Clotting cytogel

Abstract

There is provided a cytogel for tissue regeneration such as blood clotting, bone regeneration or cartilage regeneration. The cytogel has cells functionalized with a first click chemistry group and polymers functionalized with a second click chemistry group. One of the first click chemistry group or the second click chemistry group comprises a cycloalkene, a heterocycloalkene, a cycloalkyne or a heterocycloalkyne and the other comprises an azide, a tetrazine, a triazine, a pyridazine, or a nitrone. The first click chemistry group and the second click chemistry group react to form a covalent link between the cells and the polymers.

Description

CLOTTING CYTOGELCROSS-REFERENCE TO A RELATED APPLICATION

[0001] This disclosure claims priority from U. S. provisional application number 63 / 584,306 filed on November 13, 2023 which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to the field of blood clotting, and more specifically clotting with a cytogel that includes a combination of cells and polymers.BACKGROUND OF THE ART

[0003] Blood clots are essential for hemostasis and regeneration. Unfortunately, naturally occurring clotting is slow to form and mechanically weak, due to a high content of mechanically ineffective cells, low structural polymer content, and complex coagulation cascades. On the other hand, conventional strategies focused on polymer networks are inapplicable to highly cellularized materials like blood clots. These limitations pose a significant risk for life-threatening hemorrhage and limit potential applications of blood clots. Overcoming these limitations is therefore desirable to provide a blood clot with improved mechanical properties.SUMMARY

[0004] In one aspect, there is provided a cytogel comprising: cells functionalized with a first click chemistry group; and polymers functionalized with a second click chemistry group; where one of the first click chemistry group or the second click chemistry group comprises a cycloalkene, a heterocycloalkene, a cycloalkyne or a heterocycloalkyne and the other comprises an azide, a tetrazine, a triazine, a pyridazine, or a nitrone; and where the first click chemistry group and the second click chemistry group react to form a covalent link between the cells and the polymers. In some embodiments, the cytogel comprises between 2 and 50 % by volume of the cells, preferably from 20 to 40 % by volume of the cells. In some embodiments, the cytogel comprises from 1 to 2 wt. % of the polymers. The cells can be one or more of red blood cells (RBC), fibroblasts, adipose- derived stem cells, and microalgae cells. The polymers can be one or more of hyaluronic acid, fibrin, alginate, chitosan, polyacrylic acid, polyethylene glycol, polyacrylamide, poly(vinylpyrrolidone), polysialic acid, polyvinyl alcohol, gelatin, albumin, dextran, agarose. In some embodiments, the cells are red blood cells. In some embodiments, the polymer is hyaluronic acid. In some embodiments, the polymers have a size of from 50 to 250 nm. In someembodiments, the cycloalkene is a C3-C10 cycloalkene. In some embodiments, the heterocycloalkene is a 3 to 10 membered ring and the heteroatoms are selected from N, O and S. In some embodiments, the cycloalkyne is a C7-C9 cycloalkyne. In some embodiments, the heterocycloalkyne is a 7 to 9 membered ring and the heteroatoms are selected from N, O, and S. In some embodiments, one of the first click chemistry group or the second click chemistry group comprises the cycloalkene or the heterocycloalkene and the other comprises the tetrazine, the triazine or the pyridazine. In some embodiments, one of the one of the first click chemistry group or the second click chemistry group comprises the cycloalkyne and the other comprises the azide, the tetrazine, the triazine, the pyridazine or the nitrone.

[0005] In a further aspect, there is provided the use of the cytogel as defined herein for bleeding control, tissue repair or tissue regeneration or for use as an embolic agent.

[0006] In yet a further aspect, there is provided a method for controlling bleeding of a tissue, repairing the tissue, or regenerating the tissue of a subject in need thereof, the method comprising applying the cytogel as defined herein on the tissue.

[0007] In still a further aspect, there is provided a method of producing a cytogel, the method comprising: providing cells functionalized with a first click chemistry group and polymers functionalized with a second click chemistry group; and contacting the cells and the polymers to allow a click chemistry reaction to occur between the first click chemistry group and the second click chemistry group to produce a covalent bond between the cells and the polymers; where one of the first click chemistry group or the second click chemistry group comprises a cycloalkene, a heterocycloalkene, a cycloalkyne or a heterocycloalkyne and the other comprises an azide, a tetrazine, a triazine, a pyridazine, or a nitrone. The providing step can include functionalized the cells with the first click chemistry group and functionalizing the polymers with the second click chemistry group. In some embodiments, the cycloalkene group is trans-cyclooctene. In some embodiments cycloalkyne group is selected from

[0008] In some embodiments, the heterocycloalkyne group is selected from

[0009] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1A is a schematic of a cytogel formed via direct cell-crosslinking.

[0011] FIG. 1 B is a schematic of a cell aggregate culture.

[0012] FIG. 1C is a schematic of a cell encapsulation in hydrogel.

[0013] FIG. 2A is a schematic of an agglutination of negatively charged cells, carrying polysaccharides on their surface, and positively charged polymers through electrostatic interactions.

[0014] FIG. 2B is a schematic showing hydrophobic interactions of polymers carrying pendent hydrophobic groups, these hydrophobic groups insert into the lipid bilayers of cell membranes, thereby connecting cells.

[0015] FIG. 2C is a schematic of a receptor-ligand recognition with polymers decorated with peptide ligands (e.g., RGD).

[0016] FIG. 2D is a schematic of a cell surface-modified with reactive groups (e.g., azide) using metabolic labelling to crosslink polymers carrying complementary reactive groups (e.g., di benzocyclooctyne (DBCO)).

[0017] FIG. 2E is a schematic of cells that are initially modified with a click motif on surface proteins using biocompatible covalent reactions and then bioorthogonally crosslinked by polymer linkers carrying complementary reactive groups (e.g., tetrazine).

[0018] FIG. 3A is a schematic of a modification of red blood cell (RBC) surface with transcyclooctyne (TCO) functional groups through the reaction of NHS ester with cell surface primary amines.

[0019] FIG. 3B is a schematic comparing the formation of a cytogel with the modification of Fig. 3A and a native clotting that naturally occurs in vivo.

[0020] FIG. 3C is a schematic comparing a cytogel and a native clot.

[0021] FIG. 3D is a schematic showing the regeneration in vivo after the formation of a cytogel or a native clot.

[0022] FIG. 4 is a1H nuclear magnetic resonance image spectrum showing the protons attributed to tetrazine (numbered as per scheme 1).

[0023] FIG. 5A is a schematic showing RBC tagged with TCO-linked red fluorescence.

[0024] FIG. 5B is a confocal image showing an even distribution of TCO-linked red fluorescence on the RBC surface (scale bar 10 pm).

[0025] FIG. 6A is confocal image of modified RBCs (RBC-TCO) which emit no fluorescence signals.

[0026] FIG. 6B is a confocal image of native RBC incubated with fluorescent dye (Tetrazine- AF568) which have negligible fluorescence likely due to non-specific adsorption.

[0027] FIG. 7A is a graph showing measurements of fluorescent intensity from modified RBCs incubated at various concentrations of Tetrazine-AF568. The cell number was kept constant across the groups tested.

[0028] FIG. 7B is a graph showing the density of TCO on modified RBCs estimated to be 5.4 x 107per cell using a titration-based method.

[0029] FIG. 7C is a graph showing the assessment of TCO stability on modified RBCs over time. Higher fluorescent intensity indicates a greater degree of TCO retention on the RBC surface.

[0030] FIG. 8A is a graph of a rheological analysis showing the instantaneous gelation of a mixture comprising modified RBCs (RBC-TCO) and polymer linker (hyaluronic acid modified with tetrazine (HA-TZ), 1 w / v%). The resulting RBC gel exhibited a significantly higher storage modulus (G') compared to the loss modulus (G"), while the mixture of native RBCs and polymer behaved as a viscous liquid.

[0031] FIG. 8B is a graph of the rheological comparison between covalently crosslinked RBC cytogel (2 w / v% HA-TZ) and traditional gel formulations.

[0032] FIG. 9 is a graph showing representative curves of the tangent of the phase angle δ (tan δ) of blood gels formed using different strategies: blood gels formed by electrostatic (chitosan), enhanced electrostatic (polymers consisting of cationic and adjacent aromatic residues), and hydrophobic interactions (hydrophobically modified chitosan), and the RBC cytogels by formed covalent crosslinking as per the present disclosure which have a tan δ of around 0.1.

[0033] FIG. 10A is a photograph showing that the engineering blood clot (EBC) of the present disclosure effectively seals injured liver and halts bleeding

[0034] FIG. 10B is a photograph exhibiting the stretchability and adhesiveness of the EBC.

[0035] FIG. 11A is a photograph of a RBC cytogel soaked with deionized water.

[0036] FIG. 11 B is a photograph of the RBC cytogel of Fig. 11 A where the cells were lysed.

[0037] FIG. 12A is a schematic showing the formation of cytogels with a long-range of continuous cell-crosslinked network.

[0038] FIG. 12B is a graph showing the effect of RBC volume ratio on the shear moduli of cytogels. Higher volume ratios lead to larger shear moduli, which peak at a volume ratio of 33%.

[0039] FIG. 13A is a graph showing the effect of the hydrodynamic size of the polymer linker. S, M, L and XL represent HA-TZ with small, medium, large and extra-large hydrodynamic diameters, respectively. Larger polymer linkers facilitate crosslinking between adjacent cells.

[0040] FIG. 13B is a graph showing representative curves of different HA-TZ measured by dynamic laser scattering (DLS). S, M, L and XL represent the HA-TZ with small, medium, large and extra-large hydrodynamic diameters.

[0041] FIG. 13C is a bar graph showing the hydrodynamic diameter of different HA-TZ measured by DLS. Data are presented as mean ± standard deviation (SD) (n = 4).

[0042] FIG. 13D is a graph showing the storage and loss modulus (top and bottom curves respectively) for small HA-TZ of Fig. 13B.

[0043] FIG. 13E is a graph showing the storage and loss modulus (top and bottom curves respectively) for medium HA-TZ of Fig. 13B.

[0044] FIG. 13F is a graph showing the storage and loss modulus (top and bottom curves respectively) for large HA-TZ of Fig. 13B.

[0045] FIG. 13G is a photograph of a small HA-TZ of Fig. 13B which fails to crosslink RBCs into cytogels.

[0046] FIG. 13H is a photograph of a medium HA-TZ of Fig. 13B which fails to crosslink RBCs into cytogels.

[0047] FIG. 131 is a photograph of a large HA-TZ of Fig. 13B which crosslinks RBCs for form cytogels.

[0048] FIG. 14A is a graph showing the effect of HA-TZ concentration on the shear moduli of cytogels. Higher polymer concentrations lead to larger shear moduli.

[0049] FIG. 14B is a graph showing the effect of tetrazine ratio of HA-TZ on moduli of RBC cytogels. Shear moduli of RBC cytogels increases with the increasing tetrazine ratio when the RBC ratio is fixed at 33% and HA-TZ concentration is fixed at 1%.

[0050] FIG. 15A is a graph showing the hydrodynamic diameter of various polymer linkers measured using dynamic light scattering. PEG, Alg, Chi, PAAc and HA denote polyethylene glycol, alginate, chitosan, polyacrylic acid and hyaluronic acid, respectively. Data are presented as mean ± SD (n = 3).

[0051] FIG. 15B is a bar graph showing the shear moduli of the cytogels formed using polymer linkers as per Fig. 15A (G’ right bar and G” left bar for each polymer).

[0052] FIG. 16A is a photograph showing the cell pellet centrifuged during the formation of a living fibroblast cytogel with high cell density (~5 x 107cells / mL).

[0053] FIG. 16B is a photograph showing the crosslinking the human vocal fold fibroblasts (hVFFs).

[0054] FIG. 16C is a photograph showing the resulting gel formed.

[0055] FIG. 16D is a graph showing a rheological analysis showing immediate gel formation upon mixing modified hVFFs and polymer solution, with a storage modulus of 60 Pa and a loss modulus of 30 Pa (top curve storage modulus and bottom curve loss modulus).

[0056] FIG. 16E is a confocal image of LIVE / DEAD staining of cells within the 3D fibroblast cytogels.

[0057] FIG. 16F is confocal image of fibroblast gels treated with 70% ethanol used as a positive control for Fig. 16E.

[0058] FIG. 16G is a graph showing the viability assessment showing that hVFFs exhibit 89% viability within the fibroblast cytogels. Data are presented as mean ± SD (n = 5).

[0059] FIG. 17A is a photograph showing living adipose-derived stem cell (ASC) gel with high cell density (~4 x 106cells / mL) formed by cell-crosslinking in the tissue culture plate.

[0060] FIG. 17B is a confocal image of high-density cells which remained alive in the 3D ASC gels as evidenced by LIVE / DEAD assay.

[0061] FIG. 17C is a graph showing the viability of ASC (83% in the cell-crosslinked gels). Data are presented as mean ± SD (n = 3).

[0062] FIG. 18A is a photograph showing a microalgae cell pellet after centrifuge.

[0063] FIG. 18B is a photograph showing living microalgae gels with a high cell content of approximately 33%.

[0064] FIG. 18C is a graph showing a rheological analysis showing the rapid formation of microalgae cytogels through click clotting strategy, resulting in a storage modulus of 120 Pa and a loss modulus of 70 Pa.

[0065] FIG. 19A is a schematic of modified lap shear test performed on RBC cytogels.

[0066] FIG. 19B is a photograph of the test of Fig. 19A.

[0067] FIG. 19C is a graph showing representative force-displacement curves of RBC cytogels with varying cell volume ratios.

[0068] FIG. 19D is a graph showing the fracture energy of RBC cytogels correlates closely with the cell volume ratio, peaking at a volume ratio of 33%.

[0069] FIG. 19E is a graph showing that the fracture energy of RBC cytogels is independent of the concentration of HA-TZ.

[0070] FIG. 19F is a graph showing the effect of shear strain rate on the fracture energy of RBC cytogels. Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by one-way ANOVA with post hoc Tukey tests. The “ns” denotes no significant difference.

[0071] FIG. 19G is a schematic of a cyclic tensile test employed to study the toughening mechanism of RBC cytogels.

[0072] FIG. 19H is a photograph of the test of Fig. 19G.

[0073] FIG. 191 is a schematic of RBC fracture and hemoglobin release during the test of Fig.19G.

[0074] FIG. 19J is a graph showing RBC cytogels subject to two cycles of loading and unloading at varying maximum stretch. The hysteresis observed indicates the energy dissipation of RBC cytogels. The amount of released hemoglobin (Hgb) from stretched RBC cytogels is proportional to the stretch ratio, indicating that RBCs rupture and dissipate energy. Data were normalized to unstretched samples.

[0075] FIG. 19K is a graph showing the hemoglobin release in function of the maximum stretch.

[0076] FIG. 20A is schematic of the geometry of a sample used for finite element modelling simulation.

[0077] FIG. 20B is a graph showing the fracture of the RBC cytogel which is characterized by a bi-linear cohesive zone mode, characterized by parameters: cohesive strength σmax, failure displacement δf, and intrinsic cohesive energy r0.

[0078] FIG. 20C is a graph showing a material point of the RBC cytogel is subject to loading and unloading, the mechanical work per volume U is defined as the area under the nominal stressstretch curve. The energy dissipation per volume Ud is defined as the shaded area enclosed by the loading-unloading curve.

[0079] FIG. 20D is a graph showing the hysteresis ratio h = U / Ud plotted as functions of U for the RBC gel, tough gel, and porcine liver.

[0080] FIG. 21 A is a graph showing a finite element method (FEM) simulation of the fracture behavior of RBC cytogels during the modified lap-shear test, which demonstrate the energy dissipation of specimens in models with and without the Mullins effect, respectively.

[0081] FIG. 21 B is a graph showing finite element results with and without Mullins effect.

[0082] FIG. 22A is a schematic of EBC clotting with TCO-tetrazine.

[0083] FIG. 22B is a schematic of EBC clotting with DBCO-azide.

[0084] FIG. 22C is a schematic of fibrin clotting.

[0085] FIG. 22D is a graph of coagulation profiles of EBC using TCO-tetrazine ligation, measured by rheology. EBC rapidly forms a clot through “click clotting”, with the storage modulus continually increasing until reaching a plateau due to fibrin assembly. In contrast, native blood clots (NBC) form slowly.

[0086] FIG. 22E is a graph showing a comparison of clotting times between EBC and NBC.

[0087] FIG. 22F is a graph showing the gelation kinetics of RBC cytogel formed via DBCO-Azide reaction.

[0088] FIG. 23A is a confocal image of EBC showing stained RBCs and stained HA-TZ.

[0089] FIG. 23B is a confocal image showing stained fibrin.

[0090] FIG. 23C is a confocal image combining Figs. 23A-23B.

[0091] FIG. 24A is a confocal image of reconstructed 3D confocal images of EBC stained forRBC.

[0092] FIG. 24B is a confocal image of stained fibrin in the EBC of Fig. 24A.

[0093] FIG. 24C is a confocal image of stained HA-TZ in the EBC of Fig. 24A.

[0094] FIG. 24D is a confocal image showing the staining combination of Figs. 24A-24C.

[0095] FIG. 25A is a scanning electron microscopy (SEM) image showing RBCs linked together within EBC. Crosslinked RBCs deform under local stress. Scale bar 1 pm.

[0096] FIG. 25B is a SEM image of EBC with modified RBCs.

[0097] FIG. 25C is a SEM image close up of Fig. 25B.

[0098] FIG. 25D is a SEM image of EBC with unmodified RBCs.

[0099] FIG. 25E is a SEM close up of Fig. 25D.

[0100] FIG. 25F is a SEM image of a NBC.

[0101] FIG. 25G is a SEM image of a EBC cytogel.

[0102] FIG. 26A is a graph showing a representative force-displacement curves obtained from modified lap-shear tests, comparing NBC, RBC cytogel, EBC, EBCh (containing fibrin at physiological level) and a commercial hemostatic matrix, Floseal™.

[0103] FIG. 26B is a bar graph showing the fracture energy of NBC, EBC, RBC cytogel, and Floseal™.

[0104] FIG. 26C is a schematic illustration of the setup used to measure burst pressure.

[0105] FIG. 26D is a close-up schematic of Fig. 26C.

[0106] FIG. 26E is a photograph of an underformed EBC under no pressure.

[0107] FIG. 26F is a photograph of a highly deformed EBC, sustaining high pressure before burst.

[0108] FIG. 26G is a bar graph of the burst pressure of NBC, EBC, RBC cytogel, and Floseal™.

[0109] FIG. 26H is a graph showing the representative force-displacement curves of EBC and NBC under 180-degree peeling test.

[0110] FIG. 26I is a photograph showing NBC adhered to porcine skin for the peeling test of Fig. 26H.

[0111] FIG. 26J is a photograph showing EBC adhered to porcine skin for the peeling test of Fig. 26H.

[0112] FIG. 26K is a photograph showing EBCh (containing physiological fibrin content) adhered on porcine skin for the peeling test of Fig. 26H.

[0113] FIG. 26L is a bar graph showing the adhesion energy of NBC, EBC and EBCh.

[0114] FIG. 27A is photograph of a no self-supporting gel is formed upon mixing human whole blood with 2% chitosan solution at varied ratios (blood:chitosan = 1 :1).

[0115] FIG. 27B is a photograph showing aggregates are formed in the gel of Fig. 27A due to electrostatic interactions between blood cells and chitosan, but significant hemolysis occurs as well.

[0116] FIG. 27C is a photograph of the failed clotting because too much chitosan impairs blood clotting with the addition of CaCl2, which is possibly because the chitosan solution changes the pH of whole blood.

[0117] FIG. 27D is a photograph of a no self-supporting gel is formed upon mixing human whole blood with 2% chitosan solution at varied ratios (blood:chitosan = 3:1).

[0118] FIG. 27E is a photograph showing aggregates are formed in the gel of Fig. 27D due to electrostatic interactions between blood cells and chitosan, but significant hemolysis occurs as well.

[0119] FIG. 27F is a photograph of the clotting of the gel of Fig. 27D with the addition of CaCh.

[0120] FIG. 27G is a graph showing a force-displacement curve of whole blood / chitosan (3:1) clots under lap-shear test.

[0121] FIG. 27H is a graph showing the calculated fracture energy of whole blood / chitosan (3:1) clots under lap-shear test. Data are presented as mean ± SD (n = 3).

[0122] FIG. 28A is confocal image of cytocombatibility test using in vitro culture of human vocal fold fibroblasts (hVFFs) without the supplement of Fetal Bovine Serum (FBS) and without EBC.

[0123] FIG. 28B is confocal image of cytocombatibility test using in vitro culture of hVFFs without FBS and with EBC.

[0124] FIG. 28C is confocal image of cytocombatibility test using in vitro culture of hVFFs with FBS and without EBC.

