US20160279283A1 - Controllable self-annealing microgel particles for biomedical applications - Google Patents

Controllable self-annealing microgel particles for biomedical applications Download PDF

Info

Publication number
US20160279283A1
US20160279283A1 US15/179,151 US201615179151A US2016279283A1 US 20160279283 A1 US20160279283 A1 US 20160279283A1 US 201615179151 A US201615179151 A US 201615179151A US 2016279283 A1 US2016279283 A1 US 2016279283A1
Authority
US
United States
Prior art keywords
microgel particles
tissue
microgel
annealing
scaffold
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US15/179,151
Other languages
English (en)
Inventor
Donald R. Griffin
Westbrook Weaver
Tatiana Segura
Dino Di Carlo
Philip Scumpia
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
Original Assignee
University of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California filed Critical University of California
Priority to US15/179,151 priority Critical patent/US20160279283A1/en
Assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA reassignment THE REGENTS OF THE UNIVERSITY OF CALIFORNIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SEGURA, TATIANA, GRIFFIN, Donald R., DI CARLO, DINO, SCUMPIA, Philip, WEAVER, Westbrook
Publication of US20160279283A1 publication Critical patent/US20160279283A1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF CALIFORNIA LOS ANGELES
Priority to US15/701,113 priority patent/US10912860B2/en
Priority to US15/829,440 priority patent/US20180078671A1/en
Priority to US16/264,466 priority patent/US20190151497A1/en
Priority to US16/596,312 priority patent/US20200085859A1/en
Priority to US17/144,158 priority patent/US11464886B2/en
Priority to US17/935,096 priority patent/US20230190995A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/008—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/795—Polymers containing sulfur
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0019—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0028—Polypeptides; Proteins; Degradation products thereof
    • A61L26/0047—Specific proteins or polypeptides not covered by groups A61L26/0033 - A61L26/0042
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/0066—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/0085—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/009—Materials resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Definitions

