Hydrolyzable hydrogels and uses thereof

Abstract

The present disclosure provides hydrogels, and more specifically, hydrolyzable hydrogels that contain cleavable ester moieties, and their use in applications such as tissue engineering and therapeutic delivery.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 193,211, filed May 26, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to hydrogels, and more specifically, to hydrolyzable hydrogels that may be used in applications such as tissue engineering and therapeutic delivery. [Background technology]

[0003] Advances in therapeutic delivery mechanisms for the release of cargo molecules and cells are driven by research into cytocompatible biomaterials and encapsulation methods. Synthetic hydrogels, fabricated networks of cross-linked polymeric structures, have been utilized to encapsulate bioactive substances, such as growth factors and cell products, generating 3D structures that can support and regulate cell behavior, with limited effect on viability and efficacy of bioactive cargo (Guan, X., Avci-Adali, M., Alarcin, E., Cheng, H., Kashaf, SS, Li, Y., Chawla, A., Jang, HL, & Khademhosseini, A. (2017). Development of hydrogels for regenerative engineering. Biotechnology Journal, 12(5), 1600394). The tunability of mechanical properties such as stiffness and matrix integrity of these hydrogel systems provides flexibility for use in various microenvironments (Saxena, S., Hansen, C.E., & Lyon, L.A. (2014). Microgel Mechanics in Biomaterial Design. Accounts of Chemical Research, 47(8), 2426-2434, and Guan, X., Avci-Adali, M., Alarcin, E., Cheng, H., Kashaf, S.S., Li, Y., Chawla, A., Jang, H.L., & Khademhosseini, A. (2017). Development of hydrogels for regenerative engineering. Biotechnology Journal, 12(5), 1600394).Furthermore, the ability to implement degradable chemistry for manufacturing constitutes a great advantage for non-invasive regenerative medicine applications, as after degradation, the degraded components can be excreted from the body through renal filtration (Saxena, S., Hansen, C.E., & Lyon, L.A. (2014). Microgel Mechanics in Biomaterial Design. Accounts of Chemical Research, 47(8), 2426-2434, and Ulbrich, K. (1995). Synthesis of novel hydrolytically degradable hydrogels for controlled drug release. Journal of Controlled Release, 34(2), 155-165).

[0004] Hydrogel microparticles (microgels), either in suspension or as building blocks for granular bulk hydrogels, have recently emerged as an attractive platform in biomedical applications due to their highly tunable mechanical properties, injectability, and advanced tissue integration (Daly, A.C.; Riley, L.; Segura, T.; Burdick, J.A. Hydrogel Microparticles for Biomedical Applications. Nat Rev Mater 2020, 5(1), 20-43). One of the design parameters that is directly linked to the physical properties of the microgel (e.g., stiffness, mesh size, etc.) is the degradation rate. Mechanisms of degradable crosslinking of polymers can be broadly classified as enzymatic, photodegradable, hydrolytic, or combinations thereof, imparting varying degrees of control over degradation rates (Koh, J.; Griffin, DR; Archang, MM; Feng, A.-C.; Horn, T.; Margolis, M.; Zalazar, D.; Segura, T.; Scumpia, PO; Di Carlo, D. Enhanced In Vivo Delivery of Stem Cells Using Microporous Annealed Particle Scaffolds. Small 2019, 15(39), 1903147, Griffin, DR; Weaver, WM; Scumpia, PO; Di Carlo, D.; Segura, T. Accelerated Wound Healing by Injectable Microporous Gel Scaffolds Assembled from Annealed Building Blocks. Nature Mater2015,14(7),737-744, Muir,VG;Qazi,TH;Shan,J.;Groll,J.;Burdick,JAInfluence of Microgel Fabrication Technique on Granular Hydrogel Properties.ACS Biomater.Sci.Eng.2021,7(9),4269-4281, Foster,GA;Headen,DM;Gonzalez-Garcia,C.;Salmeron-Sanchez,M.;Shirwan,H.;Garcia,AJProtease-Degradable Microgels for Protein Delivery for Vascularization.Biomaterials2017,113,170-175,Photodegradable Hydrogels for Dynamic Tuning of Physical and Chemical Properties https: / / www.science.org / doi / 10.1126 / science.1169494(accessed2021-10-25), and Carleton, MM; Sefton, MV Injectable and Degradable Methacrylic Acid Hydrogel Alters Macrophage Response in Skeletal Muscle.Biomaterials2019,223,119477). Most of these methods rely on stimuli that are not easily controlled in space or time (Jo, YS; Gantz, J.; Hubbell, JA; Lutolf, M.P. Tailoring Hydrogel Degradation and Drug Release via Neighboring Amino Acid Controlled Ester Hydrolysis. Soft Matter 2009, 5(2), 440-446).

[0005] Various polymer networks and crosslinking structures can be used to tune the degradability and diffusivity of the hydrogel, allowing intricate control of the degradation rate and release profile of this platform (Jain, E., Hill, L., Canning, E., Sell, SA, & Zustiak, SP (2017)). Control of gelation, degradation and physical properties of polyethylene glycol hydrogels through the chemical and physical identity of the crosslinker. Journal of Materials Chemistry B, 5(14), 2679-2691).Several mechanisms for degradable crosslinking of hydrogels have been explored, including enzymatic, photodegradable, ester-based hydrolysis, or combinations of these, with varying degrees of control over degradation rates (Sung, B., Kim, C., & Kim, M.-H. (2015). Biodegradable colloidal microgels with tunable thermosensitive volume phase transitions for controllable drug delivery. Journal of Colloid and Interface Science, 450, 26-33; Stukel, J., Thompson, S., Simon, L., & Willits, R. (2015). Polyethlyene glycol microgels to deliver bioactive nerve growth factor: Microgels to Deliver Bioactive NGF. Journal of Biomedical Materials Research Part A, 103(2), 604-613; and Kloxin, A. M., Kasko, A. M., Salinas, C. N., & Anseth, K. S. (2009). Photodegradable Hydrogels for Dynamic Tuning of Physical and Chemical Properties.Science,324(5923),59-63).An increasingly popular degradation technique focuses on sequence-specific enzymatic degradation, whereby release relies on proteolytic gel degradation performed by cells and endogenous enzyme release (Kroger, SM, Hill, L., Jain, E., Stock, A., Bracher, PJ, He, F., & Zustiak, SP (2020). Design of Hydrolytically Degradable Polyethylene Glycol Crosslinkers for Facile Control of Hydrogel Degradation. Macromolecular Bioscience, 20(10), 2000085, and Lueckgen, A., Garske, DS, Ellinghaus, A., Mooney, DJ, Duda, GN, & Cipitria, A. (2019). Enzymatically-degradable alginate hydrogels promote cell spreading and in vivo tissue infiltration. Biomaterials, 217, 119294). By altering the amino acid sequence of the peptide crosslinker, degradation can be tailored to the type of encapsulated cells as well as the expected implantation environment. Although this method shows promise, degradation is dependent on external stimuli that cannot be easily controlled in space or time (Jo, YS, Gantz, J., Hubbell, JA, & Lutolf, MP (2009). Tailoring hydrogel degradation and drug release via neighboring amino acid-controlled ester hydrolysis. Soft Matter, 5(2), 440-446).In addition, the large amount of enzymatically cleavable peptide linkers used during production makes this an expensive method to scale up to clinically relevant sizes and has the potential to be immunogenic as degradable peptide sequences can be recognized by the host immune system (Griffin, DR, Archang, MM, Kuan, CH, Weaver, WM, Weinstein, JS, Feng, AC, Ruccia, A., Sideris, E., Ragkousis, V., Koh, J., Plikus, MV, Di Carlo, D., Segura, T., & Scumpia, PO (2020). Activating an adaptive immune response from a hydrogel scaffold imparts regenerative wound healing [Preprint]. Bioengineering). Another chemistry that is under much development is photolytic cleavage of hydrogel linkers. This method of degradation relies on an external light source through the use of photocleavable compounds as crosslinkers during fabrication (Ji, H., Xi, K., Zhang, Q., & Jia, X. (2017). Photodegradable hydrogels for external manipulation of cellular microenvironments with real-time monitoring. RSC Advances, 7(39), 24331-24337; Villiou, M., Paez, JI, & del Campo, A. (2020). Photodegradable Hydrogels for Cell Encapsulation and Tissue Adhesion. ACS Applied Materials & Interfaces, 12(34), 37862-37872; and Kloxin, A. M., Kasko, A. M., Salinas, C. N., & Anseth, K. S. (2009). Photodegradable Hydrogels for Dynamic Tuning of Physical and Chemical Properties. Science, 324(5923), 59-63).Photodegradable hydrogels have been used in different applications such as tissue adhesion, where cell-containing hydrogels are depolymerized via a controlled light source, allowing for the immediate release of cells and de-binding from the tissue (Villiou, M., Paez, JI, & del Campo, A. (2020). Photodegradable Hydrogels for Cell Encapsulation and Tissue Adhesion. ACS Applied Materials & Interfaces, 12(34), 37862-37872). This is advantageous for applications such as wound dressings and controlled cell therapy treatments where the release rate can be controlled (Villiou, M., Paez, JI, & del Campo, A. (2020). Photodegradable Hydrogels for Cell Encapsulation and Tissue Adhesion. ACS Applied Materials & Interfaces, 12(34), 37862-37872). However, given the need for patient compliance and limited tissue depth, photodegradation may not be the best option for long-term cargo release.

[0006] There is a clear need for hydrogels capable of controlled degradation in vivo, which may be useful for tissue engineering and therapeutic delivery applications. The present disclosure addresses this need, among others. Summary of the Invention

[0007] The present disclosure provides hydrogels that are hydrolytic and capable of controlled degradation in vivo.The disclosed hydrogels may prove useful in applications ranging from tissue engineering, drug delivery, and regenerative medicine.Compared to previously disclosed degradable hydrogels, such as those that use PEG-based degradable crosslinkers, the disclosed hydrogels show advantages in manufacturing due to their increased hydrophobicity and more compact size.

[0008] In one aspect, a hydrogel is provided that includes a polymer backbone crosslinked with a first crosslinker containing at least one moiety of formula I: [ka] wherein all variables are as defined herein.

[0009] In another embodiment, a process for synthesizing a hydrogel described herein includes reacting a polymer with a first crosslinker comprising at least one moiety of formula I.

[0010] Also provided is a therapeutic delivery composition comprising the hydrogel described herein and one or more therapeutic agents. Also provided is a method of delivering a therapeutic agent to a target site in a subject, the method comprising administering to the target site a therapeutically effective amount of the therapeutic agent described herein.

[0011] Further provided are cell culture media, tissue scaffolds, bioreactors, and wound dressings comprising the hydrogels described herein.

[0012] 1. A method of promoting tissue growth in a subject in need thereof, the method comprising: Identifying a target site; administering to the target site a therapeutically effective amount of a hydrogel described herein.