[0125] FIG. 28D is confocal image of cytocombatibility test using in vitro culture of hVFFs with FBS and with EBC.

[0126] FIG. 28E is a graph showing the viability analysis which indicates that cells in all groups exhibited a viability of approximately 100% (left bar graph control and right bar graph EBC).

[0127] FIG. 28F is a graph showing that cell density in the EBC group is significantly higher than that in the control when no FBS added to the media. Data are presented as mean ± SD (n = 10). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. ***p < 0.001 , ns denotes no significant difference (left bar graph control and right bar graph EBC).

[0128] FIG. 29A is a graph showing the results of a hemolysis test, a comparison of hemolysis of pristine and modified RBC resulted by the present polymer linkers and other materials, including chitosan, PBS and DI water. No hemolysis is found with the polymer linkers, while chitosan causes significant hemolysis. Data are presented as mean ± SD (n = 3).

[0129] FIG. 29B is a photograph of the tubes used for the test of Fig. 29A.

[0130] FIG. 30A is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of platelets (gpllb / gpllla). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0131] FIG. 30B is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of platelets (p-selectin). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0132] FIG. 30C is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of monocytes (CD86). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0133] FIG. 30D is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry toassess the response of monocytes (CD11 b). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0134] FIG. 30E is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of monocytes (MHCII). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0135] FIG. 30F is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of granulocytes (CD86). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0136] FIG. 30G is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of granulocytes (CD11b). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0137] FIG. 30H is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of granulocytes (MHCII). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0138] FIG. 30I is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of B lymphocytes (CD86). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0139] FIG. 30J is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry toassess the response of B lymphocytes (MHCII). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0140] FIG. 30K is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of CD4 T cells (CD44). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0141] FIG. 30L is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of CD8 T cells (CD44). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0142] FIG. 30M is a graph showing normal anticoagulated human whole blood was incubated with EBC. The incubated blood cells were then separated and analyzed using flow cytometry to assess the response of Natural Killer T cells (CD16). Data are presented as mean ± SD (n = 3). Statistical significance and P values were determined by two-sided t-test for the comparison between two groups. NS denotes no significant difference.

[0143] FIG. 31 is a schematic illustration of a rat subcutaneous implantation model used to evaluate the biocompatibility and biodegradation of EBC.

[0144] FIG. 32A is a microscopy image of a stained histological section of a tissue implanted with NBC (at day 3).

[0145] FIG. 32B is a microscopy image of the zoom-in view of the black box in Fig. 32A.

[0146] FIG. 32C is a microscopy image of a stained histological section of a tissue implanted with NBC (at day 7).

[0147] FIG. 32D is a microscopy image of the zoom-in view of the black box in Fig. 32C.

[0148] FIG. 32E is a microscopy image of a stained histological section of a tissue implanted with NBC (at day 14).

[0149] FIG. 32F is a microscopy image of the zoom-in view of the black box in Fig. 32E.

[0150] FIG. 32G is a microscopy image of a stained histological section of a tissue implanted with NBC (at day 28).

[0151] FIG. 32H is a microscopy image of the zoom-in view of the black box in Fig. 32G.

[0152] FIG. 33A is a microscopy image of a stained histological section of a tissue implanted with EBC (at day 3).

[0153] FIG. 33B is a microscopy image of the zoom-in view of the black box in Fig. 33A.

[0154] FIG. 33C is a microscopy image of a stained histological section of a tissue implanted with EBC (at day 7).

[0155] FIG. 33D is a microscopy image of the zoom-in view of the black box in Fig. 33C.

[0156] FIG. 33E is a microscopy image of a stained histological section of a tissue implanted with EBC (at day 14).

[0157] FIG. 33F is a microscopy image of the zoom-in view of the black box in Fig. 33E.

[0158] FIG. 33G is a microscopy image of a stained histological section of a tissue implanted with EBC (at day 28).

[0159] FIG. 33H is a microscopy image of the zoom-in view of the black box in Fig. 33G.

[0160] FIG. 34A is a microscopy image of a stained histological section of a tissue implanted with RBC cytogel (at day 3).

[0161] FIG. 34B is a microscopy image of the zoom-in view of the black box in Fig. 34A.

[0162] FIG. 34C is a microscopy image of a stained histological section of a tissue implanted with RBC cytogel (at day 7).

[0163] FIG. 34D is a microscopy image of the zoom-in view of the black box in Fig. 34c.

[0164] FIG. 34E is a microscopy image of a stained histological section of a tissue implanted with RBC cytogel (at day 14).

[0165] FIG. 34F is a microscopy image of the zoom-in view of the black box in Fig. 34E.

[0166] FIG. 34G is a microscopy image of a stained histological section of a tissue implanted with RBC cytogel (at day 28).

[0167] FIG. 34H is a microscopy image of the zoom-in view of the black box in Fig. 34G.

[0168] FIG. 35A is a microscopy image of a stained histological section of a tissue implanted with Floseal™ (at day 3).

[0169] FIG. 35B is a microscopy image of the zoom-in view of the black box in Fig. 35A.

[0170] FIG. 35C is a microscopy image of a stained histological section of a tissue implanted with Floseal™ (at day 7).

[0171] FIG. 35D is a microscopy image of the zoom-in view of the black box in Fig. 35C.

[0172] FIG. 35E is a microscopy image of a stained histological section of a tissue implanted with Floseal™ (at day 14).

[0173] FIG. 35F is a microscopy image of the zoom-in view of the black box in Fig. 35E.

[0174] FIG. 35G is a microscopy image of a stained histological section of a tissue implanted with Floseal™ (at day 28).

[0175] FIG. 35H is a microscopy image of the zoom-in view of the black box in Fig. 35G.

[0176] FIG. 36 is a graph showing the blinded pathological evaluation of the inflammation degree at each time point, graded on a scale: 0 (no inflammation), 1 (very mild), 2 (mild), 3 (moderate), 4 (severe), and 5 (very severe).

[0177] FIG. 37A is a graph showing the biodegradation of the implants over time.

[0178] FIG. 37B is a photograph of the NBC implant and adjacent tissues harvested at day3.

[0179] FIG. 37C is a photograph of the NBC implant and adjacent tissues harvested at day 7.

[0180] FIG. 37D is a photograph of the NBC implant and adjacent tissues harvested at day 14.

[0181] FIG. 37E is a photograph of the NBC implant and adjacent tissues harvested at day 28.

[0182] FIG. 37F is a photograph of the NBC implant and adjacent tissues harvested at day 56.

[0183] FIG. 37G is a photograph of the EBC implant and adjacent tissues harvested at day 3.

[0184] FIG. 37H is a photograph of the EBC implant and adjacent tissues harvested at day 7.

[0185] FIG. 37I is a photograph of the EBC implant and adjacent tissues harvested at day 14.

[0186] FIG. 37J is a photograph of the EBC implant and adjacent tissues harvested at day 28.

[0187] FIG. 37K is a photograph of the EBC implant and adjacent tissues harvested at day 56.

[0188] FIG. 37L is a photograph of the RBC cytogel implant and adjacent tissues harvested at day 3.

[0189] FIG. 37M is a photograph of the RBC cytogel implant and adjacent tissues harvested at day 7.

[0190] FIG. 37N is a photograph of the RBC cytogel implant and adjacent tissues harvested at day 14.

[0191] FIG. 370 is a photograph of the RBC cytogel implant and adjacent tissues harvested at day 28.

[0192] FIG. 37P is a photograph of the RBC gel implant and adjacent tissues harvested at day 56.

[0193] FIG. 37Q is a photograph of the Floseal™ implant and adjacent tissues harvested at day 3.

[0194] FIG. 37R is a photograph of the Floseal™ implant and adjacent tissues harvested at day 7.

[0195] FIG. 37S is a photograph of the Floseal™ implant and adjacent tissues harvested at day 14.

[0196] FIG. 37T is a photograph of the Floseal™ implant and adjacent tissues harvested at day 28.

[0197] FIG. 37U is a photograph of the Floseal™ implant and adjacent tissues harvested at day 56.

[0198] FIG. 38A is an image of the biodistribution of degraded products from EBC containing fluorescently-labelled HA in major organs at day 3 post subcutaneous implantation.

[0199] FIG. 38B is an image of the biodistribution of degraded products from EBC containing fluorescently-labelled HA in major organs at day 7 post subcutaneous implantation.

[0200] FIG. 38C is an image of the biodistribution of degraded products from EBC containing fluorescently-labelled HA in major organs at day 14 post subcutaneous implantation.

[0201] FIG. 38D is an image of the biodistribution of degraded products from EBC containing fluorescently-labelled HA in major organs at day 21 post subcutaneous implantation.

[0202] FIG. 38E is an image of the biodistribution of degraded products from EBC containing fluorescently-labelled HA in major organs at day 28 post subcutaneous implantation.

[0203] FIG. 38F is graph showing the average fluorescent intensity of major organs over time (for each organ at days 3, 7, 14, 21, and 28 post implantation respectively from left to right).

[0204] FIG. 38G is a microscopy image showing a histology staining of the lung.

[0205] FIG. 38H is a microscopy image showing a histology staining of the heart.

[0206] FIG. 38I is a microscopy image showing a histology staining of the liver.

[0207] FIG. 38J is a microscopy image showing a histology staining of the spleen.

[0208] FIG. 38K is a microscopy image showing a histology staining of the kidney.

[0209] FIG. 39A is a photograph of a EBC being ejected through a syringe.

[0210] FIG. 39B is a photograph of the syringe extruded EBC.

[0211] FIG. 39C is a graph showing the loss modulus and storage modulus (respectively left and right bars) of RBC cytogel prepared using modified RBCs as prepared or after 7-day storage at 4°C.

[0212] FIG. 40A is a schematic illustration of the use of EBC as a hemostatic sealant for treating noncompressible hemorrhage in a rat liver laceration model.

[0213] FIG. 40B is a schematic illustration showing the liver laceration and hemostasis.

[0214] FIG. 40C is a graph showing a comparison of blood loss between EBC and control groups in the rat liver laceration model.

[0215] FIG. 40D is a graph showing a comparison of to hemostasis between EBC and control groups in the rat liver laceration model.

[0216] FIG. 41 A is a schematic illustration of EBC used as a scaffolding material for the treatment of volumetric wounds, quickly stopping bleeding and promoting tissue regeneration in a rat liver puncture model.

[0217] FIG. 41 B is a schematic illustration of a liver puncture and regeneration.

[0218] FIG. 41 C is a schematic of the experimental plan for sample capture.

[0219] FIG. 41 D is a graph showing a comparison of blood loss between treatments in rat liver puncture model.

[0220] FIG. 41 E is a graph showing a comparison of time to hemostasis between treatments in rat liver puncture model.

[0221] FIG. 42A is a graph comparing results of a hematology test (erythrocytes) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0222] FIG. 42B is a graph comparing results of a hematology test (hemoglobin) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0223] FIG. 42C is a graph comparing results of a hematology test (hematocrit) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0224] FIG. 42D is a graph comparing results of a hematology test (mean corpuscular volume (MOV)) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0225] FIG. 42E is a graph comparing results of a hematology test (mean corpuscular hemoglobin (MCH)) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0226] FIG. 42F is a graph comparing results of a hematology test (mean corpuscular hemoglobin concentration (MCHC)) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0227] FIG. 42G is a graph comparing results of a hematology test (reticulocyte) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0228] FIG. 42H is a graph comparing results of a hematology test (platelets) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0229] FIG. 421 is a graph comparing results of a hematology test (white blood cells (WBC)) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0230] FIG. 42J is a graph comparing results of a hematology test (monocytes) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0231] FIG. 42K is a graph comparing results of a hematology test (neutrophils) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0232] FIG. 42L is a graph comparing results of a hematology test (eosinophils) of complete blood counting over 28 days of implantation (EBC left bar, Floseal™ right bar).

[0233] FIG. 43 is a graph showing blood test results showing inflammation caused by Floseal™ on Day 5 post-surgery mainly attributed to lymphocytes, while EBC did not induce systemic inflammation (EBC left bar, Floseal™ right bar).

[0234] FIG. 44A is a graph showing the serology test results (glucose) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0235] FIG. 44B is a graph showing the serology test results (urea) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0236] FIG. 44C is a graph showing the serology test results (cholesterol) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0237] FIG. 44D is a graph showing the serology test results (lipase) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0238] FIG. 44E is a graph showing the serology test results (total protein) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0239] FIG. 44F is a graph showing the serology test results (globulins) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0240] FIG. 44G is a graph showing the serology test results (albumin / globulin) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0241] FIG. 44H is a graph showing the serology test results (total bilirubin) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0242] FIG. 44I is a graph showing the serology test results (direct bilirubin) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0243] FIG. 44J is a graph showing the serology test results (indirect bilirubin) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0244] FIG. 44K is a graph showing the serology test results (alkaline phosphatase (ALP)) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0245] FIG. 44L is a graph showing the serology test results (gamma-glutamyl transferase) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0246] FIG. 44M is a graph showing the serology test results (alanine transaminase) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0247] FIG. 44N is a graph showing the serology test results (aspartate aminotransferase) after 28 days of implantation (EBC left bar, Floseal™ right bar).

[0248] FIG. 440 is a graph showing the albumin level fluctuation over the implantation period.

[0249] FIG. 45A is a photograph of a liver treated by using EBC for 28 days.

[0250] FIG. 45B is a histological image showing complete liver regeneration by using EBC on Day 28 after surgery.

[0251] FIG. 45C is a close-up of Fig. 45B.

[0252] FIG. 45D is a photograph of a liver treated by using Floseal™ for 28 days.

[0253] FIG. 45E is a histological image showing regeneration by using Floseal™ on Day 28 after surgery.

[0254] FIG. 45F is a close-up of Fig. 45E.

[0255] FIG. 46A is a photograph of an EBC implant on a liver at day 5.

[0256] FIG. 46B is a photograph of an EBC implant on a liver at day 14.

[0257] FIG. 46C is a photograph of an EBC implant on a liver at day 28.

[0258] FIG. 46D is a photograph of a Floseal™ implant on a liver at day 5.

[0259] FIG. 46E is a photograph of a Floseal™ implant on a liver at day 14.

[0260] FIG. 46F is a photograph of a Floseal™ implant on a liver at day 28.

[0261] FIG. 46G is a microscopy image of a histology section from the liver of Fig. 46A.

[0262] FIG. 46H is a microscopy image of a histology section from the liver of Fig. 46B.

[0263] FIG. 46I is a microscopy image of a histology section from the liver of Fig. 46C.

[0264] FIG. 46J is a microscopy image of a histology section from the liver of Fig. 46D.

[0265] FIG. 46K is a microscopy image of a histology section from the liver of Fig. 46E.

[0266] FIG. 46L is a microscopy image of a histology section from the liver of Fig. 46F.

[0267] FIG. 46M is a close-up of Fig. 46G.

[0268] FIG. 46N is a close-up of Fig. 46H.

[0269] FIG. 460 is a close-up of Fig. 461.

[0270] FIG. 46P is a close-up of Fig. 46J.

[0271] FIG. 46Q is a close-up of Fig. 46K.

[0272] FIG. 46R is a close-up of Fig. 46L.

[0273] FIG. 47A is a graph showing a comparison of liver regeneration rates with the use of EBC or Floseal™.

[0274] FIG. 47B is a graph showing a comparison of the assessment of the inflammation degree at the liver injury site (EBC left bar, Floseal™ right bar).

[0275] FIG. 47C is a graph showing a comparison of the occurrence rate of postoperative adhesion complications when using EBC or Floseal™.

[0276] FIG. 48A is a confocal microscopy image showing successful modification of RBCs with azide (Az) groups on the cell surface via the use of a DBCO-linked red fluorescent probe.

[0277] FIG. 48B is a confocal microscopy image showing unmodified RBCs incubated with a DBCO conjugated fluorescent probe (Cy5-DBCO) which emit negligible fluorescent signal

[0278] FIG. 49A is a graph showing the in vitro microcomputed tomography (pCT) images of 1-mL syringes containing EBC as a function of iodixanol concentration. Syringes filled with Visipaque™ 320 (Visi.) were tested for comparison.

[0279] FIG. 49B is a bar graph showing the influence of iodixanol concentration on signal intensity in HU of EBCs.

[0280] FIG. 49C is a bar graph showing the influence of the incorporation of 30% wt / v iodixanol (iod.) on the rheological properties of EBCs.

[0281] FIG. 50A is a graph showing the frequency sweep showing shear-thinning properties EBCDBCO containing 1% wt / v HA-DBCO. A frequency sweep was performed immediately after mixing (initial) and 1 hour after mixing (full gelation).

[0282] FIG. 50B is a graph showing the characteristic injection force curves of various materials in 3-mL syringes injected through a 2.4Fr microcatheter at a rate of 1 mL / min. The average value of the plateau is used to quantify the injection force.

[0283] FIG. 50C is a graph showing the characteristic injection force curves of EBCDBCO with various concentrations of HA-DBCO.

[0284] FIG. 50D is a bar graph showing the percent hemolysis of RBCs after injection for different concentrations of HA-DBCO.

[0285] FIG. 50E is a bar graph showing the injection force for different materials. EBCTz is used to represent the fully gelled state.

[0286] FIG. 50F is a bar graph showing the injection force for EBCDBCO of various HA-DBCO concentrations.

[0287] FIG. 51 A is a graph showing the characteristic force-displacement curves obtained from modified lap-shear tests, comparing EBCs, NBCs, and Onyx™ 34.

[0288] FIG. 51 B is a graph showing the fracture toughness of EBCs, NBCs, and Onyx™ 34.

[0289] FIG. 51 C is a schematic showing the 180-degree peeling test used to measure adhesion energy.

[0290] FIG. 51 D is a schematic showing adhesion of EBCs to vasculature after injection. Fibrin, a component of EBCs, is able to form covalent bonds with collagen present in the endothelial and subendothelial layers

[0291] FIG. 51 E is a bar graph showing the characteristic force-displacement curves obtained from 180-degree peeling tests, comparing adhesion of EBCs to bovine artery endothelium and subendothelium.

[0292] FIG. 51 F is a graph showing the adhesion energy of EBCs to endothelium and subendothelium layers.

[0293] FIG. 51 G is a graph showing the volumetric swelling ratios of EBCs, NBCs, and O Onyx™ 34 after incubation in PBS for 24 hours.

[0294] FIG. 52A is a graph showing the time sweep of unmodified RBCs mixed with 1% wt / v HA-DBCO, showing lack of gelation.

[0295] FIG. 52B is a SEM image showing the structure of EBCs.

[0296] FIG. 52C is a SEM image showing RBCs are linked together within EBC.

[0297] FIG. 52D is a graph showing the time sweep of EBCs using various bioorthogonal reactions showing characteristic click clotting profiles.

[0298] FIG. 52E is a graph showing the effect of polymer concentration on EBC rheological properties. EBCDBCO containing 25% v / v RBCAZ and various HA-DBCO concentrations is used as a model system.

[0299] FIG. 52F is a graph showing the effect of modified RBC content on EBC rheological properties. EBCDBCO containing 1% wt / v HA-DBCO and various RBC-Az concentrations is used as a model system

[0300] FIG. 53A is an image of polyvinyl alcohol (PVA) sacrificial mould cast in PDMS before dissolution.

[0301] FIG. 53B is an image of polydimethylsiloxane (PDMS) vasculature phantom after the dissolution of the sacrificial PVA mould in an ultrasonic water bath.

[0302] FIG. 54A is a bar graph showing Maximum embolization pressure obtained by EBC and Onyx™ from the in vitro PDMS model.

[0303] FIG. 54B is a schematic showing the two failure modes observed. Material properties, such as fracture toughness, determine how material fails during embolization.

[0304] FIG. 54C is a graph showing the Characteristic embolization pressure-time curve for EBCs showing the elastic leak failure mode.

[0305] FIG. 54D is a graph showing the characteristic embolization pressure-time curve for Onyx™ showing the rupture failure mode.

[0306] FIG. 54E is a graph showing the characteristic ex vivo embolization pressure-time curves of EBC, NBC, and Onyx™.

[0307] FIG. 54F is a bar graph showing the maximum ex vivo embolization pressures.

[0308] FIG. 55A is a graph showing the phase diagram of the failure modes for embolization in the G / pL-H / L plane. Precompression of the embolic material is fixed at A = 0.8.

[0309] FIG. 55B is a graph showing the plot of the failure criteria (Pf / Pc) as a function of G / pH (precompression is fixed at λ = 0.8).

[0310] FIG. 55C is a graph showing an Ashby plot showing the fracture toughness (G) and shear modulus (p) of various embolic materials. Dashed lines indicate where G / p is constant; lines closer to the upper-left corner indicate a larger value of G / p.

[0311] FIG. 55D is a graph showing the steady-state leaking pressure (ps) as a function of the injection flow rate the non-dimensionalized geometric parameter (H / L) is fixed at 0.075.