  • the technical field relates generally to the field of wound treatment, and in particular, the use of microgel particles and scaffolds including the particles for treating and sealing wounds and for tissue filler applications.
  • a central concept tied to the generation and regeneration of tissue is collective cell migration, a process by which entire networks of cells move together into an area of development to facilitate the formation of functional tissue.
  • researchers have sought to develop would healing agents; however, these materials display batch-to-batch variability and exhibit degradation rates that limit extended structural support for growing tissues.
  • Synthetic materials are more tunable than natural materials and their mechanical properties have been engineered to allow use with a wide range of tissue types. Despite this tunability, however, synthetic injectable biomaterials have been limited to non-porous or nanoporous scaffolds that require physical degradation for cellular migration through the material.
  • Porous synthetic hydrogels that contain pre-formed microscale interconnected pores allow greater cell mobility without the need for degradation, circumventing the trade-off between cell mobility and material stability inherent to non-porous scaffolds.
  • the typical mode of pore formation includes the toxic removal of porogens, or the degradation of encapsulated microparticles, which requires these constructs to be either cast ex vivo, preventing them from seamlessly integrating with the surrounding tissue like an injectable biomaterial or requires long-term in vivo development to resolve the porous structure.
  • STAR Sphere Templated Anigiogenic Regeneration
  • the instant inventors have identified the gold standard of the development of interconnected microporous scaffolds that allow for interconnected cell networks and collective migration without the need for scaffold degradation or invasive procedures for implantation is essential for bulk integration with the surrounding tissue.
  • these materials should facilitate collective cell migration that mediates regeneration while providing molecular cues to promote wound healing and niche recognition.
  • the instant inventors have also identified that these materials must be able to be seamlessly replaced by migrating cells and natural matrix, provide a stable structural support prior to replacement, and be easily delivered and conform to the site of injury to minimize fibrotic and inflammatory responses.
  • the technology described herein utilizes chemistry to generate tiny microgels that can be assembled into a large unit, leaving behind a path for cellular infiltration.
  • the result is a packed cluster of microscopic synthetic polymer bodies (e.g., spheres) attached at their surfaces, akin to a jar of gumballs that are stuck together.
  • the cluster creates a scaffold of microporous annealed particles (e.g., a porous gel scaffold) that fills in the wound.
  • New tissue quickly grows into the voids between the microgel particles, and as the microgel particles degrade into the body, a matrix of newly grown tissue is left where the wound once was. New tissue continues growing until the wound is completely healed.
  • microgel systems described herein represents a substantial improvement over conventional products.
  • the technologies described herein do not require added growth factors to attract cells into the material.
  • the geometry of the described microgel networks entice cells to migrate into the microgel.
  • microporous gels described herein are flowable and/or injectable and can be applied in multiple different ways, including for example topically or by injection. Injected and/or flowable microporous gels can be inserted transdermally or into deep tissue. Flowable microporous gels can also be administered topically to the dermis and other tissues.
  • the microgel particles when an annealing agent is applied to the plurality of microgel particles, the microgel particles form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • the systems, compositions, methods, and devices are specifically engineered for biomedical applications.
  • the microporous gel particles further comprise a crosslinker, wherein the crosslinker includes a matrix metalloprotease (MMP)-degradable crosslinker.
  • MMP matrix metalloprotease
  • an annealing agent comprises Factor XIIIa.
  • the annealing agent comprises Eosin Y, a free radical transfer agent, or a combination thereof.
  • the microporous gel particles comprise a crosslinker that is degradable. In certain embodiments, the microporous gel particles comprise interstitial spaces that comprise border surfaces exhibiting negative concavity. In one or more embodiments, the covalently-stabilized scaffold of microgel particles has a void volume of from about 10% to about 50%.
  • a microporous gel system for biomedical applications includes an aqueous solution containing a plurality of microgel particles formed with a biodegradable crosslinker such as a matrix metalloprotease (MMP)-degradable crosslinker and an annealing agent that when applied to the plurality of microgel particles causes the microgel particles to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • a biodegradable crosslinker such as a matrix metalloprotease (MMP)-degradable crosslinker
  • MMP matrix metalloprotease
  • a microporous gel system in another embodiment, includes a delivery device and a collection of biodegradable microgel particles contained in an aqueous solution and stored in the delivery device.
  • An annealing agent or annealing agent precursor is also stored in the delivery device.
  • the delivery device may contain a single or multiple compartments, depending on the particular embodiment employed.
  • a method of treating tissue includes delivering to the tissue an aqueous-based solution containing a plurality of microgel particles decorated with cell adhesive peptides, wherein the microgel particles are formed with a biodegradable crosslinker such as matrix metalloprotease (MMP)-degradable crosslinker.
  • MMP matrix metalloprotease
  • the plurality of microgel particles are exposed to an annealing agent that anneals the microgel particles to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • a microporous gel system for biomedical applications includes a collection of microgel particles formed by a reaction of a backbone polymer having one or more cell attachment moieties, one or more annealing components, and a biodegradable network crosslinker component.
  • the microporous gel system includes an endogenous or exogenous annealing agent that links the microgel particles together in situ via the annealing components to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • the delivery device contains an aqueous solution comprising a plurality of microgel particles and the annealing agent or an annealing agent precursor.
  • the delivery device comprises a single compartment delivery device containing the aqueous solution comprising a plurality of microgel particles and the annealing agent.
  • the delivery device comprises a multiple (e.g., double) compartment delivery device, wherein one compartment contains the aqueous solution containing plurality of microgel particles and a first annealing agent precursor and the second compartment contains the aqueous solution containing plurality of microgel particles and a second annealing agent precursor.
  • microporous gels further comprise a (MMP)-degradable crosslinker that comprises at least one D-amino acid.
  • the microgel particles comprise a (MMP)-degradable crosslinker comprises a plurality of D-amino acids.
  • a microporous gel system comprising: a delivery device; a plurality biodegradable microgel particles contained in an aqueous solution and stored in the delivery device; and an annealing agent or annealing agent precursor stored in the delivery device.
  • the microporous gel particles further comprise a collection of biodegradable microgel particles of two or more types that are contained in an aqueous solution and stored in the delivery device.
  • the delivery device comprises two compartments, biodegradable microgel particles are stored in each of the two compartments, and a first annealing precursor is stored in one compartment and a second annealing precursor is stored in the other compartment, wherein the annealing agent is formed by the presence of both the first and second annealing precursors.
  • the delivery device comprises a single compartment and the collection of biodegradable microgel particles and the annealing agent are both stored in the single compartment.
  • the annealing agent comprises a photoinitiator and a free radical transfer agent stored in the single compartment.
  • the microporous gel system further comprises a light-emitting device configured to illuminate a mixture of the collection of biodegradable microgel particles and the annealing agent.
  • the microgel particles comprise substantially monodisperse spheres.
  • the substantially monodisperse spheres have a diameter within the range of from about 30 micrometers to about 150 micrometers.
  • the microgel particles are covalently linked to another after annealing.
  • a method of treating tissue comprising: delivering to the tissue an aqueous-based solution containing a plurality of microgel particles; and exposing the plurality of microgel particles to an annealing agent that anneals the microgel particles to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • the plurality of microgel particles is decorated with cell adhesive peptides, and wherein the microgel particles are formed with a matrix metalloprotease (MMP)-degradable crosslinker.
  • MMP matrix metalloprotease
  • the annealing agent is delivered to the tissue.
  • the annealing agent is present within the tissue.
  • the method further comprises initiating the annealing of the microgel particles with exposure to light.
  • the wavelength of light is in the visible range. In some embodiments, the wavelength of light is in the infrared range.
  • the aqueous-based solution and the annealing agent are delivered simultaneously. In some embodiments, the aqueous-based solution and the annealing agent are delivered sequentially.
  • the microgel particles comprise a therapeutically active chemical compound. In certain embodiments, the microgel particles expose or elute the chemical compound to the tissue.
  • the tissue comprises a site of cosmetic reconstruction, chronic wound development, acute tissue damage, or a tissue gap caused by surgical incision.
  • the (MMP)-degradable crosslinker comprises D-amino acid.
  • a microporous gel system or device comprising: a collection of microgel particles comprising a backbone polymer having one or more cell attachment moieties, one or more annealing components, and one or more biodegradable network crosslinker components; and an endogenous or exogenous annealing agent that links the microgel particles together in situ via the annealing components to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • the backbone polymer comprises poly(ethylene glycol) vinyl sulfone.
  • the one or more cell attachment moieties comprise a RGD peptide or a fragment thereof, fibronectin or a fragment thereof, collagen or a fragment thereof, or laminin or a fragment thereof. In some embodiments, the one or more cell attachment moieties comprise a RGD peptide or a fragment thereof. In an embodiment, the one or more cell attachment moieties comprise SEQ ID NO: 3 or a fragment thereof. In further or additional embodiments, the one or more annealing components comprise a K-peptide and a Q-peptide.
  • the K-peptide comprises a Factor XIIIa-recognized lysine group and the Q-peptide comprises a Factor XIIIa-recognized glutamine group.
  • the biodegradable network crosslinker component comprises a matrix metalloprotease (MMP)-degradable crosslinker.
  • MMP matrix metalloprotease
  • the (MMP)-degradable crosslinker comprises D-amino acid.
  • the collection of microgel particles comprises microgel particles of two or more types.
  • the microgel particles of a first type comprise (MMP)-degradable crosslinker comprising D-amino acid
  • microgel particles of a second type comprise (MMP)-degradable crosslinker comprising only L-amino acid.
  • the system or device comprises a single compartment delivery device containing the collection of microgel particles and the annealing agent.
  • the system or device further comprises a double compartment delivery device, wherein one compartment contains the aqueous solution containing plurality of microgel particles and a first annealing agent precursor and the second compartment contains the aqueous solution containing plurality of microgel particles and a second annealing agent precursor, wherein the annealing agent is formed by the presence of the first and second annealing agent precursors.
  • a method of treating tissue comprising: delivering to the tissue a first layer of microgel particles decorated with cell adhesive peptides, wherein the microgel particles are formed with a biodegradable crosslinker; exposing the first layer to an annealing agent that anneals the microgel particles to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein; delivering to the tissue a second layer of microgel particles decorated with cell adhesive peptides, wherein the microgel particles are formed with a biodegradable crosslinker and wherein the microgel particles in the second layer differ in one of a physical property or chemical composition as compared to the microgel particles in the first layer; and exposing the second layer to an annealing agent that anneals the microgel particles to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • the microgel particles in the second layer have a different size. In yet additional embodiments, the microgel particles in the second layer have a different shape. In one or more embodiment, the microgel particles in the second layer have a different stiffness. In certain embodiments, the microgel particles in the second layer having a chemical component different from a chemical component in the first layer. In further or additional embodiment, the microgel particles in the second layer having a chemical component of a different concentration from the same chemical component in the first layer.
  • method of treating tissue comprising: delivering to the tissue an aqueous-based solution containing a plurality of microgel particles decorated with cell adhesive peptides, wherein the microgel particles are formed with a biodegradable crosslinker; exposing the plurality of microgel particles to an annealing agent that anneals the microgel particles to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • a method of treating tissue includes delivering to the tissue a first layer of microgel particles decorated with cell adhesive peptides, wherein the microgel particles are formed with a biodegradable crosslinker.
  • the first layer is exposed to an annealing agent that anneals the microgel particles to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • a second layer of microgel particles decorated with cell adhesive peptides is delivered to the tissue, wherein the microgel particles are formed with a biodegradable crosslinker and wherein the microgel particles in the second layer differ in one of a physical property or chemical composition as compared to the microgel particles in the first layer.
  • the second layer is exposed to an annealing agent that anneals the microgel particles to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • a method of treating tissue includes delivering to the tissue an aqueous-based solution containing a plurality of microgel particles decorated with cell adhesive peptides, wherein the microgel particles are formed with a biodegradable crosslinker.
  • the plurality of microgel particles are exposed to an annealing agent that anneals the microgel particles to form a covalently-stabilized scaffold of microgel particles having interstitial spaces therein.
  • a method of making microgel particles comprising: providing a water-in-oil droplet generating microfluidic device having a plurality of input channels leading to a common channel and a pair of oil-pinching channels intersecting with the common channel at a downstream location flowing a first pre-polymer solution containing a polymer backbone modified with oligopeptides into a first input channel; flowing a second solution containing a biodegradable crosslinker into a second input channel; flowing an oil and a surfactant into the pair of oil pinching channels to form droplets containing the first pre-polymer solution and the second solution; and collecting microgel particles formed by cross-linking of the droplets.
  • the method further comprises a third input channel interposed between the first input channel and the second input channel, wherein a third inert solution containing a pre-polymer is flowed into the third input channel.
  • the method further comprises sheathing the generated droplets with an additional pair of sheathing channels located downstream of a location where the pair of oil pinching channels intersect with the common channel, wherein the additional pair of sheathing channels carries oil and a surfactant at a higher concentration than the surfactant contained in the upstream pair of oil pinching channels.
  • the method further comprises centrifuging the collected microgel particles.
  • the method comprises reducing the free water volume content of the centrifuged microgel particles.
  • the method comprises storing the collected microgel particles for an extended period of time (e.g., months to years).
  • a method of making microgel particles includes providing a water-in-oil droplet generating microfluidic device having a plurality of input channels leading to a common channel and a pair of oil-pinching channels intersecting with the common channel at a downstream location.
  • a first pre-polymer solution containing a polymer backbone modified with oligopeptides is flowed into a first input channel.
  • a second solution containing a biodegradable crosslinker is flowed into a second input channel.
  • An oil and a surfactant are flowed into the pair of oil pinching channels to form droplets containing the first pre-polymer solution and the second solution.
  • Microgel particles are formed by cross-linking of the droplets which are then collected.
  • FIG. 1 illustrates a portion of a scaffold formed from a plurality of annealed microgel particles.
  • FIG. 2A illustrates an exemplary method of injecting microgel particles into a wound site for healing the same.
  • FIG. 2B schematically illustrates an exemplary annealing reaction between different microgel particles potentiated by linkers on the surface of the microgel particles.
  • FIG. 2C illustrates an exemplary process of tissue infiltration into a scaffold formed within a delivery site on tissue, where the boundary between the tissue and the microgels represents any interface between them, where cells can pass through the interface moving inwards from the tissue or outward toward the tissue from the microgels.
  • FIG. 3A illustrates a top down view of a microfluidic device according to one embodiment used to generate a plurality of microgel particles as part of a microporous gel system.
  • FIG. 3B illustrates a magnified view of the droplet generation region and downstream oil/surfactant pinching region (see box region in FIG. 3A ).
  • FIG. 3C illustrates magnified, perspective views of two branch channels illustrated in FIG. 3A .
  • FIG. 3D illustrates a side view of the microfluidic device of FIG. 3A according to one embodiment.
  • FIG. 3E illustrates a photograph taken of a reduction to practice of the scheme illustrated in FIG. 3B where fluorescent solution on the left contains crosslinker, the fluorescent solution on the right contains polymer and reaction buffer, and the middle stream contains an inert liquid solution to prevent mixing of left and right solutions prior to droplet segmentation.
  • Bright fluorescence between middle and right streams illustrates pH change in the middle stream due to diffusion of reaction buffer.
  • FIG. 3F illustrates a photograph of a reduction to practice of the scheme illustrated in FIG. 3B and FIG. 3E , while also showing the light microscopic view of droplet segmentation after the pinching oil streams are introduced.
  • FIG. 4A illustrates a top down view of a microfluidic device according to another embodiment used to generate a plurality of microgel particles as part of a microporous gel system.
  • FIG. 4B illustrates that in the droplet segmentation region, mineral oil with 0.25% Span® 80 pinches and segments PEG pre-gel, and downstream a 5% Span® 80 solution in mineral oil mixes and prevents downstream coalescence of microgels before complete gelation.
  • FIG. 4C illustrates droplets do not recombine during incubation in the bifurcation region and exit from the microchannel to the collection well.
  • FIG. 5 illustrates an exemplary microfluidic T-junction that may be used to generate microgel droplets according to one embodiment.
  • FIG. 6A illustrates an exemplary dispensing device in the form of a double-barreled syringe according to one embodiment.
  • FIG. 6B illustrates an exemplary dispensing device in the form of a single-barreled syringe according to another embodiment.
  • FIG. 6C illustrates an exemplary dispensing device in the form of a tube that holds the microgel particles according to one embodiment.
  • FIG. 7A illustrates hematoxylin and eosin staining (H&E staining) of tissue sections in SKH1-Hr hr mice for tissue injected with the scaffold (Microporous Annealed Particle or “MAP” scaffold) as well as the non-porous control twenty-four (24) hours after injection.
  • the scaffold Mericroporous Annealed Particle or “MAP” scaffold
  • FIG. 7C illustrate representative images of wound closure during a 5-day in vivo wound healing model in SKH1-Hr hr mice comparing the gel scaffold (left panels) to a non-porous PEG gel control (right panels).
  • FIG. 7D illustrates representative images of wound closure during 7-day in vivo BALB/c experiments.
  • the scaffolds promote significantly faster wound healing than the no treatment control, the gels lacking the K and Q peptides, the non-porous PEG gel, and faster wound healing than the precast porous gel.
  • Porous gels created ex vivo to precisely match the wound shape using the canonical, porogen-based, casting method showed appreciable wound healing rates, comparable to the scaffolds, but lacking injectability (N ⁇ 5).
  • FIG. 7E is a bar graph illustrating wound closure quantification data from BALB/c in vivo wound healing for each treatment category corresponding to FIG. 7D . All data are presented as average+/ ⁇ SEM. Statistical significance performed using standard two-tailed t-test (*: p ⁇ 0.05; **p ⁇ 0.01).
  • FIG. 7F illustrates traces of wound bed closure during 7 days in vivo for each treatment category corresponding to FIG. 7D and FIG. 7E .
  • FIG. 7G illustrates how the microgel particle-containing solution or slurry can be injected using a syringe device (e.g., 25 Gauge syringe) like that of FIG. 6A or 6B into a treatment site where the microgel conforms to the shape of the injection site (e.g., in this case a star-shaped laser cut acrylic mold) and subsequent annealing of the scaffold into the star shape.
  • a syringe device e.g., 25 Gauge syringe
  • the shape of the injection site e.g., in this case a star-shaped laser cut acrylic mold
  • FIGS. 8A and 8B illustrate stained microscopic images of damaged tissue (i.e., wound site) that has been treated with the microgel scaffold ( FIG. 8A ) and with no treatment or “sham” ( FIG. 8B ) in a mouse model twenty-one (21) days after skin excision and gel application.
  • the scar reduction enabled by the microgel scaffold can clearly be seen in FIG. 8A .
  • Squares indicate hair follicles and oil glands (sebaceous glands) in the reforming tissue after gel application to a wound. Circles indicate remaining microgel particles in the reforming tissue.
  • FIG. 8C illustrates a graph showing the epidermal thickness for the tissue treated with the sham as well as tissue treated with the gel scaffold.
  • FIG. 8D illustrates a graph showing the number of sebaceous glands for the tissue treated with the sham as well as tissue treated with the gel scaffold.
  • FIG. 8E illustrates a graph showing the number of hair follicles for the tissue treated with the sham as well as tissue treated with the gel scaffold.
  • FIG. 8F illustrates a graph showing the scar width for the tissue treated with the sham as well as tissue treated with the gel scaffold.
  • FIG. 8G illustrates a graph showing the number of milial cysts for the tissue treated with the sham as well as tissue treated with the gel scaffold.
  • FIG. 9A illustrates a graph of storage modulus as a function of time post-mixing for different gelation kinetics (pH and temperature). pH 8.25 at 25 degrees Celsius is represented by the bottom line in the graph; pH 8.8 at 25 degrees Celsius is represented by the top line in the graph; and pH 8.25 at 37 degrees Celsius is represented by the middle line in the graph.
  • FIG. 9B illustrates different hydrogel weight percentages were used to produce different stiffness materials on the x-axis.
  • the graph illustrated Storage Modulus (Pa) for various hydrogel weight percentages.
  • FIG. 9C illustrates different crosslinker stoichiometries that were used to produce different stiffness values in the resultant gel on the x-axis.
  • the graph illustrated Storage Modulus (Pa) as a function of the r-ratio of free crosslinker ends (—SH) to vinyl groups (—VS) on the PEG molecule.
  • FIG. 9D illustrates a graph of the % degradation as a function of time for both the non-porous control (bottom line of the graph) as well as a porous gel described herein (top line of the graph).
  • FIG. 9E illustrates SEM images of a scaffold annealed with FXIIIa at 200 ⁇ m (top panel) or 100 ⁇ m (bottom panel).
  • FIG. 9F illustrates SEM images of microgel particles without FXIIIa at 200 ⁇ m (top panel) or 100 ⁇ m (bottom panel). Un-annealed microgel particles are seen in FIG. 9F .
  • FIG. 10 shows a microgel fabricated using the described technique, where the surface of the microgel has been augmented with a fluorescent bovine serum albumin (BSA) protein (outer perimeter) through the use of phosphine-azide ‘click’ chemistry. Further, nanoparticles (500 nm) are embedded within the microgel during microfluidic fabrication.
  • BSA fluorescent bovine serum albumin
  • FIG. 11 illustrates an exemplary method of treating damaged tissue using the microporous gel system described herein.
  • Microgel particles are applied (top panel), optionally, an applicator is utilized (second panel), annealing of microgel particles is initiated to form a scaffold (third panel) and improved wound healing is observed (bottom panel).
  • FIG. 12A illustrates fluorescent images demonstrating the formation of 3D cellular networks during six days of culture in porous gel scaffolds in vitro as well as non-porous gels after 6 days.
  • 350 Pa bulk modulus identical to porous gel scaffolds
  • 600 Pa microscale modulus matched to individual microgels.