[0013] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0014] [Figure 1A]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 1B]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 1C]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 1D]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 1E]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 1F]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 1G]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 1H]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 1I]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 1J]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 1K]We show that hydrolytically degradable microgels can be fabricated by the addition of an ester-containing dithiol crosslinker. Figure 1A) PEG-4MAL macromers are modified with linear PEG-FITC and segmented through a flow-focusing microfluidic chip with a continuous phase containing small dithiol molecules, DTT, and EGBMA. This results in monodisperse microgels that can be tracked with fluorescence. Scale bar 1 mm. Figure 1B-E) Size distribution of microgels based on EGBMA concentration in the oil phase. Insets represent the intensity of individual microgels after fabrication, showing similar modification of the macromer backbone with a linear PEG-FITC tracker with a minimum of n=36 pooled from three independent microfluidic runs. Figure 1F-G) Swelling of the microgels in aqueous buffer is directly proportional to the molar concentration of the EGBMA linker in the crosslinked phase, with a minimum of n=6 per sample. Figure 1H) Tracking of released PEG-FITC in solution depends on the EGBMA concentration in the microgels. Figure 1I) Day 3 images of microgels deformed by applied pressure in a tapered microcapillary. Figure 1J) Shear stress vs. strain after 3 days of incubation in aqueous buffer for confined microgels fabricated with various concentrations of EGBMA, n=6, over 60 points in total. 1K) Quantification of shear modulus of all microgel formulations after different times of exposure to aqueous buffer, n=6. Unless otherwise stated, all data are presented as mean ± sem, swelling data were analyzed using a mixed effects model with Tukey correction for multiple comparisons, and shear modulus was analyzed by two-way ANOVA with Tukey correction for multiple comparisons. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. [Figure 2A]We show that in the absence of adhesive and inflammatory cues, microgel co-culture with monocytes does not induce activation. Cell survival after 48 h of incubation does not reveal any changes due to the presence of microparticles in the co-culture. Expression of markers CD45, F4 / 80, CD206 is comparable across all groups tested, d-insets represent fold expression of CD206 relative to all cells expressing F4 / 80 in the co-culture. Minimum n=6, all data are presented as mean±sem. Data were analyzed by one-way ANOVA with Tukey correction for multiple comparisons. [Figure 2B] We show that in the absence of adhesive and inflammatory cues, microgel co-culture with monocytes does not induce activation. Cell survival after 48 h of incubation does not reveal any changes due to the presence of microparticles in the co-culture. Expression of markers CD45, F4 / 80, CD206 is comparable across all groups tested, d-insets represent fold expression of CD206 relative to all cells expressing F4 / 80 in the co-culture. Minimum n=6, all data are presented as mean±sem. Data were analyzed by one-way ANOVA with Tukey correction for multiple comparisons. [Figure 2C] We show that in the absence of adhesive and inflammatory cues, microgel co-culture with monocytes does not induce activation. Cell survival after 48 h of incubation does not reveal any changes due to the presence of microparticles in the co-culture. Expression of markers CD45, F4 / 80, CD206 is comparable across all groups tested, d-insets represent fold expression of CD206 relative to all cells expressing F4 / 80 in the co-culture. Minimum n=6, all data are presented as mean±sem. Data were analyzed by one-way ANOVA with Tukey correction for multiple comparisons. [Figure 2D]We show that in the absence of adhesive and inflammatory cues, microgel co-culture with monocytes does not induce activation. Cell survival after 48 h of incubation does not reveal any changes due to the presence of microparticles in the co-culture. Expression of markers CD45, F4 / 80, CD206 is comparable across all groups tested, d-insets represent fold expression of CD206 relative to all cells expressing F4 / 80 in the co-culture. Minimum n=6, all data are presented as mean±sem. Data were analyzed by one-way ANOVA with Tukey correction for multiple comparisons. [Figure 3A] We show that the degradation of subcutaneous microgel implants is directly proportional to the concentration of EGBMA linker. Figure 3A) Scheme of microgel fabrication with near-infrared PEG linker and injection into dorsal subcutaneous pocket. Representative images of implant pockets at different time points after injection and explant. Figure 3B) Mean normalized radiant efficiency for all formulations at 1 month and after explant (points after vertical dashed line). Figure 3C-E) Quantification of normalized radiant efficiency at 0, 9, and 25 days after implantation. All data are presented as mean ± s.d., with a minimum of n = 5 recipients for DTT and n = 10 for all other groups. P values ​​were calculated using one-way ANOVA with Dunnett's multiple comparison analysis, **p<0.05, ***p<0.0005, ****p<0.0001. [Figure 3B] We show that the degradation of subcutaneous microgel implants is directly proportional to the concentration of EGBMA linker. Figure 3A) Scheme of microgel fabrication with near-infrared PEG linker and injection into dorsal subcutaneous pocket. Representative images of implant pockets at different time points after injection and explant. Figure 3B) Mean normalized radiant efficiency for all formulations at 1 month and after explant (points after vertical dashed line). Figure 3C-E) Quantification of normalized radiant efficiency at 0, 9, and 25 days after implantation. All data are presented as mean ± s.d., with a minimum of n = 5 recipients for DTT and n = 10 for all other groups. P values ​​were calculated using one-way ANOVA with Dunnett's multiple comparison analysis, **p<0.05, ***p<0.0005, ****p<0.0001. [Figure 3C] We show that the degradation of subcutaneous microgel implants is directly proportional to the concentration of EGBMA linker. Figure 3A) Scheme of microgel fabrication with near-infrared PEG linker and injection into dorsal subcutaneous pocket. Representative images of implant pockets at different time points after injection and explant. Figure 3B) Mean normalized radiant efficiency for all formulations at 1 month and after explant (points after vertical dashed line). Figure 3C-E) Quantification of normalized radiant efficiency at 0, 9, and 25 days after implantation. All data are presented as mean ± s.d., with a minimum of n = 5 recipients for DTT and n = 10 for all other groups. P values ​​were calculated using one-way ANOVA with Dunnett's multiple comparison analysis, **p<0.05, ***p<0.0005, ****p<0.0001. [Figure 3D] We show that the degradation of subcutaneous microgel implants is directly proportional to the concentration of EGBMA linker. Figure 3A) Scheme of microgel fabrication with near-infrared PEG linker and injection into dorsal subcutaneous pocket. Representative images of implant pockets at different time points after injection and explant. Figure 3B) Mean normalized radiant efficiency for all formulations at 1 month and after explant (points after vertical dashed line). Figure 3C-E) Quantification of normalized radiant efficiency at 0, 9, and 25 days after implantation. All data are presented as mean ± s.d., with a minimum of n = 5 recipients for DTT and n = 10 for all other groups. P values ​​were calculated using one-way ANOVA with Dunnett's multiple comparison analysis, **p<0.05, ***p<0.0005, ****p<0.0001. [Figure 3E]We show that the degradation of subcutaneous microgel implants is directly proportional to the concentration of EGBMA linker. Figure 3A) Scheme of microgel fabrication with near-infrared PEG linker and injection into dorsal subcutaneous pocket. Representative images of implant pockets at different time points after injection and explant. Figure 3B) Mean normalized radiant efficiency for all formulations at 1 month and after explant (points after vertical dashed line). Figure 3C-E) Quantification of normalized radiant efficiency at 0, 9, and 25 days after implantation. All data are presented as mean ± s.d., with a minimum of n = 5 recipients for DTT and n = 10 for all other groups. P values ​​were calculated using one-way ANOVA with Dunnett's multiple comparison analysis, **p<0.05, ***p<0.0005, ****p<0.0001. [Figure 4A] Figure 1 shows that myeloid cell mobilization and polarization is modulated by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of myeloid markers CD11b, F4 / 80, MHCII, and CD206 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 4B] Figure 1 shows that myeloid cell mobilization and polarization is modulated by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of myeloid markers CD11b, F4 / 80, MHCII, and CD206 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 4C]Figure 1 shows that myeloid cell mobilization and polarization is modulated by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of myeloid markers CD11b, F4 / 80, MHCII, and CD206 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 4D] Figure 1 shows that myeloid cell mobilization and polarization is modulated by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of myeloid markers CD11b, F4 / 80, MHCII, and CD206 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 4E] Figure 1 shows that myeloid cell mobilization and polarization is modulated by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of myeloid markers CD11b, F4 / 80, MHCII, and CD206 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 4F]Figure 1 shows that myeloid cell mobilization and polarization is modulated by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of myeloid markers CD11b, F4 / 80, MHCII, and CD206 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 4G] Figure 1 shows that myeloid cell mobilization and polarization is modulated by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of myeloid markers CD11b, F4 / 80, MHCII, and CD206 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 5A] Figure 1 shows that lymphocyte cell recruitment is controlled by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of lymphocyte markers CD3, CD4, CD8, CD25, and PD-1 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 5B]Figure 1 shows that lymphocyte cell recruitment is controlled by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of lymphocyte markers CD3, CD4, CD8, CD25, and PD-1 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 5C] Figure 1 shows that lymphocyte cell recruitment is controlled by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of lymphocyte markers CD3, CD4, CD8, CD25, and PD-1 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 5D] Figure 1 shows that lymphocyte cell recruitment is controlled by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of lymphocyte markers CD3, CD4, CD8, CD25, and PD-1 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 5E] Figure 1 shows that lymphocyte cell recruitment is controlled by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of lymphocyte markers CD3, CD4, CD8, CD25, and PD-1 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 5F] Figure 1 shows that lymphocyte cell recruitment is controlled by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of lymphocyte markers CD3, CD4, CD8, CD25, and PD-1 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 5G] Figure 1 shows that lymphocyte cell recruitment is controlled by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of lymphocyte markers CD3, CD4, CD8, CD25, and PD-1 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 5H] Figure 1 shows that lymphocyte cell recruitment is controlled by the degradation of synthetic microgel implants 7 days after injection. Flow cytometry analysis and quantification of lymphocyte markers CD3, CD4, CD8, CD25, and PD-1 from subcutaneous implant pockets containing different formulations of non-degradable and degradable microgels. All data are presented as mean ± sem, with a minimum of n = 4 recipients. P values ​​were calculated using one-way ANOVA and corrected for multiple comparisons by controlling for false discovery rate. [Figure 6A]We show that cytokine responses to implantable synthetic microgels are dynamic and dominated by IFN-γ responses that may vary with the degradation potential of the implantable material. Figure 6A) Principal component analysis of 32 cytokines measured in implant tissues from animals that received different synthetic microgel formulations. Arrow colors and directions indicate the contribution to each dimension of the PCA. Figure 6B) Cytokine correlations for all measured cytokines assessed using Pearson's correlation coefficient. Figure 6C) Left: Pearson correlations, dendrograms, and hierarchical clustering of cytokines based on cytokine names showing module membership. Figure 6D) Correlation plots for all cytokines against IFN-γ. Figure 6E-F) Box plots show cytokine concentrations of GM-CSF and IL-4 with raw values ​​plotted on a log10 scale. Minimum n=6 per recipient. P-values ​​presented are from estimated marginal mean (EMM) comparisons. [Figure 6B] We show that cytokine responses to implantable synthetic microgels are dynamic and dominated by IFN-γ responses that may vary with the degradation potential of the implantable material. Figure 6A) Principal component analysis of 32 cytokines measured in implant tissues from animals that received different synthetic microgel formulations. Arrow colors and directions indicate the contribution to each dimension of the PCA. Figure 6B) Cytokine correlations for all measured cytokines assessed using Pearson's correlation coefficient. Figure 6C) Left: Pearson correlations, dendrograms, and hierarchical clustering of cytokines based on cytokine names showing module membership. Figure 6D) Correlation plots for all cytokines against IFN-γ. Figure 6E-F) Box plots show cytokine concentrations of GM-CSF and IL-4 with raw values ​​plotted on a log10 scale. Minimum n=6 per recipient. P-values ​​presented are from estimated marginal mean (EMM) comparisons. [Figure 6C]We show that cytokine responses to implantable synthetic microgels are dynamic and dominated by IFN-γ responses that may vary with the degradation potential of the implantable material. Figure 6A) Principal component analysis of 32 cytokines measured in implant tissues from animals that received different synthetic microgel formulations. Arrow colors and directions indicate the contribution to each dimension of the PCA. Figure 6B) Cytokine correlations for all measured cytokines assessed using Pearson's correlation coefficient. Figure 6C) Left: Pearson correlations, dendrograms, and hierarchical clustering of cytokines based on cytokine names showing module membership. Figure 6D) Correlation plots for all cytokines against IFN-γ. Figure 6E-F) Box plots show cytokine concentrations of GM-CSF and IL-4 with raw values ​​plotted on a log10 scale. Minimum n=6 per recipient. P-values ​​presented are from estimated marginal mean (EMM) comparisons. [Figure 6D] We show that cytokine responses to implantable synthetic microgels are dynamic and dominated by IFN-γ responses that may vary with the degradation potential of the implantable material. Figure 6A) Principal component analysis of 32 cytokines measured in implant tissues from animals that received different synthetic microgel formulations. Arrow colors and directions indicate the contribution to each dimension of the PCA. Figure 6B) Cytokine correlations for all measured cytokines assessed using Pearson's correlation coefficient. Figure 6C) Left: Pearson correlations, dendrograms, and hierarchical clustering of cytokines based on cytokine names showing module membership. Figure 6D) Correlation plots for all cytokines against IFN-γ. Figure 6E-F) Box plots show cytokine concentrations of GM-CSF and IL-4 with raw values ​​plotted on a log10 scale. Minimum n=6 per recipient. P-values ​​presented are from estimated marginal mean (EMM) comparisons. [Figure 6E]We show that cytokine responses to implantable synthetic microgels are dynamic and dominated by IFN-γ responses that may vary with the degradation potential of the implantable material. Figure 6A) Principal component analysis of 32 cytokines measured in implant tissues from animals that received different synthetic microgel formulations. Arrow colors and directions indicate the contribution to each dimension of the PCA. Figure 6B) Cytokine correlations for all measured cytokines assessed using Pearson's correlation coefficient. Figure 6C) Left: Pearson correlations, dendrograms, and hierarchical clustering of cytokines based on cytokine names showing module membership. Figure 6D) Correlation plots for all cytokines against IFN-γ. Figure 6E-F) Box plots show cytokine concentrations of GM-CSF and IL-4 with raw values ​​plotted on a log10 scale. Minimum n=6 per recipient. P-values ​​presented are from estimated marginal mean (EMM) comparisons. [Figure 6F] We show that cytokine responses to implantable synthetic microgels are dynamic and dominated by IFN-γ responses that may vary with the degradation potential of the implantable material. Figure 6A) Principal component analysis of 32 cytokines measured in implant tissues from animals that received different synthetic microgel formulations. Arrow colors and directions indicate the contribution to each dimension of the PCA. Figure 6B) Cytokine correlations for all measured cytokines assessed using Pearson's correlation coefficient. Figure 6C) Left: Pearson correlations, dendrograms, and hierarchical clustering of cytokines based on cytokine names showing module membership. Figure 6D) Correlation plots for all cytokines against IFN-γ. Figure 6E-F) Box plots show cytokine concentrations of GM-CSF and IL-4 with raw values ​​plotted on a log10 scale. Minimum n=6 per recipient. P-values ​​presented are from estimated marginal mean (EMM) comparisons. [Figure 7]We show that hydrolysis-sensitive ethylene linkers are used for microparticle crosslinking to fabricate degradable droplet microfluidic-based microgels for therapeutic delivery. The tunability and degradability afforded by ester-based degradation in vivo modulates immune cell infiltration to the implant site and immune polarization of the host. [Figure 8] 1H NMR spectra of the prepared PEG-4MAL macromer and microgels. [Figure 9A] The experimental setup for capillary micromechanics is shown. Figure 9A) Tapered glass micropipettes (Fivephoton Biochemicals) had the following dimensions: tip inner diameter = 50 μm, base outer diameter = 1.5 mm, length = 5.5 cm, taper style = long. A high-precision pressure regulator (Elveflow) applied pressure to the micropipette containing the microgel. To promote optimal flow dynamics, the micropipette was immersed in 1% BSA. The microgel will deform until equilibrium is reached, when the external applied pressure is balanced with the internal elastic stress. A microscope (EVOS) under the micropipette tip acquired images (10x), which were subsequently analyzed with ImageJ. Figure 9B) Microgel shape within the tapered region. The microgel was in contact with the wall by the mean radius, R-band, and the mean length, L-band. The taper angle is θ. As the pressure p increases, the L-band increases and the R-band decreases. Elastic properties were calculated from these measurements as previously described (Wyss et al, Soft Matter, 2010). Figure 9C) Image series of a microgel deforming in response to increasing pressure. [Figure 9B]The experimental setup for capillary micromechanics is shown. Figure 9A) Tapered glass micropipettes (Fivephoton Biochemicals) had the following dimensions: tip inner diameter = 50 μm, base outer diameter = 1.5 mm, length = 5.5 cm, taper style = long. A high-precision pressure regulator (Elveflow) applied pressure to the micropipette containing the microgel. To promote optimal flow dynamics, the micropipette was immersed in 1% BSA. The microgel will deform until equilibrium is reached, when the external applied pressure is balanced with the internal elastic stress. A microscope (EVOS) under the micropipette tip acquired images (10x), which were subsequently analyzed with ImageJ. Figure 9B) Microgel shape within the tapered region. The microgel was in contact with the wall by the mean radius, R-band, and the mean length, L-band. The taper angle is θ. As the pressure p increases, the L-band increases and the R-band decreases. Elastic properties were calculated from these measurements as previously described (Wyss et al, Soft Matter, 2010). Figure 9C) Image series of a microgel deforming in response to increasing pressure. [Figure 9C]The experimental setup for capillary micromechanics is shown. Figure 9A) Tapered glass micropipettes (Fivephoton Biochemicals) had the following dimensions: tip inner diameter = 50 μm, base outer diameter = 1.5 mm, length = 5.5 cm, taper style = long. A high-precision pressure regulator (Elveflow) applied pressure to the micropipette containing the microgel. To promote optimal flow dynamics, the micropipette was immersed in 1% BSA. The microgel will deform until equilibrium is reached, when the external applied pressure is balanced with the internal elastic stress. A microscope (EVOS) under the micropipette tip acquired images (10x), which were subsequently analyzed with ImageJ. Figure 9B) Microgel shape within the tapered region. The microgel was in contact with the wall by the mean radius, R-band, and the mean length, L-band. The taper angle is θ. As the pressure p increases, the L-band increases and the R-band decreases. Elastic properties were calculated from these measurements as previously described (Wyss et al, Soft Matter, 2010). Figure 9C) Image series of a microgel deforming in response to increasing pressure. [Figure 10] Figure 1 shows the in vitro cytotoxicity of RAW264.7 macrophage cells treated with all microgel formulations (degradable and non-degradable). The graph represents cell viability during 7 days of co-culture as determined by Alamar Blue assay. Data represent the mean standard deviation of the mean (n=4). No statistical differences were found by one-way ANOVA. [Figure 11A] We show that in the absence of adhesion and inflammatory signals, microgel co-culture with monocytes does not induce activation. 11A-D) Cell viability after 96 h of incubation does not reveal any changes due to the presence of microparticles in the co-culture. Expression of markers CD45, F4 / 80, CD206 is consistent across all groups tested. 11D-Inset represents the fold expression of CD206 relative to all cells expressing F4 / 80 in the co-culture. All data are presented as mean ± sem, n=3. Data were analyzed by one-way ANOVA with Tukey correction for multiple comparisons. [Figure 11B] We show that in the absence of adhesion and inflammatory signals, microgel co-culture with monocytes does not induce activation. 11A-D) Cell viability after 96 h of incubation does not reveal any changes due to the presence of microparticles in the co-culture. Expression of markers CD45, F4 / 80, CD206 is consistent across all groups tested. 11D-Inset represents the fold expression of CD206 relative to all cells expressing F4 / 80 in the co-culture. All data are presented as mean ± sem, n=3. Data were analyzed by one-way ANOVA with Tukey correction for multiple comparisons. [Figure 11C] We show that in the absence of adhesion and inflammatory signals, microgel co-culture with monocytes does not induce activation. 11A-D) Cell viability after 96 h of incubation does not reveal any changes due to the presence of microparticles in the co-culture. Expression of markers CD45, F4 / 80, CD206 is consistent across all groups tested. 11D-Inset represents the fold expression of CD206 relative to all cells expressing F4 / 80 in the co-culture. All data are presented as mean ± sem, n=3. Data were analyzed by one-way ANOVA with Tukey correction for multiple comparisons. [Figure 11D] We show that in the absence of adhesion and inflammatory signals, microgel co-culture with monocytes does not induce activation. 11A-D) Cell viability after 96 h of incubation does not reveal any changes due to the presence of microparticles in the co-culture. Expression of markers CD45, F4 / 80, CD206 is consistent across all groups tested. 11D-Inset represents the fold expression of CD206 relative to all cells expressing F4 / 80 in the co-culture. All data are presented as mean ± sem, n=3. Data were analyzed by one-way ANOVA with Tukey correction for multiple comparisons. [Figure 12A]Box plots showing cytokine concentrations with raw values ​​plotted on a log10 scale and estimated marginal mean (EMM) comparisons for all time points are provided. n=6 per group. [Figure 12B] Box plots showing cytokine concentrations with raw values ​​plotted on a log10 scale and estimated marginal mean (EMM) comparisons for all time points are provided. n=6 per group. [Figure 12C] Box plots showing cytokine concentrations with raw values ​​plotted on a log10 scale and estimated marginal mean (EMM) comparisons for all time points are provided. n=6 per group. [Figure 12D] Box plots showing cytokine concentrations with raw values ​​plotted on a log10 scale and estimated marginal mean (EMM) comparisons for all time points are provided. n=6 per group. [Figure 12E] Box plots showing cytokine concentrations with raw values ​​plotted on a log10 scale and estimated marginal mean (EMM) comparisons for all time points are provided. n=6 per group. [Figure 12F] Box plots showing cytokine concentrations with raw values ​​plotted on a log10 scale and estimated marginal mean (EMM) comparisons for all time points are provided. n=6 per group. [Figure 13A] Box plots showing cytokine concentrations with raw values ​​plotted on a log10 scale and estimated marginal mean (EMM) comparisons for all time points are provided. n=6 per group. [Figure 13B] Box plots showing cytokine concentrations with raw values ​​plotted on a log10 scale and estimated marginal mean (EMM) comparisons for all time points are provided. n=6 per group. [Figure 13C] Box plots showing cytokine concentrations with raw values ​​plotted on a log10 scale and estimated marginal mean (EMM) comparisons for all time points are provided. n=6 per group. [Figure 13D]Box plots showing cytokine concentrations with raw values ​​plotted on a log10 scale and estimated marginal mean (EMM) comparisons for all time points are provided. n=6 per group. [Figure 14] H&E staining 30 days after microgel implant was injected into the dorsal subcutaneous space. [Figure 15] Immunohistochemical evaluation of dorsal microgel implants 30 days after injection. Samples were stained for pan-macrophage marker CD68 (red) and nuclear marker DAPI (blue). Microgel area is represented by white dashed line. Inset shows a representative image at 20x magnification of the area surrounding the microgel. Scale bars, inset 20 μm, 10x image 50 μm. [Figure 16A] Degradable hydrogel properties in vitro and in vivo are shown: Figure 16A) In vivo tracking of hydrogels implanted within the subcutaneous space of mice; Figure 16B) IVIS imaging demonstrating microgel localization and changes in fluorescence intensity over time. [Figure 16B] Degradable hydrogel properties in vitro and in vivo are shown: Figure 16A) In vivo tracking of hydrogels implanted within the subcutaneous space of mice; Figure 16B) IVIS imaging demonstrating microgel localization and changes in fluorescence intensity over time.

[0015] Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The following description of the present disclosure is provided as an enabling teaching of the present disclosure in its best, currently known embodiment. Many modifications and other embodiments will occur to those skilled in the art related to the disclosed compositions and methods having the benefit of the teachings presented in the foregoing description and the associated drawings. It is therefore to be understood that the present disclosure is not limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the appended claims. Those skilled in the art will recognize many variations and adaptations of the aspects described herein. These variations and adaptations are intended to be included within the teachings of the present disclosure and encompassed by the scope of the claims herein.

[0017] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0018] As would be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure.

[0019] Any recited method may be carried out in the order of events recited or in any other order that is logically possible. That is, unless expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Thus, no order is intended to be inferred in any respect from a method claim unless the claim or specification specifically states that the steps are to be limited to a specific order. This holds true for any possible implicit basis for interpretation, including any obvious meaning derived from the arrangement of steps or operational flow, grammatical construction or punctuation, or logical matters regarding the number or type of aspects described herein.

[0020] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publications provided herein may be different from the actual publication dates and may be independently confirmed.

[0021] It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the disclosed compositions and methods belong.Terms as defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of the present specification and related art, and should not be interpreted in an ideal or overly formal sense unless expressly defined herein.

[0022] Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in this disclosure.

[0023] As used herein, the term "comprising" should be interpreted as specifying the presence of the stated feature, integer, step, or component as referred to, but does not exclude the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Additionally, each of the terms "by," "comprising," "comprises," "comprised of," "including," "includes," "included," "involving," "involves," "involved," and "such as" are used in their open, non-limiting sense and may be used interchangeably. Additionally, the term "comprising" is intended to include examples and aspects encompassed by the terms "consisting essentially of" and "consisting of." Similarly, the term "consisting essentially of" is intended to include examples encompassed by the term "consisting of."

[0024] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell," "a tissue," or "a hydrogel" includes, but is not limited to, two or more such cell, tissue, or hydrogel.

[0025] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is herein disclosed as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it can be understood that the particular value forms a further aspect. For example, if the value "about 10" is disclosed, then "10" is also disclosed.