[0312] FIG. 55E is a graph showing the steady-state leaking pressure (ps) as a function of the non-dimensionalized geometric parameter (H / L). The injection flow rate (Q) is fixed at 10 mL / min.

[0313] FIG. 55F is a graph showing the pressure-time curves of EBC in vitro embolization at various geometric parameters. For H / L = 0.0125.

[0314] FIG. 56A is a schematic showing the various gelation rates of the different clotting systems used in EBCs.

[0315] FIG. 56B is a graph showing time sweep of G’ on EBCs containing various ratios of bioorthogonal ligation pairs.

[0316] FIG. 56C is a bar graph showing the time to full gelation (defined as the time it takes for G’ to reach 95% of its plateau value) of EBCs containing various ratios of bioorthogonal ligation pairs.

[0317] FIG. 56D is an image of PDMS vasculature phantom after injection of EBCDBCO containing 1% wt / v HA-DBCO showing depth of penetration.

[0318] FIG. 56E is an image of PDMS vasculature phantom after injection of EBCTz containing 1% wt / v HA-Tz showing the depth of penetration.

[0319] FIG. 57A is graph showing the cell viability after 24 hours, calculated as the as the percentage of live cells to total cells in the sample.

[0320] FIG. 57B is a graph showing the cell viability after 24 hours, calculated as the as the percentage of live cells to total cells in the sample

[0321] FIG. 57C is a bar graph showing the hemolysis assay results. Values represent the mean ± SD (n = 3). P values were determined by one-way analysis of variance (ANOVA) with Tukey post hoc comparisons.

[0322] FIG. 58A is a graph showing in vitro degradation of EBC and NBC incubated in bovine plasma supplemented with tPA. Degradation is calculated as the percentage of wet-mass remaining compared to the initial wet-mass.

[0323] FIG. 58B is a graph showing the absorbance values of the supernatant at 540 nm was used as a measure of clot degradation. Values represent the mean ± SD (n = 3).

[0324] FIG. 58C is a graph showing in vitro degradation of EBC and Onyx incubated in PBS supplemented with hyaluronidase.

[0325] FIG. 59A is an in vivo post-surgery pCT image of the rat abdominal artery (proximal).

[0326] FIG. 59B is an in vivo post-surgery pCT image of the rat abdominal artery (distal).

[0327] FIG. 59C is a 3D reconstruction of pCT images of the rat abdominal artery.

[0328] FIG. 60A is an image of the baseline digital subtraction angiography (DSA) of the right gastroepiploic artery (porcine model)

[0329] FIG. 60B is an image of the post-embolization DSA of the right gastroepiploic artery (porcine model).

[0330] FIG. 60C is an image of 7-day post-embolization DSA of the right gastroepiploic artery (porcine model).

[0331] FIG. 60D is an image of the baseline DSAofthe right lateral hepatic artery (porcine model)

[0332] FIG. 60E is an image of the post-embolization DSA of the right lateral hepatic artery (porcine model).

[0333] FIG. 60F is an image of 7-day post-embolization DSA of the right lateral hepatic artery (porcine model).

[0334] FIG. 60G is an image of the baseline DSA of the splenic artery (porcine model)

[0335] FIG. 60H is an image of the post-embolization DSA of the splenic artery (porcine model).

[0336] FIG. 60I is an image of 7-day post-embolization DSA of the splenic artery (porcine model).

[0337] FIG. 61A is baseline digital angiography (DA) image showing embolization of the gastroepiploic artery.

[0338] FIG. 61 B is a post-embolization DA image (before contrast) showing embolization of the gastroepiploic artery.

[0339] FIG. 61 C is a post-embolization DSA image (after contrast) showing embolization of the gastroepiploic artery.

[0340] FIG. 61 D is a 7-day post-embolization DA image showing embolization of the gastroepiploic artery.

[0341] FIG. 62A is baseline DA image showing embolization of the splenic artery.

[0342] FIG. 62B is a post-embolization DA image (before contrast) showing embolization of the splenic artery.

[0343] FIG. 62C is a post-embolization DSA image (after contrast) showing embolization of the splenic artery.

[0344] FIG. 62D is a 7-day post-embolization DA image showing embolization of the splenic artery.

[0345] FIG. 63A is a histological microscopy image showing the occluded artery at day 1 showing the degradation of the RBC crosslinker.

[0346] FIG. 63B is a histological microscopy image showing the occluded artery at day 7 showing the degradation of the RBC crosslinker.

[0347] FIG. 63C is a histological microscopy image showing the degradation of TERC within an artery 7 days after embolization.DETAILED DESCRIPTION

[0348] The term “engineered cytogel” or “cytogel” for short refers to the gels of the present disclosure which combine functionalized cells and polymers into a cytogel by covalently linking the cells and polymers. One example of a cytogel is an RBC cytogel. The term “RBC cytogel” as used herein refers to embodiments where the cells in the cytogel that were functionalized are specifically RBCs and the polymers are biocompatible long-chain polymers, for example hyaluronic acid. Another example of a cytogel is the engineered blood clot (EBC). The term “engineered blood clot” or EBC as used herein refers to embodiments where the RBC cytogel was interpenetrated with a fibrin network.

[0349] There is accordingly provided a cytogel for blood clotting, the cytogel combines cells and polymers that are functionalized with click chemistry groups to form covalent bonds between the cells and polymers. It should be noted however that, although the present cytogels were designed for blood clotting and embolisms, they can also be applied in cartilage regeneration and bone regeneration.

[0350] The combination of click chemistry and clotting is referred to herein as a “click clotting” strategy. The present click clotting strategy can instantaneously crosslink living cells into cytogels via bioorthogonal click reactions. In one example, this strategy allows to directly crosslink red blood cells into cytogels and implement it within blood clots. As demonstrated in the Example 1 below, the resulting engineered cytogel exhibits a tunable clotting speed which can be instantaneous (< 5 seconds) in some cases or slower in others. The cytogel, more specifically the EBC, also demonstrated an enhanced fracture toughness (13-fold) and improved adhesion properties (4-fold) compared to native blood clots. The experiments and computational modelling revealed a toughening mechanism based on cell rupture. In vivo experiments showed that EBC and the other tested cytogels outperformed clinically used products in treating non-compressiblehemorrhage. Specifically, the present cytogel can halt hemorrhage, promote tissue regeneration, induce minimal inflammation and foreign body responses, and avoid postoperative adhesion. Furthermore, the present strategy is applicable to a range of cells and polymers as well be described in greater details below. The present cytogels are therefore useful for bleeding control, tissue repair and tissue regeneration.

[0351] The present cytogels comprise cells functionalized with a first click chemistry group and polymers functionalized with a second click chemistry group. The first click chemistry group is a cycloalkene, heterocycloalkene, cycloalkyne or heterocycloalkyne click chemistry group that reacts with the second click chemistry group comprising a nitrogen that reacts with the double bond of the cycloalkene or heterocycloalkene, or the triple bond of the cycloalkyne or heterocycloalkyne to form a covalent link between the two click chemistry groups. Alternatively, the second click chemistry group is a cycloalkene, heterocycloalkene, cycloalkyne or heterocycloalkyne click chemistry group and the first click chemistry group comprises a nitrogen that reacts with the double bond of the cycloalkene or heterocycloalkene, or the triple bond of the cycloalkyne or heterocycloalkyne to form a covalent link between the two click chemistry groups. The click chemistry group comprising the nitrogen is for example an azide, a tetrazine, a triazine, a pyridazine, or a nitrone. Thus, the “first” and “second” click chemistry groups are interchangeable.

[0352] The term “cycloalkene” as used herein can be defined as a C3-C10 cycloalkene group, comprising a carbon - carbon double bond that is sterically available. Preferably the carbon atoms of the carbon - carbon double bond are not substituted. The C3-C10 cycloalkene group can be substituted or unsubstituted. Examples of a C3-C10 include but are not limited to cyclopropene, cyclobutene, cyclopentene, cyclohexene, cycloheptene, cyclooctene (cis or trans), cyclononene, and cyclodecene. Examples of cycloalkene containing compounds that can be functionalized onto a cell surface and / or a polymer include trans-cyclooctene — NHS ester (TCO-NHS), TCO-NHS with a polyethylene glycol spacer arm (PEG) such as TCO-PEGn-NHS ester where n is an integer of from 1 to 15, preferably 4 to 12, trans-cyclooctene — COOH, and trans-cyclooctene-PEGn- COOH where n is an integer of from 1 to 15, preferably 4 to 12.TCO-NHS ester, specifically (E)-Cyclooct-4-enyl 2,5- dioxo-1-pyrrolidinyl carbonate.

[0353] The TCO-NHS ester as shown above reacts by carbodiimide chemistry with a primary amine on the surface of the cell or the polymer as per Scheme 1. A similar reaction can be leveraged for functionalizing TCO-PEGn-NHS ester or trans-cyclooctene — COOH on the cell surface or on the polymer. Moreover, similar reactions can be performed with other compounds to provide the cycloakene, heterocycloalkene, cycloalkyne or heterocycloalkyne on the surface of the cells or on the polymers.Scheme 1.

[0354] The term “heterocycloalkene” refers to a three to ten membered ring containing at least one heteroatom and the remainder being carbon atoms, the heterocycloalkene comprising a carbon - carbon double bond that is sterically available. Preferably the carbon atoms of the carbon - carbon double bond are not substituted. The heteroatoms are for example one or more of N, S or O. The heterocycloalkene can be substituted or unsubstituted.

[0355] The term “cycloalkyne” as used herein refers to a C7-C9 carbon ring comprising a carbon - carbon triple bond. The cycloalkyne can be substituted or unsubstituted. Preferably the cycloalkyne is cyclooctyne. Examples of cycloalkyne include but are not limited to: ce(OCT)Polymer / Cell Surface

[0356] The term “heterocycloalkyne” refers to a 7 to 9 membered ring containing at least one heteroatom and the remainder being carbon atoms and comprising a carbon - carbon triple bond. The heteroatoms are for example one or more of N, S or O. The heterocycloalkyne can be substituted or unsubstituted. Examples of heterocycloalkyne include but are not limited to:(DIBAC-PEGn-amine where n is an integer from 1 to 6, preferably from 2 to 4)

[0357] The optional spacer can be as illustrated in the examples above. Suitable spacers can include polyethylene glycol (e.g. PEG4) and alkane radicals optionally substituted and optionally interrupted by N, O or phenyl.

[0358] One particular example of the click chemistry between the two functional groups is cells modified with trans-cyclooctene (TOO) and polymers modified with tetrazine. The reaction between TOO and tetrazine is an inverse electron demand Diels-Alder (I EDDA) reaction, typically a dienophile reacting with a tetrazine. Another example is cells modified with an azide and polymers modified with dibenzocyclooctyne (DBCO) such as sulfo-DBCO amine (shown below). This reaction is a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, typically a strained cyclooctyne reacting with an aliphatic azide or a pendant azide. The I EDDA and SPAAC reactions (which can be done with alternative functional groups as explained herein) are preferable due to their reaction kinetics and compatibility with living cells.(sulfo-DBCO amine)

[0359] In some embodiments, when the polymers are functionalized with a click chemistry group comprising a cycloalkene or a heterocycloalkene, the cells are functionalized with a tetrazine, a triazine or a pyridazine. On the other hand, when the cells are functionalized with a click chemistry group comprising a cycloalkene or a heterocycloalkene, the polymers are functionalized with a tetrazine, triazine or a pyridazine. The azide group can also react with the cycloalkene or heterocycloalkene with Deep Eutectic Solvents (DES). In some embodiments, when the polymers are functionalized with a click chemistry group comprising a cycloalkyne or a heterocycloalkyne, the cells are functionalized with a click chemistry group comprising a diazenediyl (-N=N-). Alternatively, when the cells are functionalized with a click chemistry group comprising a cycloalkyne or a heterocycloalkyne, the polymers are functionalized with a click chemistry group comprising a diazenediyl (-N=N-). The diazenediyl containing click chemistry group is preferably an azide, a tetrazine, a triazine or a pyridazine. The tetrazine, triazine or pyridazine are optionally substituted by -COOCH3, -NHBoc, -NHAc, -SO2CH3, -SMe or-OMe. The azide can be a pendant azide group: -N=N+=N’.

[0360] Strain-promoted alkyne-nitrone cycloaddition (SPANG) reactions are also contemplated herein. Accordingly, when the polymers are functionalized with a click chemistry group comprising a cycloalkyne or a heterocycloalkyne, the cells are functionalized with a click chemistry group comprising a nitrone. When the cells are functionalized with a click chemistry group comprising a cycloalkyne or a heterocycloalkyne, the polymers are functionalized with a click chemistry group comprising a nitrone. The nitrone is of the formula R-C=N+(R’)-O_where R and R’ are alkyl or combine together to form a heterocycloalkyl. The alkyl is preferably an optionally substituted Ci-Ce alkyl or is H. The heterocycloalkyl is preferably a 5 membered or 6 membered ring. One example of a linear nitrone is R-C=N+(Me)-O'. Exemplary cyclic nitrones are as illustrated below. Cyclic nitrones are preferred as they are more stable in aqueous solvents.

[0361] In general, when one of the cells or the polymers is functionalized with a cycloalkene, heterocycloalkene, cycloalkyne, or heterocycloalkyne, the other is functionalized with a suitable click chemistry group comprising a nitrogen that reacts with the double bond or the triple bond to form a covalent link between the two groups.

[0362] The cytogels are preferably characterized by a cell content of from 2 to 50 %, from 3 to 48 %, from 5 to 45 %, from 10 to 40 %, or from 20 to 40 % by volume. Any cell that is immune compatible with the target clotting site can be used for the cytogels of the present disclosure. The cell types include RBC, fibroblasts, adipose-derived stem cells, microalgae cells, and non- pathogenic bacteria such as E. coli. In some embodiments, the RBCs are O negative red blood cells. In particular, cells extracted from an individual can be used to form the cytogel which is then applied on that same individual. One example could be to functionalize whole blood obtained from the individual including all cell types present in the blood. This application is useful in remote locations, war zones and emergency settings. Similarly, a cytogel made with O negative RBCs is universally compatible with other blood types and can also be used in emergency settings.

[0363] In at least some embodiments, the cells are provided as “alive cells” to form a “living network”. In such embodiments or in other embodiments, the cytogels are preferably characterized by a polymer concentration of from 0.5 to 5 wt. %, from 0.5 to 4 wt. %, from 0.5 to 3 wt. %, from 0.5 to 2 wt. %, from 1 to 4 wt. %, from 1 to 3 wt. % or from 1 to 2 wt. %. The polymeris a cytocompatible polymer that generally can have a size that is 30 to 70 fold smaller than the diameter of the selected cells, for example, a size of from 50 to 250 nm. In one exemplary embodiment, the cells are RBC and the polymer size is 120-150 nm. Examples of polymers include but are not limited to hyaluronic acid, fibrin, alginate, chitosan, polyacrylic acid, polyethylene glycol, polyacrylamide, poly(vinylpyrrolidone), polysialic acid, polyvinyl alcohol, gelatin, albumin, dextran, agarose and the like.

[0364] The cytogels are formed by bioorthogonal crosslinking of cells and polymers, which is a chemistry process that is independent of the type and functions of the cells and polymers. When modifying cells, the surface amine group can be used as targets, however hydroxyl and carboxyl and thiol groups can also be used. The amines are preferred because the amines are more ubiquitous on the surface of mammalian cells and bacterial cells. The role of the polymers is structural and therefore is independent of the exact chemistry of the polymer. The polymers serve as physical chains to link cells together into the cytogel.

[0365] The present cytogel can be formulated as an embolic material for endovascular embolization procedures. This is because the cytogel has demonstrated both biocompatibility and improved mechanical properties. In such embodiments, the cytogel can be referred to as an Engineered Blood Clots (EBC) when in the context of a temporary embolic agent. The embolic material can be used in invasive surgical methods or in trauma to stop the bleeding of arteries.EXAMPLE 1Materials

[0366] Methyltetrazine amine (1011), methyltetrazine acid (1125), sulfo-DBCO amine (1227), trans-cyclooctyne (TCO) - polyethylene glycol 4 (PEG4) - N-Hydroxysuccinimide (NHS) ester (TCO-PEG4-NHS) (A137), Azido-PEG4-NHS ester (AZ103), AZDye™ 405 cadaverine (1418), AZDye™ 568 tetrazine (1363), and Cyanine (Cy) 5 dibenzocyclooctyne (DBCO) (A130) were purchased from Click Chemistry Tools (Scottsdale, USA). Hyaluronic acid (HA, molecular weight (Mw) - 2000 kDa) was purchased from Lyphar Biotech (Xi’an, China). Alginate (high molecular weight, 1-1 G) was purchased from KIMICA Corporation (Tokyo, Japan). Chitosan (high molecular weight, 419419), polyacrylic acid (PAAc, Mw -1250 kDa, 306215), N-(3-Dimethylaminopropyl)- N’-ethylcarbodiimide hydrochloride (EDC, 03450), N-hydroxysuccinimide (130672), fluoresceinamine (201626), 2-(N-Morpholino)ethanesulfonic acid (MES) hydrate (M8250), sodium hydroxide solution (S2770) sodium chloride (NaCI, S9888), dimethyl sulfoxide (DMSO, 276855),calcium chloride (CaCh), dialysis tubing (molecular weight cut off (MWCO) of 14 kDa, D9527) and citrate-phosphate-dextrose solution with adenine (CPDA, C4431) were purchased from Sigma- Aldrich (St. Louis, USA). Normal human red blood cells (pooled sex, CPDA anticoagulant) and normal human whole blood (pooled gender, CPDA anticoagulant) were purchased from BiolVT (Westbury, USA).Cell surface modification

[0367] RBC surface was decorated with TCO groups or azide groups via carbodiimide chemistry as explained below. For the TCO modification, human RBCs of 1 mL were firstly washed for three times with cold phosphate buffered saline (PBS) (9 mL, pH=7.4) and centrifuged (1000 x g, 5 min). The RBCs were then resuspended in 5 mL of PBS. A stock solution of TCO- PEG4-NHS ester (400 pL) was added to 3.6 mL PBS, and immediately added to the RBC suspension. The mixture was incubated for 40 minutes at 37 °C. Unreacted TCO-PEG4-NHS was removed by gradient wash of RBCs with cold PBS and centrifuge (about 4 °C). The supernatant was replaced by PBS after each centrifuge with increasing volumes of 2, 4, 6, 8 mL, followed by another three complete PBS washes. Using the same protocol, the RBC surface was also decorated with the click moiety azide by incubating the cells with Azido-PEG4-NHS ester. The presence and distribution of TCO-PEG4-NHS on RBCs were both detected by a fluorescent probe, AZDye™ 568 Tetrazine. The modified RBCs were incubated with AZDye™ 568 Tetrazine (2 pg / mL in PBS) for 5 minutes at room temperature, and washed with PBS three times. The stained cells were imaged using a confocal laser scanning microscope (Zeiss, LSM710).Characterization of modified RBC

[0368] The amount of TCO on RBC surface was quantified with flow cytometry. Modified RBCs of a fixed amount (20 pL of RBCs) were added into PBS with varying concentrations of AZDye™ 568 Tetrazine, making the ratio of tetrazine groups per RBC between 106and 108, followed by incubation at room temperature for 5 minutes. After washing with PBS three times to remove unattached AZDye™ 568 Tetrazine, the fluorescence intensity of RBCs was measured using flow cytometry (BD LSR Fortessa). The saturated tetrazine number for each RBC was estimated from a transition point on the curve of fluorescence to tetrazine / RBC number ratios. The tetrazine number further informed the number of TCO on RBCs, as the reaction efficiency of TCO-tetrazine was approximated to be 100%. The in vitro stability of TCO was characterized by monitoring the TCO number presenting on cells over time. The modified RBCs were stored inCPDA solution at 4 °C for different periods of time. The stored cells were incubated with excessive AZDye™ 568 Tetrazine (108tetrazine per RBC) for 5 minutes at room temperature. After being washed with PBS for three times, flow cytometry was performed as described above.Polymer synthesis and characterization

[0369] A range of polymers, including hyaluronic acid (HA), chitosan, alginate, polyacrylic acid and polyethylene glycol, were modified with the click tetrazine (TZ) moiety via the standard carbodiimide chemistry. First, 0.5% w / v HA was dissolved in stirred buffer containing 0.1 M MES and 0.3 M NaCI pH 6.0. Then, EDC and NHS were both added at 2.5 x molar excess of the carboxylic acid groups of HA. Methyltetrazine amine was added at 0.25, 0.50, or 0.75 mmol per gram of HA for reaction of 24 h at room temperature. The solution was dialyzed in 14 kDa MWCO dialysis tubing for 5 days against a decreasing salt gradient from 150 mM to 0 mM NaCI in deionized (DI) water and then freeze-dried. The modifications of alginate (0.5% w / v) and polyacrylic acid (0.3% w / v) were conducted with similar protocols by adding methyltetrazine amine at 0.75 mmol per gram of polymer in MES buffer. The modification of chitosan (0.3% w / v) was conducted by adding methyltetrazine acid at 0.75 mmol per gram of polymer in MES buffer. The modification of 4-arm PEG amine (2% w / v) was conducted by adding methyltetrazine acid at 1.5 x molar excess of the amine group in MES buffer. HA was also modified with a different click moiety dibenzocyclooctyne (DBCO) by the reaction of HA and sulfo-DBCO amine by using the same EDC / NHS protocol. The resulting polymers were analyzed by1H nuclear magnetic resonance spectrometry (1H NMR, 400 MHz Varian Mercury) with deuterium oxide (D2O) as solvent to obtain the degree of substitution (DS) of the tetrazine group.