  • FIG. 12B illustrates a graph of cell survival twenty-four (24) hours post annealing is greater than 93% across three cell lines representing different human tissue types.
  • HDF Human dermal fibroblasts
  • AhMSC Adipose-derived human mesenchymal stem cells
  • BMhMSC Bone marrow-derived human mesenchymal stem cells.
  • FIG. 13A illustrates an exemplary method for combining living cells with preformed microgel particles prior to annealing.
  • the microgel particles are annealed to one another, entrapping the living cells within the interconnected microporous network created upon microgel annealing.
  • FIGS. 13B-D are photographic images illustrating that microgel particle solutions combined with living cells are moldable to macro-scale shapes, and can be injected to form complex shapes that are maintained after annealing.
  • FIG. 13B illustrates an exemplary in vitro syringe injection.
  • FIG. 13C illustrates an exemplary in vitro shape molding.
  • FIG. 13D illustrates an exemplary in vitro annealed scaffold.
  • FIG. 13E illustrates microgel particles are moldable to macro-scale shapes and can be performed in the presence of live cells (indicated by arrows pointing to fluorescent HEK-293T cells).
  • FIG. 14A illustrates a graph showing that varying sizes of microgel particles can be synthesized over a range of frequencies of production in an exemplary embodiment.
  • FIG. 14B illustrates that providing a high inlet pressure to each solution inlet (where the oil inlets are exceeding 30 Psi) enables an increase in production frequency in another exemplary embodiment.
  • FIG. 14C illustrates a graph showing high precision fabrication of microgel building blocks allows creation of defined gel scaffolds. Different building block sizes allow for deterministic control over resultant micro-porous network characteristics, presented here as median pore sizes+/ ⁇ standard deviation (SD).
  • SD standard deviation
  • a solid microgel scaffold for biomedical applications such as wound healing is disclosed that is formed when a plurality of microgel particles are annealed to one another in an annealing reaction.
  • the annealing reaction in one aspect of the subject matter described herein forms covalent bonds between adjacent microgel particles.
  • the scaffold in the post-annealed state, the scaffold forms a three-dimensional structure that conforms to the site of application or delivery. Because of the imperfect packing of the microgel particles, the annealed scaffold formed from the particles includes interstitial spaces formed therein where cells can migrate, bind, and grow.
  • the formed scaffold structure is porous upon annealing in the wound or other delivery site (unlike the non-porous solid scaffold provided by fibrin-based products).
  • This porosity includes the interstitial spaces mentioned above as well as nanoscopic pores that may be created or formed in the particles themselves.
  • the micro-porosity of the scaffold structure allows for high diffusivity of nutrients, cell growth and differentiation factors, as well as cell migration, ingrowth, and penetration.
  • the microporosity of the scaffold provides for accelerated healing or improved therapeutic delivery of drugs or medicaments over conventional fibrin glue, hyper-branched polymers, or polymers with degradable crosslinker options, because of the enhanced cell migration through interstitial spaces while maintaining overall scaffold integrity.
  • One advantage of the subject matter described herein beyond methods such as the STARTM technology is that the formation of a scaffold occurs in vivo, allowing it to completely fill the desired space and be tuned to bind (chemically or otherwise) to the surrounding tissue.
  • the pre-delivery formation of the microgel particles allows for controlled mechanical tunability of the resultant formed scaffold to match the properties of the surrounding tissue. These capabilities result in a better seal and overall integration with the tissue. Greater integration results in decreased possibility of material failure and enhanced long-term regeneration. This also helps prevent contamination from the environment.
  • the microporous nature of the annealed scaffold is beneficial to reduce immune foreign body response to the scaffold.
  • FIG. 1 illustrates a portion of the formed three dimensional scaffold 10 that is formed by a plurality of annealed microgel particles 12 .
  • the scaffold 10 includes interstitial spaces therein 14 that are voids that form micropores within the larger scaffold 10 .
  • the interstitial spaces 14 have dimensions and geometrical profiles that permit the infiltration, binding, and growth of cells.
  • the microporous nature of the scaffold 10 disclosed herein involves a network of interstitial spaces or voids 14 located between annealed microgel particles 12 that form the larger scaffold structure.
  • the interstitial spaces or voids 14 created within the scaffold 10 exhibit negative concavity (e.g., the interior void surface is convex).
  • FIG. 1 illustrates a portion of the formed three dimensional scaffold 10 that is formed by a plurality of annealed microgel particles 12 .
  • the scaffold 10 includes interstitial spaces therein 14 that are voids that form micropores within the larger scaffold 10 .
  • the interstitial spaces 14 have dimensions
  • FIG. 1 illustrates an exemplary void 14 with void walls 16 exhibiting negative concavity.
  • the negative concavity is caused because the microgel particles 12 that are annealed to one another are generally or substantially spherical in shape in one preferred embodiment. This allows for the packing of microgel particles 12 that, according to one embodiment, produces a low void volume fraction between about 10% and about 50% and, in another embodiment between about 26% to about 36%. While the void volume fraction is low, the negative concavity exhibited in certain embodiments within the network of voids 14 provides a relatively high surface area to void volume for cells to interact with. For a given volume of cells, they would then, on average, be exposed to even more and larger surfaces (e.g., on the void walls 16 ) to interact within the network of voids in the scaffold 10 .
  • the void network consists of regions where microgel surfaces are in close proximity (e.g., near neighboring annealed microgel particles 12 ) leading to high surface area adhesive regions for cells to adhere and rapidly migrate through, while neighboring regions further in the gaps between microgel particles 12 have a larger void space that can enable cell and tissue growth in this space. Therefore the combined adjacency of the tight void areas and more spacious void gaps is expected to have a beneficial effect on tissue ingrowth and regrowth, compared to either entirely small voids or all larger voids.
  • the negative concavity results due to the spherical shape of the microgel particles 12 .
  • the microgel particles 12 might not be spherical in shape.
  • Other non-spherical shapes may still be used in the scaffold 10 .
  • the scaffold 10 is formed by microgel particles 12 that are secured to one another via annealing surfaces 17 .
  • the annealing surfaces 17 are formed either during or after application of the microgel particles 12 to the intended delivery site.
  • the scaffold 10 may be used for various applications, including a variety of medical applications such as military field medicine, medical trauma treatment, post-surgical closure, burn injuries, inflammatory and hereditary and autoimmune blistering disorders, etc.
  • the scaffold 10 is used as a tissue sealant (e.g., an acute wound-healing substance, surgical sealant, topical agent for partial thickness, full thickness, or tunneling wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, donor skin graft sites, post-Moh's surgery, post-laser surgery, podiatric wounds, wound dehiscence, abrasions, lacerations, second or third degree burns, radiation injury, skin tears, and draining wounds, and the like).
  • tissue sealant e.g., an acute wound-healing substance, surgical sealant, topical agent for partial thickness, full thickness, or tunneling wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, donor skin graft
  • FIGS. 2A-2C illustrate an embodiment, where the scaffold 10 is used to treat a wound site 100 formed in tissue 102 of a mammal.
  • the scaffold 10 is used for immediate treatment of acute wounds.
  • the scaffold 10 provides several benefits, including a rapid method to seal wounds 100 , prevent trans-epidermal water loss, provide cells or medication(s), and enhance the healing of skin wounds (e.g., surgical sites, burn wounds, ulcers) to provide more natural tissue development (e.g., avoiding the formation of scar tissue).
  • One particular benefit of the scaffold 10 is the ability of the scaffold 10 to reduce or minimize the formation of scar tissue.
  • the scaffold 10 provides a more effective alternative to tissue glues and other current injectable tissue fillers and adhesives.
  • microgel particles 12 are delivered to the wound site 100 followed by the initiation of the annealing reaction to anneal the microgel particles 12 to one another to form the scaffold 10 .
  • the wound site 100 is sealed by the scaffold 10 and as time progresses, the wound site 100 is healed into normal tissue (see also FIG. 11 ).
  • FIG. 2B illustrates how adjacent microgel particles 12 (particle A and particle B) undergo chemical or enzymatic initiation of the annealing reaction to form an annealing surface 17 between microgel particles 12 .
  • FIG. 2C illustrates a magnified view illustrating how the scaffold 10 acts as a structural support yet permits the tissue infiltration and biomaterial resorption due to the porous nature of the scaffold 10 .
  • a cell 106 is illustrated infiltrating the interstitial spaces formed within the scaffold 10 .
  • the scaffold 10 may also be used in a regenerative capacity, for example, applied to tissue for burns, acute and chronic wounds, and the like.
  • the scaffold 10 is used for chronic wounds.
  • chronic wounds where the normal healing process is inhibited, the scaffold 10 can be used not only to seal wounds, but also to remove excess moisture, and apply medication(s), including cellular therapies that can assist in promoting the normal wound healing process.
  • injection of the microgel particles 12 directly into the dermis via needle or cannula may be used to improve tissue contour, tissue loss, or tissue displacement.
  • cells used in regenerative medicine can grow within the microgel particles 12 .
  • cells e.g., mesenchymal stem cells, fibroblasts, etc.
  • cells may be included as a therapy by initially polymerizing the cells (1-20 cells) within microgel particles, or cells may be initially adhered to microgel particles, or cells may be introduced with the microgel particle solution (non-adhered), prior to annealing in situ in tissue.
  • the scaffold 10 may also be used for in vitro tissue growth, three-dimensional (3D) matrices for biological science studies, and cosmetic and dermatologic applications.
  • cancer cells could be seeded along with the microgel precursors and once annealed could allow for rapid 3D growth of tumor spheroids for more physiologically-relevant drug testing without the need for matrix degradation as would be required for other 3D culture gels (e.g., Matrigel®).
  • Matrigel® 3D culture gels
  • Epidermal layers can form over the surface of a scaffold 10 , which could allow testing of drugs or cosmetics on a more skin-like substitute compared to animal models.
  • Previous 3D culture materials either can enable cell seeding within the gel uniformly through the volume, but not maintain cell-cell contacts because of the lack of porosity, or create porosity but require cells to be seeded following fabrication and migrate into the scaffold.
  • the precursor materials prior to final annealing is flowable and can be delivered as paste, slurry, or even injected to the delivery site of interest.
  • Other injectable hydrogels can provide a scaffold for in situ tissue regrowth and regeneration, however these injected materials require gel degradation prior to tissue reformation limiting their ability to provide physical support.
  • the injectable microporous gel system described herein circumvents this challenge by providing an interconnected microporous network for simultaneous tissue reformation and material degradation.
  • Microfluidic formation enables substantially monodisperse microgel particles 12 to form into an interconnected microporous annealed particle scaffold 10 (in one aspect of the subject matter described herein), thereby enabling the controlled chemical, physical, and geometric properties of the microgel particles 12 (e.g., building blocks), to provide downstream control of the physical and chemical properties of the assembled scaffold 10 .
  • cells incorporated during scaffold 10 formation proliferate and form extensive three-dimensional networks within forty-eight (48) hours.
  • the injectable gel system that forms the scaffold 10 facilitates cell migration resulting in rapid cutaneous tissue regeneration and tissue structure formation within five (5) days.
  • the combination of microporosity and injectability achieved with the scaffolds 10 enables novel routes to tissue regeneration in vivo and tissue creation de novo.
  • FIG. 2A illustrates the scaffold 10 formed within a wound site 100 .
  • Successful materials for tissue regeneration benefit from precisely matching the rate of material degradation to tissue development. If degradation occurs too quickly then insufficient scaffolding will remain to support tissue ingrowth. Conversely, a rate that is too slow will prevent proper tissue development and can promote fibrosis and/or immune rejection.
  • Tuning of degradation rates based on local environment has been approached using hydrolytically and enzymatically degradable materials.
  • decoupling loss of material mechanical stability with cellular infiltration has proven extremely challenging. Promotion of cellular infiltration into the material can also be approached using a lightly crosslinked matrix, however this often results in mechanical mismatch with surrounding tissues and poor material stability.
  • the hydrogel degradation rate can be tuned by altering the polymeric backbone identity or crosslinking density, matching the rates of degradation and tissue formation.
  • these techniques can be tuned to address specific applications of injectable hydrogels, they do not provide a robust pathway to achieve bulk tissue integration that does not rely on loss of material stability.
  • microporous gel system and the resulting scaffold 10 that is created as described herein circumvents the need for material degradation prior to tissue ingrowth by providing a stably linked interconnected network of micropores for cell migration and bulk integration with surrounding tissue.
  • the microporous gel system achieves these favorable features by, according to one embodiment, using the self-assembly of microgel particles 12 as “building blocks” or “sub-units” formed by microfluidic water-in-oil droplet segmentation.
  • the microgel particles 12 formed in this manner are substantially monodisperse.
  • the microgel particles 12 can be injected and molded into any desired shape. Lattices of microgel particles 12 are then annealed to one another via surface functionalities to form an interconnected microporous scaffold 10 either with or without cells present in the interconnected porous networks.
  • the scaffold 10 preferably, in one embodiment, includes covalently linked microgel particles 12 that form a three-dimensional scaffolding 10 for tissue regeneration and ingrowth.
  • the microporous gel system provides an ideal biomaterial scaffold for efficient cellular network formation in vitro and bulk tissue integration in vivo.
  • the modular microporous gel system also provides mechanical support for rapid cell migration, molecular cues to direct cell adhesion, and resorption during and after tissue regeneration.
  • the chemical, physical, and geometric properties of the microgel particles 12 can be predictably and uniformly tailored, allowing for downstream control of the properties of the emergent scaffolds 10 .
  • novel building block-based approach in which robustly achieved imperfect self-assembly is desirable to achieve microporosity fundamentally changes the use and implementation of hydrogels as tissue mimetic constructs, providing a philosophical change in the approach to injectable scaffolding for bulk tissue integration.
  • the microporous gel system uses microgel particles 12 having diameter dimensions within the range from about 5 ⁇ m to about 1,000 ⁇ m.
  • the microgel particles 12 may be made from a hydrophilic polymer, amphiphilic polymer, synthetic or natural polymer (e.g., poly(ethylene glycol) (PEG), poly(propylene glycol), poly(hydroxyethylmethacrylate), hyaluronic acid (HA), gelatin, fibrin, chitosan, heparin, heparan, and synthetic versions of HA, gelatin, fibrin, chitosan, heparin, or heparan).
  • PEG poly(ethylene glycol)
  • poly(propylene glycol) poly(hydroxyethylmethacrylate)
  • HA hyaluronic acid
  • gelatin fibrin, chitosan, heparin, heparan
  • synthetic versions of HA gelatin, fibrin, chitosan, heparin, or heparan
  • the microgel particle 12 is made from any natural (e.g., modified HA) or synthetic polymer (e.g., PEG) capable of forming a hydrogel.
  • a polymeric network and/or any other support network capable of forming a solid hydrogel construct may be used.
  • Suitable support materials for most tissue engineering/regenerative medicine applications are generally biocompatible and preferably biodegradable.
  • suitable biocompatible and biodegradable supports include: natural polymeric carbohydrates and their synthetically modified, crosslinked, or substituted derivatives, such as gelatin, agar, agarose, crosslinked alginic acid, chitin, substituted and cross-linked guar gums, cellulose esters, especially with nitrous acids and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins, and keratins; vinyl polymers such as poly(ethyleneglycol)acrylate/methacrylate/vinyl sulfone/maleimide/norbornene/allyl, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes; and mixtures or copolymers of the above classes, such as graft copolymers obtained by initializing polymerization of
  • biocompatible and biodegradable polymers are available for use in therapeutic applications; examples include: polycaprolactone, polyglycolide, polylactide, poly(lactic-co-glycolic acid) (PLGA), and poly-3-hydroxybutyrate. Methods for making networks from such materials are well-known.
  • the microgel particles 12 further include covalently attached chemicals or molecules that act as signaling modifications that are formed during microgel particle 12 formation.
  • Signaling modifications includes the addition of, for example, adhesive peptides, extracellular matrix (ECM) proteins, and the like.
  • Functional groups and/or linkers can also be added to the microgel particles 12 following their formation through either covalent methods or non-covalent interactions (e.g., electrostatic charge-charge interactions or diffusion limited sequestration).
  • Crosslinkers are selected depending on the desired degradation characteristic. For example, crosslinkers for the microgel particles 12 may be degraded hydrolytically, enzymatically, photolytically, or the like. In one particular preferred embodiment, the crosslinker is a matrix metalloprotease (MMP)-degradable crosslinker.
  • MMP matrix metalloprotease
  • crosslinker sequences are hydrolytically degradable natural and synthetic polymers consisting of the same backbones listed above (e.g., heparin, alginate, poly(ethyleneglycol), polyacrylamides, polymethacrylates, copolymers and terpolymers of the listed polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes).
  • the crosslinkers are synthetically manufactured or naturally isolated DNA oligos with sequences corresponding to: restriction enzyme recognition sequences, CpG motifs, Zinc finger motifs, CRISPR or Cas-9 sequences, Talon recognition sequences, and transcription factor-binding domains.
  • any of the crosslinkers from the listed embodiments one are activated on each end by a reactive group, defined as a chemical group allowing the crosslinker to participate in the crosslinking reaction to form a polymer network or gel, where these functionalities can include: cysteine amino acids, synthetic and naturally occurring thiol-containing molecules, carbene-containing groups, activated esters, acrylates, norborenes, primary amines, hydrazides, phosphenes, azides, epoxy-containing groups, SANPAH containing groups, and diazirine containing groups.
  • a reactive group defined as a chemical group allowing the crosslinker to participate in the crosslinking reaction to form a polymer network or gel
  • these functionalities can include: cysteine amino acids, synthetic and naturally occurring thiol-containing molecules, carbene-containing groups, activated esters, acrylates, norborenes, primary amines, hydrazides, phosphenes, azides, epoxy-containing groups, SANPA
  • the chemistry used to generate microgel particles 12 allows for subsequent annealing and scaffold 10 formation through radically-initiated polymerization.
  • This includes chemical-initiators such as ammonium persulfate combined with Tetramethylethylenediamine.
  • photoinitators such as Irgacure® 2959 or Eosin Y together with a free radical transfer agent such as a free thiol group (used at a concentration within the range of 10 ⁇ M to 1 mM) may be used in combination with a light source that is used to initiate the reaction as described herein.
  • a free thiol group may include, for example, the amino acid cysteine, as described herein.
  • peptides including a free cysteine or small molecules including a free thiol may also be used.
  • Another example of a free radical transfer agent includes N-Vinylpyrrolidone (NVP).
  • microgel particle 12 formation chemistry allows for network formation through initiated sol-gel transitions including fibrinogen to fibrin (via addition of the catalytic enzyme thrombin).
  • Functionalities that allow for particle-particle annealing are included either during or after the formation of the microgel particles 12 .
  • these functionalities include ⁇ , ⁇ -unsaturated carbonyl groups that can be activated for annealing through either radical initiated reaction with ⁇ , ⁇ -unsaturated carbonyl groups on adjacent particles or Michael and pseudo-Michael addition reactions with nucleophilic functionalities that are either presented exogenously as a multifunctional linker between particles or as functional groups present on adjacent particles.
  • This method can use multiple microgel particle 12 population types that when mixed form a scaffold 10 .
  • microgel particle 12 of type X presenting, for example, nucleophilic surface groups
  • microgel particle 12 type Y presenting, for example, ⁇ , ⁇ -unsaturated carbonyl groups.
  • functionalities that participate in Click chemistry can be included allowing for attachment either directly to adjacent microgel particles 12 that present complimentary Click functionalities or via an exogenously presented multifunctional molecule that participates or initiates (e.g., copper) Click reactions.
  • the annealing functionality can include any previously discussed functionality used for microgel crosslinking that is either orthogonal or similar (if potential reactive groups remain) in terms of its initiation conditions (e.g., temperature, light, pH) compared to the initial crosslinking reaction.
  • initiation conditions e.g., temperature, light, pH
  • the subsequent annealing functionality can be initiated through temperature or photoinitiation (e.g., Eosin Y, Irgacure®).
  • the initial microgels may be photopolymerized at one wavelength of light (e.g., ultraviolent with Irgacure®), and annealing of the microgel particles 12 occurs at the same or another wavelength of light (e.g., visible with Eosin Y) or vice versa.
  • annealing moieties can include non-covalent hydrophobic, guest/host interactions (e.g., cyclodextrin), hybridization between complementary nucleic acid sequences or nucleic acid mimics (e.g., protein nucleic acid) on adjoining microgel particles 12 , or ionic interactions.
  • An example of an ionic interaction would consist of alginate functionality on the microgel particle surfaces that are annealed with Ca2+. So-called “A+B” reactions can be used to anneal microgel particles 12 as well.
  • A+B reactions can be used to anneal microgel particles 12 as well.
  • two separate microgel types are mixed in various ratios (between 0.01:1 and 1:100 A:B) and the surface functionalities of type A react with type B (and vice versa) to initiate annealing.
  • reaction types may fall under any of the mechanisms listed herein.
  • the microgel particles 12 are fabricated using either microfluidic or millifluidic methods, generating deterministic microgel particle length scales with small variability and in high throughput (e.g., frequencies greater than 10 particles/second).
  • the coefficient of variation of the microgel particle 12 length scale e.g., diameter
  • Milli- or microfluidics allow for uniform, pre-determined, concise material properties to be included pre-, in-, and post-formation of microgel particles 12 .
  • the microfluidic/millifluidic production mechanism allows for ease of scaling-up production as well as good quality control over chemical composition and physical characteristics of the microgel particles 12 .
  • the millifluidic and/or microfluidic technologies for microgel particle 12 generation are easily scalable processes to create large amounts of material for commercial needs, while maintaining high accuracy and precision in microgel particle 12 characteristics. Moreover, this is all accomplished at low cost in comparison to other technologies involving electrospinning or large-scale fibrin purification.
  • microgel particles 12 are formed using automated fluidic methods relying on water-in-oil emulsion generation. This includes microfluidic or millifluidic methods utilizing glass/PDMS, PDMS/PDMS, glass/glass, or molded/cast/embossed plastic chips to create water in oil droplets with a size distribution variation that is less than 35%.
  • FIGS. 3A-3F illustrates one embodiment of a microfluidic that is used to generate the microgel particles 12 .
  • the microfluidic device 20 is formed in a substrate material 22 such as PDMS which may include another substrate material 24 (e.g., glass) that is bonded the substrate 22 .
  • the microfluidic device 20 includes a first inlet 26 , a second inlet 28 , and a third inlet 30 .
  • the third inlet 30 is interposed between the first inlet 26 and the second inlet 28 .
  • the first inlet 26 is coupled to a solution containing a 4-arm poly(ethylene glycol) vinyl sulfone (PEG-VS) backbone (20 kDa) that has been pre-modified with oligopeptides for cell adhesive properties (e.g., RGD) and surface/tissue annealing functionalities (e.g., K and Q peptides).
  • PEG-VS poly(ethylene glycol) vinyl sulfone