[0026] When a range is expressed, a further embodiment includes from the one particular value and / or to the other particular value. For example, when a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase "from x to y" includes ranges from "x" to "y", as well as ranges greater than "x" and less than "y". Ranges can also be expressed as upper limits, e.g., "about x, y, z, or less", and should be interpreted to include the specific ranges of "about x", "about y", and "about z", as well as the ranges "less than x", "less than y", and "less than z". Similarly, the phrase "about x, y, z, or more" should be interpreted to include the specific ranges of "about x", "about y", and "about z", as well as the ranges "greater than x", "greater than y", and "greater than z". In addition, the phrase "about "x" to "y", where "x" and "y" are numerical values, includes "about "x" to about "y".

[0027] It should be understood that such range formats are used for convenience and brevity and should be interpreted flexibly to include not only the numerical values ​​explicitly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. To illustrate, a numerical range of "about 0.1% to about 5%" should be interpreted not only to include the values ​​of about 0.1% to about 5% explicitly recited, but also to include the individual values ​​(e.g., about 1%, about 2%, about 3%, and about 4%) and subranges within the indicated range (e.g., about 0.5% to about 1.1%, about 5% to about 2.4%, about 0.5% to about 3.2%, and about 0.5% to about 4.4%, as well as other possible subranges).

[0028] As used herein, the terms "about," "approximately," "at or about," and "substantially" mean that the amount or value in question may be an exact value or a value that provides an equivalent result or effect as recited in the claims or taught herein. That is, it is understood that the amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximated and / or larger or smaller, as desired, to provide an equivalent result or effect, reflecting tolerances, conversion factors, rounding, measurement errors, and the like, and other factors known to those of skill in the art. In some circumstances, values ​​that provide an equivalent result or effect cannot be reasonably determined. In such cases, as used herein, unless otherwise indicated or inferred, "about" and "at or about" are generally understood to mean nominal values ​​that exhibit a variation of ±10%. In general, a quantity, size, formulation, parameter, or other quantity or characteristic is "about," "approximately," or "at or about," whether or not it is expressly stated as such. When "about," "approximately," or "at or about" is used prior to a quantitative value, it is understood that the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0029] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or have an effect on undesired symptoms, but generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition used; the age, weight, general health, sex, and diet of the patient; the timing of administration; the route of administration; the excretion rate of the specific composition used; the duration of treatment; drugs used in combination with or simultaneously with the specific composition used, and similar factors that are within the knowledge and opinion of the medical practitioner and may be well known in the medical field. When treating a particular disease or condition, in some cases, the desired response may be to inhibit the progression of the disease or condition. This may include only slowing down the progression of the disease temporarily. However, in other cases, it may be desirable to permanently stop the progression of the disease. This can be monitored by periodic diagnostic methods known to those skilled in the art for any particular disease. The desired response to the treatment of a disease or condition may also be to delay or even prevent the onset of the disease or condition.

[0030] For example, it is well within the skill of the art to start a dose of the composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for administration. Consequently, a single dose composition can contain such amounts or submultiples thereof to make up a daily dose. In the event of any contraindications, the dosage can be adjusted by the individual physician. It is generally preferred that the maximum dose of the pharmacological agent of the present invention (alone or in combination with other therapeutic agents), i.e., the highest safe dose according to sound medical judgment, be used. However, one skilled in the art will understand that a patient may insist on a lower or tolerated dose for medical reasons, psychological reasons, or virtually any other reason.

[0031] Response to a therapeutically effective dose of the disclosed compositions can be measured by determining the physiological effect of the treatment or drug, such as a reduction or absence of disease symptoms following administration of the treatment or pharmacological agent. Other assays are known to those of skill in the art and can be used to measure the level of response. The amount of treatment can be varied, for example, by increasing or decreasing the amount of the disclosed compositions, by changing the disclosed compositions administered, by changing the route of administration, by changing the timing of administration, etc. Dosages can vary and can be administered in one or more doses per day for one or several days. Guidance can be found in the literature for appropriate dosages of a given class of pharmaceutical agent.

[0032] As used herein, the term "prophylactically effective amount" refers to an amount effective for preventing the development or onset of a disease or condition.

[0033] As used herein, the term "prevent" or "preventing" refers to hindering, avoiding, eliminating, forestalling, blocking, or impeding something from happening, especially by prior action. Where reduce, inhibit, or prevent are used herein, it is understood that the use of the other two words is also expressly disclosed unless specifically indicated otherwise.

[0034] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and is meant to include cases where the event or circumstance occurs and cases where it does not occur.

[0035] As used interchangeably herein, a "subject," "individual," or "patient" may refer to a vertebrate organism, such as a mammal (e.g., a human). A "subject" may also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably a human, and components thereof.

[0036] As used herein, the terms "treating" and "treatment" may generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be, but is not necessarily, prophylactic in that it prevents or partially prevents a disease, condition, or state thereof, such as tissue loss. The effect may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom, or side effects caused by the disease, disorder, or condition. The term "treatment" as used herein may include any treatment of a disorder in a subject, particularly a human, and may include any one or more of the following: (a) preventing a disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it, (b) inhibiting a disease, e.g., arresting its onset, and (c) relieving a disease, e.g., alleviating or ameliorating a disease and / or its symptoms or state. As used herein, the term "treatment" may refer to therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with a disease and / or those in whom a disorder is to be prevented. As used herein, the term "treating" can include inhibiting a disease, disorder, or condition, e.g., preventing its progression, as well as alleviating a disease, disorder, or condition, e.g., causing regression of a disease, disorder, and / or condition. Treating a disease, disorder, or condition can also include improving at least one symptom of a particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating a subject's pain by administration of a painkiller, even if such an agent does not treat the cause of the pain.

[0037] As used herein, a "dose," "unit dose," or "dosage" may refer to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or pharmaceutical composition thereof calculated to produce a desired response or responses associated with its administration.

[0038] As used herein, "therapeutic" can refer to treating, curing, and / or ameliorating a disease, disorder, condition, or side effect, or reducing the rate of progression of a disease, disorder, condition, or side effect.

[0039] Compounds are described using standard nomenclature. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0040] The compounds described herein include other isomers, such as enantiomers, mixtures of enantiomers, diastereomers, tautomers, racemates, and rotamers, as if each were specifically described, unless otherwise indicated or excluded by context. It should be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be in either the (R-) or (S-) configuration. The compounds provided herein may be enantiomerically pure or may be either diastereomeric or enantiomeric mixtures. It should be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. Thus, one skilled in the art will recognize that administration of the (R-) form of a compound is equivalent to administration of the (S-) form of the compound for compounds that undergo epimerization in vivo. Unless stated to the contrary, formulas having chemical bonds shown only as solid lines and not as wedges or dashed lines contemplate each possible isomer, e.g., each enantiomer, diastereomer, and meso compound, as well as mixtures of isomers such as racemic or scalemic mixtures.

[0041] A dash ("-") that is not between two letters or symbols is used to indicate the point of attachment of a substituent. For example, -(C=O)NH2 is attached through the carbon of the keto (C=O) group.

[0042] The term "substituted" as used herein means that any one or more hydrogens on the specified atom or group are replaced with a moiety selected from the indicated group, provided that the normal valence of the specified atom is not exceeded, and the resulting compound is stable. For example, if the substituent is oxo (i.e., =O), two hydrogens on the atom are replaced. For example, a pyridyl group substituted by oxo is a pyridine. Combinations of substituents and / or variables are only permissible if such combinations result in stable compounds or useful synthetic intermediates. A stable active compound refers to a compound that can be isolated and formulated into a dosage form that has a shelf life of at least one month. A stable manufacturing intermediate or precursor of an active compound is stable if it does not decompose within the time required for reaction or other use. A stable moiety or substituent is one that does not decompose, react, or fall apart within the time required for use. Non-limiting examples of unstable moieties are those that combine heteroatoms in unstable configurations, which are typically known and identifiable to those skilled in the art.

[0043] Any suitable group may be present at the "substituted" or "optionally substituted" positions which will form a stable molecule and satisfy the desired objectives of the present invention, including, but not limited to, alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycle, aldehyde, amino, carboxylic acid, ester, ether, halo, hydroxy, keto, nitro, cyano, azido, oxo, silyl, sulfoxo, sulfonyl, sulfone, sulfoxide, sulfonylamino, or thiol.

[0044] "Alkyl" is a straight or branched saturated aliphatic hydrocarbon group. In certain embodiments, alkyl is C1-C2, C1-C3, or C1-C6 (i.e., the alkyl chain can be 1, 2, 3, 4, 5, or 6 carbons in length). As used herein, a specified range refers to an alkyl group with the length of each member of the range described as an independent species. For example, as used herein, C1-C6 alkyl is intended to refer to an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms, each of which is described as an independent species, and as used herein, C1-C4 alkyl is intended to refer to an alkyl group having 1, 2, 3, or 4 carbon atoms, each of which is described as an independent species. C0-C n When alkyl is used herein in conjunction with another group, such as (C3-C7 cycloalkyl)C0-C4 alkyl, or -C0-C4(C3-C7 cycloalkyl), the indicated group, in this case cycloalkyl, is either directly linked by a single covalent bond (C0 alkyl) or linked by an alkyl chain, in this case 1, 2, 3, or 4 carbon atoms. Alkyl can also be linked through other groups, such as heteroatoms, such as -O-C0-C4 alkyl(C3-C7 cycloalkyl). Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentane, 3-methylpentane, 2,2-dimethylbutane, and 2,3-dimethylbutane. In one embodiment, the alkyl group is optionally substituted as described herein.

[0045] "Cycloalkyl" refers to a saturated monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings may be linked together in a fused or bridged fashion. Non-limiting examples of typical cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. In one embodiment, the cycloalkyl group is optionally substituted as described herein.

[0046] "Alkenyl" refers to a straight or branched chain aliphatic hydrocarbon group having one or more carbon-carbon double bonds that may occur at any stable point along the chain, and each of the bonds is independently either cis or trans. Non-limiting examples include C2-C4 alkenyl and C2-C6 alkenyl (i.e., having 2, 3, 4, 5, or 6 carbons). As used herein, the specified ranges refer to alkenyl groups with each member of the range described as an independent species, as described above for the alkyl moiety. Examples of alkenyl include, but are not limited to, ethenyl and propenyl. In one embodiment, the alkenyl group is optionally substituted as described herein.

[0047] "Alkynyl" is a straight or branched chain aliphatic hydrocarbon group having one or more carbon-carbon triple bonds that may occur at any stable point along the chain, such as C2-C4 alkynyl or C2-C6 alkynyl (i.e., having 2, 3, 4, 5, or 6 carbons). As used herein, the specified ranges refer to alkynyl groups with each member of the range described as an independent species, as described above for the alkyl moiety. Examples of alkynyl include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, and 5-hexynyl. In one embodiment, the alkynyl group is optionally substituted as described herein.

[0048] "Alkoxy" refers to an alkyl group as defined above covalently bonded through an oxygen bridge (-O-). Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, 2-butoxy, tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy. Similarly, an "alkylthio" or "thioalkyl" group refers to an alkyl group as defined above with the indicated number of carbon atoms covalently bonded through a sulfur bridge (-S-). In one embodiment, an alkoxy group is optionally substituted as described herein.

[0049] "Alkanoyl" is an alkyl group as defined above covalently bonded via a carbonyl (C=O) bridge. The carbonyl carbon is included in the number of carbons, for example, a C2 alkanoyl is a CH3(C=O)- group. In one embodiment, the alkanoyl group is optionally substituted as described herein.

[0050] "Halo" or "halogen" independently refer to either fluoro, chloro, bromo, or iodo.

[0051] An aryl group refers to an aromatic group containing only carbon in the aromatic ring or rings. In one embodiment, an aryl group contains 1-3 separate or fused rings, 6-14 or 18 ring atoms, and does not contain heteroatoms as ring members. Where indicated, such aryl groups may be further substituted with carbon or non-carbon atoms or groups. Such substitution may include fusion to a 4-7 or 5-7 membered saturated or partially unsaturated cyclic group, optionally containing 1, 2, or 3 heteroatoms independently selected from N, O, B, P, Si, and S, to form, for example, a 3,4-methylenedioxyphenyl group. Aryl groups include, for example, phenyl and naphthyl, including 1-naphthyl and 2-naphthyl. In one embodiment, the aryl group is pendant. An example of a pendant ring is a phenyl group substituted with a phenyl group. In one embodiment, the aryl group is optionally substituted as described herein.

[0052] The term "heterocycle" refers to saturated and partially saturated heteroatom-containing ring radicals, where the heteroatoms may be selected from N, O, and S. The term heterocycle includes monocyclic 3- to 12-membered rings, as well as bicyclic 5- to 16-membered ring systems, which may include fused, bridged, or spiro bicyclic systems. It does not include rings containing -OO-, -OS-, and -SS- moieties. Examples of saturated heterocyclic groups include saturated 4- to 7-membered monocyclic groups containing 1-4 nitrogen atoms [e.g., pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, azetidinyl, piperazinyl, and pyrazolidinyl]; saturated 4- to 6-membered monocyclic groups containing 1-2 oxygen atoms and 1-3 nitrogen atoms [e.g., morpholinyl]; and saturated 3- to 6-membered monocyclic groups containing 1-2 sulfur atoms and 1-3 nitrogen atoms [e.g., thiazolidinyl]. Examples of partially saturated heterocyclic radicals include, but are not limited to, dihydrothienyl, dihydropyranyl, dihydrofuryl, and dihydrothiazolyl. Examples of partially saturated and saturated heterocyclic groups include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl, pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl, thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[1,4]dioxanyl, indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl, isochromanyl, chromanyl, 1,2-dihydroquinolyl, 1,2,3,4-tetrahydro-isoquinolyl, 1,2, Examples of heterocyclic rings include, but are not limited to, 3,4-tetrahydro-quinolyl, 2,3,4,4a,9,9a-hexahydro-1H-3-aza-fluorenyl, 5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinol, 3,4-dihydro-2H-benzo[1,4]oxanyl, benzo[1,4]dioxanyl, 2,3,-dihydro-1H-benzo[d]isothazol-6-yl, dihydropyranyl, dihydrofuryl, and dihydrothiazolyl. Bicyclic heterocycles include groups in which a heterocyclic radical is fused to an aryl radical and the point of attachment is the heterocyclic ring. Bicyclic heterocycles also include heterocyclic radicals fused to a carbocyclic radical.Representative examples include, but are not limited to, partially unsaturated fused heterocyclic groups containing 1 to 5 nitrogen atoms, such as indoline and isoindoline, partially unsaturated fused heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, partially unsaturated fused heterocyclic groups containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, and saturated fused heterocyclic groups containing 1 to 2 oxygen atoms or sulfur atoms.

[0053] "Heteroaryl" refers to a stable monocyclic, bicyclic, or polycyclic aromatic ring containing 1 to 4, or in some embodiments 1, 2, or 3, heteroatoms selected from N, O, S, B, and P (typically selected from N, O, and S), with the remaining ring atoms being carbon, or a stable bicyclic or tricyclic ring system containing at least one 5-, 6-, or 7-membered aromatic ring containing 1 to 4, or in some embodiments 1 to 3 or 1 to 2 heteroatoms selected from N, O, S, B, or P, with the remaining ring atoms being carbon. In one embodiment, the only heteroatom is nitrogen. In one embodiment, the only heteroatom is oxygen. In one embodiment, the only heteroatom is sulfur. Monocyclic heteroaryl groups typically have 5 to 6 ring atoms. In some embodiments, the bicyclic heteroaryl group is an 8-10 membered heteroaryl group, i.e., a group containing 8 or 10 ring atoms in which one 5-, 6-, or 7-membered aromatic ring is fused to a second aromatic or non-aromatic ring, and the point of attachment is the aromatic ring. If the total number of S and O atoms in the heteroaryl group exceeds 1, these heteroatoms are not adjacent to one another. In one embodiment, the total number of S and O atoms in the heteroaryl group is 2 or less. In another embodiment, the total number of S and O atoms in the heteroaryl group is 1 or less. Examples of heteroaryl groups include, but are not limited to, pyridinyl, imidazolyl, imidazopyridinyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, triazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.

[0054] In one aspect, a hydrogel is provided that includes a polymer backbone crosslinked with a first crosslinker containing at least one moiety of formula I: [ka] During the ceremony, m and n are independently 1 or 2; A is C2-C 10 is alkyl, [ka] is the attachment point for the moiety in the first crosslinker.

[0055] In some embodiments of formula I, m is 1. In some embodiments of formula I, m is 2. In some embodiments of formula I, n is 1. In some embodiments of formula I, n is 2.

[0056] In some embodiments of formula I, A is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, or C 10 In some embodiments of formula I, A is selected from ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

[0057] In some embodiments, the moiety of formula I is [ka] may be selected from:

[0058] The polymer backbone may be composed of any polymer or combination of polymers used in the preparation of hydrogels. As known in the art, a hydrogel is a polymer network formed by crosslinking one or more multifunctional molecules or polymers. The resulting polymer network is hydrophilic and swells in an aqueous environment, thus forming a gel-like material, i.e., a hydrogel. Typically, a hydrogel comprises a backbone bound to a crosslinker (such as the first crosslinker described herein).

[0059] Hydrogels are characterized by their water insolubility, hydrophilicity, high water absorption, and swelling properties. The molecular components, units, or segments of hydrogels are characterized by a significant portion of hydrophilic components, units, or segments, such as segments capable of hydrogen bonding, or segments capable of having ionic or dissociative species, such as acids (e.g., carboxylic acids, phosphonic acids, sulfonic acids, sulfinic acids, phosphinic acids, etc.), bases (e.g., amine groups, proton-accepting groups, etc.), or other groups that develop ionic properties when immersed in water (e.g., sulfonamides). The classes of acrylic polymers or polymer chains that contain or terminate with acryloyl groups (and to a lesser extent methacryloyl groups), as well as oxyalkylene units (such as polyoxyethylene chains and polyoxyethylene / polyoxypropylene copolymer chains), are also well recognized as hydrophilic segments that may be present within hydrophilic polymers. Representative water insoluble polymer compositions are provided below, but the entire class of hydrogel materials known in the art may be used to various degrees. The polymers described below and containing acidic groups may optionally be partially or fully neutralized with an alkali metal base, either on the monomer or polymer or both.