[0370] The high molecular weight HA-TZ (XL, extra-large) was synthesized as described above using the pristine HA. Lower molecular weight HA-TZ was prepared by the enzymatic hydrolysis using hyaluronidase. Briefly, pristine HA-TA was dissolved in 0.1 M pH 6.0 MES buffer at 0.5% w / v. Bovine testicular hyaluronidase (400-1000 U / mg, Sigma-Aldrich, H3506) was added to the solution at 0.5 mg / mL and left for reaction under stirring at 37 °C. The reaction was terminated at predetermined time points by being immersed in boiling water for 5 minutes followed by cooling in ice / water bath. Reaction of 20 min, 40 min and 60 min results in the large (L), medium (M), and small (S) HA-TZ, respectively. The solution was filtered, dialysed and freeze-dried to achieve the HA-TZ with varied molecular weight. The hydrodynamic radius of HA-TZ was measured by dynamic light scattering (DLS) at the concentration of 0.5 mg / mL. Molecular weightof the resulting HA-TZ was in the range of 20-2000 kDa (small 20-200, medium 200-600, large 600-1200, XL 1200-2000 (estimated values)).Synthesis of fluorescently labelled polymers

[0371] The resulting hyaluronic acid modified with tetrazine (HA-TZ) was further labelled with fluorescein to yield the fluorescent polymer HA-TZ-F for subcutaneous implantation and biodistribution studies. Briefly, the synthesized HA-TZ (10% DS of TZ) was dissolved in 0.1 M pH 6.0 MES buffer at 0.5% w / v. Fluoresceinamine at 2% molar ratio of carboxylic acid group of HA was dissolved in DMSO. EDC and NHS were both added to HA solution at equal molar of carboxylic acid group, and stirred for 30 min. The fluoresceinamine solution was added dropwise to HA solution under stirring, then left for 24 h reaction at room temperature. The solution was firstly dialyzed against 1 :1 ethanokwater for 2 days, and then water for 3 days followed by lyophilization. HA-TZ was also labelled with AZDye™ 405 for confocal imaging. Briefly, AZDye™ 405 Cadaverine was added to 0.5% w / v HA-TZ solution by 1 % molar ratio (amine to carboxyl) with 2.5 x molar excess EDC and NHS. The reaction was conducted for 24 hours protected from light at room temperature, followed by dialysis and lyophilization, to yield the labelled polymer HA- TZ-405.Preparation of RBC cytogels via click clotting

[0372] The synthesized polymers were dissolved in PBS at various concentrations ranging from 1% to 3% (w / v). Modified RBCs were collected into one syringe after centrifuge, then quickly mixed with the polymer solution in the other syringe until homogeneous. For example, 1.5% (w / v) HA-TZ solution mixing with modified RBCs at 1 :2 by volume results in 1% (w / v) final polymer concentration and 33% (v / v) RBC ratio in the prepared gel. The resulting mixture (RBC cytogels) was immediately ejected from the syringe for a variety of tests and applications. Various cells including human vocal fold fibroblasts (hVFFs), human adipose-derived stem cells (ASCs), and microalga C. reinhardtii (+) were surface-modified with TCO and then mixed with HA-TZ solution to achieve cytogels using the same method for preparing RBC cytogels.Preparation of EBC

[0373] EBC was prepared by forming a cellular network of RBCs integrated with a fibrous polymer network of fibrin and other blood components. Human whole blood was firstly processed by substituting native RBCs with the modified RBCs. To initiate the native and click clottingsimultaneously, whole blood was recalcified by using calcium chloride (CaCh, final concentration 30 mM), and then mixed with equal volume of polymer solution with varied concentration (e.g., 2% w / v HA-TZ).Rheological measurement

[0374] The mixed gels were immediately ejected from the syringe onto the bottom plate of a torsional rheometer equipped with 20 mm flat upper plate geometry (DHR-2, TA Instruments). Mineral oil was applied to the gel periphery to prevent the gel from dehydration during test. The storage and loss moduli (G’ and G”) of gels made of various polymer species, degree of TZ substitution, polymer concentration, and RBC ratio were measured using 0.1 % strain at 1 Hz.Confocal imaging

[0375] To visualize the structure of EBC, the RBCs was labelled with Cy5 by click chemistry, and the synthesized fluorescent polymer HA-TZ-405 and Fibrinogen-Alexa Fluor 488 Conjugate were used. First, human RBCs were grafted with TCO and Azide by sequence. The modified RBCs were then incubated with DBCO-Cy5 to be labelled with fluorescence via DBCO-Azide ligation. Labelled RBCs with TCO moiety were re-suspended with platelet-rich-plasma with the addition of Fibrinogen-Alexa™ Fluo 488 (5%) and calcium chloride, then mixed with HA-TZ-405 solution to form EBC. Unmodified RBCs were used as control. The structure was imaged by using confocal laser scanning microscopy (Zeiss, LSM710) under the DAPI, AF488 and Cy5 channels. Stack images were also obtained to construct the three-dimensional structure of EBC by using the software ZEISS Zen.Scanning electron microscopy (SEM) imaging

[0376] The morphology of materials was observed using a field emission scanning electron microscope (FE450, FEI) with the accelerating voltage of 10 kV. The prepared EBC and other materials including un-crosslinked EBC, NBC, and RBC gel were fixed with 4% paraformaldehyde, then dehydrated with gradient ethanol (30-100%). The dry samples were coated 4 nm platinum using a high-resolution sputter coater (ACE600, Leica) to increase surface conductivity.Fracture toughness measurement

[0377] Fracture toughness of RBC cytogels and other EBCs was measured by modified lap shear tests. To prepare RBC cytogels of defined shape yet with ultrafast gelation kinetics, the modified RBCs and polymer precursors were pre-cooled at 4 °C fridge for 30 minutes before mixing to slow down the reaction rate. The mixture was immediately poured into a polytetrafluoroethylene (PTFE) mold of 20 *15 x1.5 mm3and covered with a PTFE sheet. After 10 min at room temperature, the RBC gel was taken out of molds and glued with two thin polyethylene terephthalate (PET) films on each side using Krazy Glue. The length of overlapping joint is 20 mm. An initial edge crack of 5 mm was made with blade in the middle of each sample. The samples were tested using an Instron machine (model 5965) equipped with a 10 N load cell. The displacement rate was set at 2 mm min-1, unless otherwise specified. EBC samples were prepared in the mold, incubated at 37 °C for 1 hour to complete native clotting, and tested using the same method. For comparison, native blood clots (NBC), Floseal™ hemostatic matrix (Baxter) infused with native blood clots, and blood / chitosan clots were prepared and measured using the same method.Tensile test

[0378] To measure the tensile behavior under cyclic loading, the RBC cytogels were prepared into strips in PTFE mold with the dimension of 30 x 8 x 1.5 mm3. The accurate size of samples was measured after demolding. Samples were tested by an Instron machine (10 N load cell) with the displacement rate of 3 mm min-1. Four tensile cycles were applied to samples at each stretch ratio. The nominal stress was obtained by dividing the force by the cross-sectional area. The nominal strain was obtained by dividing the change in length by the original length.Measurement of hemoglobin released from ruptured RBCs

[0379] Hemoglobin was released from stretched RBC cytogels due to the cell rupture. The release amount was quantified for each RBC cytogel underwent tensile tests. Briefly, after tensile test, RBC cytogels were cut into cubes and weighed for the mass (around 50 mg). The gel pieces were immersed into 2 mL PBS and incubated overnight at 4 °C fridge. After centrifuge, the absorbance at 540 nm of the supernatant was measured by using a microplate reader (Synergy HTX, Agilent). Results were reported by normalizing the absorbance to the mass of RBC cytogels.Finite element modeling of lap shear test

[0380] To study the toughening mechanism based on the Mullins effect, a finite element model was developed to simulate the modified lap shear test of the RBC cytogels. An initial crack is introduced to an RBC cytogel, which is deformable and dissipative. The RBC cytogel is damaged upon loading, e.g., by ruptures of red blood cells. Such damages lead to the loss of modulus of the RBC cytogel, giving rise to the softening behavior along the unloading path and forming a hysteresis loop. This behavior is referred to as the Mullins effect. Here, the combined neo-Hookean and modified Ogden-Roxburgh model was used to capture the Mullins effect. In the model, the softening behavior is empirically described by a pseudo-elastic energy function W (F,η): (1)

[0381] where W is the free energy without the Mullins effect; <Φ(η ) is the damage function in the form of with Wmaxbeing the maximumstrain energy density stored in the material before unloading and erf being the error function; r, m , and β are the Mullins coefficients that characterize the energy dissipation as detailed below. In Eqn. (1), F is the deformation gradient tensor and η is a damage variable, which can be expressed as:(2)

[0382] where Wmaxis the maximum strain energy stored in the material uploading, and W is the strain energy without considering Mullins effect. The neo-Hookean free energy function was adopted for W’.(3)

[0383] whereΛi is the / 1hprincipal stretch and is the shear modulus. The shear modulus is taken to be μ =0.88 kPa, estimated by the slope of the shear stress-shear strain curve. The gel is assumed to be incompressible so the Young’s modulus is three times the shear modulus, E = 3μ.

[0384] Due to the Mullins effect, only part of the strain energy becomes recoverable upon unloading, while the rest is dissipated due to the Mullins effect. The area of the hysteresis loop isthe dissipated energy density defined by UD and the shaded area under the loading path is the total mechanical work done on a unit volume before unloading defined by U. Based on the modified Ogden-Roxburgh model, the hysteresis ratio is defined as:(4)

[0385] Upon evaluating the UD and U from the data, h was established as a function of U for the RBC cytogels. By fitting Eqn. (4) to the data, the Mullins coefficients for the RBC cytogels were obtained, which will be input into finite element modelling to capture the energy dissipation during fracture process. Compared to other dissipative materials, the RBC cytogels show higher energy dissipation capacity at lower mechanical energy input, indicated by the high h values at small U values.

[0386] The fracture of the RBC cytogel was modelled using a bilinear triangular cohesive zone model, which has been widely implemented to study the coupling between the interfacial properties and the bulk deformation. The intrinsic fracture toughness Gowas defined using the interfacial strength Smaxand the maximum separation distance 5maxvia: (5)

[0387] The interfacial strength Smaxis difficult to measure precisely. Smaxwas treated as a fitting parameter and vary it between ~1 kPa and ~10 kPa. This range is chosen according to the representative range of adhesive strength reported for tissue adhesives using the lab-shear test.

[0388] The commercial FE package ABAQUS (2016, SIMULIA) was used to carry out the finite element simulations. The problem was simplified to be two-dimensional by adopting a planestrain condition. Rigid backings were added to the bottom and the top surfaces in the simulation. The top backing was subjected to a constant velocity boundary condition in the direction parallel to the interface. The bottom surface is constrained in a frictionless manner, while one node near the left boundary is pinned to prevent the rigid body motion. The fracture energy can thus be determined using the force-displacement curve F(δ).(6)

[0389] where A is the bonded area. The bulk of the RBC cytogel was modelled with CPE4R element, and the interface was modelled with COH2D4 element. The explicit solver in ABAQU S was used to simulate the lap shear process.

[0390] It was found that the combination of r0= 3Jm-2and Smax= 15.4 kPa yields T « 15 Jrrr2and showed a good agreement between the experimental and computational forcedisplacement curves of lap shear test. The Mullins effect was then removed in the fine element (FE) model and it was found that the crack propagated at a much lower critical displacement and force, yielding a lower fracture toughness T « r0= 3 Jm- 2. Comparing the FE models with and without the Mullins effect, the one with Mullins effect dissipates considerable amount of energy during the gel deformation, causing a higher fracture toughness. The use of FE models revealed the Mullins effect caused by the RBC rupture as being the key toughening mechanism in RBC cytogels.Adhesion energy measurement

[0391] T-peel test was performed with an Instron machine (Model 5965, 10-N load cell) to measure the adhesion energy between blood clots and biological tissues. Fresh porcine skin from a local grocery store was cut into strips and the skin surface was cleaned with deionized water. An elastomer mold with the inner dimension of 30 mm in length, 10 mm in width and 1.5 mm in thickness was attached onto the skin with Vaseline applied to the interface for complete sealing. Precursor solutions of EBC or recalcified human whole blood were poured into the molds. A porous fabric saturated with recalcified whole blood was put on whole blood specimens as a rigid backing. The samples were left for coagulation in a sealed and humidified chamber at 37 °C for 1 hour. The elastomer molds were carefully removed without disturbing the clot / tissue specimens. Thin PET films were adhered to EBC and skin by using Krazy™ glue as rigid backing. An initial crack of 5 mm was created between the clot and skin. The specimens were then gripped to the Instron machine and peeled at a displacement of 100 mm min-1. The adhesion energy of the specimen was calculated by dividing two times of the average force at the plateau (Favg) by the width of the specimen:Burst pressure measurement

[0392] A burst chamber was manufactured according to the ASTM F2392: Standard test method for burst strength of surgical sealants. Collagen casing was used as a model tissue. A biopsy punch was used to create a 3-mm diameter hole in the center of collagen casing. Tested materials (EBC, NBC, RBC cytogels and FlosealTM-blood clot gels) were prepared into disk shape with 15 mm in diameter and 3 mm in thickness. The clot samples were glued to collagen casing using Krazy™ glue in order to test the cohesive burst pressure of materials. A home-made elastomer ring was placed onto samples to avoid leaking of the burst chamber. During the test, a syringe filled with PBS was connected to both the burst chamber and a pressure gauge. A syringe pump was used to feed the PBS to the chamber. The peak pressure before the cohesive failure was recorded and used as the burst pressure.In vitro biodegradation

[0393] RBC cytogels prepared at one batch were cut into small pieces. Several pieces of gels were randomly selected and measured the weight before and after lyophilization. The ratio of dry / wet weight of RBC cytogels was calculated. Meanwhile, one piece of pristine RBC cytogel with measured accurate mass was added to 2 mL PBS containing 0.1 % (w / v) hyaluronidase (400- 1000 ll / rng, Sigma-Aldrich, H3506) and 0.1% (w / v) sodium azide as a preservative. The RBC cytogels were incubated at 37 °C with agitation. At pre-determined time intervals, the samples were washed three times with PBS, lyophilized, and then measured the remaining dry weight. The initial dry weight of each sample can be determined by the calculated dry / wet ratio. Percentage of remaining dry weight to initial dry weight of gels was calculated and reported.In vitro blood compatibility

[0394] Hemolysis test was performed as the first indicator of blood compatibility. To evaluate whether the polymer or cell-crosslinking will cause hemolysis, 100 pL of 1 % HA-TZ solution was mixed 100 pL native RBCs or modified RBCs (RBC-TCO) for 15 minutes. 10 mL PBS was then added to the mixture and then incubated at 37 °C for 30 minutes. For comparison, chitosan solution was also mixed with RBCs in the same way. 100 pL native RBCs was added to DI water to achieve complete hemolysis as the positive control. 100 pL native RBCs was added to PBS as the negative control. The mixtures were centrifuged (1000 g, 5 min), and the absorbance at 540 nm of the supernatant was measured by using a microplate reader. Percent hemolysis wascalculated according to the following equation: % hemolysis=(AbsSamPie- AbSnegative) / AbSpositive* 100%.

[0395] Interaction with platelets and white blood cells was checked as the second indicator of blood compatibility. Briefly, one piece of EBC (around 10 * 10 * 1.5 mm3) was incubated with 10 mL normal human whole blood under agitation at 37 °C for 10 minutes. EBC was then removed to obtain the EBC-conditioned whole blood. The blood was then diluted with an equal volume of PBS (pH 7.4) and separated by density gradient centrifugation over Lymphoprep™ (Stemcell Technologies). The mononuclear cell layer was collected, washed twice in PBS with 2% fetal bovine serum (FBS), and counted using a trypan blue exclusion method. Cells were divided into triplicates of 2 x 106cells for flow cytometry staining. Cells were pre-incubated with Human TruStain FcX™ (BioLegend) for Fc receptor blocking for 20 minutes on ice. The cells were then stained for surface-markers in PBS with 2% FBS for 20 minutes on ice, using the following antibodies: BUV737-conjugated anti-human CD3 (UCHT1 , BD Biosciences); BV785-conjugated anti-human CD4 (RPA-T4, BioLegend); allophycocyanin (APC) - Cy7 - conjugated anti-human CD8a (HIT8a, BioLegend); Fluorescein isothiocyanate (FITC) - conjugated anti-human CD20 (2H7, BioLegend), anti-human CD41 (HIP8, BioLegend); BUV395-conjugated anti-human CD56 (NCAM16.2, BD Biosciences); BV650-conjugated anti-human CD14 (M5E2, BioLegend); PE- Cy7-conjugated anti-human HLA-DR (L243, BioLegend); APC-conjugated anti-human CD86 (BU63, BioLegend), anti-human CD41 / CD61 (A2A9 / 6, BioLegend); PE-conjugated anti-human CD16 (B73.1 , BioLegend), anti-human P-selectin (AK4, BioLegend); Peridinin-Chlorophyll- Protein (PerCP) Cy5.5 - conjugated anti-human CD44 (IM7, BioLegend); and Pacific Blue- conjugated anti-human CD11b (ICRF44, BioLegend). Viability Dye eFluor™ 506 (eBioscience) was used to discriminate dead cells. Compensation was performed with BD™ CompBeads (BD Biosciences). The data were acquired on BD Fortessa and analyzed with FACS Diva (BD Biosciences) or FlowJo (Tree Star) software.In vitro cytocompatibility

[0396] The cytotoxicity of EBC was evaluated using immortalized human vocal fold fibroblasts, following the International Organization for Standardization (ISO) 10993-5: Tests for in vitro cytotoxicity. For every 200 mg of EBC, 1 mL of Dulbecco’s Modified Eagle’s medium (DMEM) was added for extraction. Meanwhile, 20,000 cells per well were seeded in a 96-well plate. The extracts, after a 24-hour incubation period, were collected and supplemented 1% penicillin-streptomycin, and with or without 10% fetal bovine serum. The culture medium withinthe 96-well plate was then replaced with the supplemented EBC extracts. Completed pristine DMEM was used as the control. The cells were then cultured for 24 h inside an incubator, in an environment at 37 °C, 95% relative humidity, and 5% CO2 atmosphere. Cell viability was assessed using a Live / Dead viability kit (Invitrogen, L3224), following the protocol of the manufacturer. Confocal laser scanning microscopy (Zeiss, LSM710) was used for investigation. Live cells were visualized in green and dead cells in red. The images were analyzed using Imaged™ software (National Institutes of Health, NIH). Cell viability was defined as the percentage of live cells in the sample, while the cell density of a sample was used to measure cell proliferation.In vivo biocompatibility, biodegradation and biodistribution

[0397] All in vivo experiments were approved by the McGill University Animal Care Committee(Protocol # 2019-8098) and performed according to the guidelines of the Canadian Council on Animal Care. Female Sprague Dawley rats (250-300 g) were purchased from Charles River Laboratories (Wilmington, USA) and used for all the in vivo studies.

[0398] Dorsal subcutaneous implantation was used to evaluate the in vivo biocompatibility, biodegradation and biodistribution of RBC cytogel and EBC. Autologous whole blood from the rat saphenous vein was collected to prepare the RBC cytogel and EBC samples for implantation. The rat was restrained, and the hind leg was immobilized in the extended position. The collection site was shaved with an electric clipper and cleaned with 70% alcohol. Blood was collected into a vial containing CPDA anticoagulant by using a 23-gauge needle from the lateral saphenous vein. Blood flow is stopped by applying pressure with sterile gauze to achieve hemostasis. The collected whole blood was processed, and then combined with the fluorescently labelled polymer (HA-TZ-F) to fabricate RBC cytogel and EBC. Samples were prepared into a disk shape of 6 mm in diameter and 1.5 mm in thickness using aseptic techniques. For implantation, rats were anesthetized using isoflurane (4% isoflurane in oxygen) in an induction chamber. Anesthesia was maintained at 2% isoflurane using a nose cone during the surgery. A volume of 1 mL of saline was injected subcutaneously. Dorsal hair was removed, and the rats were placed over a heating pad for the during of the surgery. The subcutaneous space was accessed by a 1 cm skin incision per implant in the rat’s back. Blunt dissection was performed from the incision point towards the shoulder blades of the rat to create subcutaneous space for implantation. RBC cytogel and EBC were implanted into the subcutaneous spaces, and whole blood clot (NBC) and Floseal™ were used as control. Four implants were placed per rat, and the skin incisions were closed with sutures. On Day 3, 7, 14, 28, 42, and 56, rats were euthanized by 5% isoflurane induction followedby CO2 inhalation. Subcutaneous regions of interest were excised. The size of the implants was measured and normalized to the initial size to represent the in vivo degradation. Excised samples were then fixed in 4% paraformaldehyde solution and embedded in paraffin for histological analysis to evaluate the biocompatibility.

