microporous gel systems, uses of a plurality of microgel particles in an aqueous solution and an ann

专利摘要:
A microporous gel system for certain applications, including biomedical applications, includes an aqueous solution containing a plurality of microgel particles including a biodegradable crosslinking agent. In some respects, the microgel particles act as gel building blocks that anneal with each other to form a covalently stabilized scavenger of microgel particles having interstitial spaces within them. In certain aspects, the annealing of the microgel particles occurs after exposure to an annealing agent that is endogenously present or is exogenously added. In some embodiments, the annealing of the microgel particles requires the presence of an initiator such as exposure to light. In particular embodiments, the chemical and physical properties of the gel building blocks can be controlled to allow control downstream of the resulting assembled sequester. In one or more modalities, the cells are able to quickly infiltrate the interstitial spaces of the mounted sequester.
公开号:BR112017000813B1
申请号:R112017000813-0
申请日:2015-07-17
公开日:2021-03-16
发明作者:Donald R. Griffin；Westbrook Weaver；Tatiana Segura；Dino Di Carlo；Philip Scumpia
申请人:The Regents Of The University Of California；
IPC主号:

专利说明:

Related applications
[001] This application claims priority to U.S. Provisional Patent Application Nos. 62 / 025,844 deposited on July 17, 2014, 62 / 059,463 deposited on October 3, 2014, and 62 / 103,002 deposited on January 13, 2015. Priority is claimed under 35 USC § 119. Requests for above-mentioned patents are hereby incorporated by reference as if presented completely here. Technical Field
[002] The technical field generally refers to the field of wound treatment, and in particular, the use of microgel particles and supports, including particles for the treatment and sealing of wounds and applications of tissue fillers. Background
[003] A central concept linked to the generation and regeneration of tissues is the collective migration of cells, a process by which entire networks of cells move together to a development area to facilitate the formation of functional tissue. Researchers have sought to develop wound healing agents; however, these materials exhibit batch to batch variability and degradation rates that limit extended structural support for growing tissues. Synthetic materials are more tunable than natural materials and their mechanical properties have been modified to allow use with a wide range of fabric types. Despite this harmony, however, synthetic injectable biomaterials have been limited to non-porous or nanoporous supports that require physical degradation for cell migration through the material. Porous synthetic hydrogels that contain pre-formed microscale interconnected pores allow greater cell mobility without the need for degradation, bypassing the balance between cell mobility and material stability inherent in non-porous supports. The typical way of pore formation includes the removal of toxic porogens, or the degradation of encapsulated microparticles, which implies that these constructs are fused ex vivo, preventing them from integrating perfectly with the surrounding tissue as an injectable bio-material. or requiring long-term in vivo development to resolve the porous structure. For example, Healionics Corporation has developed a self-describing technology such as Spherical Model Anigenogenic Regeneration (STAR) in which STAR supports are formed by sintering together a matrix of packaged granules of controlled size, molding a polymer within the interstitial space between the granules , and the granules dissolving to obtain a network of interconnected spherical voids. As noted above, however, these conventional processes require the removal of toxic porogens. summary
[004] Human skin wounds are an increasing threat to public health and the economy and are very difficult to treat. Doctors, when treating skin wounds, try to keep the area moist because dry wounds heal much more slowly than wet wounds. To achieve this, doctors often use ointments to fill the wound, as well as fill a hole with new padding. However, these and other conventional methods for wound healing do not provide an ideal support to allow new tissue to grow. As a result, the growth of new tissue, if any, is relatively slow and fragile which leads to longer cure times, as timely healing is still possible.
[005] In the context of curing tissue engineering, the present inventors have identified the gold standard for the development of interconnected microporous supports that allow interconnected cellular networks and collective migration, without the need for support degradation or invasive implant procedures, quantity integration with the surrounding tissue is essential. In fact, to be more effective, the present inventors have identified that these materials must facilitate collective cell migration that mediates regeneration, providing molecular signals to promote wound healing and niche recognition. In addition, the present inventors have also identified that these materials must be able to be easily replaced by migration cells and natural matrix, providing a stable structural support before replacement, and being easily delivered and conforming to the injury site to minimize fibrotic and inflammatory responses.
[006] Provided here are systems, compositions, methods and devices that apply these principles and provide a biomaterial that promotes rapid tissue regeneration, maintaining the structural support of the tissue surrounding a wound. In fact, the present inventors have achieved solutions to unmet and long-acting medical needs in the field of tissue engineering using microgel-based, injectable or fluid-sized material chemistry and microfluid manufacturing of uniform spherical building blocks -mes, including, for example, building blocks the thickness of a human hair.
[007] The technology described here uses chemistry to generate small microgels that can be assembled in a large unit, leaving behind a path for cell infiltration. The result is a packaged set of microscopic synthetic polymer organisms (for example, spheres) stuck to their surfaces, similar to a bottle of the gum balls that are stuck together. The set creates a support of annealed microporous particles (for example, a porous gel support) that fills the wound. New tissue grows rapidly in the voids between the microgel particles, and as the microgel particles degrade in the body, a recently grown tissue matrix is left, where it once was. New tissue continues to grow until the wound is completely healed.
[008] The microgel systems described here represent a substantial improvement over conventional products. For example, the technologies described here do not require added growth factors to attract cells into the material. The geometry of the microgel networks described attracts cells to migrate to the microgel.
[009] The present inventors have demonstrated that the described microgels can promote the growth of new cells and the formation of networks of cells linked at previously invisible rates. For example, during in vivo studies, significant tissue regeneration was seen in the first 48 hours, with much more healing over five days compared to conventional materials in use today.
[0010] The technologies described here are useful for a wide variety of applications. For example, the disseminated microgel technology can be used for wound applications, including acute damage, such as surgical lacerations and wound closure, and also more chronic applications such as diabetic ulcers and large burn area wounds. The hydrogel holders described here can also be useful in trauma situations, such as battlefields or emergency rooms.
[0011] Described here, in certain respects, are systems, compositions, methods and devices comprising a microporous gel comprising an aqueous solution comprising a plurality of microgel particles and a crosslinker, including, for example , a biodegradable crosslinker. Microporous gels described herein are flowable and / or injectable and can be applied in a number of different ways, including, for example, topically or by injection. Flowable and / or injected microporous gels can be inserted transdermally or into deep tissues. Flowable microporous gels can also be administered topically to the dermis and other tissues.
[0012] In one aspect, when an annealing agent is applied to the plurality of microgel particles, the microgel particles form a covalently stabilized support of microgel particles having interstitial spaces in it. In certain applications, systems, compositions, methods and devices are specifically designed for biomedical applications. In some embodiments, the microporous gel particles further comprise a crosslinker, wherein the crosslinker includes a degradable matrix metalloprotease (MMP) crosslinker. In one or more embodiments, an annealing agent comprises Factor XIIa. In other or additional embodiments, the annealing agent comprises Eosin Y, a free radical transfer agent, or a combination thereof.
[0013] In some embodiments, microgel systems, compositions, methods and devices still comprise a light source configured to illuminate a mixture of a plurality of microgel particles and the annealing agent. In one or more embodiments, the microporous gel particles comprise cell-adherent peptides exposed on their surface. In some embodiments, the microporous gel particles comprise a K Peptide. In additional or supplementary embodiments, the microporous gel particles comprise a K peptide comprising a recognized Xylla Factor lysine group. In some embodiments, the microporous gel particles comprise a Q peptide. In some embodiments, the Q peptide comprises a recognized factor XIIIa glutamine group. In certain embodiments, the microporous gel particles comprise a crosslinker that is degradable. In certain embodiments, the microporous gel particles comprise interstitial spaces that comprise edge surfaces that exhibit negative concavity. In one or more embodiments, the covalently stabilized microgel particle support has an empty volume of about 10% to about 50%.
[0014] In one embodiment, a microporous gel system for biomedical applications includes an aqueous solution that contains a plurality of microgel particles formed with a biodegradable crosslinker, such as a degradable metalloprotease matrix (MMP) crosslinker and an annealing agent that , when applied to the plurality of microgel particles causes the microgel particles to form a covalently stabilized support of microgel particles having interstitial spaces in it.
[0015] In another embodiment, a microporous gel system includes a delivery device and a set of biodegradable micro-gel 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.
[0016] In another embodiment, a tissue treatment method includes delivering to the tissue an aqueous-based solution that contains a plurality of microgel particles decorated with cell adhesive peptides, wherein the microgel particles are formed with the a biodegradable crosslinker, such as degradable matrix metalloprotease (MMP) crosslinker. The plurality of mi-crogel particles are exposed to an annealing agent that anneals the microgel particles to form a covalently stabilized support of microgel particles having interstitial spaces therein.
[0017] In another embodiment, a microporous gel system for biomedical applications includes a set of microgel particles formed by a reaction of a main structure polymer having one or more cell-binding portions, one or more annealing components, and a cross-linking component of biodegradable mesh. The microporous gel system includes an endogenous or exogenous annealing agent that bonds the microgel particles together in situ through the annealing components to form a covalently stabilized support of microgel particles having interstitial spaces therein.
[0018] In another aspect, described here, are systems, compositions, methods and devices comprising a delivery device or mechanism and microporous gel. In certain embodiments, the delivery device contains an aqueous solution comprising a plurality of microgel particles and the annealing agent or an annealing agent precursor. In one or more embodiments, the delivery device comprises a single compartment delivery device containing the aqueous solution comprising a plurality of microgel particles and the annealing agent. In one or more embodiments, the delivery device comprises a multiple compartment (e.g., double) delivery device, wherein one compartment contains the aqueous solution containing a plurality of microgel particles and a first precursor of annealing agent and the second compartment contains the aqueous solution containing a plurality of microgel particles and a second precursor of annealing agent. In certain embodiments, microporous gels further comprise a degradable (MMP) crosslinker comprising at least one D amino acid. In additional or supplementary embodiments, the particles comprise a degradable (MMP) reticulating microgel comprising a plurality of D amino acids.
[0019] In yet another aspect, described here is a microporous gel system comprising: a delivery device; a plurality of biodegradable microgel particles contained or an aqueous solution and stored in the delivery device; and an annealing agent or annealing agent precursor stored in the delivery device. In one or more embodiments, the microporous gel particles further comprise a set of biodegradable microgel particles of two or more types, which are contained in an aqueous solution and stored in the delivery device. In certain embodiments, the delivery device comprises two compartments, biodegradable microgel particles are stored in each of the two compartments, and a first re-cooking precursor is stored in one compartment and a second annealing pre-cursor is stored in the other compartment, where the annealing agent is formed by the presence of both the first and second annealing precursors. In one or more embodiments, the delivery device comprises a single compartment and the set of biodegradable microgel particles and the re-baking agent are both stored in the single compartment. In still other or additional embodiments, the annealing agent comprises a photoinitiator and a free radical transfer agent stored in the single compartment. In an additional or supplementary modality, the microporous gel system further comprises a light-emitting device configured to illuminate a mixture of the biodegradable microgel assembly and the annealing agent. In certain embodiments, the microgel particles comprise substantially monodispersed spheres. In one or more embodiments, the substantially monodispersed spheres have a diameter within the range of about 30 micrometers to about 150 micrometers. In additional or supplementary embodiments, the microgel particles are covalently bonded to one another after annealing.
[0020] Provided here in another aspect is a tissue treatment method, 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 support of microgel particles having interstitial spaces therein. In some embodiments, the plurality of microgel particles is decorated with cell adhesive peptides, and wherein the microgel particles are formed with a degradable metalloprotease matrix (MMP) crosslinker. In one or more embodiments, the annealing agent is delivered to the fabric. In some modalities, the annealing agent is present inside the fabric. In still additional modalities, the method still comprises initiating the annealing of the microgel particles with exposure to light. In some modalities, the wavelength of light is in the visible range. In some embodiments, the wavelength of light is in the infrared range. In one or more embodiments, the water-based solution and the annealing agent are delivered simultaneously. In some embodiments, the aqueous-based solution and the annealing agent are delivered sequentially. In still additional or supplementary embodiments, the microgel particles comprise a therapeutically active chemical compound. In certain embodiments, the microgel particles expose or elute the chemical compound to the tissue. In one or more modalities, the tissue comprises a cosmetic reconstruction site, chronic wound development, acute tissue damage, or a tissue opening caused by a surgical incision.
[0021] In another aspect, a microporous gel system or device is provided which comprises: a set of microgel particles comprising a polymer of main structure having one or more cell binding portions, one or more annealing components, and a or more components of biodegradable network crosslinker; and an endogenous or exogenous annealing agent that binds the microgel particles together in situ by means of the annealing components to form a covalently stabilized support of microgel particles having interstitial spaces therein. In certain embodiments, the main structure polymer comprises poly (ethylene glycol) vinyl sulfone. In one or more embodiments, the one or more cell-binding moieties comprise an 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-binding moieties comprise an RGD peptide, or fragment thereof. In one embodiment, the one or more cell-binding moieties comprise SEQ ID NO: 3 or a fragment thereof. In additional or supplementary embodiments, the one or more annealing components comprise a K peptide and a Q peptide. In certain embodiments, the K peptide comprises a recognized Xyla Factor lysine group and the Q peptide comprises a recognized Xllla Factor glutamine group. In some embodiments, the cross-linking component of a biodegradable network comprises a degradable metallopro-tease (MMP) matrix cross-linker. In one or more embodiments, the degradable (MMP) crosslinker comprises amino acid D. In certain embodiments, the set of microgel particles comprises microgel particles of two or more types. In one or more embodiments, microgel particles of the first type comprise degradable (MMP) crosslinker comprising amino acid D, and mi-crogel particles of a second type consist of degradable (MMP) crosslinker comprising only amino acid L. In one or more embodiments, the system or device comprises a single compartment delivery device containing the set of microgel particles and the annealing agent. In one or more embodiments, the system or device further comprises a double compartment delivery device, in which one compartment contains the aqueous solution containing a plurality of microgel particles and a first precursor of annealing agent and the second compartment contains the aqueous solution containing a plurality of microgel particles and a second annealing agent precursor, wherein the annealing agent is formed by the presence of the first and second agent precursors.