[0060] Some representative polymers that may comprise the polymer backbone include, but are not limited to, polyacrylic acid, polymethacrylic acid, polymaleic acid, copolymers thereof, and alkali metal and ammonium salts thereof; graft copolymers of starch and acrylic acid, starch and saponified acrylonitrile, starch and saponified ethyl acrylate, and saponified acrylate-vinyl acetate copolymers; polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl alkyl ethers, polyethylene oxide, polyacrylamide, and copolymers thereof; copolymers of maleic anhydride and alkyl vinyl ethers; and subpolymers of saponified starch graft copolymers of acrylonitrile, acrylate esters, vinyl acetate, and starch graft copolymers of acrylic acid, methacrylic acid, and maleic acid.

[0061] In some embodiments, the polymer backbone may include a biopolymer. In some embodiments, the biopolymer may be functionalized or modified in such a manner as to provide functionality that allows crosslinking with a first crosslinker. Representative examples of biopolymers that may be used include, but are not limited to, collagen, gelatin, fibrin, hyaluronic acid, elastin, pectin, agarose, glycoaminoglycan, alginate, cellulose, DNA, RNA, or functionalized derivatives thereof.

[0062] In some embodiments, the polymer backbone comprises poly(ethylene glycol) or a functionalized derivative thereof. Representative examples of such polymer backbones include poly(ethylene glycol) (PEG), poly(ethylene glycol)-di-acrylate (PEG-DA), multi-arm poly(ethylene glycol)-acrylate (PEG-Ac), poly(ethylene glycol)-dithiol (PEG-diSH), poly(ethylene glycol) divinyl sulfone (PEG-diVS), multi-arm poly(ethylene glycol) vinyl sulfone (PEG-VS), poly(ethylene glycol)-di-methacrylate (PEG-DMA), multi-arm poly(ethylene glycol)-methacrylate (PEG-Mac), poly(ethylene glycol)-di-allyl ether (PEG-diAE), multi-arm ... The polymers may be formed from polymers including, but not limited to, poly(ethylene glycol)-allyl ether (PE-AD), poly(ethylene glycol)-di-vinyl ether (PEG-diVE), multi-arm poly(ethylene glycol)-vinyl ether (PEG-VE), poly(ethylene glycol)-di-maleimide (PEG-diMI), multi-arm poly(ethylene glycol)-maleimide (PEG-MI), poly(ethylene glycol)-di-norborene, multi-arm poly(ethylene glycol)norborene, poly(ethylene glycol)-vinyl carbonate, multi-arm poly(ethylene glycol)-vinyl carbonate, and polyethylene glycol oligofumarate.

[0063] In some particular embodiments, the polymer backbone may be formed from a multi-arm poly(ethylene glycol)-maleimide.

[0064] The above exemplary polymers may be crosslinked either during or after polymerization using a first crosslinker described herein and optionally one or more additional crosslinkers. Crosslinking may be performed using methods known to those skilled in the art, such as, for example, by initiation in the presence of radiation via a radical initiator.

[0065] The first crosslinker comprises at least two moieties capable of reacting with the polymer backbone. The polymer backbone itself has active groups available to react with at least two moieties of the first crosslink to form covalent bonds. It is generally understood that the presence of the moiety of formula I in the first crosslinker, when crosslinked, results in the observed hydrolysis properties of the hydrogels provided herein.

[0066] In some embodiments, the first crosslinker comprises a compound of formula II: [ka] During the ceremony, X 1 and X 2 is independently selected at each occurrence from moieties capable of reacting with the polymer backbone; L 1 and L 2 is independently selected at each occurrence from the linking portion; m, n, and A are defined as in claim 1.

[0067] In some embodiments of Formula II, m is 1. In some embodiments of Formula II, m is 2. In some embodiments of Formula II, n is 1. In some embodiments of Formula II, n is 2.

[0068] In some embodiments of Formula II, A is C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, or C 10 In some embodiments of Formula II, A is selected from ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3-dimethylbutyl.

[0069] In some embodiments, the compound of formula II is [ka] is selected from.

[0070] X 1 and X 2 Each independently may be any suitable functional moiety capable of reacting with an active group or moiety such as may be found in the polymer backbone. Representative examples of such groups include, but are not limited to, halo, hydroxy, amino, thiol, carboxylic acid, ester, and the like. In some embodiments, the group X 1 and X 2 may each independently include a polymerizable group, such as an oxiranyl, acryloyl, or methacryloyl group. In certain embodiments, X 1 and X 2 are -SH, respectively.

[0071] L 1 and L 2 each independently represents a moiety X 1 and X 2 to the corresponding carbonyl group to which it is attached, or any other suitable linking moiety. 1 and L 2 can be independently selected from C1-C6 alkyl, C1-C6 heteroalkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, 3-8 membered monocyclic or bicyclic heterocycle, 6-10 membered monocyclic or bicyclic aryl, 5-10 membered monocyclic or bicyclic heteroaryl, or any suitable combination thereof, each of which can be optionally substituted as described herein.

[0072] In some embodiments, L 1 and L 2 are each independently C1-C 10 alkyl, for example, selected from methylene, ethylene, propylene, or butylene. In certain embodiments, L 1 and L 2 are each methylene.

[0073] In certain embodiments, the first cross-linking agent comprises ethylene glycol bis(mercaptoacetate).

[0074] In some embodiments, the polymer backbone is further crosslinked with a second crosslinker. In such embodiments, the second crosslinker is typically hydrolytically stable, i.e., does not contain the moiety of formula I found in the first crosslinker, or any other moiety that may be hydrolytically cleaved under the conditions in which the hydrogel is intended to be used. In certain embodiments, the second crosslinker may include dithiothreitol (DTT).

[0075] In embodiments including a second crosslinker, the degradation of the hydrogel may be tunable by varying the molar ratio of the first crosslinker to the second crosslinker. In some embodiments, the molar ratio of the first crosslinker to the second crosslinker can be in the range of about 100:1 to about 1:100, e.g., about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:15, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. One skilled in the art can easily understand that a higher molar ratio of the first crosslinker to the second crosslinker will increase hydrolysis and shorten the degradation time, and a lower molar ratio of the first crosslinker to the second crosslinker will decrease hydrolysis time and increase degradation time.

[0076] The degradation products of the hydrogel should be substantially biocompatible, i.e., do not substantially adversely affect the living body or other body tissues, or cells, either at the site where the hydrogel is placed or any other part of the body. Methods for assessing the biocompatibility of materials are well known.

[0077] In some embodiments, the hydrogels described herein may contain bioactive agents capable of modulating the function and / or characteristics of cells. For example, the bioactive agents may be capable of modulating the function and / or characteristics of cells dispersed on or within the hydrogel. Alternatively, or in addition, the bioactive agents may be capable of modulating the function and / or characteristics of endogenous cells surrounding, for example, a hydrogel implanted in a tissue defect, and directing the cells into the defect. The at least one bioactive agent may include, for example, polynucleotides and / or polypeptides encoding or including transcription factors, differentiation factors, growth factors, or combinations thereof. The at least one bioactive agent may also include any agent capable of tissue formation, destruction, and / or targeting a specific disease state (e.g., cancer).Representative examples of such bioactive agents include chemotactic agents, various proteins (such as short-term peptides, bone morphogenic proteins, collagens, glycoproteins, and lipoproteins), cell attachment mediators, biologically active ligands, integrin binding sequences, various growth and / or differentiation agents and fragments thereof, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factors (e.g., bFGF), platelet-derived growth factor (PDGF), insulin-like growth factors (e.g., IGF-1, IGF-II), and transforming growth factors (e.g., TGF-β ... I-III), parathyroid hormone, parathyroid hormone-related peptides, bone morphogenetic proteins (e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-12, BMP-13, BMP-14), transcription factors, e.g., sonic hedgehog, growth differentiation factors (e.g., GDF5, GDF6, GDF8), recombinant human growth factors (e.g., MP52 and MP-52 variant rhGDF-5), cartilage-derived morphogenetic proteins (CDMP-1, CDMP-2, CDMP-3), specific morphogenetic proteins (e.g., CDMP-2, CDMP-3), Examples of suitable polypeptides include, but are not limited to, small molecules that affect upregulation of long factor, tenascin-C, hyaluronic acid, chondroitin sulfate, fibronectin, decorin, thromboelastin, peptides derived from thrombin, heparin binding domains, heparin, heparan sulfate, polynucleotides, DNA fragments, DNA plasmids, MMPs, TIMPs, interfering RNA molecules such as siRNAs, oligonucleotides, proteinaceous proteins, glycoproteins, glycosaminoglycans, and DNA encoding shRNAs.

[0078] In some embodiments, the hydrogels described herein may contain a therapeutic agent that may be used in treating a condition or disorder in a subject in need of such treatment. The term "therapeutic agent" includes any synthetic or naturally occurring biologically active compound or composition of matter that, when administered to an organism (either human or non-human animal), induces a desired pharmacological, immunogenic, and / or physiological effect by local and / or systemic action. Thus, the term encompasses compounds or chemicals traditionally considered to be drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, genetic constructs, and the like. Examples of therapeutic agents include those that have a Merck Index of 14 or higher, such as 5-hydroxytryptamine, 5-hydroxybutyric acid ... th Edition), the Physician's Desk Reference(64 th The Pharmacological Basis of Therapeutics (12 Edition) thThese include, without limitation, pharmaceutical agents, vitamins, mineral supplements, substances used in the treatment, prevention, diagnosis, cure, or mitigation of a disease or illness, substances that affect the structure or function of the body, or prodrugs that become biologically active or more active after being placed in a physiological environment. For example, the term "therapeutic agent" includes, but is not limited to, adjuvants, anti-infectives such as antibiotics and antivirals, analgesics and analgesic combinations, anorexics, anti-inflammatory agents, anti-epileptics, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergics and cholinomimetics, antimuscarinics and muscarinic agents, antiandrenergics, antiarrhythmics, antihypertensives, hormones and nutrients, anti-arthritic agents, antiasthmatics, anticonvulsants, antistaminergics, antiemetics, antineoplastic agents, antipruritics, antipyretics, antispasmodics, cardiovascular agents (including calcium channel blockers, beta blockers, and beta agonists), antihypertensives, diuretics, vasodilators, central nervous system stimulants, cough suppressants, antihistamines, antispasmodics ... and cold remedies, decongestants, diagnostic agents, bone growth stimulants and bone resorption inhibitors, immunosuppressants, muscle relaxants, psychostimulants, sedatives, tranquilizers, proteins, peptides and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced), as well as compounds or compositions for use in all of the major therapeutic areas, including, but not limited to, nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), including both double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules, and other biologically active macromolecules such as, for example, proteins and enzymes. The drugs may be biologically active agents used in medical applications, including veterinary medicine, and in agriculture, such as plants, as well as other areas.

[0079] Hydrogels may be injectable and / or implantable, or may be in the form of membranes, sponges, gels, solid scaffolds, spun fibers, woven or nonwoven meshes, nanoparticles, microparticles, or any other desired configuration.

[0080] In another embodiment, the hydrogel can include at least one cell dispersed on or within the hydrogel. For example, the cells can be fully or partially encapsulated within the hydrogel. The cells can include any progenitor cells, such as, for example, totipotent, pluripotent, or multipotent stem cells, as well as any of their lineage progeny cells, including more differentiated cells. The cells can be autologous, xenogeneic, allogeneic, and / or allogeneic. If the cells are not autologous, it may be desirable to administer immunosuppressants to minimize immune rejection. The cells used can be primary cells, expanded cells, or cell lines, and can be dividing or non-dividing cells. The cells can be expanded ex vivo prior to introduction into or onto the hydrogel. For example, autologous cells can be expanded in this manner if sufficient numbers of viable cells cannot be harvested from the host subject. Alternatively, or additionally, the cells can be tissue fragments, including tissues with some internal structure. The cells can be primary tissue explants and preparations thereof, cell lines (including transformed cells), or host cells.

[0081] In some embodiments, cells can refer to any progenitor cell, such as totipotent stem cells, pluripotent stem cells, and multipotent stem cells, as well as their lineage progeny cells, including more differentiated cells. The terms "stem cell" and "progenitor cell" are used interchangeably herein. Cells can be derived from embryonic, fetal, or adult tissues. Examples of progenitor cells can include totipotent stem cells, pluripotent stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells, neural stem cells, pancreatic stem cells, cardiac stem cells, embryonic stem cells, embryonic germ cells, neural crest stem cells, kidney stem cells, liver stem cells, lung stem cells, hemangioblasts, and endothelial progenitor cells. Additional exemplary progenitor cells can include dedifferentiated chondrogenic cells, chondrogenic cells, umbilical cord blood stem cells, multipotent adult progenitor cells, myogenic cells, osteogenic cells, tenogenic cells, ligamentogenic cells, adipogenic cells, and dermatogenic cells.

[0082] The hydrogel can be formed with at least one cell and / or bioactive agent. For example, a plurality of cells can be distributed on or within the hydrogel in a substantially uniform manner, or alternatively, different cells or different densities and / or spatial distributions of the same cells are distributed within different portions of the hydrogel. The cells can be seeded before or after crosslinking of the polymer backbone. Alternatively, the hydrogel can be incubated in a solution of at least one bioactive agent after crosslinking of the polymer backbone.

[0083] Generally, cells are introduced into the hydrogel in vitro or in vivo. The cells may be mixed with the hydrogel and cultured in an appropriate growth (or storage) medium to ensure cell viability. If the hydrogel is to be implanted for in vivo use after in vitro seeding, for example, sufficient growth medium may be provided to ensure cell viability during in vitro culture prior to in vivo application. Once the hydrogel is implanted, the nutritional requirements of the cells may be met by the circulating fluid of the host subject.

[0084] Any available method may be used to introduce cells into the hydrogel. For example, cells may be injected into the hydrogel (e.g., in combination with growth medium) or introduced by other means such as pressure, vacuum, infiltration, or manual mixing. Alternatively, or additionally, cells may be a layer on the hydrogel, or the hydrogel may be immersed in a cell suspension, allowing the cells to be incorporated into or to be maintained under sufficient conditions and time for them to bind to the hydrogel. In general, it is desirable to avoid excessive manual manipulation of the cells to minimize cell death during the impregnation procedure. For example, in some situations, it may not be desirable to manually mix or knead the cells with the hydrogel, although such an approach may be useful if a sufficient number of cells survive the procedure. Cells may also be introduced into the hydrogel in vivo by simply placing the hydrogel in a subject adjacent to a source of desired cells. Bioactive agents, if contained in the hydrogel, may be released from the hydrogel, which may also mobilize local cells, cells in the circulation, or cells at a distance from the site of implantation or injection.

[0085] The number of cells introduced into the hydrogel will vary based on the intended use of the hydrogel and the type of cells used. For example, if dividing autologous cells are being introduced by injection or mixing into the hydrogel, a smaller number of cells can be used. Alternatively, if non-dividing cells are being introduced by injection or mixing into the hydrogel, a larger number of cells may be required. The hydrogel can be either hydrated or freeze-dried prior to the addition of the cells. For example, the hydrogel can be in a freeze-dried state prior to the addition of the cells to rehydrate the cells and allow the cells to settle in the hydrogel.

[0086] The hydrogels described herein can be used in a variety of biomedical applications, including tissue engineering, drug delivery applications, and regenerative medicine. In one example, the hydrogels described herein can be used to promote tissue growth in a subject. One step of the method can include identifying a target site. The target site can include a tissue defect where promotion of new tissue is desired. The target site can also include a disease location (e.g., a tumor). Methods for identifying tissue defects and disease locations are known in the art and can include various imaging modalities, such as, for example, CT, MRI, and X-ray. After identifying the target site, the hydrogel can be administered to the target site. The hydrogel can then be loaded into a syringe or other similar device and injected or implanted into the tissue defect. Upon injection or implantation into the tissue defect, the hydrogel can be formed into the shape of the tissue defect using tactile means. Alternatively, the hydrogel can be formed into a specific shape prior to implantation into the subject. After implantation, cells can begin to migrate from the hydrogel into the tissue defect, express growth and / or differentiation factors, and / or promote cell expansion and differentiation. In addition, the presence of the hydrogel in the tissue defect can promote the migration of endogenous cells surrounding the tissue defect into the hydrogel. Upon implantation, the moiety of formula I can be hydrolyzed. The hydrolysis of this moiety can occur at a controlled rate, resulting in the controlled degradation of the hydrogel. This degradation can create space for cell growth and deposition of new extracellular matrix to replace the hydrogel.

[0087] As used herein, the term "tissue" can refer to an aggregate of cells having substantially the same function and / or morphology within a multicellular organism. A "tissue" is typically an aggregate of cells of the same origin, but can be an aggregate of cells of different origins. The cells can have substantially the same or substantially different functions and can be of the same or different types. A "tissue" can include, but is not limited to, an organ, part of an organ, bone, cartilage, skin, neurons, axons, blood vessels, cornea, muscle, fascia, brain, prostate, breast, endometrium, lung, pancreas, small intestine, blood, liver, testes, ovaries, cervix, colon, stomach, esophagus, spleen, lymph nodes, bone marrow, kidney, peripheral blood, embryonic, or ascites tissue.

[0088] Further provided are kits for carrying out the methods described herein. By "kit" is intended any article of manufacture (e.g., a package or container) that contains at least one reagent, such as any one of the compositions described herein. The kit may be promoted, distributed, or sold as a unit for carrying out the methods described herein. In addition, the kit may contain a package insert that describes the kit and methods for its use. Any or all of the kit reagents may be provided in a container that protects them from the external environment, such as a sealed container or pouch.

[0089] Also disclosed are kits comprising the compositions disclosed herein in one or more containers. The disclosed kits can optionally include a pharma- ceutically acceptable carrier and / or diluent. In one embodiment, the kit includes one or more other components, supplements, or adjuvants described herein. In one embodiment, the kit includes instructions or packaging that describe how to administer the compositions of the kit. The containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and can be of any suitable size, shape, or configuration. In one embodiment, the compositions disclosed herein are provided in the kit as solids. In another embodiment, the compositions disclosed herein are provided in the kit as liquids or solutions. In one embodiment, the kit includes an ampoule or syringe that contains the compositions described herein in liquid or solution form.

[0090] The present disclosure also provides the following embodiments of the invention provided herein.