[0399] On Day 3, 7, 14, 21 and 28, vital organs including heart, lungs, liver, kidneys and spleen were harvested. The biodistribution of degradation product (i.e. HA-TZ-F) was evaluated by visualizing the fluorescence in those organs using the I VIS spectrofluorophotometer in vivo imaging. To determine systemic toxicity, on Day 28, vital organs were collected, formalin-fixed, and paraffin-embedded for further histological analysis.In vivo rat hemorrhage model of liver incision

[0400] For hemostatic sealing of the deep incisional hepatic injury, the rats were anesthetized using isoflurane (4% isoflurane in oxygen) in an induction chamber. Anesthesia was maintained at 2% isoflurane using a nose cone during the surgery. A volume of 1 mL of saline was injected subcutaneously. Abdominal hair was removed, and the rats were placed over a heating pad for the duration of the surgery. The liver was exposed via a laparotomy. A laceration wound of 7 mm in length and 3 mm in depth was made to the liver using a #11 scalpel. Immediately after wiping off the blood using a gauze, around 500 pL of EBC or commercial hemostatic matrix Floseal™ was applied to the injury site. The time to hemostasis was recorded by a stopwatch. The amount of blood loss until bleeding stops was determined by measuring the weight of blood lost and absorbed to a pre-weighed gauze. After the surgery, the rats were euthanized by 5% isoflurane induction followed by CO2 inhalation.In vivo rat hemorrhage model of liver puncture

[0401] Following the same procedures of anesthesia and laparotomy, a volumetric injury of 4 mm in diameter and 3 mm in depth was made to the liver using a biopsy punch. Immediately after wiping off the blood using a gauze, around 500 pL of EBC or commercial hemostatic matrix Floseal™ was injected onto the wound bed. The time to hemostasis was recorded by a stopwatch. The amount of blood loss until bleeding stops was determined by measuring the weight of blood lost and absorbed to a pre-weighed gauze. After hemostasis was achieved, exposed liver was placed back, the abdominal incision was closed with sutures, and the animals were left to recover. On Day 5, 10, 14 and 28 after the surgery, blood was collected from saphenous vein. Whole blood was used for hematology test and serum was used for blood chemistry test. On Day 5, 14 and28, the rats were euthanized by 5% isoflurane induction followed by CO2 inhalation. Livers with the hemostatic materials were excised and fixed in 4% paraformaldehyde solution for 48 hours for histological analysis.Histology and analysis

[0402] Fixed tissue samples were placed into 70% ethanol and submitted for histological processing and hematoxylin and eosin stain (H&E) staining at the Histology Core at McGill University. Z.-H.G. is the pathologist-in-chief at the University of British Columbia examined all histological sections.Statistical analysis

[0403] A sample size of N > 3 was used for all experiments. Data is shown as mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA and post hoc Tukey tests for multiple comparisons or Student’s t-tests for comparison between two groups (Prism 9). P values < 0.05 were considered statistically significant.Cytogel design

[0404] Highly cellularized materials, characterized by an overwhelming cell content, encompass a wide range of materials, including blood clots, biological tissues, engineered tissues, cell aggregates and organoids. Developing highly cellularized materials with superior mechanical performance has remained a challenge. Blood clots are one example. Blood clots contain approximately 45% red blood cells (RBC) by volume that have long been considered as “bystanders” for the formation and mechanics of blood clots. As the content of mechanically ineffective cells is much higher than the structural polymer fibrin (0.57-1.15% by volume), native blood clots are inherently weak and vulnerable to fracture. Also, they take minutes to form due to slow coagulation processes. These factors have limited the performance and application scope of blood clots. They cannot stop bleeding under severe hemorrhage, causing 43% and 90% of deaths following traumatic injury in civilian and military settings, respectively. Moreover, failed formation, rupture, and detachment of blood clots at wound sites substantially impair regeneration of various tissues including bone, cartilage, muscle, liver, and periodontal tissues.

[0405] Traditional strategies to improve mechanical properties of biomaterials are generally focused on designing polymer networks. They are thus inapplicable to highly cellularized materials due to the deficiency of structural polymers, and the sensitivity of cells and associatedbiological processes. For blood clots, enhancing fibrin crosslinking and platelet contraction stiffens clots but sacrifices their stretchability and toughness. In addition, the slow formation of blood clots (fibrin assembly) cannot be bypassed by introducing coagulation factors. Alternatively, leveraging the abundant cells (e.g., RBCs in blood clots) can potentially circumvent these challenges. However, previously reported approaches, despite the improvement in clotting speed, either lead to mechanically weak gels or have issues of hemolysis and disturbed fibrin formation. Examples include blood gelation via agglutinating RBCs using positively charged polymers or with the addition of hydrophobic or aromatic moieties. To engineer blood clots and other highly cellularized materials, an improved strategy should integrate cells into mechanically strong and tough gels in a bioorthogonal manner so that the cells could serve as mechanically effective units, while maintaining their biological functions.

[0406] It is provided herein the design and characterization of cytogels, composed of high- density crosslinked cells as building elements, and establish their application in engineered blood clots (EBCs) to enhance hemostasis and regeneration. The cytogels developed herein exhibited superior biomechanical performance, distinguishing them from conventional hydrogels where the mechanical behavior is primarily governed by the polymeric network, as well as cell aggregates that possess limited mechanical properties and require lengthy preparation procedures (Figs. 1A- 1C). To form the tough cytogels, a strategy referred to herein “click clotting” was used. The high- density cells were crosslinked with a small amount of polymer linkers through bioorthogonal click reactions. Unlike other approaches reliant on electrostatic interactions, hydrophobic insertion, or receptor-ligand recognition, the present strategy enables rapid and effective mechanical integration and toughening of cells (Figs. 2A-2E). In one example, RBCs and the TCO-tetrazine reaction was used as a non-limitative model system (Figs. 3A-3B). It was presently demonstrated the successful formation of RBC cytogels, despite the low structural polymer content. By implementing click clotting in native blood, it was also demonstrated that ultrafast-clotting EBCs composed of interpenetrating RBC and fibrin networks can be formed (Fig. 3B). The specific structure and cells’ energy dissipation capacity confer EBCs with remarkable fracture resistance (Fig. 3C). With the inherent regenerative activity, EBCs could outperform existing biomaterials by simultaneously enabling effective hemostasis and regeneration (Fig. 3D).Formation of cytogels

[0407] To form cytogels, two design criteria were considered: (i) covalent linkages should be formed bioorthogonally between cells and polymer linkers; (ii) a long-range connected cellnetwork should be constructed by sufficient linkages using proper cell density, polymer size and concentration. The design strategy is exemplified by the RBC gel as a model system. To satisfy the first design criterion, the click reaction of trans-cyclooctyne (TCO) / tetrazine (TZ) was chosen, which is a bioorthogonal and ultrafast to form covalent linkages. Instead of inserting click motifs into the fluid-like lipid bilayer of cell membranes, TCO groups were conjugated covalently to membrane proteins, some of which (e.g., BAND 3) were tethered to the underlying cytoskeleton (spectrin network) and can provide robust anchorage (Fig. 3A). The TZ groups were grafted to long-chain polymers such as hyaluronic acid (HA) via carbodiimide chemistry (Scheme 1 and Fig. 4). Confocal imaging confirmed the presence and even distribution of TCO on the RBC surface (Figs. 5A-5B) and the lack of fluorescence for unmodified RBCs (Figs. 6A-6B). Quantitively, a high density of TCO was measured at 5.4 x 107per RBC and the TCO was stable on the RBC surface over 21 days (Figs. 7A-7C). The degree of substitution of HA (TZ to carboxyl) was 4-10% dependent on the feed ratio of TZ (Table 1).Scheme 1.A = the repeating unit of polymers, such as saccharide unit, amino acid, acrylic unit, ethylene glycol , etc.B = the click chemistry group including tetrazine, azide, TCO, DBCO, etc.R and R' are reactive functional groups including amine, carboxylic acid, aldehyde, thiol, hydroxyl and click chemistry groups. They reaction can be achieved by carbodiimide chemistry, Schiff base chemistry, Michael addition, click chemistry, etc.Table 1. Degree of substitution of tetrazine calculated from1H NMR

[0408] Mixing the modified RBCs (RBC-TCO) and polymer linkers (HA-TZ) resulted in cytogels via click clotting in less than 5 seconds. The plateaued storage modulus G' of 200 Pa was much larger than the loss modulus G" of 25 Pa (Fig. 8A), whereas the mixture of unmodified RBCs and polymer linkers remained as liquid. The RBC cytogels were stiffer as shown with much higher G', and more elastic over a wide frequency range, compared to viscoelastic blood gels formed by electrostatic and / or hydrophobic interactions (Fig. 8B). All the viscoelastic blood gels feature a similar tan 5 of ~0.5, in contrast to the present RBC gel with tan 5 of -0.1 (Fig. 9). Moreover, the RBC gel and the resulting EBC are highly deformable (Figs. 10A-10B). These mechanical properties were absorbed to the effective integration of the cytoskeleton via protein- mediated covalent linkage: even though cell membrane is lysed, the proteins tethered to the cytoskeleton still serve as crosslinkers to maintain gel integrity (Figs. 11A-11 B).

[0409] To meet the second criterion of long-range connectivity (Fig. 12A), the volume fraction of RBCs, the size and concentration of polymer linkers were systematically varied. When less than 2% by volume of cells were included (1.3x108cell / mL), it was found that the cells were too sparse to form a long-range network. With increasing cell content, the shear modulus increased to a peak value at a cell volume ratio of 33% (2.1 x109cell / mL) (Fig. 12B). Even higher cell content (50%) leads to a decrease of modulus possibly due to poor mixing of the over-crowded cells and the polymer linkers. Using the optimal cell density, the effect of polymer linkers was further studied. Interestingly, a preferred hydrodynamic size (33 - 53 nm) was determined, which decides whether or not the polymer linker can crosslink cells into gels (Figs. 13A-13I). This hydrodynamic size relates to the electro-repulsive distance between cells. The hydrodynamic size should be larger than the distance to allow gel formation. This effect is applicable to other cells because cell surface are mostly negatively charged. However, the hydrodynamic size will vary based on the cell type and cell size. Considering that the polymer linker can only react with modified cells andthat a finite gap (over 20 nm) exists between RBCs due to the electrostatic repulsion of negatively charged cell membranes (Fig. 12A), it was rationalized that large polymer linkers can mend the gap between cells. Besides the polymer size, the concentration and TZ ratio of polymer linkers determines the linkage density among cells and thus the shear moduli of RBC cytogels (Figs. 14A-14B). Altogether, it is presently demonstrated, in one example, that proper RBC volume ratio (33%), polymer size (137 nm), and polymer concentration (1-2%) jointly resulted in sufficient linkages, which were suitable for the long-range continuous network formation of cytogels.

[0410] The present strategy to form cytogels is broadly applicable to different polymers and cells when the aforementioned criteria are met. Screening various polymer linkers, were formed cytogels with alginate, chitosan and polyacrylic acid of large hydrodynamic sizes (Figs. 15-15B). Also, the present design strategy was validated with various cells, including fibroblasts, adipose- derived stem cells, and microalgae cells. (Figs. 16A-16G, 17A-17C, and 18A-18C). The present strategy can rapidly assemble cells into mechanically robust living materials with high cell content and high cell viability (>89%), which technically are challenging and time consuming to achieve using conventional methods. The cytogels can be used to engineer tissues and living constructs with tailored functionality.Mechanical properties of tough cytogels

[0411] The design of RBC cytogels endow them with unique mechanical responses. Despite previous knowledge on cell crosslinking, the mechanism and mechanics of cell-crosslinked gels had remained poorly understood. As the cells are covalently integrated and highly dissipative, it was hypothesized that the cells could contribute substantially to the fracture properties of RBC cytogels. To test this point, the fracture energy of the RBC cytogels was characterized using modified lap-shear tests (Figs. 19A-19C). The measured fracture energy showed a nonmonotonic trend with respect to the cell volume ratio (Fig. 19D), which was consistent with that of the shear modulus (Fig. 12B). Specifically, the fracture energy peaks at the RBC volume ratio of 33%, comparable to the RBC content in native whole blood. Despite the viscoelastic nature of cells, the fracture energy was not sensitive to the concentration of polymer linkers (Fig. 19E). The result implies that the fracture of cell-crosslinked gels is dominated by cell rupture, which is different from conventional polymeric hydrogels whose fracture toughness increase with increasing polymer content. In support of this point, a weak dependence on loading rate was also found (Fig. 19F). As the energy required to rupture an individual cell becomes the limiting factor,the macroscopic fracture energy is closely related to cell volume rather than the polymer concentration.

[0412] To further investigate the mechanism underlying the fracture of RBC cytogels, the dissipative property of the gels was evaluated with cyclic tensile tests (Figs. 19G-19I). The RBC cytogels were subjected to two loading-unloading cycles at three different maximum stretches. The hysteresis (the area between loading and unloading paths) was significant during the first cycle but disappeared in the following cycles (Fig. 19J), reminiscent of Mullins effect in elastomers and tough hydrogels. This characteristic was presumably attributed to the rupture of RBCs, dissipating energy under large deformation and causing permanent damage. As the rupture of RBCs would release intracellular hemoglobin, the amount of hemoglobin released from the RBC gel post-test was further measured. A linear correlation was observed between released hemoglobin and stretch ratio (Fig. 19K).

[0413] Based on the mechanical testing results, a finite element model was developed to simulate the fracture of RBC cytogels as described above. A cohesive zone model was used to simulate the crack tip opening and the Mullins model to capture the energy dissipation in the bulk (Figs. 20A-20D). Parameterized with the cyclic test results, the finite element model with the Mullins effect captures the force-displacement curve and the fracture energy calculated from the lap-shear tests (Figs. 21A-21 B). By contrast, the model without the Mullins effect predicts a much lower fracture energy, signifying the role of energy dissipation in toughening RBC cytogels, which is deduced to be due to cell rupture.Design and mechanics of engineered blood clots

[0414] As the formation of cytogels is bioorthogonal, click clotting could work in unison with native coagulation cascades in whole blood. The resulting clots, termed engineered blood clots (EBC), exhibit instantaneous clotting and enhanced mechanical properties. To test so, EBC were formed by mixing RBC-TCO in anticoagulated whole blood and HA-TZ in PBS or platelet-rich- plasma (PRP) solutions with calcium added. Given the distinct reaction kinetics, rapid solidification was expected due to RBC crosslinking and subsequent stiffening by the formation of a fibrin network. The former can be readily tuned by varying the biorthogonal reaction (Figs. 22A-22C). With the ultrafast TCO-TZ reaction, Fig. 22D shows the storage modulus (G’) well exceeds the loss modulus (G”) instantaneously at the beginning, and gradually increases to a plateau of 250 Pa, indicating both clotting mechanisms take place. The measured gelation time(i.e., the time when G’ > G”) of EBC was significantly smaller than that of native blood clots (NBC), which is beneficial for rapid hemorrhage control (Fig. 22E). The clotting time can be tuned with other click reactions, for instance, dibenzocyclooctyne (DBCO)-azide reaction, which leads to a clotting time around 2-3 minutes (Fig. 22F). To this end, the present strategy of forming tough cytogels is amendable to various click reactions tailorable for specific applications requiring varied time windows.

[0415] To verify the formation of the RBC network and fibrin network, the microstructure of EBC was characterized with confocal imaging and scanning electron microscopy. The individual components were labelled, including RBCs, HA-TZ and fibrin, with fluorescence. Figs. 23A-23C shows RBCs with red fluorescence clustered by the blue HA-TZ, proving the click clotting of the RBC network. A green fibrous network interpenetrated with the RBC network reflects the formation of fibrin. The interpenetrating network of EBC was further revealed in the reconstructed 3D confocal images (Figs. 24A-24D). SEM images show the cell-cell adhesion via polymer crosslink in EBC, while unmodified RBCs did not adhere to each other within the blood clots (Figs. 25A-25G). These results substantiate excellent compatibility between click clotting and native coagulation mechanism and full structural integration of RBCs into the EBC.

[0416] The structure of EBC was expected to result in high fracture toughness. To test this point and compare EBC with NBC and a commercial hemostat Floseal™ (Baxter), modified lapshear tests were performed to measure their fracture energy (Fig. 26A). The fracture energy of EBC (25 J m-2) is 10 times and 8 times higher than NBC and Floseal™, respectively (Fig. 26B). By raising the fibrin content to its physiological level, EBCh were made with even higher fracture energy 34 J rrr2. In addition, the burst pressure of different materials was measured using a burst chamber setup (Figs. 26C-26F). Both RBC cytogels and EBC achieve much higher burst pressure, compared to NBC and Floseal™ (Fig. 26G). Specifically, the burst pressure obtained with EBCh (59 mmHg) is around 3 times that of NBC (20 mmHg). Given the coupling between bulk and adhesion properties, it was hypothesized that the present design could enhance the adhesion energy of EBC on biological tissues. Peeling tests were performed (Figs. 26H-26K) and measured the adhesion energy of EBC to be 6.3 J nr2, around 5 times higher than that of native clots (Fig. 26L).

[0417] The present strategy is advantageous over previously reported methods using chitosan to crosslink RBCs, which lead to brittle clots, hemolysis, or inconsistent clotting (Figs. 27A-27H). EBCs outperform many biological tissues (e.g., liver, spleen) and polymeric hydrogels(e.g., gelatin, alginate) at the similar content of structural polymers. The high toughness of EBC is attributed to the cell-based toughening mechanism and the interpenetrating network structure, an analogue of double-network gels. Crosslinked RBCs dissipate considerable energy through membrane rupture when EBC is stressed, while the fibrin network maintains physical integrity. Further, as the matrix of EBC is more dissipative and fibrin and platelets enable interfacial bonding with tissues, the EBC affords tougher bioadhesion. The excellent mechanical properties, together with ultrafast clotting kinetics, make EBC useful for various applications such as surgical sealants, drug / cell delivery carriers and adhesive scaffolds.Biocompatibility and biodegradability of EBC

[0418] To evaluate the translation potential of EBC, a series of tests were conducted including in vitro experiments to evaluate cytocompatibility and hemocompatibility, as well as in vivo rodent experiments to assess biocompatibility and biodegradability. First, immortalized human vocal fold fibroblasts were cultured with EBC extract for 24 hours. The LI VE / DEAD assay indicated excellent cytocompatibility of EBC, with approximately 100% cell viability (Figs. 28A-28F). Moreover, EBC demonstrated the ability to promote cell proliferation when serum supplement is absent in the media, thanks to the regenerative properties of blood clots, containing growth factors and cytokines. These results suggest that EBC are suitable to serve as a bioactive matrix to facilitate cell growth.

[0419] Regarding hemocompatibility, the impact of EBC on hemolysis, platelet function, and immune cell responses was investigated within the blood. The results showed that the cytogel induced no hemolysis, in contrast to chitosan, a commonly used hemostatic agent, which induced significant hemolysis (Figs. 29A-29B). Further, EBC did not adversely affect platelets and leukocytes in circulation, as it did not activate or destroy these blood components, including platelets, monocytes, granulocytes, B lymphocytes and NK cells. Notably, there was minimal activation of MHCII monocytes, CD4 T cells and CD8 T cells (Figs. 30A-30M). The excellent blood compatibility of EBC can be attributed to its native components and bioorthogonal crosslinking, making it suitable for blood-contacting applications such as hemostatic sealants and embolization agents.

[0420] To further confirm the biocompatibility of EBC, subcutaneous implantation was performed in rodent models and compared it with NBC, RBC cytogel, and Floseal™ (Fig. 31). Histological analysis was conducted on explanted samples on Days 3, 7, 14, and 28. On Day 3post-implantation, all materials led to minimal presence and invasion of lymphocytes and plasmocytes, indicating mild levels of inflammation (degree 1 or 2) (Figs. 32A-32H, 33A-33H, 34A-34H, and 35A-35H). Importantly, the inflammatory response to EBC and RBC cytogels was reduced to no or very mild levels of inflammation (degree 0 or 1) on Day 7 and thereafter (Fig. 36). In contrast, Floseal™ exhibited more inflammatory reactions, with increased fibrosis and numbers of lymphocytes, plasmocytes, fibroblasts, and macrophages, resulting in moderate inflammation that persisted on Day 14. Overall, EBC demonstrated excellent biocompatibility comparable to autologous materials.