  • the PEG-VS backbone may be prefunctionalized with 500 ⁇ M K-peptide (Ac-FKGGERCG-NH 2 [SEQ ID NO: 1]) (Genscript), 500 ⁇ M Q-peptide (Ac-NQEQVSPLGGERCG-NH 2 [SEQ ID NO: 2]), and 1 mM RGD (Ac-RGDSPGERCG-NH 2 [SEQ ID NO: 3]) (Genscript).
  • the solution input to the first inlet 26 may contain about 5% (on a weight basis) modified PEG-VS contained in a buffer of 0.3 M triethanolamine (Sigma), pH 8.25.
  • the second inlet 28 is coupled to a solution containing the crosslinker, which in one embodiment, is an 12 mM di-cysteine modified Matrix Metallo-protease (MMP) (Ac-GCRDGPQGIWGQDRCG-NH 2 [SEQ ID NO: 4]) substrate (Genscript).
  • MMP Matrix Metallo-protease
  • the MMP substrate was pre-reacted with 10 ⁇ M Alexa-fluor 647-maleimide (Life Technologies). Of course, in practical applications, the use of the fluorescent probe is not needed. All solutions can be sterile filtered through a 0.2 ⁇ m Polyethersulfone (PES) membrane in a Luer-lock syringe filter.
  • PES Polyethersulfone
  • K-peptides refer to those peptides that contain therein a Factor XIIIa recognized lysine group.
  • Q-peptides refer to those peptides that contain therein a Factor XIIIa recognized glutamine group.
  • peptide sequences beyond those specifically mentioned above may be used. The same applies to the RGD peptide sequence that is listed above.
  • the third inlet 30 is coupled to an aqueous solution containing 5% by weight of PEG-VS (unmodified by K, Q, or RGD peptides).
  • the aqueous PEG-VS solution is preferably viscosity-matched with the PEG-VS solution introduced via the first inlet 26 and can be used to control the pH of the crosslinker solution and to inhibit crosslinking until droplet formation.
  • the aqueous PEG-VS solution acts as a barrier that prevents any material diffusive mixing of reactive solutions upstream of the droplet generation region. This significantly increases the lifespan of the device before fouling occurs.
  • 3E and 3F illustrate how the inert liquid solution prevents mixing of left and right solutions prior to droplet segmentation. Note that the method of making the microgel particles 12 will also work with omitting the third inlet 30 , and adjusting peptide/crosslinker concentrations accordingly, yet the lifespan of the device will not be as long.
  • the first inlet 26 , second inlet 28 , and third inlet 30 are connected to, respectively, channels 32 , 34 , 36 .
  • the channels intersect at junction 38 and are carried in a common channel 40 .
  • the fourth inlet 42 is provided in the device and is coupled to an oil phase that contains a surfactant (e.g., 1% SPAN® 80 by volume although other surfactants can be used).
  • the fourth inlet 42 is connected to two channels 44 , 46 that intersect at junction 48 at a downstream region of the common channel 40 .
  • the junction 48 in the device 20 is where the aqueous-based droplets are formed that include the PEG-VS component and the crosslinker.
  • a fifth inlet 50 is provided that is coupled to another oil phase that contains a surfactant at a higher volumetric percentage than that connected to the fourth inlet 42 .
  • the fifth inlet 50 can be connected to an oil phase containing 5% SPAN® 80 by volume. Again, other surfactants besides SPAN® 80 could also be used.
  • the fifth inlet 50 is connected to two channels 52 , 54 that intersect at junction 56 in a pinching orientation as illustrated.
  • the common channel 40 continues to a series of progressively branching branch channels 58 .
  • the branch channels 58 permit continuous flow of the microgel particles 12 through individual parallel channels where local environmental conditions can be optionally controlled. For example, temperature of the individual branch channels 58 can be controlled to regulate crosslinking conditions for the microgel particles 12 .
  • the branch channels 58 may be illuminated with light to control light-activated reactions.
  • the microgel particles 12 may be removed from the device 20 using the outlet 59 . It should be understood, however, that regulation of the temperature of the branch channels 58 or the use of light activation is entirely optional as the crosslinking reaction may occur just through the passage of time when the device is operated at or around ambient temperatures.
  • the first inlet 26 , second inlet 28 , third inlet 30 , fourth inlet 42 , and fifth inlet 50 are connected, respectively, to fluid lines 26 ′, 28 ′, 30 ′, 42 ′, and 50 ′ that connect to a pumping device 51 or multiple pumping devices 51 that pumps respective fluids into the correspondingly connected inlets 28 , 28 , 30 , 42 , 50 .
  • the pumping device 51 may include separate pumps tied to each different fluid. Examples of types of pumps that may be used include syringe pumps or other pumps commonly used in connection with microfluidic devices.
  • the pumping device 51 uses regulated pressurized gas above a fluid reservoir to pump fluid at the desired flow rate(s) through the device.
  • FIGS. 4A-4C illustrate an alternative embodiment of a microfluidic device 60 that is used to generate the microgel particles 12 .
  • the microfluidic device 60 includes first inlet 62 , a second inlet 64 , a third inlet 66 , and a fourth inlet 68 .
  • the first inlet 62 is coupled to a modified PEG-VS source such as that described above.
  • the second inlet 64 is coupled to a crosslinking agent.
  • the third inlet 66 is coupled to a source containing oil and a surfactant.
  • the fourth inlet 68 is coupled to a source containing oil and a surfactant at a higher concentration than that coupled to the third inlet 66 .
  • the first inlet 62 and the second inlet 64 are coupled to respective channels 70 , 72 that lead to a common channel 74 .
  • the third inlet 66 is coupled to a pair of channels 76 , 78 that intersect with the common channel 74 at a junction 80 (best seen in FIG. 4B ) where droplet generation occurs (droplets will form the microgel particles 12 upon reaction).
  • the fourth inlet 68 is coupled to a pair of channels 82 , 84 that intersect with the common channel 74 at a downstream location 86 (best seen in FIG. 4B ) with respect to junction 80 .
  • the device 60 includes a series of progressively branching branch channels 88 which are similar to those described in the context of the embodiment of FIGS. 3A-3C .
  • Microgel particles 12 passing through branch channels 88 may collected in a collection chamber 90 or the like which can be removed from the device 60 .
  • Fluid is delivered to the device 60 using fluid lines and a pumping device as described previously in the context of the embodiment of FIGS. 3A-3C .
  • the fluidic conditions that lead to microgel particle 12 formation include, in one embodiment, on-chip mixing of a PEG-based and crosslinker-based aqueous solutions, where one part contains base polymer and the other contains the crosslinking or initiating agent.
  • a PEG-based and crosslinker-based aqueous solutions where one part contains base polymer and the other contains the crosslinking or initiating agent.
  • FIGS. 3A-3C there is a three-input mixing which includes the aforementioned components plus the addition of the aqueous-based inert stream.
  • These PEG and crosslinker solutions are mixed at either a 1:1 volumetric ratio, or another controllable ratio (controlled by relative flow rates into the device) up to 1:100.
  • the ratios of the oil and total aqueous flow rates are controlled to determine a specific size microgel particle 12 , where these ratios can range from 4:1 (aqueous: oil) down to 1:10 (aqueous:oil).
  • the chip device 20 is designed to have three aqueous-based solutions combined to form the microgel particles 12 , wherein the base polymer and crosslinking/initiating agent are separated by a non-reactive solution upstream of the droplet generator to prevent reaction of solutions and fouling of the chip over time in the region upstream of droplet generation.
  • the portion of non-reactive solution should be equal to or less than base and cross-linker solutions, from 1 to 0.05 times of the volume rate of the other solutions. This embodiment can thus improve the reliability and lifetime of chips used for microgel generation.
  • cells can be introduced into either of the two or three introduced aqueous solutions to enable encapsulation of these cells (single cells or clusters of 2-20 cells per particle) within microgel particles 12 such that encapsulated cells can produce factors to enhance wound healing or cell ingrowth.
  • FIGS. 3A-3D and 4A-4C illustrate different embodiments of a microfluidic device 20 , 60 that may be used to generate the microgel particles 12
  • the microfluidic flow path may include a ‘T-junction’ architecture such as that illustrated in FIG. 5 .
  • the microfluidic device 92 includes a junction formed between a first channel 94 that carries the aqueous phase while a second channel 96 includes the oil phase. Droplets 97 are formed and carried via an outlet channel 98 (which may be the same as the first or second channels 94 , 96 ).
  • different droplet formation configurations may be used to generate the microgel particles 12 .
  • the device may generate droplets 97 using the gradient of confinement due to non-parallel top and bottom walls such as that disclosed in Dangla et al., Droplet microfluidics driven by gradients of confinement, Proc Natl Acad Sci USA, 110(3): 853-858 (2013), which is incorporated by reference herein.
  • the channel surfaces should be modified such that the aqueous phase is non-wetting, which can include a fluorination of the surface, or converting the surfaces to become hydrophobic or fluorophilic, either by a covalent silane-based treatment or another non-specific adsorption based approach.
  • a plastic polymer containing fluorophilic groups comprises the chip material and can be combined with the previously mentioned surface coatings or without a surface coating.
  • the oil used in the preferred embodiment should be either a mineral oil (paraffin oil) supplemented with a non-ionic surfactant, vegetable oil supplemented with an ionic surfactant, or a fluorinated oil supplemented with a fluorinated surfactant (or any combination of these two oil/surfactant systems).
  • mineral oil paraffin oil
  • vegetable oil supplemented with an ionic surfactant
  • fluorinated oil supplemented with a fluorinated surfactant (or any combination of these two oil/surfactant systems).
  • a preferred embodiment of the microfluidic system for microgel particle 12 generation includes a low concentration of surfactant in the initial pinching oil flow (1% or less) that creates droplets followed by addition of an oil+surfactant solution from a separate inlet that is merged with the formed droplet and oil solution and contains a higher level of surfactant (up to 10 times or even 50 times higher than the initial surfactant). This is illustrated, for example, in the embodiments of FIGS. 3A-3D and 4A-4C .
  • the two oil pinching flows have the same concentration of surfactant.
  • there is not a second pinching oil flow and only the flow-focusing oil flow to generate droplets.
  • there is no second pinching oil flow and only the t-junction oil flow is used to generate droplets.
  • the t-junction droplet junction may optionally be combined with a second focusing oil inlet with equal or greater surfactant concentration.
  • microgel particles 12 are extracted from the oil phase using either centrifugation through an aqueous phase, or filtration through a solid membrane filtration device. For example, filtration may be used to reduce the volume of free aqueous solution holding the microgel particles 12 (free volume). In one embodiment, the aqueous free volume is less than about 35% of the total volume.
  • microgel particle generation is carried out in a milli- or microfluidic platform, generating stocks of relatively monodisperse microgel particles 12 that are then mixed at desired ratios to obtain deterministic distributions and ratios of microgel particle 12 sizes. Ratios of microgel particle 12 sizes can be controlled precisely to control pore structure, or chemical properties in a final annealed scaffold 10 with stoichiometric ratios from: 1:1, 10:1, or exceeding 100:1.
  • microgel particles 12 via a water-in-oil system can also be carried out using sonic mixing methods or a rotating vortex. These latter methods generate polydisperse populations of microgel particles 12 with size ranges from 100 nanometers to 500 micrometers. These particles can then be filtered using porous filters, microfluidic filtration, or other techniques known in the art to obtain a narrower size distribution of microgel particles 12 (e.g., coefficient of variation less than 50%).
  • the component microgel particles 12 of different shapes can be fabricated using stop flow lithography, continuous flow lithography, and other methods to create shaped particles that rely on shaping flows (see Amini et al. International Publication No.
  • microgel particles 12 are non-spherical with long and short dimensions that can vary between 5 and 1000 micrometers.
  • Shaped particles can also be fabricated by generating spherical particles in a water in oil emulsion, followed by extrusion of said particles through microfabricated constrictions that have length scales smaller than the diameter of the particle.
  • the previously spherical particles adopt the shape of the constriction as they transition to a gel and retain that shape as they gel in the constriction by any of the crosslinker reactions listed above. The gels retain that shape after exiting the microfabricated construction.
  • Shaped particles can allow for additional control of pores, overall porosity, tortuosity of pores, and improved adhesion within the final scaffold formed by microgel particle 12 annealing.
  • the microgel particles 12 are either modified covalently or not (e.g., inclusion spatially within by diffusion) to provide biologically active molecules (e.g., small molecule drugs, antibiotics, peptides, proteins, steroids, matrix polymers, growth factors, antigens, antibodies, etc.).
  • biologically active molecules e.g., small molecule drugs, antibiotics, peptides, proteins, steroids, matrix polymers, growth factors, antigens, antibodies, etc.
  • Inclusion of signaling molecules after formation of the microgel particle 12 may be accomplished through passive diffusion, surface immobilization (permanent or temporary), and/or bulk immobilization (permanent or temporary).
  • nanoparticles are included in the initial pre-polymer solution and incorporated in the microgel particles 12 during initial polymerization or gelation, and the nanoparticles may include biologically active molecules for sustained or rapid release and delivery.
  • microgel particles 12 containing free primary amines can be modified with NHS-Azide.
  • To this set of microgel particles 12 can be added a protein modified with a NHS-phosphine, resulting in surface-coating of the microgel particles 12 with the modified protein.
  • FIG. 10 illustrates an embodiment in which a microgel particle 12 has nanoparticles embedded therein and a surface that has been modified with a protein using Click chemistry.
  • the microgel particles 12 (which can be a homogeneous or heterogeneous mixture) may be applied to a desired location (in vitro, in situ, in vivo).
  • the desired location on mammalian tissue 102 can include, for example, a wound site 100 or other site of damaged tissue.
  • the microgel particles 12 can be introduced alone in an aqueous isotonic saline solution or slurry (with preferably 30-99% volume fraction of microgel particles 12 , and less preferably 1-30% volume fraction).
  • microgel particles 12 can be introduced along with cells as single-cells or aggregates with cell to particle ratios from 10:1 to create dense cell networks within the final annealed scaffold 10 or 1:100 or even 1:1000 to create sparsely seeded scaffolds 10 with cells that produce soluble factors useful for regeneration.
  • microgel particles 12 can be cultured with cells at a low volume fraction of particles ( ⁇ 10%) for a period of time in cell-permissive media to promote adhesion to the individual microgel particles 12 .
  • These composite cell-adhered microgel particles 12 can be introduced as the active component that would anneal to form a microporous cell-seeded scaffold 10 , which may be beneficial to enhance the speed of regenerative activity.
  • microgel particles 12 Desired in vitro locations to introduce microgel particles 12 include well plates (e.g., 6-well, 96-well, 384-well) or microfluidic devices to form 3D microporous culture environments for cells following annealing, and enable subsequent biological assays or high-throughput screening assays with more physiologically-relevant 3D or multi-cellular conditions.
  • microgel particle 12 solutions can be pipetted into wells or introduced via syringe injection followed by introduction of an annealing solution or triggering of annealing photochemically.
  • a solution of microgel particle 12 solution could be mixed with a slow acting annealing solution (annealing occurring over 10-30 min) before delivery.
  • In situ locations include external wound sites (e.g., cuts, blisters, sores, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, donor skin graft sites, post-Moh's surgery sites, post-laser surgery sites, podiatric wounds, wound dehiscence, abrasions, lacerations, second or third degree burns, radiation injury, skin tears and draining wounds, etc.).
  • external wound sites e.g., cuts, blisters, sores, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, donor skin graft sites, post-Moh's surgery sites, post-laser surgery sites, podiatric wounds, wound dehiscence, abrasions, lacerations, second or third degree burns, radiation injury, skin tears and draining wounds, etc.
  • the microgel particle solution may be used to heal other epithelial surfaces (i.e., urothelial (bladder and kidney), aerodigestive (lung, gastrointestinal), similarly to skin epithelium (i.e., stomach or duodenal ulcer; following penetrating trauma to the lung, bladder or intestinal fistulas, etc.). Additionally, the microgel particle solution can be applied to other tissues through a catheter or cannula, such as nervous tissue and cardiac tissue where tissue ingrowth would be beneficial to prevent scarring and to facilitate regenerative healing following injury, such as after spinal cord trauma, cerebral infarction/stroke, and myocardial infarction.
  • tissue ingrowth would be beneficial to prevent scarring and to facilitate regenerative healing following injury, such as after spinal cord trauma, cerebral infarction/stroke, and myocardial infarction.
  • microgel particle containing solution can be stored separately from an annealing solution and be mixed during introduction (a method analogous to epoxy adhesives) to prevent premature initiation of the annealing reaction before entry into a wound site 100 .
  • FIG. 6A illustrates one such embodiment of a delivery device 110 that includes a first barrel 112 , a second barrel 114 , and a plunger 116 that is used to dispense the solution containing the microgel particles 12 from each barrel 112 , 114 .
  • the first barrel 112 contains microgel particles 12 and thrombin at a concentration ranging from 0.1 to 5 U/ml and the second barrel 114 contains the microgel particles 12 and FXIII at a concentration of 0.1 to 1,000 U/ml).
  • both barrels 112 , 114 there is a 1 to 1 volume fraction of K and Q peptide containing microgel particles 12 where the concentration of K and Q peptides range from 10-1,000 ⁇ M in the microgel particles 12 .
  • the thrombin upon mixing the thrombin activates the FXIII (to form FXIIIa) and the resultant FXIIIa is responsible for surface annealing and linking of the K and Q peptides on the adjacent microgel particles 12 .
  • the two barrels 112 , 114 can contain two separate microgel particle 12 types with annealing moieties that require the combination to initiate cross-linking.
  • An alternative storage and delivery method would be in a single barrel syringe 110 as illustrated in FIG. 6B or a multi-use or single-use compressible tube as illustrated in FIG. 6C (e.g., similar to toothpaste or antibiotic ointment) in which the microgel particle slurry can be squeezed out to a desired volume and spread over the wound site 100 and then annealed through exposure to light, where the active agent for photochemistry is Eosin-Y at a concentration of 100 ⁇ M although concentrations within the range of 10 ⁇ M-1 mM will also work.
  • Eosin-Y is accompanied with a radical transfer agent which can be, for example, a chemical species with a free thiol group.
  • a radical transfer agent includes cysteine or peptides including cysteine(s) described herein (e.g., used at a concentration of 500 ⁇ M).
  • the light should be delivered via a wide spectrum white light (incandescent or LED), or a green or blue LED light. A flashlight, wand, lamp, or even ambient light may be used to supply the white light. Exposure should occur between 0.1 seconds and 1000 seconds, and the intensity of light should range between 0.01 mW/cm 2 to 100 mW/cm 2 at the site of annealing.
  • light-mediated annealing can be accomplished using a UV light (wavelengths between 300-450 nm), where the agent for photochemistry is IRGACURE® 2959, at a concentration of 0.01% w/v to 10% w/v.
  • the exposure time should be between 0.1 seconds and 100 seconds, with a light intensity of 0.1 mW/cm 2 to 100 mW/cm 2 at a site of annealing.
  • microgel precursors 12 would be stored in opaque (opaque with respect to wavelength range that initiates annealing) syringe or squeeze tubes 110 containers prior to use.
  • Desired in situ locations include internal cuts and tissue gaps (e.g., from surgical incisions or resections), burn wounds, radiation wounds and ulcers, or in cosmetic surgery applications to fill the tissue location and encourage tissue ingrowth and regeneration rather than the fibrotic processes common to contemporary injectables.
  • tissue gaps e.g., from surgical incisions or resections
  • burn wounds e.g., from radiation wounds and ulcers
  • cosmetic surgery applications to fill the tissue location and encourage tissue ingrowth and regeneration rather than the fibrotic processes common to contemporary injectables.
  • the microgel particle slurry can be spread using a sterile applicator to be flush with the wound or mounded within and around the wound site 100 (within the wound and 2 mm to 1 cm beyond the original wound extents) to create an annealed scaffold that extends beyond the wound site 100 or tissue defect to provide additional protection, moisture, and structure to support tissue regeneration.
  • An annealing process is initiated through the application of a stimulus (e.g., radical initiator, enzyme, Michael addition, etc.) or through interactions with a stimulus that is already present at the site of application of the microgel particles 12 that interacts with functional groups on the surface of the microgel particles 12 , forming a solid contiguous highly porous scaffold 10 formed from the annealed (linked) microgel particles 12 .
  • a stimulus e.g., radical initiator, enzyme, Michael addition, etc.
  • the annealing process can allow for fusion of the scaffold 10 to the surrounding tissue, providing an effective seal, a local medication and/or cell delivery device, a vascularized scaffold for in vivo sensing, and a better path to tissue regeneration.
  • the annealing process allows for on-site/on-demand gel formation (which is ideal for in vitro and in vivo applications), for example delivery through a small incision to a minimally-invasive surgical site or through injection by a needle or through a catheter or cannula.
  • the scaffold 12 may comprise of homogeneous or heterogeneous populations of microgel particles 12 .
  • the heterogeneous populations of microgel particles 12 may vary in physical (e.g., in size, shape, or stiffness) or vary in chemical composition (e.g., varied ratios of degradable linkers, or L- or D-amino acids to modify degradation rate, varied annealing moieties, cell adhesive moieties, or loading of microgels 12 with bioactive molecules or nanoparticles).
  • the heterogeneous composition of the final annealed scaffold 10 can be random or structured in layers of uniform composition to create gradients in micro-porous structures (by varying microgel particle 12 sizes in layers, for example) or gradients of chemical composition (by layers of microgel particles 12 with different composition or bio-active molecule loading). Gradients may be useful in directing cell ingrowth and tissue regeneration in vivo, or development of tissue structures in vitro. Gradients in microgel particle 12 composition could be achieved by delivering sequential slurries of a gel of a single composition, followed by annealing, and then subsequent delivery of the next gel of a second composition, followed by annealing which links the new layer of microgels to the previous layer, until a desired number of layers have been accumulated.
  • each layer can be controlled using the volume of slurry injected and area of the injection site.
  • An alternative embodiment to achieve gradients is to load a multi-barrel syringe applicator such as that illustrated in FIG. 6A with different microgel compositions in each of the barrels. Each of the barrels are simultaneously compressed and feed to the nozzle 120 in layered sheets.
  • the nozzle 120 itself of the syringe applicator can be non-circular or rectangular to create a layered slurry of multiple composition that is injected to a site in a ribbon-like structure, which can then be annealed in this arrangement. Formation of the structurally contiguous annealed scaffold 10 may be achieved through radical, enzymatic or chemical (e.g., Click chemistry) processes.
  • annealing occurs through surface chemistry interactions between microgel particles 12 once they are ready to be placed at the delivery site.
  • the process occurs through radical-initiated annealing via surface polymerizable groups (e.g., radical initiation by photo-sensitive radical initiators, etc.).
  • the process occurs through enzymatic chemistry via surface presented enzymatically-active substrates (e.g., transglutaminase enzymes like Factor XIIIa).
  • the process occurs through covalent coupling via Michael and pseudo-Michael addition reactions.
  • This method can use multiple microgel particle population types that when mixed form a solid scaffold 10 (e.g., microgel particle 12 type A presenting, for example, nucleophilic surface groups and microgel particle 12 type B presenting, for example, ⁇ , ⁇ -unsaturated carbonyl groups).
  • the process occurs through Click chemistry attachment.
  • this method can use heterogeneous microgel particle 12 populations that when mixed form a solid microporous gel.
  • annealing may be achieved using light (for example, either white light or UV light) to initiate a chemical reaction between molecules on the gel surfaces, mediated by a light activated molecule in solution in and around (or directly covalently liked to) the microgels as described herein.
  • the microgel particles 12 include a PEG based polymeric backbone in combination with an enzymatically degradable crosslinker to allow for bioresorbability.