[0091] Embodiment 1. A hydrogel comprising a polymer backbone crosslinked with a first crosslinker containing at least one moiety of formula I, [ka] During the ceremony, m and n are independently 1 or 2; A is C2-C 10 is alkyl, [ka] is an attachment point for a moiety within the first crosslinker, the hydrogel. The hydrogel of embodiment 1, wherein embodiment 2.m is 1. The hydrogel of embodiment 1, wherein embodiment 3.m is 2. Embodiment 4. The hydrogel according to any one of embodiments 1 to 3, wherein n is 1. Embodiment 5. The hydrogel of any one of embodiments 1 to 3, wherein n is 2. Embodiment 6. A hydrogel according to any one of embodiments 1 to 5, wherein A is selected from C2-C8 alkyl, C2-C6 alkyl, and C2-C4 alkyl. Embodiment 7. The hydrogel of any one of embodiments 1 to 6, wherein A is a C2 alkyl. Embodiment 8. The hydrogel of any one of embodiments 1 to 7, wherein the polymer backbone comprises poly(ethylene glycol) or a functionalized derivative thereof.

[0023] Embodiment 9. The polymer backbone is selected from the group consisting of poly(ethylene glycol) (PEG), poly(ethylene glycol)-di-acrylate (PEG-DA), multi-arm poly(ethylene glycol)-acrylate (PEG-Ac), poly(ethylene glycol)-dithiol (PEG-diSH), poly(ethylene glycol) divinyl sulfone (PEG-diVS), multi-arm poly(ethylene glycol) vinyl sulfone (PEG-VS), poly(ethylene glycol)-di-methacrylate (PEG-DMA), multi-arm poly(ethylene glycol)-methacrylate (PEG-Mac), poly(ethylene glycol)-di-allyl ether (PEG-diAE), multi-arm poly(ethylene glycol)-aryl ether (PEG-aryl ether), ... 9. The hydrogel of any one of the preceding claims, comprising a polymer selected from poly(ethylene glycol)-vinyl ether (PE-AD), poly(ethylene glycol)-di-vinyl ether (PEG-diVE), multi-arm poly(ethylene glycol)-vinyl ether (PEG-VE), poly(ethylene glycol)-di-maleimide (PEG-diMI), multi-arm poly(ethylene glycol)-maleimide (PEG-MI), poly(ethylene glycol)-di-norborene, multi-arm poly(ethylene glycol)norborene, poly(ethylene glycol)-vinyl carbonate, multi-arm poly(ethylene glycol)-vinyl carbonate, and polyethylene glycol oligofumarate, or a combination thereof. Embodiment 10. The hydrogel of any one of embodiments 1 to 9, wherein the polymer backbone comprises a multi-arm poly(ethylene glycol)-maleimide. Embodiment 11. The hydrogel of any one of embodiments 1 to 10, wherein the first crosslinker comprises m+n moieties capable of reacting with the polymer backbone, where m and n are as defined in embodiment 1. Embodiment 12. The first crosslinker comprises a compound of formula II, [ka] During the ceremony, X 1 and X 2 is independently selected at each occurrence from moieties capable of reacting with the polymer backbone; L 1 and L 2 is independently selected at each occurrence from the linking portion; 12. The hydrogel according to any one of the preceding embodiments, wherein m, n, and A are defined as in embodiment 1. Embodiment 13.X 1 and X 2 and each is -SH. Embodiment 14.L 1 and L 2 But independently, at each occurrence, C1-C 10 14. The hydrogel according to embodiment 12 or 13, wherein the alkyl group is selected from alkyl, aryl, aryl, arylsulfuric acid ... Embodiment 15. The hydrogel of any one of embodiments 1 to 14, wherein the first crosslinker comprises ethylene glycol bis(mercaptoacetate). Embodiment 16 The hydrogel of any one of embodiments 1 to 15, wherein the first crosslinker is hydrolyzable. Embodiment 17. The hydrogel of any one of embodiments 1 to 16, wherein the polymer backbone is further crosslinked with a second crosslinker. Embodiment 18 The hydrogel of embodiment 17, wherein the second crosslinker is hydrolytically stable. Embodiment 19. The hydrogel of embodiment 17 or 18, wherein the second crosslinker comprises dithiothreitol (DTT). Embodiment 20. The hydrogel of any one of embodiments 17 to 19, wherein the degradation of the hydrogel is adjustable by varying the molar ratio of the first crosslinker to the second crosslinker. Embodiment 21. The hydrogel of any one of embodiments 1 to 20, wherein the hydrogel is injectable and / or implantable. Embodiment 22. The hydrogel according to any one of embodiments 1 to 21, wherein the hydrogel is in the form of a membrane, a sponge, a gel, a solid scaffold, a spun fiber, a woven or nonwoven mesh, a nanoparticle, or a microparticle. Embodiment 23. A hydrogel according to any one of embodiments 1 to 22, further comprising at least one cell. Embodiment 24. A process for synthesizing a hydrogel according to any one of embodiments 1 to 23, comprising reacting a polymer with a first crosslinker comprising at least one moiety of formula I, [ka] wherein all variables are as defined in embodiment 1. Embodiment 25. The first crosslinker comprises a compound of formula II, [ka] During the ceremony, X 1 and X 2 is independently selected at each occurrence from moieties capable of reacting with the polymer backbone; L 1 and L 2 is independently selected at each occurrence from the linking portion; 25. The process of embodiment 24, wherein m, n, and A are defined as in embodiment 1. Embodiment 26 The process of embodiment 24 or 25, wherein the first crosslinker comprises ethylene glycol bis(mercaptoacetate). Embodiment 27. The process of any one of embodiments 24 to 26, further comprising reacting the hydrogel with a second crosslinker, wherein the second crosslinker is hydrolytically stable. Embodiment 28 The process of embodiment 27, wherein the second cross-linking agent comprises dithiothreitol (DTT). Embodiment 29. A therapeutic delivery composition comprising the hydrogel of any one of embodiments 1 to 23 and one or more therapeutic agents. Embodiment 30. The therapeutic delivery composition of embodiment 29, wherein the one or more therapeutic agents may be selected from cells, proteins, antibodies, nucleic acids, growth factors, or drugs. Embodiment 31. A cell culture medium comprising a hydrogel according to any one of embodiments 1 to 23. Embodiment 32. A tissue scaffold comprising a hydrogel according to any one of embodiments 1 to 23. Embodiment 33. A bioreactor comprising the hydrogel according to any one of embodiments 1 to 23. Embodiment 34. A wound dressing comprising a hydrogel according to any one of embodiments 1 to 23. Embodiment 35. A method of promoting tissue growth in a subject in need thereof, comprising: Identifying a target site; and administering to the target site a therapeutically effective amount of the hydrogel of any one of embodiments 1-23. Embodiment 36 The method of embodiment 35, wherein the target site comprises a tissue defect in which promotion of new tissue is desired. Embodiment 37. The method of embodiment 35 or 36, wherein the target site is identified using an imaging modality. Embodiment 38. The method of embodiment 37, wherein the imaging modality is selected from CT, MRI, or X-ray. Embodiment 39. The method of any one of embodiments 35 to 38, wherein the hydrogel is injected or implanted into the target site. Embodiment 40. A method of delivering a therapeutic agent to a target site in a subject, the method comprising administering to the target site a therapeutically effective amount of a therapeutic delivery composition described in embodiment 29 or 30. Embodiment 41 The method of embodiment 40, wherein the target site is associated with a disease state or condition. Embodiment 42 The method of embodiment 40 or 41, wherein the target site is a tumor. Embodiment 43. The method of any one of embodiments 40 to 42, wherein the target site is identified using an imaging modality. Embodiment 44. The method of embodiment 43, wherein the imaging modality is selected from CT, MRI, or X-ray. Embodiment 45. The method of any one of embodiments 40 to 44, wherein the hydrogel is injected or implanted into the target site.

[0092] Although several embodiments of the present disclosure have been described, it should be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0093] By way of non-limiting illustration, examples of certain specific embodiments of the present disclosure are provided below. EXAMPLES

[0094] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how the compositions, articles, and / or methods claimed herein are made and evaluated, are intended to be purely exemplary of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, temperature is in degrees Celsius or at ambient temperature, and pressure is at or near atmospheric pressure.

[0095] Example 1. Hydrolytically Degradable Microgels with Tunable Mechanical Properties Modulate the Host Immune Response The application of ester-containing linkers provides a degradation mechanism based on hydrolytic cleavage of ester bonds. Degradation can be controlled by polymer content, macromer molecular weight, crosslink density, and hydrophobicity of the ester-labile linker (Jo, YS; Gantz, J.; Hubbell, JA; Lutolf, MPTailoring Hydrogel Degradation and Drug Release via Neighboring Amino Acid Controlled Ester Hydrolysis. Soft Matter 2009, 5(2), 440-446, and Zustiak, SP; Leach, JBHydrolytically Degradable Poly(Ethylene Glycol) Hydrogel Scaffolds with Tunable Degradation and Mechanical Properties. Biomacromolecules 2010, 11(5), 1348-1357). In contrast to hydrogels that involve enzymatic degradation, hydrogels developed via this approach are degradable via hydrolysis, allowing for consistent degradation profiles that depend solely on the tunable physical, mechanical, and chemical properties of the hydrogel (Jo, YS; Gantz, J.; Hubbell, JA; Lutolf, M.P. Tailoring Hydrogel Degradation and Drug Release via Neighboring Amino Acid Controlled Ester Hydrolysis. Soft Matter 2009, 5(2), 440-446).Bulk gels have been engineered with hydrolyzable crosslinkers before (Jo, YS; Gantz, J.; Hubbell, JA; Lutolf, M.P. Tailoring Hydrogel Degradation and Drug Release via Neighboring Amino Acid Controlled Ester Hydrolysis. Soft Matter 2009, 5(2), 440-446, and Hunckler, MD; Medina, JD; Coronel, MM; Weaver, JD; Stabler, CL; Garcia, A.J. Linkage Groups within Thiol-Ene Photoclickable PEG Hydrogels Control In Vivo Stability. Advanced Healthcare Materials 2019, 8(14), 1900371), but this modality has not yet been translated to microgels fabricated via microfluidic-based polymerization.

[0096] The ability to incorporate degradability into hydrogel networks constitutes a great advantage for regenerative medicine and immune engineering applications, as the durability and mechanical properties of the material modulate the tissue response to the implant. The immune response to the biomaterial ultimately determines the fate of the implanted material, whether it is incorporated into the local tissue or sequestered by the foreign body response (FBR). First, after implantation of the biomaterial, an inflammatory type 1 injury response driven by the proinflammatory mediators IFNγ and TNFα occurs in the vicinity of the material. Pro-regenerative biomaterials then promote the upregulation of M2 (CD206) through IL-4 signaling. +) drive a shift towards a type 2 immune response, promoting macrophage polarization and T helper 2 cell infiltration (Developing a pro-regenerative biomaterial scaffold microenvironment requires T helper 2 cells https: / / www.science.org / doi / 10.1126 / science.aad9272(accessed 2022-02-11)). Meanwhile, the host response to synthetic implants is typically driven primarily by mononuclear phagocytes (Sussman, E. M.; Halpin, M. C.; Muster, J.; Moon, R. T.; Ratner, B. D. Porous Implants Modulate Healing and Induce Shifts in Local Macrophage Polarization in the Foreign Body Reaction. Ann Biomed Eng 2014, 42(7), 1508-1516, and Mikos, A. G., et al. Host response to tissue engineered devices Adv Drug Deliv Rev 1998,33(1-2):111-139) and other cells involved in executing type 3 immune responses (Chung, L.; Maestas, DR; Lebid, A.; Mageau, A.; Rosson, GD; Wu, X.; Wolf, MT; Tam, AJ; Vanderzee, I.; Wang, X.; Andorko, JI; Zhang, H.; Narain, R.; Sadtler, K.; Fan, H.; Cihakova, D.; Le Saux, CJ; Housseau, F.; Pardoll, DM; Elisseeff, JHInterleukin 17 and Senescent Cells Regulate the Foreign Body Response to Synthetic Material Implants in Mice and Humans. Sci Transl Med 2020,12(539),eaax3799). Activated macrophages and Th17 cells secrete TGFβ and other factors that recruit fibroblasts and promote their differentiation into myofibroblasts, driving fibrosis at the implant surface.Recently, more complex interactions between different immune populations of various phenotypes have been implicated in response to biomaterials (Doloff, JC; Veiseh, O.; de Mezerville, R.; Sforza, M.; Perry, TA; Haupt, J.; Jamiel, M.; Chambers, C.; Nash, A.; Aghlara-Fotovat, S.; Stelzel, JL; Bauer, SJ; Neshat, SY; Hancock, J.; Romero, NA; Hidalgo, YE; Leiva, IM; Munhoz, AM; Bayat, A.; Kinney, BM; Hodges, HC; Miranda, RN; Clemens, MW; Langer, R. The Surface Topography of Silicone Breast Implants Mediates the Foreign Body Response in Mice, Rabbits and Humans. Nat Biomed Eng. 2021,5(10),1115-1130, and Witherel,CE;Sao,K.;Brisson,BK;Han,B.;Volk,SW;Petrie,RJ;Han,L.;Spiller,KLRegulation of Extracellular Matrix Assembly and Structure by Hybrid M1 / M2 Macrophages.Biomaterials 2021,269,120667). However, this characterization has mostly been of bulk hydrogel implants, with relatively little work on the effect of degradation on microgel implantation and local tissue response.

[0097] Herein, a fabrication approach based on flow-focusing droplet generation is presented that generates monodisperse hydrolytic microgels with modular mechanical and degradation profiles upon the introduction of a labile ethylene linker, ethylene glycol bis(mercaptoacetate) (EGBMA). It is demonstrated that controlled hydrogel degradation profiles can be achieved by tuning the ester concentration within the hydrogel microparticles via the addition of various molar concentrations of EGBMA to the non-degradable linker in the continuous flow phase. The addition of EGBMA did not affect macrophage polarization in vitro but promoted degradation in vivo. In addition, the impact of degradability on tissue responses is characterized to microgel suspension implants. It is demonstrated that controlling the degradation profile of the microgel suspension can modulate the type 1 immune response to the implant.

[0098] Addition of ester-containing dithiol molecules produces hydrolyzable microgels Hydrolytically degradable microparticles (i.e., microgels) were fabricated by droplet segmentation using a flow-focusing microfluidic device as previously reported (Headen, D. M.; Aubry, G.; Lu, H.; Garcia, A. J. Microfluidic-Based Generation of Size-Controlled, Biofunctionalized Synthetic Polymer Microgels for Cell Encapsulation. Advanced Materials 2014, 26(19), 3003-3008). PEG-4MAL macromers were functionalized with linear PEG-FITC via Michael-type addition for particle tracking prior to segmentation in the microfluidic device. The aqueous phase (containing the functionalized macromers) was pumped into droplets generated by the microfluidic device, which were then covalently crosslinked with a continuous phase of oil containing small dithiol molecules, dithiothreitol (DTT) and ethylene glycol bis(mercaptoacetate) (EGBMA). The addition of EGBMA allowed the incorporation of hydrolytically unstable ester linkers (Figure 1A and Table 1). The concentration of EGBMA was varied from 0.25, 0.5 to 1.0 mM in the continuous crosslinking phase, while the concentration of DTT was kept constant at 15 mM. 1 H molecular diffusion nuclear magnetic resonance (NMR) revealed the absence of maleimide groups in the crosslinked PEG-4MAL macromers, indicating that the maleimide groups within the microgel droplets reacted efficiently after on-chip crosslinking (Figure 8). [Table 1]

[0099] Maintaining the flow rate constant for all conditions for a device with a 200 μm nozzle resulted in monodisperse microgel particles with a mean diameter of 208 μm (CV 8%) when performing only DTT in the crosslinking phase (Figure 1B). Addition of EGBMA to the crosslinking solution produced a monodisperse microgel population (Figures 1C-1E, CV < 10% for all groups), with microgels ranging from 219 to 270 μm in diameter. EGBMA crosslinked microgels were 4, 10, and 33% larger in diameter 4 hours after fabrication compared to DTT-only microgels, and there was significant swelling in the highest concentration EGBMA group compared to the DTT control (Figures 1F and 1G, p < 0.0001 DTT vs. 1.0 mM EGBMA). Macromer functionalization with PEG-FITC was equivalent across groups, as seen by mean fluorescence intensity measurements of microgels after fabrication (Figure 1B-1E), indicating that the differences in swelling can be attributed to the presence of the EGBMA linker, rather than the availability of maleimide groups for crosslinking. By day 30, the DTT-crosslinked microgels had reached an equilibrium size 6% higher than the initial microgel size, while the EGBMA / DTT microgels with the middle and highest concentrations of the labile crosslinker had swollen 26% and 46% larger than their initial size, respectively (Figure 1G, p=0.01 DTT vs. 0.5 mM EGBMA, p<0.001 DTT vs. 1.0 mM EGBMA). Microgel degradation was also assessed by tracking the amount of PEG-FITC released into solution, as PEG-FITC is covalently attached to the PEG-4MAL macromer and can only be released from the hydrogel network by hydrolysis of EGBMA. The PEG-FITC release results are consistent with the swelling experiments, whereby the 1.0 mM EGBMA crosslinked microgels release PEG-FITC at a faster rate than the lower EGBMA concentrations, while the completely non-degradable control releases small amounts of trapped PEG-FITC, followed by the absence of PEG-FITC present in solution as expected.

[0100] Finally, the effect of EGBMA on the mechanical properties of the resulting microgels was determined by pressure-induced deformation through a tapered microcapillary (Wyss, H.M.; Franke, T.; Mele, E.; Weitz, D.A. Capillary Micromechanics: Measuring the Elasticity of Microscopic Soft Objects. Soft Matter 2010, 6(18), 4550-4555) (Figures 9A-9C). The microgels are deformed by the pressure difference across the microgel located at the end of the microcapillary. As the pressure difference increases, the microgel undergoes a radial compressive strain and an axial elongation (Figure 1I). The shear stress and strain can be determined using the taper angle, edge contact length, and average diameter when the microgel is at equilibrium (Figure 1J). Calculation of the shear modulus G at this equilibrium state demonstrated that there was no difference in the elasticity of the microgels 4 hours after fabrication, with values ​​for all groups tested ranging from 20 to 22 kPa. After 72 h in solution, the shear modulus decreased with increasing EGBMA concentration in the microgels, with the most degradable linker group (Figure 1K, p<0.0001 vs. DTT) showing a reduction in modulus from 20 kPa to 14 kPa (28% reduction). By day 7, the most degradable linker group had a 61% (8 kPa) decrease in modulus, a behavior explained by a reduction in crosslink density due to hydrolysis of the ester linker. Taken together, these data do not demonstrate differences in effective crosslink density immediately after fabrication, but the elastic properties of the microgels exhibit a time-dependent decrease based on EGBMA hydrolysis and loss of network crosslinks. Furthermore, the results provide evidence of the consistency of the flow-focusing microfluidic platform in producing physically and mechanically homogenous microgels.