[0421] The biodegradation of EBC was also evaluated through subcutaneous implantation in rats for 8 weeks (Figs. 37A-37U). On Day 3 post-implantation, significant swelling was observed in Floseal™ and RBC gel, while EBC exhibited minimal swelling. This can be attributed to the fibrin network of EBC, which can resist the swelling of hydrophilic polymers. These implants, while maintaining overall integrity, gradually decreased in size over time. EBC and RBC gel degraded significantly slower and completely absorbed within 8 weeks, in contrast to NBC and Floseal™ (Figs. 37B-37U). The different degradation profiles can be explained by the availability and activity of enzymes that can degrade gelatin and hyaluronic acid. Prolonged degradation of EBC can extend the duration of implants in vivo. Furthermore, the biodistribution of EBC degradation products were investigated using fluorescent labelling and IVIS imaging. As expected, the biodistribution mapping over 28 days revealed the liver and kidney as the main clearance pathways (Figs. 38A-38K). Histological analysis showed no evidence of systematic toxicity in major organs on Day 28, suggesting the long-term safety of EBC for implantation.EBC promotes hemostasis and regeneration

[0422] The combination of excellent mechanical and biological properties of EBC makes it as a promising solution for hemostasis, tissue repair and regeneration. EBC can be easily delivered using common syringes and catheters and offers high usability. Moreover, its precursors can be stored in fridge before usage, as modified RBCs stored in a regular refrigerator for 7 days showed no significant changes in shear moduli (Figs. 39A-39C). Together with the clinically use of human blood and the TCO-TZ chemistry, the present EBC offers usability, stability, and suitability for clinical use.

[0423] The efficacy of EBC was initially evaluated as an injectable hemostatic sealant for managing noncompressible hemorrhage, which is an unmet clinical challenge associated with>35% of trauma deaths. Using a rat liver deep incision hemorrhage model, the hemostatic efficacy of EBC was compared to Floseal™. Incisions of 7 mm in length and 3 mm in depth were created and then EBC or Floseal™ were applied to the bleeding liver (Figs. 40A-40B). The instantaneous clotting ability of EBC allowed to quickly seal the bleeding site. Additionally, the in-situ formed EBC exhibited toughness, stretchability, and adhesiveness, preventing clot rupture or detachment (Fig. 8B). The results demonstrated that EBC significantly reduced blood loss from 1813 mg to 26 mg (Fig. 40C) and decreased bleeding time from 265 s to 5.4 s (Fig. 40D). In contrast, Floseal™ absorbed the blood and slowly formed mechanically weak clots, resulting in 145 mg of blood loss and 90.6 s of bleeding time. The excellent performance of EBC can be attributed to its ultrafast gelation, high fracture toughness, and strong adhesion.

[0424] To further examine the hemostatic and regenerative capacity of EBC, the EBC was used it as a scaffolding material for treating volume defects in a rat liver hemorrhage model. A tissue defect was created measuring 4 mm in diameter and 3 mm in depth, and then applied EBC or Floseal™ to the wound for hemostasis and subsequent regeneration (Figs. 41A-41C). Similar to the previous test, EBC significantly reduced both blood loss and bleeding time (Figs. 41 D-41 E). Although there was no significant difference in hemostatic efficacy compared to Floseal™, that EBC could better promote regeneration and minimize foreign body reactions. This assumption is based on the use of autologous blood and a minimal amount of biodegradable HA in EBC.

[0425] To test this hypothesis, systemic responses and local regeneration profiles were monitored through blood tests and histological analysis over time. The blood test results indicated no effect on RBCs, but an increase in platelets and white blood cells was observed in the Floseal™ group on Day 5 post-surgery (Figs. 42A-42L). The increase in inflammation-related cells was primarily linked to lymphocytes (Fig. 43). Furthermore, physiological parameters related to organ function were examined, including glucose, urea, cholesterol, lipase, total protein, globulins, bilirubin, albumin, alkaline phosphatase (ALP), y-glutamyltransferase (GGT), alanine transaminase (ALT), and aspartate transaminase (AST) (Figs. 44A-44O). Most parameters remained within normal levels, indicating normal organ functions despite the liver defect and the presence of biomaterials. Notably, albumin levels showed a slight decrease on Day 5 and recovery on Day 10, with the EBC group consistently exhibiting higher albumin levels compared to the Floseal™ group (Fig. 440). This difference may be attributed to variations in liver regeneration rates. These results suggest that the foreign materials applied locally to the injury sites caused systemic inflammation, while EBC did not significantly affect blood cell parameters due to its excellent biocompatibility.

[0426] The local liver regeneration profiles were further examined through visual examination and histological analysis, revealing significant differences between EBC and Floseal™ in promoting tissue regeneration (Figs. 45A-45F). Digital photos showed injured livers gradually regenerated with the degradation of EBC or Floseal™. On Day 14, a visible capsule with newly formed blood vessels was observed in the Floseal™ group, indicating a typical foreign body response (Figs. 46A-46R). EBC fully regenerated the injured liver to its pristine state on Day 28, whereas scars were found on the Floseal™ administrated livers (Figs. 45A-45F). When examining microscope images of the explants, it was found that EBC greatly promoted liver regeneration with a fast regeneration rate (78% on Day 5, 82% on Day 10, and around 100% on Day 28), while Floseal™ led to much slower regeneration (20% on Day 5, 51 % on Day 10, and 84% on Day 28) (Fig. 47A). Histological analysis of inflammation levels showed that EBC led to very mild to mild inflammation within the two weeks post-injury (Fig. 47B), resembling the native wound healing process and facilitating tissue regeneration. In contrast, Floseal™ caused severe inflammation, as evidenced by a significant accumulation of lymphocytes and some plasmocytes around the material, which is consistent with the hematology test results and likely to impair regeneration. Additionally, a thick fibrosis capsule was observed surrounding Floseal™, whereas there was no fibrosis observed with EBC, further highlighting the advantages of the cytogels formed with autologous cells over foreign body responses in engineered materials. Moreover, EBC significantly reduced the occurrence of postoperative adhesion complications compared to Floseal™ (17% vs. 83%, Figs. 45A-45F and Fig. 47C). Overall, EBC, as a native scaffolding material, can promote tissue regeneration with minimal inflammation and foreign body responses, and prevent postoperative adhesions, outperforming the clinically used products.Conclusions

[0427] To summarize, it is herein reported the design and mechanics of cytogels created through the click clotting strategy, and its application as EBC for treating non-compressible hemorrhage. By incorporating crosslinked RBCs and a small amount of polymer linkers, the clotting process of EBC was remarkably accelerated to less than five seconds, while enhancing fracture toughness, burst pressure, and adhesion energy by factors of 13, 3, and 5, respectively. The present comprehensive investigations, including experiments and modelling, indicate that crosslinked RBCs play a pivotal role in enhancing the dissipation and toughening properties of the clots. The resulting EBCs exhibit significant potential for hemostasis and tissue regeneration, while avoiding adverse effects such as inflammation, foreign body reactions, and postoperative adhesions. Furthermore, the utilization of self-supply human blood, combined with the inherentbiocompatibility and bioactivities of blood clots, makes them a versatile and affordable solution for various therapeutic applications, encompassing bleeding control, embolization, immune modulation, and regenerative medicine. The present work establishes the design and application of tough cytogels, underscores the prospects of engineered blood clots for future clinical utilization, and opens up various avenues for engineered living materials with unprecedented properties.EXAMPLE 2

[0428] The clotting cytogel can also be used as an embolic material. The properties of an embolic material is that it should (1) be radiopaque so that it is visible on common imaging modalities such as angiography, computed tomography (CT) imaging, magnetic resonance imaging (MRI), and ultrasound (US) imaging, (2) not cause imaging artifacts or distortions, (3) be easily injectable through clinically used microcatheters and should be non-adhesive to the catheter material to prevent trapping of the catheter within the vasculature, (4) have adequate mechanical properties so that it is able to form a stable occlusion which is resistant to fragmentation and migration, (5) be tunable to allow for use in various applications, (6) be highly biocompatible to prevent potential adverse complications, (7) allow for cell proliferation and promote the healing of the diseased vessel, and (8) be biodegradable. The clotting cytogel fulfills these properties and thus can be used as an embolic agent.Design and Synthesis

[0429] The design of EBCs was inspired by native blood clots (NBCs), which serve as natural hemostatic agents to halt excessive bleeding during injuries. NBCs possess many advantages that are not seen in today’s clinically used embolic materials. Clots form a temporary barrier, sealing the vessel from bleeding and allowing the tissue repair process to begin. They also create a framework for new tissue to grow into, forming a scaffold for wound healing. However, while NBCs have many advantages when it comes to embolization, they also have many drawbacks which has limited their use in clinical interventional radiology. Here NBCs were further engineered, forming Engineered Blood Clots (EBCs), which possess the intrinsic advantages of NBCs but are designed to be easily used in an interventional radiology clinical setting. To form EBCs that can be effectively used for embolization the design combines the native clotting process with bioorthogonal crosslinking of red blood cells and biodegradable polymer linkers. This results in a dual-network, cellular-based hydrogel that demonstrates excellent mechanical properties andbiocompatibility. The choice of bioorthogonal crosslinking is an important one because it ensures high biocompatibility and will not disrupt the biological system. This contrasts with the current clinically used treatments, such as Onyx™ or n-BCA, which require harsh chemicals or solvents for gelation. Further, the dual-network structure of EBCs and the high deformability of RBCs contribute to their superior mechanical properties. Resulting in a robust embolic material which can form a stable occlusive seal that is mechanically resistant to fragmentation.

[0430] Hyaluronic acid (HA) was chosen as the polymer linker due to its high biocompatibility, degradation in vivo, and long hydrodynamic size which allows for effective linking of the cells. Carboxyl groups present on HA were modified with click chemistry functional groups (either DBCO or Tz) using EDC / NHS chemistry.1H NMR was employed to confirm the successful modification of the resulting polymers and quantify the degree of substitution of the click moiety.Functionalized Hyaluronic Acid Synthesis

[0431] Hyaluronic acid (HA, MW ~ 2000 kDa) used for EBC formation was purchased from Lyphar Biotech. Carboxyl groups on HA were modified with a click chemistry functional group — either dibenzocyclooctyne (DBCO) or tetrazine (Tz) — using EDC / NHS carbodiimide chemistry following previously established protocols (Scheme 2). First, sodium hyaluronate was dissolved at a concentration of 0.5% wt / v in a 100 mM MES buffer solution (0.3 M NaCI, pH 6.0). EDC and NHS (Sigma-Aldrich, 03450 and 130672, respectively) were added at 2.5-times molar excess of the carboxyl groups of HA, followed by either sulfo DBCO-amine or methyltetrazine amine (Click Chemistry) at 0.75 mmol per gram of HA. The reaction was stirred at room temperature for 24 hours and subsequently transferred to 14 kDa MWCO dialysis tubing (Sigma-Aldrich, D9527) and dialyzed for 5 days against a decreasing salt gradient from 150 mM to 0 mM NaCI (150 mM , 100 mM, 50 mM, 0 mM, 0 mM, for 24 hours per solution). To obtain sterilized HA, the solution was subsequently dialyzed in a 50% v / v ethanol solution for 24 hours followed by DI water for 2 days (changing the solution every 24 hours). After dialysis, the solution was frozen at - 80°C overnight and lyophilized (Labconco™ Freezone 4.5L - 84°C) for 48 hours.Scheme 2.sulfo DBCO-amine

[0432] Scheme 2 shows the synthesis of HA-DBCO via carbodiimide chemistry. Through the use of EDC and NHS, the amine group of sulfo DBCO-amine is conjugated to the carboxyl group of HA, forming HA-DBCO. A similar reaction is used to synthesize HA-Tz, except methyltetrazine- amine is used instead of sulfo DBCO-amine.Functionalized Hyaluronic Acid1H NMR Characterization

[0433] The resulting functionalized polymers were characterized using1H nuclear magnetic resonance spectrometry (1H NMR, 400 MHz Varian Mercury). Deuterium oxide (D2O) was used as the solvent and the results were analyzed to obtain the degree of substitution (DS%) of the DBCO or Tz functional group to carboxyl groups on HA. Chemical shift values were reported in parts per million (ppm). The degree of substitution (DS%) was calculated by the following equation:

[0434] where lcand Ncare the integral value and number of protons for the click moieties ( δ =7.27 - 7.51 ppm for DBCO, and 8 =7.47 - 7.57 ppm or 8 =8.40 - 8.30 ppm for Tz). IHA and NHA are those for the N-acetyl methyl groups on HA ( 8 =1 .9 - 2.0 ppm).

[0435] Proton peaks between 7.27 - 7.51 ppm account for the 8 hydrogens on the aromatic rings of the DBCO moiety. The peak at 1.9 - 2.0 ppm corresponds to the 3 hydrogens of the N- acetyl methyl (-CH3) groups on HA. The peaks between 7.47 - 7.57 ppm and 8.40 - 8.30 ppm correspond to 2 hydrogens each on the phenyl ring of the Tz moiety. The degree of substitution was calculated to be 8 - 14% for both HA-DBCO and HA-Tz.Red Blood Cell Surface Modification

[0436] RBC membranes were modified with the complementary click motif through carbodiimide chemistry — either Az for HA-DBCO or TCO for HA-Tz. Pegylated labelling reagents containing the click motif are linked to an NHS ester moiety which covalently binds to primary amines located on RBC surface membrane proteins. Confocal imaging was used to confirm the presence and even distribution of these click motifs on the surface of RBC-Az modified cells (Fig. 48A), whereas negligible fluorescent signal was detected on unmodified RBCs (Fig. 48B).

[0437] RBCs used for cell surface modification were obtained from bovine whole blood in CPD anticoagulant (Lampire Biological Laboratories, 7200804). RBC surfaces were modified with the complementary click chemistry motif through a reaction between primary amines located on the cell surface and NHS esters containing the click moiety either Az for HA-DBCO or TCO for HA-Tz. First, citrated whole blood was centrifuged (1000g, 5 min) to separate the RBCs from the plasma. The separated RBCs were then thoroughly washed three times with cold PBS (9:1 PBS to RBC ratio) and centrifuged to remove the supernatant. Following washing, RBCs were resuspended in PBS at a 9:1 PBS to RBC ratio followed by the addition of a stock solution (25 mg / mL dissolved in DMSO) of either Azido-PEG4-NHS Ester or TCO-PEG4-NHS Ester (Click Chemistry Tools, AZ103 and A137 respectively) to achieve a final concentration of 1 mg / mL. The solution was then incubated at 37°C for 45 minutes to allow the reaction to complete. Following incubation, the solution was centrifuged, and the supernatant was gradually replaced with fresh PBS as to slowly remove the DMSO to prevent hemolysis from occurring, followed by another three complete PBS washes and the complete removal of the supernatant.Modified Red Blood Cell Confocal Imaging

[0438] RBC surface modification was observed using confocal imaging. Az-modified RBCs were suspended in PBS at a 9:1 PBS to RBC ratio and incubated with a Cy5 DBCO fluorescent probe (Click Chemistry Tools, A130) at a concentration of 10 pg / mL for 1 hour at 37°C (see Fig.5A as per Example 1). Following incubation, the modified cells were thoroughly washed with PBS three times to remove the unreacted fluorescent probe. The fluorescently labelled RBCs were then diluted in PBS at a 100:1 PBS to RBC ratio and imaged using a confocal laser scanning microscope (Zeiss, LSM710).Preparation of EBCs

[0439] EBCs used for embolization were prepared by forming a crosslinked cellular network of RBCs, integrated with the native fibrin network and other blood components. Functionalized HA was dissolved overnight in Visipaque™ 320 (GE Healthcare, V-560) at various concentrations ranging from 1% to 4% (wt / v). Modified whole blood was obtained by substituting native RBCs with modified RBCs suspended in an equal volume of platelet-rich plasma (PRP) obtained by centrifuging whole blood at 300g for 15 minutes. To initiate the native and click clotting simultaneously, modified blood was recalcified using a calcium chloride (CaCh) solution at a final concentration of 30 mM. Equal volumes of modified blood and the HA solution were then quickly mixed in syringes until fully homogeneous. For example, an initial 2% wt / v HA-DBCO solution mixed with an equal volume of modified blood results in a final polymer concentration of 1 % wt / v and a 25% v / v cell ratio.Scanning Electron Microscopy (SEM)

[0440] The morphology of EBC was observed using a field emission scanning electron microscope (FE450™, FEI) with an accelerating voltage of 10 kV under various magnifications. EBC samples were fixed with 4% paraformaldehyde and subsequently dehydrated using an increasing ethanol gradient from 30% to 100% (v / v). The dried samples were then coated under 4 nm of platinum, using a high-resolution sputter coater (ACE600™, Leica), to increase surface conductivity.Native Blood Clot Formation

[0441] Native blood clots (NBCs) were formed by recalcifying using a CaCh solution at a final concentration of 30 mM. Following recalcification, the blood clot was incubated at 37°C for 1 hour and sealed to maintain humidity during the coagulation process.Rheological Analysis

[0442] All rheological measurements were made using the Discovery™ HR-2 rheometer (TA Instruments) with a 20-mm steel parallel plate and 1-mm gap at a temperature of 37°C, to mimic physiological temperatures. Mineral oil was applied around the circumference of the plate to prevent material dehydration during testing. The Storage modulus (G’), loss modulus (G”), and gelation kinetics were measured using a time sweep over 3600s at 0.1% shear strain and 1 Hz. The time to full gelation was defined as the time it takes for the storage modulus to reach 95% of the plateau value. A frequency sweep was performed at frequencies from 0.001 to 100 Hz at 0.5% shear strain.Radiopacity

[0443] Computed tomography (CT) imaging of EBC samples was performed using a Bruker Skyscan™ 1172 microCT system. Samples containing 0, 10, 20, or 30 (% wt / v) of iodixanol were loaded into 1-mL syringes and loaded into the microCT scanner. Syringes containing Visipaque™ 320 were tested for comparison. The acquisition was realized using the following parameters: 75 kV and 420 mA. The radiopacity of the different EBC compositions was evaluated using NRECON™ 3D reconstruction software (Bruker) by calculating the mean X-ray attenuation in Hounsfield units (HU).

[0444] One important design parameter when developing an embolic material is its radiopacity. A material must be radiopaque to allow for visibility of the material by the interventionalist during the procedure. To do so, the radiopaque agent iodixanol (Visipaque™ 320, 652 mg / mL of iodixanol) was incorporated into EBCs. Iodixanol is an iodine-based, non-ionic, isosmotic radiocontrast agent that is visible on the common imaging modalities, such as angiography, CT imaging, and MRI. Further, iodixanol does not cause imaging artifacts, which are associated with tantalum powders or metallic coils, and impair the evaluation of the diseased area on follow-up imaging. The use of iodixanol as a radiopaque agent in hydrogel results in nonpermanent visibility due to a rapid release of the contrast agent after injection. Non-permanent visibility is suitable for clinical applications as radiopacity is only required during the embolization procedure, while longterm radiopacity could affect follow-up imaging. The radiopacity of EBCs with varying concentrations of iodixanol was evaluated in vitro, using microCT imaging (Fig. 49A). Concentrations exceeding 30% wt / v iodixanol led to notable declines in EBC mechanical properties. However, the incorporation of 30% wt / v iodixanol provided sufficient signal intensityfor satisfactory visibility and did not significantly impact EBC mechanical properties (Fig. 49B), which were marginally stiffer compared to those of NBCs (Fig. 49C). EBCs with a 30% wt / v iodixanol composition were used for subsequent tests.Injection force measurement

[0445] An injection force test was performed to measure the injectability of EBCs through clinically used microcatheters. A 3-mL syringe (BD Luer-Lock™ tip, 309657) containing 1 mL of material was fixed to the lower gripper of a mechanical tester (Instron™ model 5965). The syringe plunger was depressed by a compression plate connected to a 1-kN load cell at a constant flow rate of 1 mL / min. The samples were injected through 150 cm 2.4Fr SuperCrossa™ microcatheters (Teleflex™, 5305) into a PBS solution. The injection force, which was defined as the plateau force, and the cohesion of the material after injection were both analyzed. Injection of Onyx™ 34 (Medtronic) and Visipaque™ 320 were used as references.