  • the PEG-based polymeric backbone is a 4-arm poly(ethylene glycol) vinyl sulfone (PEG-VS) backbone pre-modified with oligopeptides for cell adhesive properties (e.g., RGD) and surface annealing functionalities (e.g., K and Q peptides) and the cross-linker is a matrix metalloprotease (MMP)-degradable cross-linker.
  • PEG-based polymeric backbone is a 4-arm poly(ethylene glycol) vinyl sulfone (PEG-VS) backbone pre-modified with oligopeptides for cell adhesive properties (e.g., RGD) and surface annealing functionalities (e.g., K and Q peptides)
  • MMP matrix metalloprotease
  • microgel particles 12 are formed by a water-in-oil emulsion. Gelation of the microgel particles 12 occurs upon combination of PEG solution with cross-linker solution (followed shortly by partitioning into microgel droplets before completion of gelation). A variety of substrates, including peptide ligands, can be further added for enhanced bioactivity. In one embodiment, scaffold formation is accomplished by addition and activation of radical photo-initiator to the purified microgel particles 12 to induce chemical cross-linking.
  • scaffold formation is accomplished by the use and/or activation of an endogenously present or exogenously applied transglutaminase enzyme, Factor XIII, to the purified microgel particles 12 that have been modified with two peptide ligands either pre-formation, during formation, or post-formation to induce enzymatic cross-linking.
  • scaffold formation is accomplished using a combination of the aforementioned radical and enzymatic methods.
  • porous scaffolds provide for greater access for live cells due to the freedom of movement through the pores (i.e., not requiring degradation to allow penetration like all current and previous non-porous and nano-porous scaffolds).
  • FIG. 7B shows that when implanting and annealing a scaffold 10 in a skin wound in vivo, significantly enhanced cell invasion and tissue-structure in growth was observed after 5 days when compared to a non-porous gel of the same material as seen in FIG. 7B .
  • FIG. 7A illustrates H&E staining of tissue sections in SKH1-Hr hr mice for tissue injected with the scaffold 10 (identified as MAP scaffold) as well as the non-porous control 24 hours after injection.
  • FIG. 7C illustrate representative images of wound closure during a 5 day in vivo wound healing model in SKH1-Hr hr mice.
  • FIG. 7D illustrates representative images of wound closure during 7 day in vivo BALB/c mice experiments.
  • FIG. 7E illustrates wound closure quantification data from BALB/c in vivo wound healing.
  • the scaffolds 10 promote significantly faster wound healing than the no treatment control, the non-porous PEG gel, and the gels lacking the K and Q peptides.
  • Porous gels created ex vivo to precisely match the wound shape using the canonical, porogen-based, casting method showed appreciable wound healing rates, comparable to the scaffolds 10 , but lacking injectability (N ⁇ 5).
  • FIG. 7F illustrates traces of wound bed closure during 7 days in vivo for each treatment category corresponding to FIG. 7D .
  • therapeutic agents applied to the microgel particles 12 or the scaffold 10 can be released slowly or rapidly, and the scaffold 10 has the ability to break down over a pre-determined period of time either from hydrolysis, proteolysis, or enzymolysis, depending on the intended treatment (e.g., if it is being used to treat a chronic wound, a more stable cross-linker that degrades slowly over time is used).
  • the annealing quality of the microgel scaffold 10 allows the scaffold 10 to function as a tissue sealant (e.g., acute wounds, surgical closure, etc.), and the filling of different molded shapes that are clinically useful to mimic tissues.
  • tissue sealant e.g., acute wounds, surgical closure, etc.
  • FIG. 7G illustrates how the microgel particle containing solution or slurry can be applied using a syringe device like that of FIG. 6A or 6B into a treatment site where the microgel conforms to the shape of the injection site (e.g., in this case a star-shaped site) and subsequent annealing of the scaffold 10 into the star shape.
  • a syringe device like that of FIG. 6A or 6B into a treatment site where the microgel conforms to the shape of the injection site (e.g., in this case a star-shaped site) and subsequent annealing of the scaffold 10 into the star shape.
  • the rate of degradation of the microgel scaffolds 10 By adjusting the rate of degradation of the microgel scaffolds 10 the scar forming or regenerative response in a wound can be modified.
  • the degradation rate of the microgel scaffolds 10 was modified by using D-instead of L-amino acids in the MMP-degradable crosslinker. Adjusting the ratio of microgel particles 12 with D- or L-chirality in the crosslinker adjusted the rate of degradation in the tissue. Scaffolds 10 made from mixtures of D and L crosslinked microgels (at a 1:1 ratio) resulted in gels present in the tissue 21 days after injection, however in the D-only gels, there was no remaining gel left after 21 days in vivo.
  • FIGS. 8A-8G show the effects of scar reduction when using a 1:1 mixture of D:L, as compared directly to a no treatment wound. Dermal thickness is doubled and scar size is reduced by 25% in the 1:1 D:L gel treatment. Additionally, six (6) times more hair follicles and sweat glands are present in the gel-treated case, compared to the no treatment case.
  • a microfluidic water-in-oil emulsion approach was used to segment a continuous pre-gel aqueous phase into uniform scaffold building blocks as described herein.
  • Generating microgel particles 12 as building blocks serially at the microscale, rather than using the typical vortex and sonication-based approaches allowed tight control over the formation environment and ultimate material properties of the emergent scaffold 10 .
  • microfluidic generation of microgel particle “building blocks” is a readily scalable process: a practical requirement for wide adoption and use.
  • the resultant microgel particles 12 were composed of a completely synthetic hydrogel mesh of poly(ethylene)glycol-vinyl sulfone (PEG-VS) backbones decorated with cell-adhesive peptide (RGD [SEQ ID NO: 3]) and two transglutaminase peptide substrates (K [SEQ ID NO: 1] and Q [SEQ ID NO: 2]).
  • PEG-VS poly(ethylene)glycol-vinyl sulfone
  • RGD cell-adhesive peptide
  • K transglutaminase peptide substrates
  • the microgel particles 12 were crosslinked via Michael type addition with cysteine-terminated matrix metalloprotease-sensitive peptide sequences that allowed for cell-controlled material degradation and subsequent resorption.
  • the microgel particles 12 were purified into an aqueous solution of isotonic cell culture media for storage and when used to form a gel were annealed to one another via a non-canonical amide linkage between the K and Q peptides mediated by activated Factor XIII (FXIIIa), a naturally occurring enzyme responsible for stabilizing blood clots.
  • FXIIIa activated Factor XIII
  • This enzyme-mediated annealing process allowed incorporation of living cells into a dynamically forming scaffold 10 that contained interconnected microporous networks.
  • a slurry of the microgel particles 12 can be delivered via syringe application, ultimately solidifying in the shape of the cavity in which they are injected.
  • FIG. 9A illustrates how the annealing kinetics can be altered by the adjustment of pH and temperature.
  • the annealing environment chosen for this experiment was pH 8.25 and a temperature of 37° C.
  • microgel particles 12 By tuning the microgel particle size and composition a diverse set of assembled scaffolds 10 were able to be generated.
  • microgel particles 12 By using microgel particles 12 from 30 to 150 ⁇ m in diameter, networks with median pores diameters ranging from ⁇ 10 to ⁇ 35 ⁇ m were achieved).
  • Different PEG weight percentages and crosslinker stoichiometries were screened to demonstrate a range of easily achievable storage moduli from ⁇ 10 to 1000 Pa that spans the stiffness regime necessary for mammalian soft tissue mimetics.
  • FIG. 9B illustrates different hydrogel weight percentages were used to produce different stiffness materials.
  • FIG. 9B illustrates different hydrogel weight percentages were used to produce different stiffness materials.
  • FIG. 9C illustrates different crosslinker stoichiometries (r-ratio of crosslinker ends (—SH) to vinyl groups (—VS)) that were used to produce different stiffness values in the resultant gel.
  • FIG. 9D illustrates a graph of the % degradation as a function of time for both the non-porous control as well as the inventive porous gel described herein. Degradation kinetics of particle-based, porous gel and the non-porous are shown for equal volumes of gels in vitro. The particle-based, porous gels degrade faster than non-porous gel due to higher surface area to volume ratios and faster transport through the microporous gel.
  • FIG. 9E illustrates SEM images of a scaffold annealed with FXIIIa.
  • FIG. 9F illustrates SEM images of microgel particles 12 without FXIIIa. Un-annealed particles are seen in FIG. 9F .
  • HDF Dermal Fibroblasts
  • AhMSC Adipose-derived Mesenchymal Stem Cells
  • BMhMSC Bone Marrow-derived Mesenchymal Stem Cells
  • Cells incorporated into the scaffold began to exhibit spread morphology 90 minutes following the onset of annealing. After two (2) days in culture, all observed cells within the scaffolds exhibited a completely spread morphology, which continued through day six (6). Importantly, an extensive network formation for all cell lines was observed by day two (2). Cell networks increased in size and complexity through the entirety of the experiment. The BMhMSCs were of particular note, as their expansive network formation and slower proliferation rate indicated that these cells were able to spread to extreme lengths, forming highly interconnected cellular networks within the microporous scaffolds.
  • the scaffold was able to sustain the formation of what appeared to be a complete hair follicle with adjoining sebaceous gland within the wound bed resembling the structure of these glands in the uninjured skin.
  • the scaffolds While investigating the seamless interface provided by the injectable scaffolds differences were observed in both overall and immune cell quantities at day one (1). After one (1) day post-injection, the scaffolds contained significantly higher numbers of cells within the scaffold than their non-porous bi-lateral controls. This corroborated the greater ease of cell mobility previously observed in our in vitro network formation experiments. Further, the scaffold and its surrounding tissue contained a significantly lower number of polymorphonuclear cells when compared to the non-porous bi-lateral control of the same mouse. This result indicated an overall lower initial innate immune response to the scaffolds at day one (1).
  • the annealed, microgel particle-based scaffolds represent a new class of injectable biomaterial that introduces microscale interconnected porosity through robustly achieved imperfect self-assembly and annealing of individual building blocks.
  • This approach allows control of micro-scale and hierarchical macro-scale properties through deterministic chemical composition and microfluidic particle generation. Both incorporated live cells and surrounding host tissue are able to immediately infiltrate the scaffold without the need for material degradation, a feat never before accomplished using injectable scaffolds.
  • the injectable microgel particles completely filled the tissue void, providing a seamless boundary with the surrounding tissue.
  • the interconnected microporosity of the resulting scaffold promoted cellular migration at the wound site that resulted in greater bulk integration with the surrounding tissue while eliciting a reduced host immune response, in comparison to an injectable non-porous control. Ultimately this led to faster healthy tissue reformation than with similarly comprised injectable non-porous gels.
  • This gel system presents a fundamental change in the approach to bottom-up modular biomaterials by utilizing the negative space of lattice formation to promote the development of complex three-dimensional networks on time scales previously unseen using current hydrogel technologies.
  • the “plug and play” nature of this strategy allows the incorporation of a wide range of already established materials (e.g., fibrin), signals (e.g., growth factors), and cell populations (e.g., stem cells).
  • Complex combinations of building blocks with deterministic chemical and physical properties may enable tissue regeneration in a range of distinct physiological niches (e.g., neural, cardiac, skin, etc.), where particle-annealed scaffolds are tailored to each niche via their building block properties.
  • the unique combination of microporosity, injectability, and modular assembly inherent to scaffolds has the potential to alter the landscape of tissue regeneration in vivo and tissue creation de novo.
  • Microfluidic water-in-oil droplet generators were fabricated using soft lithography as previously described. Briefly, master molds were fabricated on mechanical grade silicon wafers (University wafer) using KMPR 1025 or 1050 photoresist (Microchem). Varying channel heights were obtained by spinning photoresist at different speeds, per the manufacturer's suggestions. Devices were molded from the masters using poly(dimethyl)siloxane (PDMS) SYLGARD® 184 kit (Dow Corning). The base and crosslinker were mixed at a 10:1 mass ratio, poured over the mold, and degassed prior to curing for 6 hours at 65° C.
  • PDMS poly(dimethyl)siloxane
  • Channels were sealed by treating the PDMS mold and a glass microscope slide (VWR) with oxygen plasma at 500 mTorr and 75 W for 15 seconds. Immediately after channel sealing, the channels were functionalized by injecting 100 ⁇ l of a solution of RAIN-X® and reacting for 20 minutes at room temperature. The channels were then dried by air followed by desiccation overnight.
  • VWR glass microscope slide
  • Droplets were generated using a microfluidic water-in-oil segmentation system as illustrated in FIGS. 3A-3F and 4A-4C .
  • the aqueous phase is a 1:1 volume mixture of two parts: (i) a 10% w/v 4arm PEG-VS (20 kDa) in 300 mM triethanolamine (Sigma), pH 8.25, prefunctionalized with 500 ⁇ M K-peptide (Ac-FKGGERCG-NH 2 [SEQ ID NO: 1]) (Genscript), 500 ⁇ M Q-peptide (Ac-NQEQVSPLGGERCG-NH 2 [SEQ ID NO: 2]), and 1 mM RGD (Ac-RGDSPGERCG-NH 2 [SEQ ID NO: 3]) (Genscript) and (ii) an 8 mM (12 mM for the three-inlet device) di-cysteine modified Matrix Metallo-protease (MMP) (Ac-GCRDGPQGIWGQDRCG-NH 2 [S
  • the mixture was then extracted and purified by overlaying the oil solution onto an aqueous buffer of HEPES buffered saline pH 7.4 and pelleting in a table top centrifuge at 18000 ⁇ g for 5 mins.
  • the microgel-based pellet was washed in HEPES buffered saline pH 7.4 with 10 mM CaCl 2 and 0.01% w/v Pluronic F-127 (Sigma).
  • the microgel aqueous solution was then allowed to swell and equilibrate with buffer for at least 2 hours at 37° C.
  • microgel particles were pelleted by centrifugation at 18000 ⁇ g for five minutes, and the excess buffer (HEPES pH 7.4+10 mM CaCl 2 ) was removed by aspiration and drying with a cleanroom wipe. Subsequently, microgel particles were split into aliquots, each containing 50 ⁇ l of concentrated building blocks. An equal volume of HEPES pH 7.4+10 mM CaCl 2 was added to the concentrated building block solutions. Half of these include Thrombin (Sigma) to a final concentration of 2 U/ml and the other half includes FXIII (CSL Behring) to a final concentration of 10 U/ml. These solutions were then well mixed and spun down at 18000 ⁇ g, followed by removal of excess liquid with a cleanroom wipe (American Cleanstat).
  • Annealing was initiated by mixing equal volumes of the building block solutions containing Thrombin and FXIII using a positive displacement pipet (Gilson). These solutions were well mixed by pipetting up and down, repeatedly, in conjunction with stirring using the pipet tip. The mixed solution was then pipetted into the desired location (mold, well plate, mouse wound, etc.).
  • a macroscale (50 ⁇ L) non-porous gel was generated with the same chemical composition.
  • a 30 ⁇ L solution of 2 ⁇ PEG-VS+peptides (RGD, K, and Q peptides) dissolved in 0.3 M TEOA was combined with 30 ⁇ L of 2 ⁇ MMP-1 crosslinker dissolved in water.
  • the mixture was quickly vortexed and 50 ⁇ L of the mixture was placed between two 8 mm rheological discs at a spacing of 1 mm (Anton Paar Physica MCR301 Rheometer). The storage modulus was then measured over a period of 20 minutes (2.5 Hz, 0.1% strain).
  • an amplitude sweep (0.01-10% strain) was performed to find the linear amplitude range for each. An amplitude within the linear range was chosen to run a frequency sweep (0.5-5 Hz).
  • This mixture was allowed to partially anneal for 10 minutes before removal of top glass slide and placement in a humidified incubator at 37° C. for 90 minutes.
  • the scaffolds were then placed into HEPES buffered saline (pH 7.4) overnight to reach equilibrium.
  • the samples were then placed between two 8 mm discs on the rheometer and tested identically to the pre-annealed microgel particles.
  • microgel particles were covalently linked after addition of FXIIIa
  • SEM was used to directly visualize scaffolds.
  • Microgel particle mixtures were either treated with FXIIIa (10 U/ml) or with buffer only.
  • the building block solutions were placed onto a 1 ⁇ 1 in silicon wafer piece, and dried in an SEM (Hitachi 54700) high vac chamber (1 ⁇ 10 ⁇ 3 mTorr).
  • Building blocks with or without FXIIIa were then visualized using 10 kV (10 mA max) on either 200 ⁇ or 500 ⁇ as seen in FIGS. 9D and 9F .
  • HEK293T cells constitutively expressing GFP via lentiviral transfection were maintained in DMEM (Life Technologies) supplemented with 10 ⁇ g/ml puromycin.
  • DMEM fetal mesenchymal calf serum
  • BMhMSC bone marrow-derived human mesenchymal stem cells
  • AhMSC adipose-derived human mesenchymal stem cells
  • All cell lines were maintained according to manufacturer's specifications (before and after incorporation into porous or non-porous gels). Specifically, for the MSC populations reduced-serum, basal medium (Life Technologies) was used to retain stemness.
  • particle-based scaffolds were annealed with microgel particles as described above, with the addition of cell suspensions to the building block solutions prior to annealing.
  • cell suspensions were prepared at a final concentration of 25 ⁇ 10 6 cells/ml in respective culture media un-supplemented with serum.
  • 2 ⁇ l of cell suspension was added to 50 ⁇ l of microgel particle mixture containing FXIII and combined with 50 ⁇ l of microgel particle mixture containing Thrombin (500 cells/ ⁇ l of gel). This mixture was injected into the corner of a coverslip-bottom PDMS well. The well top was covered with a second coverslip and the ⁇ gel/cell mixture was allowed to undergo annealing for 90 minutes at 37° C.
  • HEK-293-T cells were incorporated into a star-shaped mold by mixing cells with microgel particles (as described above) and pipetting 5 ⁇ l of the mixture into the center of the mold Immediately following, microgel particles without cells were pipetted in the remainder of the mold, and annealed as described above.
  • Proliferation was assessed by counting the number of cell nuclei present in the particle-based scaffold constructs after 0, 2, 4, and 6 days of culture in vitro. Nuclei were stained with a 2 ⁇ g/ml DAPI solution in 1 ⁇ PBS for 2 hours, followed by visualization on a Nikon C2 using the 405 nm LED laser. Specifically, each scaffold was imaged by taking 55 z slices in a 150 ⁇ m total z height and compressing every 5 slices into a maximum intensity projection (MIP) image. Nuclei in the MIPs were enumerated using a custom MATLAB® script, counting the total number of cells.
  • MIP maximum intensity projection
  • each time point z-stack images of three separate microgel scaffolds were analyzed, where each z-stack image measured a total volume of 1270 ⁇ 1270 ⁇ 150 ⁇ m 3 (or ⁇ 280 nL).
  • the 90 minute counts lead to a calculation of ⁇ 525 cells/ ⁇ l of gel, consistent with the experimental amount added (500 cells/ ⁇ l of gel).
  • the constructs were prepared, grown, and fixed as above.
  • the scaffolds were blocked with 1% BSA in 1 ⁇ PBS for 1 hour at room temperature, followed by staining for f-actin via a Rhodamine-B conjugate of phalloidin (Life Technologies) for 3 hours at room temperature.
  • the scaffolds were then washed with 1% BSA in 1 ⁇ PBS, followed by counterstaining with a 2 ⁇ g/ml DAPI solution in 1 ⁇ PBS for 1 hour at room temperature.
  • Imaging was performed as with proliferation imaging, with the exception of using a 40 ⁇ magnification water immersion lens. Total heights of image stacks were 130 ⁇ m, with the total number of slices at 260 (volume captures ⁇ 15 nL).
  • Either non-porous or porous hydrogel including 10 U/ml FXIIIa was injected into wound beds, allowed to undergo gelation for 10 minutes, followed by subsequent covering of the wound by a stretchy gauze wrap to prevent animal interaction. The mice were then separated into individual cages. Pain medication was administered subcutaneously every 12 hours for the next 48 hours (for Day 1 sacrifices pain killer was administered once after surgery).
  • the skin of the back was removed using surgical scissors and the wound site was isolated via a 10 mm biopsy punch.
  • the samples were immediately fixed using 4% formaldehyde at 4° C. (overnight) followed by transfer to ethanol and embedding of the sample into a paraffin block.
  • the blocks were then sectioned at 6 ⁇ m thickness by microtome (Leica) and underwent Hematoxylin and Eosin (H&E) staining.
  • H&E Hematoxylin and Eosin
  • the average of 3 HPFs from different sections of the wound were examined.
  • the total number of leukocytes/HPF within 0.2 mm of the hydrogel at the wound edge was quantified and averaged for each wound type.
  • the leukocyte count for each wound was compared to its bilateral control on the same animal and the relative difference was recorded as a fraction of each animal's overall immune response. This comparison was possible because each animal had one wound injected with the microgel scaffold and one wound with the non-porous control.
  • Closure fraction was determined by comparing the pixel area of the wound to the pixel area within the 10 mm center hole of the red rubber splint. Closure fractions were normalized to Day 0 for each mouse/scaffold type ( FIG. 7B ).
  • the samples were immediately submerged in TISSUE-TEK® Optimal Cutting Temperature (OCT) fluid and frozen into a solid block with liquid nitrogen.
  • OCT Optimal Cutting Temperature
  • the blocks were then cryo-sectioned at 25 ⁇ m thickness by cryostat microtome (Leica) and kept frozen until use.
  • the sections were then fixed with paraformaldehyde for 30 minutes at room temperature, hydrated with PBS, and kept at 4° C. until stained.
  • Sections were stained with primary antibodies overnight at 4° C., and subsequently washed with 3% NGS in 1 ⁇ PBST. Secondary antibodies were all prepared in 3% NGS in 1 ⁇ PBST at a dilution of 1:100. Sections were incubated in secondary antibodies for 1 hour at room temperature, and subsequently washed with 1 ⁇ PBST. Sections were counterstained with 2 ⁇ g/ml DAPI in 1 ⁇ PBST for 30 mins at room temperature. Sections were mounted in Antifade Gold mounting medium.
  • FIG. 11 illustrates one example of method of treating damaged tissue 102 .
  • FIG. 11 illustrates a wound site 100 formed in tissue 102 of a mammal.
  • a delivery device 110 e.g., tube as illustrated
  • an aqueous solution is used to deliver the microgel particles 12 to the wound site 100 .
  • an optional applicator 122 is used to spread the microgel particles 12 into and over the wound site 100 .
  • the applicator 122 is also used to make the upper, exposed surface of the microgel particles 12 generally flush with the surface of the tissue 102 .
  • the applicator 122 can also be used to make the upper, exposed surface of the microgel particles 12 mounded or elevated with respect to the surface of the tissue 102 to allow for increased structure for cellular ingrowth and prevention of a depressed tissue interface upon full healing.
  • annealing of the microgel particles 12 is initiated to form the scaffold 10 of annealed microgel particles 12 .
  • a light source 124 in the form of a flashlight is used to illuminate a mixture of microgel particles 12 , a photoinitiator (e.g., Eosin Y), and a free radical transfer agent (e.g., RGD peptide).
  • a photoinitiator e.g., Eosin Y
  • RGD peptide free radical transfer agent
  • other annealing modalities as described herein may also be used.
  • the annealing reaction illustrated in FIG. 11 causes the formation of a covalently-stabilized scaffold 10 of microgel particles 12 having interstitial spaces therein.
  • Cells 106 (as seen in FIG. 2C ) from the surrounding tissue 102 then begin to infiltrate the spaces within the scaffold 10 , grow, stimulate, and ultimately effectuate the healing process of the tissue 102 .
  • a bandage or moist dressing is optionally placed over the scaffold-filled wound to protect it from damage during the healing process.
  • the scaffold 10 has degraded and the tissue 102 has returned to a healed state.
  • an in vitro cell morphology and proliferation model was developed using three human cell lines: Dermal Fibroblasts (HDF), Adipose-derived Mesenchymal Stem Cells (AhMSC), and Bone Marrow-derived Mesenchymal Stem Cells (BMhMSC).
  • HDF Dermal Fibroblasts
  • AhMSC Adipose-derived Mesenchymal Stem Cells
  • BMhMSC Bone Marrow-derived Mesenchymal Stem Cells
  • microgel particles 12 can be combined and mixed with a solution of living cells 106 prior to annealing to create a microporous scaffold 10 that contains living cells 106 residing in the microporous network and dispersed either homogenously or heterogeneously within the macroscopic annealed gel scaffold 10 as seen in FIG. 13A .
  • the microgel particles 12 can be purified into an aqueous solution of isotonic cell culture media for storage and when used to form a porous gel were annealed to one another via a non-canonical amide linkage between the K and Q peptides mediated by activated Factor XIII (FXIIIa), a naturally occurring enzyme responsible for stabilizing blood clots.
  • FXIIIa activated Factor XIII
  • This enzyme-mediated annealing process allowed incorporation of living cells 106 into a dynamically forming porous scaffold 10 that contained interconnected microporous networks.
  • a slurry of the microgel particles 12 can be delivered via syringe application ( FIG. 13A ), ultimately solidifying in the shape of the cavity in which they are injected as seen in FIGS. 13B-E .
  • Microfluidic fabrication of the microgel particles 12 enables deterministic control over the microgel size and production frequency as illustrated in FIG. 14A .
  • the pressure that is applied to the inlets of the microfluidic system 20 determines the frequency of microgel production ( FIG. 14B ).
  • porous microgel scaffolds 10 created using different size microgel particles 12 have distinct porous characteristics, such as the median pore size within the network as seen in FIG. 14C .