[0101] Microgel degradation and by-products do not induce monocyte activation in vitro To evaluate the effect of hydrolysis products of EGBMA / DTT crosslinked hydrogels on cell viability and activation, the RAW264.7 mouse macrophage cell line was grown in the presence of different microgel formulations for 7 days. The presence of the microgels and manufacturing by-products (e.g., any encapsulated DTT or hydrolysis by-products) were not toxic to this cell line (Figure 10).These results are consistent with previous studies demonstrating no toxicity associated with DTT crosslinking of encapsulated cells or cells co-cultured in fully crosslinked microgels. (Headen, D.M.; Aubry, G.; Lu, H.; Garcia, A.J. Microfluidic-Based Generation of Size-Controlled, Biofunctionalized Synthetic Polymer Microgels for Cell Encapsulation. Advanced Materials 2014, 26(19), 3003-3008; Coronel, M.M.; Martin, K.E.; Hunckler, M.D.; Barber, G.; O'Neill, E.B.; Medina, J.D.; Opri, E.; McClain, C.A.; Batra, L.; Weaver, J.D.; Lim, H.S.; Qiu, P.; Botchwey, E.A.; Yolcu, E.S.; Shirwan, H.; Garcia, A.J. Immunotherapy via PD-L1-Presenting Biomaterials Leads to Long-Term Islet Graft Survival.Science Advances 2020, and Headen, DM; Woodward, KB; Coronel, MM; Shrestha, P.; Weaver, JD; Zhao, H.; Tan, M.; Hunckler, MD; Bowen, WS; Johnson, CT; Shea, L.; Yolcu, ES; Garcia, AJ; Shirwan, H. Local Immunomodulation with Fas Ligand-Engineered Biomaterials Achieves Allogeneic Islet Graft Acceptance.Nature Materials 2018,17(8),732-739).

[0102] To evaluate the effect of microgels on immune cell polarization in vitro, we set up a co-culture system containing primary monocytes derived from bone marrow of C57BL / 6J mice with different formulations of cross-linked microgels. Polystyrene beads (PS) of similar size (200 μm) and the same concentration per well were included as negative controls, as they have been shown not to induce cell activation (Moore, MW; Cruz, AR; LaVake, CJ; Marzo, AL; Eggers, CH; Salazar, JC; Radolf, JD Phagocytosis of Borrelia Burgdorferi and Treponema Pallidum Potentiates Innate Immune Activation and Induces Gamma Interferon Production. Infection and Immunity 2007, 75(4), 2046-2062). Additionally, an IL-4 polarized M2 regulatory phenotype was included as a positive control since exposure of macrophages to bulk PEG hydrogels has been shown to shift cell polarization to a regulatory phenotype in the absence of adhesive and inflammatory signals (Lynn, AD; Bryant, SJP Henotypic Changes in Bone Marrow Derived Murine Macrophages Cultured on PEG-Based Hydrogels and Activated by Lipopolysaccharide. Acta Biomater 2011,7(1),123-132). Although the microparticle-containing groups exhibited similar cell viability after 48 h of coculture with all microgel formulations or PS (Figure 2A), the addition of IL-4 resulted in an increase in cell numbers in the coculture. No changes in the expression of CD45, F4 / 80, and the regulatory marker CD206 were observed in the presence of PS or PEG-based microgels after 2 days of coculture (Figures 2B-2D). In addition, the overall expression of these markers was comparable after 4 days of co-culture (Figure 11A-D).These findings demonstrate that changes in the elastic properties and size of the microgels or degradation products do not induce any phenotypic changes in macrophage marker expression under non-inflammatory conditions in vitro.

[0103] Subdermal microgel implantation results in controlled degradation in vivo To test the ability of EGBMA / DTT crosslinked hydrogels to degrade in vivo, microgels were fabricated as above, but the PEG-FITC tracker was replaced by linear PEG of the same molecular weight containing a near-infrared dye for in vivo tracking. Microgels were injected into subcutaneous pockets on the backs of albino mice (to avoid attenuation of signal detection due to melanin pigmentation (Curtis, A.; Calabro, K.; Galarneau, J.-R.; Bigio, IJ; Krucker, T. Temporal Variations of Skin Pigmentation in C57Bl / 6 Mice Affect Optical Bioluminescence Quantitation. Mol Imaging Biol 2011, 13(6), 1114-1123)). Degradation of the microgels was tracked via in vivo fluorescence imaging (IVIS) (Figure 3A). Normalized radiant efficiency tracking over time demonstrates a decrease in the fluorescent signal that is dependent on the EGBMA concentration (Figure 3B). Notably, a decrease in signal intensity was observed in the DTT crosslinked group, but intensity values ​​were comparable to those at day 1 post-explant, and no degradation was observed in this group as expected (Figure 3B, post-explant values ​​after dashed line, p=0.44). No differences in fluorescent signal between the microgel formulations were observed at day 1 post-injection (Figure 3C), but by day 9, the fluorescent signal was significantly lower in the microgel formulations containing the medium and highest concentrations of EGBMA crosslinker (Figure 3D, p=0.008, p=0.0001 vs. DTT, respectively). At day 25, the signal intensity was 26%, 17%, and 12% of the original signal, a decrease directly proportional to the EGBMA linker concentration (Figure 3E, p=0.0004, p<0.0001, p<0.0001 vs. DTT). Thus, IVIS imaging confirms that EGBMA crosslinked microgels degrade in vivo, and this degradation occurs over a period of several weeks.

[0104] Degradation properties modulate immune responses to microgels in vivo It was hypothesized that the degradation and changes in mechanical properties of the microgels due to swelling and hydrolysis of the ester-containing linker would affect the immune response to the implant. All four different microgel formulations were injected into subcutaneous dorsal pockets of BALB / cJ mice and tissues were collected on day 7 for multiparametric flow analysis. This time point was chosen because it demonstrated differences in mechanical properties and degradation profiles between the materials being tested. To further evaluate how the dynamic changes in degradability and mechanical properties affect the cellular environment, the analysis focused on the comparison between the non-degradable (DTT) and the three different degradable formulations. Compared to the degradable microgels containing 1.0 mM EGBMA, in microgels crosslinked with DTT, myeloid cell populations (CD45+CD11b+) predominated at the injection site (Figures 4A-4B, p=0.018). Phenotyping of subpopulations within the myeloid compartment revealed differences in the presence of F4 / 80+ macrophages as a function of microgel crosslinker formulation (Figures 4C-4D, p=0.008). Measuring activation of this cell population based on cellular expression of major histocompatibility complex class II (MHCII) showed no differences between the microgel formulations (Figures 4E-4F), although the intensity expression of this marker was greatest in the DTT crosslinked group when compared to the most degradable microgel formulation (Figure 4F, p=0.03 DTT vs. 1.0 mM EGBMA). Further analysis of macrophage polarization using M1 and M2-associated markers, CD86 and CD206, showed an increased presence of M2-polarized F4 / 80-expressing macrophages in the non-degradable DTT group compared to all other degradable formulations (Figure 4G), indicating that the tissue response to non-degradable, non-phagocytic microgels was dominated by an M2-like phenotype at this time point and that this polarization could be modulated by the material degradation profile. The effect of the microgel formulations was next examined on the recruitment of T cells to the injection site. DTT-crosslinked microgels recruited greater numbers of CD3+ cells to the implant pocket compared to degradable microgels (Figures 5A-5B). This lymphocyte response was dominated by CD4 helper cells, which were elevated in the presence of non-degradable microgels (Figures 5C-5D).Expression of activation markers CD25 and PD-1 (Figures 5E-5H) was also affected by the microgel formulation, with increased upregulation of surface expression of these markers observed in the non-degradable group compared to the degradable microgels. Overall, the enhanced presence of CD4 cells combined with increased M2-like cell phenotype in the DTT group demonstrates an interplay between these two cell populations in the tissue response to non-degradable PEG-based microgels. Furthermore, microgel degradation profiles, including changes in mechanical properties, can modulate the recruitment and phenotype of specialized cell subpopulations that alter the host tissue response to biomaterial implants.

[0105] The microgel-induced cytokine environment is dynamic and governed by IFN-γ expression. To better understand the interplay of the immune environment with the microgel degradation profile, multiplexing techniques were performed to investigate the cytokine and chemokine (hereafter referred to as cytokine) milieu that regulates T cell recruitment and macrophage polarization following microgel injection. In addition, given the high level of correlation between cytokines and the potential confounding factor of mouse age on cytokine release (4 week difference between first and last time points analyzed), a modular cytokine analysis method, CytoMod, was implemented to provide some context between cytokine clustering and observed cellular phenotypes as opposed to evaluating individual cytokines at different time points (Cohen, L.; Fiore-Gartland, A.; Randolph, AG; Panoskaltsis-Mortari, A.; Wong, S.-S.; Ralston, J.; Wood, T.; Seeds, R.; Huang, QS; Webby, RJ; Thomas, PG; Hertz, TA Modular Cytokine Analysis Method Reveals Novel Associations With Clinical Phenotypes and Identifies Sets of Co-Signaling Cytokines Across Influenza Natural Infection Cohorts and Healthy Controls. Frontiers in Immunology 2019,10,1338 and Distinct inflammatory profiles. distinguishes COVID-19 from influenza with limited contributions from cytokine storm https: / / www.science.org / doi / 10.1126 / sciadv.abe3024(accessed 2021-10-25)).Principal component analysis (PCA) of grouped cytokines identified two directions in the cytokine profile, with most of the variation in cytokine levels being determined in one direction by the cytokines IFN-γ and IL-2 and in the orthogonal direction by G-CSF (Figure 6A, vector direction and color represent contribution to PCA). Cytokine similarity across all subjects was defined by their Pearson correlation coefficients (Figure 6B), whereby, using unsupervised hierarchical clustering, five cytokine modules were identified (Figure 6C). Statistically significant correlations were found in module 1, consisting of cytokines and chemokines involved in inflammation and Th polarization responses (IFN-γ, IL-2, IL-4, IL-17, IL-10, IL-6, MIG, RANTES, M-CSF, LIX). Cytokine specific scores were calculated between cytokine levels and the mean cytokine matrix of all subjects, which determined IFN-γ as the driving cytokine consistent with the previous PCA analysis. Correlation plots within this module show statistically significant positive correlations between most cytokines (IL-2, IL-10, IL-6, LIX, M-CSF) and IFN-γ. Thus, conditions with high expression of IFN-γ were relatively more likely to exhibit high concentrations of these other cytokines (Figure 6D).

[0106] Direct comparison of raw cytokine values ​​for all conditions at all time points evaluated showed a dynamic profile, with EGBMA-containing formulations exhibiting lower mean cytokine levels for most cytokines evaluated (Figures 6E-6F, 12A-12F, 13A-13D). Non-degradable microgels led to increased expression of GM-CSF and G-CSF early after injection (Figures 6E, 12A-12F). By day 7, expression of other chemokines involved in immune cell recruitment such as M-CSF and monokines induced by IFN-γ (MIG) was reduced in the highest EGBMA degradable linker group compared to non-degradable controls (Figures 12A-12F and 13A-13D). Expression of type 1-associated cytokine TNF-α and IFN-γ driver IL-2 was also reduced in tissues exposed to degradable microgels (Figures 12A-12F and 13A-13D). Notably, expression of the key type 2 cytokine IL-4 was dependent on the microgel formulation, with higher levels of IL-4 observed at all time points for non-degradable microgels compared to all degradable formulations (Figure 6F). In addition, no differences in expression of the angiogenic factor VEGF were observed between formulations at the time points examined (Figures 12A-12F). These results, combined with multi-parametric flow analysis of cell phenotypes at the injection site, indicate that the response to PEG-based non-degradable microgels is driven by type 1 immunity with some cross-regulation by the type 2-driving cytokine IL-4. Importantly, this response can be modulated by the introduction of labile ester groups that hydrolyze in vivo. Tissue responses to microgel injections in the backs of albino mice were generally mild, with cellular infiltration surrounding the implant periphery (Figure 14). Notably, none of the implants showed signs of encapsulation or cyst formation. Cellular deposition around the periphery of the microgel implant was evident at the host-implant border 4 weeks after implantation (Figure 15). Qualitative evaluation of the images revealed a higher cell density around the implants for implants containing non-degradable microgels (DTT) compared to all formulations of degradable microgels.Immunohistochemistry for the pan-macrophage marker CD68 demonstrated increased presence of CD68 expression in the DTT and 0.25 and 0.5 mM EGBMA conditions compared to the condition containing the highest concentration of EGBMA (1.0 mM EGBMA). These results are consistent with flow cytometric assessment of myeloid populations (Figures 4A-4G).

[0107] Observations and Conclusions Strategies to impart degradability to microgels have utilized degradable chains in the polymer backbone or labile crosslinker units to allow cleavage by either hydrolysis, enzymatic reaction, or dissolution. Previously, protease-degradable microgels have been generated for the delivery of angiogenic factors (Foster, GA; Headen, DM; Gonzalez-Garcia, C.; Salmeron-Sanchez, M.; Shirwan, H.; Garcia, AJ Protease-Degradable Microgels for Protein Delivery for Vascularization. Biomaterials 2017, 113, 170-175). These microgels were formed implementing droplet microfluidics, but required custom microfluidic device design given the limited solubility of the crosslinked peptide in the continuous phase. Here, a fabrication strategy is presented that utilizes ester hydrolysis to modulate the degradation of crosslinked PEG-4MAL microgels. In contrast to previous approaches, this strategy can be implemented in the same microfluidic device previously designed for the fabrication of non-degradable microgels, since the labile crosslinker units can be added to the oil-crosslinked phase. Thus, this strategy allows for the tuning of the degradation properties of the microgel product by simply adjusting the crosslinker supply.

[0108] Hydrogel degradation was monitored by assessing changes in physical and mechanical properties, including swelling, release of the PEG-FITC tag, and elastic modulus. Changes in these parameters were directly related to the EGBMA crosslinker content and thus the number of hydrolyzable groups. Immediately after fabrication, microgels synthesized with the highest concentration of labile ester junctions swelled to approximately 140% of the size of the non-degradable microgel control, but no notable difference in elastic modulus was observed at this time point. This is explained by the fact that multiple ester bonds must be cleaved to fully release the PEG-4MAL macromers. Indeed, measurable changes in elastic modulus were first observed after 72 h in aqueous buffer, when sufficient crosslinks had been cleaved and the PEG-4MAL macromers were dissolved in aqueous media. The differences were more pronounced with time and directly proportional to the EGBMA content, demonstrating the tunability of this approach.Although not tested in this example, it is known that hydrophobicity and the presence of carbon units between the ester and thiol can affect the rate of ester hydrolysis (Zustiak, SP; Leach, JB Hydrolytically Degradable Poly(Ethylene Glycol) Hydrogel Scaffolds with Tunable Degradation and Mechanical Properties. Biomacromolecules 2010, 11(5), 1348-1357; Jo, YS; Gantz, J.; Hubbell, JA; Lutolf, MPTailoring Hydrogel Degradation and Drug Release via Neighboring Amino Acid Controlled Ester Hydrolysis. Soft Matter 2009, 5(2), 440-446; and Schoenmakers, RG; van de Wetering, P.; Elbert, DL; Hubbell, JA The Effect of the Linker on the Hydrolysis Rate of Drug-Linked Ester Bonds. Journal of Controlled Release 2004, 95(2), 291-300). Thus, the implementation of linkers with hydrophobic units between the ester and thiol groups, or modification of the polymer density, may provide additional control over the degradation of hydrogels synthesized by this approach without any obvious impact on the fabrication technique.

[0109] Material degradability is highly desirable for biomaterial platforms, but can result in undesirable toxicity and immune activation responses that hinder their applicability in the clinic. In this example, no significant effect on cell viability was observed in any of the microgel formulations tested, demonstrating that the presence of microgels or their degradation by-products does not result in toxicity-induced cell death at doses up to about 3 microgels / μL. In addition, the size of the microgels (>200 μm) implemented in this example should prevent them from being phagocytosed by macrophages (Champion, JA; Mitragotri, S. Role of Target Geometry in Phagocytosis. PNAS 2006, 103(13), 4930-4934), however, degradation by-products and partial internalization would result in macrophage polarization.Alterations in mechanical cues have demonstrated effects on M1-like macrophage activation (Patel, NR; Bole, M.; Chen, C.; Hardin, CC; Kho, AT; Mih, J.; Deng, L.; Butler, J.; Tschumperlin, D.; Fredberg, JJ; Krishnan, R.; Koziel, H. Cell Elasticity Determines Macrophage Function. PLOS ONE 2012, 7(9), e41024 and Fereol, S.; Fodil, R.; Labat, B.; Galiacy, S.; Laurent, V. M.; Louis, B.; Isabey, D.; Planus, E. Sensitivity of Alveolar Macrophages to Substrate Mechanical and Adhesive Properties. Cell Motility 2006,63(6),321-340), and changes in surface chemistry have been shown to have a higher impact on M2-like polarization (Thiols Decrease Human Interleukin(IL)4 Production and IL-4-Induced Immunoglobulin Synthesis. J Exp Med 1995,182(6),1785-1792, and Li,Z.;Bratlie,K.M.How Cross-Linking Mechanisms of Methacrylated Gellan Gum Hydrogels Alter Macrophage Phenotype.ACS Appl.Bio Mater.2019,2(1),217-225). In this example, neither the presence of EGBMA cross-linking units nor changes in mechanical properties affected M2-associated CD206 marker expression in vitro. Most of the unreacted cross-linking molecules were removed from the microgel suspension by centrifugation / washing steps. Furthermore, at the microgel-to-cell ratios implemented, the concentration of linker molecules in solution did not affect the expression of this marker due to hydrolysis.