[0022] In an additional aspect, a tissue treatment method is described which comprises: delivering to the tissue a first layer of microgel particles decorated with cell adhesive peptides, in which the microgel particles are formed with a biodegradable reticulant ; exposing the first layer of a baking agent that anneals the microgel particles to form a covalently stabilized substrate of microgel particles having interstitial spaces within them; delivering to the tissue a second layer of microgel particles decorated with cell adhesive peptides, in which the microgel particles are formed with a biodegradable crosslinker and in which the microgel particles in the second layer differ in one from a physical property or chemical composition, compared to the microgel particles of the first layer; and exposing the second layer to an annealing agent that anneals the microgel particles to form a covalently stabilized support of microgel particles having interstitial spaces therein. In one or more embodiments, the microgel particles in the second layer are of a different size. In still further embodiments, the microgel particles in the second layer have a different shape. In one or more embodiments, the micro-gel particles in the second layer have a different stiffness. In certain embodiments, chemical components differ from a chemical component in the first layer. In another or additional embodiment, the microgel particles in the second layer have a chemical component of a different concentration than the same chemical component in the first layer.
[0023] In another aspect, a tissue treatment method is provided, comprising: delivering to the tissue a water-based solution containing a plurality of microgel particles decorated with cell adhesive peptides, in which the micro-particle particles gel 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 support of microgel particles having interstitial spaces therein.
[0024] In another embodiment, a tissue treatment method includes delivering to the tissue a first layer of microgel particles decorated with cell adhesive peptides, in which 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 support of microgel particles having interstitial spaces therein. A second layer of microgel particles decorated with cell adhesive peptides is delivered to the tissue, in which the microgel particles are formed with a biodegradable crosslinker and in which the microgel particles in the second layer differ in one from a physical property or composition. compared to the microgel particles in the first layer.
[0025] In another embodiment, a tissue treatment method includes delivering to the tissue a water-based solution that contains a plurality of microgel particles decorated with cell adhesive peptides, in which the microgel particles are formed with a biodegradable crosslinker. The plurality of microgel particles is exposed to an annealing agent that anneals the microgel particles to form a covalently stabilized support of microgel particles having interstitial spaces therein.
[0026] In yet an additional aspect, described is a method of preparing microgel particles which comprises: providing a microfluidic device that generates droplets of water in oil having a plurality of inlet channels leading to a common channel and a pair oil compression channels intersecting with the common channel at a downstream location that flows a first prepolymer solution containing a main structure of the oligopeptide-modified polymer to a first inlet channel; flow a second solution containing a biodegradable crosslinker to a second inlet channel; an oil and a surfactant flow into the pair of oil compression channels to form droplets containing the first solution and the second prepolymer solution; and collect the microgel particles formed by crosslinking the droplets. In another mode, the method also comprises a third input channel interposed between the first input channel and the second input channel, in which a third inert solution containing a prepolymer is drained into the third input channel. Entrance. In one or more modalities, the method also involves plating the droplets generated with an additional pair of plating channels located downstream from a location, where the pair of oil compression channels intersect with the common channel, in which the additional pair of plating channels carries oil and a surfactant in a higher concentration than the surfactant contained in the upstream pair of oil compression channels. In one embodiment, the method also comprises centrifuging the collected microgel particles. In another aspect, the method comprises reducing the free water volume content of the centrifuged microgel particles.
[0027] In yet another embodiment, a method of preparing microgel particles includes providing a microfluidic device for generating water-in-oil droplets that has a plurality of inlet channels leading to a common channel and a pair of oil compression channels intersecting with the common channel at a downstream location. A first prepolymer solution containing an oligopeptide-modified polymer backbone is poured into a first inlet channel. A second solution containing a biodegradable crosslinker is poured into a second inlet channel. An oil and a surfactant are poured into the pair of oil compression channels to form droplets that contain the first solution and the second prepolymer solution. Microgel particles are formed by crosslinking the droplets which are then collected.
[0028] Other objectives, characteristics and advantages of the present invention will become apparent to those skilled in the art from the detailed description that follows. It should be understood, however, that the detailed description and the specific examples, although indicating some modalities of the present description, are given by way of illustration and not by way of limitation. Many variations and modifications within the scope of this disclosure can be made without departing from the spirit of the same, and the disclosure includes all of these modifications. In addition, aspects of one modality can be used in other different modalities. Brief Description of Drawings
[0029] The new features of the invention are presented with particularity in the appended claims. A better understanding of the characteristics and advantages of the present invention will be obtained by reference to the following detailed description which presents illustrative modalities, in which the principles of the invention are used, and the accompanying drawings, of which:
[0030] FIG. 1 illustrates a portion of a support formed from a plurality of annealed microgel particles.
[0031] FIG. 2A illustrates an exemplary process of injecting microgel particles into a wound site for healing.
[0032] FIG. 2B schematically illustrates an exemplary annealing reaction between different microgel particles enhanced by binders on the surface of the microgel particles.
[0033] FIG. 2C illustrates an exemplary process of tissue infiltration into a support formed within a delivery site on the tissue, where the boundary between the tissue and the microgels represents any interface between them, where cells can pass through the moving interface into the fabric or out towards the fabric from the microgels.
[0034] FIG. 3A illustrates a top-down view of a microfluidic device according to an embodiment used to generate a plurality of microgel particles as part of a microporous gel system.
[0035] FIG. 3B illustrates an enlarged view of the droplet generation region and downstream oil / surfactant compression region (see box region in FIG. 3A).
[0036] FIG. 3C illustrates perspective views, enlarged of two branch channels illustrated in FIG. 3A.
[0037] FIG. 3D illustrates a side view of the microfluidic device of FIG. 3A according to an embodiment.
[0038] FIG. 3E illustrates a photograph taken of a reduction for the practice of the scheme illustrated in FIG. 3B, where the fluorescent solution on the left side contains crosslinker, the fluorescent solution on the right contains polymer and reaction buffer, and the medium flow contains an inert liquid solution to avoid mixing the left and right solutions before segmenting the droplet. Bright fluorescence between the right and middle flows illustrates the pH change in the medium flow due to the diffusion of reaction buffer.
[0039] FIG. 3F illustrates a photograph of a reduction for the practice of the scheme illustrated in FIG. 3B and FIG. 3E, at the same time, showing the light microscopic view of the droplet segmentation after the oil compression streams are introduced.
[0040] 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.
[0041] FIG. 4B illustrates that, in the droplet segmentation region, mineral oil with 0.25% Span® 80 compresses and pre-gel PEG segments, and downstream a 5% Span® 80 solution in mixtures of mineral oil and prevents coalescence of microgels downstream before complete gelation.
[0042] FIG. 4C illustrates that droplets do not recombine during incubation in the bifurcation region and leave the microchannel for the collection well.
[0043] FIG. 5 illustrates an exemplary microfluidic T-junction that can be used to generate microgel droplets according to one embodiment.
[0044] FIG. 6A illustrates an exemplary delivery device in the form of a two-tube syringe according to an embodiment.
[0045] FIG. 6B illustrates an exemplary delivery device in the form of a single-barrel syringe according to another embodiment.
[0046] FIG. 6C illustrates an exemplary delivery device in the form of a tube that holds microgel particles according to one embodiment.
[0047] FIG. 7 A illustrates hematoxylin and eosin staining (H&E staining) of tissue sections in SKH1-Hrhr mice for tissues injected with the support (Microporous Annealed Particles or "MAP" support), as well as the non-porous control twenty-four (24) hours after the injection.
[0048] FIG. 7B illustrates a wound closure graph (%) as a function of days post-injection. This graph shows that over a (5) five-day period there is no statistically significant improvement in wound closure rates for the use of supports compared to bilateral non-porous controls (N = 5).
[0049] FIG. 7C illustrates representative images of wound closure in a 5-day in vivo wound healing model in SKH1-Hrhr mice comparing the gel holder (left panels) with a non-porous PEG gel control (right panels).
[0050] FIG. 7D illustrates representative images of wound closure in 7-day in vivo BALB / c experiments. After 7 days in vivo, supports promote significantly faster wound healing than untreated control, gels lacking K and Q peptides, non-porous PEG gel, and faster healing than porous gel precast. Porous gels created ex vivo to precisely match the shape of the wounds using the canonical molding method, based on a porogen agent, showed appreciable wound healing rates, comparable to the supports, but lacking injectability (N> 5).
[0051] FIG. 7E is a bar graph illustrating quantification data for wound closure of BALB / c wound healing in vivo for each treatment category that corresponds to FIG. 7D. All data are presented as mean +/- SEM. Statistical significance performed using standard two-tailed t-test (*: p <0.05; ** p <0.01).
[0052] FIG. 7F illustrates traces of wound bed closure for 7 days in vivo for each treatment category that corresponds to FIG. 7D and FIG. 7E.
[0053] FIG. 7G illustrates that the solution containing microgel particles or slurry can be injected using a syringe device (for example, a 25 gauge syringe) like that of FIG. 6A or 6B at a treatment site where the microgel conforms to the shape of the injection site (for example, in this case an acrylic star-shaped laser cut mold) and subsequent annealing of the star-shaped support.
[0054] FIGS. 8A and 8B illustrate stained microscopic images of damaged tissue (eg, wound site) that was treated with the microgel holder (FIG. 8A) and with no treatment or "sham" (FIG. 8B) in a twenty mouse model and one (21) days after skin excision and gel application. The scar reduction allowed by the gel micro-seeker can be clearly seen in FIG. 8A. Squares indicate the hair follicles and sebaceous glands (sebaceous glands) in the reconstitution tissue after applying the gel to a wound. Circles indicate microgel particles remaining in the reconstitution tissue.
[0055] FIG. 8C illustrates a graph showing the thickness of the epidermis for the tissue treated with the simulacrum as well as the tissue treated with the gel support.
[0056] FIG. 8D illustrates a graph showing the number of sebaceous glands for the tissue treated with the simulacrum as well as the tissue treated with the gel support.
[0057] FIG. 8E illustrates a graph showing the number of hair follicles for the tissue treated with the simulacrum as well as the tissue treated with the gel support.
[0058] FIG. 8F illustrates a graph showing the scar width of the tissue treated with the sham as well as the tissue treated with the gel support.
[0059] FIG. 8G illustrates a graph showing the number of millial cysts for the tissue treated with the simulacrum as well as the tissue treated with the gel support.
[0060] FIG. 9 A illustrates a storage module graph as a function of post-mixing time, during 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 to 25 degrees Celsius, is represented by the upper line in the graph; and pH 8.25 at 37 degrees Cel-sius, is represented by the middle line in the graph.
[0061] FIG. 9B illustrates different percentages by weight of hydrogel that were used to produce different stiffness materials on the x-axis. The illustrated storage module (Pa) graph for various percentages by weight of hydrogel.
[0062] FIG. 9C illustrates different cross-link stoichiometries that were used to produce different values of stiffness in the resulting gel on the x-axis. The graph illustrated the storage module (Pa) as a function of the ratio of free crosslinker ends (-SH) to vinyl groups (-VS) on the PEG molecule.
[0063] FIG. 9D illustrates a graph of% degradation as a function of time, both for the non-porous control (bottom line of the graph), as well as a porous gel described here (top line of the graph).
[0064] FIG. 9E shows SEM images of a substrate annealed with FXIIIa at 200 μm (top panel) or 100 μm (bottom panel).
[0065] FIG. 9F illustrates SEM images of microgel particles without FXIIIa at 200 μm (top panel) or 100 μm (bottom panel). Non-annealed microgel particles are seen in FIG. 9F.
[0066] FIG. 10 shows a microgel manufactured using the described technique, in which the surface of the microgel was increased with a fluorescent bovine serum albumin (BSA) protein (outer perimeter) through the use of phosphine-azide "click" chemistry . In addition, nanoparticles (500 nm) are embedded within the microgel during the manufacture of microfluids.
[0067] FIG. 11 illustrates an exemplary process for treating damaged tissue using the microporous gel system described herein. Microgel particles are applied (upper panel), optionally, an applicator is used (second panel), annealing of microgel particles is initiated to form a support (third panel) and improvement of wound healing is observed (lower panel).
[0068] FIG. 12A illustrates fluorescent images demonstrating the formation of 3D cell networks during six days of culture in porous gel holders in vitro as well as non-porous gels after 6 days. (350 Pa: volume module identical to porous gel supports, 600 Pa: microscale module combined with individual microgels).
[0069] FIG. 12B illustrates a graph where cell survival twenty-four (24) hours after annealing is greater than 93% over three cell lines representing different types of human tissue. HDF: human dermal fibroblasts, AhMSC: human mesenchymal stem cells derived from adipose, BMh-MSC: human mesenchymal stem cells derived from bone marrow.
[0070] FIG. 13 A illustrates an exemplary method for combining living cells with preformed microgel particles before re-cooking. The microgel particles are annealed to each other, trapping living cells within the interconnected microporous network created after microgel annealing.
[0071] FIGS. 13B-D are photographic images that illustrate that solutions of microgel particles combined with living cells are moldable into macroscale shapes, and can be injected to form complex shapes that are maintained after annealing. FIG. 13B illustrates exemplary in vitro syringe injection. FIG. 13C illustrates an exemplary in vitro shaped impression. FIG. 13D illustrates an exemplary in vitro annealed support. FIG. 13E illustrates that mi-crogel particles are moldable in macroscale forms and can be performed in the presence of living cells (indicated by arrows pointing to fluorescent HEK-293T cells).
[0072] FIG. 14A illustrates a graph showing that various microgel particle sizes can be synthesized across a range of production frequencies of an exemplary embodiment.
[0073] FIG. 14B illustrates that providing a high inlet pressure for each solution inlet (where the oil inlets are greater than 30 psi) allows an increase in the frequency of production in another exemplary mode.
[0074] FIG. 14C illustrates a graph showing that high precision fabrication of microgel building blocks allows the creation of defined gel supports. Different building block sizes allow deterministic control over the characteristics of the resulting microporous mesh, presented here as medium pore sizes +/- standard deviation (SD). Detailed Description of the Illustrated Modalities
[0075] In the description of the preferred modality, reference is made to the attached drawings that form a part of it, and in which a specific modality in which the material described here can be practiced is shown by way of illustration. It should be understood that other modalities can be used and structural changes can be made without departing from the scope and spirit of the inventive theme described here. In addition, various aspects of different modalities can be used with other modalities described herein without departing from the scope of the invention.