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Materials Engineering (AREA)
  • Dermatology (AREA)
  • Transplantation (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Dispersion Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Preparation (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Processes Of Treating Macromolecular Substances (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Media Introduction/Drainage Providing Device (AREA)
US15/179,151 2014-07-17 2016-06-10 Controllable self-annealing microgel particles for biomedical applications Abandoned US20160279283A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
US15/179,151 US20160279283A1 (en) 2014-07-17 2016-06-10 Controllable self-annealing microgel particles for biomedical applications
US15/701,113 US10912860B2 (en) 2014-07-17 2017-09-11 Controllable self-annealing microgel particles for biomedical applications
US15/829,440 US20180078671A1 (en) 2014-07-17 2017-12-01 Controllable self-annealing microgel particles for biomedical applications
US16/264,466 US20190151497A1 (en) 2014-07-17 2019-01-31 Controllable self-annealing microgel particles for biomedical applications
US16/596,312 US20200085859A1 (en) 2014-07-17 2019-10-08 Therapeutic polymer gel system to promote healing and prevent fibrosis at a wound or surgical site
US17/144,158 US11464886B2 (en) 2014-07-17 2021-01-08 Controllable self-annealing microgel particles for biomedical applications
US17/935,096 US20230190995A1 (en) 2014-07-17 2022-09-24 Controllable self-annealing microgel particles for biomedical applications