[0446] Another important parameter in the design of an embolic material is the injectability. An embolic material must be injectable through long microcatheters but undergo fast gelation to form a stable, robust occlusion upon injection. The rheological properties of a material will dictate its injectability. In particular, a material’s shear-thinning properties are an important aspect of designing injectable materials. EBCs exhibit shear-thinning behaviour; the complex viscosity decreases as the frequency increases (Fig. 50A). To understand how the shear-thinning properties change over time, a frequency sweep was performed on EBCs immediately after mixing and after 1 hour, allowed to fully gel. Both EBCs (initial and after full gelation) exhibited shear-thinning behaviours. This is crucial to ensure continuous delivery throughout the injection window. To test the injectability of EBCs the force required to inject the material through a 2.4Fr microcatheter was measured. The applied force increases until it reaches a plateau force, defined as the injection force (Fig. 50B and 50C). At this point, the material would begin to extrude from the catheter. EBCs formed a continuous, cohesive stream upon injection from 2.4Fr and 5Fr catheters. Rupture of RBCs after injection through a 2.4Fr microcatheter was negligible (< 10% hemolysis and < 5% for polymer concentrations under 2% wt / v) (Fig. 50D). The injection force was measured for both EBCDBCO and EBCTZ, and compared to that of clinically used materials Onyx™ 34 and Visipaque™ 320 (Fig. 50E). EBCTZ can be considered to represent the fully gelled state. While EBCs have a larger injection force than that of Onyx™ and Visipaque™ it is still considered to be injectable. Even the fully gelled form, EBCTZ, is still injectable, but with some difficulties. Decreasing the polymer concentration makes EBC easier to inject (Fig. 50F), however,it also results in reduced mechanical properties. 1 % wt / v HA-DBCO was chosen as the ideal comprise between injectability and sufficient mechanical properties. However, the polymer concentration can be varied depending on the application or desired outcomes.Fracture Toughness Measurement

[0447] A modified lap shear test was performed to measure the fracture toughness of EBCs. Samples were injected into a 20x15x1.5 mm3PTFE mold and covered with a PTFE sheet. EBC and NBC samples were sealed and incubated at 37°C for 1 hour to allow for full gelation. After gelation, samples were removed from the molds and glued to two thin polyethylene terephthalate (PET) films on each side as rigid backings. An initial edge crack of 5 mm was introduced in the middle of each sample. The specimens were loaded vertically onto an Instron™ machine equipped with a 10-N load cell with a displacement rate set at 2 mm / min. The fracture toughness ( r ) was calculated as the total work (Wt) (i.e. the area under the force-displacement curve) divided by the fracture surface area (A):

[0448] The fracture surface area (A) is the product of the sample width and the total crack length. The total crack length is defined as the sample length minus the initial crack length. The fracture surfaces were examined post-testing to confirm cohesive failure. The fracture toughness of NBCs and Onyx™ 34 were measured for comparison.Enhanced Fracture Toughness

[0449] A requirement of the ideal embolic material is the ability to form a robust, stable occlusion, resistant to fragmentation or migration. Current clinically used liquid embolic agents, such as Onyx™ or n-BCA, are extremely fragile which can result in fracture of the material after injection. Other materials, such as Gelfoam™ or autologous blood clots, are injected as fragments which result in a distal embolization. However, uneven distribution of particle sizes limits the reproducibility and predictability of the embolization. EBCs are injected as a cohesive mass, relying on a combination of native fibrin formation and strong covalent crosslinking to form tough, dual-network cytogel in situ. This structure of EBCs promises high resistance against fracture. To test this point, the fracture toughness of EBCs was measured using a modified lap-shear test (Fig. 51A). Fracture toughness of EBCs (55.94 J / m2) were enhanced around 8-fold compared to thatof NBCs (6.06 J / m2) and Onyx (6.91 J / m2) (Fig. 51 B). The enhanced fracture toughness of EBCs can be attributed to its unique structure. The two interpenetrating networks work symbiotically, highly elastic, crosslinked RBCs dissipate energy, while structural integrity is maintained through the fibrin network.Adhesion Measurement

[0450] A 180-degree peeling test was performed to measure the adhesion energy between EBCs and various materials. Fresh bovine arteries (Montpak International, Terrebonne, Canada) were cut along the length of the vessel to create a flat surface. Samples were injected into a 30x10x1.5 mm3elastomer mold which was applied to the inner lumen of the arteries. For adhesion tests on the subendothelial layer, the endothelium was delicately removed through gentle rubbing with a cotton thread. Samples were sealed and incubated at 37°C for 1 hour to allow for full gelation. No compression was applied to the specimens. After gelation, the elastomer molds were carefully removed, and thin PET films were glued to the EBC and backside of the vessel as rigid backings. An initial crack of 5 mm was introduced between the EBC and the blood vessel. The specimens were attached to an Instron™ machine equipped with a 10-N load cell with a displacement rate set at 100 mm / min (Fig. 51 C). The test was repeated to measure the adhesion between EBCs and a thin nylon film (McMaster-Carr, 8539K192). The adhesion energy (G) was calculated as two-times the plateau force (F-, / ,0 / 1) divided by the width of the specimen (w):Endovascular Adhesion

[0451] Another important mechanical property to consider for embolization procedures is the adhesiveness of a material. This pertains to the ability of the embolic agent to adhere to both the endovascular surface and the delivery device during the operation. Onyx™ is advertised as a nonadhesive embolic agent because it does not adhere to the catheter, thereby reducing the risk of catheter entrapment within the vessel. In contrast, the highly adhesive n-BCA embolic agents can reflux around the catheter and trap it within the vasculature, leading to serious complications. Ideal embolic agents should strongly adhere to the vasculature to reinforce occlusion and reduce migration, but not to the catheter. This criterion could be met with EBCs, wherein fibrin and platelets enable the selective adhesion to the vasculature. Specifically, fibrin can form covalentbonds with collagen, the main component of the subendothelial layer, that is exposed during vascular injury (Fig. 51 D). Platelets have also shown to adhere to the endothelium which can then bind with fibrin / fibrinogen.

[0452] The adhesion of EBCs was tested to various substrates using a 180-degree peeling test (Fig. 51 E). EBCs were able to form an interfacial bond with the endothelial layer of a bovine artery (Fig. 51 F). However, this resulted in relatively small adhesion energies (3.58 J / m2). The adhesion energy was enhanced around 2-fold (8.31 J / m2) when the adhesion was tested between EBCs and the subendothelial layer (Fig. 51 F). The adhesion of between EBCs and the subendothelial layer was measured to model a situation where embolization would occur on an inflamed or damaged vessel, such as in the case of hemorrhage control. While EBCs demonstrated adhesion towards the vasculature they were non-adhesive towards catheters. The adhesion between EBCs and nylon, a commonly used material in the fabrication of microcatheters, was measured to test potential catheter adhesion. However, EBC adhesion with nylon could not withstand the force of gravity, so the exact adhesion energy was below the detection limit of this method (Fig. 51 F).Volumetric Swelling Measurement

[0453] Material samples were prepared into disk shapes 5 mm in diameter and 1.5 mm in height, immersed in PBS in a sealed Petri dish, and incubated at 37°C. After 24 hours the swollen diameters and heights were measured using a digital caliper. Swelling was quantified using the volumetric swelling ratio, which was calculated as the volume of the swollen state (7S) divided by the initial sample volume (70:Swelling Properties

[0454] Material swelling is another important aspect of embolization, as it ensures a tight occlusive seal of the artery. The volumetric swelling ratio (VSR) was measured to understand the swelling mechanics of EBCs (Fig. 51 G). Samples were formed in pre-determined sizes and incubated in PBS for 24 hours before measuring the VSR. NBCs have a VSR < 1 due to the clot contraction, driven by activated platelets and causing compaction of the fibrin network along with compression of the embedded RBCs. However, EBCs do not demonstrate clot contraction eventhough they contain both platelets and a fibrin network. Unlike NBCs, the fibrin network does not solely dictate the structure of EBCs — as activated platelets are unable to contract the interconnected RBC-polymer network. EBCs demonstrate slight swelling (VSR = 1.16) in PBS. Onyx™ swells much more than both NBCs and EBCs (VSR = 1.83). This is important because Onyx™ is non-adhesive so expansion of the material after injection — due to EVOH precipitation — is what ensures an occlusive seal.Gelation Mechanisms and Kinetics

[0455] Mixing of functionalized polymer linkers and modified RBCs results in the instant formation of a cellular crosslinked cytogel. Both bioorthogonal reactions explored — HA-DBCO + RBC-Az and HA-Tz + RBC-TCO — resulted in an initial storage modulus (G’O) which was larger than the initial loss modulus (G”0); while mixing unmodified RBCs and HA results in a G’ which is similar to G”, behaving as a viscous solution (Fig. 52A). The structure was examined by SEM showing the cellular-based nature of the material (Fig. 52B). Due to the large size difference between RBCs (8 pm: diameter) and the HA polymer linkers (150 nm: effective diameter), HA acts as a cellular glue, which covalently binds the cells together (Fig. 52C). Click-modified cell surface proteins can bind and crosslink with multiple HA linkers, resulting in an interconnected RBC network in conjunction with the native fibrin network.

[0456] To understand the gelation kinetics of the click clotting process the bioorthogonal and native clotting processes were uncoupled. A rheological time sweep was performed on EBCs without the addition of Ca2+ions, to prevent the fibrin network formation (Fig. 52D). These results demonstrate the gelation occurring solely from the bioorthogonal reaction between the HA polymer linkers and the modified RBCs. Here, two separate bioorthogonal reactions were demonstrate with distinct reaction kinetics. The IEDDA reaction involving HA-Tz and RBC-TCO exhibits extremely fast kinetics, resulting in a nearly instantaneous gelation of EBCTz. In contrast, the SPAAC reaction between HA-DBCO and RBC-Az demonstrates much slower kinetics. Specifically, EBCDBCO undergoes a more gradual gelation process, showing a gradual increase of G’ before reaching a plateau value.

[0457] Further, rheological tests were conducted of varying cell-polymer mixtures to probe the effect of cell - polymer interactions on EBC formation. Because the functionalized HA is acting as a cellular crosslinking agent, varying the density of cell - polymer crosslinks will dramatically influence the rheological properties. By increasing the HA-DBCO concentration in EBCDBCO anincrease in G’ was observed (Fig. 52E), while G” remained generally constant. It follows that increasing the polymer concentration results in a stiffer, more elastic gel. Interestingly, a critical polymer concentration of 0.5% wt / v was found which resulted in a sufficient number of linkages to form a stable gel. Polymer concentrations of less than 0.5% w / v resulted in poor rheological properties for the present conditions tested. Varying the RBC density results in a similar influence on the rheological properties (Fig. 52F).

[0458] It was found that the elastic properties plateau at cell densities of 25% v / v. Further increases in cell densities may result in over-crowding of RBCs and poor mixing with HA. A cell density of 25% v / v corresponds to mixing equal volumes of functionalized HA with modified blood — since blood contains around 50% hematocrit. Further, it was found that cell densities of less than 5% v / v resulted in poor rheological properties, as cells are too sparse to form an interconnected network.In Vitro Vasculature Flow Model

[0459] In vitro vasculature phantoms were fabricated by casting PDMS (Sylgard™ 184, Dow Corning) around sacrificial PVA molds. Sacrificial molds were 3D-printed (Ender-3™ V2, Creality) from water-soluble PVA filaments (PVA 3D Printer Filament, Monoprice). After printing, the molds were briefly immersed in warm water to smooth the surface. PDMS (10:1 base to crosslinker ratio) was then degassed in a vacuum chamber before being poured over the 3D-printed molds. After casting, the assembly was placed in an oven at 60°C for 3 hours to allow for full curing of the PDMS (Fig. 53A). After curing, the assembly was placed in an ultrasonic water bath at 50°C until the PVA was fully dissolved (Fig. 53B).

[0460] The vasculature phantoms were connected to a biomimetic pulsatile pump (TrandoMed™, EDUP120) which simulates the activity of the human heart. The phantoms were connected to the pump via silicone tubes, 3 mm in diameter, representing a feeding artery and a draining vein. A third silicone tube was also connected proximal to the vasculature phantoms via a Y-connector, to allow for pressure release after embolization. The vasculature phantom was specifically designed to model an AVM, where the deep penetration of the embolic material into the nidus is required; the size of the phantom was modeled to be physiologically relevant. The vasculature phantom had vessels of various diameters, from 1 mm to 3 mm, with a total volume of 2652 mm3. The system was perfused with a water-glycerol solution (3:2 ratio of water to glycerol) with the temperature set to 37°C. To mimic physiological conditions, the pulsatile pumpparameters were set as follows: pulsation frequency — 60 bpm; pressure at pulsation output end — 90 mmHg; flow at pulsation output end — 220 mL / min. A 2.4Fr microcatheter (SuperCrossa™ 5305, Teleflex) was inserted into the system via a hemostatic Y-adapter proximal to the phantom. EBC was injected into the system to embolize the phantom vasculature until the material reached the draining vein or reflux occurred into the feeding artery. Following embolization, the depth of penetration of the embolic material into the vasculature phantom was analyzed.In Vitro Occlusion Model

[0461] An in vitro occlusion test was performed to evaluate the embolic properties of EBCs. PDMS tubes (3 mm inside diameter and 1.5 mm thickness) were used as blood vessel phantoms. The diameter of the PDMS phantom was chosen to match that of a small artery which would be typically targeted for embolization. A water-glycerol solution, consisting of a 60:40 (% v / v) ratio of water to glycerol, was used as a blood-mimicking fluid due to its similar viscosity to blood. The water-glycerol solution was flowed through the tubes with the use of a syringe pump. A 5Fr catheter (Cook Medical, G13794) was fed into the tubing to inject the EBC directly into the PDMS vessel phantom. The catheter was removed, and the flow rate of the syringe pump was set to 10 mL / min. The pressure was monitored upstream with a pressure gauge. The pressure-time curves, including the maximum embolization pressure, were recorded. The tests were repeated with NBCs and Onyx™ 34 as comparisons. The test was also repeated using bovine arteries (average diameter: 4.23 mm ± 0.44 mm), Montpak International, Terrebonne, Canada) as the occlusion vessel. The arteries were connected to the syringe pump with the use of zip-ties. Anticoagulated bovine whole blood was flowed through the system and the pressure was monitored upstream.In Vitro Embolization Model

[0462] An in vitro embolization model was designed to test whether EBCs could form a stable occlusion, resistant to physiological blood pressures without fragmentation or displacement. The model consisted of PDMS tubes (diameter: 3 mm) as vessel phantoms and a water-glycerol solution with a viscosity matching that of blood (dynamic viscosity: 5 cP). A syringe pump was used to flow the water-glycerol solution through the PDMS vessel phantom, while a pressure gauge monitored the pressure of occlusion. Both EBCs and Onyx were able to successfully embolize the vessel phantoms at superphysiological pressures (p > 120 mmHg) — demonstrating their ability to effectively form an occlusive seal, blocking blood flow (Fig. 54A). EBCs with 1 % wt / v HA-DBCO were able to form a stable occlusion at pressures over 3-times the systolic bloodpressure (403.26 mmHg). Importantly, the failure modes for these materials are drastically different, which could be attributed to their different mechanical properties (Fig. 54B). EBCs are soft and tough and fail by elastic leak — this is when the seal fails entirely by elastic deformation, meaning the seal leaks without any material damage. This is demonstrated by the embolization pressure-time curve (Fig. 54C), where EBCs initially fail at a maximum leak initiation pressure (p , and then plateau at a steady-state leaking pressure (ps). The embolic material undergoes no fragmentation or migration within the PDMS tube, therefore, the seal can reform if the pressure is lowered to below that of ps. This is demonstrated in Fig. 54C, where the pressure is completely removed, and the seal is allowed to reform. psis highly repeatable cycle-to- cycle, while pi is reduced after the first cycle — this is due to fluid trapped at the interface between gel and PDMS reducing the friction. The elastic deformation leading to the elastic leak failure can be seen through cusping of the EBC at the upstream face of the material; it can also be observed that the initial leak path and the steady-state leaking, where the EBC remains stable within the PDMS phantom. The material was removed from the PDMS phantom after testing to reveal it remains completely intact, with no observable material damage.

[0463] Conversely, Onyx™ is stiff and brittle, and fails by rupture — which results in permanent damage and potential fragmentation of the material. While the maximum embolization pressure of Onyx™ is similar to that for EBCs (Fig. 54A), the failure mode is drastically different. After the failure of Onyx™, there is no steady-state leaking pressure that occurs — the pressure drops rapidly to zero (Fig. 54D). This indicates no elastic leak occurs and the failure is entirely due to irreversible material damage. In this case, the seal cannot be reformed, and fracture of the material occurs. This can be observed through images showing the fragmentation of the material during testing, and after the material was removed from the PDMS phantom.

[0464] Comparable results were observed from ex vivo occlusion studies with explanted bovine arteries. It is worth noting a larger drop in pressure from ptto ps, for the EBC occlusion, than what is observed in the in vitro, PDMS model (Fig. 54E). This is potentially due to greater adhesion and friction between the EBC and bovine endovascular layer, as well as the elastic deformation of the artery due to pressure build-up. In this case, EBCs were able to form a stable occlusion at superphysiological pressures greater than that of NBCs, but comparable to Onyx™ (Fig. 54F).Elastic Leak for Reliable Embolization

[0465] The failure behaviour of an embolic material is an important consideration as it relates to the design of an embolic agent. Failure of an embolic material by elastic leak provides a more reliable occlusion as it acts as a safety valve of sorts, allowing for pressure release without irreversible material damage — which can cause fragmentation of the embolic agent and possible off-target complications. A seal fails by elastic leak when the critical pressure for elastic leak is larger than the rupture failure pressure (Pc> Pf). The critical pressure for elastic leak (Pc) and the rupture failure pressure (Pf) are theoretically derived from equations, from which the criteria for failure by elastic leak is defined as:

[0466] where r is the fracture toughness, is the shear modulus, H is the seal diameter, and A is a dimensionless measure of precompression. Fig. 55A shows a phase diagram of the failure modes for embolization. At a fixed precompression A and given a set of geometric parameters, the mechanical properties of a seal will determine the failure mode — by elastic leak or by rupture. H / L represents the non-dimensionalized geometric parameter (L is the seal length) while r / pL represents the non-dimensionalized material parameter.

[0467] The term Pc / Ptrepresents the failure mode of embolization. When Pc / Pf< 1 , the seal fails by elastic leak. Conversely, when Pc / Pf> 1 the seal will fail by rupture (Fig. 55B). For a given precompression and vessel diameter, there exists a critical r / ^ ratio — termed ( r / / z)c— below which the embolic material will fail by rupture rather than elastic leak. It follows that the r / p ratio should be optimized for an embolic material if resistance to fragmentation is desired. Fig. 55C shows an r -p Ashby plot of the embolic materials tested in this study. The shear modulus was determined from the material’s storage modulus (a measure of elastic response) which is analogous to the shear modulus in purely elastic materials. EBCs exhibit a r / p ratio greater than both NBCs and Onyx™, giving them the ability to provide a more reliable embolization, resistant to fragmentation, setting them apart from stiff and brittle materials.

[0468] The in vitro occlusion experiments were repeated while varying the injection rate and the geometric parameters. Herein it is reported that the steady-state leaking pressure (ps) and not the leakinitiation pressure (pi), as the critical elastic leak pressure (Pc) predicts steady-state leaking; ps= Pc. From Fig. 55D, pswas determined as insensitive to changes in the injection rate,agreeing with the theoretical model and experimental. In particular, it was expected that psto depend only on the material and geometric parameters of the system. Indeed, by varying the geometric parameter H / L a change in pswas observed (Fig. 55E). By changing the diameter of the PDMS tube, while fixing the seal length, it was observed that increasing H / L reduces ps, while decreasing H / L increases ps. At a value of H / L = 0.125, no steady-state leaking is observed (the pressure drops to zero) indicating elastic leak did not occur (Fig. 55F).Tunable Gelation Kinetics

[0469] The varied considerations of the embolization procedure underscore the need for a versatile, tunable material system. Among the factors clinicians consider when selecting an embolic material for a particular application is its penetration depth. Certain applications call for deep penetration into the vasculature, while others require a material to be more mechanically robust immediately upon injection. For instance, in preoperative procedures for liver resections, where temporary embolization is used to induce hypertrophy of the future liver remnant, precise penetration into specific liver vasculature is important. Here the tunability of EBCs’ gelation kinetics was demonstrated using a range of bioorthogonal and natural reactions (Fig. 56A). The bioorthogonality of the RBC crosslinking process allows to combine this with the native clotting cascade. When combining EBCTZ with PRP and Ca2+ions, instantaneous gelation was observed via RBC Tz - TCO crosslinking, followed by a gradual stiffening due to the fibrin network formation (Fig. 56B). By substituting the ultrafast Tz-TCO reaction with the slower DBCO-Az crosslinking (EBCDBCO) the gelation kinetics can be slowed (Fig. 56B). Further, as both reactions are highly selective, they are mutually orthogonal. As such, utilizing these two reactions in combination provides further control over the gelation kinetics. Combining equal volumes of HA-Tz and HA- DBCO, and by extension equal volumes of RBC-TCO and RBC-Az, results in gelation kinetics falling between the extremes of EBCTZ and EBCDBCO. For all tested conditions, the initial storage modulus (G’O) exceeds the initial loss modulus (G”0), indicating instant gelation. In this case, the gelation kinetics was quantified by the time to full gelation — defined as the time it takes for G’ to reach 95% of its plateau value. The time to full gelation ranges from around 5 minutes for EBCTZ to around 18 minutes for EBCDBCO (Fig. 56C).

[0470] The gelation time was expected to influence the penetration depth. To measure the depth of penetration of the different EBC formulations, an in vitro vasculature model was used, where PDMS phantoms were connected to a pulsatile pump set to emulate physiological conditions. EBCs were injected through 2.4Fr microcatheters proximal to the phantom modelsand the depth of penetration was observed. After injection, slow-gelling EBCDBCO can flow deep into the vasculature, reaching the draining vein (Fig. 56D). Conversely, fast-gelling EBCTZ was deposited directly at the catheter tip and did not flow due to the instantaneous click clotting (Fig. 56E). After injection EBCTz would reflux around the catheter, forming a proximal plug in the feeding artery.Cytotoxicity

[0471] Cytotoxicity tests were performed using immortalized human vocal fold fibroblasts following ISO 10993-5 standards. Extracts were prepared by incubating EBC in cell culture medium for 24 hours, while 20 000 cells per well were seeded in a 96-well plate. For every 200 mg of EBC, 1 mL of Dulbecco’s Modified Eagle’s medium (DMEM) was added. Following incubation, extracts were supplemented with 1% penicillin-streptomycin and either with or without 10% fetal bovine serum (FBS). The culture medium within the 96-well plate was then replaced with the supplemented extracts. Completed pristine DMEM was used as a positive control and extract of Onyx™ 34 was used as a comparison. The cells were cultured for 24 hours inside an incubator, in an environment at 37 °C, 95% relative humidity, and 5% CO2 atmosphere. Cell viability was assessed using a Live / Dead viability kit (Invitrogen, L3224), following the protocol of the manufacturer. Confocal laser scanning microscopy (Zeiss, LSM710) was used for investigation. Live cells were visualized in green and dead cells in red. The images were analyzed using Imaged™ software (National Institutes of Health). Cell viability was defined as the percentage of live cells in the sample, while the cell density of a sample was used to measure cell proliferation.Cytocompabitlity and Cell Proliferation

[0472] In vitro tests were conducted to evaluate the safety and efficacy of EBCs for in vivo use. Cytotoxicity of EBCs was evaluated by culturing immortalized human vocal fold fibroblasts (hVFFs) with EBC extract media for 24 hours. Cells cultured in DMEM were used as the positive control and cells cultured in Onyx™ extract media were used as a comparison. Tests were repeated for all samples with and without the addition of fetal bovine serum (FBS), to evaluate a material’s ability to support cell proliferation. A live / dead assay showed high cell viability (> 97%) for all samples, with the exception of Onyx™, which had much lower cell viability — 26.7% without FBS and 31.8% with FBS (Fig. 58A). The low cell viability result from Onyx™ can be explained from the use of DMSO, a cytotoxic organic solvent, which is dissipated into its surroundings afterthe injection of the material. Complications, such as vasospasm and systemic toxicity, have been associated with the use of DMSO in Onyx™. Conversely, EBCs demonstrate the ability to substantially promote cell proliferation even when FBS supplement is absent in the media. Results without FBS show a significant increase in cell density after 24 hours when cells are cultured in EBC extract, compared with the control (Fig. 58B). The regenerative capacity of EBCs illustrates its potential as a bioactive material that can improve healing outcomes when used in embolization procedures. To demonstrate the hemocompatibility of EBCs, assays were conducted in accordance with the ISO 10993-4 protocol to assess their hemolytic potential. EBCs exhibited relatively low hemolysis (3.62%), comparable to NBCs (2.38%) and clinically used metallic coils (2.06%) (Fig. 58C). A hemolysis rate below 5% is considered permissible for blood-contacting materials; the hemolysis induced by a gelatin slurry was significantly higher (6.83%).In Vitro Degradation

[0473] In vitro degradation assays were performed to measure the fibrinolysis and enzymatic degradation of EBCs. To measure the effect of fibrinolysis, EBCs and NBCs were incubated in plasma supplemented with tissue plasminogen activator (tPA). All samples were first washed thoroughly in PBS to remove excess Ca2+ions. After, samples were prepared of the same size and weighed using an analytical balance at time 0. Plasma was obtained by centrifugation of bovine whole blood at 1000g for 5 minutes. 10 pg / mL of tPA (Sigma-Aldrich, T0831) was added to the plasma. The samples were sealed and incubated in the supplemented plasma at 37°C under gentle agitation (100 rpm). At pre-determined time points, the plasma was removed, and the remaining material was weighed. At each time point the plasma was replaced with fresh supplemented plasma.

[0474] EBCs were incubated in PBS containing hyaluronidase to measure the enzymatic degradation. The degradation of Onyx™ 34 was measured for comparison. 10 pg / mL of hyaluronidase (bovine testicular hyaluronidase, Sigma-Aldrich, H3506) and 0.01 % wt / v sodium azide (NaN3, Sigma-Aldrich, S2002) were added to PBS. The samples were incubated in the supplemented PBS, sealed, and placed at 37°C under gentle agitation (100 rpm). At predetermined time points, the PBS was removed, and the remaining material was weighed. At each time point the PBS was removed and replaced with fresh supplemented PBS.In Vitro Biodegradation

[0475] Material degradation is an important aspect of temporary embolization as it ensures normal blood flow can be restored once the underlying issue has been addressed. The use of autologous blood clots for embolization was associated with particularly fast degradation and subsequent recanalization. An in vitro degradation assay was performed to measure the effect of fibrinolysis on EBCs, compared to NBCs. Materials were incubated in bovine plasma supplemented with tPA, which activates the fibrinolytic system, degrading NBCs and the associated fibrin network. EBCs, however, are resistant to fibrinolysis, showing minimal degradation after 6 hours, while NBCs show significant clot lysis (Fig. 58A and 58B) — only -40% of the mass remains after 6 hours. Further, EBCs can maintain structural integrity even after degradation of the fibrin network and complete RBC lysis, strictly through HA crosslinked through proteins tethered to the cell’s cytoskeleton. However, EBCs are biodegradable through an enzymatic process. Hyaluronidase, which is present within human plasma, mediates the degradation of the HA component of EBCs. EBCs fully degrade within 10 days when incubated in PBS supplemented with hyaluronidase, compared to Onyx™, which remains even after 14 days (Fig. 58C); Onyx™ is not biodegradable and results in a permanent embolization.Rat Abdominal Artery Embolization

[0476] In vivo rat embolization procedures were approved by the McGill University Animal Care Committee and performed according to the guidelines of the Canadian Council on Animal Care. Female Sprague Dawley rats (250a - 300g, n = 4) were purchased from Charles River Laboratories (Wilmington, USA). Initially, rats were anesthetized using isoflurane (4% isoflurane in oxygen) in an induction chamber. Anesthesia was maintained at 2% isoflurane using a nose cone during the surgery. After anesthesia, the rats were placed in a supine position for the duration of the surgery. Initially, before the surgery, the hindlimb skin color was observed and recorded. Abdominal hair was removed, and the area was cleaned with an antiseptic solution. A 1 cm incision was made longitudinally along the abdomen of the rat and the abdominal artery was exposed. 100 pL of EBC was injected distally into the artery using a 30G needle. After injection, the incision was closed with a 5-0 polypropylene suture. Throughout the surgery, the hindlimb skin color was observed. Post-surgery, rats were euthanized and subjected to microCT scanning imaging (Mediso™ nanoScan PET / CT) to confirm embolization. All images were recorded and analyzed using Imaged™ software to produce three-dimensional (3D) images.In Vivo Embolization

[0477] To assess the safety and efficacy of EBCs as an embolic material, an in vivo experiment was conducted wherein the abdominal artery of a rat was embolized using EBC before testing in a larger porcine model. The study aimed to test the feasibility of injecting EBCs into small vessels, its mechanical stability post-injection, and its retention at the injection site without causing nontarget embolization. 100 pL of EBCDBCO containing 1% wt / v HA-DBCO was injected distally into the abdominal artery of rats. EBCs were easily injectable through 1-mL syringes with a 30G needle. Following injection, the hindlimbs of the rats exhibited a gradual darkening and a noticeable decrease in temperature. Within 10 minutes after injection the colour of the rats’ hindlimbs were visibly darker. This indicates an interruption of hindlimb perfusion signifying successful embolization and occlusion of the artery. Following the procedure, the animals were euthanized. Radiopacity of EBCs and absence of non-target distal migration or fragmentation was confirmed through post-procedure microCT imaging (Mediso nanoScan PET / CT). Hyperdense spots were visible near the proximal side of the injection, while an absence of hyperdense spots near the distal side indicates a stable occlusion without fragmentation or distal migration after embolization with EBCs (Figs. 59A-59B). This result is further illustrated by the 3D reconstructions of the microCT images which show that the EBC remains at the injection site (Fig. 59C).Porcine Embolization Model

[0478] In vivo porcine embolization procedures were conducted according to the guidelines of the Canadian Council on Animal Care and approved by the Institutional Animal Protection Committee at the University of Montreal Hospital Research Centre (CRCHUM). Pigs (n = 2) were acclimatized for seven days prior to the experiment. Two days before the procedure, blood was harvested from the animals to allow for preparation of the embolic agent. Pigs were fasted (food only) the day before the experiment. For the procedure, all animals received xylazine (2 mg / kg) and ketamine (25 mg / kg, intramuscularly) as preanesthetic medication. Anesthesia was induced using propofol (1 .66 mg / kg, intravenously) and maintained throughout the procedure by isoflurane inhalation. After the induction of anesthesia, femoral access was obtained through US guidance. A 5Fr sheath was positioned, followed by the catheterization of the hepatic artery using a 4Fr Cobra catheter. A supraselective catheterization of a segmental artery was performed using a 2.8Fr microcatheter. Digital subtraction angiography (DSA) of the segmental artery was performed to opacify the vessels under fluoroscopic guidance. Then, each segmental artery was embolized using EBCs. Following embolization, a DSA was performed to assess arterialocclusion. The splenic artery; one segmental artery of each renal artery (lower pole); a segmental gastric artery; and a left and right segmental hepatic artery were all embolized following the same methodology. Following the procedure, animals were recovered from anesthesia and survived for 7 days before euthanasia. Prior to euthanasia, animals were re-anesthetized for angiography and terminal imaging.

[0479] Specifically, six distinct arterial sections were targeted for embolization: the stomach, liver (left and right lobes), spleen, and left and right kidneys (inferior pole). EBCDBCO containing a total polymer content of 1 % wt / v HA-DBCO was used as the embolic material. Two days prior to the procedure blood was collected from the pigs; EBCs were formed using the autologous blood components of each animal. Before the procedure the pigs were anesthetized and intubated; the femoral artery was catheterized under US guidance using a 4Fr Cobra catheter. The catheter was then advanced to the common hepatic artery. A supraselective catheterization of the gastroepiploic artery was then performed using a 2.8Fr Progreat microcatheter (Teurmo Interventional Systems) and a baseline digital subtraction angiography (DSA) was obtained (Fig. 60A). Contrast backflow into the gastroduodenal and the superior pancreaticoduodenal artery was observed. After injection of EBC, DSA acquisition demonstrated an immediate and complete embolization of the right gastroepiploic artery — beginning from approximately 15 mm from the gastroduodenal bifurcation (Fig. 60B). Opacification of the superior pancreaticoduodenal and the gastroduodenal arteries was identical to the DSA acquired before embolization suggesting no nontargeted embolization of these segments. The microcatheter was then advanced into the right lateral hepatic artery and a baseline DSA was acquired (Fig. 60D). The right lateral hepatic artery was visible; some backflow of contrast in the left hepatic artery was observed. The right lateral hepatic artery was embolized using EBC; DSA demonstrated a complete embolization of the right lateral hepatic artery (Fig. 60E). The microcatheter was then advanced into the splenic artery; a baseline DSA was acquired (Fig. 60G). Following injection of EBC, the splenic artery was completely embolized (Fig. 60H). The left lateral hepatic artery and the lower polar arteries of both the left and right kidneys were also embolized following the same procedure. Following the procedure, the animals were allowed to recover from anesthesia and survived for 7 days before euthanasia. Prior to euthanasia, animals were reanesthetized for angiography. At the 7-day postembolization mark, all the embolized arteries exhibited either partial or complete recanalization (Fig. 60C, 60F, and 60I). This recanalization suggests that EBC is suitable for temporary vascular occlusion applications, rather than providing a long-term or permanent solution. Following euthanasia, the targeted tissues were analyzed for signs of occlusion. Thenecropsy revealed necrosis of the targeted organs compatible with ischemia due to occlusion of the embolized arteries. Remnants of the EBC was found in the gastroepiploic artery, compatible with the partial recanalization observed from the 7-day post-embolization DSA. The appearance of the EBC was translucent, rather than opaque suggesting an intact HA gel and lysis of the RBC crosslinkers.

[0480] To further validate the safety and efficacy of the Tough Engineered Radiopaque Clots (TERCs) developed with the EBC for future clinical use, in the porcine embolization model, six arterial sections were targeted for embolization: stomach, liver (left and right lobes), spleen, and left and right kidneys (inferior pole). Two days prior to the procedure, blood was collected from the porcine and used the autologous blood to prepare TERCs. The gastroepiploic artery was catheterized and a baseline digital angiography (DA) was obtained. After injection of TERC, digital subtracted angiography (DSA) showed an immediate and complete embolization of the right gastroepiploic artery (Figs. 61A-61 D). Stagnation of contrast at the proximal end of the gel demonstrates a complete occlusion of the targeted segment immediately following embolization. The visibility of the gel can be seen on the DA after the injection of TERC, but before the injection of the contrast agent. Opacification of the superior pancreaticoduodenal and the gastroduodenal arteries was identical to the baseline DA acquired before embolization suggesting no non-targeted embolization of these segments. The microcatheter was then advanced into the splenic artery and a baseline DA was acquired. Following injection of TERC, the splenic artery was completely embolized (Figs. 62A-62D). The left lateral and right lateral hepatic arteries, and the lower polar arteries of both the left and right kidneys were also embolized following the same procedure. It was noted that TERCs are easily injectable and the clotting process allows for a sufficient time window for operation.

[0481] Following the procedure, the animals were recovered from anesthesia and survived for 7 days. Prior to euthanasia, animals were re-anesthetized for angiography. One animal had to be sacrificed at the 1-day time point due to a pulmonary edema and fluid overload induced by a concomitant renal failure post embolization. At the 7-day post-embolization mark, all embolized arteries exhibited either partial or complete recanalization (Figs. 61A-61 D, 62A-62D). This recanalization suggests that TERC is suitable for temporary embolization applications, rather than providing a permanent solution. Following euthanasia, the targeted tissues were analyzed for signs of occlusion. Remnants of TERC was found in the gastroepiploic artery in agreement with the partial recanalization observed from the 7-day post-embolization DSA. The appearance of TERC was translucent, rather than opaque, and histological sections at the 7-day mark showsthe absence of RBCs in the occluding material, suggesting an intact HA gel and clearance of the RBC crosslinkers (Figs. 63A-63B).

[0482] Histological analysis of the embolized arteries revealed the extent of recanalization and inflammation induced by TERCs. While the gel remained in the arteries at the 7-day time point, there was significant evidence of material degradation and vessel recanalization (Fig. 63C). Recanalization was especially evident in larger arteries. Some smaller arteries around the site of embolization remain occluded. Histological analysis of the inflammation levels demonstrated that TERCs led to a very mild or lack of inflammatory response within the artery or arterial walls 7 days post-embolization, evidenced by the lack of immune cells in and around the remaining material. Additionally, embolization with TERCs did not result in fibrosis of the target arteries or remodeling of the vessel with fibrotic connective tissue, suggesting TERC could be used to induce a temporary embolization rather than result in a permanent occlusion.Statistical Analysis

[0483] A sample size of n S? 3 was used for all experiments. Data are shown as mean ± SD. Statistical analysis was performed using one-way ANOVA and post hoc Tukey tests for multiple comparisons or Student’s t-tests for comparison between two groups. P values < 0.05 were considered statistically significant.Discussion

[0484] The present example showed that the cytogel of the present disclosure can be used as a biomaterial for temporary endovascular embolization procedures and combines biocompatibility with superior mechanical properties. Taking a systematic approach, the design and optimization of EBCs was successfully carried out based on clinical needs using in vitro models. By characterizing various EBC formulations, the optimal material system was identified that offers reliable occlusion and tunability of physical properties. In vitro findings were further validated through both rat and porcine in vivo models, confirming the successful use of EBCs for temporary applications. Moreover, the study of the mechanical properties of EBCs provided valuable insights regarding the mechanics of embolization.

Claims

WHAT IS CLAIMED IS:

1. A cytogel comprising: cells functionalized with a first click chemistry group; and polymers functionalized with a second click chemistry group; wherein one of the first click chemistry group or the second click chemistry group comprises a cycloalkene, a heterocycloalkene, a cycloalkyne or a heterocycloalkyne and the other comprises an azide, a tetrazine, a triazine, a pyridazine, or a nitrone; and wherein the first click chemistry group and the second click chemistry group react to form a covalent link between the cells and the polymers.

2. The cytogel of claim 1 , comprising between 2 and 50 % by volume of the cells.

3. The cytogel of claim 2, comprising from 20 to 40 % by volume of the cells.

4. The cytogel of any one of claims 1 to 3, comprising from 1 to 2 wt. % of the polymers.

5. The cytogel of any one of claims 1 to 4, wherein the cells are one or more of red blood cells (RBC), fibroblasts, adipose-derived stem cells, and microalgae cells.

6. The cytogel of any one of claims 1 to 5, wherein the polymers are one or more of hyaluronic acid, fibrin, alginate, chitosan, polyacrylic acid, polyethylene glycol, polyacrylamide, poly(vinylpyrrolidone), polysialic acid, polyvinyl alcohol, gelatin, albumin, dextran, agarose.

7. The cytogel of any one of claims 1 to 6, wherein the cells are red blood cells.

8. The cytogel of any one of claims 1 to 7, wherein the polymer is hyaluronic acid.

9. The cytogel of any one of claims 1 to 8, wherein the polymers have a size of from 50 to250 nm.

10. The cytogel of any one of claims 1 to 9, wherein the cycloalkene is a C3-C10 cycloalkene.

11. The cytogel of any one of claims 1 to 10, wherein the heterocycloalkene is a 3 to 10 membered ring and the heteroatoms are selected from N, O and S.

12. The cytogel of any one of claims 1 to 11 , wherein the cycloalkyne is a C7-C9 cycloalkyne.

13. The cytogel of any one of claims 1 to 12, wherein the heterocycloalkyne is a 7 to 9 membered ring and the heteroatoms are selected from N, O, and S.

14. The cytogel of any one of claims 1 to 8, wherein one of the first click chemistry group or the second click chemistry group comprises the cycloalkene or the heterocycloalkene and the other comprises the tetrazine, the triazine or the pyridazine.

15. The cytogel of any one of claims 1 to 8, wherein one of the one of the first click chemistry group or the second click chemistry group comprises the cycloalkyne and the other comprises the azide, the tetrazine, the triazine, the pyridazine or the nitrone.

16. Use of the cytogel as defined in any one of claims 1 to 15 for bleeding control, tissue repair or tissue regeneration.

17. Use of the cytogel as defined in any one of claims 1 to 15 as an embolic agent.

18. A method for controlling bleeding of a tissue, repairing the tissue, or regenerating the tissue of a subject in need thereof, the method comprising applying the cytogel as defined in any one of claims 1 to 15 on the tissue.

19. A method of producing a cytogel, the method comprising: providing cells functionalized with a first click chemistry group and polymers functionalized with a second click chemistry group; and contacting the cells and the polymers to allow a click chemistry reaction to occur between the first click chemistry group and the second click chemistry group to produce a covalent bond between the cells and the polymers; wherein one of the first click chemistry group or the second click chemistry group comprises a cycloalkene, a heterocycloalkene, a cycloalkyne or a heterocycloalkyne and the other comprises an azide, a tetrazine, a triazine, a pyridazine, or a nitrone.

20. The method of claim 19, wherein the step of providing comprises functionalizing cells with a first click chemistry group and functionalizing polymers with a second click chemistry group.

21. The method of claim 19 or 20, wherein the cycloalkene group is trans-cyclooctene.

22. The method of claim 19 or 20, wherein the cycloalkyne group is selected from00 k23. The method of claim 19 or 20, wherein the heterocycloalkyne group is selected from

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