[0076] In one aspect of the matter described here, a solid microgel support 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 - ment. The annealing reaction, in one aspect of the matter described herein, forms covalent bonds between the adherent microgel particles. For example, in the post-annealed state, the support forms a three-dimensional structure that conforms to the application or delivery site. Because of the imperfect packaging of the micro-gel particles, the annealed support formed from the particles includes interstitial spaces formed therein where cells can migrate, clump together, and grow. The support structure formed is porous under annealing at the wound or other delivery site (unlike the solid non-porous support provided by fibrin-based products). This porosity includes the interstitial spaces mentioned above, as well as nanoscopic pores that can be created or formed in the particles themselves. The microporosity of the support structure allows high diffusivity of nutrients, cell growth and differentiation factors, as well as cell migration, internal growth, and penetration. The microporosity of the support provides accelerated healing or delivery of improved therapeutic drugs or medications over conventional fibrin glue, hyper-branched polymers, or degradable polymers with crosslinking options, because of increased cell migration through spaces interstitials, maintaining the overall integrity of the support. In addition, but not limited to biometric and natural materials, the degradation profile and physical properties (for example, hardness, internal diffusivity, etc.) are improved, for example, by having a wide range available and a wider matrix of therapeutically active biological or chemical signals that can be included within the material (eg, antibiotics, steroids, growth factors, and the like can be loaded onto the support). In addition, the release or elution of drugs, compounds, or other material to release or control biological activity, in certain modalities, can be tuned by modifying the desired biomaterial. The signal compounds or molecules discussed above can be exposed to tissue during the healing process or degradation of the support. Signal compounds or molecules can also be released or eluted in the affected area after the initial placement of the support at the delivery site.
[0077] An advantage of the matter described here in addition to methods such as STAR ™ technology is that the formation of a support occurs in vivo, allowing it to completely fill the desired space and be tuned to connect (chemically or otherwise) to the surrounding tissue. In addition, the pre-delivery formation of the microgel particles allows controlled mechanical tuning of the resulting formed support to match the properties of the surrounding tissue. These capabilities result in better sealing and total integration with the fabric. Greater integration results in a decrease in the possibility of material failure and greater long-term regeneration. This also helps to avoid contamination of the environment. In addition, the microporous nature of the annealed support is beneficial in reducing the response to a foreign body immune to the support.
[0078] FIG. 1 illustrates a portion of the formed three-dimensional support 10 which is formed by a plurality of annealed microgel particles 12. Support 10 includes in it interstitial spaces 14 which are empty spaces that form micropores within the major support 10. Interstitial spaces 14 have dimensions and geometric profiles that allow the infiltration, connection and growth of cells. It should be appreciated that the microporous nature of the support 10 disclosed herein involves a network of interstitial spaces or voids 14 located between the annealed microgel particles 12 that form the larger support structure. In one embodiment, the interstitial spaces or voids 14 created within the support 10 exhibit negative concavity (for example, the interior void is convex). FIG. 1 illustrates an exemplary void 14 with empty walls 16 exhibiting negative concavity. The negative concavity is caused because the microgel particles 12 that are annealed with each other are, in general or substantially spherical in a preferred embodiment. This allows for the packaging of microgel particles 12 which, according to one embodiment, produce a low void fraction between about 10% and about 50% and, in another embodiment, between about 26% to about 36% . While the fraction of void volume is low, the negative concavity exhibited in certain embodiments within the void network 14 provides a relatively high surface area for void volume for cells to interact. For a given volume of cells, which would then, on average, be exposed to even more and larger surfaces (for example, in the void walls 16) to interact with the network of voids in the support 10.
[0079] It is important to note that the void network consists of regions where the microgel surfaces are in close proximity (for example, annealed microgel particles nearby 12) that lead to the adhesive regions of high surface area for the cells to adhere and they quickly migrate, while additional neighboring regions in the spaces between the microgel particles 12 have a larger void space that can allow the growth of cells and tissues in this space. Consequently, the combined adjacency of the tighter empty areas and the broader empty areas is expected to have a beneficial effect on tissue growth and regrowth, compared to either entirely small or all larger voids.
[0080] Note that in the above-described embodiment, the negative concavity results are due to the spherical shape of the microgel 12 particles. In other embodiments, the microgel 12 particles may not be spherical in shape. Other non-spherical shapes can still be used on the support 10. Still referring to FIG. 1, the support 10 is formed by microgel particles 12 which are fixed to each other by means of annealing surfaces 17. As explained in the present document, the annealing surfaces 17 are formed, either during or after the application of the particles microgel 12 at the intended delivery site.
[0081] Support 10 can be used for various applications, including a variety of medical applications, such as military field medicine, medical traumatic treatment, post-surgical closure, burn injuries, inflammatory bullous disorders, and autoimmune disorders, and hereditary, etc., In one or more modalities, support 10 is used as a tissue sealant (for example, an acute wound healing substance, surgical sealant, partial-thickness, full-thickness topical agent, or encapsulation, pressure ulcers, venous ulcers, diabetic ulcers, chronic venous ulcers, donor skin graft sites, post-Moh surgery, post-laser surgery, podiatric wounds, wound dehiscence, abrasions, lacerations, second or third degree burns, radiation damage, skin tears and drainage wounds, and the like). FIGS. 2A-2C illustrate one embodiment, where support 10 is used to treat a wound site 100 formed in tissue 102 of a mammal. In certain embodiments, support 10 is used for the treatment of immediate acute wounds. In acute wounds, support 10 provides several benefits, including a quick method to seal wounds 100, prevent loss of transepidermal water, provide cells or medication (s), and improve healing of skin wounds (eg, surgery sites , burn wounds, ulcers) to promote the development of more natural tissue (for example, preventing the formation of scar tissue). A particular advantage of support 10 is the ability of support 10 to reduce or minimize the formation of scar tissue. Support 10 provides a more effective alternative to fabric glues and other current injectable fabric fillers and adhesives.
[0082] As can be seen in FIG. 2A, microgel particles 12 are delivered to wound site 100, followed by the initiation of the annealing reaction to anneal microgel particles 12 to one another to form support 10. As seen in FIG. 2A, wound site 100 is sealed by support 10 and as time progresses, wound site 100 is healed for normal tissue (see also Fig. 11). FIG. 2B illustrates how adjacent microgel particles 12 (particles A and particles B) undergo chemical or enzymatic initiation of the annealing reaction in order to form a annealing surface 17 between the microgel particles 12. Fig 2C illustrates an enlarged view that illustrates how the support 10 acts as a structural support, yet allows tissue infiltration and biomaterial resorption due to the porous nature of the support 10. A cell 106 is illustrated by infiltrating the interstitial spaces formed within the support 10.
[0083] Support 10 can also be used in a regenerative capacity, for example, applied to tissue by burns, acute and chronic wounds, and the like. In one embodiment, support 10 is used for chronic wounds. In chronic wounds, where the normal healing process is inhibited, support 10 can be used not only to seal wounds, but also to remove excess moisture, and to apply medication (s), including cell therapies that can help in promoting the normal wound healing process. In the case of applications of tissue fillers for the loss of volume related to aging, lipoatrophy, lipodystrophy, dermal scar, or superficial or deep rhytides, injection of microgel particles 12 directly into the dermis through the needle or cannula can be used to improve tissue contour, tissue loss, or tissue displacement. Because cells used in alternative medicine can grow within microgel 12 particles, cells (eg, mesenchymal stem cells, fibroplasts, etc.) can be included as a therapy by initially polymerizing the cells (1-20 cells) within of the microgel particles, or cells can be initially adhered to microgel particles, or cells can be introduced with the microgel particle solution (not adhered), before annealing in situ to the tissue.
[0084] Support 10 can also be used for in vitro tissue growth, three-dimensional (3D) matrices for biological science studies, and cosmetic and dermatological applications. For example, cancer cells can be seeded together with microgel precursors and once annealed they could allow rapid 3D growth of tumor spheroids for testing more physiologically relevant drugs without the need for matrix degradation, as would be required for other 3D culture gels (for example, Matrigel®). It is expected that the rapid ability to form contacts between cells in the 3D matrix of the annealed gel will increase the growth and formation of microwovens from a single cell type or from several types of cells that can be used for screening test drugs or cosmetics. Epidermal layers can form on the surface of a support 10, which could allow testing of drugs or cosmetics, in a substitute more similar to the skin compared to animal models. Previous 3D culture materials can both allow cells to be sown within the gel evenly throughout the volume, but do not maintain cell-cell contacts, due to lack of porosity, or create porosity, but require cells to be seeded in the manufacturing sequence and migrate into the support.
[0085] As explained here, while the annealed support 10 generally forms a defined structure, the precursor materials before the final annealing is fluid and can be delivered as a paste, suspension, or even injected to the delivery site of interest. Other injectable hydrogels can provide support for tissue regrowth and regeneration in situ, however these injected materials require gel degradation prior to tissue reforming limiting their ability to provide physical support. The microporous injectable gel system described here circumvents this challenge by providing an interconnected microporous network for simultaneous tissue reform and de-gradation of the material.
[0086] Microfluidic formation allows substantially monodisperse microgel particles 12 to form on an interconnected microporous annealed particle support 10 (in one aspect of the matter described here), thereby allowing the controlled chemical, physical and geometric properties microgel particles 12 (for example, building blocks), to provide downstream control of the physical and chemical properties of the mounted support 10. In vitro formation, the cells incorporated in the support 10 proliferate and form extensive three-dimensional networks within forty-three eight (48) hours. In vivo, the injectable gel system that forms the support 10 facilitates cell migration, resulting in rapid tissue regeneration and skin tissue formation within five (5) days. The combination of microporosity and injectability achieved with supports 10 allows new avenues for regeneration in vivo and the creation of new tissue.
[0087] FIG. 2A illustrates the support 10 formed within a wound site 100. Successful materials for the benefit of tissue regeneration from the precise combination of the rate of material degradation for tissue development. If degradation occurs too quickly, then insufficient supports will remain to support the internal growth of tissue. On the other hand, a rate that is too slow will prevent proper tissue development and can promote fibrosis and / or immune rejection. Adjustment of degradation rates based on the local environment was addressed using hydrolytically and enzymatically degradable materials. However, the loss of dissociation from mechanical stability of material with cellular infiltration proved extremely challenging. Promotion of cell infiltration into the material can also be addressed using a slightly cross-linked matrix, however, this often results in poor mechanical combination with the surrounding tissues and poor material stability. Alternatively, the rate of degradation of the hydrogel can be adjusted by changing the identity of the polymeric backbone or crosslinking density, combining the rates of degradation and tissue formation. Although these techniques can be adjusted to address specific injectable hydrogel applications, they do not provide a robust path to achieve bulky tissue integration that does not depend on loss of material stability.
[0088] Each wound site is unique in its chemical and physical degradation requirements for functional tissue regeneration, requiring a material strategy that is robust for a variety of challenging environments. The microporous gel system and the resulting support 10 that is created as described herein avoids the need for material degradation prior to internal tissue growth by providing a stably linked interconnected network of micropores for cell migration and mass integration with the surrounding fabric. The microporous gel system achieves these favorable characteristics when, according to one modality, it uses the self-assembly of microgel particles 12 as "building blocks" or "subunits", formed by water droplet segmentation in micro-fluids. According to one embodiment, the microgel particles 12 formed in this way are substantially monodispersed. The microgel particles 12 can be injected and molded in any desired shape. Grids of microgel particles 12 are then hybridized to each other via surface functionalities to form an interconnected microporous support 10 either with or without cells present in the interconnected porous networks. The support 10 preferably, in one embodiment, includes microgel particles co-covalently bonded 12 that form a three-dimensional support 10 for tissue regeneration and internal growth.
[0089] By combining injectability and microporosity, the microporous gel system provides an ideal biomaterial support for an efficient formation of cellular network in vitro and integration of voluminous tissue in vivo. The modular microporous gel system also provides mechanical support for rapid cell migration, molecular signals for direct cell adhesion, and reabsorption during and after tissue regeneration. Through the manufacture of microfluids, the chemical, physical, and geometric properties of microgel particles 12 can be predictable and uniformly adapted, allowing control downstream of the properties of emerging supports 10. The block-based approach new construction 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 shift in the approach to injectable supports for mass tissue integration.
[0090] In one aspect of the matter described here, the microporous gel system uses microgel 12 particles having diameter dimensions within the range of about 5 μm to about 1000 μm. Microgel particles 12 can be made from a hydrophilic polymer, amphiphilic polymer, synthetic or natural polymer (for example, poly (ethylene glycol) (PEG), poly (propylene glycol), poly (hydroxyethyl methacrylate) , hyaluronic acid (HA), gelatin, fibrin, chitosan, heparin, heparan, and synthetic versions of HA, gelatin, fibrin, chitosan, heparin, or heparan). In one embodiment, microgel particles 12 are made from any natural (eg, modified HA) or synthetic (eg, PEG) polymers capable of forming a hydrogel. In one or more modalities, a polymeric network and / or any other support network capable of forming a solid hydrogel construct can be used. Supporting materials suitable for most tissue engineering / regenerative medicine applications are generally biocompatible and preferably biodegradable. Examples of suitable biocompatible and biodegradable supports include: natural polymeric carbohydrates and their synthetically modified, cross-linked, or substituted derivatives, such as gelatin, agar, agarose, cross-linked alginic acid, chitin, substituted and cross-linked guar gums, esters cellulose, especially with nitric acids and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural nitrogen-containing polymers, such as proteins and their derivatives, including cross-linked or modified gelatins and keratin; vinyl polymers such as poly (ethylene glycol) acrylate / vinyl methacrylate / maleimide / norborene / ally, polyacrylamides, polymethacrylates, copolymers and terpolymers of the polycondensates above, such as polyesters, polyamides, and other polymers, such as poly -rethanes; and mixtures or copolymers of the above classes, such as graft copolymers obtained by initiating the polymerization of synthetic polymers into a pre-existing natural polymer. A variety 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 acid. Methods for producing networks of such materials are well known.