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US201462025844P 2014-07-17 2014-07-17
US201462059463P 2014-10-03 2014-10-03
US201562103002P 2015-01-13 2015-01-13
PCT/US2015/040962 WO2016011387A1 (en) 2014-07-17 2015-07-17 Controllable self-annealing microgel particles for biomedical applications
US15/179,151 US20160279283A1 (en) 2014-07-17 2016-06-10 Controllable self-annealing microgel particles for biomedical applications

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/040962 Continuation WO2016011387A1 (en) 2014-07-17 2015-07-17 Controllable self-annealing microgel particles for biomedical applications

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/701,113 Division US10912860B2 (en) 2014-07-17 2017-09-11 Controllable self-annealing microgel particles for biomedical applications

Publications (1)

Publication Number Publication Date
US20160279283A1 true US20160279283A1 (en) 2016-09-29

Family

ID=55079098

Family Applications (7)

Application Number Title Priority Date Filing Date
US15/179,151 Abandoned US20160279283A1 (en) 2014-07-17 2016-06-10 Controllable self-annealing microgel particles for biomedical applications
US15/701,113 Active US10912860B2 (en) 2014-07-17 2017-09-11 Controllable self-annealing microgel particles for biomedical applications
US15/829,440 Abandoned US20180078671A1 (en) 2014-07-17 2017-12-01 Controllable self-annealing microgel particles for biomedical applications
US16/264,466 Abandoned US20190151497A1 (en) 2014-07-17 2019-01-31 Controllable self-annealing microgel particles for biomedical applications
US16/596,312 Abandoned US20200085859A1 (en) 2014-07-17 2019-10-08 Therapeutic polymer gel system to promote healing and prevent fibrosis at a wound or surgical site
US17/144,158 Active 2035-10-31 US11464886B2 (en) 2014-07-17 2021-01-08 Controllable self-annealing microgel particles for biomedical applications
US17/935,096 Abandoned US20230190995A1 (en) 2014-07-17 2022-09-24 Controllable self-annealing microgel particles for biomedical applications

Family Applications After (6)

Application Number Title Priority Date Filing Date
US15/701,113 Active US10912860B2 (en) 2014-07-17 2017-09-11 Controllable self-annealing microgel particles for biomedical applications
US15/829,440 Abandoned US20180078671A1 (en) 2014-07-17 2017-12-01 Controllable self-annealing microgel particles for biomedical applications
US16/264,466 Abandoned US20190151497A1 (en) 2014-07-17 2019-01-31 Controllable self-annealing microgel particles for biomedical applications
US16/596,312 Abandoned US20200085859A1 (en) 2014-07-17 2019-10-08 Therapeutic polymer gel system to promote healing and prevent fibrosis at a wound or surgical site
US17/144,158 Active 2035-10-31 US11464886B2 (en) 2014-07-17 2021-01-08 Controllable self-annealing microgel particles for biomedical applications
US17/935,096 Abandoned US20230190995A1 (en) 2014-07-17 2022-09-24 Controllable self-annealing microgel particles for biomedical applications

Country Status (10)

Country Link
US (7) US20160279283A1 (zh)
EP (1) EP3169372A4 (zh)
JP (3) JP6651500B2 (zh)
KR (4) KR102614915B1 (zh)
CN (2) CN106714854B (zh)
AU (1) AU2015289474B2 (zh)
BR (1) BR112017000813B1 (zh)
CA (1) CA2955357A1 (zh)
IL (2) IL282559B (zh)
WO (1) WO2016011387A1 (zh)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9803085B2 (en) 2008-09-24 2017-10-31 Wright Asphalt Products Company System and method for high throughput preparation of rubber-modified asphalt cement
WO2018136205A1 (en) 2016-12-29 2018-07-26 Tempo Therapeutics, Inc. Methods and systems for treating a site of a medical implant
US10233120B2 (en) 2008-04-30 2019-03-19 Wright Advanced Asphalt Systems System and method for pre-treatment of rubber-modified asphalt cement, and emulsions thereof
US20200085859A1 (en) * 2014-07-17 2020-03-19 The Regents Of The University Of California Therapeutic polymer gel system to promote healing and prevent fibrosis at a wound or surgical site
WO2021174008A1 (en) * 2020-02-28 2021-09-02 University Of Florida Research Foundation Compositions, methods, kits, and systems relating to charge-neutral microgels for 3d cell culture and printing
US11129790B2 (en) 2017-05-19 2021-09-28 Northeastern University Chemo-enzymatic site-specific modification of peptides and proteins to form cleavable conjugates
US11931480B2 (en) 2016-02-16 2024-03-19 The Regents Of The University Of California Microporous annealed particle gels and methods of use

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201514788D0 (en) * 2015-08-20 2015-10-07 Ecole Polytech Malleable scaffold material and uses thereof
US20210196863A1 (en) * 2016-02-02 2021-07-01 The Regents Of The University Of California Hydrogel for endogenous neuroprogenitor cell recruitment
EP3439697B1 (en) * 2016-04-08 2024-06-05 The Regents of the University of California Hydrogel for engineered immune response to d-chirality peptides
CA3030558A1 (en) * 2016-07-12 2018-01-18 Northeastern University Single cell fluorescence in situ hybridization in microfluidic droplets
JP6345736B2 (ja) * 2016-07-15 2018-06-20 国立研究開発法人科学技術振興機構 液滴安定化装置、液滴分取装置及びそれらの方法
US11131673B2 (en) 2017-04-27 2021-09-28 Northeastern University Live single-cell bioassay in microdroplets
US11926091B2 (en) 2018-03-27 2024-03-12 UNITED STATES OF AMERICA has certain rights in the invention from DOE Grant No. DE-SC0008581 In situ partially degradable separation interface for fabrication of complex near net shape objects by pressure assisted sintering
EP3866867A4 (en) * 2018-10-18 2022-07-13 The Regents of the University of California PROCESS FOR THE PREPARATION OF MODULAR HYDROGELS FROM MACROMOLECULES WITH ORTHOGONAL PHYSICOCHEMICAL REACTION
WO2021113812A1 (en) * 2019-12-06 2021-06-10 University Of Virginia Patent Foundation Injectable micro-annealed porous scaffold for articular cartilage regeneration
JP2021171180A (ja) * 2020-04-21 2021-11-01 株式会社ユニバーサルエンターテインメント 遊技機
US20230293438A1 (en) * 2020-07-16 2023-09-21 The Regents Of The University Of California Injectable drug-releasing microporous annealed particle scaffolds for treating myocardial infarction
US11879118B2 (en) * 2020-11-10 2024-01-23 C.C. Imex Gel tray for bacteria transformation lab
AU2022283426A1 (en) * 2021-05-27 2023-12-07 The Regents Of The University Of California Superporous gel matrix for encapsulation of cells
KR20230059380A (ko) * 2021-10-26 2023-05-03 강원대학교산학협력단 피부 복원용 3차원 스캐폴드
WO2023192339A1 (en) * 2022-03-29 2023-10-05 The Penn State Research Foundation Granular hydrogel bioinks with preserved interconnected porosity and methods for making and using the same
WO2024015656A1 (en) * 2022-07-11 2024-01-18 The Regents Of The University Of California Cell-microgel encapsulation in injectable formulations
WO2024030599A1 (en) * 2022-08-04 2024-02-08 Duke University Technologies for scaffold and void space analysis of granular particle scaffolds
WO2024036108A1 (en) * 2022-08-09 2024-02-15 Bionaut Labs Ltd. Devices, systems, and methods for the treatment of malignant neoplasm disorders using controlled release devices

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080193536A1 (en) * 2006-08-14 2008-08-14 Alireza Khademhosseini Cell-Laden Hydrogels