[0110] Despite several strategies reported for degradable PEG-based hydrogels, there are few reports that the in vivo degradation rate can be several orders of magnitude different from the in vitro degradation rate (Hunckler, MD; Medina, JD; Coronel, MM; Weaver, JD; Stabler, CL; Garcia, AJ Linkage Groups within Thiol-Ene Photoclickable PEG Hydrogels Control In Vivo Stability. Advanced Healthcare Materials 2019, 8(14), 1900371; Amer, LD; Bryant, SJ The in Vitro and in Vivo Response to MMP-Sensitive Poly(Ethylene Glycol) Hydrogels. Ann Biomed Eng 2016, 44(6), 1959-1969; and Browning, MB; Cereceres, SN; Luong, PT; Cosgriff-Hernandez, EM Determination of the in Vivo Degradation Mechanism of PEGDA Hydrogels. J Biomed Mater Res A 2014,102(12),4244-4251). Indeed, protease-cleavable formulations that have been shown to degrade rapidly in culture do not degrade after implantation (Amer, LD, Bryant, SJ The in Vitro and in Vivo Response to MMP-Sensitive Poly(Ethylene Glycol) Hydrogels. Ann Biomed Eng 2016,44(6),1959-1969).Similarly, differences in degradation rates have also been observed in hydrolysis, whereby gradual hydrolysis rates in vitro did not match the rapid degradation observed in vivo (Hunckler, MD; Medina, JD; Coronel, MM; Weaver, JD; Stabler, CL; Garcia, AJ Linkage Groups within Thiol-Ene Photoclickable PEG Hydrogels Control In Vivo Stability. Advanced Healthcare Materials 2019,8(14),1900371). Here, using near-infrared dye-labeled microgels, it was demonstrated that DTT / EGBMA crosslinked microgels degrade in vivo, with degradation times extending to weeks. This is consistent with in vitro studies and degradation rates observed with other ester-containing bulk PEG hydrogels (Zustiak, SP; Leach, JB Hydrolytically Degradable Poly(Ethylene Glycol) Hydrogel Scaffolds with Tunable Degradation and Mechanical Properties. Biomacromolecules 2010,11(5),1348-1357). In subsequent studies, it will be important to evaluate how the addition of biological factors (e.g., adhesion ligands, encapsulated cells, or therapeutic agents) alters the rate of ester hydrolysis in these microgels.

[0111] Finally, tissue response as a function of degradability was evaluated in a subcutaneous dorsal model. This site offers an easily accessible location capable of holding a significant microgel implant volume. Furthermore, multiple independent microgel suspensions can be injected into different quadrants of the dorsum, thus allowing the use of the same animal as its own internal positive control. Multiparametric flow analysis demonstrated a degradation-dependent immune response with enhanced presence of myeloid and T cells, especially CD4+, in the non-degradable formulations, consistent with other studies showing T helper cells driving the response to synthetic material implants (Chung, L.; Maestas, DR; Lebid, A.; Mageau, A.; Rosson, GD; Wu, X.; Wolf, MT; Tam, AJ; Vanderzee, I.; Wang, X.; Andorko, JI; Zhang, H.; Narain, R.; Sadtler, K.; Fan, H.; Cihakova, D.; Le Saux, CJ; Housseau, F.; Pardoll, DM; Elisseeff, JHInterleukin 17 and Senescent Cells Regulate the Foreign Body Response to Synthetic Material Implants in Mice and Humans. Sci Transl Med 2020,12(539),eaax3799, and Chan,T.;Pek,EA;Huth,K.;Ashkar,AACD4+T-Cells Are Important in Regulating Macrophage Polarization in C57BL / 6 Wild-Type Mice.Cellular Immunology 2011,266(2),180-186). Further evaluation of the cytokine milieu provided further insight into the diversity and complexity of the immune response.In contrast to reports of IL-17-driven immune responses to synthetic bulk implants (Chung, L.; Maestas, DR; Lebid, A.; Mageau, A.; Rosson, GD; Wu, X.; Wolf, MT; Tam, AJ; Vanderzee, I.; Wang, X.; Andorko, JI; Zhang, H.; Narain, R.; Sadtler, K.; Fan, H.; Cihakova, D.; Le Saux, CJ; Housseau, F.; Pardoll, DM; Elisseeff, JHInterleukin 17 and Senescent Cells Regulate the Foreign Body Response to Synthetic Material Implants in Mice and Humans. Sci Transl Med 2020, 12(539), eaax3799), we found that injection of a synthetic microgel suspension resulted in a significant expression of IFN-γ that remained elevated for up to 4 weeks. Notably, unsupervised clustering of cytokine correlations identified IFN-γ as the dominant response driving cytokine communication. IFN-γ is one of the canonical cytokines driving type 1 immune responses (Tuzlak, S.; Dejean, A.S.; Iannacone, M.; Quintana, F.J.; Waisman, A.; Ginhoux, F.; Korn, T.; Becher, B. Repositioning TH Cell Polarization from Single Cytokines to Complex Help. Nat Immunol 2021, 22(10), 1210-1217), which is mainly produced by activated T cells and promotes M1 polarization through STAT1 phosphorylation (Kak, G.; Raza, M.; Tiwari, B.K. Interferon-Gamma (IFN-γ): Exploring Its Implications in Infectious Diseases. Biomolecular Concepts 2018, 9(1), 64-79).Evaluation of macrophage cell responses immediately after implantation of microgel suspensions revealed phenotypic characteristics (i.e., CD206 expression) that resembled a more M2-like phenotype. This phenotypic plasticity demonstrates changes in the microenvironment that lead to repolarization of IFN-γ-activated macrophages. Given that M1 polarization can prime a transition to a distinct M2 phenotype in response to IL-4, it should be investigated whether this repolarization is indeed due to the presence of other cytokines in the immune response (i.e., persistence of IL-4) (O'Brien, EM; Spiller, KL Pro-Inflammatory Polarization Primes Macrophages to Transition into a Distinct M2-like Phenotype in Response to IL-4. Journal of Leukocyte Biology n / a(n / a)). In addition, these studies were performed in BALB / cJ mice, which have been shown to have a genetic predisposition toward M2 polarization. Therefore, the microgel-induced M2 phenotype cannot be generalized until it is tested in other strains (Chan, T.; Pek, EA; Huth, K.; Ashkar, AACD4+T-Cells Are Important in Regulating Macrophage Polarization in C57BL / 6 Wild-Type Mice. Cellular Immunology 2011,266(2),180-186).

[0112] Although type 1 cytokines appeared to be the primary driver of local tissue responses, this example indicates a reciprocal IL-4-driven response that should be further explored. Even 30 days after injection, observable changes in IL-4 secretion were evident in the non-degradable implants. Modulation of this immune response was possible by imparting a degree of degradability to the microgel platform, particularly in that the hydrolyzable microgels experienced a reduction in IL-4 secretion and a corresponding reduction in the presence of CD206+ macrophages compared to the non-degradable microgels. Even minimal incorporation of a labile ester crosslinker that did not result in complete degradation in the time frame examined (i.e., 0.25 mM EGBMA) provided differences in cellular and cytokine profiles, indicating that the degradation profile of the material has a profound impact on the tissue response.

[0113] Although not explored in this example, a range of parameters such as material shape, size, surface texture, stiffness, and charge may influence host-implant interactions and subsequent immune recognition and development of FBR (Doloff, JC; Veiseh, O.; de Mezerville, R.; Sforza, M.; Perry, TA; Haupt, J.; Jamiel, M.; Chambers, C.; Nash, A.; Aghlara-Fotovat, S.; Stelzel, JL; Bauer, SJ; Neshat, SY; Hancock, J.; Romero, NA; Hidalgo, YE; Leiva, IM; Munhoz, AM; Bayat, A.; Kinney, BM; Hodges, HC; Miranda, RN; Clemens, MW; Langer, R. The Surface Topography of Silicone Breast Implants Mediates the Foreign Body Response in Mice, Rabbits and Humans. Nat Biomed Eng 2021,5(10),1115-1130, Veiseh,O.;Doloff,JC;Ma,M.;Vegas,AJ;Tam,HH;Bader,AR;Li,J.;Langan ,E.;Wyckoff,J.;Loo,WS;Jhunjhunwala,S.;Chiu,A.;Siebert,S.;Tang,K.;Hollister-Lock,J.;Ar esta-Dasilva, S.; Bochenek, M.; Mendoza-Elias, J.; Wang, Y.; Qi, M.; Lavin, DM; Chen, M.; Dholakia, N.; Thakrar, R.; Lacik, I.; Weir, GC; Oberholzer, J.; Greiner, DL; Langer, R.; Shape-Dependent Foreign Body Immune Response to Materials Implanted in Rodents and Non-Human Primates.Nature Mater 2015,14(6),643-651; and Blakney,AK;Swartzlander,MD;Bryant,SJThe Effects of Substrate Stiffness on the in Vitro Activation of Macrophages and in Vivo Host Response to Poly(Ethylene Glycol)-Based Hydrogels.J Biomed Mater Res A 2012,100(6),1375-1386. For example, the FBR from spherical agarose microgels is modulated by the implant shape and size, with larger spherical implants activating lower FBRs compared to smaller implants (Veiseh,O.;Doloff,JC;Ma,M.;Vegas,AJ;Tam,HH;Bader,AR;Li,J.;Langan,E.;Wyckoff,J.;Loo,WS;Jhunjhunwala,S.;Chiu,A.;Si ebert, S.; Tang, K.; Hollister-Lock, J.; Aresta-Dasilva, S.; Bochenek, M.; Mendoza-Elias, J.; Wang, Y.; Qi, M.; Lavin, D M;Chen,M.;Dholakia,N.;Thakrar,R.;Lacik,I.;Weir,GC;Oberholzer,J.;Greiner,DL;Langer,R.;Anderson,DGSize-and Shape-Dependent Foreign Body Immune Response to Materials Implanted in Rodents and Non-Human Primates.Nature Mater 2015,14(6),643-651). Similarly, chemical modification of PEG hydrogels with hydrophilic materials can modulate the FBR by reducing protein absorption and cell attachment (Jansen, LE; Amer, LD; Chen, EY-T.; Nguyen, TV; Saleh, LS; Emrick, T.; Liu, WF; Bryant, SJ; Peyton, SRZwitterionic PEG-PC Hydrogels Modulate the Foreign Body Response in a Modulus-Dependent Manner. Biomacromolecules 2018, 19(7), 2880-2888). It is increasingly recognized that key material properties such as stiffness have a profound impact on driving cell behavior (Blakney, A.K.; Swartzlander, M.D.; Bryant, S.J. The Effects of Substrate Stiffness on the in Vitro Activation of Macrophages and in Vivo Host Response to Poly(Ethylene Glycol)-Based Hydrogels. J Biomed Mater Res A 2012, 100(6), 1375-1386, and Irwin, E.F.; Saha, K.; Rosenbluth, M.; Gamble, L.J.; Castner, D.G.; Healy, K.E. Modulus-Dependent Macrophage Adhesion and Behavior. Journal of Biomaterials Science, Polymer Edition 2008, 19(10), 1363-1382). A stiffness-driven inflammatory response to PEG hydrogels has been reported previously and is thought to be related to increased immune cell adhesion to stiffer surfaces (Blakney, AK; Swartzlander, MD; Bryant, SJ The Effects of Substrate Stiffness on the in Vitro Activation of Macrophages and in Vivo Host Response to Poly(Ethylene Glycol)-Based Hydrogels. J Biomed Mater Res A 2012,100(6),1375-1386). This response has recently been attributed to the mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4), independent of other biochemical cues (Goswami, R.; Arya, RK; Sharma, S.; Dutta, B.;Stamov,DR;Zhu,X.;Rahaman,SOMechanosensing by TRPV4 Mediates Stiffness-Induced Foreign Body Response and Giant Cell Formation.)Science Signaling 14(707),eabd4077). As the microgel implants used in this study swell significantly during hydrolytically mediated degradation, we cannot exclude that this dynamic shift in size after implantation, together with the reduction in stiffness, leads to the modulation of cell infiltration observed in this example. It would be interesting to separate these two parameters in non-degradable implants to evaluate the specific effect of size of DTT-crosslinked microgels on FBR. .

[0114] In summary, this example presents a cost-effective approach to imparting microgels with degradable features from segmented PEG-4MAL macromers via droplet microfluidics. Microgels with ester-labile crosslinking junctions degrade easily in vitro and in vivo. Furthermore, the degradation profile influences the immune response to the implant, with reduced type 1-associated cytokines and cells present when degradable microgels are delivered. The simplicity of this strategy and the efficiency of hydrolysis of the resulting microgel population make this approach attractive for regenerative medicine and drug delivery applications.

[0115] experiment Microfluidic Device Fabrication: PDMS microfluidic devices were prepared as previously reported (Headen, D. M.; Aubry, G.; Lu, H.; Garcia, A. J. Microfluidic-Based Generation of Size-Controlled, Biofunctionalized Synthetic Polymer Microgels for Cell Encapsulation. Advanced Materials 2014, 26(19), 3003-3008). Briefly, PDMS was molded using soft lithography and a SU8 master with the microfluidic device pattern and heated to 110°C for 20 min. The resulting PDMS microfluidic device was removed from the wafer, bonded to a glass slide, and heated at 70°C overnight.

[0116] PEG-4MAL microgel fabrication: A flow-focusing microfluidic device with a 200 μm nozzle was used to form polymer droplets. The aqueous phase consisted of 5% w / v PEG-4Mal (20 KDa, Laysan Bio) previously reacted with thiol-PEG-FITC (1 kDa, Nanocs). The co-flow shield phase consisted of mineral oil (Sigma) with 2% SPAN80 (Sigma). The crosslinker phase contained an emulsion of mineral oil / SPAN80 with DTT (Thermo) at a concentration of 15 mM. To render the microgels degradable, various amounts of EGBMA (Sigma) were added to the crosslinker phase at concentrations of 0.25, 0.5, and 1.0 mM. After fabrication, the microgels were extracted from the oil phase by centrifugation and washed with a 2% bovine serum albumin (Sigma) / PBS (corning) solution.

[0117] Microgel sizing and swelling: Characterization of the crosslinking phase relative to the size of the microgels was measured after fabrication using a Biotek Cytation spectrophotometer. Triplicate 50 μL samples were placed in glass bottom 6-well plates. Quantitative fluorescence intensity of each microgel was recorded for all samples. Droplet diameter was measured using the cell analysis plugin of Cytation Gen software. For swelling studies, 1000 microgels were placed in 1 mL PBS and placed in an incubator. 50 μL samples were taken daily and measured as described above. For FITC tracking studies, 1000 microgels were placed in 1 mL PBS and the solution was changed daily. Collected supernatant fluorescence was measured using a Cytation3 plate reader.

[0118] Mechanical testing of microcapillaries: The elastic properties of the microgels were determined using pressure-driven capillary micromechanics (Wyss, HM; Franke, T.; Mele, E.; Weitz, D. Capillary Micromechanics: Measuring the Elasticity of Microscopic Soft Objects. Soft Matter 2010, 6(18), 4550-4555). At various time points (day 0, 3, 7), microgels were inserted into the end of a tapered glass micropipette (Fivephoton Biochemicals) that had been pre-coated with 1% (w / v) BSA in PBS. A high-precision pressure regulator (Elveflow) was attached to the end of the micropipette and pressure was applied at various intervals (0, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 40, 50, 60 kPa). When the microgels reached equilibrium (no longer moved within the micropipette when external pressure was in equilibrium with internal elastic stress), images were taken under a microscope (10x; EVOS) and parameters were measured using ImageJ.

[0119] Viability assessment: RAW264.7 cells were co-cultured with 10,000 microgels for 7 days. Cell metabolic activity was measured via AlamarBlue (Invitrogen). Assays were performed at different time points (1, 2, 4, and 7 days). After 4 hours of incubation, 100 μL of supernatant was transferred into wells of a 96-well plate and OD was measured using a Cytation3 imaging reader (Biotek) at wavelengths of 570 nm and 600 nm.

[0120] Co-culture of bone marrow-derived macrophages: Bone marrow was isolated from femurs and tibias of 6-week-old male C57BL / 6J mice. Bones were cleaned from soft tissue and cut on one side to expose the bone marrow, inverting them into a 200 μL pipette tip cut to fit into a 1.5 mL Eppendorf tube. The bones were then centrifuged at 10,000×g for 15 seconds to pellet the bone marrow at the bottom of the Eppendorf tube. The bones were discarded and the cells were then resuspended in RBC lysis buffer (Biolegend 420302) to remove red blood cells. The cells were then washed with MACS buffer (DPBS pH 7.2, 0.5% BSA, 2 mM EDTA) and monocytes were isolated using a Monocyte Isolation Kit (BM), Mouse (Miltenyi Biotec 130-100-629), and LS columns (Miltenyi Biotec 130-042-401).

[0121] Monocytes were cultured in low attachment plates for 6 days in RPMI1640 medium (Gibco11875-085) supplemented with 10% heat-inactivated fetal bovine serum, 1% pen / strep, and 20ng / mL mouse M-CSF (Biolegend574804). Cells were harvested and seeded with microparticles at a ratio of 1:10 (10,000 cells / 1000 microgels per well). M2 control macrophages were cultured in medium supplemented with both 20ng / mL mouse M-CSF and 20ng / mL mouse IL-4 (Biolegend574304). After 48 and 96 h of co-culture, cells were harvested and stained using the following markers: live dead (Zombie Violet, BioLegend423113), CD45 (PE-Texas Red, BioLegend103146), CD11b (PercpCy5.5, BioLegend101228), F4 / 80 (FITC, BioLegend123108), and CD206 (PECy7, BioLegend141720). Samples were analyzed on a FACS-AriaIIIu flow cytometer (BD Biosciences).

[0122] Implantation of microgels into mice: All animal procedures were performed under a protocol approved by the Georgia Tech IACUC and in accordance with National Institutes of Health guidelines (IACUC approved protocol number A100326). Microgels were injected subepidermally into 8-12 week old BALB / cJ mice. A 100 μL injection consisted of approximately 3000 particles of non-degradable or degradable hydrogel. All four conditions were injected into the same animal at independent sites to reduce any variability due to inherent biological differences between animals.

[0123] In vivo tracking of microgels: Macromers were functionalized with 1KDa PEG labeled with AlexaFluor750NHS ester (Thermo Fisher). Immediately after fabrication, 3000 microgels were injected under the epidermis in 100 μL of saline. Signal intensity and distribution were monitored longitudinally using an IVIS SpectrumCT imaging system (Perkin-Elmer). Data were analyzed using Living Image software. Regions of interest (ROIs) were drawn in the defined pocket areas and quantified using radiant efficiency [p / s / sr] / [μW / cm2]. ROIs maintained the same size for the pockets of each group at all time points and were appropriately sized to contain the fluorescent signal for each area to ensure that imaging data between individual donors were comparable across time. Intensity measurements were normalized to day 0 values.

[0124] Multiparametric flow analysis of tissue responses: Tissue samples were obtained with a 12 mm biopsy punch and digested with Accumax solution (Sigma) for 60 min at 37° C. The digested tissue was passed through a 40 μm strainer and then washed twice with 1×PBS. Cells were washed and stained for live / dead (Zombie violet, BioLegend423113) and surface stained with the following myeloid markers: CD45 (PE-Texas Red, BioLegend103146), CD11b (PercpCy5.5, BioLegend101228), F4 / 80 (FITC, BioLegend123108), CD11c (BV785, BioLegend117335), MHCII (APC-Cy7, BioLegend107652), CD86 (APC, BioLegend105012), CD206 (PECy7, BioLegend141720). and lymphoid markers: CD45 (BV711, BioLegend), CD3 (BV510, BioLegend100233), CD4 (APC, BioLegend100412), CD8 (PercpCy5.5, BioLegend100732), CD25 (PECy7, BioLegend102016), and PD-1 (PE Texas Red, BioLegend135227). Flow cytometry was performed on a BD Aria and analyzed with FCS express.

[0125] Cytokine analysis: Microgels were injected subcutaneously beneath the epidermis as described above. At set time points, tissue was removed using a 12 mm biopsy punch around the injection site. Samples were then placed in RIPA buffer containing protease inhibitors (Thermo). Samples were sonicated and centrifuged at 10,000×g for 10 min at 4°C to remove debris. Supernatants were frozen in liquid nitrogen and stored at −80°C until analysis. Samples were analyzed using the Milliplex MAP Mouse Cytokine / Chemokine 32-plex assay (Millipore, MCYTMAG) on a Magpix multiplexing instrument (Luminex) according to the manufacturer's instructions. PCA was performed on all samples using the “prcomp” function in R and visualized using the “factoextra” package. Cytokine correlations were investigated using CytoMod (Cohen, L.; Fiore-Gartland, A.; Randolph, A.; Panoskaltsis-Mortari, A.; Wong, S.-S.; Ralston, J.; Wood, T.; Seeds, R.; Huang, Q.S.; Webby, R.J.; Thomas, P.G.; Hertz, T.A. Modular Cytokine Analysis Method Reveals Novel Associations With Clinical Phenotypes and Identifies Sets of Co-Signaling Cytokines Across Influenza Natural Infection Cohorts and Healthy Controls. Frontiers in Immunology 2019,10,1338 and Distinct inflammatory profiles distinguish COVID-19 from influenza with limited contributions from cytokine storm https: / / www.science.org / doi / 10.1126 / sciadv.abe3024(accessed For the correlation analysis, the detection limit was set to a value below the detection limit.Log. 10 Multivariate linear regression with concentrations were modeled as a function of time. Pairwise differences in estimated marginal means between conditions were assessed using the “emmeans” package in R, and Tukey's method was used to adjust for multiple comparisons.

[0126] Immunohistochemistry: After euthanasia on day 30, tissue was removed using a 12 mm biopsy punch around the injection site, which was then fixed overnight in 10% formalin solution. Samples were then dehydrated in graded ethanol solutions, cleared in xylene, and paraffin embedded. Sections were cut at 10 μm, and slides were stained using hematoxylin and eosin (H&E) and IHC for macrophage pan marker CD68 (abcam, ab125212), and a nuclear stain (DAPI, Invitrogen D1306).

[0127] Statistical Analysis All experiments were performed on biological replicates. Sample size for each experimental group and statistical tests used to determine significant differences between groups with post-hoc tests where appropriate are reported in the appropriate figure legends. Exact p-values ​​or significance symbol meanings are indicated in the legends. Data were analyzed using Graphpad Prism v9 (GraphPad Inc.). For cytokine analysis, concentration data were log-transformed for normalization and analyses were performed in R using the hclust, glm, and lmmeans packages. Experiments were unblinded and no randomization was used.

[0128] Example 2. Degradable microgels Hydrogel crosslinking with ester-containing linkers offers a degradation mechanism focused on hydrolytic cleavage of ester bonds. Degradation can be controlled by the polymer content of the ester-labile linker, molecular weight, and crosslink density (Zustiak, SP, & Leach, JB (2010). Hydrolytically Degradable Poly(Ethylene Glycol) Hydrogel Scaffolds with Tunable Degradation and Mechanical Properties. Biomacromolecules, 11(5), 1348-1357). In contrast to enzyme-dependent hydrogels, the hydrogels developed by this approach are degradable by hydrolysis, allowing for a controlled and consistent degradation profile that depends solely on the tunable physical, mechanical, and chemical properties of the hydrogel (Sung, B., Kim, C., & Kim, M.-H. (2015). Biodegradable colloidal microgels with tunable thermosensitive volume phase transitions for controllable drug delivery. Journal of Colloid and Interface Science, 450, 26-33, and Stukel, J., Thompson, S., Simon, L., & Willits, R. (2015). Polyethlyene glycol microgels to deliver bioactive nerve growth factor: Microgels to Deliver Bioactive NGF. Journal of Biomedical Materials Research Part A, 103(2), 604-613).Importantly, this degradation method does not prevent the implementation of cysteine-terminated adhesives or peptides (Stukel, J., Thompson, S., Simon, L., & Willits, R. (2015). Polyethlyene glycol microgels to deliver bioactive nerve growth factor: Microgels to Deliver Bioactive NGF. Journal of Biomedical Materials Research Part A, 103(2), 604-613). Its use in drug delivery and tissue engineering has proven effective in providing long-term sustained release profiles (Zustiak, SP, & Leach, JB (2010). Hydrolytically Degradable Poly(Ethylene Glycol) Hydrogel Scaffolds with Tunable Degradation and Mechanical Properties. Biomacromolecules, 11(5), 1348-1357; Pradal, C., Grondahl, L., & Cooper-White, JJ (2015). Hydrolytically Degradable Polyrotaxane Hydrogels for Drug and Cell Delivery Applications. Biomacromolecules, 16(1), 389-403; and Davis KA, & Anseth, KS (2002). Controlled Release from Crosslinked Degradable Networks; Critical Reviews in Therapeutic Drug Carrier Systems, 19(4-5), 385-424). Herein, we present a fabrication mechanism to generate monodisperse hydrolytic hydrogels based on flow-focusing droplet generation with tunable release and degradation profiles that relies on the introduction of labile ethylene linkers.By tuning the labile ester chemistry of the hydrogel structure through the addition of varying amounts of ethylene linkers and non-degradable thiol linkers, we are able to develop controlled hydrogel degradation profiles. In addition, we characterize the effects of changes in mechanical properties and degradative by-products on cell phenotypes in vitro and in vivo.

[0129] method Fabrication of microfluidic devices. PDMS microfluidic devices were constructed from the addition of 184 silicone elastomer and 184 silicone elastomer curing agent. The silicone mixture was then placed onto a silicon wafer consisting of the microfluidic device pattern and heated to 110 °C for 20 min. The resulting PDMS microfluidic device was removed from the wafer, bonded to a glass slide, and heated at 70 °C overnight.

[0130] 4-arm poly(ethylene glycol) microgel structures. Appropriate amounts of PEG-4Mal (20 KDa 4-arm polyethylene glycol from Laysan Bio), PEG-biotin, and DTT (dithiothreitol) were weighed out. A 10 mM DPBS / HEPES (Dulbecco's Phosphate Buffered Saline / 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) solution was made and used to suspend the PEG-biotin. This new solution of DPBS / HEPES solution and PEG-biotin was then used to resuspend the PEG-4Mal that was flowing through the lines to the microfluidic device. In addition, DTT and DPBS / HEPES solutions were made. A 2% Span 80 / mineral oil solution was also made, and 395 μl of DTT in DPBS / HEPES was added to 5 mL of the 2% Span 80 / mineral oil solution. For the degradable microgels, the calculated concentration of degradable thiol linker was added to a solution of DTT and 2% SPAN80 / mineral oil. Following the creation of the solutions, three syringe pumps were set up and the microfluidic device was prepared for creation. One pump was set up to push oil through the device (1 μl / min), another pump was set up to push PEG through the device (5 μl / min), and the final pump was used to push DTT solution through the device (35 μl / min). A collection line filled with dPBS and 1% BSA (bovine serum albumin) was set up from the collection bath in the device to the collection tube. After preparing the device, the lines were set up and the three solutions were passed through the microfluidic device. After the pumps and lines were passed through the device for approximately 45 minutes, the collection tube was placed in a centrifuge for 5 minutes and a series of washes were performed to remove the DTT and oil from the collection and allow the microgels to collect at the bottom of the tube.

[0131] Microgel degradation. Approximately 200 microgels were placed into each well of a 48-well plate and incubated over several days. Every day, the number of microgels in each well was counted and analyzed for swelling using an LED microscope. After analyzing the microgels, DPBS was added to each well and they were returned to the incubator.

[0132] Semi-quantitative analysis of protein encapsulation. Western blot transfer was performed to analyze the ability of the hydrogels to capture proteins. Semi-quantitative analysis of the microgels was performed via gel electrophoresis and chemiluminescence imaging techniques to determine whether the microgels were ready for implantation.

[0133] Implantation of microgels into mice. Microgels were injected subepidermally into 8-12 week old Balb / C mice. A 100uL injection consisted of approximately 3000 non-degradable or degradable hydrogels. Non-degradable microgels consisted of DTT crosslinker and no degradable thiol linker, whereas degradable microgels consisted of a mixture of DTT crosslinker and 0.25mM, 0.5mM, or 1mM thiol linker.

[0134] Tracking the microgels. Using the IVIS imaging system, we were able to image the microgels and view their fluorescence. Over the course of several days, the microgel signaling was tracked and the degradation rate was recorded.

[0135] Example 3. Hydrolyzable hydrogels for therapeutic delivery Hydrogels are increasingly used in regenerative medicine to deliver drugs or biological therapeutics because they are modular, biocompatible, and can be engineered to have controllable mechanical properties. Degradable hydrogels are increasingly fabricated using sequence-specific enzymatic degradation of peptides incorporated into the hydrogel. Although this degradation method is cytocompatible, the low solubility of peptides in oil and the need for large volumes of peptide solution limit the synthesis of monodisperse degradable hydrogels using microfluidic devices. Here, hydrolytic hydrogels with tunable degradation are reported based on a labile chemical response to endogenous stimuli (i.e., hydrolysis).

[0136] method Hydrogel particles (microgels) were fabricated in a microfluidic water-in-oil droplet generator as previously described (Headen et al. Microsystems & Nanoengineering 4.1(2018):1-9). The polymers were pre-functionalized with 1 kDa SH-PEG-FITC for in vitro tracking or SH-PEG-AF750 for in vivo imaging. The microgels were cross-linked with solutions containing dithiothreitol (DTT) or a mixture of DTT and the degradable linker ethylene glycol bis-mercaptoacetate in different molar ratios. The microgels were injected under the skin of mice for in vivo tracking. Briefly, 8-12 week old Balb / C mice were injected with 100 uL of approximately 3000 non-degradable hydrogels (DTT cross-linked) or degradable hydrogels (mixtures of DTT and 0.25 mM, 0.5 mM, or 1 mM degradable linkers).

[0137] result Degradation of the microgels was followed by measuring the swelling ratio and the presence of PEG-FITC linker in microgel cultures at 37 °C in dPBS. The swelling ratio was found to be related to the molar concentration of the degradable linker at the time of fabrication (Figure 1F). Highly degradable hydrogels swelled approximately 40% compared to non-degradable hydrogels (p<0.0001) within 4 hours after fabrication. By 1 month after culture, 1 mM degradable hydrogels had expanded to 60% of the size of DTT non-degradable hydrogels (P<0.0039). Initially, no difference in size was observed between the DTT, 0.25 mM, and 0.5 mM groups, but by day 30, there was a 30% increase in size compared to non-degradable gels (p<0.0001, p<0.019, respectively). Degradation of the hydrogel would result in cleavage of the PEG-FITC linker from the PEG-4MAL backbone (Figure 1B). Therefore, the fluorescence intensity of the cultures was followed over time to determine the presence of PEG-FITC in the solution. As observed in the swelling studies, higher fluorescence intensity was in the 1 mM degradable hydrogel group (p<0.001 vs. DTT). No significant differences were observed when comparing the least degradable gels to the DTT crosslinked hydrogels. To assess in vivo degradation, the microgels were monitored using an IVIS imaging system (Figure 16A-B). Immediately after injection, a strong signal was detected in all groups. By day 1, the signal had decayed by approximately 38% in all groups, which may be due to the presence of free dye during production rather than degradation. By day 3, the signal had decreased to 33% in the 1 mM hydrogel group, although a constant signal remained at the DTT injection site. By day 10, no detectable signal was observed in the highly degradable groups, while no change was observed in the DTT microgel group.

[0138] conclusion A microfluidic water-in-oil droplet generator can be implemented to prepare monodisperse hydrolytic hydrogels. Implementing an ethylene linker together with a nondegradable thiol linker allows for controllable and sustained material degradation in vitro and in vivo.

[0139] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of some aspects of the claims, and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Furthermore, although only certain representative compositions and method steps disclosed herein have been specifically described, other combinations of compositions and method steps are also intended to fall within the scope of the appended claims even if not specifically recited. Thus, although combinations of steps, elements, components, or elements may be explicitly referred to herein, other combinations of steps, elements, components, and elements are included even if not explicitly stated.

[0140] As used herein, the term "comprising" and variations thereof are used synonymously with the term "including" and variations thereof, and are open and non-limiting terms. Although the terms "comprising" and "including" are used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" can be used instead of "comprising" and "including" to provide more specific embodiments of the present invention, and are also disclosed. Except in the examples, or unless otherwise indicated, all numbers expressing amounts of ingredients, reaction conditions, and the like used in the specification and claims should at least be understood and should not be construed in light of the number of significant digits and ordinary rounding approaches, without attempting to limit the application of the doctrine of equivalents to the scope of the claims.

Claims

1. A hydrogel comprising a polymer backbone crosslinked with a first crosslinking agent containing at least one moiety of Formula I, 【Chemical 1】 wherein, m and n are independently 1 or 2, A is C ２ -C １０ is alkyl, 【Chemical 2】 is a bonding point for said moiety within said first crosslinking agent, the hydrogel.

2. The hydrogel according to claim 1, wherein said polymer backbone comprises poly(ethylene glycol) or a functionalized derivative thereof.

3. The polymer backbone is selected from poly(ethylene glycol) (PEG), poly(ethylene glycol)-di-acrylate (PEG-DA), multi-arm poly(ethylene glycol)-acrylate (PEG-Ac), poly(ethylene glycol)-dithiol (PEG-diSH), poly(ethylene glycol) divinyl sulfone (PEG-diVS), multi-arm poly(ethylene glycol) vinyl sulfone (PEG-VS), poly(ethylene glycol)-di-methacrylate (PEG-DMA), multi-arm poly(ethylene glycol)-methacrylate (PEG-Mac), poly(ethylene glycol)-di-allyl ether (PEG-diAE), multi-arm poly(ethylene glycol)-allyl ether (PE-AD), poly(ethylene glycol)-di-vinyl ether (PEG-diVE), multi-arm poly(ethylene glycol)-vinyl ether (PEG-VE), poly(ethylene glycol)-di-maleimide (PEG-diMI), multi-arm poly(ethylene glycol)-maleimide (PEG-MI), poly(ethylene glycol)-di-norbornene, multi-arm poly(ethylene glycol) norbornene, poly(ethylene glycol-vinyl carbonate, multi-arm poly(ethylene glycol)-vinyl carbonate, and polyethylene glycol oligofumarate, or a combination thereof. The hydrogel according to claim 1.

4. The hydrogel according to claim 1, wherein said polymer backbone comprises multi-arm poly(ethylene glycol)-maleimide.

5. The hydrogel according to claim 1, wherein said first crosslinking agent comprises m + n moieties capable of reacting with said polymer backbone, and m and n are as defined in claim 1.

6. The first crosslinking agent comprises a compound of Formula II, 【Chemical 3】 wherein, X １ and X ２ are each independently selected, at each occurrence, from moieties capable of reacting with the polymer backbone L １ and L ２ are each independently selected from the connecting portions at each occurrence, The hydrogel according to claim 1, wherein m, n, and A are defined as in claim 1.

7. X １ and X ２ are each —SH, and L1 and L2 are each independently selected from C1-C10 alkyl each time they appear, the hydrogel according to claim 6.

8. The hydrogel according to claim 1, wherein the first crosslinking agent comprises ethylene glycol bis(mercaptoacetate).

9. The hydrogel according to claim 1, wherein the first crosslinking agent is hydrolyzable.

10. The hydrogel according to claim 1, wherein the polymer backbone is further crosslinked with a second crosslinking agent.

11. The hydrogel according to claim 10, wherein the second crosslinking agent is hydrolytically stable.

12. The hydrogel according to claim 10, wherein the second crosslinking agent comprises dithiothreitol (DTT).

13. The hydrogel according to claim 1, wherein the hydrogel is injectable and / or transplantable.

14. The hydrogel according to claim 1, wherein the hydrogel is in the form of a membrane, sponge, gel, solid scaffold, spun fiber, woven or non-woven mesh, nanoparticle, or microparticle.

15. The hydrogel according to claim 1, further comprising at least one cell.

16. A process for synthesizing a hydrogel according to any one of claims 1 to 15, comprising reacting a polymer with a first crosslinking agent comprising at least one moiety of formula I, [Chemical Formula 4] wherein all variables are as defined in claim 1.

17. A therapeutic delivery composition comprising a hydrogel according to any one of claims 1 to 15 and one or more therapeutic agents.

18. The therapeutic delivery composition according to claim 17, wherein the one or more therapeutic agents can be selected from cells, proteins, antibodies, nucleic acids, growth factors, or drugs.

19. An agent for use in a method of doing so in a subject in need of promoting tissue growth, comprising a hydrogel according to any one of claims 1 to 15, wherein the method comprises identifying a target site, and administering a therapeutically effective amount of the hydrogel to the target site.

20. An agent for use in a method of delivering a therapeutic agent to a target site in a subject, comprising a therapeutic delivery composition according to claim 17, wherein the method comprises administering a therapeutically effective amount of the composition to the target site.

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