 2.63, C = 3.5 x 10’4) fabricated with varying light exposure during printing. (FIG. 18 A) 'H-NMR of photopolymer solutions formed by dissolving hydrogels (W = 2.63, C = 0) in the absence of crosslinker fabricated with either low light exposure (undercure, 2 s per layer) or high light exposure (overcure, 18 s per layer) during printing. Undercuring results in lower monomer conversion (-44%), whereas overcuring achieves near complete consumption (-94%). Shaded areas indicate peaks corresponding to acrylamide (Am) and polyacrylamide (PAm). (FIG. 18B) Hydrodynamic diameter (dh) measured for photopolymers (W = 2.63, C = 0) formed via undercuring, optimal (6 s per layer) curing, and overcuring, all in the absence of crosslinker. The reduction in hydrodynamic diameter by overcuring suggests lower chain lengths, likely due to excessive initiation points as a result of longer light exposure per layer. (FIG. 18C) Conversion of C=C peak (cc=c, %) as a function of irradiant energy for DLP resin (W = 2.63, C = 3.5 x 10’4). Shaded areas indicate time windows for different levels of light exposure. (FIG. 18D) Fluorescence micrographs of as-printed hydrogels with varying light exposure conditions. Both undercure and overcure conditions result in poor fidelity and low resolution of the printed object compared to optimal curing (which matches the CAD dimensions). Mechanical properties of hydrogels fabricated via DLP or CLEAR with (FIGs. 18E, 18F) undercure or (FIGs. 18G, 18H) overcure during printing. (FIGs. 18E. 18G) Tensile modulus (ET) and (FIGs. 18F, 18H) work of fracture (Wf) for DLP and CLEAR printed hydrogels. Improvements in mechanical properties due to CLEAR are best realized when conversion within the printed object is low (such as with undercure), which allows CLEAR to introduce higher concentrations of polymers that result in entanglement. To that end, no significant difference is seen between hydrogels obtained with DLP or CLEAR in the case of overcuring. Although overcuring layers with DLP can improve the tensile modulus due to high monomer conversion, it is important to note that this approach is not feasible for printing of high-resolution structures or negative features. These results further emphasize the importance of the CLEAR approach to improve properties while retaining the ability to obtain high-resolution structures. Data are reported as mean ± SD; n > 3; ns = not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

[0028] FIGs. 19A-19G show CLEAR printing technique outperforms traditional approaches of improving green strength (e.g., light flood cure or thermal post-curing). Polyacrylamide hydrogels (W = 2.63, C = 3.5 x 1 O’4) are fabricated with (i) DLP alone, (ii) DLP followed by light flood cure (15 mW cm’2, 5 min), (iii) DLP with incorporation of thermal initiator, VA- 044 (5 mM) within precursor resin followed by thermal post cure at 45 °C for 2 h, and (iv) CLEAR approach. (FIG. 19 A) FT-IR spectra (demonstrating acrylamide C=C peak) of thin films of DLP resin without and with post-cure (light, thermal) and CLEAR resin recorded before and after exposure to conditions that mimic print settings. Intensity of the acrylamide peak reduces but remains in the case of DLP alone, highlighting the low monomer conversion. However, the C=C peak disappears in the case of post-curing and CLEAR. (FIG. 19B) Representative tensile stress (o) - stretch (A) curves, (FIG. 19C) tensile modulus (ET), (FIG. 19D) work of fracture (Wf), (FIG. 19E) polymer content (<p), (FIG. 19F) compressive modulus (Ec) for hydrogels fabricated with DLP, post-cure (light, thermal), and CLEAR. (FIG. 19G) Conversion of acrylamide monomer (cAm) measured via 'H-NMR of photopolymer solutions obtained by dissolving hydrogels (W = 2.63, C = 0) in the absence of crosslinker printed with DLP without and with post-curing (light, thermal), or printed with CLEAR. Data are reported as mean ± SD; n > 4; ns = not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0029] FIGs. 20A-20F show parameters that influence CLEAR printing of highly entangled hydrogels (W = 2.63, C = 3.5 x 10'4). (FIG. 20A) Representative tensile stress (o) - stretch (X) curves, (FIG. 20B) tensile modulus (ET), (FIG. 20C) work of fracture (Wf) for CLEAR printed hydrogels cured in a nitrogen-purged and sealed container or under ambient air. The presence of oxygen in air can inhibit the redox initiation as part of CLEAR and have an adverse effect on mechanical properties. (FIG. 20D) Representative o - A. curves, (FIG. 20E) ET, (FIG. 20F) Wf for CLEAR printed hydrogels cured in a nitrogen-purged and sealed container at 4 °C, room temperature (RT, 21 - 24 °C), and 37 °C. ET and Wf are insensitive to the curing temperature within the range studied here; however, the rates of redox initiation would be affected by the temperature with the reaction reaching complete conversion faster at an elevated temperature. Data are reported as mean ± SD; n > 4; ns = not significant; *p < 0.05; **p < 0.01; ****p < 0.0001.

[0030] FIGs. 21 A-21G show the effect of introducing a wait period (IWAIT) between the addition of redox initiator and light exposure during DLP on the mechanical properties of CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 10'4). (FIGs. 21B, 21E) Representative tensile stress (ct) - stretch (A) curves, (FIGs. 21C, 21F) tensile modulus (ET), and (FIGs. 21D, 21G) work of fracture (Wf) for CLEAR printed hydrogels with varying wait periods with different redox initiator concentration (FIGs. 21B-21D) APS (0.05 wt.%), TEMED (0.1 wt.%) and (E-G) APS (0.02 wt.%), TEMED (0.04 wt.%). # denotes inability to print hydrogels due to spontaneous redox mediated gelation of precursor resin in the vat. The operating window for print completion after addition of redox initiators can var7between 18 minutes to 3 hours depending on the rate of reaction controlled by the concentration of added initiators. Statistical comparisons of all groups to IWAIT (0 min) shown on the graph. Data are reported as mean ± SD; n > 3; ns = not significant.

[0031] FIG. 22 shows the effect of light irradiation (visible light, 20 mW cm'2) on CLEAR resin for highly entangled hydrogels (W = 2.63, C = 3.5 x 10'4) containing redox initiators: APS (0.05 wt.%), TEMED (0. 1 wt.%) in the absence of photoinitiator. Representative photorheology (storage modulus G', closed circles and loss modulus G", open circles) of precursor over time. Shaded region indicates light exposure. Redox initiators alone do not undergo photoinitiation at the light intensity and concentration investigated in the current study.

[0032] FIGs. 23A-23C show the investigation of the shelf-life of the resin over 28 days. The base formulation of the CLEAR resin containing monomer, crosslinker, photoinitiator, and photoabsorber is stored away from light at room temperature for up to 28 days and appropriate quantities of redox initiators are mixed with the resin immediately before printing. (FIG. 23A) Timeline of base resin storage along with (FIG. 23B) Tensile modulus (ET) and (FIG. 23C) work of fracture (Wf) for CLEAR printed (W = 2.63, C = 3.5 x 10’5) hydrogels from resins stored for various times. No significant differences in mechanical properties are observed between CLEAR printed highly entangled hydrogels formed with resins stored for over 28 days. Data are reported as mean ± SD; n = 4; ns = not significant.

[0033] FIGs. 24A-24G show the influence of redox initiator concentration on time to spontaneous gelation and mechanical performance of CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 10‘4). (FIG. 24A) Photographs demonstrating sol-gel transition of precursor resin (with varying redox initiator concentration) to hydrogel over the duration of 2 h. Increasing redox initiator concentration results in faster gelation. (FIG. 24B) Representative rheology (storage modulus G'. closed circles and loss modulus G", open circles) of precursors with different redox initiator concentrations over time. Induction of gel point (as measured through rheology ) is faster due to reduced radical diffusion and lower oxygen inhibition as compared to a vat of resin. (FIG. 24C) Representative stress (a ) - stretch (X) curves of samples stretched uniaxially to rupture and (FIG. 24D) tensile modulus (ET), and (FIG. 24E) work of fracture (Wf) for hydrogels fabricated with different redox initiator concentrations via the CLEAR approach. Modulus and work of fracture are insensitive to the range of redox initiator concentrations used. (FIG. 24F) Conversion of acrylamide (C=C, cc=c) over time solely through redox initiation (in the absence of light). Redox initiator formulation D was chosen for representation. (FIG. 24G) Photographs demonstrating sol-gel transition of precursor resin (with redox initiator formulation D) in the presence of hexane or mineral oil, indicating that these do not interfere with the redox reaction and hence can be employed as solvents to wash uncured resin from the printed structure as part of the CLEAR method. Redox initiator formulation, A: APS (0.02 wt.%), TEMED (0.04 wt.%), B: APS (0.03 wt.%), TEMED (0.06 wt.%), C: APS (0.04 wt.%), TEMED (0.08 wt.%), and D: APS (0.05 wt.%), TEMED (0. 1 wt.%). Data are reported as mean ± SD; n > 3; ns = not significant

[0034] FIGs. 25A-25J show the investigation of the reusability of CLEAR resin. Upon addition of redox initiators to the CLEAR resin, the reaction is initiated. While the rate of the reaction can be controlled by tuning the concentration of initiator, the resin can undergo spontaneous gelation, which limits its reusability. Here, two potential methods to inhibit the redox reaction when needed are explored, thereby empowering the user with control over when the redox reaction is desired. (FIG. 25A) Schematic representation of the CLEAR resin reuse via freezing at -20 °C overnight. Unused CLEAR resin (containing redox initiator) after printing can be frozen to quench the redox reaction and then thawed for further use. (FIG. 25B) Photographs depicting that thawed CLEAR resin can remain stable (i.e., can still flow) at least up to 7 days without spontaneous gelation. (FIG. 25C) Representative tensile stress (G ) - stretch ( ) curves, (FIG. 25D) tensile modulus (Er), and (FIG. 25E) work of fracture (Wf) for CLEAR printed (W = 2.63. C = 3.5 x 10’5) hydrogels from fresh or reused resin. (FIG. 25F) Schematic representation of the CLEAR resin reuse by storing the resin under oxygen. Unused CLEAR resin (containing redox initiator) after printing can be purged and sealed with oxygen, which inhibits the redox reaction. (FIG. 25 G) Photographs depicting that CLEAR resin stored under oxygen in a sealed container can remain stable at least up to 7 days without spontaneous gelation. (FIG. 25H) Representative tensile stress (a) - stretch ( ) curves, (FIG. 251) tensile modulus (ET), and (FIG. 25 J) work of fracture (Wr) CLEAR printed (W = 2.63, C = 3.5 x 10’5) hydrogels from fresh or reused resin. No significant differences in mechanical properties are observed between printed highly entangled hydrogels formed with fresh or reused resins. The same redox initiator [APS (0.02 wt.%), TEMED (0.04 wt.%)] formulation is used for all experiments and additional redox initiator is added to the CLEAR resin before reuse for printing. Data are reported as mean ± SD: n > 3; ns = not significant

[0035] FIGs 26A-26D show CLEAR processing of highly entangled hydrogels (W = 2.63. C = 3.5 x 10'4) into 3D structures. (FIG. 26A) Photographs of hydrogels with various shapes such as (i) chess pieces and (ii) knotted and entangled structures. Scale bars: 10 mm. (FIG. 26B) Photographs of hydrogels processed into porous shapes like (i) bucky ball and (ii) gyroid. (FIG. 26C) Fluorescence micrographs of (i) macroporous square, (ii) hexagonal lattice, and (iii) snow flake architecture. Fluorescence micrographs of (iv) CU logo and (v) aperiodic monotile. These multi-component structures (outline, infill, blocks) containing fluorophores (gray or yellow) are printed separately and assembled together. Photograph of (vi) device with perfusable serpentine channel. Scale bars: 5 mm and 10 mm. (FIG. 26D) Photographs demonstrating the ability of hydrogel to sustain twisting, knotting, and stretching without rupture. Scale bar: 10 mm.

[0036] FIGs. 27A-27D show optimization of print parameters and fabrication of hydrogels (W = 2.63, C = 3.5 x 1 O'4) with open channels via DLP or CLEAR. (FIG. 27 A) Maximum intensity projection images of square patterns formed with variable light exposure time (t) to identify the critical energy dose required to reach gel point during DLP for precursor resin containing different concentrations of photoabsorber. (FIG. 27B) CAD rendering of square channels in a vertical orientation parallel with the print direction. After printing, hydrogels are submerged in water to swell to equilibrium. Fluorescence images of constructs printed with DLP or CLEAR with same light energy dose. (FIG. 27C) CAD rendering of square channels in a horizontal orientation perpendicular to the light projection axis. After printing, hydrogels are submerged in water to swell to equilibrium. Fluorescence images of constructs printed with DLP or CLEAR with the same light energy’ dose. Scale bar: 5 mm. (FIG. 27D) Quantification of measured channel length or height for DLP or CLEAR printed hydrogels. Dotted line indicates the desired dimension based on the CAD model. The deviation of observed from expected (ideal) dimensions can be attributed to swelling of hydrogels. Data are reported as mean ± SD; n > 3.

[0037] FIGs. 28A-28B show CLEAR processing can be extended to the fabrication of highly entangled hydrogels (W = 2.63, C = 3.5 x 1 O'4) through different projection workflows. Photograph of auxetic octet-truss lattice printed with (FIG. 28A) layer-by-layer and (FIG. 28B) continuous projection method. Layer-by-layer fabrication involves the conventional method of lowering the build plate into the resin vat, irradiation of 2D sliced image, followed by upward movement of the build plate to allow- the resin to flow back in. This process is then repeated until the entire 3D structure is printed. Continuous fabrication involves steady, upw ard movement of the build plate w ithout low ering of the build plate in betw een layers, with continuous projection of sliced images. Hence, continuous fabrication is faster and may be better suited for CLEAR given the shelf-life of the resin after addition of redox initiators. Scale bar: 10 mm.

[0038] FIGs. 29A-29B show’ the investigation of operating time window’ for integration strength of CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 1 O'4) containing redox initiators (FIG. 29A) APS (0.05 wt.%), TEMED (0. 1 wt.%) or (FIG. 29B) APS (0.02 wt.%), TEMED (0.04 wt.%). t denotes elapsed time between CLEAR printing of hydrogels and annealing them together. It is important to note that the operating time window depends on the concentration of redox initiators and the time required for printing of the object that needs to be annealed later. The time window is dictated by the degree of monomer conversion achieved at a given time and therefore the operating window can be extended through use of low concentration of redox initiators which can slow down the reaction. # denotes extremely low integration making it difficult to measure via 180° peel test. Data are reported as mean ± SD; n = 4; ns = not significant, ***p < 0.001; ****p < 0.0001.

[0039] FIGs. 30A-30C show CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 1 O'4) can be readily sutured to ex vivo tissues. (FIG. 30A) Photograph of macroporous patch sutured (simple, interrupted) to porcine skin with high strength as seen by the ability of the patch to stretch without fracture at the suture zone. Suture: 4-0 monofilament polypropylene (non-absorbable). Scale bar: 1 cm. (FIG. 30B) Quantification of suture retention strength of CLEAR printed highly entangled hydrogels. Data are reported as mean ± SD; n = 4. (FIG. 30C) Photograph of patch sutured on both ends to porcine skin and subjected to physiologically relevant stretch, highlighting that the sutured hydrogels are compliant with dynamic movements of tissues. Scale bar: 1 cm.

[0040] FIGs. 31A-31C show CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 1 O'4) can be dried and re-hydrated without adversely affecting the mechanical properties. (FIG. 31 A) Photograph of macroporous lattice in its swollen, freeze-dried, and air-dried states. Scale bar: 10 mm. Comparison of (FIG. 3 IB) Tensile modulus (ET) and (FIG. 31C) work of fracture (Wf) of hydrogels swollen or rehydrated after freeze- or air-drying. Data are reported as mean ± SD; n > 3; ns = not significant.

[0041] FIGs. 32A-32B show CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 1 O'4) are cytocompatible without the need for multiple washing steps. (FIG. 32A) Maximum intensity projection fluorescence micrographs of human mesenchymal stromal cells (hMSCs) seeded in contact with no hydrogel (control), DLP, and CLEAR printed hydrogels. Live (calcein AM, green) and Dead (ethidium homodimer- 1, red). Scale bar: 100 mm. (FIG. 32B) Quantification of cell viability at day 1 after treatment. The hydrogels are placed in contact with cells directly after the DLP or CLEAR process is complete. Low cell viability in the case of DLP can be attributed to leaching of unreacted, cytotoxic acrylamide (monomer) and N.Nc-niethylenebistacrylamide) (crosslinker), whereas the CLEAR approach results in complete conversion of monomers to prevent cytotoxicity. Data are reported as mean ± SD; n > 3; ns = not significant; ****p < 0.0001.

[0042] FIGs. 33A-33C show the effect of solvent on swelling and mechanical properties of CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 10'4). (FIG. 33 A) Representative tensile stress (o) - stretch (X) curves, (FIG. 33B) tensile modulus (ET), (FIG. 33C) work of fracture (Wf) for CLEAR printed hydrogels submerged in deionized, ultrapure water (FLO), Dulbecco’s Phosphate Buffered Saline (PBS), or Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (10%) until equilibrium. Physiological buffers (pH. salinity, osmolarity) do not negatively impact mechanical properties of printed hydrogels, establishing their potential use for biomedical applications. Data are reported as mean ± SD; n > 4; ns = not significant, *p < 0.05.

[0043] FIGs. 34A-34C show CLEAR printed highly entangled hydrogels with extremely low covalent crosslinker concentration (W = 2.63, C = 3.5 x IO’3) exhibit higher mechanical properties than those with DLP alone. (FIG. 34A) Representative tensile stress (o) - stretch (A) curves, (FIG. 34B) tensile modulus (ET), (FIG. 34C) work of fracture (Wf) for DLP and CLEAR printed hydrogels. Data are reported as mean ± SD; n > 4; ***p < 0.001; ****p < 0.0001.

[0044] FIGs. 35A-35I show the influence of crosslinker concentration, type of crosslinker, and t pe of monomer on the mechanical properties of CLEAR printed highly entangled hydrogels (W = 2.63). (FIG. 35A) Representative tensile stress (o) - stretch ( ) curves, (FIG. 35B) tensile modulus (ET), (FIG. 35C) work of fracture (Wf) for CLEAR printed hydrogels with varying N, Atymethylenebis(acrylamide) (BisAm, crosslinker) concentration from 0.005 wt.% (C = 3.5 x 10'5) to 0.1 wt.% (C = 7.1 x 10’4). (FIG. 35D) Representative o - curves, (FIG. 35E) ET. (FIG. 35F) Wf for CLEAR printed hydrogels with a variety of crosslinkers. GelMA: gelatin methacrylamide (0.5 wt.%), MeHA: methacrylated hyaluronic acid (Mw = 70 kDa, Degree of substitution = 50%, 0.25 wt.%), PEGDA: poly(ethylene glycol) diacrylate (Mw = 10 kDa, 0.5 wt.%), GH: guest-host inclusion complex formed with A-(l- Adamantyl)acrylamide (10 mM) and 6-Acrylamido-P-cyclodextrin (10 mM). (FIG. 35G) Representative a - X curves, (FIG. 35H) ET, (FIG. 351) Wf for CLEAR printed hydrogels with a variety of co-monomers in a 3: 1 acry lamide to co-monomer molar ratio and BisAm as crosslinker (C = 3.5 x 10'4). NIP Am: A-isopropylacrylamide, HEA: A-Hydroxyethyl acrylamide, THA: N- [Tris(hydroxymethyl)methyl] acrylamide, DMAPS: 3-[(3- Acrylamidopropyl)dimethylammonio]propane-1 -sulfonate. Data are reported as mean ± SD; n > 3.

[0045] FIGs. 36A-36C show the mechanical characterization of CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 10'4) with the same crosslink fraction but varying molecular weights of poly(ethylene glycol) (PEGDA, crosslinker). Quantification of (FIG. 36A) tensile modulus (Er), (FIG. 36B) tensile stretch at failure (If), (FIG. 36C) work of fracture (Wf) for different hydrogels. Increasing crosslinker molecular weight (at fixed crosslinker molar concentration) can likely result in an increase in overall network mesh size, resulting in a decrease in hydrogel modulus. Interestingly, PEGDA (M= 10 000 Da) employed as crosslinker has a molecular weight higher than the entanglement molecular weight of PEG. Data are reported as mean ± SD; n = 3; ns = not significant; *p < 0.05; **p < 0.01.

[0046] FIGs. 37A-37I show CLEAR printed hydrogels (C = 3.5 x 10'4) exhibit higher mechanical properties than those with DLP across a wide range of monomer concentrations: (FIGs. 37A-37C) W = 11.9, (FIGs. 37D-37F) W = 9.2, and (FIGs. 37G-37I) W = 4.8. (FIGs. 37A, 37D, 37G) Representative tensile stress (o) - stretch (A) curves, (FIGs. 37B, 37E, 37H) tensile modulus (ET), (FIGS. 37C, 37F, 371) work of fracture (Wr) for DLP and CLEAR printed hydrogels. The photoinitiator (0.1 wt.%) concentration is kept constant for all experiments. In the case of CLEAR resin, redox initiator formulation [APS (0.05 wt.%), TEMED (0.1 wt.%)] is employed. Data are reported as mean ± SD; n > 3; *p < 0.05; **p < 0.01; ***p < 0.001.

[0047] FIGs. 38A-38D show CLEAR printed thiol-ene hydrogels exhibit superior mechanical properties than those with DLP alone. (FIG. 38A) Schematic depiction of CLEAR for printing of thiol-ene hydrogels. Low light exposure (hv) is correlated with lower conversion on the DLP; however, dark polymerization at room temperature as part of CLEAR likely improves conversion of unreacted macromers and thus improves mechanical properties. (FIG. 38B) Representative tensile stress (o) - stretch (A) curves, (FIG. 38C) tensile modulus (ET), and (FIG. 38D) work of fracture (Wf) for DLP and CLEAR printed hydrogels. The precursor resin contains macromer (8-arm 10 kDa PEG modified with norbomene, 25 wt.%), crosslinker (DTT, 1 : 1 thiol: norbomene stoichiometry), photoinitiator (LAP, 0.1 wt.%), photoabsorber (tartrazine). In addition to the base formulation, redox initiators [APS (0.05 wt.%), TEMED (0. 1 wt .%)] are added to the CLEAR resin. Data are reported as mean ± SD; n = 4; **p < 0.01.

[0048] FIGs. 39A-39E show CLEAR printing can be extended to elastomers and leveraged to form highly entangled hydroxyethyl acrylate (HEA) elastomers with mechanical properties far superior to those obtained with DLP alone or UV flood cure. (FIG. 39A) Photographs demonstrating sol-gel transition of precursor resin (with varying redox initiator concentration) over the duration of 3.5 h. Increasing redox initiator concentration results in faster gelation. Redox initiator formulation, A: BPO (0.3 wt.%), DMA (8.8 mM), B: BPO (0.35 wt.%), DMA (10.3 mM), and C: BPO (0.4 wt.%), DMA (12 mM) (FIG. 39B) Representative rheology (storage modulus G', closed circles and loss modulus G", open circles) of precursor with redox initiator formulation ‘B’ over time. Time to gel point as measured via rheology is faster due to reduced radical diffusion and low oxygen inhibition in a thin film as compared to a vat of resin. (FIG. 39C) Representative tensile stress (a) - stretch ( / .) curves, (FIG. 39D) tensile modulus (ET), (FIG. 39E) work of fracture (Wf) for DLP, UV post-cured, and CLEAR printed elastomers. Data are reported as mean ± SD; n > 4; *p < 0.05; **p < 0.01; ****p < 0.0001.

[0049] FIGs. 40A-40C show CLEAR printing of highly entangled hydroxy ethyl acrylate (HEA) elastomers in the absence of solvent. (FIG. 40 A) Representative tensile stress (a) - stretch ( ) curves, (FIG. 40B) tensile modulus (ET). and (FIG. 40C) work of fracture (Wf) for DLP, UV post-cured, and CLEAR printed elastomers. Further improvements in mechanical properties of entangled elastomers can be realized through CLEAR than UV post-cure or DLP alone. Photoinitiator (TPO), photoabsorber (Sudan I), and redox initiators (BPO / DMA in the case of CLEAR) are dissolved directly in the solution of HEA monomer. Data are reported as mean ± SD; n > 3; ns = not significant; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0050] FIGs. 41A-41C show' mechanical characterization of CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 10'4) with constant total monomer fraction but varying molar ratios of acrylamide to acryhc acid (co-monomers). Quantification of (FIG. 41 A) tensile modulus (ET), (FIG. 41B) tensile stretch at failure (If), (FIG. 41C) w ork of fracture (Wf) for different hydrogels. CLEAR printed highly entangled poly(acrylic acid) hydrogels are weaker than the highly entangled poly(acrylamide) hydrogels at the same crosslink fraction. However, optimal modulus-work of fracture balance is achieved with a precursor solution containing 1 : 1 molar ratio of acrylamide to acrylic acid to form hydrogels. Statistical comparisons of all groups to [Am]: [Ac] = 1 :0 shown on the graph. Data are reported as mean ± SD; n > 3; ns = not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0051] FIGs. 42A-42C show7CLEAR printed highly entangled adhesive hydrogels formed with mixtures of acrylamide and acrylic acid (1 :1 molar equivalent) monomer (W = 2.63, C = 3.5 x 10'4) exhibit higher mechanical properties than those with DLP alone. (FIG. 42A) Representative tensile stress (o) - stretch ( ) curves, (FIG. 42B) tensile modulus (ET), (FIG. 42C) work of fracture (Wf) for DLP and CLEAR printed hydrogels. Data are reported as mean ± SD; n > 4; ****p < 0.0001.

[0052] FIGs. 43A-43E show the assessment of hysteresis of CLEAR printed highly entangled adhesive hydrogels (W = 2.63. C = 3.5 x 1 O’4). Representative tensile stress (c> ) - stretch (X) curves for hydrogels stretched to a maximum stretch of (FIG. 43 A) 1.5, (FIG. 43B) 2.0, and (FIG. 43C) 3.0. Quantification of (FIG. 43D) hysteresis energy (Uhys) and (FIG. 43E) hysteresis for hydrogels at various maximum stretch. Shaded area in (FIG. 43E) denotes the typical range of hysteresis observed in double network hydrogels (e.g., alginate Alg / polyacrylamide PAm hydrogels). Data are reported as mean ± SD; n = 4; **p < 0.01; ***p < 0.001 ; ****p < 0.0001 .

[0053] FIGs. 44A-44B show CLEAR printed highly entangled adhesive hydrogels (W = 2.63, C = 3.5 x 1 O’4) are cytocompatible. (FIG. 44A) Maximum intensity projection fluorescence micrographs of human mesenchymal stromal cells (hMSCs) seeded with conditioned media from no hydrogel (control) and CLEAR printed adhesive hydrogels. Live (calcein AM, green) and Dead (ethidium homodimer-1, red). Scale bar: 100 mm. (FIG. 44B) Quantification of cell viability at day 1 after treatment. Data are reported as mean ± SD; n = 3; ns = not significant.

[0054] FIGs. 45A-45B show the effect of bridging polymer on the interfacial toughness of CLEAR printed highly entangled adhesive hydrogels (W = 2.63, C = 3.5 x 1 O'4) adhered to ex vivo porcine heart tissue. (FIG. 45A) Photograph of 180° peel test of CLEAR printed adhesive hydrogel adhered to heart tissue with chitosan. Scale bar: 1 cm. (FIG. 45B) Quantification of interfacial toughness ( int) with and without chitosan (bridging polymer) to facilitate adhesion of the hydrogel onto heart tissue. Data are reported as mean ± SD; n > 4; *p < 0.05.

[0055] FIGs. 46A-46G show robust adhesion of CLEAR printed highly entangled adhesive hydrogels (W = 2.63, C = 3.5 x 1 O’4) to ex vivo tissues. Photographs of hydrogels adhered to diverse freshly isolated porcine tissues including (FIG. 46A) heart, (FIG. 46B) lung, (FIG. 46C) stomach, (FIG. 46D) small intestine. (FIG. 46E) fl exor / extensor tendon before (i) or during (ii) 180° Peel test and before (iii) or during (iv) Lap Shear test. Scale bars: 10 mm. Representative force / width as a function of displacement plots for measurement of hydrogel adhesion to tissues through (FIG. 46F) 180° peel and (FIG. 46G) lap shear test.

[0056] FIGs. 47A-47C show strength of adhesion measurement of CLEAR printed highly entangled adhesive hydrogels (W = 2.63, C = 3.5 x 1 O’4) to ex vivo tissues. (FIG. 47 A) Quantification of adhesive shear strength (assessed through lap shear test) to various freshly isolated porcine tissues. Data are reported as mean ± SD; n > 4. (FIG. 47B) Photograph of CLEAR printed highly entangled hydrogel processed in the form of a porous patch adhered to ex vivo porcine heart. (FIG. 47C) Photograph demonstrating the ability of a CLEAR printed porous patch to sustain low and high stretch while adhered to porcine heart tissue. Scale bars: I cm.

[0057] FIGs. 48A-48C show CLEAR printed highly entangled adhesive hydrogels (W = 2.63, C = 3.5 x 1 O’4) can be processed into macroporous lattice structures that conform to curved tissues ex vivo and are compliant with the dynamic movement of the tissue simulated through manual pumping of phosphate buffered saline (PBS) through the intestine. Photograph of porous hydrogel patch attached onto (FIG. 48A) (i, ii) collapsed or empty intestine, (FIG. 48B) (i, ii) filled with PBS. Scale bars: 3 cm. Hydrogel patch can (FIG. 48C) adapt (i) before and (ii) after a dynamic, peristaltic expansion when adhered to porcine small intestine ex vivo. Scale bars: 1 cm.

[0058] FIGs. 49A-49C show CLEAR printed highly entangled adhesive hydrogels (W = 2.63, C = 3.5 x 1 O’4) can be press-fit into ex vivo cartilage defects as demonstrated through (FIG. 49 A) photograph of printed hydrogel disc adhered into cartilage freshly isolated from a bovine knee joint. Scale bar: 2 mm (FIG. 49B) Quantification of integration strength measured via push-out indentation tests to investigate the strength of adhesion of a press-fit hydrogel. Data are reported as mean ± SD: n = 6. (FIG. 49C) Photograph of porous hydrogel lattice press-fit into an ex vivo defect created on a bovine femoral condyle. Scale bar: 10 mm.

[0059] FIGs. 50A-50D show spatial programming of adhesion through 3D ridges on CLEAR printed highly entangled adhesive hydrogels (W = 2.63. C = 3.5 x 1 O'4) applied onto ex vivo porcine heart tissue. (FIG. 50A) Peeling of hydrogels containing 3D ridges with tunable spacing demonstrating strong adhesion on ex vivo porcine heart tissue. (FIG. 5 OB) Representative force (F) - displacement (d) curves showing stepwise peeling away of ridges. (FIG. 50C) Quantification of steady state peel force for ridges with different spacing. 1 :x refers to a 1 mm ridge and x mm spacing between the ridges. Increase in the spacing results in a subsequent decrease in force required for debonding. Data are reported as mean ± SD; n = 5; ns = not significant; **p < 0.01; ***p < 0.001. (FIG. 50D) Photograph of porcine heart tissue after micro-contact imprinting of ridges to create spatial patterns. This method of sculpting can be useful to integrate sensors or infill cells and therapeutics within grooves. Scale bars: 10 mm. FIGs. 51A-51B show CLEAR printed highly entangled elastomers without adhesive hydrogel layer exhibit poor adhesion strength to ex vivo tissues. Photographs of porous elastomer mesh that lacks the ability to adhere onto porcine stomach as seen (FIG. 51 A) during and (FIG. 5 IB) after the 90° peel test. Scale bar: 10 mm.

[0060] BRIEF SUMMARY OF THE INVENTION

[0061] In one aspect, a method of printing a three-dimensional (3D) object is provided. The method includes: providing a container comprising a photocurable resin, wherein the photocurable resin comprises: i) at least one prepolymer, ii) optionally at least one crosslinking agent; iii) at least one photoinitiator; and iv) at least one non-photoresponsive initiator; waiting for a waiting period, wherein during the waiting period the at least one prepolymer polymerizes and the at least one photoinitiator is not activated; exposing at least one layer of the photocurable resin to a photocuring light; and printing the 3D object.

[0062] In one aspect, this method displays high monomer conversion at room temperature without the need for additional stimuli such as light or heat after printing and enables additive manufacturing of highly entangled hydrogels and elastomers that exhibit 4 to 7-fold higher extension energies in comparison to traditional DLP.

[0063] DETAILED DESCRIPTION OF THE INVENTION

[0064] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0065] Throughout this document, values expressed in a range format 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. For example, a range of "‘about 0. 1% to about 5%’?or “about 0. 1% to 5%" should be interpreted to include notjust about 0.1% to about 5%, but also the individual values (e.g, 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "‘about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0066] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B ” In addition, it is to be understood that the phraseology or terminology7employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting: information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference

[0067] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0068] Definitions

[0069] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0070] The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8. 0.7, 0.6, 0.5, 0.4. 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term “substantially free of’ can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4. 3.5, 3, 2.5, 2, 1.5, 1, 0.9. 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0071] The term “organic group” as used herein refers to any carbon-containing functional group. Examples can include an oxygen-containing group such as an alkoxy group, aryloxy group, aralkyloxy group, oxo(carbonyl) group; a carboxyl group including a carboxylic acid, carboxylate, and a carboxylate ester: a sulfur-containing group such as an alkyl and and sulfide group; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R. C(O)C(O)R, C(O)CH2C(O)R. C(S)R. C(O)OR. OC(O)R. C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2. (CH2)O-2N(R)C(O)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (Ci-Cioo)hydrocarbyl. wherein R can be hydrogen (in examples that include other carbon atoms) or a carbon-based moiety, and wherein the carbon-based moiety can be substituted or unsubstituted.

[0072] The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The substitution can be direct substitution, whereby the hydrogen atom is replaced by a functional group or substituent, or an indirect substitution, whereby an intervening linker group replaces the hydrogen atom, and the substituent or functional group is bonded to the intervening linker group. A non-limiting example of direct substitution is: RR-H RR-C1, wherein RR is an organic moiety / fragment / molecule. A non-limiting example of indirect substitution is: RR-H RR- (LL)zz-Cl, wherein RR is an organic moiety / fragment / molecule, LL is an intervening linker group, and ‘zz‘ is an integer from 0 to 100 inclusive. When zz is 0, LL is absent, and direct substitution results. The intervening linker group LL is at each occurrence independently selected from the group consisting of -H, -O-. -OR, -S-. -S(=O)-, -S(=O)2-. -SR, -N(R)-, - NR2, -CR=, -C=, -CH2-, -CHR-, -CR2-, -CH3, -C(=O)-, -C(=NR)-, and combinations thereof. (LL)zz can be linear, branched, cyclic, acyclic, and combinations thereof.

[0073] The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g. , F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and ar l sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCFs, R, O (oxo), S (thiono), C(O), S(O). methylenedioxy, ethylenedioxy, N(R)2, SR, SOR. SO2R, SO2N(R)2, SO3R. C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R. C(0)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)O-2N(R)C(0)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)C0N(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(0)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (Ci-Cioo)hydrocarbyL alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroar l, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.

[0074] The term “alkyl’" as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n- butyl, n-pentyl, n-hexyl, n-heptyl, and n-oct l groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term "‘alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0075] The term “alkenyl” as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or. in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, -CH=C=CCH2, -CH=CH(CH3), - CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

[0076] The term “alkynyl” as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to - C=CH. -C =C(CH3). -C =C(CH2CH3). -CH2C=CH. -CH2C=C(CH3). and -CH2C=C(CH2CH3) among others.

[0077] The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is bonded to a hydrogen forming a “formyl” group or is bonded to another carbon atom, which can be part of an alkyl, aryl, aralk l cycloalkyl, cycloalkydalkyl, heterocyclyl, heterocyclylalkyl, heteroary l, heteroary lalkyl group or the like. An acyl group can include 0 to about 12, 0 to about 20. or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group can include double or triple bonds within the meaning herein. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning herein. A nicotinoyl group (pyridy 1-3 -carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoy l, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group. An example is a trifluoroacetyl group.

[0078] The term “cycloalky l” as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbomyl, adamantyl, bornyl, campheny 1, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to. decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbomyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term “cycloalkenyl” alone or in combination denotes a cyclic alkenyl group.

[0079] The term “heterocycloalkyl” as used herein refers to a cycloalkyl group as defined herein in which one or more carbon atoms in the ring are replaced by a heteroatom such as O. N, S, P, and the like, each of which may be substituted as described herein if an open valence is present, and each may be in any suitable stable oxidation state.

[0080] The term “aryl” as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to. phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyL naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, ary l groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.

[0081] The term “aralkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyd and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an ary 1 group as defined herein.

[0082] The term “heterocyclyl” as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O. and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. The term heterocyclyl includes rings where a CH2 group in the ring is replaced by one or more C=O groups, such as found in cyclic ketones, lactones, and lactams. Examples of heterocyclyl groups containing a C=O group include, but are not limited to. - propiolactam, y-butyrolactam, 5- valerolactam, and s-caprolactam. as well as the corresponding lactones. A heterocyclyl group designated as a C'2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a Cr-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl. azaindolyl. indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with groups such as those listed herein.

[0083] The term “heteroaryl” as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroary l group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. A heterocyclyl ring designated Cx ycan be any ring containing ‘x’ members up to ‘y’ members, including all intermediate integers between ‘x‘ and ‘y' and that contains one or more heteroatoms, as defined herein. In a ring designated Cx-y. all non-heteroatom members are carbon. Heterocyclyl rings designated Cx-ycan also be polycyclic ring systems, such as bicyclic or tricyclic ring systems. Heteroary l groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindoly l. indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl. isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein.

[0084] Additional examples of ary l and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl. N- hydroxytriazolyl, N-hydroxyimidazolyl. anthracenyl (1-anthracenyl, 2-anthracenyl, 3- anthracenyl), thiophenyl (2 -thienyl, 3-thienyl), furyl (2-furyl, 3-furyl) , indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-l-yl, l,2,3-triazol-2-yl l,2,3-triazol-4-yl. l,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2 -thiazolyl, 4- thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4- pyridazinyl, 5 -pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6- quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1 -isoquinolyl, 3-isoquinolyL 4-isoquinolyl, 5- isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b] furanyl, 5-benzo[b]furanyl, 6-benzo[b] furanyl, 7- benzo[b] furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2.3- dihydro-benzo [bl furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2.3-dihydro-benzo[b]furanyl),

[0085] 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2- benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6- benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3- dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro- benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro- benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl,

[0086] 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl,

[0087] 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1 -benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5 -benzimidazolyl. 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1- benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5 -benzothiazolyl, 6-benzothiazolyl,

[0088] 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b.f|azepine (5H-dibenz[b,f] azepin- 1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,1 l-dihydro-5H-dibenz[b,f| azepine (10,1 l-dihydro-5H-dibenz[b,f|azepine-l-yl,

[0089] 10,1 l-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,1 l-dihydro-5H-dibenz[b,f|azepine-3-yl.

[0090] 10,1 l-dihydro-5H-dibenz[b,f|azepine-4-yl, 10,1 l-dihydro-5H-dibenz[b,f|azepine-5-yl), and the like.

[0091] The term “heterocyclylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclyl alkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl ethyl, and indol-2-yl propyl.

[0092] The term “heteroarylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.

[0093] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy. isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy. cyclopenty loxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where tw o adjacent atoms of a structure are substituted therewith.

[0094] The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N (group)? wherein each group can independently be FI or non-H, such as alky l, aryl, and the like. Amines include but are not limited to R-NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylammes and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylary lamines, alkyldiarylamines, triary lamines, and the like. The term “amine” also includes ammonium ions as used herein.

[0095] The term “amino group” as used herein refers to a substituent of the form -NEE, - NHR, -NR2, -NR? . wherein each R is independently selected, and protonated forms of each. except for -NR3 . which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An ‘Amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group includes a monoalkylamino. dialkylamino, and trialkylamino group.

[0096] The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0097] The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, polyhalo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl. 1,1 -dichloroethyl, 1.2-di chloroethyl, l,3-dibromo-3,3- difluoropropyl, perfluorobutyl, and the like.

[0098] The terms “epoxy-functional” or “epoxy-substituted” as used herein refers to a functional group in which an oxygen atom, the epoxy substituent, is directly attached to two adjacent carbon atoms of a carbon chain or ring system. Examples of epoxy-substituted functional groups include, but are not limited to, 2,3-epoxypropyI. 3.4-epoxybutyl, 4,5- epoxypentyl, 2,3-epoxypropoxy. epoxypropoxypropyl, 2-glycidoxyethyl, 3-glycidoxypropyl, 4-glycidoxybutyl, 2-(glycidoxycarbonyl)propyl, 3-(3,4-epoxycylohexyl)propyl, 2-(3,4- epoxycyclohexyljethyl, 2-(2,3-epoxycylopentyl)ethyl, 2-(4-methyl-3.4- epoxycyclohexyljpropyl, 2-(3.4-epoxy-3-methylcylohexyl)-2-methylethyl, and 5,6- epoxyhexyl.

[0099] The term “monovalent” as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond.

[0100] The term “hydrocarbon” or “hydrocarbyl” as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups.

[0101] As used herein, the term “hydrocarbyl” refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and can be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. Hydrocarbyl groups can be shown as (Ca- Cbjhydrocarby I. wherein a and b are integers and mean having any of a to b number of carbon atoms. For example. (Ci-C4)hydrocarbyl means the hydrocarbyl group can be methyl (C 1), ethyl (C2), propyl (C3), or butyl (C4), and (Co-Cb)hydrocarbyl means in certain embodiments there is no hydrocarbyl group.

[0102] The term “solvent” as used herein refers to a liquid that can dissolve a solid, liquid, or gas. Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids.

[0103] The term “independently selected from” as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase “X1, X2, and X3are independently selected from noble gases” would include the scenario where, for example, X1, X2, and X3are all the same, where X1, X2, and X3are all different, where X1and X2are the same but X3is different, and other analogous permutations.

[0104] The term “room temperature” as used herein refers to a temperature of about 15 °C to 28 °C.

[0105] The term “standard temperature and pressure” as used herein refers to 20 °C and 101 kPa.

[0106] The term “monomer” refers to any discreet chemical compound that is polymerized when subjected to suitable conditions. Non-limiting examples of conditions that can cause a monomer to become polymerized include, electromagnetic radiation (e.g, UV, IR, and visible light) and thermal radiation (e.g, heating).

[0107] As used herein, the term “polymer” refers to a molecule composed of repeating structural units typically connected by covalent chemical bonds. The term “polymer” is also meant to include the terms copolymer and oligomers. In certain embodiments, a polymer comprises a backbone (i.e., the chemical connectivity7that defines the central chain of the polymer, including chemical linkages among the various polymerized monomeric units) and a side chain (i.e.. the chemical connectivity that extends away from the backbone).

[0108] As used herein, the term “photoinitiator” refers to a molecule that creates reactive species (such as for example, free radicals, cations or anions) when exposed to electromagnetic radiation such as UV or visible light.

[0109] As used herein, the term “polymerization” or “crosslinking” refers to at least one reaction that consumes at least one functional group in a monomeric molecule (or monomer), oligomeric molecule (or oligomer) or polymeric molecule (or polymer), to create at least one chemical linkage between at least two distinct molecules (e.g, intermolecular bond), at least one chemical linkage within the same molecule (e.g, intramolecular bond), or any combinations thereof. A polymerization or crosslinking reaction may consume between about 0% and about 100% of the at least one functional group available in the system. In certain embodiments, polymerization or crosslinking of at least one functional group results in about 100% consumption of the at least one functional group. In other embodiments, polymerization or crosslinking of at least one functional group results in less than about 100% consumption of the at least one functional group.

[0110] As used herein, a “non-photoresponsive initiator” is a reagent or group of reagents that generates free radicals but does not require light of any wavelength to produce free radicals. Examples of non-photoresponsive initiators include redox initiators and thermal initiators.

[0111] Methods of Manufacturing

[0112] Entanglements are ubiquitous in nature with examples across length scales from chromosomal DNA to dynamic tangling of worms. Polymer entanglements in melts have been well-studied, however, the role of entanglements on fatigue and fracture of polymer networks is only recently appreciated. Unlike chemical crosslinks, entanglements act as nonpermanent crosslinks that can slide past one another under stress to simultaneously increase material modulus and toughness without embrittlement or hysteresis.

[0113] DLP is a bottom-up vat photopolymerization method that allows high speed fabrication of 3D objects via layer-by-layer projection of 2D sliced images. Despite the promise of DLP, processing parameters such as light-attenuating additives to control resolution, oxygen inhibition, and diffusion limitations of the radical mediated chain growth reaction can introduce gradients in monomer conversion that reduce the overall concentrations of long polymer chains and resulting strength of the printed object (Fig. IB, Figs. 8A-8B). To overcome this mechanical performance-processability conflict, provided herein in various embodiments is a one-step, Continuous-curing after Light Exposure Aided by Redox initiation (CLEAR) process towards formation of highly entangled polymer networks using vat photopolymerization methods. CLEAR allows spatial light illumination (i.e., photopolymerization) to set the shape of the object while a complementary redox reaction (i.e.. dark polymerization) allows unreacted monomer within the object to slowly reach complete conversion, which enables high concentrations of polymer chains that can entangle (Fig. 1C). The continuous curing process occurs at room temperature without the need for additional steps that involve light or heat.

[0114] Provided herein is a method to print 3D objects that, in various embodiments, include gels and elastomers that contain long, highly entangled polymer chains. Without being bound by theory, these entangled polymers result in physical interactions between polymers that stiffen the material but allow for the polymer chains to slide past one another to dissipate forces to improve the material toughness, in contrast to conventional non-entangled polymer networks that exhibit failure when loaded.

[0115] In various embodiments, the method provides for 3D printing of hydrogels and elastomers using digital light processing (DLP). DLP utilizes a liquid resin to form the resulting 3D objects, and is known as a vat photopolymerization technique. This resin is subjected to a light from a projector that cures the resin in a layer-by-layer process. In various embodiments, the methods described herein can print parts upside down (known as bottom-up printing) or right-side-up (known as top-down printing).

[0116] In various embodiments a method of printing a three-dimensional (3D) object is provided. In certain embodiments, the method includes providing a container comprising a photocurable resin, wherein the photocurable resin comprises: i) at least one prepolymer, ii) optionally at least one crosslinking agent; iii) at least one photoinitiator; and iv) at least one non-photoresponsive initiator.

[0117] In certain embodiments, the method includes waiting for a waiting period, wherein during the waiting period the at least one prepolymer polymerizes and the at least one photoinitiator is not activated. In certain embodiments, the waiting for a waiting period is before the photocuring. In certain embodiments, the waiting for a waiting period is after the photocuring, such as for example, a waiting period after a layer of the prepolymer has been polymerized, or after the last layer of the prepolymer has been polymerized.

[0118] In certain embodiments, the at least one prepolymer comprises at least two copolymers.

[0119] In certain embodiments, the method includes exposing at least one layer of the photocurable resin to a photocuring light.

[0120] In certain embodiments, the method includes printing the 3D object.

[0121] The container can be any suitable container, vessel, or vat used for vat polymerization, and can be made of a suitable material that is transparent to light, such as quartz, plastic, glass, and the like. The container can be of any regular geometric solid shape such as a cube, rectangular cube, sphere, cylinder, polygon cylinder, and the like.

[0122] In various embodiments, the at least one prepolymer is at least one chosen from acrylamide, methacrylamide, acrylic acid, methacrylic acid. N-isopropylacrylamide, N- isopropylmethacrylamide, 2-hydroxy ethyl acrylate, 2 -hydroxy ethyl methacrylate, N- [tris(hydroxymethyl)methyl |acrylamide. acrylic acid N-hydroxysuccinimide ester, methacrylic acid N-hydroxysuccinimide ester, ethyl acrylate, ethyl methacrylate. 2- acrylamido-2-methyl-l-propanesulfonic acid, and [2-(methacryloyloxy)ethyl]dimethyl-(3- sulfopropyl)ammonium hydroxide, ethyl vinyl sulfone, and phenyl vinyl sulfone.

[0123] In various embodiments, the at least one crosslinking agent is at least one chosen from N,N'-methylenebis(acrylamide), gelatin methacrylamide, gelatin acrylamide, poly(ethylene glycol) diacrylate, Poly(ethylene glycol) dimethacrylate, acrylated hyaluronic acid, methacrylated hyaluronic acid, and tricyclo[5.2.1.02,6]decanedimethanol diacrylate.

[0124] In various embodiments, the at least one photoinitiator is at least one chosen from acetophenone, benzophenone, 2-phenylacetophenone. 2,2-dimethoxy-2-phenylacetophenone, Bis(2,4,6-trimethylbenzoyl)-phenylphosphineoxide. 2-Hydroxy-4'-(2 -hydroxy ethoxy)-2- methylpropiophenone, 2-methyl-(4-methylthienyl)-2-morpholinyl-l -propan- 1 -one, Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, Ethyl (2,4,6-trimethylbenzoyl) phenyl phosphinate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LzkP (lithium phenyl-2,4,6- trimethylbenzoylphosphmate). TPO (dipheny 1(2,4, 6-trimethylbenzoyi)phosphine oxide). BAPO (phenylbi s(2,4,6-trimethylbenzoyl)phosphine oxide). Eosin Y. Riboflavin, Ru(bpy)s / SPS, VA-044 (2,2!-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride), VA-086 (2,2'-azobis 2-methyl-N-(2-hydroxyethyl)propionamide]), and 1907 (2-methyl-4'- (meUiyltbio)-2-morpholinopropiophenone).

[0125] In various embodiments, the at least one non-photoresponsive initiator is a redox initiator or a thermal initiator. In various embodiments, the redox initiator is BPO / DMA, tris [4-(diethylamino)phenyl]amine / bis-(4-t-butylphenyl)-iodonium hexafluorophosphate, MPP / BPO, or APS / TEMED. In various embodiments, the photocurable resin further comprises a photoabsorber.

[0126] In various embodiments, the waiting period is 1 to 360 minutes. In various embodiments, the waiting period is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260. 270, 280, 290, 300, 310, 320, 330, 340, 350, or 360 minutes. Without being bound by theory, during the waiting period, the non-photoresponsive initiator, such as a redox initiator or a thermal initiator, reacts or is otherwise activated to create a reactive species (such as free radicals) to promote polymerization of the monomer / prepolymer. Further, without being bound by theory, the polymerization of the monomer / prepolymer by the redox initiator or a thermal initiator followed by photopolymerization is believed to provide highly entangled polymers with superior properties, as described herein. The term “at least one photoinitiator is not activated’' means that the photoinitiator in the photocurable resin, such as any of the photoinitiators described herein, is not subject to light or any other kind of electromagnetic radiation or other environment that would cause it to produce free radicals capable of initiating or continuing polymerization of the prepolymer.

[0127] In various embodiments, exposing is for a period of about 0.5 s to about 30 minutes. In various embodiments, the exposing is for a period of about 0.5, 1, 2, 3, 4, 5, 6. 7 ,8, 9, 10, 11. 12. 13. 14, 15, 16, 7. 18, 19, 20, 30, 40, 50, or 60 seconds, or about I. 2, 3, 4.5 ,6, 7, 8. 9. 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 minutes. The intensity of the photocuring light during the exposing (exposure period) can be constant or can vary during the exposure period.

[0128] In various embodiments, the photocuring light has a wavelength of about 350 nm to about 1200 nm. In various embodiments, the photocuring light has a wavelength of about

[0129] 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520,

[0130] 530, 540, 550, 560, 570, 580, 590, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800,

[0131] 820, 840. 860, 880, 900, 920, 940. 960, 980, 1000, 1020, 1040, 1060. 1080, 1100, 1120,

[0132] 1140, 1 160, 1180, or 1200 nm.

[0133] In various embodiments, the photocuring light has an intensity of about 1 mW / cm2to about 3 W / cm2In various embodiments, the photocuring light has an intensity' of about 1, 2, 3, 4, 5,. 6, 7, 8, 9, 10, 20, 30. 40, 50, 60, 70, 80, 90, or about 100 mW / cm2. In various embodiments, the photocuring light has an intensity of about 150, 200, 250, 300. 350, 400. 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1 100, 1 150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, or about 3000 mW / cm2.

[0134] In various embodiments, the printing does not comprise a flood cure. Advantageously, and without being bound by theory, the present method can provide a fully cured 3D object after the final layer of the prepolymer has polymerized with the photocuring light. Aside from cleaning the object with air or solvent as necessary, no final bulk / flood cure is necessary for the 3D object to have the properties described herein. In various embodiments, after the final layer of the prepolymer has polymerized (post-printing) with the photocuring light, the cured 3D object is allowed to remain at room temperature for a period of about 10 s to about 12 hours.

[0135] In various embodiments, the exposing includes digital light processing (DLP). Although DLP printing is described herein, the present method can be used with stereolithographic methods such as additive manufacturing and other forms of stereolithography, volumetric additive manufacturing, or any other additive manufacturing technique that involves exposure to light.

[0136] In various embodiments, the tensile modulus of the printed 3D object according to the methods herein is at least 50% greater than the tensile modulus of a printed 3D object not subjected to the step of waiting. In various embodiments, the tensile modulus of the printed 3D object according to the methods herein is at least 10, 20, 30. 40. 50. 60. 70. 80. 90, 100, 110, 120, 130, 140, or 150% greater than the tensile modulus of a printed 3D object not subjected to the step of waiting. Not being subjected to the step of waiting refers to a monomer / prepolymer not being subject to reaction with a non-photoresponsive initiator, such as a redox initiator or a thermal initiator for the periods of time described herein.

[0137] In various embodiments, the water content of the printed 3D object according to the methods herein is at least 1.1 to 10 times smaller than the water content of a printed 3D object not subjected to the step of waiting. In various embodiments, the water content of the printed 3D object according to the methods herein is at least 1.1, 1.5, 2. 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times smaller than the water content of a printed 3D object not subjected to the step of waiting.

[0138] In various embodiments, the friction coefficient of the printed 3D obj ect according to the methods herein is at least 1.1 to 100 times greater than the friction coefficient of a printed 3D object not subjected to the step of waiting. In various embodiments, the friction coefficient of the printed 3D object according to the methods herein is at least 1 . 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than the friction coefficient of a printed 3D object not subjected to the step of waiting.

[0139] In various embodiments, the at least one prepolymer is at least one chosen from alginate, hyaluronic acid, polyethylene glycol), gelatin, chitosan, poly vinyl alcohol, polyacrylamide, polyacrylic acid, polyallylamine, dextran, keratin, silk fibroin, collagen, fibrin, elastin, glycogen covalently functionalized by at least one group selected from the group consisting of acrylamide, methacrylamide, acrylic acid, methacrylic acid, N- isopropylacrylamide, N-isopropylmethacrylamide, 2-Hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, N-[Tris(hydroxymethyl)methyl]acrylamide, acrylic acid N-hydroxysuccinimide ester, methacrylic acid N-hydroxysuccinimide ester, ethyl acrylate, ethyl methacrylate, ethyl vinyl sulfone, phenyl vinyl sulfone, 2-acrylamido-2-methyl-l -propanesulfonic acid, and [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide.

[0140] In various embodiments, a cured object produced by the method described herein is provided. The cured object is, in various embodiments, the 3D object obtained after the photopolymerizing light exposure has completed or is stopped. In various embodiments, the cured object includes a hydrogel. In various embodiments, the cured object is a hydrogel. In various embodiments, the cured object is printed from a macromer prepolymer as described herein. In various embodiments, the cured object is bioadhesive. In various embodiments, the cured object is a hydrogel that is bioadhesive. The term “bioadhesive” refers to the ability of the cured object to adhere to tissue from an organism, both in vitro and in vivo. The tissue can be any tissue, such as muscle, tendon, bone, skin, endothelial tissue, organ tissue, and the like. The tissue can be from any suitable source, including animals and humans. In various embodiments, the tissue is human tissue.

[0141] In various embodiments, the cured object is a patterned hydrogel. The pattern can be a three-dimensional pattern of any geometry, for example a series of ridges and valleys. The patterns can also be formed in the interior of the cured object. In various embodiments, the formed pattern is a plurality7of voids (spaces lacking the cured material and containing air) in the cured object, making the cured object porous. The voids can be randomly scattered throughout the interior of the object or they can be regular and repeating. When the cured object is a sheet of material, one side of the material can have a different pattern than the other side of the material. In various embodiments, the object includes a first surface and a second surface, wherein the first surface requires a larger peeling force to remove the object from a surface than the second surface. In various embodiments, the object includes a plurality of surfaces, such as a first, second, third, fourth, fifth, sixth, seventh surface, and the like. Any of the plurality of surfaces, such as the first surface and second surface referred to herein can be an external surface (in contact with air / atmosphere) or an internal surface. Thus, the internal surface can be an internal layer, such as a first layer or a second layer. In various embodiments, the larger peeling force is a force that is at least, greater than, or equal to 1. 1 to 100 times greater on the first side as compared to the second side. In various embodiments, the larger peeling force is a force that is at least, greater than, or equal to 1. 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. 24. 25, 26, 27, 28, 29, 30. 31. 32. 33, 34, 35, 36, 37, 38, 39, 40, 41, 42. 43. 44. 45. 46. 47, 48, 49, 50, 51, 52, 53, 54,

[0142] 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,

[0143] 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 times greater on the first side as compared to the second side.

[0144] In various embodiments, the interfacial adhesion and shear strength of the printed 3D object according to the methods herein is at least 2 to 20 times higher than the interfacial adhesion and shear strength of a printed 3D object not subjected to the step of waiting. In various embodiments, the interfacial adhesion and shear strength of the printed 3D object according to the methods herein is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times higher than the interfacial adhesion and shear strength of a printed 3D object not subjected to the step of waiting.

[0145] In various embodiments, the 3D object includes a hydrogel, or is a hydrogel. In various embodiments, printing the 3D object further includes annealing the printed 3D object to at least a portion of a non-polymeric material. In some embodiments, the non-polymeric material is a not a hydrogel. In various embodiments, the non-polymeric material is a semiconductor material. The non-polymeric material is not particularly limited and includes, in non-limiting examples, electronic sensors and microfluidic circuits.

[0146] Monomer s / Prepolymers

[0147] In various embodiments, suitable monomers for use with these methods are thermoset monomers and prepolymers. The term "monomer" and “prepolymer"’ are used herein interchangeably to refer to a resin or other substance that has not undergone any polymerization as a result of a photoinitiator or a non-photoactive initiator as described herein. Suitable monomers / prepolymers include acry lic resins, polyesters, vinyl esters, vinyl sulfones, epoxy functional resins, isocyanate resins, phenolic resins, amino resins, furan resins, polybenzoxazines. Suitable acrylic resins include, but are not limited to, acrylamide, methacrylamide, acrylic acid, methacrylic acid, N-isopropylacrylamide, N- isopropylmethacrylamide, 2-hydroxyethyl acrylate, 2 -hydroxy ethyl methacry late, N- [tris(hydroxymethyl)methyl]acrylamide, acrylic acid N-hydroxysuccinimide ester, methacrylic acid N-hydroxysuccinimide ester, ethyl acrylate, ethyl methacrylate. 2- acrylamido-2-methyl-I-propanesulfonic acid, and [2-(methacryloyloxy)ethyl]dimethyl-(3- sulfopropyljammonium hydroxide. Suitable vinyl sulfones include ethyl vinyl sulfone and phenyl vinyl sulfone. When these monomers / prepolymers are subjected to the methods herein, a thermoset polymer is produced. In various embodiments, the 3D printed object is fully cured at the end of the process and does not require a flood cure. The monomer / prepolymer can be about 30 to about 99% (w / w) of the pre-polymerized resin. The monomer / prepolymer can be 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59. 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84. 85. 86. 87. 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% (w / w) of the pre-polymerized resin. Crosslinking Agents

[0148] In various embodiments, the method includes the use of at least one crosslinking agent. Suitable crosslinking agents include, but are not limited to, N,N'- methylenebis(acrylamide), gelatin methacrylamide, gelatin acrylamide, poly(ethylene glycol) diacrylate (average molecular weight ranging from 250 Da- 200,000 Da), Poly(ethylene glycol) dimethacrylate (average molecular weight ranging from 250 Da- 200,000 Da), acrylated hyaluronic acid, methacrylated hyaluronic acid, tricyclo[5.2.1.02,6]decanedimethanol di acrylate, and the like. In various embodiments, each of these crosslinkers can independently be used in an amount of about 0.001 to about 2 wt.% relative to the amount of the monomer / prepolymer. In various embodiments, each of these crosslinkers can independently be used in an amount of about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 wt.% relative to the amount of monomer / prepolymer.

[0149] Photoactive Agents

[0150] In various embodiments, the method includes the use of at least one photoactive agent. Photoactive agents include photoinitiators and photoabsorbers. Suitable photoinitiators include, but are not limited to, acetophenone, benzophenone, 2- phenylacetophenone, 2,2-dimethoxy-2-phenylacetophenone, Bis(2.4.6-trimethylbenzoyl)- phenylphosphineoxide, 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 2-methyl- (4-methylthienyl)-2-morpholinyl-l -propan- 1 -one, Diphenyl(2,4,6- trimethylbenzoyl)phosphine oxide, Ethyl (2,4.6-trimethylbenzoyl) phenyl phosphinate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP, TPO, BAPO, Eosin ¥, Riboflavin,

[0151] Ru(bpy / SPS, VA-044, VA-086, 1907, 12959

[0152] Suitable photoabsorbers include, but are not limited to, to tartrazine, Sudan I, curcumin, anthocyanin, Tinuvin CarboProtect.

[0153] In various embodiments, the photoactive agent be used in an amount of about 0.001 to about 5 wt.% relative to the amount of the monomer / prepolymer. In various embodiments, the photoactive agent can be used in an amount of about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4. 1.5, 1.6, 1.7, 1.8, 1.9. 2, 3, 4, or 5 wt.% relative to the amount of monomer / prepolymer.

[0154] Non-photoresponsive Initiator

[0155] In various embodiments, the non-photoresponsive initiator is a redox initiator. A redox initiator can be used to initiate polymerization relying on the free radicals producing during an oxidation-reduction reaction. The type of redox initiator that can be used in the methods herein is not particularly limited. Redox initiators are typically present as a reagent pair, in which one reagent is an oxidizing agent and one reagent is a reducing agent. Suitable redox initiators include BPO (benzoyl peroxide) / DMA (N,N-dimethylaniline). The BPO and DMA can be mixed in any suitable ratio, such as from 10: 1 BPO:DMA to 1 : 10 BPO:DMA. In various embodiments, the redox initiator is (tris [4-(diethylamino)phenyl]amine / bis-(4-t- butylphenyl)-iodonium hexafluorophosphate, which can be mixed in any suitable ratio such as from 10:1 to 1: 10. In various embodiments, the redox initiator is N-(4- methoxyphenyl)pyrrolidine (MPP) / BPO, which can be mixed in any suitable ratio such as from 10: 1 to 1 : 10. In various embodiments, the redox initiator is APS (ammonium persulfate) / TEMED (N,N,N',N'-tetramethyl ethylenediamine), which can be mixed in any suitable ratio such as from 10: 1 to 1:10. In various embodiments, the oxidizing and reducing agents in the redox initiator are present in a 1 : 1 ratio. In various embodiments, each of pair of reagents in the redox initiator can be independently present in an amount of about 0.001 to about 1% (w / w) of the pre-polymerized resin. In various embodiments, each of pair of reagents in the redox initiator can be independently present in an amount of about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04. 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2. 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or about 1% (w / w) relative to the amount of monomer / prepolymer. In various embodiments, the non-photoresponsive initiator is a thermal initiator. Thermal initiators are compounds that generate radicals or cations upon exposure to heat. The type of thermal initiator that can be used in the methods herein is not particularly limited. Suitable thermal initiators include, but are not limited to tert-amyl peroxybenzoate, benzoyl peroxide, 2,2’-azobis(isobutyronitrile) (AIBN), l,r-Azobis(cyclohexanecarbonitrile), 1,1- Bis(tert-butylperoxy)cyclohexane, tert-Butyl hydroperoxide, tert-Butyl peracetate, cumene hydroperoxide, and the like. In various embodiments, the thermal initiator can be present in an amount of about 0.001 to about 1% (w / w) of the pre-polymerized resin. In various embodiments, the thermal initiator can be present in an amount of about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or about 1% (w / w) relative to the amount of monomer / prepolymer.

[0156] Macromers

[0157] In various embodiments, biopolymers modified with any of the monomers / prepolymers described herein (macromers) can be used as the initial material to be polymerized. Biopolymers such as, but not limited to, alginate, hyaluronic acid, poly(ethylene glycol)-branched and linear versions, gelatin, chitosan, poly vinyl alcohol, polyacrylamide, polyacrylic acid, polyallylamine, dextran, keratin, silk fibroin, collagen, fibrin, elastin, glycogen can be modified with various chemical groups, including but not limited to, acrylamide, methacrylamide, acrylic acid, methacrylic acid, N- Isopropylacrylamide, N-Isopropylmethacrylamide, 2-Hydroxyethyl acrylate, 2-Hydroxyethyl methacry late, N-| tris(hydroxy methy I )methyl | acry lamide, acrylic acid N-hydroxysuccinimide ester, methacrylic acid N-hydroxysuccinimide ester, ethyl acrylate, ethyl methacrylate, ethyl vinyl sulfone, phenyl vinyl sulfone, 2-acrylamido-2-methyl-l -propanesulfonic acid, [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide to provide materials that can be used in the method described herein. The molecular these macromers can range from 250 Daltons to 20 M Daltons. In various embodiments, the macromer can be about 30 to about 99% (w / w) of the pre-polymerized resin. In various embodiments, the macromer can be 30, 31, 32, 33, 34, 35, 36. 37. 38. 39. 40. 41. 42. 43, 44, 45, 46, 47, 48, 49, 50, 51, 52. 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% (w / w) of the pre-polymerized resin.

[0158] In various embodiments, polymer chain growth is initiated prior to 3D printing by activating the non-photoresponsive initiator(s), which starts slowly growing polymer chains. At some point, the resin is exposed to light with a defined pattern using vat photopolymerization to define a 3D object and areas not exposed to light are removed (e.g., washing, physical removal). The non-photoresponsive initiator continues growing polymer chains after 3D printing, leading to high molecular weight polymers within the 3D printed object. Since polymer chains are growing slowly the resin is only briefly exposed to light, a structure of a highly resolved object is created while still achieving high molecular weight polymers.

[0159] There are many applications where a tough 3D printed material are useful. In various embodiments, the methods described herein can be used for the spatial patterning of adhesive and non-adhesive materials through 1) structural characteristics that physically enable spatial tissue adhesion (e.g. grooves where the material only contacts and adheres to tissue at protruding features) or 2) multi-material printing to spatially separate polymers with adhesive and non-adhesive properties within the composite printed object. Similar techniques can be employed to spatially pattern conductive polymers within a composite print. Along with tissue adhesion, the long polymer chains obtained through this method provide a water-rich layer on the surface of the 3D printed material that lowers the friction coefficient far below conventional hydrogels, which should have important applications for preventing wear between tissues and materials.

[0160] EXAMPLES

[0161] Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein.

[0162] Materials and Methods

[0163] All chemicals were obtained from Millipore-Sigma unless stated otherwise and the specific product numbers (#) are listed where applicable. Gelatin methacrylamide (GelMA) was purchased from Allevi (3D Systems). Methacrylate-modified hyaluronic acid (MeHA) was synthesized using a previously established protocol. Briefly, HA (5 g, Lifecore Biomedical) was dissolved in deionized water (0.2 L) under ice-cold conditions. Methacrylic anhydride (19.3 g, 20 equivalent, #276685, Sigma) was added dropwise to the solution and pH was maintained at 8.0-9.0 throughout the 5 h reaction. The solution was transferred to dialysis tubing (Spectra Por, 6-8 kDa cutoff) and dialyzed against deionized water for 10 d, frozen at -80 °C, and lyophilized. Dry polymer was stored under inert nitrogen at -20 °C. N- (l-Adamantyl)acrylamide and 6-Acrylamido-P-cyclodextrin were acquired from TCI Chemicals. Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, photoinitiator) was purchased from Colorado Photopolymer Solutions (Arkema Inc.).

[0164] 3D printing of highly entangled hydrogels

[0165] Acrylamide (Am, 60 wt.%, #A8887, Sigma), N.Nc-melhylene bisacrylamide (BisAm, 0.005 to 0.1 wt.%, M7279. Sigma), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 0.1 wt .%), tartrazine (TTz, 1 mM, T0388, Sigma) and deionized (DI) water were used as monomer, crosslinker, photoinitiator, photoabsorber, and solvent, respectively, to form the DLP resin. Stock solutions of BisAm and LAP were prepared at 2 wt.% in DI water. W was defined as the water to monomer molar ratio, C as the crosslinker to monomer molar ratio, and I as the photoinitiator to monomer molar ratio. Am monomer was dissolved with specific volumes of water, crosslinker, and photoinitiator to prepare a precursor of desired W (= 2.63), C (= 3.5 x 10'5to 7 x 10'4), and / (= 3.8 x 10'4to 9.4 x 10'4). To dissolve a high concentration of Am monomer in DI water, the solution was vortexed, heated at 40 °C to 45 °C for 5 min to 10 min and the temperature was maintained at ~25 °C during printing to prevent precipitation of Am from the resin. CLEAR resin was formulated with the same components as above with the addition of redox initiators: ammonium persulfate (APS, 0.02 wt.% to 0.05 wt.%, #A3678, Sigma) and N,N.N',N'-tetramethylethylenediamine (0.04 wt.% to 0. 1 wt.%, #E6758, Sigma). To fabricate degradable highly entangled hydrogels, disulfide based crosslinker [N,W-bis(acryloyl)cystamine; BAC, #044132-03, Thermo Scientific] was used. Stock solutions of BAC were freshly prepared at 2 wt.% in acetic acid immediately before use.

[0166] 3D printing of hydrogels was conducted on a Lumen Alpha DLP printer (35 pm xy pixel resolution, Volumetric Inc., USA). Briefly, DLP or CLEAR resin was dispensed into the poly(dimethyl siloxane) PDMS vat, and the layers were crosslinked sequentially with light exposure (5.5 s to 7.5 s, 20 mW cm2. 100 pm layer thickness). After printing was complete, 3D printed hydrogels were removed from the build platform and either (i) directly immersed in DI water (DLP) or (ii) stored in a nitrogen-purged sealed container for 1 h to 3 h at room temperature (21 °C to 25 °C) to allow' removal or complete conversion of unreacted monomer (CLEAR). For studies with under- or over-curing on the printer, the light exposure during DLP was varied, including undercure (2 s, 20 mW cm2. 100 pm layer thickness) and overcure (18 s. 20 mW cm2. 100 pm layer thickness). For comparison to traditional postprocessing methods, DLP printed hydrogels were either subjected to (i) visible light flood cure (5 min., 10 mW cm2. Exfo Omnicure Vis S1000 lamp with 400 nm to 500 nm bandpass filter) or (ii) thermal curing (2 h. 45 °C) of printed hydrogels containing 2,2'-Azobis[2-(2- imidazolin-2-yl)propane] dihydrochloride (VA-044 thermal initiator, Fujifilm, 5 mM) within the precursor solution. Once the curing was complete, all hydrogels were submerged in DI water to allow them to reach equilibrium before any further testing. To optimize the photoabsorber concentration to control the light penetration, DLP and CLEAR resins containing varying amounts of tartrazine (0 mM to 1.5 mM) were exposed to a range of irradiation conditions (varying exposure time, intensity = 20 mW cm2J on the printer to determine the critical exposure time as well as print resolution.

[0167] Photoabsorber (1.5 mM) and photoinitiator (0.25 wt.%) concentrations were adjusted for printing of 3D objects with negative features to avoid inadvertent over-polymerization of voids in the z- and xy-direction. Additionally, the redox initiator concentrations were altered to APS (0.02 wt.% to 0.03 wt.%) and TEMED (0.04 wt.% to 0.06 wt.%) to enable printing of tall structures (> 4 mm z-height) without spontaneous gelation of the resin in the vat. 3D printed objects with or without negative features were wiped with an absorbent pad or gently blown with nitrogen on the surface to remove excess / uncured resin during CLEAR curing. For intricate topologies or highly porous structures, printed hydrogels were rinsed with hexane (#HX0290, Sigma) along with mechanical agitation via vortex mixing to flush out uncured resin. Standard protocols for washing of 3D printed parts before flood cure can be readily adapted. Post-washing. 3D printed parts were held in a sealed, nitrogen-purged container for 1 h to 3 h at room temperature (21 °C to 25 °C) to allow' CLEAR curing to reach completion.

[0168] Multi-material CLEAR printing was undertaken following a simple print-pause-print method that involves switching between different resins while building up a 3D structure. Briefly, the build plate was lowered into a vat containing resin 1 and printing was performed as described earlier. After the first few- layers w ere printed, the process w as paused, the build plate withdrawn from the vat, and the resin 1 was replaced with resin 2. To avoid crosscontamination and minimize mixing between resins, a simple cleaning method was employed to wick away excess, unreacted resin within or around the printed structure using absorbent wipes. The build plate can be programmed to be lowered to the same height as the previous layer (xy) or to a new height to print sequential layers (z). After the projection of remaining sliced images was turned off, the print w as complete. The resulting multi-material structure was subjected to curing and then swollen in DI water. Various fluorescent tracers were added within same highly entangled hydrogel precursor solutions to formulate distinct resins and highlight the broad design freedoms afforded by this fabrication strategy to introduce heterogeneity’ within individual layers (co-planar; xy) and across sequential layers (stacked; z).

[0169] To anneal printed hydrogels through CLEAR, hydrogels (precursor containing redox initiators) were fabricated as described, rinsed wi th hexane, and assembled by placing them together with careful alignment of desired features at various times after printing. Gentle compression (£ 1 kPa, 2 min.) was applied upon contact to enable attachment and the assembly was placed in a sealed, nitrogen-purged container to facilitate complete curing. Assembled hydrogels were swollen after annealing and the integration toughness was measured via a 180-degree peel test (6 mm min’1).

[0170] CAD models for solid shapes or complex geometries used in the study were either designed in Fusion 360 (Autodesk, USA) or downloaded from online repositories (NIH 3D print exchange and MakerBot Thingiverse). To aid visualization, printed hydrogels were soaked in tartrazine (20 mM, for photographs). To enable visualization through fluorescence microscopy, fluorescein isothiocyanate (FITC)-labeled dextran (0.5 mg niL1. M = 2 MDa, #FD20000S, Sigma) or methacryloxy ethyl thiocarbamoyl rhodamine B (20 mM, Polysciences) was added to the precursor resin before 3D printing. For visualization of lumens within printed hydrogels, 2 -hydroxy ethyl agarose (0.5 wt%, 37 °C, #A4018, Sigma) containing rhodamine B isothiocyanate-labeled dextran (M = 0.5 MDa) was perfused into the channel and the agarose solution allowed to gel to prevent diffusion of fluorescent tracer before imaging. Z-stack fluorescence images were acquired on a confocal microscope (Nikon Eclipse Ti2 with AXR scanner) equipped with a 2x objective lens. Images were analyzed using ImageJ (NIH) and linear adjustment of brightness / contrast was applied to the entire image. The background within certain images was manually segmented using Photoshop editor (Adobe Inc., USA).

[0171] Assessment of conversion through real time FT-IR

[0172] FTIR spectrometer (Nicolet 6700 FTIR spectrometer with an MCT / A detector), equipped with a horizontal transmission stage, was used along with potassium bromide (KBr) plates (International Crystal Laboratories). Briefly, resin containing acrylamide (monomer), N.Nc-methylenebisacrylamide (crosslinker), LAP (photoinitiator, 0.1 wt.%), tartrazine (photoabsorber, 1 mM) with or without APS (0.05 wt.%) / TEMED (0.1 wt.%) (redox initiators) was added between the plates and the pathlength was set to allow greater than 90% transmission of light. Distinct C=C peaks corresponding to acrylamide appeared between 1026 and 924 cm'1. A delay time of 30 seconds was introduced in the beginning to determine the initial peak area. The samples were irradiated with visible light (20 mW cmzExfo Omnicure Vis S1000 lamp with 400 nm to 500 nm bandpass filter) for 10 seconds to mimic the energy delivered per layer during 3D printing. The disappearance of the C=C (between 1026 and 924 cm'1) peak and the appearance of C-H (2992 to 2888 cm'1) were used to track and determine conversion of acrylamide over 120 min (number of scans = 2 per min., resolution = 4).

[0173] Assessment of conversion through quantitative1H-NMR

[0174] Conversion of acrylamide to poly(acrylamide) within printed hydrogels was characterized by 'H-NMR (400 MHz, Bruker). Briefly, rectangular hydrogels without BisAm crosslinker (W = 2.63, C = 0) were printed (one layer or ten layers, 100 pm thickness) using DLP without and with post-cure (light or thermal) and CLEAR (as described earlier) and dissolved in deuterium oxide (D2O, Cambridge Isotope Laboratories) at 15 mg mL'1. After dissolution, dimethyl sulfone (6H. 3. 1-3.3, #41867, Sigma) was added to the polymer solution as an internal standard (10 mg mL'1), the spectra were recorded, and the concentration of acrylamide (3H, 8 = 5.6 - 6.5) and poly(acrylamide) (2H, 8 = 1.4 - 1.9) within the sample relative to internal standard were measured to calculate conversion.

[0175] Mechanical characterization

[0176] Photo-rheological characterization w as performed on a Discovery' HR20 rheometer (TA Instruments) (equipped with 20 mm diameter parallel plate geometry', 100 pm gap, 25 °C) to obtain the gelation profile through oscillatory shear time sweeps (time to gel point defined as the crossover between storage, G' and loss modulus, G") in the presence of visible light (20 mW cm2) or 405 nm light (15 mW cm2. Alpha Prototy pe Projector, Volumetric Inc ). Rheological properties of fully swollen printed hydrogels were measured on a Discovery HR20 rheometer (TA Instruments) (equipped with 8 mm diameter sand-blasted parallel plate geometry, 25 °C) via oscillatory frequency sweeps (0. 1 rad s ' to 100 rad s '. 0.1% strain) and oscillatory amplitude sweeps (1 rad s '. 0.01% to 100% strain). The swollen samples were cut into 8 mm discs and secured w ith an axial force (0. 1 N to 0. 15 N). All samples were circumferentially covered with mineral oil (#M8410, Sigma) after loading to avoid drying or evaporation of water during the rheological tests. The viscosity of polymer solutions was measured as an average viscosity at low' shear rate via flow- sweeps (0.01 s'1to 100 s'1).

[0177] For unconfined compression testing (Q800 DMA, TA Instruments, force ramp = 0.5 N min '). hydrogels were 3D printed into 5 mm diameter cylindrical discs and swollen to equilibrium. Compressive moduli were determined from the linear elastic region (10-20% strain) of the stress-strain curves. For uniaxial tensile testing (RSA-G2 DMA, TA Instruments), hydrogels were 3D printed into rectangular samples (15 x 5 x 1.5 mm3), swollen to equilibrium, and stretched until failure (stretch rate = 0.05 s'1). Samples were secured with sandpaper covered clamps to avoid slip under tension. Nominal stress was defined as the force on the deformed gel divided by the cross-sectional area of the undeformed sample, stretch was defined as the ratio of the length of deformed to the length of undeformed samples, and work of fracture was quantified as the area under the stress-strain curves for samples without a precut crack. Tensile moduli (slope from 5-15% strain), stressstretch at failure, and work of fracture were determined using TA Instrument Analysis software.

[0178] Hysteresis energy (Uhys) was determined by the area between the loading and unloading curve of the hydrogel for various degree of stretch. Hysteresis (Hys = Uhys / W ) was defined as the ratio of irreversible work to total work done by extension. For suture retention measurements, USP 4-0 non-absorbable sutures (Monofilament Polypropylene, Walter Products) were employed using the needle provided by the manufacturer and passed through the printed, swollen hydrogel specimen (suture bite centered with respect to the sample width) and closed into a loop by multiple knots. The samples were preloaded until the suture thread was taut, then subjected to uniaxial tension (RSA-G2 DMA, 6 mm min ') and stretched until suture pullout. Suture retention strength was quantified as the ratio of suture load to the product of suture diameter and sample thickness. Mass swelling ratios (Qm) were calculated as the ratio of the swollen gel mass (ms) to that of dry polymer after lyophilization (md). Polymer fraction of hydrogels in the equilibrium sw ollen state was determined using equation 1 : where v-i is the specific volume of water (^=1 mL / g) and v2is the specific volume of polyacrylamide (v2=0.768 mL / g)

[0179] To investigate the rate of diffusion, diffusive permeability of a small molecule fluorescent tracer (calcein, M = 622.5 Da) w as quantified for DLP- and CLEAR-printed hydrogels. Briefly, printed hydrogels containing channels (500 mm) were first equilibrated in DI water and positioned on the confocal microscope (Nikon Eclipse Ti2 with AXR scanner equipped with a 2 / objective lens. 4* magnification) such that the entire diameter of the channel was within the field of view. After aligning the sample, any residual liquid was removed, and the channel was perfused with fluorescent dye. Time-lapse imaging was performed (6 min duration, 3 s interval) to track the outward diffusion of dye from the channel and acquired image sets were used to quantify diffusive permeability using a custom MATLAB script.

[0180] Assessment of intensity weighted mean hydrodynamic diameter through dynamic light scattering (DLS)

[0181] The hydrodynamic diameter of polymers within printed hydrogels was characterized by DLS (Malvern ZetaSizer Nano-ZS). Briefly, rectangular hydrogels (1 mm thick) without BisAm crosslinker (W = 2.63, C = 0) were printed (6 s exposure, 100 pm thickness) using DLP or CLEAR (as described earlier) and dissolved in DI water at 15 mg mL’1. After dissolution, the samples were diluted to 0. 1 mg mL’1and filtered through 0.45 pm nylon syringe filters prior to testing. Aqueous solution of acrylamide monomer (1 mg mL’1) was first tested to confirm that monomer does not scatter light and does not contribute to measured signal in samples that may contain unreacted monomer.

[0182] Assessment of molecular weight through gel permeation chromatography (GPC

[0183] The molecular weights of polymers were characterized by triple detection GPC (Viscotek TDA302 and GPCmax) equipped with SB-806, SB-802 HQ columns, refractive index, light scattering, and viscometer detectors. Briefly, rectangular hydrogels (1 mm thick) without BisAm crosslinker (W = 2.63, C = 0) were printed (6 s exposure, 100 pm thickness) using DLP or CLEAR (as described earlier) and dissolved in DI water at 15 mg mL’1. After dissolution, the samples were diluted in 0.1 M NaNCh at a concentration of 0.469 mg mL’1, left to dissolve overnight on a shaker at room temperature and filtered through a 0.45 pm nylon syringe filter prior to testing. The mobile phase w as 0.1 M NaNCh in DI water at a flow rate of 0.7 mL min’1at 35 °C. GPC results were calculated using a dn / dc value of 0. 1650 mL g’1. The GPC system w as calibrated using PEG and Dextran standards before testing and three injections were performed per sample. All GPC characterization was carried out by a third-party contract research organization (PolyAnalytik Inc.. Canada). Atomic force microscopy (AFM) -nanoindentation of printed highly entangled hydrogels

[0184] Highly entangled hydrogels were 3D printed (10 x 2 x 3 mm3) through the DLP or CLEAR process as described earlier and fully swollen in DI water until equilibrium. Swollen hydrogels were soaked in 30% sucrose solution for 6 h, followed by embedding in Tissue- Tek O.C.T (#4583, Avantor) compound within a disposable cryomold and frozen overnight at -20 °C. Frozen samples were cryo-sectioned into 60 mm thick slices using a Cryostat (Leica Biosystems). Sections were hydrated and washed multiple times to remove O.C.T. compound. The sections were physically adhered (with z-x plane facing the cantilever) to a wet PVDF membrane (0.22 mm hydrophilic, #GVWP04700, Sigma) attached to a glass slide. Force map data were acquired on Asylum Research MFP3D-Bio AFM with contact mode cantilever (nominal spring constant = 0.2 N m1. measured spring constant = 0.37 N m1with a 12 mm diameter spherical glass tip). Samples were kept hydrated in water during tests and the immersed cantilever optical lever sensitivity was calibrated in water with a thermal method. Specimens were scanned over a 90 mm x 90 mm area, with 50 points along the sample z-direction by 10 points along the sample x-direction. Due to sample roughness exceeding vertical range of the AFM, some measurement points were unsuccessful, but some full-length z-traces were still acquired. Measurements were made at 2 Hz with 23 nN trigger force. A 2-point Hertz model (for a spherical probe) was applied to the measured forcedisplacement curves to obtain modulus values. Representative heat maps of modulus as a function of z line scan were plotted to visualize the differences between DLP and CLEAR- printed hydrogels.

[0185] 3D printing of highly entangled elastomers

[0186] Hydroxy ethyl acrylate (HEA, 292818), polyethylene glycol diacrylate (PEGDA, M = 250 Da, 0.5 wt.%, 475629), 2,4,6-trimethylbenzoyldiphenyl phosphine oxide (TPO, 0.1 wt.%, TCI), Sudan-I (0.75 mM, 103624) and N.Nc-dmnethy I formamide (DMF, 227056) were used as monomer, crosslinker, photoinitiator, photoabsorber, and solvent, respectively. CLEAR resin included redox initiators compatible with organic solvent, namely: benzoyl peroxide (BPO, 0.3-0.4 wt.%, 517909) and N.N-dimethylaniline (DMA, 8.8 - 12 mM. #515124, Sigma). Solvent free elastomer resins were formulated by dissolving dry solids (TPO, Sudan-I, BPO) directly in HEA. DLP or CLEAR resin was dispensed in the vat and the print was conducted through sequential projection of sliced images (4 s, 20 mW cm2. 100 pm layer thickness). Post-printing, the objects were stored in a nitrogen-purged, sealed container overnight and away from light. To study the effect of post-processing, DLP printed elastomers were additionally exposed to light right after printing through a commercial UV flood cure system (Any cubic) for 20 min. After complete curing, all printed elastomers were rinsed with isopropyl alcohol (IP A) for 30 min on a shaker to remove any unreacted monomer. Elastomers (without any solvent) were then subjected to uniaxial tensile loading (RSA-G2, 0.25 s'1) until fracture to determine tensile modulus, stress-stretch at failure, and work of fracture as described earlier.

[0187] 3D printing of highly entangled adhesive hydrogels

[0188] Acrylamide (Am) and acry lic acid (Ac, #147230, Sigma), N,N -methylene bisacrylamide (Bis Am, 0.05 wt.%), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 0. 1 wt.%), tartrazine (1 mM) and deionized (DI) water were used as monomer, crosslinker, photoinitiator, photoabsorber, and solvent, respectively to form precursor resin. CLEAR resin was formulated with the addition of redox initiators: APS (0.05 wt.%) and TEMED (0.1 wt.%). Hydrogels were CLEAR printed as detailed earlier (6 s, 20 mW cm2). followed bycuring in a sealed, nitrogen-purged container, and allowed to reach equilibrium swelling before testing. All uniaxial tensile testing were performed on rectangular hydrogel strips that were swollen to equilibrium before testing unless noted otherwise. For 3D printing of hydrogels with discrete regions, multi-material printing was undertaken as described earlier using resin containing (i) acrylamide monomer (slippery, non-adhesive) and (ii) acrylamide and acrylic acid monomers (sticky, adhesive). Fabrication of elastomer-hydrogel hybrid for tissue adhesion was also undertaken through multi -material CLEAR printing, wherein the highly entangled elastomer resin w as first used to build a 3D structure while the adhesive hydrogel resin was used for the last few (-400 mm) layers. Adhesion was qualitatively tested in comparison with 3D elastomer structures without adhesive hydrogel layers.

[0189] 3D printing of thiol-ene hydrogels

[0190] Eight-arm polyethylene glycol modified with norbomene (8-arm lOkDa PEG-NB) was synthesized using carbic anhydride. Briefly, 5 g of 8-arm PEG-OH (lOkDa, 0.5 mmol. JenKem), 0.4104 g cis-5-norboenene-endo-2,3-dicarboxylic anhydride (2.5 mmol, carbic anhydride, #247634, Sigma), 61 mg of 4-dimethylaminopyridine (0.5 mmol, DMAP, #D1450, TCI) and 41.5 mL anhydrous THF (#401757, Sigma) were added to a round bottom flask and mixed at 60 °C for 12 hours. Another 0.4104 g of carbic anhydride and 61 mg of DMAP were added, and the reaction was continued for another 24 hours. The product was precipitated in cold diethyl ether, washed, and dried under vacuum. (8-arm 10 kDa PEG-NB, 25 wt.%), crosslinker (dithiothreitol DTT, 1: 1 thiol: norbomene stoichiometry ), photoinitiator (LAP, 0. 1 wt.%), photoabsorber (tartrazine, 1 mM) were used to form precursor resin. In addition to the base formulation, redox initiators [APS (0.05 wt.%), TEMED (0.1 wt.%)] were added to the CLEAR resin. Hy drogels were CLEAR printed as detailed earlier (6 s, 20 mW cm2). followed by curing in a sealed, nitrogen-purged container for 2 hours, and tested without swelling.

[0191] Assessment of tissue adhesion

[0192] Freshly isolated porcine tissues were obtained from Animal Technologies Inc. (USA) and used for all ex vivo tests. The adhesion performance of printed hydrogels to tissues was quantified through measurements of interfacial toughness (or adhesion energy) and shear strength via 180-degree peel test and lap shear tests, respectively. Rectangular (10 x 25 x 1.5 mm3) sheets of highly entangled adhesive hydrogels were 3D printed and swollen before use. Rigid polyethy lene terephthalate (PET) films (Transparency sheets, Apollo Inc.) were applied using Krazy glue (i.e. cyanoacrylate) to act as stiff backings for all hydrogels and tissues. Both hydrogels and tissues were soaked in Dulbecco’s phosphate buffered saline (DPBS) before adhesion. Chitosan solution (1 wt.%, #419419, Sigma) was prepared by dissolving high molecular weight chitosan (0.5 g, >75% deacety lated, M = 310 000 Da to 375 000 Da) in DI water (50 mL), pH adjusted to 4.5 using acetic acid, and stirred overnight. Excess PBS from the hydrogel surface was gently blown with nitrogen, chitosan solution was applied to the surface, hydrogels were placed onto the desired tissue, and slight compression (0.5 kPa to 1 kPa, £ 5 min.) was employed to facilitate attachment.

[0193] Adhered hydrogels were either sprayed with PBS or covered with PBS-soaked towels within a sealed plastic bag to avoid dehydration before testing. The free ends of the tissue and hydrogel were clamped and subjected to unidirectional tensile peeling (RSA-G2 DMA) at a loading rate of 90 mm min'1. Interfacial toughness was calculated as two times the plateau value of the ratio of the force and width, whereas shear strength was estimated as the ratio of maximum force to the adhesion area. For adhesion of 3D patterned hydrogels, chitosan solution was gently brushed on the region of interest and the hydrogel was applied onto tissues. The peeling rate for 3D printed hydrogel ridges and metamaterial tissue adhesives was held constant at 60 mm min'1. To determine integration strength of printed adhesive hydrogels with cartilage, push-out indentation tests were employed. Articular cartilage explants (outer diameter = 6 mm) were harvested from juvenile bovine femoral condyles (Research 87, Boston, MA). The explants were trimmed down to ~2.8 mm thickness using a tissue slicer (Roboz Surgical Instrument Co.). Cartilage annuli were then formed using 4 mm dermal punches to excise the inner cores. Next, adhesive hydrogel discs (4 mm diameter) were CLEAR printed and swollen, and then chitosan solution was applied to the periphery of the discs. These discs were press-fit into the cartilage annuli with slight compression (0.5 kPa to 1 kPa, £ 5 min.) to facilitate attachment. Cartilage integration strength was assessed immediately after press-fitting using a custom 3.8 mm indenter to measure the force required to push the hydrogel out of the cartilage annulus (12 mm min'1). Integration strength was calculated as the ratio of maximum force to the adhered area at the interface (equals product of the height and circumference of the hydrogel disc).

[0194] Cell culture and cytocompatibility

[0195] Human mesenchymal stromal cells (hMSCs, PromoCell) were used for all in vitro cytocompatibility experiments. The culture media was comprised of high-glucose Dulbecco's modified Eagle's medium (#D5796, Thermo Fischer Scientific) supplemented with 10% v / v fetal bovine serum (Gibco), and 1% v / v penicillin-streptomycin (#15070063, Thermo Fischer Scientific). DLP or CLEAR printed highly entangled hydrogel specimens were fabricated as described earlier, rinsed with PBS to remove unreacted resin from the surface of the hydrogels, and incubated in media. Cells were seeded (density: 7500 cells cm2) on 24 well glass bottom dishes (Cellvis), incubated with hydrogel conditioned media and imaged after 24 h. To assess viability, cells were incubated with calcein AM (2 mM), ethidium homodimer-1 (4 mM), and Hoechst 33342 (1 : 1000) at 37 °C for 30 min following manufacturer’s protocol (Live / Dead™ Cytotoxicity Kit, #L3224, Thermo Fisher Scientific). Cell number was calculated based on the total number of nuclei identified through Hoechst 33342 positive stain (binary mask. Otsu threshold) from maximum projection images analyzed on ImageJ (NIH). Cell viability was calculated as the number of live cells divided by the total number of cells within a single maximum projection image.

[0196] Statistical analysis

[0197] All data are reported as mean ± standard deviation with n > 3. All statistics were conducted using Prism 9 (GraphPad Inc., USA). Comparisons between two experimental groups were performed using Student’s / -tests (a = 0.05, significance determined at / ? < 0.05) and comparisons between more than two groups were performed using a one-way analysis of variance (ANOVA) with post hoc Tukey's multiple comparison test. Example 1: Protocol for DLP Printing

[0198] Acrylamide monomer (30 to 80 wt.%) was dissolved in water along with crosslinker (bisacrylamide, 0.001 to 2 wt.%), photoinitiator (LAP, 0.005 to 2 wt.%), and photoabsorber (tartrazine, 0 to 4 mM) to form a resin solution. Redox initiator (ammonium persulfate APS 0.01 to 0.1 wt.% / N,N,N',N'-Tetramethyl ethylenediamine TEMED 0.01 to 0.1 wt.%) was added to the resin right before 3D printing. The resin was dispensed into a PDMS vat, and the layers were crosslinked with light exposure (6 s. 20 mW cm2). The redox initiator concentration can be tuned to allow a print window ranging from 10 minutes to 6 hours and avoid premature resin crosslinking due to redox initiation alone. After print completion, the structures were taken off the build plate and cleaned (either through mechanical agitation, rinsing with hexane / mineral oil, or air-drying) to remove excess / uncured monomer especially for structures containing pores, negative features, hollow channels. The structures were placed in a sealed container (purged with nitrogen) at temperature (18 to 45 deg C) for 15 minutes to 3 hours to achieve complete curing.

[0199] Example 2: Protocol for DLP Printing of bioadhesive materials and adhesion

[0200] Acrylamide and acrylic acid monomer solutions (total monomer concentration of 30 to 80 wt.%, with molar ratios of acry lamide: acrylic acid from 0.1% to 100% ) were dissolved in water along with crosslinker (bisacrylamide, 0.001 to 2 wt.%), photoinitiator (LAP, 0.005 to 2 wt.%). and photoabsorber (tartrazine, 0 to 4 mM) to form a resin solution. Redox initiator (ammonium persulfate APS 0.01 to 0.1 wt .% / N,N,N',N'-Tetramethyl ethylenediamine TEMED 0.01 to 0.1 wt.%) was added to the resin right before 3D printing. The resin was dispensed into a PDMS vat. and the layers were crosslinked with light exposure (6 s, 20 mW cm2). The redox initiator concentration can be tuned to allow a print window ranging from 10 minutes to 6 hours and avoid premature resin crosslinking due to redox initiation alone. After print completion, the structures were taken off the build plate and cleaned (either through mechanical agitation, rinsing with hexane / mineral oil, or airdrying) to remove excess / uncured monomer especially for structures containing pores, negative features, hollow channels. The structures were placed in a sealed container (purged with nitrogen) at temperature (18 to 45 deg C) for 15 minutes to 3 hours to achieve complete curing.

[0201] Example 3: Protocol for adhering bioadhesive materials

[0202] To adhere the bioadhesive material, excess water is removed from the surface of the printed object with an absorbent pad or through gently blowing air or nitrogen gas. The bioadhesive material is then applied to the target region on the tissue, and force is applied (-1-50% compressive strain) for 1 second to 5 minutes, to facilitate adhesion.

[0203] Bioadhesive co-polymer 3D printed objects are inherently adhesive to tissues and organs through hydrogen boding afforded by the carboxylic acid groups on the acrylic acid monomer that is incorporated into the co-polymer during printing. Additionally, bioadhesion can be stabilized by coating the surface of the 3D printed object with a cationic polymer solution (e.g. chitosan solution with concentration ranging from 0.01 wt% to 6 wt%) before adhering the object to tissues. The cationic polymer diffuses into the 3D printed object and once the object is applied to the tissue the cationic polymer also diffuses into the tissue where pH change triggers hydrogen bonding to occur between the cationic polymer and itself and the 3D printed object or tissue. This creates an entangled polymer bridge between the 3D printed object and the tissue. This process was carried out in the subsequent figures.

[0204] Example 4: Protocol printing and adhering materials with spatially patterned adhesion

[0205] To create a patterned adhesion object, CAD files can be generated to make desired geometry. As a proof of concept, a CAD file was created that contains a slab of material with periodic ridges and valleys on one side of the material. The ridges and valley width could range from 0.5 mm to 1000 mm. the height of the ridge can be from 0.5 mm to 5 cm. although this is completely dictated by the 3D printer capabilities and could be smaller or larger depended on resolution and build platform size. To specifically adhere the ridges of the material, chitosan solutions as described above, can be applied to the surface of the ridge or where adhesion is desired.

[0206] Example 5: Combining Light and Dark Polymerization for 3D Printing

[0207] The CLEAR protocol for processing of hydrogels suitable for use in tissue repair, drug deliver}', and biomedical devices is provided herein. As a non-limiting example, acrylamide was chosen as a model monomer and bis-acrylamide at low concentrations as the crosslinker. When formed via casting with very slow reaction rates, hydrogels contain dense entanglements and sparse crosslinks, thereby achieving both high modulus and high toughness (Figs. 9A-9D). For printing, the DLP resin contains a mixture of monomer, crosslinker, photoinitiator, and photoabsorber while the CLEAR resin comprises the same components with the addition of redox initiators before 3D printing (Table 1). ’H-NMR spectra of thin (100 mm) films prepared from the DLP and CLEAR resins in the absence of crosslinker indicate the disappearance of acrylamide and emergence of poly(acrylamide) (-98% conversion) for CLEAR, whereas -95% of the acrylamide remains unreacted for DLP (Fig. ID).

[0208] 5 Table 1: Summary of precursor solutions employed towards fabrication of (i) highly entangled hydrogel, (ii) highly entangled elastomer, and (iii) highly entangled adhesive hydrogel. When measured with FTIR, light exposure of thin films (matching the same energy of 0.2 J cm'2delivered to a single layer during printing), results in nearly 20% conversion for both DLP and CLEAR resins during irradiation (Fig. IE). While conversion remains constant after irradiation for DLP, it steadily increases to 100% for CLEAR as the redox reaction (dark polymerization) completely consumes unreacted monomers. This dark polymerization is also reflected through an increase in storage modulus, G' with elapsed time for CLEAR, compared to DLP wherein G' remains unchanged after light irradiation (Fig. IF).

[0209] DLP and CLEAR hydrogel resins are 3D printed into solid objects and swollen until equilibrium. Hydrogels printed with CLEAR exhibit nearly 2- and 4-fold increases in elastic modulus (Er) and work of fracture (Wf) under tension, respectively, over those printed with DLP. and match properties to those obtained with casting (Fig. 2A-C, Figs. 10A-10B). CLEAR printed hydrogels sustain nominal stresses of up to 250 kPa (which corresponds to a true tensile strength of nearly 1 MPa), whereas DLP printed hydrogels are substantially weaker and fail at low tensile elongation, which is further illustrated through the abi li ty of CLEAR printed hydrogel rings to lift weight at a nominal stress of nearly 200 kPa (Fig. 2D).

[0210] Moreover, the ET and Wf of CLEAR printed hydrogels are independent of the stretch rate over two orders of magnitude, implying near-perfect elasticity (Figs. 1 1A-11C). Theoretical predictions for the elasticity7of CLEAR printed hydrogels suggests 11- to 15-fold higher levels of entanglements relative to covalent crosslinks. CLEAR printed hydrogels also exhibit increased mechanical properties in compression and under shear, as well as increased polymer content (after swelling) (Figs. 12A-12F), slower diffusion of a fluorescent probe (Figs. 13A-13B) relative to DLP, likely a result of increased polymer chain entanglements or effective crosslinks due to improved monomer conversion within the CLEAR hydrogels. When mapped at the nanoscale through atomic force microscopy (AFM). CLEAR-printed hydrogels exhibit a 3.5-fold higher modulus in comparison to those fabricated with DLP (Fig. 2E), which is observed throughout the entire depth of the z-layer, meaning that CLEAR drives the entirety7of the printed part to reach higher conversion and not just the outermost xy- layers. All testing is performed on swollen hydrogels, which better represents the environment in which hydrogels are used, yet this is not always the case in reported hydrogels and can dramatically reduce their mechanical performance (Figs. 14A-14D). Despite being 3D printed and swollen, the ET and Wf of CLEAR printed highly entangled single network hydrogels match or exceed reported single network hydrogels (Fig. 2F, Table 2). Table 2: Comparison of CLEAR-printed highly entangled single network hydrogels with previously reported single network hydrogels.

[0211] PEGDA refers to polyethylene glycol) diacrylate, GelMA refers to gelatin methacryloyl. MeHA and NorHA refer to methacrylate and norbornene modified hyaluronic acid. GH refers to guest-host supramolecular crosslinks, Am denotes acry lamide, PLL refers to poly(L-lysine) diacrylatc as a crosslinker, NaSS refers to sodium p-styrenesulphonate, MPTC refers to 3-(methacryloylamino)propyl- trimethylammonium chloride, CD denotes cyclodextrin (CD) rings. ‘Swollen’ indicates the hydrogels were allowed to reach equilibrium swelling before mechanical testing while ‘as prepared’ refers to hydrogels that were tested without swelling. It is important to note that the comparison is limited to single network hydrogels and does not include composite, phase-separated, dual-crosslinked, or microarchitected hydrogels with hierarchical structures.

[0212] Direct experimental characterization of entanglements at atomistic scale is nontrivial. To indirectly measure this, hydrogels are printed without crosslinkers, where their stability during swelling is due to entanglements. The rate of dissolution is much slower for CLEAR printed hydrogels than for DLP, as enhanced entanglement prolongs dissolution (22) (Figs. 14A-14D). The higher degree of entanglement with CLEAR is also marked by a substantial increase in polymer solution viscosity7and a shift in the crossover frequency (Figs. 15A-15D).

[0213] When measured via gel permeation chromatography and dynamic light scattering, the molecular weight and hydrodynamic radius of polymer chains is similar between DLP and CLEAR (Figs. 16A-16B, Table 3). This further highlights that dense entanglements within CLEAR printed hydrogels may indeed be due to higher polymer concentrations and not chain length. Specifically, increased monomer conversion combined with an unusually low amount of water results in chain crowding within CLEAR printed networks. To ensure limited network defects within CLEAR printed hydrogels that may influence entanglements and mechanics, a degradable crosslinker [N,N'-bis(acryloyl)cystamine] is introduced. In the presence of a reducing agent, the disulfide-based crosslinks degrade, and the network undergoes reverse gelation, indicating the absence of undesired chemical crosslinks between polymer chains (Figs. 17A-17B).

[0214] Table 3: Summary of molecular weights for photopolymers fabricated by dissolving hydrogels (W = 2.63, C = 0) in the absence of crosslinker printed with DLP or CLEAR as measured by gel permeation chromatography.

[0215] 1Mn denotes the number-average molecular weight;2MWindicates the weight-average molecular weight; and3MPrepresents the peak molecular weight. a,b,c indicate three independent samples prepared whereas the reported molecular weight values are average of three injections per sample.

[0216] Lastly, to further illustrate that changes in material properties are due to entanglements and not increased conversion alone, printed layers are either under- or overcured during DLP (Figs. 18A-18H). While CLEAR improves mechanics with under-curing (low conversion), there is no mechanical improvement when CLEAR is used with over- curing (high conversion). Despite high conversion, over-curing fails to improve the work of fracture, likely due to increased initiation points that reduce effective entanglements and would not be feasible for printing of high-resolution structures or negative features (Figs. 18A-18H).

[0217] A comparison was made of CLEAR to DLP printed hydrogels that have undergone traditional post-curing (e.g., light, heat). Although post-curing DLP printed hydrogels with light (Wf = 287 kJ m'3) or heat (Wf = 327 kJ m'3) results in considerable improvements in the work of fracture over DLP alone, hydrogels fabricated with CLEAR (Wf = 542 kJ m'3) exhibit superior values (Figs. 19A-19G). Further, polymer content, modulus, and strength of CLEAR hydrogels outperform these existing methods of post-curing (Figs. 19A-19G). despite similar monomer conversions. Unlike CLEAR, wherein the initiation and propagation of chains occur in a slow, steady manner, light or thermal methods of post-curing are often more rapid, which may lead to kinetic trapping of polymer chains and low conformational entropy that results in a lower work of fracture. The precise control of temperature with thermal curing could potentially overcome this limitation. Additionally, post-curing with light can introduce mechanical heterogeneities due to non-uniform light penetration, while thermal curing can be difficult to adopt for hydrogels due to water evaporation.

[0218] Taken together, the increase in strength of CLEAR printed hydrogels can be attributed to (i) higher polymer content due to enhanced conversion that enables a higher ability to carry load, and (ii) higher density of chain entanglements that function as crosslinks under low strains by transferring tension between chains but slip at high strains to prevent catastrophic failure. There are, however, numerous factors (presence of oxygen, temperature, wait time) that influence the redox reaction within CLEAR (Figs. 20A-20F, Figs. 21 A-21 G) and that this current approach is thus beneficial mainly for systems that use free-radical polymerizations and exhibit low conversion during printing.

[0219] Although there are kinetically driven differences between light and dark polymerization, the latter can occur continuously under ambient conditions (Fig. 22). One challenge is that while the base resin (monomer, crosslinker, photoinitiator, photoabsorber) is shelf-stable for at least 28 days (Figs. 23A-23C), the long-term storage after addition of redox initiators is limited. The pot life (i.e., the operating time window once the redox initiators are added) for the CLEAR resin can be tuned from 20 minutes to greater than 3 hours by decreasing the redox initiator concentration, without changing the resulting mechanical properties (i.e., ET and Wf) (Figs. 24A-24G). Although this presents a drawback to reusing the resin, alternative methods (e.g., freeze-thawing or storage of resin under oxygen to quench the redox reaction) may be adopted to address this limitation (Figs. 25A-25J). Example 6: CLEAR Printing of High Resolution and Diverse 3D Structures

[0220] CLEAR printing can help resolve the mechanical performance-processability conflict of highly entangled hydrogels. Unlike soft or brittle hydrogels that may be challenging to process due to their inability to support their own weight or recovery from the build plate, highly entangled hydrogels can be processed into structurally complex materials. CLEAR allows the generation of varied topologies (e.g., features that mimic trabecular bone, knotted geometries, gyroid with interconnected voids, macroporous lattice structures) or open channels within hydrogels (Fig. 3 A, Figs. 26A-26D). Both DLP and CLEAR achieve high print resolution and fidelity (-400 mm nominal xy- and z- negative feature size, even after swelling) and the dark polymerization does not negatively impact resolution (Figs. 27A- 27D). CLEAR can also be used for multi-material printing to create heterogeneous regions in z- and xy-directions (Fig. 3B), as well as into porous geometries that conform to curved surfaces leading to materials that are ‘stiff-yet-flexible’ (Fig. 3C).

[0221] Further, CLEAR printed hydrogels can be shaped into auxetic structures such as a metamaterial lattice (e.g., octet truss) (Fig. 3D) or a coil (Fig. 3E) that can sustain compressive or tensile loading with minimal hysteresis (demonstrated through the ability’ of the structures to recover their original shape without plastic deformation upon removal of load). Lastly, CLEAR can be modified for a continuous printing approach to further accelerate times for processing (Figs. 28A-28D), motivated by prior advances in lithography printing.

[0222] There is also great interest in the integration of dissimilar printed components into single constructs (e.g., embedding electronic sensors, integrating microfluidic circuits), which is challenging with traditional printing. Here, CLEAR is useful as an annealing technique to assemble 3D printed hydrogel devices without the need for any additional reagents or substrate functionalization (Fig. 3F). The ongoing redox reaction (via CLEAR) results in the formation of an entangled polymer network at the interface when two (or more) hydrogels are brought in contact.

[0223] This approach provides robust mechanical interlocking, resulting in a high integration strength (650 J m‘2) that is similar to the fracture toughness of the hydrogel itself. It is important to note that the integration strength decreases with time prior to contact (between immediate to 4 hours) and the time window to achieve high integration strength is dependent on the concentration of redox initiators and the time required for printing the desired object (Figs. 29A-29B). In one embodiment, an enclosed serpentine reservoir is formed via the annealing of hydrogels with open channels. There are numerous other properties (e.g., ability to suture to tissues, dehydrate and rehydrate, and cytocompatibility) that motivate the use of CLEAR-printed hydrogels in biomedicine (Figs. 30A-30C, Figs. 31A-31C, Figs. 32A-32B, Figs. 33A-33C). The versatility of CLEAR can also be leveraged towards printing of a wide variety' of hydrogel formulations with varying chemical composition (Figs. 34A-34C, Figs. 35A-35I). These span across a range of crosslinker concentrations as well as various types of monomers and crosslinkers to improve the functionality (e.g., mechanical properties, temperature-responsiveness, supramolecular interactions, degradation, antifouling) of highly entangled hydrogels, while simultaneously achieving ET and Wf values as high as 140 kPa and 1.5 MJ m’3, respectively (Figs. 35A-35I, Figs. 36A-36C). Further, the benefit of CLEAR to improve mechanical properties advances beyond highly entangled hydrogels and can be extended to traditional hydrogels formed via chain-growth (e.g., dilute acrylamide solutions) (Figs. 37A-37I) or step-growth (e.g., thiol-norbomene) reactions (Figs. 38A-38D).

[0224] In various embodiments, CLEAR can also be implemented with various non-aqueous polymer chemistries both in the presence or absence of solvent (Figs. 39A-39D, Figs. 40A- 40C). As a non-limiting example, CLEAR can be used in the printing of highly entangled elastomers (Figs. 39A-39D, Table 1). Specifically, hydroxyethyl acrylate is used as a model monomer due to its low vapor pressure and the low glass transition temperature of the resulting polymer. CLEAR printed highly entangled elastomers have a higher ET of 1.9 MPa (2-fold higher) and Wf of 25 MJ m‘3(6.5-fold higher) than those printed with DLP. When DLP is combined with a common post-processing method (i.e., UV flood cure) for printed elastomers, the ET (1.5 MPa) and Wf (10 MJ m"3) values increase, but to levels inferior to CLEAR. Additionally, CLEAR printed elastomers exhibit a nominal strength of 2.5 MPa (corresponding to a true strength of 38.5 MPa), which is greater than DLP printing alone (true strength of 4.9 MPa) or with UV post-curing (true strength of 13.2 MPa).

[0225] Example 7: Spatial Programming of Tissue Adhesion with CLEAR Printing

[0226] Hydrogels are being developed as versatile tissue adhesives due to their tunability in physical properties and ability to deliver therapeutics, yet the adhesion of stiff but brittle hydrogels is difficult as interfacial cracks are prone to cause cohesive failure. Thus, improved material toughness is desired to dissipate mechanical energy' and limit crack propagation. Several approaches (e.g., interpenetrating polymer networks, composite hydrogels of soft and stiff regions) have been described to toughen hydrogels, however, these systems often require multi-step fabrication and exhibit pronounced mechanical hysteresis. Highly entangled hydrogels provide a solution due to their single network design and the ability to 3D print predefined geometries (Fig. 4A). To aid in adhesion through the formation of interfacial bonds, carboxylic acid groups are incorporated within highly entangled hydrogels (Figs. 41A-41C). CLEAR printed highly entangled adhesive hydrogels (1 : 1 molar ratio acrylamide: acrylic acid) exhibit high ET of 220 kPa and Wr of 774 kJ tn'3, nearly 6-fold and 4.6-fold higher, respectively, than those printed with DLP alone (Figs. 42A- 42C), as well as low hysteresis (12.6% at a stretch of 2) (Figs. 43A-43D) and high human mesenchymal stromal cell viability (> 95%) (Figs. 44A-44B). Robust adhesion enabled by physical crosslinking (i.e., hydrogen bonds, electrostatic interactions) is observed when the hydrogels are applied to ex vivo porcine heart tissue and the stability of adhesion is further strengthened through mechanical interlocking with a well-established bridging polymer (e.g., chitosan) (Fig. 45 A-45B). CLEAR printed adhesive hydrogels exhibit high interfacial toughness (Gint averaging from 265 to 895 J m'2) and shear strength (up to 50 kPa) values when applied to a variety of wet porcine tissues (e.g., heart, lung, stomach, small intestine, tendon) ex vivo (Fig. 4C, Figs. 46A-46G, Figs. 47A-47C). For instance, the interfacial toughness (up to 1410 J m'2) attained with CLEAR printed, highly entangled, single network hydrogels is similar to that of cast double-network hydrogels (400 to 1700 J m’2) and is far higher than cast regular, single network hydrogels (10 to 400 J m'2) using the same method of adhesion.

[0227] Tough hydrogels can be rapidly fabricated into desired shapes with features that would be challenging to attain through traditional casting or molding. For example, 3D printing enables the processing of hydrogels into auxetic structures that endow large bending compliance to conform to the curved surfaces of organs (e.g., porcine heart), even in the presence of blood (Fig. 4C). The properties of CLEAR printed adhesive hydrogels also allow for maintained adhesion even under large deformations without debonding or rupture (Figs. 47A-47C), wrapping around intestinal tissue while allowing peristaltic movements (simulated through flow of PBS ex vivo) without constriction (Figs. 48A-48C) and being press-fit into cartilage defects ex vivo with a strong lateral integration of nearly 43 kPa (Figs. 49A-49C). CLEAR printing of open grooves within highly entangled adhesive hydrogels facilitates formation of enclosed channels directly at a tissue interface without leakage, which could be used for local delivery of therapeutics or to deliver reagents to trigger detachment (Fig. 4D). Furthermore, CLEAR enables printing of hydrogels with tunable adhesion realized through 3D ridges where the strength of adhesion is spatially programmed by altering ridge spacing (i.e., increasing ridge spacing by a factor of two reduces the steady-state peeling force from nearly 2.9 N to 1.7 N) (Figs. 50A-50D). Metamaterial patterns (e.g., through inclusion of nonlinear cuts) of adhesion can be used to suppress crack propagation. CLEAR printing of hydrogels with programmed nonlinear cuts allows directional adhesion, where the forward direction entraps the propagating cracks and steers them to the reverse direction to achieve a high, steady force (Fs = 2.2 ± 0.6 N) of adhesion, whereas peeling in the reverse direction requires only 0.4 N (about 5-fold lower) force (Fig. 4E). This approach enhances adhesion and peel force 2-fold as compared to unpattemed hydrogels without altering material composition.

[0228] It can be challenging to tailor the spatial adhesivity of hydrogels to ensure reversibility if desired for certain applications (e.g., removable wound dressings). Although patterning can be achieved with ultrasound, or modification of substrates, these approaches are limited in 3D complexity7, whereas multi-material CLEAR printing is a simple approach to discretize ‘sticky’ (acrylamide and acry lic acid) and ‘slippery7’ (acrylamide) regions in the z- or xy-directions. When applied to tissue, the slippery region within the CLEAR printed patch is unable to adhere, which facilitates easy removal without damage to the underlying substrate, whereas the sticky regions ensure patch bonding to the tissue (Fig. 4F). Multimaterial CLEAR printing can also be used to bond high water content hydrogels to elastomers, which overcomes challenges of casting and molding approaches. Multi-material CLEAR printing also allows the integration of an adhesive hydrogel layer with an elastomer, thereby forming functional hydrogel-elastomer hybrids that are tissue adhesive and overcome challenges with low permeability of bridging polymer into non-swollen elastomers (Fig. 4G, Figs. 51A-51B). These examples demonstrate the untapped potential of 3D printing and CLEAR, which can open avenues for the design of next-generation hydrogel-based tissue adhesives for the sensing, monitoring, and management of human health.

[0229] Example 8: Role of initiator concentration in obtaining effective entanglements

[0230] Highly entangled hydrogels are formed with unusually low amounts of water, crosslinker, and initiator, as well as high monomer concentrations. These formulations result in crowding of formed polymer chains. Within these hydrogels, the crosslinker concentration is kept low to form a network where chain entanglements greatly outnumber chemical crosslinks. Since the crosslinker concentration is low, a low amount of initiator is required to create long chains and form a polymer network with a low fraction of dangling ends. In contrast, a high amount of initiator may result in shorter chains and the formation of a polymer network with a high fraction of dangling ends (Figs. 7A-7H). Although the density of entanglements is nearly identical in these two example polymer networks formed at low and high initiator concentrations, the density’ of effective entanglements that are trapped by the polymer network differs. Entanglements that are trapped by chemical crosslinks (in the case of low initiator concentration) can more effectively bear load. However, entanglements on dangling ends that are not trapped (in the case of high initiator concentrations) can disentangle and may fail to withstand loads under tension.

[0231] Example 9: Estimation of the relative ratio of entanglements to covalent crosslinks within CLEAR printed highly entangled hydrogels

[0232] As a first approximation towards the prediction and mechanistic explanation of the presence of entanglements, rubberlike elasticity theory was utilized previously to predict the modulus of the CLEAR printed highly entangled hydrogels: where, E is the Young’s modulus, u represents Poisson’s ratio (assumed to be 0.5). R is the ideal gas constant (8.314 J mol'1K'1), T is temperature (298.15 K), f is the junction functionality7(assumed to be 4), Nj is the degree of polymerization between junctions, g is the frequency of chain-end defects, ra is the density of polymer (assumed to be 1.302 for polyacrylamide). Mr is the molecular weight of the repeating unit (71.08 g mol'1), fo is the initial polymer volume fraction while fs is the swollen polymer volume fraction. It is assumed that all crosslinkers are incorporated within the polymer network, each crosslinker is an end of four chains, and the network is free of chain-end defects.

[0233] In the case of CLEAR printed highly entangled hydrogels (W = 2.63, C = 3.5 x 1 O'4), the degree of polymerization between junctions (Nj) can be defined as (2C)'1» 1429, while the initial and swollen polymer volume fractions are 0.60 and 0.12. This results in a predicted modulus of 16.7 kPa, whereas the experimentally measured Young’s modulus (via uniaxial stretch) is nearly 222 ± 35 kPa. This suggests that the discrepancy in predicted and measured moduli can be attributed to the contribution of entanglements as crosslinks and that the theoretical degree of polymerization between covalent and physical crosslinks to achieve the measured elastic modulus would be nearly 93 to 128. This estimates that there are roughly 11 to 15-times more entanglements within the network relative to covalent crosslinks. This relative ratio of entanglements to covalent crosslinks is even higher in an alternate formulation (W = 2.63, C = 3.5 x 10'5). In this case, Nj » 14286, whereas the initial and swollen polymer volume fractions are 0.60 and 0.03, which amounts to a predicted modulus of 1.05 kPa. The experimentally measured Young’s modulus is nearly 57 ± 12 kPa. The theoretical degree of polymerization to achieve the measured elastic modulus would be nearly 218 to 335, which suggests roughly 43 to 66-fold more entanglements than covalent crosslinks. Interestingly, for a regular polyacrylamide hydrogel (W = 22, C = 3.5 x 10'4) with an initial polymer volume fraction of 0.15, the predicted elastic modulus is 3.33 kPa, whereas the measured elastic modulus is 2.56 ± 0.97 kPa. The predicted and measured values are in agreement, highlighting the absence of additional crosslinks in the form of entanglements.

[0234] Example 10: Theoretical photoinitiator consumption during DLP and post-processing

[0235] Theoretical consumption was calculated assuming first-order kinetics of the photolysis reaction:

[0236] I and Io are the photoinitiator (LAP) concentrations at time ‘t’ and time t = 0, respectively, f is the quantum yield of LAP (assumed to be unity ), J is the incident light intensity (20 mW cm'2), e is the molar extinction coefficient of LAP at 405 nm (25 M'1cm'1), u is the frequency of light. NA is the Avogadro’s number, and h is the Planck constant. Solving this, k = 3.893 x 10'3s'1. In an ideal case without light attenuation or scattering and considering 6 s light exposure per layer during DLP, I / Io = 0.977, suggesting that roughly 2.3% of the photoinitiator undergoes decomposition. On the contrary, in the case of traditional casting of hydrogels, the precursor solution is typically exposed to light for at least 300 s. This suggests I / Io = 0.311 and about 68.9% of the photoinitiator gets consumed. For a solution at a specific photoinitiator concentration, constant light exposure for a long period of time (e.g., in the case of casting or light-based post-curing) results in a higher level of photoinitiator decomposition and excessive initiation events which can adversely affect polymer chain lengths when compared to shorter exposure times.

[0237] Example 11: Influence of various processing parameters on CLEAR-printed hydrogel properties

[0238] It is important to understand the various parameters that can be altered during the CLEAR printing process and how they influence final material properties. First, the presence of oxygen (as compared to a nitrogen-purged environment) during the curing of the printed part has an adverse effect on ET and Wr of CLEAR printed hydrogels, whereas there is no significant impact of temperature (within the range of 4 to 37 °C) on hydrogel mechanics (Figs. 20A-20F). This is due to the influence of oxygen on the radical polymerization during the redox initiation. Next, the introduction of a wait period (between the addition of redox initiators to the resin and light exposure during 3D printing) also does not negatively impact the mechanical performance of CLEAR printed hydrogels (Figs. 21 A-21G). This finding is dependent on the redox initiator concentration, which allows for tens of minutes at a high concentration and even several hours at a low concentration without changes to the CLEAR- printed hydrogel properties. This will greatly expand the working times after resin mixing, to support various processing scenarios during manufacturing. Finally, redox initiators alone do not undergo initiation upon light irradiation, highlighting the kinetically driven differences between the light and dark polymerizations within CLEAR (Fig. 22).

[0239] Example 12: Potential uses of CLEAR-printed highly entangled hydrogels in biomedicine

[0240] There are a wide range of uses for CLEAR-printed hydrogels in biomedical applications based on their properties. For example, the ability of the hydrogels to suppress crack growth allows them to be sutured to tissues (e.g., ex vivo porcine skin) and resistant to tear when subjected to tensile deformation (Figs. 30A-30C). This is challenging for most hydrogels used in biomedicine. Further, macroporous CLEAR printed hydrogel scaffolds / patches can be dehydrated (air- or freeze-dried) and rehydrated without negatively impacting their mechanical performance, which is important for storage and long shelf-lives (Figs. 31 A-31 C). With regards to cytotoxicity, CLEAR printed hydrogel -conditioned media supports the high viability (> 95%) of human mesenchymal stromal cells (hMSCs) after 24 h culture, comparable to control media, when tested immediately after printing (Figs. 32A- 32B). This is in contrast to conditioned media from DLP printed hydrogels when tested immediately after printing, which results in low cell viability (< 10%), likely due to leaching of unreacted monomer, crosslinker, or photoinitiator which are know n to be cytotoxic. CLEAR printed hydrogels also retain their excellent mechanical properties when swollen in phosphate buffered saline (PBS) or Dulbecco's modified Eagle’s medium (DMEM), highlighting their stability under physiological pH and osmolarity (Figs. 33A-33C).

[0241] Example 13: Varying type of monomer and crosslinker to form highly entangled hydrogels with enhanced functionality

[0242] Polyacrylamide hydrogels were primarily used to illustrate the CLEAR printing process; however, it is useful to introduce other chemistries within the networks to expand their functionality' and use. To illustrate this, CLEAR technology was assessed to see if similarly impart improved mechanical properties to other highly entangled hydrogel formulations. A wide range of C and fixed I were explored to obtain CLEAR printed hydrogels with modulus spanning from 56 to 291 kPa and work of fracture from 0.15 to 1.64 MJ m'?(Figs. 35A-35I).

[0243] Furthermore, in an effort to extend the utility’ of CLEAR and improve the functionality of highly entangled hydrogels, a library of different crosslinkers were explored including gelatin methacrylamide (GelMA), methacry late modified hyaluronic acid (MeHA), poly(ethylene glycol) di acrylate (PEGDA) of varying molecular weights, and guest-host inclusion complex as well as a variety of different monomers that include N- isopropylacrylamide (NIP Am), JV-Hydroxyethyl acrylamide (HEA), N- [Tris(hydroxymethyl)methyl]acrylamide (THA), 3-[(3-acrylamidopropyl)dimethylammonio] propane- 1 -sulfonate (DMAPS) (Figs. 35A-35I, Figs. 36A-36C). These stiff yet tough highly entangled hydrogels can be useful in a wide range of applications. For instance, incorporation of GelMA or MeHA can allow enzymatic degradation while PEGDA can allow slow hydrolytic degradation, thereby making the hydrogels suitable for controlled release of therapeutic payload. Further, use of NIP Am can endow hydrogels with temperature responsiveness making them attractive for soft robotics, whereas HEA, THA, and DMAPS can provide anti-fouling capability and low immunogenicity needed for tissue implants.

[0244] The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary' skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.

[0245] Enumerated Embodiments

[0246] The following enumerated embodiments are provided, the numbering of which is not to be construed as conveying importance.

[0247] Embodiment 1 provides a method of printing a three-dimensional (3D) object, the method comprising: providing a container comprising a photocurable resin, wherein the photocurable resin comprises: i) at least one prepolymer, ii) optionally at least one crosslinking agent; iii) at least one photoinitiator; and iv) at least one non-photoresponsive initiator; waiting for a waiting period, wherein during the waiting period the at least one prepolymer polymerizes and the at least one photoinitiator is not activated; exposing at least one layer of the photocurable resin to a photocuring light; and printing the 3D object.

[0248] Embodiment 2 provides the method of embodiment 1, wherein the at least one prepolymer is at least one chosen from acrylamide, methacrylamide, acry lic acid, methacrylic acid, N-isopropylacrylamide, N-isopropylmethacrylamide, 2 -hydroxy ethyl acry late, 2- hydroxy ethyl methacrylate, N-[tris(hydroxymethyl)methyl]acrylamide. acrylic acid N- hydroxysuccinimide ester, methacrylic acid N-hydroxysuccinimide ester, ethyl acrylate, ethyl methacry late, 2-acrylamido-2-methyl-l -propanesulfonic acid, and [2- (methacryloyloxy)ethy l]dimethy l-(3-sulfopropyl)ammonium hydroxide, ethyl vinyl sulfone, and phenyl vinyl sulfone.

[0249] Embodiment 3 provides the method of any one of embodiments 1-2, wherein the at least one crosslinking agent is at least one chosen from N,N'-methylenebis(acrylamide), gelatin methacrylamide, gelatin acrylamide, poly(ethylene glycol) di acrylate. poly(ethylene glycol) dimethacrylate, acrylated hyaluronic acid, methacrylated hyaluronic acid, and tricyclo[5.2. 1.02, 6] decanedimethanol diacrylate.

[0250] Embodiment 4 provides the method of any one of embodiments 1-3, wherein the at least one photoinitiator is at least one chosen from acetophenone, benzophenone, 2- phenylacetophenone, 2,2-dimethoxy-2-phenylacetophenone, Bis(2,4,6-trimethylbenzoyl)- phenylphosphineoxide, 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone. 2-methyl- (4-methylthienyl)-2-morpholinyl-l -propan- 1 -one, Diphenyl(2,4,6- trimethylbenzoyljphosphine oxide. Ethyl (2,4,6-trimethylbenzoyl) phenyl phosphinate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP, TPO, BAPO, Eosin Y, Riboflavin, Ru(bpy)s / SPS, VA-044, VA-086, and 1907.

[0251] Embodiment 5 provides the method of any one of embodiments 1-4, wherein the at least one non-photoresponsive initiator is a redox initiator or a thermal initiator.

[0252] Embodiment 6 provides the method of embodiment 5. wherein the redox initiator is BPO / DMA, tris [4-(diethy laminojpheny 1] amine / bis-(4-t-buty lphenyl)-iodonium hexafluorophosphate, MPP / BPO, or APS / TEMED.

[0253] Embodiment 7 provides the method of any one of embodiments 1-6. wherein the photocurable resin further comprises a photoabsorber.

[0254] Embodiment 8 provides the method of any one of embodiments 1-7, wherein the waiting period is about 1 to about 360 minutes.

[0255] Embodiment 9 provides the method of any one of embodiments 1-8, wherein the exposing is for a period of about 0.5 s to about 30 minutes.

[0256] Embodiment 10 provides the method of any one of embodiments 1-9. wherein the photocuring light has a wavelength of about 350 nm to about 600 nm.

[0257] Embodiment 11 provides the method of any one of embodiments 1-10, wherein the photocuring light has an intensity of about 1 mW / cm2to about 3 W / cm2.

[0258] Embodiment 12 provides the method of any one of embodiments 1-11, wherein the printing does not comprise a flood cure.

[0259] Embodiment 13 provides the method of any one of embodiments 1-12, wherein the exposing comprises digital light processing (DLP).

[0260] Embodiment 14 provides the method of any one of embodiments 1-13, wherein the tensile modulus of the printed 3D object is at least 50% greater than the tensile modulus of a printed 3D object not subjected to the step of waiting.

[0261] Embodiment 15 provides the method of any one of embodiments 1-14, wherein the at least one prepolymer is at least one chosen from alginate, hyaluronic acid, poly(ethylene glycol), gelatin, chitosan, poly vinyl alcohol, polyacrylamide, polyacrylic acid, polyallylamine, dextran, keratin, silk fibroin, collagen, fibrin, elastin, glycogen covalently functionalized by at least one group selected from the group consisting of acrylamide, methacrylamide, acrylic acid, methacrylic acid, N-isopropylacrylamide, N- isopropylmethacrylamide, 2-Hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, N- [Tris(hydroxymethyl)methyl] acrylamide, acrylic acid N-hydroxy succinimide ester, methacrylic acid N-hydroxy succinimide ester, ethyl acrylate, ethyl methacrylate, ethyl vinyl sulfone, phenyl vinyl sulfone, 2-acrylamido-2-methyl-l -propanesulfonic acid, and [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide.

[0262] Embodiment 16 provides the method of any one of embodiments 1-15, wherein the 3D object comprises a hydrogel.

[0263] Embodiment 17 provides the method of any one of embodiments 1-16, wherein printing the 3D object further comprises annealing the printed 3D object to at least a portion of a non-polymeric material.

[0264] Embodiment 18 provides the method of embodiment 17, wherein the non-polymeric material comprises a semi-conductor. Embodiment 19 provides the method of any one of embodiments 1-18, wherein the photocurable resin is substantially free of solvent.

[0265] Embodiment 20 provides a cured object produced by the method of any one of embodiments 1-19. Embodiment 21 provides the cured object of embodiment 20, wherein the object comprises a hydrogel.

[0266] Embodiment 22 provides the cured object of embodiment 21. wherein the hydrogel is bioadhesive.

[0267] Embodiment 23 provides the cured object of any one of embodiments 21-22, wherein the hydrogel is a patterned hydrogel.

[0268] Embodiment 24 provides the cured object of any one of embodiments 20-23, wherein the object comprises a first surface and a second surface, and wherein the first surface requires a larger peeling force to remove the object from a surface than the second surface.

Claims

CLAIMSWhat is claimed is:

1. A method of printing a three-dimensional (3D) object, the method comprising: providing a container comprising a photocurable resin, wherein the photocurable resin comprises: i) at least one prepolymer, ii) optionally at least one crosslinking agent; iii) at least one photoinitiator; and iv) at least one non-photoresponsive initiator; waiting for a waiting period, wherein during the waiting period the at least one prepolymer polymerizes and the at least one photoinitiator is not activated; exposing at least one layer of the photocurable resin to a photocuring light; and printing the 3D object.

2. The method of claim 1 , wherein the at least one prepolymer is at least one chosen from acrylamide, methacrylamide, acrylic acid, methacrylic acid, N-isopropylacrylamide, N- isopropylmethacrylamide, 2-hydroxy ethyl acrylate, 2 -hydroxy ethyl methacry late, N- [tris(hydroxymethyl)methyl] acr lamide, acrylic acid N-hydroxy succinimide ester, methacrylic acid N-hydroxysuccinimide ester, ethyl acrylate, ethyl methacrylate. 2- acrylamido-2-methyl-l -propanesulfonic acid, and [2-(methacryloyloxy)ethyl]dimethyl-(3- sul fopropyljammonium hydroxide, ethyl vinyl sulfone, and phenyl vinyl sulfone.

3. The method of claim 1, wherein the at least one crosslinking agent is at least one chosen from N,N'-methylenebis(acrylamide), gelatin methacrylamide, gelatin acrylamide, poly(ethylene glycol) diacrylate, poly(ethylene glycol) dimethacrylate, acrylated hyaluronic acid, methacrylated hyaluronic acid, and tricyclo[5.

2. 1.026] decanedimethanol diacrylate.

4. The method of claim 1, wherein the at least one photoinitiator is at least one chosen from acetophenone, benzophenone, 2-phenylacetophenone, 2,2-dimethoxy-2- phenylacetophenone, bis(2,4,6-trimethylbenzoyl)-phenylphosphineoxide, 2-hydroxy-4'-(2- hydroxyethoxy)-2-methylpropiophenone, 2-methyl-(4-methylthienyl)-2-morpholinyl-l- propan-l-one, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, ethyl (2,4,6- trimethylbenzoyl) phenyl phosphinate, lithium phenyl-2,4,6-trimethylbenzoylphosphinate,LAP, TPO, BAPO, Eosin Y, Riboflavin, Ru(bpy)3 / SPS, VA-044, VA-086, and 1907.

5. The method of claim 1. wherein the at least one non-photoresponsive initiator is a redox initiator or a thermal initiator.

6. The method of claim 5, wherein the redox initiator is BPOZDMA, tris [4- (diethylamino)phenyl]amine / bis-(4-t-butylphenyl)-iodoni urn hexafluorophosphate.MPP / BPO, or APS / TEMED.

7. ’The method of claim 1, wherein the photocurable resin further comprises a photoabsorber.

8. The method of claim 1, wherein the waiting period is about 1 to about 360 minutes.

9. The method of claim 1, wherein the exposing is for a period of about 0.5 s to about 30 minutes.

10. The method of claim 1, wherein the photocuring light has a wavelength of about 350 nm to about 600 nm.1 1 . The method of claim 1 , wherein the photocuring light has an intensity of about 1 mW / cm2to about 3 W / cm2.

12. The method of claim 1, wherein the printing does not comprise a flood cure.

13. The method of claim 1, wherein the exposing comprises digital light processing (DLP).

14. The method of claim 1, wherein the tensile modulus of the printed 3D object is at least 50% greater than the tensile modulus of a printed 3D object not subjected to the step of waiting.

15. The method of claim 1, wherein the at least one prepolymer is at least one chosen from alginate, hyaluronic acid, polyethylene glycol), gelatin, chitosan, poly vinyl alcohol,polyacrylamide, polyacrylic acid, polyallylamine, dextran, keratin, silk fibroin, collagen, fibrin, elastin, glycogen covalently functionalized by at least one group selected from the group consisting of acrylamide, methacrylamide, acrylic acid, methacrylic acid, N- isopropylacrylamide, N-isopropylmethacrylamide, 2-Hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, N-[Tris(hydroxymethyl)methyl]acrylamide, acrylic acid N-hydroxysuccinimide ester, methacrylic acid N-hydroxysuccinimide ester, ethyl acrylate, ethyl methacrylate, ethyl vinyl sulfone, phenyl vinyl sulfone, 2-acrylamido-2-methyl-l -propanesulfonic acid, and [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide.

16. The method of claim 1, wherein the 3D object comprises a hydrogel.

17. The method of claim 1, wherein printing the 3D object further comprises annealing the printed 3D object to at least a portion of a non-polymeric material.

18. The method of claim 1 , wherein the non-polymeric material comprises a semiconductor.

19. The method of claim 1, wherein the photocurable resin is substantially free of solvent.

20. A cured object produced by the method of claim 1.

21. The cured object of claim 20, wherein the object comprises a hydrogel.

22. The cured object of claim 21, wherein the hydrogel is bioadhesive.

23. The cured object of claim 21, wherein the hydrogel is a patterned hydrogel.

24. The cured object of claim 21, wherein the object comprises a first surface and a second surface, and wherein the first surface requires a larger peeling force to remove the object from a surface than the second surface.

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