Family Cites Families (80)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4726877A (en) * 1986-01-22 1988-02-23 E. I. Du Pont De Nemours And Company Methods of using photosensitive compositions containing microgels
US4753865A (en) 1986-01-22 1988-06-28 E. I. Du Pont De Nemours And Company Photosensitive compositions containing microgels
US5213580A (en) 1988-08-24 1993-05-25 Endoluminal Therapeutics, Inc. Biodegradable polymeric endoluminal sealing process
US5843156A (en) 1988-08-24 1998-12-01 Endoluminal Therapeutics, Inc. Local polymeric gel cellular therapy
US5575815A (en) 1988-08-24 1996-11-19 Endoluminal Therapeutics, Inc. Local polymeric gel therapy
US5124188A (en) 1990-04-02 1992-06-23 The Procter & Gamble Company Porous, absorbent, polymeric macrostructures and methods of making the same
EP0627911B1 (en) 1992-02-28 2000-10-25 Board Of Regents The University Of Texas System Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers
JP3156955B2 (ja) * 1995-07-12 2001-04-16 大日精化工業株式会社 連結されたミクロゲル粒子の製造方法及びそれで処理された物品
US6066325A (en) * 1996-08-27 2000-05-23 Fusion Medical Technologies, Inc. Fragmented polymeric compositions and methods for their use
US8603511B2 (en) 1996-08-27 2013-12-10 Baxter International, Inc. Fragmented polymeric compositions and methods for their use
US6063061A (en) * 1996-08-27 2000-05-16 Fusion Medical Technologies, Inc. Fragmented polymeric compositions and methods for their use
US20020064546A1 (en) 1996-09-13 2002-05-30 J. Milton Harris Degradable poly(ethylene glycol) hydrogels with controlled half-life and precursors therefor
US5854382A (en) * 1997-08-18 1998-12-29 Meadox Medicals, Inc. Bioresorbable compositions for implantable prostheses
US6316522B1 (en) 1997-08-18 2001-11-13 Scimed Life Systems, Inc. Bioresorbable hydrogel compositions for implantable prostheses
US6007833A (en) 1998-03-19 1999-12-28 Surmodics, Inc. Crosslinkable macromers bearing initiator groups
US6410044B1 (en) 1998-03-19 2002-06-25 Surmodics, Inc. Crosslinkable macromers
US7547445B2 (en) 1998-03-19 2009-06-16 Surmodics, Inc. Crosslinkable macromers
US6152943A (en) 1998-08-14 2000-11-28 Incept Llc Methods and apparatus for intraluminal deposition of hydrogels
ES2435847T3 (es) 1998-10-23 2013-12-23 Polyheal Ltd. Composiciones de microesferas para curar heridas
US7615373B2 (en) * 1999-02-25 2009-11-10 Virginia Commonwealth University Intellectual Property Foundation Electroprocessed collagen and tissue engineering
DK2093245T3 (da) 1999-08-27 2012-06-04 Angiodevice Internat Gmbh Biocompatible polymer device
ES2312495T3 (es) 2000-11-17 2009-03-01 Virginia Commonwealth University Intellectual Property Foundation Colageno electroprocesado.
US6878384B2 (en) 2001-03-13 2005-04-12 Microvention, Inc. Hydrogels that undergo volumetric expansion in response to changes in their environment and their methods of manufacture and use
US6669827B2 (en) 2001-06-18 2003-12-30 Austin & Neff, L.L.C. Systems and methods for affecting the ultra-fast photodissociation of water molecules
GB0115320D0 (en) * 2001-06-22 2001-08-15 Univ Nottingham Matrix
US20040258731A1 (en) 2001-11-21 2004-12-23 Tsuyoshi Shimoboji Preparation approriate for cartilage tissue formation
US7745532B2 (en) 2002-08-02 2010-06-29 Cambridge Polymer Group, Inc. Systems and methods for controlling and forming polymer gels
US20040147016A1 (en) 2002-09-30 2004-07-29 Rowley Jonathan A. Programmable scaffold and methods for making and using the same
US8574204B2 (en) 2002-10-21 2013-11-05 Angiodynamics, Inc. Implantable medical device for improved placement and adherence in the body
CA2517204A1 (en) * 2003-03-13 2004-09-23 Eugenia Kumacheva Method of producing hybrid polymer-inorganic materials
EP1652019A2 (en) 2003-06-30 2006-05-03 Nupower Semiconductor, Inc. Programmable calibration circuit for power supply current sensing and droop loss compensation
WO2005032418A2 (en) 2003-10-01 2005-04-14 University Of Washington Novel porous biomaterials
US20050119762A1 (en) 2003-11-03 2005-06-02 Peter Zilla Hydrogel providing cell-specific ingrowth
AU2005318097A1 (en) * 2004-12-22 2006-06-29 Kuros Biosurgery Ag Michael-type addition reaction functionalised peg hydrogels with factor XIIIA incorporated biofactors
WO2007044669A2 (en) 2005-10-07 2007-04-19 Lonza Walkersville, Inc. Engineered biological matrices
GB2431104A (en) 2005-10-10 2007-04-18 Univ Greenwich Microgel particles grafted to a substrate
US9132208B2 (en) * 2008-08-07 2015-09-15 Lifenet Health Composition for a tissue repair implant and methods of making the same
US8029575B2 (en) 2005-10-25 2011-10-04 Globus Medical, Inc. Porous and nonporous materials for tissue grafting and repair
EP2347774B1 (en) * 2005-12-13 2017-07-26 The President and Fellows of Harvard College Scaffolds for cell transplantation
EP1808187A1 (en) * 2006-01-11 2007-07-18 Straumann Holding AG Cell selective implant surface with controlled release of bioactive agents
CN101410098B (zh) 2006-01-23 2012-01-18 耶路撒冷希伯来大学伊森姆研究发展公司 包括含亲脂性药物的纳米胶囊的微球
US20070212385A1 (en) 2006-03-13 2007-09-13 David Nathaniel E Fluidic Tissue Augmentation Compositions and Methods
DE102006040772A1 (de) 2006-08-31 2008-03-20 Kist-Europe Forschungsgesellschaft Mbh Polymermatrix, Verfahren zu deren Herstellung sowie deren Verwendung
GB0701896D0 (en) * 2007-02-01 2007-03-14 Regentec Ltd Composition
US8277832B2 (en) * 2007-10-10 2012-10-02 The University Of Kansas Microsphere-based materials with predefined 3D spatial and temporal control of biomaterials, porosity and/or bioactive signals
US20090294049A1 (en) 2008-06-02 2009-12-03 Medtronic Vascular, Inc. Biodegradable Adhesive Hydrogels
JP6363320B2 (ja) 2008-06-16 2018-07-25 ファイザー・インク 薬剤を装填したポリマーナノ粒子及びその製造方法と使用方法
US8557288B2 (en) 2008-08-15 2013-10-15 Washington University Hydrogel microparticle formation in aqueous solvent for biomedical applications
US20120015440A1 (en) 2008-09-08 2012-01-19 Tokyo University Of Science Educational Foundation Administrative Org. Spheroid composite, spheroid-containing hydrogel and processes for production of same
US11090387B2 (en) 2008-12-22 2021-08-17 The Trustees Of The University Of Pennsylvania Hydrolytically degradable polysaccharide hydrogels
WO2011012715A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Biodegradable polyethylene glycol based water-insoluble hydrogels
CA2781518C (en) 2009-11-25 2016-08-23 Healionics Corporation Granules of porous biocompatible materials
GB201002862D0 (en) * 2010-02-19 2010-04-07 Univ Manchester Microgel compositions
EP2555810B1 (en) 2010-04-08 2018-08-22 Healionics Corporation Implantable medical devices having microporous surface layers and method for reducing foreign body response to the same
US20110256628A1 (en) 2010-04-20 2011-10-20 The University Of Washington Through Its Center For Commercialization Adaptive tissue engineering scaffold
US8524215B2 (en) 2010-08-02 2013-09-03 Janssen Biotech, Inc. Absorbable PEG-based hydrogels
EP2624873B1 (en) * 2010-10-06 2019-12-04 President and Fellows of Harvard College Injectable, pore-forming hydrogels for materials-based cell therapies
US9522344B2 (en) 2010-11-18 2016-12-20 The Regents Of The University Of California Method and device for high-throughput solution exchange for cell and particle suspension
US20130233420A1 (en) 2010-11-18 2013-09-12 The Regents Of The University Of California Particle focusing systems and methods
US9234171B2 (en) 2010-12-08 2016-01-12 Rutgers, The State University Of New Jersey Stem cell differentiation using novel light-responsive hydrogels
US20120202263A1 (en) 2011-02-03 2012-08-09 The Trustees Of The University Of Pennsylvania Bioactive Macromers and Hydrogels and Methods for Producing Same
WO2012155110A1 (en) * 2011-05-11 2012-11-15 Massachusetts Institute Of Technology Microgels and microtissues for use in tissue engineering
US20130143056A1 (en) 2011-06-08 2013-06-06 Surmodics, Inc. Photo-vinyl linking agents
WO2013033717A1 (en) 2011-09-02 2013-03-07 The Regents Of The University Of California Enzyme responsive nanocapsules for protein delivery
WO2013049404A2 (en) 2011-09-30 2013-04-04 The Regents Of The University Of California Devices and methods for programming fluid flow using sequenced microstructures
US20150104427A1 (en) 2011-11-10 2015-04-16 The Regents Of The University Of California Enzyme-assisted spatial decoration of biomaterials
KR101820792B1 (ko) * 2011-12-20 2018-01-23 한온시스템 주식회사 차량 공조장치용 컨트롤러
US9974886B2 (en) 2012-08-08 2018-05-22 Nanyang Technological University Methods of manufacturing hydrogel microparticles having living cells, and compositions for manufacturing a scaffold for tissue engineering
WO2014039245A1 (en) 2012-09-07 2014-03-13 The Regents Of The University Of California Method of creating hydrogels through oxime bond formation
US9364543B2 (en) 2012-10-24 2016-06-14 Indiana University Research And Technology Corporation Visible light curable hydrogels and methods for using
EP2801613A1 (en) 2013-05-08 2014-11-12 Ecole Polytechnique Fédérale de Lausanne (EPFL) Arrays of discrete cell culture microenvironments, methods of making such arrays and uses thereof
US9381217B2 (en) 2013-09-09 2016-07-05 Georgia Tech Research Corporation Microgels for encapsulation of cells and other biologic agents
US20150290362A1 (en) 2014-04-10 2015-10-15 Georgia Tech Research Corporation Hybrid fibrin-microgel constructs for tissue repair and regeneration
CN106714854B (zh) 2014-07-17 2020-09-04 加利福尼亚大学董事会 用于生物医学应用的可控的自退火微凝胶颗粒
WO2016096054A1 (en) 2014-12-19 2016-06-23 Ecole Polytechnique Federale De Lausanne (Epfl) Method and device for mixing two streams of droplets
US20160303281A1 (en) 2015-04-17 2016-10-20 Rochal Industries, Llc Composition and kits for pseudoplastic microgel matrices
US20200305773A1 (en) 2016-01-21 2020-10-01 The Regents Of The University Of California Device and method for analyte sensing with microporous annealed particle gels
US20210196863A1 (en) 2016-02-02 2021-07-01 The Regents Of The University Of California Hydrogel for endogenous neuroprogenitor cell recruitment
WO2017142879A1 (en) 2016-02-16 2017-08-24 The Regents Of The University Of California Methods for immune system modulation with microporous annealed particle gels
CN110446508A (zh) 2016-12-29 2019-11-12 泰普治疗公司 用于治疗医疗植入物部位的方法和系统

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080193536A1 (en) * 2006-08-14 2008-08-14 Alireza Khademhosseini Cell-Laden Hydrogels

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Du et al., Directed assembly of cell-laden microgels for fabrication of 3D tissue constructs, PNAS, vol. 105(28):9522-9527 (July 15, 2008) *
Ergenc et al., Recent Advances in the Modeling of PEG Hydrogel Membranes for Biomedical Applications, Biomedical Applications, Biomedical Engineering, Trends in Materials Science, Mr Anthony Laskovski (Ed.), ISBN: 978-953-307-513-6, Chapter 14, pages 307-346 (2011) *
Jia et. al., Hyaluronic Acid-Based Microgels and Microgel Networks for Vocal Fold Regeneration, Biomacromolecules, 2006, 7 (12), pp 3336–3344) *
Park et al., Bovine Primary Chondrocyte Culture in Synthetic Matrix Metalloproteinase-Sensitive Poly(ethylene glycol)-Based Hydrogels as a Scaffold for Cartilage Repair, Tissue Engineering, vol. 10(3/4/):515-522 (2004) *
Sala et al., Microstructured Polymer Films and Matrices for Tissue Engineering, Poster Session, University of Zurich, Institute for Biomedical Engineering, 1 page with 2 pages of citation info, 2007, also available at http://www.lbb.ethz.ch/Publications/Posters/CCMXposter.pdf (last visited 12/21/2016) *
Thorne et al., Microgel applications and commercial considerations, Colloid. Polym. Sci., vol. 289:625-646 (2011) *
Turturro et al., MMP-Sensitive PEG Diacrylate Hydrogels with Spatial Variations in Matrix Properties Stimulate Directional Vascular Sprout Formation, PLoS ONE 8(3): e58897. doi:10.1371/journal.pone.0058897, pages 1-14 (Mar. 12, 2013) *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10233120B2 (en) 2008-04-30 2019-03-19 Wright Advanced Asphalt Systems System and method for pre-treatment of rubber-modified asphalt cement, and emulsions thereof
US9803085B2 (en) 2008-09-24 2017-10-31 Wright Asphalt Products Company System and method for high throughput preparation of rubber-modified asphalt cement
US10093804B2 (en) 2008-09-24 2018-10-09 Wright Asphalt Products Company System and method for high throughput preparation of rubber-modified asphalt cement
US11464886B2 (en) 2014-07-17 2022-10-11 The Regents Of The University Of California Controllable self-annealing microgel particles for biomedical applications
US10912860B2 (en) 2014-07-17 2021-02-09 The Regents Of The University Of California Controllable self-annealing microgel particles for biomedical applications
US20200085859A1 (en) * 2014-07-17 2020-03-19 The Regents Of The University Of California Therapeutic polymer gel system to promote healing and prevent fibrosis at a wound or surgical site
US11931481B2 (en) 2016-02-16 2024-03-19 The Regents Of The University Of California Microporous annealed particle gel system
US11931480B2 (en) 2016-02-16 2024-03-19 The Regents Of The University Of California Microporous annealed particle gels and methods of use
US10668185B2 (en) * 2016-12-29 2020-06-02 Tempo Therapeutics, Inc. Methods of manufacturing injectable microgel scaffolds
US20200222593A1 (en) * 2016-12-29 2020-07-16 Tempo Therapeutics, Inc. Systems to promote healing at a site of a medical device
EP3562523A4 (en) * 2016-12-29 2020-09-30 Tempo Therapeutics, Inc. PROCESSES AND SYSTEMS FOR THE TREATMENT OF A MEDICAL IMPLANT SITE
US10576185B2 (en) * 2016-12-29 2020-03-03 Tempo Therapeutics, Inc. Systems to promote healing at a site of a medical device
AU2017394923B2 (en) * 2016-12-29 2022-07-14 Tempo Therapeutics, Inc. Methods and systems for treating a site of a medical implant
CN110446508A (zh) * 2016-12-29 2019-11-12 泰普治疗公司 用于治疗医疗植入物部位的方法和系统
US20190321797A1 (en) * 2016-12-29 2019-10-24 Tempo Therapeutics, Inc. Methods of manufacturing injectable microgel scaffolds
WO2018136205A1 (en) 2016-12-29 2018-07-26 Tempo Therapeutics, Inc. Methods and systems for treating a site of a medical implant
US11129790B2 (en) 2017-05-19 2021-09-28 Northeastern University Chemo-enzymatic site-specific modification of peptides and proteins to form cleavable conjugates
WO2021174008A1 (en) * 2020-02-28 2021-09-02 University Of Florida Research Foundation Compositions, methods, kits, and systems relating to charge-neutral microgels for 3d cell culture and printing

Also Published As

Publication number Publication date
CA2955357A1 (en) 2016-01-21
IL250092B (en) 2021-05-31
KR20220104071A (ko) 2022-07-25
IL282559A (en) 2021-06-30
AU2015289474A1 (en) 2017-02-02
US11464886B2 (en) 2022-10-11
CN106714854A (zh) 2017-05-24
JP2017522113A (ja) 2017-08-10
BR112017000813B1 (pt) 2021-03-16
JP7188779B2 (ja) 2022-12-13
US20230190995A1 (en) 2023-06-22
AU2015289474B2 (en) 2019-12-05
US20200085859A1 (en) 2020-03-19
US20180078671A1 (en) 2018-03-22
CN106714854B (zh) 2020-09-04
BR112017000813A2 (pt) 2017-12-05
JP2020075150A (ja) 2020-05-21
CN111939316A (zh) 2020-11-17
IL282559B (en) 2022-07-01
KR20170031741A (ko) 2017-03-21
CN111939316B (zh) 2022-08-05
KR102614915B1 (ko) 2023-12-19
KR102264607B1 (ko) 2021-06-14
JP2022177012A (ja) 2022-11-30
JP6651500B2 (ja) 2020-02-19
WO2016011387A1 (en) 2016-01-21
US10912860B2 (en) 2021-02-09
KR102421923B1 (ko) 2022-07-18
EP3169372A4 (en) 2018-03-21
EP3169372A1 (en) 2017-05-24
US20190151497A1 (en) 2019-05-23
KR20210072133A (ko) 2021-06-16
US20170368224A1 (en) 2017-12-28
US20210138105A1 (en) 2021-05-13
IL250092A0 (en) 2017-03-30
KR20230173741A (ko) 2023-12-27

Similar Documents

Publication Publication Date Title
US11464886B2 (en) Controllable self-annealing microgel particles for biomedical applications
Van Vlierberghe et al. Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review
Babu et al. Controlling structure with injectable biomaterials to better mimic tissue heterogeneity and anisotropy
Meco et al. Impact of elastin-like protein temperature transition on PEG-ELP hybrid hydrogel properties
US20190060522A1 (en) Natural Polymer-Derived Scaffold Material and Methods for Production Thereof
Wang et al. Minimally invasive co-injection of modular micro-muscular and micro-vascular tissues improves in situ skeletal muscle regeneration
Sanz-Horta et al. Technological advances in fibrin for tissue engineering
Werner et al. Modulating extracellular matrix at interfaces of polymeric materials
Veernala et al. Cell encapsulated and microenvironment modulating microbeads containing alginate hydrogel system for bone tissue engineering
US20230293438A1 (en) Injectable drug-releasing microporous annealed particle scaffolds for treating myocardial infarction
US10849988B2 (en) Hydrogel for engineered immune response to D-chirality peptides
Vedaraman 3D artificial extracellular matrices for directed in vitro cell growth
Tan Delivery of highly proliferative co-cultured skin cells in 3D GelMA core-shell microspheres: in vitro studies
Kyburz Bio-Functionalized PEG Hydrogels to Study and Direct Mesenchymal Stem Cell Migration and Differentiation
Ndreu-Halili Cell/Tissue Microenvironment Engineering and Monitoring in Tissue Engineering, Regenerative Medicine, and In Vitro Tissue Models

Legal Events

Date Code Title Description
AS Assignment

Owner name: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, CALIF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GRIFFIN, DONALD R.;WEAVER, WESTBROOK;SEGURA, TATIANA;AND OTHERS;SIGNING DATES FROM 20160610 TO 20160622;REEL/FRAME:039296/0280

AS Assignment

Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:UNIVERSITY OF CALIFORNIA LOS ANGELES;REEL/FRAME:041011/0608

Effective date: 20161213

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION