(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/019078 Al 4 February 2016 (04.02.2016) P O P C T (51) International Patent Classification: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, C09D 11/04 (2006.01) B33Y 10/00 (201 5.01) HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, A61F 2/00 (2006.01) B33Y 30/00 (201 5.01) KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (21) International Application Number: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, PCT/US20 15/042764 SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (22) International Filing Date: TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. 30 July 2015 (30.07.2015) (84) Designated States (unless otherwise indicated, for every (25) Filing Language: English kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, (26) Publication Language: English TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, (30) Priority Data: TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, 62/030,903 30 July 2014 (30.07.2014) US DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, (71) Applicant: TUFTS UNIVERSITY [US/US]; Ballou Hall, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, Medford, Massachusetts 021 55 (US). GW, KM, ML, MR, NE, SN, TD, TG). (72) Inventors: JOSE, Rodrigo R.; 121A Boston Ave, Med Declarations under Rule 4.17 : ford, Massachusetts 02 155 (US). OMENETTO, Fiorenzo; — as to applicant's entitlement to apply for and be granted a 503 Concord Ave, Lexington, Massachusetts 02421 (US). patent (Rule 4.1 7(H)) KAPLAN, David; 46 Pond Street, Concord, Massachu setts 01742 (US). — as to the applicant's entitlement to claim the priority of the earlier application (Rule 4.1 7(in)) (74) Agents: CAHILL, John J. et al; Choate, Hall & Stewart LLP, Two International Place, Boston, Massachusetts Published: 021 10 (US). — with international search report (Art. 21(3)) (81) Designated States (unless otherwise indicated, for every — before the expiration of the time limit for amending the kind of national protection available): AE, AG, AL, AM, claims and to be republished in the event of receipt of AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, amendments (Rule 48.2(h)) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, 00 o © v © (54) Title: THREE DIMENSIONAL PRINTING OF BIO-INK COMPOSITIONS (57) Abstract: 3D printing of biopolymer-based inks provides for manufacturing a broad range of products with desirable p ope ties. A print nozzle may be charged to form a cone-shaped ink droplet to result in increased resolution, more reliable contact with ir- regular surfaces, and a mechanism to control contacting the ink to the print surface. THREE DIMENSIONAL PRINTING OF BIO-INK COMPOSITIONS GOVERNMENT SUPPORT [0001] This invention was made with government support under grant no. 3P4 1EB002520-09S 1, awarded by the National Institutes of Health. The United States Government has certain rights in the invention. RELATED APPLICATIONS [0002] This international patent application claims the benefit of priority under 35 U.S.C. 119(e) of U.S. provisional patent application No. 62/030,903, filed July 30, 2014, entitled "THREE DIMENSIONAL PRINTING OF ΒΙΟ -ΓΝΚ COMPOSITIONS", the contents of which is hereby incorporated by reference in its entirety herein. BACKGROUND [0003] Three-dimensional printing is a type of computer-based printing that creates a three-dimensional object by progressively depositing material onto a substrate (i.e., a printable surface). The concept of three-dimensional printing has been around for over thirty years, but availability of the technology has been limited commercially until the last several years. In many current three-dimensional printing systems, an ink-jet-type printer is used to serially print a material such as a thermoplastic, a metal alloy, or a plaster as layers of particles or three- dimensional dots on the substrate. Computer-control of the location and number of such layers can direct so-called "additive manufacturing" of a designed article. SUMMARY [0004] The present invention encompasses a recognition that certain biological compositions are particularly suitable for use as inks in printing technologies (e.g., ink-jet and/or 3D printing technologies), and can be valuably employed to generate biocompatible three dimensional ("3D") structures with surprising and beneficial attributes (e.g., structural and/or physical properties). [0005] The present invention provides, among other things, certain biologically-based ink compositions (herein, "bio-ink compositions"), as well as articles and/or devices that are engineered and fabricated from such compositions. In certain embodiments, provided bio-ink compositions are self-curing. In certain embodiments, provided bio-ink compositions are substantially free of organic solvent. In some embodiments, provided bio-ink compositions are characterized in that, upon printing, they cure to form a crystallized layer that is substantially insoluble in water so that the crystallized layers do not dissolve, denature, and/or decompose when exposed to subsequent printed layers. Thus, in many embodiments, provided bio-ink compositions display material and/or chemical features that are suitable for use as 3D-printable inks. [0006] Implementations of the invention are useful for a wide range of applications, including but not limited to: medical/surgical devices, imaging, optoelectronics, photonics, therapeutics, biomedical and tissue engineering, synthetic biology, drug delivery, and a variety of consumer products. The present invention also provides methods of preparing bio-ink compositions, methods of printing, and improved printing apparatus. [0007] In some embodiments, bio-ink compositions for use in accordance with the present invention are printed, extruded, and/or deposited on a surface. In accordance with some embodiments, micro-scale, nano-scale, and pico-scale level printed structures are fabricated on the surface of a printable substrate from certain bio-ink compositions disclosed herein. [0008] In some embodiments, when printed, extruded, and/or deposited bio-ink compositions form crystallized layers. In some embodiments, crystallized layers of bio-ink compositions are defined by a repeating secondary structure, such as an alpha-helix or a beta- sheet and/or hydrogen bonding. [0009] In some embodiments, such printed structures include two-dimensional ("2D") structures. [0010] In some embodiments, bio-ink compositions are characterized in that when formed, resultant crystallized layers are substantially insoluble. In some embodiments, substantially insoluble layers do not dissolve, degrade, denature, and/or decompose when exposed to solvents or additional printed layers. In some embodiments, substantially insoluble layers do not dissolve, degrade, denature, and/or decompose once transferred physiological environments, simulated physiological environments, or completely submersed in solvent, for example water/phosphate buffered saline (PBS). [0011] In some embodiments, 3D structures form when layers of bio-ink compositions ink are printed, extruded, and/or deposited atop previous layers. In some embodiments, printable bio-ink compositions for use in accordance with the present invention form 3D structures when individual layers are serially printed, extruded, and/or deposited on a printable substrate and without a need to machine, mill, or mold patterns in solid materials to form such 3D structures. [0012] In some embodiments, bio-ink compositions for use in accordance with the present invention self-cure. In some embodiments, bio-ink compositions that self-cure do not require damaging cure mechanisms yet produce robust structures. In some embodiments, bio- ink compositions substantially concurrently self-cure upon printing, extruding, and/or depositing on a printable surface. In some embodiments, a short drying and/or curing time occurs after printing, extruding, and/or depositing of a bio-ink composition. In some embodiments, a short drying and/or curing time occurs between printing of subsequent layers. In some embodiments, a short drying and/or curing time is in a range between about 0.1 seconds and about 600 seconds. In some embodiments, drying time is dependent on a layer thickness. In some embodiments, drying time is dependent on a volume of ink. In some embodiments, drying time is dependent on environmental factors. In some embodiments, environmental factors include, for example, temperature and/or humidity. [0013] In some embodiments, bio-ink compositions for use in accordance with the present invention that are printed, extruded, and/or deposited generate 3D structures that possess more consistent geometry and more regular features, including sharp angles and clean edges. In some embodiments, 3D structures formed from bio-ink compositions for use in accordance with the present invention have consistent geometry and/or more regular features that are more easily achievable and can be maintained during exposure to subsequent printings, solvents, and/or physiological environments. [0014] In some embodiments, bio-ink compositions are characterized in that when formed, resultant crystallized layers are partially soluble when exposed to solvents or additional printed layers. In