[0091] In one or more embodiments, the microgel particles 12 further include covalently bound chemicals or molecule cells that act as signaling modifications that are formed during the formation of the microgel particle 12. Signaling modifications include the addition of, for example, adhesive peptides, extracellular matrix proteins (ECM), and the like. Functional groups and / or binders can also be added to microgel particles 12 after their formation, either through covalent methods or non-covalent interactions (for example, electrostatic charge-charge interactions or limited diffusion sequestration). Crosslinkers are selected depending on the desired degradation characteristic. For example, crosslinkers for microgel particles 12 can be degraded hydrolytically, enzymatically, photolytically, or the like. In a particularly preferred embodiment, the crosslinker is a degradable metalloprotease matrix (MMP) crosslinker.
[0092] Examples of such crosslinkers are peptides manufactured synthetically or naturally isolated with sequences that correspond to MMP-1 target substrate, MMP-2 target substrate, MMP-9 target substrate, random sequences, Omi target sequences, protein sequences heat shock target, and any of these sequences listed with all or some amino acids being chirality D or chirality L. In another embodiment, the crosslinking sequences are hydrolytically natural and degradable synthetic polymers, consisting of the same main structures listed above (for example, heparin, alginate, poly (ethylene glycol), polyacrylamides, polymethacrylates, copolymers and terpolymers of the listed polycondensates, such as polyesters, polyamides and other polymers, such as polyurethanes).
[0093] In another embodiment, crosslinkers are DNA oligos manufactured synthetically or naturally isolated with sequences that correspond to: restriction sequences of enzyme recognition, CpG motifs, zinc finger motifs, CRISPR or CAS-9 sequences, claw recognition sequences, and transcription factor binding domains. Any of the crosslinkers of the listed modalities are activated at each end by a reactive group, defined as a chemical group allowing the crosslinker to participate in the crosslinking reaction to form a polymer or gel network, in which these functionalities may include: amino - cysteine acids, synthetic and naturally occurring molecules containing thiol, groups containing carbene, activated esters, acrylates, norbenes, primary amines, hydrazides, phosphenes, azides, containing epoxy groups, groups containing SANPAH, groups containing diiazirine.
[0094] In one embodiment, the chemistry used to generate microgel particles 12 allows subsequent annealing and support formation 10 through radical-initiated polymerization. This includes chemical initiators, such as ammonium persulfate combined with tetraethylethylenediamine. Alternatively, photoinitiators such as Irgacure® 2959 or Eosin Y in conjunction with a free radical transfer agent, such as a free thiol group (used in a concentration within the range of 10 μM to 1 mM) can be used in combination with a light source that is used to initiate the reaction, as described herein. An example of a free thiol group can include, for example, the amino acid cysteine, as described herein. Of course, peptides including a free cysteine or small molecules, including a free thiol, can also be used. Another example of a free radical transfer agent includes N-vinylpyrrolidone (NVP).
[0095] Alternatively, Michael and pseudo-Michael addition reactions, including α, β-unsaturated carbonyl groups (eg acrylates, vinylsulfones, maleimides, and the like) to a nucleophilic group (eg, thiol , amine, aminoxy) can be used for annealing microgel particles 12 to form the support 10. In another alternative embodiment, chemically formed microgel particle 12 allows the formation of the network through initiated sol-gel transitions including fibrinogen into fibrin (through the addition of the catalytic thrombin enzyme).
[0096] The functionalities that allow particle-particle hybridization are included, either during or after the formation of microgel 12 particles. In one or more modalities, these functionalities include α, β-unsaturated carbonyl groups, which can be activated for annealing, either through a radical-initiated reaction with carbonyl groups α, β-unsaturated in adjacent particles or addition reactions of Michael and pseudo-Michael with nucleophilic functionalities that, or are presented exogenously as a multifunctional binding agent between particles or as functional groups present in adjacent particles. This method can use several types of microgel particle population 12 which, when mixed together, form a support 10. For example, microgel particles 12 of type X having, for example, nucleophilic surface groups can be used with microgel particles 12 type Y with, for example, α, β-unsaturated carbonyl groups. In another modality, the participating click chemistry functionalities can be included allowing the connection, either directly to the adjacent mi-crogel particles 12 that present complementary click functionalities or through an exogenously presented multifunctional molecule that participates or initiates (eg copper) reactions cli-que.
[0097] The annealing functionality can include any previously discussed functionality used for microgel crosslinking that is orthogonal or similar (if the potential reactive groups remain) in terms of their initiation conditions (for example, temperature, light, pH) compared to the first crosslinking reaction. For example, if the initial crosslinking reaction consists of a Michael addition reaction that is temperature dependent, the subsequent annealing functionality can be initiated through temperature or photoinitiation (for example, eosin Y, Irgacure®). As another example, the initial microgels can be light-cured at a wavelength of light (for example, ultraviolent with Irgacurre®), and the annealing of microgel particles 12 occurs at the same or another wavelength of light (for example , visible with Eosina Y) or vice versa. In addition to annealing with covalent coupling reactions, the annealing portions may include non-covalent hydrophobic, guest / host interactions (eg, cyclodextrin), hybridization between complementary nucleic acid sequences or imitations of nucleic acid (eg, nucleic acid protein) in adjacent microgel particles 12, or ionic interactions. An example of an ionic interaction would consist of alginate functionality on the surfaces of the microgel particles that are annealed with Ca2 +. The so-called "A + B" reactions can be used for annealing microgel 12 particles. In this embodiment, two separate types of microgel (Type A and Type B) are mixed in varying proportions (between 0.01: 1 and 1: 100 of A: B) and type A surface features react with type B (and vice versa) to initiate annealing. These types of reactions can fall into any of the mechanisms listed here.
[0098] In one embodiment, microgel particles 12 are manufactured using either microfluidic or milifluidic methods, generating deterministic microgel particle length scales with little variability and high yield (for example, frequencies greater than 10 particles / second). The coefficient of variation in the length of the microgel particle scale 12 (for example, diameter) can be within 35% or more preferably within 15% and even more preferably within 5% of the length scale average. Mili or microfluids allow uniform, predetermined, concise material properties to be included pre-, in, and post-formation of microgel particles 12. In addition, the microfluidic / milifluidic production mechanism allows for increased production ease scale as well as good quality control over the chemical composition and physical characteristics of microgel 12 particles. Millifluidic and / or microfluidic technologies for generating microgel particles 12 are easily scalable processes to create large amounts of material for commercial needs, maintaining high precision and accuracy in the characteristics of microgel particles 12.
[0099] In one embodiment, microgel particles 12 are formed using automated fluidic methods that depend on the generation of water-in-oil emulsion. This includes microfluidic or milifluidic methods using glass / PDMS, PDMS / PDMS, glass / glass, or molded / modulated / embossed plastic chips to create droplets of water in oil with a smaller size distribution range to 35%.
[00100] FIGS. 3A-3F illustrate an embodiment of a microfluidic device 20 which is used to generate the microgel particles 12. The microfluidic device 20 is formed from a substrate material 22 such as PDMS, which may include another substrate material 24 (for example , glass) which is attached to the substrate 22. In this embodiment, the microfluidic device 20 includes a first inlet 26, a second inlet 28, and a third inlet 30. As seen in FIG. 3A, the third entry 30 is interposed between the first entry 26 and the second entry 28. In this embodiment, the first entry 26 is coupled to a solution containing a main structure of 4 poly (ethylene glycol) vinylsulfone (PEG-VS) (20 kDa) that have been pre-modified for oligopeptides with cell adhesive properties (eg, RGD) and surface / tissue annealing features (eg, peptides, K and Q). The main structure of PEG-VS can be pre-functionalized with 500 μM of peptide K (Ac-FKGGERCG-NH2 [SEQ ID NO: 1]) (Genscript), 500 μM of peptide Q (Ac-NQEQVSPLGGERCG-NH2 [SEQ ID NO: 2]), and 1 mM RGD (Ac-RGD SP GERCG-NH2 [SEQ ID NO: 3]) (Genscript). The solution inlet with the first inlet 26 may contain about 5% (by weight) of modified PEG-VS contained in a 0.3 M triethanolamine buffer (Sigma), pH 8.25. Second entry 28 is coupled to a solution containing the crosslinker, which, in one embodiment, is a 12 mM substrate of a modified di-cysteine metalloprotease (MMP) matrix (Ac-GCRDGPQGIWGQDRCG-NH2 [SEQ ID NO: 4] ) (Genscript). In experiments carried out using fluorescent imaging, the MMP substrate was pre-reacted with 10 μM Alexa-Fluor 647-maleimide (Life Technologies). Of course, in practical applications, it is not necessary to use the fluorescent probe. All solutions can be sterilized by filtration through a 0.2 μm polyethersulfone (PES) membrane in a Luer-lock syringe filter.
[00101] As used herein, K peptides refer to peptides that contain a recognized factor XIIIa lysine group therein. As used herein, Q peptides refer to peptides that contain a recognized factor XIIIa glutamine group within them. Thus, peptide sequences in addition to those specifically mentioned above can be used. The same applies to the RGD peptide sequence that is listed above.
[00102] The third entry 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 combined with the PEG-VS solution introduced through the first port 26 and can be used to control the pH of the crosslinking solution and to inhibit crosslinking until droplet formation. Because the third inlet 30 is interposed between the first inlet 26 and the second inlet 28 of the aqueous PEG-VS solution, it acts as a barrier that prevents any diffuser mixture of material from reactive solutions upstream of the droplet generation region. This significantly increases the life of the device before fouling occurs. FIGS. 3E and 3F illustrate how the inert liquid solution prevents mixing of the left and right solutions before the segmentation droplet.
[00103] With reference to Figs. 3A, 3B, and 3C, the first input 26, second input 28, and third input 30 are connected to, respectively, channels 32, 34, 36. The channels intersect at junction 38 and are carried out on a common channel 40. The fourth entry 42 is provided in the device and is coupled to an oil phase that contains a surfactant (for example, SPAN® 80 at 1% by volume, although other surfactants can be used). The fourth inlet 42 is connected to two channels 44, 46 which intersect at junction 48 with a region downstream of common channel 40. Junction 48 in device 20 is where water-based droplets are formed that include the PEG- VS and the crosslinker. The droplet contents are subjected to mixing and will form microgel particles 12 by gelation, which in this mode is a function of ambient temperature and the passage of time. In this device, a fifth inlet 50 is provided, which is coupled to another oil phase that contains a surfactant at a volumetric percentage higher than that connected to the fourth inlet 42. For example, the fifth inlet 50 can be connected to an oil phase containing 5% SPAN® 80 by volume. Again, surfactants other than SPAN® 80 could also be used. The fifth inlet 50 is connected to two channels 52, 54 which intersect at junction 56 in a press orientation as illustrated.
[00104] Common channel 40 continues for a series of branching channels progressively branching 58. Branch channels 58 allow the continuous flow of microgel particles 12 through the individual parallel channels where local environmental conditions can be optionally controlled. For example, the temperature of individual branching channels 58 can be controlled to regulate the crosslinking conditions for microgel particles 12. Likewise, branching channels 58 can be illuminated with light to control reactions activated by light. Microgel particles 12 can be removed from device 20 via outlet 59. It should be understood, however, that the temperature regulation of branching channels 58 or the use of light activation is entirely
[00105] As best seen in FIG. 3D, first inlet 26, second inlet 28, third inlet 30, fourth inlet 42, and fifth inlet 50 are connected, respectively, for fluid lines 26 ', 28', 30 ', 42' and 50 'that are connect to a pumping device 51 or multiple pumping devices 51 that pumps respective fluids to the correspondingly connected inlets 28, 28, 30, 42, 50. Pumping device 51 can include separate pumps connected to each different fluid. Examples of types of pumps that can be used include syringe pumps or other pumps commonly used in connection with microfluidic devices. In one aspect, the pumping device 51 uses the gas under regulated pressure above a fluid reservoir to pump the fluid to the desired rate (s) through the device.
[00106] FIGS. 4A-4C illustrate an alternative embodiment of a microfluidic device 60 that is used to generate the microgel particles 12. In this alternative embodiment, unlike the embodiment of FIGS. 3A-3C, there is no third port 30 that carries an aqueous solution that is used to separate the PEG and cross-linking components before droplet generation. Instead, in this fashion, the microfluidic device 60 includes a first inlet 62, a second inlet 64, a third inlet 66, and a fourth inlet 68. The first inlet 62 is coupled to a source of PEG-VS modified as described above. Second entry 64 is coupled to a crosslinker. 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 linked to the third inlet 66. In this embodiment, the first inlet 62 and the second inlet 64 are coupled to the respective channels 70, 72 that lead to a common channel 74. The third input 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 the generation of droplets (droplets) occurs will form microgel particles 12 after reaction). The fourth entry 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. As seen in FIG. 4A, device 60 includes a series of branching channels that progressively branch 88, which are similar to those described in the context of the embodiment of FIGS. 3A-3C. The microgel particles 12 that pass through the branch channels 88 can be collected in a collection chamber 90 or the like, which can be removed from the device 60. The fluid is supplied to the device 60 using fluid lines and a device pumping as described above, in the context of the embodiment of FIGS. 3A-3C.
[00107] The fluidic conditions that lead to the formation of microgel particles 12 include, in one embodiment, mixing on the chip of aqueous solutions based on PEG and the reticulant base, in which one part contains base polymer and the other contains the crosslinking agent or initiator. It is clear that, in the embodiments of FIGS. 3A-3C, there is a mixture of three inlets, which includes the above mentioned components in addition to the addition of the inert flow to the aqueous base. These PEG and crosslinker solutions are mixed in any volumetric ratio of 1: 1, or another controllable ratio (controlled by relative flow rates in the device) of up to 1: 100 The ratio of total aqueous and oil flow rates they are controlled to determine a microgel particle 12 of specific size, where these proportions can vary from 4: 1 (aqueous: oil) to 1:10 (aqueous: oil).
[00108] As explained above, in the embodiment of FIGS. 3A- 3D, the chip device 20 is designed to have three water-based solutions combined to form the microgel particles 12, in which the base polymer and crosslinking agent / initiator are separated by a non-reactive solution upstream of the generator droplet to prevent the solution reaction and chip encrustation over time in the region upstream of the droplet generation. In this configuration, the portion of the non-reactive solution should be equal to or less than the base and cross-linking solutions, from 1 to 0.05 times the volume rate of the other solutions. This modality can, thus, improve the reliability and useful life of chips used for the generation of microgel. In addition, in this or the previous embodiment, cells can be introduced into any of the two or three aqueous solutions introduced to allow encapsulation of these cells (single cells in groups of 2-20 cells per particle) within microgel particles 12 such that cells encapsulated cells can produce factors to improve wound healing or internal cell growth.
[00109] While FIGS. 3A-3D and 4A-4C illustrate different modalities of a microfluidic device 20, 60 that can be used to generate microgel particles 12, in an alternative embodiment, the microfluid flow path may include a 'T-junction' architecture as illustrated in FIG. 5. In this embodiment, 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 transported through an outlet channel 98 (which can be the same as the first or second channels 94, 96). Alternatively, different droplet configurations can be used to generate microgel 12 particles. For example, the device can generate droplets 97 using the confinement gradient due to non-parallel upper and lower walls such as the one disclosed in Dangla et al., microfluidic droplets driven by confinement gradients, Proc Natl Acad Sci USA 110 (3): 853-858 (2013), which is incorporated here by reference.
[00110] In microfluidic devices described above, the channel surfaces must be modified in such a way that the aqueous phase is non-wet, which may include a fluorination of the surface, or convert the surfaces to become hydrophobic or fluorophilic, either by a treatment based on covalent silane or another approach based on non-specific adsorption. Alternatively, a plastic polymer containing fluorophilic groups comprises the chip material and can be combined with the aforementioned surface coatings or without a surface coating. In addition, the oil used in the preferred embodiment must be either a mineral oil (paraffin oil) supplemented with a non-ionic surfactant, vegetable oil enriched with an ionic surfactant, or a fluorinated oil supplemented with a fluorinated surfactant (or any combination of these two oil / surfactant systems). These microfluidic or millifluidic methods generate monodisperse populations (coefficient of variation less than 35%) of microgel 12 particles at rates equal to or greater than 10 Hz, where the collection is carried out manually (by hand) or using automated fluid handling systems. To avoid the coalescence of microgel particles 12 before completing the crosslinking reaction, sufficient surfactant is necessary to stabilize the pre-gel droplets, however, high levels of surfactant also destabilize the process of generating droplets. Therefore, a preferred embodiment of the microgel particle microfluidic generation system 12 includes a low concentration of surfactant in the initial compression oil flow (1% or less) that creates droplets followed by the addition of an oil + surfactant solution from a separate entrance that is fused with the droplet and oil solution formed 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.
[00111] In another alternative modality, the two oil compression streams have the same concentration of surfactant. In yet another embodiment, there is no second flow of compressed oil, and only the flow of focusing oil in flow to generate droplets. In addition, as explained above, in some alternative embodiments, there is no second compression oil flow and only the t-junction oil flow is used to generate droplets. Of course, the t-junction droplet junction can optionally be combined with a second focusing oil inlet with equal or greater surfactant concentration.
[00112] After formation, 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 can be used to reduce the volume of free aqueous solution that holds microgel particles 12 (free volume). In one embodiment, the aqueous free volume is less than about 35% of the total volume. In another modality, for the generation of intentionally polydispersed populations, microgel particle generation is performed on a mili or micro fluidized platform, generating stocks of relatively monodispersed microgel 12 particles that are then mixed in desired proportions to obtain distributions deterministic and microgel 12 particle size proportions 12. Microgel 12 particle size proportions can be precisely controlled to control pore structure, or chemical properties with a final stoichiometric proportion of: 1: 1, 10: 1, or greater than 100: 1.
[00113] Alternatively, generation of microgel particles 12 via a water-in-oil system can also be performed using sonic mixing methods or a rotating vortex. These latter methods generate polydispersed populations of microgel 12 particles with ranges in size from 100 nanometers to 500 micrometers. These particles can then be filtered using porous filters, microfluid filtration, or other techniques known in the art to obtain a narrower size distribution of microgel 12 particles (for example, the coefficient of variation of less than 50 %). Alternatively, component 12 microgel particles in different shapes can be manufactured using stop flow lithography, continuous flow lithography, and other methods to create particles with a shape that is based on flow formation (see Amini et al., International Publication No. WO / 2013/049404, which is hereby incorporated by reference) combined with UV-initiated polymerization through a mask that defines the shape. In this case, the microgel particles 12 are non-spherical with long and short dimensions, which can vary between 5 and 1000 micrometers. Conforming particles can also be manufactured through the generation of spherical particles in a water-in-oil emulsion, followed by extrusion through said particles through micro-fabricated constrictions that have length scales smaller than the particle diameter. The previously spherical particles take 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 crosslinking reactions listed above. The gels retain this shape after leaving the microfabricated construction. Particles that conform can allow additional control of the pores, and improved adhesion within the final support formed by the annealing of mi-crogel particles 12.
[00114] In one or more embodiments, microgel particles 12 are modified covalently or not (eg, inclusion spatially within by diffusion) to provide biologically active molecules (eg, small molecule drugs, antibiotics) (peptides, proteins, steroids, matrix polymers, growth factors, antigens, antibodies, etc.). The inclusion of signaling molecules after the formation of the microgel particle 12 can be carried out through passive diffusion, surface immobilization (temporary or permanent), and / or large amounts of immobilization (temporary or permanent).
[00115] In another embodiment, nanoparticles are included in the initial prepolymer solution and incorporated into microgel particles 12 during initial polymerization or gelation, and nanoparticles can include biologically active molecules for release and sustained delivery or fast. In another embodiment, microgel particles 12, which contain free primary amines (included as part of an oligopeptide containing lysine) can be modified with NHS-azide. To this set of microgel particles 12, a protein modified with an NHS-phosphine can be added, resulting in the coating surface of the microgel particles 12 with the modified protein. FIG. 10 illustrates a modality in which a microgel particle 12 has nanoparticles incorporated into it and a surface that has been modified with a protein using click chemistry.
[00116] After production and optional modification, microgel particles 12 (which can be a homogeneous or heterogeneous mixture) can be applied to a desired site (in vitro, in situ, in vivo). The desired site on mammalian tissue 102 may include, for example, an injury site 100 or other site of damaged tissue. The microgel particles 12 can be introduced by themselves, in an aqueous isotonic saline solution or suspension (preferably with 30-99% volume fraction of microgel particles 12, and preferably less than 1-30% fraction volume). Alternatively, microgel 12 particles can be introduced together with the cells as individual cells or aggregated with cells of 10: 1 particle proportions to create dense cell networks within the final annealed support of 10 or 1: 100 or even 1: 1000 for create sparsely seeded substrates 10 with cells that produce soluble factors useful for regeneration. In another embodiment, microgel particles 12 can be cultured with cells at a low fraction of particle volume (<10%) for a period of time in permissive cell medium to promote adhesion to individual microgel particles 12 These microgel particles adhered to a composite cell 12 can be introduced as the active component that would anneal to form a support sown in a microporous cell 10, which can be beneficial in increasing the rate of regenerative activity. Desired in vitro locations for introducing microgel particles 12 include well plates (for example, 6 wells, 96 wells, 384 wells) or fluidic devices to form 3D microporous culture microenvironments for cells after annealing, and to allow subsequent biological assays or high-performance screening tests with more physiologically relevant or multicellular 3D conditions. For in vitro introduction, solutions of microgel particles 12 can be pipetted into wells or introduced by syringe injection followed by the introduction of an annealing solution or triggering photochemically annealing. Alternatively, a solution of microgel particle solution 12 can be mixed with a slow-acting annealing solution (annealing occurring over 10-30 min) before delivery. In situ locations include external wound sites (for example, cuts, blisters, wounds, pressure ulcers, venous ulcers, diabetic ulcers, chronic vascular ulcers, donor skin graft sites, post-Moh surgery sites, surgery sites post-laser, podiatric wounds, wound dehiscence, abrasions, lacerations, second or third degree burns, radiation damage, skin tears and drainage wounds, etc.). Since the epidermis is an epithelial structure, the microgel particle solution can be used to heal other epithelial surfaces (urothelial fissures (bladder or intestine, etc.), aerodigestive (lung, gastrointestinal), similar to python skin (ie stomach or duodenal ulcer; after penetrating trauma to lung, bladder or intestine fistulas. etc.). In addition, the microgel particle solution can be applied to other tissues through a catheter or cannula, such as nervous system tissue and cardiac tissue where internal tissue growth would be beneficial to prevent scarring and to facilitate regenerative healing after an injury, such as after spinal cord trauma, cerebral palsy / stroke and myocardial infarction.
[00117] For in situ introduction, solution containing microgel particles can be stored separately from an annealing solution and mixed during introduction (a method analogous to the epoxy adhesive) to prevent premature initiation of the annealing reaction before entry on a site of wound 100.
[00118] In another, the two solutions can be stored inside a syringe or tube clamping applicator with two pipes of equal or unequal diameters, such that when the plunger of the syringe has the tube compressed or squeezed it itself - simultaneously delivers both microgel particles 12 and the hybridization solution to the correct stoichiometry. FIG. 6A illustrates such an embodiment of a delivery device 110, which includes a first barrel 112, a second barrel 114 and a plunger 116 that is used to deliver the solution containing each barrel microgel 12 particles 112, 114. For example, the first pipe 112 contains microgel particles 12 and thrombin at a concentration ranging from 0.1 to 5 U / ml and the second pipe 114 contains microgel particles and FXIII 12 at a concentration of 0.1 to 1000 U / mL). In both pipes 112, 114 there is a fraction of volume 1 to 1 of microgel particles 12 containing peptide K and Q where the concentration of the peptide k and Q range of 1- - 1000 μM in microgel particles 12. In the modality , when mixing the thrombin activates FXII (to form FXIIIa) and the resulting FXIIIa is responsible for annealing and surface binding of K and Q peptides in adjacent microgel particles 12.
[00119] Alternatively, the two pipes 112, 114 may contain two types of separate microgel particles 12 with annealing portions that require the combination to initiate crosslinking. An alternative method of storage and delivery would be in a single-barrel syringe 110, as illustrated in FIG. 6B or a multipurpose or single-use compressible tube, as illustrated in FIG. 6C (for example, similar to toothpaste or antibiotic ointment), in which the microgel particle paste can be squeezed to a desired volume and spread over the wound site 100 and then annealed by exposure to light, where the active agent for photochemistry is Eosin-Y at a concentration of 100 μM although concentrations within the range of 10 μM - 1 mM also work. Preferably, Eosin Y is accompanied with a radical transfer agent, which can be, for example, a chemical species with a free thiol group. An example of such a transfer agent includes cysteine or peptide radicals including cysteine (s) described herein (for example, used at a concentration of 500 µM). The light must be delivered through a broad spectrum of white light (incandescent or LED), or a green or blue LED light. The flashlight, wand, lamp, or even the ambient light can be used to provide white light. Exposure should take place between 0.1 seconds and 1000 seconds, and the light intensity should vary between 0.01 mW / cm2 100 mW / cm2 at the hybridization site. In another embodiment, light-mediated annealing can be performed using UV light (wavelengths between 300-450 nm), where the photochemistry agent is IRGACURE® 2959, at a concentration of 0.01% w / va 10% w / v. The exposure time should be between 0.1 seconds and 100 seconds, with a light intensity of 0.1 mW / cm2 to 100 mW / cm2 at an annealing site. For modalities in which the initiated annealing light is used, microgel precursors 12 would be stored in an opaque syringe (opaque with respect to the wavelength range that initiates re-cooking) or squeeze tube containers 110 before of use. Desired in situ locations include internal cuts and tissue openings (for example, from surgical incisions or resections), burn wounds, radiation wounds and ulcers, or in plastic surgery applications to fill the tissue site and stimulate internal tissue growth and regeneration instead of the fibrotic processes common to contemporary injectables.
[00120] Delivery using double or single-barrel syringes is also suitable for this indication, as well as hybridization using a photoactivation and a source of UV or white light that can be inserted into the surgical site. For both in situ and in vivo applications, the microgel particle paste can be distributed using a sterile applicator to be aligned with the wound or stacked inside and around the wound site 100 (inside the wound and 2 mm to 1 cm beyond the original wound extensions) to create an annealed support that extends beyond the wound site 100 or tissue defect to provide additional protection, moisture, and structure to withstand tissue regeneration.
[00121] An annealing process is initiated through the application of a stimulus (for example, radical initiator, enzyme, Michael addition, etc.) or through interactions with a stimulus that is already present at the application site. microgel particles 12 that interact with functional groups on the surface of microgel particles 12, forming a contiguous highly porous solid support 10 formed from the annealed (bonded) microgel particles 12. If used in the fabric, the annealing process can allow the fusion of support 10 for the surrounding tissue, providing an effective seal, a local medication and / or cell delivery device, a vascularized support for in vivo detection, and a better path for tissue regeneration. The annealing process allows 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 via injection with a needle or through a catheter or cannula. Support 12 may comprise homogeneous or heterogeneous populations of microgel particles 12. As discussed, heterogeneous populations of microgel particles 12 may vary in physical composition (for example, in size, shape, or stiffness) or vary with -chemical position (for example, varying proportions of degradable ligands, or amino acids L- or D- to modify the rate of de-gradation, varied portions of annealing, portions of cell adhesion, or loading 12 microgels with molecules bioactive or non-particulates). The heterogeneous composition of the final annealed support 10 can be random or structured in layers of uniform composition to create gradients in microporous structures (varying sizes of microgel particles 12 in layers, for example) or gradients of chemical composition ( by layers of micro-gel particles 12 with different composition or bioactive molecule loading). Gradients can be useful in targeting internal cell growth and tissue regeneration in vivo, or the development of tissue structures in vitro. Gradients in microgel particle composition 12 could be achieved by delivering the sequential pastes of a gel of a single composition, followed by annealing, and then later delivery of the next gel of a second composition, followed by annealing that bonds the new layer of microgels to the previous layer, until a desired number of layers has accumulated. The thickness of each layer can be controlled using the volume of paste injected and the area of the injection site. An alternative way of achieving gradients is to load a multichannel syringe applicator such as that illustrated in FIG. 6A with different microgel compositions in each of the pipes. Each of the pipes is compressed and fed to the nozzle 120 in layered sheets at the same time. The nozzle 120 of the syringe applicator itself can be non-circular or rectangular, to create a multi-layered layered slurry, which is injected into one place in a tape-shaped structure, which can then be annealed in the present arrangement. . Formation of the structurally contiguous annealed support 10 can be achieved through radical, enzymatic or chemical processes (for example, click chemistry).
[00122] In one or more modalities, annealing occurs through surface chemical interactions between microgel particles 12 once they are ready to be placed at the delivery site. In one embodiment, the process takes place through annealing initiated by radical through polymerizable surface groups (for example, initiation of radical by initiators by photosensitive radicals, etc.). In another modality, the process occurs through enzymatic chemistry through enzymatically active substrates presented on the surface (for example, transglutami-nase enzymes, as factor XIIIa). In another embodiment, the process takes place through covalent bonding via Michael and pseudo-Michael addition reactions. This method can use various types of microgel particle populations that, when mixed together, form a solid support 10 (eg presentation of type A microgel particles 12, for example, nucleophilic groups and surface presentation of type 12 microgel particles. B, for example, carbonyl groups α, β-unsaturated). In another mode, the process takes place through the click chemistry link. Likewise, this method can use heterogeneous microgel 12 particle populations that when mixed together form a solid microporous gel. In another embodiment, annealing can be achieved using light (for example, either white light or UV light) to initiate a chemical reaction between molecules on gel surfaces, mediated by a light molecule activated in solution and in surrounding (or directly covalently attached to) the microgels, as described herein.
[00123] In a preferred embodiment, microgel particles 12 include a polymeric backbone of PEG based on combination with an enzymatically degradable crosslinker to allow bioreabsorbance. In certain embodiments, the polymer structure based on PEG is a main structure of 4 poly (ethylene glycol) vinylsulfone (PEG-VS) structures previously modified with oligopeptides for cell adhesive properties (eg RGD) and functionalities of surface annealing (for example, K and Q peptides) and the crosslinker is a degradable crosslinking matrix metalloprotease (MMP).
[00124] In one or more embodiments, microgel particles 12 are formed by a water-in-oil emulsion. Gelation of microgel particles 12 occurs after combining the PEG solution with a crosslinker solution (followed briefly by partitioning into microgel droplets before the gelation is completed). A variety of substrates, including peptide ligands, can be added for enhanced bioactivity. In one embodiment, support formation is carried out by adding and activating the photoinitiator of radicals to the purified microgel 12 particles to induce chemical cross-linking. In another embodiment, support formation is achieved through the use and / or activation of a transglutaminase enzyme endogenously present or exogenously applied, Factor XIII, for the purified microgel 12 particles that have been modified with two peptide ligands or pre-formation, during formation, or enzymatic post-formation to induce cross-linking. In a separate embodiment, support formation is achieved using a combination of enzymatic and radical methods mentioned above.
[00125] The resulting support 10 of the material presently disclosed provides advantages over current porous support technologies due to the ability to form a fully interconnected microporous support in vivo. In general, porous matrices provide greater access for living cells because of the freedom of movement through the pores (that is, they do not need degradation to allow penetration like all current and previous non-porous and nanoporous substrates). For example, when implanting and restoring a support 10 in a skin wound in vivo, significantly increased cell invasion and growing tissue structure were observed after 5 days when compared to a non-porous gel of the same material, as seen in FIGS. 7B. FIG. 7A illustrates H&E staining of tissue sections in SKH1-Hrh mice injected with tissue for 10 (identified as MAP support), as well as non-porous control 24 hours after injection. FIG. 7B illustrates a wound closure graph (%) as a function of days post-injection. This graph shows that in a period of five (5) days there is no statistically significant improvement in wound wound rates for the use of supports 10 when compared to bilateral non-porous controls (N = 5). FIG. 7C illustrate representative images of wound closure during a five-day wound healing model in vivo in SKH1-Hrhr mice. FIG. 7D illustrates representative images of wound closure during 7-day BALB / c mice experiments in vivo. FIG. 7E illustrates quantification data for wound closure from wound healing of BALB / c mice in vivo. After 7 days in vivo, supports 10 promoted significantly faster wound closure than no treatment controls, non-porous PEG gel, and gels lacking the K Q peptide. Porous gels created ex vivo to precisely match the format of wounds using the canonical method, based on porogens, of casting showed appreciable rates of wound healing, comparable to supports 10, but lacking injectability (N> 5). FIG. 7F illustrates traces of wound bed closure for 7 days in vivo for each treatment category corresponding to fig. 7D.
[00126] In addition, therapeutic agents applied to microgel particles 12 or support 10 can be released slowly or quickly, and support 10 has the ability to break down over a predetermined period of time, either from hydrolysis , proteolysis, or enzymolysis, depending on the treatment it is intended for (for example, if it is being used to treat a chronic wound, a more stable crosslinker that slowly degrades over time is used). In addition, the annealing quality of the microgel holder 10 allows the holder 10 to function as a tissue sealant (for example, acute wounds, surgical closure, etc.), and the filling of different molded shapes that are clinically useful for imitating tissues. FIG. 7G illustrates how the microgel particle containing solution or suspension can be applied using a syringe device such as that of FIGS. 6A or 6B at a treatment site where the microgel conforms to the shape of the injection site (for example, in this case a star-shaped site) and subsequent annealing of the support 10 into a star shape.
[00127] By adjusting the rate of degradation of the mi-crogel 10 supports forming the regenerative or scarring response in a wound can be modified. In one embodiment, the rate of degradation of microgel holders 10 has been modified using D amino acids instead of L in the degradable MMP crosslinker. Adjust the proportion of microgel particles 12 with chirality D- or L in the reticulant adjusted to the rate of degradation in the tissue. The supports 10 made from mixtures of crosslinked microgels D and L (with a proportion of 1: 1) resulted in gels present in the tissue 21 days after injection, however in the gels only D, there was no gel remaking after 21 days in vivo. The tissue healing and scarring response also depends on the D: L stoichiometry, and thus the rate of degradation. FIGS. 8A-8G show the effects of scar reduction when using a 1: 1 mixture of D: L, as compared directly to an untreated wound. Dermal thickness is doubled and the scar size is reduced by 25% when treated with 1: 1 D: L gel. In addition, six (6) times more hair follicles and sweat glands are present in the case treated with gel, compared to the case of no treatment. Experimental
[00128] A microfluidic water-in-oil emulsion approach was used to segment a continuous pre-gel aqueous phase into uniformly supported building blocks, as described herein. Generating microgel particles 12 as microscale serial building blocks, instead of using the typical vortex and sonication-based approaches, allowed tight control over the formation environment and the properties of the final materials of the emerging support 10. By adjusting the flow rates of both the pre-gel solution and the compression oil flow, as well as the geometry of the microfluidic channel, a range of microgel particle sizes was created with low polydispersity. Although the manufacturing method was in series, it remained practical in its high-yielding nature, with generation rates ranging from 250 Hz for larger particles (> 100 μm) to -1200 Hz for small particles (~ 15 μm). This translates to approximately 100 μl of pre-swollen gel every 50 min for a single device. This approach ultimately resulted in particles that were highly monodisperse, both physically and chemically. Microfluidic generation of microgel particle "building blocks" is an easily scalable process: a practical requirement for wide adoption and use.
[00129] The resulting microgel particles 12 were made of a completely synthetic hydrogel mesh of main poly (ethylene) glycol-vinyl sulfone (PEG-VS) structures decorated with the cell adhesive peptide (RGD [SEQ ID NO: 3 ]) and two transglutaminase peptide substrates (K [SEQ ID NO: 1] and Q [SEQ ID NO: 2]). The microgel 12 particles were cross-linked through the type of Michael addition with peptide sequences sensitive to cysteine-terminated matrix metalloprotease that allowed cell-controlled material degradation and subsequent reabsorption.
[00130] The microgel particles 12 were purified in an aqueous solution of isotonic cell culture media for storage and, when used to form a gel, were annealed to each other through a non-canonical amide bond between the peptides - K and Q deos mediated by activated Factor XIII (FXIIIa), a naturally occurring enzyme responsible for the stabilization of blood clots. This annealing process mediated by enzyme, allowed the incorporation of living cells in a support of dynamic formation 10 that contained interconnected microporous networks. Following the addition of FXIIIa, but prior to annealing support, a suspension of the microgel particles 12 can be delivered through the application of rubber, ultimately solidifying into the shape of the cavity in which they are injected. FIG. 9A illustrates how the annealing kinetics can be altered by adjusting the pH and temperature. The annealing environment chosen for this experiment was pH 8.25 and a temperature of 37 ° C.
[00131] The structural changes that lead to a further three-fold increase in the storage module in the annealed gels were observed after the addition of FXIIIa for the microgel 12 particles. Annealing was confirmed as necessary for the formation of support through observation of high vacuum SEM, in which, when dehydrating the supports, they adopted a highly stretched mesh, whereas interconnection blocks without FXIIIa separate into individual spherical granules (FIG.9E).
[00132] By adjusting the microgel particle size and composition a diverse set of mounted supports 10 could be generated. By using microgel 12 particles from 30 to 150 μm in diameter, with pore networks of medium diameters ranging from 10 ~ to 35 ~ μm was obtained). Different percentages of PEG weight and crosslinking stoichiometries have been screened to demonstrate a range of easily realized storage modules from ~ 10 to 1000 Pa, which extends from the rigidity regime required for mammalian soft tissue imitations. FIG. 9B illustrates that different percentages by weight of hydrogel were used to produce different rigidity materials. FIG. 9C illustrates different crosslinking stoichiometries (ratio of crosslinking ends (-SH) to vinyl groups (-VS)) that were used to produce different values of rigidity in the resulting gel. FIG. 9D illustrates a graph of degradation in% as a function of time, both for non-porous control, as well as the inventive porous gel described here. Degradation kinetics, porous and non-porous gel are shown by equal volumes of gels in vitro. Porous, particle-based gels degrade more quickly than non-porous gel due to the larger surface area for faster volume and transport rates through microporous gel. The degradation was carried out using 1: 1000 TrypLE®, resulting in higher concentrations than protease in a wound bed and faster degradation kinetics. FIG. 9E shows SEM images of an FXIIIa annealed holder. FIG. 9F illustrates SEM images of microgel 12 particles without FXIIIa. Unhealed particles are seen in FIG. 9F.
[00133] In order to assess the ability of the support generated to support cell growth and network formation, an in vitro cell morphology and proliferation model using three human cell lines was developed. These included: dermal fibroblasts (HDF), adipose-derived mesenchymal stem cells (Ah-MSC) and bone marrow-derived mesenchymal stem cells (BMhMSC). A single cell suspension was dynamically incorporated into an annealed FXIIIa gel. The three cell lines showed high cell viability (> 93%), after twenty-four (24) hours of culture inside the support. The HDF and AhMSC cell lines demonstrated continued proliferation over a six-day culture period with doubling times of 1.5 and 2 days, respectively. BMhMSCs were observed to undergo proliferation, as well, however, with an estimated calculated doubling time of ~ 12 days.
[00134] The cells incorporated in the support began to exhibit propagation morphology 90 minutes after the start of annealing. After two (2) days in culture, all cells observed inside the supports exhibited a completely spread morphology, which continued until day six (6). Importantly, a vast training network for all cell lines was observed for day two (2). Cellular networks increased in size and complexity throughout the experiment. BMhMSCs were of particular interest, as well as their extensive network formation and the slower proliferation rate indicated that these cells were able to spread to extreme lengths, forming highly interconnected cellular networks on microporous supports. In particular, cells that were grown in non-porous gels with identical chemical properties (5% by weight, G = 600 Pa of gel) and mechanical properties (4.5% by weight, G '= 350 Pa of gel) maintained viability but did not exhibit any appreciable network formation, even after six days in culture.
[00135] It was assumed that the ability of the supports to allow both cell proliferation and the formation of the convenient network in vitro was indicative of an ability to withstand cell migration in vivo and the integration of bulky tissue within the scope of the support. To test this hypothesis, a healing model of murine skin wounds was used, directing a tissue of interest to previous implanted porous biomaterials. Importantly, the contraction of the wound was prevented using a sutured rubber splint that limited the closure of internal tissue growth, better simulating the human healing response. Because of the injectability of the support based on microgel particles, the microgel particles were able to be delivered directly to the wound site, then annealing in situ via exogenous FXIIIa. This provided a uniform interface, simultaneously linking "building blocks" of microgel particles with each other, as well as residues of endogenous lysine and glutamine present in the surrounding tissue. Likewise, a uniform interface for chemically identical, non-porous bilateral control was observed. Despite its similar interface, the support generated resulted in significantly faster wound closure than non-porous controls (60% versus 100% wound area remaining after 5 days, respectively) when injected into the wound. wounds of CLR: SKH1-H rh mice as seen in FIG. 7B.
[00136] The disparities in wound closure rates have led to the investigation of differences in tissue responses to non-porous gel and injectable parts. The support injection using the microgel particles resulted in extensive re-epithelialization of the wound after five (5) days in vivo. Keratin-5 + cells were observed with stratified squamous morphology on the apical surface of the support, however no cells (keratin-5 + or otherwise) were observed after the border of the non-porous wound. Importantly, the support was able to support the formation of what appeared to be a complete hair follicle with an adjacent sebaceous gland within the wound bed similar to the structure of these glands in the uninjured skin. In addition, other examples of large keratin-5 + tissue structures were observed within the support including tubular structures and epithelial invaginations. It is assumed that, together, these results are an indication of collective migration of a higher order (that is, the movement of multicellular clusters in concert) contributing to the regeneration of the epidermis. Although the cells were able to infiltrate bilateral non-porous controls (as indicated by DAPI staining), no evidence of re-epithelialization or skin tissue was found after five (5) days in vivo.
[00137] Through a more in-depth investigation, it was found that the support promoted mass integration via complex formation of the vascular network in vivo. After five (5) days, both endothelial cells and support pericytes were present within the support, while only individual branches of endothelial cells without support pericytes were present in the bilateral non-porous controls. The presence of co-located endothelial cells and pericytes was evidence of the formation of a network of mature vessels. To our knowledge, this is the first example of early petition migration (<7 days) in a synthetic injectable material or porous support implanted without the inclusion of exogenous growth factors.
[00138] While investigating the perfect interface provided by the injectable supports, differences were observed in both global and immune cell quantities in one day (1). After one (1) day post-injection, the supports contained a significantly greater number of cells within the support than their bilateral non-porous controls. This corroborated the greater ease of cell mobility previously observed in our in vitro network formation experiments. In addition, the support and its surrounding tissue contained a significantly smaller number of polymorphonuclear cells when compared to the bilateral non-porous control of the same mouse. This result indicated a lower initial global innate immune response for supports on day one (1). After five (5) days post-injection, the lower fractions of CD11b + Cells (activated leukocytes) were present both in the surrounding tissue and within the support in relation to the non-porous controls, indicating a sustained lower level of the inflammatory immune response, from according to what was observed in the ex vivo microporous supports built and implanted. Combined, these two results support a geometric component currently little explored for immunological stimulation from chemically identical injectable bi-materials.
[00139] The support based on annealed microgel particles, represents a new class of injectable biomaterial that introduces interconnected microscale porosity through achieved robust imperfect self-assembly and annealing of individual building blocks. This approach allows the control of hierarchical properties of microscale and macroscale through the deterministic chemical composition and generation of microfluidic particles. Both incorporated living cells and surrounding host tissue are able to immediately infiltrate the support without the need for material degradation, a feat never achieved before using injectable supports.
[00140] In vivo, the injectable microgel particles completely filled the tissue void, providing a seamless boundary with the surrounding tissue. The interconnected microporosity of the resulting support promoted cell migration at the wound site that resulted in greater mass integration with the surrounding tissue while highlighting a reduced host immune response, compared to an injectable non-porous control. Ultimately, this led to the reformulation of healthy tissue faster than similarly understood injectable non-porous gels.
[00141] This gel system presents a fundamental change in the approach of modular bottom - top biomaterials, using the negative space of network formation to promote the development of complex three - dimensional networks in unprecedented time scales using current hydrogel technologies. The "Plug and Play" nature of this strategy allows for the incorporation of a wide variety of already established materials (for example, fibrin), signals (for example, growth factors), and cell populations (for example, stem cells) . Complex combinations of building blocks with deterministic physical and chemical properties and can allow tissue regeneration in a variety of distinct physiological niches (eg, neural, cardiac, skin, etc.), where annealed particle supports are adapted for each niche through its building block properties. The unique combination of microporosity, injectability and modular assembly inherent in supports has the potential to change the shape of tissue regeneration in vivo and creation and tissue again.
[00142] Water droplet generators in microfluidic oil were manufactured using smooth lithography, as previously described. Briefly, master molds were manufactured on mechanical grade silicon wafers (University wafer) using KMPR 1025 or 1050 photoresist (Microchem). Different channel heights were obtained by rotating photoresist at different speeds, at the suggestion of the manufacturer. Devices were molded with the masters using Sylgard® 184 (Dow Corning) poly (dimethyl) siloxane (PDMS) kit. The base and the crosslinker were mixed in a 10: 1 mass ratio, poured over the mold, and degassed before curing for 6 hours at 65 ° C. Channels were sealed by treating the mol of PDMS and a glass microscope slide (VWR) with oxygen plasma at 500 mTorr and 75 W for 15 seconds. Immediately after sealing the channel, the channels were functionalized by injecting 100 μl 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.
[00143] The 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) 10% w / v 4arm PEG-VS (20 kDa) in 300 mM triethanolamine (Sigma), pH 8.25, pre-functionalized with 500μM of K peptide (Ac-FKGGERCG-NH2 [SEQ ID NO: 1]) (Genscript), 500 μM of Q peptide (Ac-NQEQVSPLGGERCG-NH2 [SEQ ID NO: 2]), and 1 mM of RGD (Ac- RGD SP GERCG-NH2 [SEQ ID NO: 3]) (Genscript) and (ii) an 8 mM di-cysteine modified metalloprotease (MMP) matrix (12 mM for the three input device) (Ac-GCRDGPQGIWGQDRCG-NH2 [SEQ ID NO: 4]) (Genscript) substrate pre-reacted with 10 μM Alexa-Fluoro 647-maleimide (Life Technologies). All solutions were sterilized by filtration through a 0.2 μm polyethersulfone (PES) membrane in a Leur-Lok syringe filter before use in the segmentation system.
[00144] Generation was carried out at 37 ° C in an incubated microscope phase (NIKON® Eclipse Ti) for real-time monitoring of microgel quality. The aqueous inlet solutions do not sensitively mix until droplet segmentation (Peclet number> 10). The oily phase was a heavy mineral oil (Fisher) supplemented with 0.25% v / v of SPAN® 80 (Sigma-Aldrich). Downstream of the segmentation region, a second oil inlet with a high concentration of SPAN® 80 (5% v / v) was added and mixed with the flow droplet emulsion. Ultimately, the microgel-in-oil mixture came out in a large well (12 mm in diameter, volume of ~ 1 mL), where the microgel particles cured at 37 ° C for a minimum of 1 hour. The mixture was then extracted and purified by overlaying the oil solution in an aqueous buffer of HEPES-buffered saline at pH 7.4 and pelletizing in a table top centrifuge at 18000 xg for 5 minutes. The microgel-based pellet was washed in HEPES buffered saline solution at pH 7.4 with 10 mM CaCl2 and 0.01% w / v Pluronic F-127 (Sigma). The aqueous microgel solution was then allowed to swell and equilibrate with buffer for at least 2 hours at 37 ° C.
[00145] To determine the operational regime of droplet segmentation, the operation of the device was monitored in real time using a high-speed camera (Phantom), followed by image analysis for the size and polydispersity measure (using the software ImageJ), as well as the frequency of segmentation (Fan-tasma PC2). For stable droplet segmentation on this platform: (i) start all flows simultaneously (both aqueous flows and both oil flows) at 5 μl / min until all air has been discharged from the device, (ii) turn down the aqueous flow rates at the desired general volumetric rate (aqueous flow rate between 1.5 and 2 μL / minute and oil flow rates between 1 and 5 μL / minute for 5 minutes, (iii) aspirate all accumulated liquid the collection well to guarantee collection of monodisperse μgels, and (iv) perform generation.
[00146] Balanced and completely swollen "building block" microgel particles were pelleted by centrifugation at 18000 xg for five minutes, and the excess buffer (HEPES pH 7.4 + 10 mM CaCl2) was removed by aspiration and drying with a cleaning cloth. Subsequently, the microgel particles were divided into aliquots, each containing 50 μl of concentrated building blocks. An equal volume of HEPES pH 7.4 + 10 mM Cacl2 was added to the concentrated building block solutions. Half of these include thrombin (Sigma) at a final concentration of 2U / mL and the other half includes FXIII (CSL Behring) at a final concentration of 10 U / ml. These solutions were then well mixed and centrifuged at 18000 xg, followed by removing the excess liquid with a cleaning cloth (American Cleanstat).
[00147] Annealing was initiated by mixing equal volumes of building block solutions containing thrombin and FXIII using a positive displacement pipette (Gilson). These solutions were mixed well by pipetting up and down, repeatedly, together, with stirring, using the tip of the pipette. The mixed solution was then pipetted to the desired location (mold, well plate, mouse wound, etc.).
[00148] To determine the gelation kinetics for each microgel, a macroscale (50 μL) of non-porous gel was generated with the same chemical composition. A 30 μl solution of 2X PEG-VS + peptides (RGD, K, Q peptides) dissolved in 0.3 M TEOA was combined with 30 μL of 2X MMP-1 crosslinker dissolved in water. The mixture was rapidly vortexed and 50 μL of the mixture was placed between two 8 mm rheological discs with a spacing of 1 mm (Anton Paar Physica Rheometer MCR301). The storage module was then measured over a period of 20 minutes (2.5 Hz, 0.1% deformation).
[00149] To determine the mass storage module of the pre-annealed microgel particles and post-annealed support an amplitude scan (0.01-10% strain) was performed to find the linear amplitude range for each. An amplitude within the linear range was chosen to perform a frequency scan (0.5-5Hz). For pre-annealed microgel particles, 50 μl of microgel particles (5% by weight of PEG-VS 4-arm PM = 20 kDa, r = 0.8 MMP-1 crosslinker, with concentrations of peptides synthetic 250 μM synthetic K, 250 μM synthetic Q, 500 μM synthetic RGD) was injected between two 8 mm rheological discs with a spacing of 1 mm. For the measurement of post-annealed support, 50 μl of microgel particles (N = 3) was first pipetted (5% by weight of PEG-VS 4-arm PM = 20 kDa, r = 0.8 MMP-1 of cross-linking agent, with synthetic peptide concentrations of 250 μM synthetic K, 250 μM synthetic Q, 500 μM synthetic RGD) enriched with FXIIIa, final concentration of 5 U / ml, and thrombin, 1 U / ml of final concentration, between two glass slides. This mixture was partially annealed for 10 minutes before removing the upper glass plate and placing it in a humidified incubator at 37 ° C for 90 minutes. The supports were then placed in HEPES-buffered saline (pH 7.4) during the night to achieve equilibrium. The samples were then placed between two 8 mm discs in the rheometer and tested in the same way as the pre-annealed microgel particles.
[00150] To determine the average pore size in the annealed microgel supports, microgel particle stock solutions of different sizes were used to hybridize three separate supports from each (9 supports in total), as described above. Using a Nikon Ti eclipse equipped with a confocal C2 laser LED, individual slices were taken on each gel, separated by 50 μm between each slice (10 slices per gel, with 30 total slices for each gel type). These images were then analyzed using a custom script recorded in MATLAB®, to identify the pore regions and calculate the size of each in px2. Each individual pore size was then used to calculate the average pore size for that gel, and converted to μm2 using the pixel to convert μm from the original microscopy image (0.31μm / px). These areas were then converted to a characteristic length measurement by forcing the areas into a circle, and calculating the characteristic diameter of those circles. For 30 μm of mi-crogel particles, mean pore diameter was about 12 μm. For 100 μm of microgel particles, the average pore diameter was about 19 μm. For 150 μm of microgel particles, the average pore diameter was about 37 μm. Note that the interstices or empty spaces are continuous and not similar to those of the well-defined spherical open regions connected by circular pores as produced by means of leaching via microparticles or reverse opal gel manufacturing methods, however, referring to if at a pore diameter it is useful to simply describe the length scale of the empty spaces.
[00151] To determine whether the microgel particles were covalently bound after the addition of FXIIIa, SEM was used to directly visualize supports. Mixtures of microgel particles were treated with FXIIIa (10 U / ml) or buffer alone. Subsequently, the building block solutions were placed on a 1 x 1 piece of silicon wafer, and dried in a SEM high vacuum chamber (Hitachi S4700) (1x103 mTorr). Building blocks with or without FXIIIa were then visualized using 10 kV (10 mA max) of either 200x or 500x as can be seen in FIGS. 9D and 9F.
[00152] HEK293T cells constitutively expressing GFP through lentiviral transfection were maintained in DMEM (Life Technologies) supplemented with 10 μg / ml of puromycin. Three cell lines were used for in vitro experiments: human dermal fibroblasts (HDF, Life Technologies), human mesenchymal stem cells derived from bone marrow (BMhMSC, Life Technologies), and human mesenchymal stem cells derived from adipose tissue (AhMSC, Life Technologies). All cell lines were maintained according to the manufacturer's specifications (before and after incorporation into porous or non-porous gels). Specifically, for MSC populations in reduced serum, basal medium (Life Technologies) was used to retain severity.
[00153] For the quantification of cell proliferation and visualization of the formation of the network in porous supports in vitro, supports based on particles were annealed with microgel particles, as described above, with the addition of cell suspensions for the building block solutions before annealing. For each cell line, cell suspensions were prepared at a final concentration of 25 x 106 cells / ml in respective culture media not supplemented with serum. Subsequently, 2 μl of cell suspension was added to 50 μl of the microgel particle mixture and containing FXIII combined with 50 μl of the microgel particle mixture containing thrombin (500 cells ^ l of gel). This mixture was injected into the corner of a sliding cover bottom PDMS well. The top of the well was covered with a second sliding layer and the μgel-cell mixture was allowed to undergo annealing for 90 min at 37 ° C.
[00154] After annealing is completed, the upper sliding cap was removed, and the appropriate complete culture medium was added to the PDMS well. For the time point of day 0, 4% PFA was added directly to the PDMS wells and allowed to set overnight at 4 ° C. Other cells were cultured at 5% CO2 and 37 ° C for the indicated times (2, 4, and 6 days), at which time they were washed once with 1X PBS and fixed with 4% PFA overnight at 4 ° C. ° C. HEK-293-T cells were incorporated into a star-shaped mold by mixing the cells with microgel particles (as described above) and pipetting 5 μl of the mixture into the center of the mold. Immediately thereafter, the microgel particles without cells were pipetted into the remainder of the mold, and annealed as described above.
[00155] Proliferation was assessed by counting the number of cell nuclei present in the particle-based support constructs after 0, 2, 4, and 6 days of in vitro culture. The cores were stained with a 2 μg / ml DAPI solution in 1X PBS for 2 hours, followed by visualization of a Nikon C2 using the 405 nm LED laser. Specifically, each support was photographed taking 55 z slices at a total z height of 150 μm and compressing each 5 slices into a maximum intensity projection image (MIP). MIP cores were enumerated using a customized MATLAB® script, counting the total number of cells. For each time point, images from stack z of three separate microgel holders were analyzed, where each image from stack z measured a total volume of 1270 x 1270 x 150 μm3 (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).
[00156] For the visualization of cellular network formation within microgel supports in vitro, constructs were prepared, cultured, and fixed as described above. The supports were blocked with 1% BSA in 1X PBS for 1 hour at room temperature, followed by staining for f-actin by means of a rhodamine-B phalloidin conjugate (Life Technologies) for 3 hours at room temperature. The supports were then washed with 1% BSA in 1X PBS, followed by counterstaining with a 2 μg / ml DAPI solution in 1X PBS for 1 hour at room temperature. The visualization was performed as in proliferation imaging, with the exception of the use of a 40x magnification water immersion lens. Total heights of image stacks was 130 μm, with the total number of layers at 260 (volume captures ~ 15 nl).
[00157] PEG-VS supports (5% by weight of PEG-VS 4-arm PM = 20 kDa, r = 0.8 MMP-1 crosslinker, with synthetic peptide concentrations of 250 μM synthetic K [SEQ ID NO: 1], 250 μM synthetic Q [SEQ ID NO: 2], 500 μM synthetic RGD [SEQ ID NO: 3]) were used to encapsulate cells (500 cells ^ L). The cell lines used were the same as in microgel support experiments. Gels were formed for 20 minutes (TEOA 0.3 M, pH 8.25) before being placed in appropriate media. The gels were fixed after predetermined time points (T = 90 minutes, 2 days, 4 days, and 6 days), using PFA overnight at 4 ° C, washed and stored in PBS. The gels were stained as in microgel holders. All samples were stored at 4 ° C in PBS with P / S when not in immegem. The visualization was performed using a NIKON® C2 confocal exactly as in the microgel holder in in vitro experiments.
[00158] CLR: SKH1-Hrhr mice (Charles River Laboratories) (N = 6 per test) were anesthetized with isofluorane (1.5% for 10 minutes), followed by nail cutting and analgesic injection (buprenorphine, 60 μL per 20 g to 0.015 μg / μL). The skin was stretched and a 4 mm biopsy perforator was used to create identical circular wounds on the rear of the mouse. The wound periphery was protected using a rubber splint sewn through 7-8 stitches around the skin to prevent the wound from closing by contraction. Non-porous or porous hydrogel including 10 U / ml of FXIIIa was injected into the wound beds, allowing them to gel for 10 minutes, followed by posterior wound dressing by an elastic 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 to kill the pain were administered once after surgery).
[00159] On Day 1, mice (N = 6) were sacrificed by overdosing with isofluorane, followed by displacement of the posterior spine. The dorsal skin was removed using surgical scissors and the wound site was isolated using a 10 mm biopsy perforator. The samples were immediately fixed with 4% formaldehyde at 4 ° C (overnight), followed by transfer to ethanol and incorporating the sample in a paraffin block. The blocks were sectioned at 6 μm thick per microtome (Leica) and were stained with Hematoxylin and Eosin (H & E). For quantification of cell infiltration within the hydrogels and immune response around the hydrogels, a series of 3 random high-power (40X) fields (HPF) were examined for each section. The samples were analyzed for cell infiltration (> 0.1 mm in the gel) by counting the total number of cells of any type in the injected hydrogels (N = 5 with a sum of cells in 3 sections analyzed by wound) ). More than 95% of the cells that infiltrated the gels were neutrophils. To measure the immune response, an 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 edge of the wound was quantified and averaged for each type of wound. The leukocyte count for each wound was compared with its bilateral control in the same animal and the relative difference was recorded as a fraction of the general immune response of each animal. This comparison was possible because each animal had a wound injected with the support of a microgel and a wound with the non-porous control.
[00160] The wounds were photographed daily after closing the wounds. Each wound site was photographed using a high resolution camera (NIKON® COOLPIX®). Closing fraction was determined by comparing the pixel area of the wound to the pixel area within the 10 mm central hole of the red rubber splint. Closing fractions were normalized to Day 0 for each mouse / support type (FIG. 7B).
[00161] On day 5, mice (N = 6) were sacrificed and tissue collected on day 1. The samples were immediately submerged in fluid of ideal cutting temperature TISSUE-TEK® (OCT) and frozen in a solid block with liquid nitrogen. The blocks were then cryo-sectioned at a thickness of 25 μm per 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 maintained at 4 ° C until they were stained.
[00162] Slides containing sections of tissue were either blocked with 3% normal goat serum (NGS) in 1X PBS + 0.05% Tween-20 (PBST) and blocked or simultaneously permeabilized with 0.2% Triton® X -100 in 3% NGS in 1X PBST for sections stained with anti-keratin-5 antibody only. The sections were then washed in 3% NGS in 1X PBST. Primary antibody dilutions were prepared as follows in 3% NGS in 1X PBST: Anti-mouse mouse CD11b (BD Pharmingen) - 1: 100 Anti-mouse mouse PECAM-1 (BD Pharmingen) - 1: 100 Antibody mouse NG2 ( Millipore) - 1: 100 Keratin-5 from anticoelho mouse (Covance, Inc.) - 1: 250
[00163] The sections were stained with primary antibodies overnight at 4 ° C, and subsequently washed with 3% NGS in 1X PBST. Secondary antibodies were all prepared in 3% NGS in 1X PBST at a dilution of 1: 100. The sections were incubated in secondary antibodies for 1 hour at room temperature, and subsequently washed with 1X PBST. The sections were contrasted with 2 μg / ml DAPI in 1X PBST for 30 minutes at room temperature. The sections were assembled using Antifade Gold mountings.
[00164] Confocal Z stack images acquired from tissue sections from day 5 of both non-porous tissue blocks and microgel structure were compressed into MIPs, followed by separation into individual images corresponding to each of the laser channels ( i.e., gel, DAPI, CD11b). The image of the gel channel was used to trace the edge of the gel - tissue interface using the Adobe illustrator. The width of this line has been increased by 75 μm for both the tissue and into the gel from the interface (150 μm in total thickness). The new ends of this line were then used to cut the original DAPI and CD11b images, to capture only the areas corresponding to +/- 75 μm from the tissue gel interface. These images were then imported into ImageJ, and covered to merge the DAPI and CD11b channels into a single image. This image was analyzed using the ImageJ cell counter plugin, in which both the total number of nuclei was quantified, as well as the total number of CD11b + cells. Finally, the fraction of nuclei with a corresponding CD11b + signal was reported both within the tissue and within the gel.
[00165] FIG. 11 illustrates an example of a method of treating damaged tissue 102. FIG. 11 illustrates a wound site 100 formed in tissue 102 of a mammal. In operation 500, a delivery device 110 (for example, tube, as shown) that contains the suspension of microgel particles 12 contained in an aqueous solution in it is used to deliver microgel particles 12 to wound site 100. In Then, as seen in operation 510, an optional applicator 122 is used to spread the microgel particles 12 into and through the wound site 100. Applicator 122 is also used to stain the exposed, upper surface of the microgel particles. 12, generally flush with the surface of tissue 102. Applicator 122 can also be used to stain the top, exposed surface of microgel particles 12 stacked or elevated relative to the surface of tissue 102 to allow for greater structure for growth internal cell and preventing an interface with the compressed tissue after complete healing. Then, as seen in operation 520, the annealing of the microgel particles 12 is initiated to form the support 10 of annealed microgel particles 12. In this particular example, a light source 124 in the form of a flashlight is used to illuminate a mixture of microgel particles 12, a photoinitiator (for example, eosin Y), and a free radical transfer agent (for example, RGD peptide). Of course, other methods of annealing can also be used, as described herein. The annealing reaction illustrated in FIG. 11 causes the formation of a covalently stabilized support 10 of microgel particles 12 having interstitial spaces in them. The cells 106 (as seen in FIG. 2C) from the surrounding tissue 102, then begin to infiltrate the spaces within the support 10, grow, stimulate, and finally effect the healing process of tissue 102. In one embodiment, after the annealing reaction, a wet bandage or bandage is optionally placed over the wound filled with support to protect it from damage during the healing process. After a period of time has elapsed, as illustrated in operation 530, support 10 is degraded and tissue 102 is returned to a cured state.
[00166] In order to assess the ability of the porous gel support to support cell growth and network formation, an in vitro cell morphology and proliferation model was developed using three human cell lines: dermal fibroblasts (HDF), cells adipose tissue-derived mesenchymal stem cells (AhMSC) and bone marrow-derived mesenchymal stem cells (BMh-MSC). A single cell suspension was dynamically incorporated into an annealed porous gel support FXIIIa. The three cell lines exhibited high cell viability (> 93%, FIG. 12B) after twenty-four (24) hours of culture inside the porous gel support.
[00167] The cells incorporated in the porous gel support began to exhibit propagation morphology ninety (90) minutes after the appearance of annealing. After two (2) days in culture, all the cells observed inside the porous gel supports exhibited a completely spread morphology, which continued until day six. Importantly, a vast training network for all cell lines was observed on day two. Cellular networks have increased in size and complexity across the entire experience. BMhMSCs were of particular interest, as their expansive network formation and slower proliferation rate indicated that these cells were able to spread to extremes, forming highly interconnected cell networks in microporous supports as seen in FIG . 12A.
[00168] The microgel particles 12 can be combined and mixed with a solution of live cells 106 before annealing to create a microporous support 10 containing live cells 106 that reside in the microporous network and dispersed either homogeneously or heterogeneously within the macroscopic annealed gel holder 10 as seen in FIG. 13A.
[00169] The microgel particles 12 can be purified to an aqueous solution of isotonic cell culture media for storage and, when used to form a porous gel, they are re-fired together through a non-canonical amide bond between K and Q peptides mediated by activated factor XIII (FXIIIa), a naturally occurring enzyme responsible for stabilizing blood coagulates. This enzyme-mediated annealing process allowed the incorporation of living cells 106 into a porous support of dynamic formation 10 that contained interconnected microporous networks. Following the addition of FXIIIa, but prior to annealing support, a suspension of the microgel particles 12 can be delivered via syringe application (Fig. 13A), ultimately solidifying into the shape of the cavity in which they are injected as can be seen in FIGS. 13B-E.
[00170] Microfluidic manufacture of microgel particles 12 allows deterministic control over microgel size and frequency production 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). In addition, porous microgel holders 10 created using microgel particles of different sizes 12 have distinct porous characteristics, such as the average pore size within the network, as seen in fig. 14C.
[00171] While modalities have been shown and described, several modifications can be made without departing from the scope of the inventive concepts described here. The matter described herein, therefore, should not be limited, except for the following claims and their equivalents.

权利要求:
Claims (28)
[0001]
1. Microporous gel system, characterized by the fact that it comprises: an aqueous solution comprising a plurality of spherical microgel particles having a diameter in the range of 30 μm to 1000 μm; and an annealing agent which, when applied to the plurality of spherical microgel particles, causes the spherical microgel particles to form a covalently stabilized scavenger of spherical microgel particles having interconnected interstitial spaces within them where the middle pore diameter is at least 12 μm.
[0002]
Microporous gel system according to claim 1, characterized in that the spherical microgel particles still comprise a degradable crosslinking agent.
[0003]
Microporous gel system according to claim 2, characterized in that the degradable crosslinking agent comprises a degradable crosslinker with matrix metalloprotease (MMP).
[0004]
4. Microporous gel system according to claim 1, characterized by the fact that the annealing agent comprises Factor XIIIa.
[0005]
Microporous gel system according to claim 1, characterized in that the annealing agent comprises Eosin Y, a free radical transfer agent, or a combination thereof.
[0006]
Microporous gel system according to claim 5, characterized in that it further comprises a light source configured to illuminate a mixture of the plurality of spherical microgel particles and the annealing agent.
[0007]
7. Microporous gel system according to claim 1, characterized in that the spherical microgel particles comprise adhesive peptides from cells exposed on a surface thereof.
[0008]
8. Microporous gel system according to claim 1, characterized in that the spherical microgel particles comprise a K peptide.
[0009]
Microporous gel system according to claim 8, characterized in that the K peptide comprises a lysine group recognized by Factor XIIIa.
[0010]
10. Microporous gel system according to claim 1, characterized in that the spherical microgel particles comprise a Q peptide.
[0011]
11. Microporous gel system according to claim 10, characterized in that the Q peptide comprises a group of glutamine recognized by Factor XIIIa.
[0012]
12. Microporous gel system according to claim 1, characterized by the fact that the interconnected interstitial spaces comprise edge surfaces exhibiting negative concavity.
[0013]
13. Microporous gel system according to claim 1, characterized in that the covalently stabilized scavenger of spherical microgel particles has an empty volume of 10% to 50%.
[0014]
14. Microporous gel system according to claim 1, characterized in that it still comprises a delivery device.
[0015]
Microporous gel system according to claim 14, characterized in that the delivery device contains the aqueous solution comprising a plurality of spherical microgel particles and the annealing agent or precursor of the annealing agent.
[0016]
Microporous gel system according to claim 15, characterized in that the delivery device comprises a single compartment delivery device containing the aqueous solution comprising a plurality of spherical microgel particles and the annealing agent.
[0017]
17. Microporous gel system according to claim 16, characterized in that the delivery device comprises a double compartment delivery device, wherein one compartment contains the aqueous solution containing a plurality of spherical microgel particles and a first annealing agent precursor and the second compartment contains the aqueous solution containing the plurality of spherical microgels and a second annealing agent precursor.
[0018]
18. Microporous gel system according to claim 3, characterized in that the degradable crosslinking agent (MMP) comprises at least one D amino acid.
[0019]
19. Microporous gel system according to claim 18, characterized in that the spherical microgel particles comprise a degradable crosslinking agent (MMP) comprising a plurality of D amino acids.
[0020]
20. Microporous gel system according to claim 1, characterized in that the spherical microgel particles are present in a volumetric fraction of 30-99% in the aqueous solution.
[0021]
21. Microporous gel system according to claim 1, characterized by the fact that the spherical microgel particles have a minimum storage module of 10 Pa.
[0022]
22. Microporous gel system according to claim 1, characterized by the fact that the interconnected interstitial spaces are defined by an average pore diameter of 12 μm to 37 μm.
[0023]
23. Microporous gel system according to claim 1, characterized by the fact that it is capable of undergoing annealing in 30 minutes or less.
[0024]
24. Microporous gel system according to claim 1, characterized in that it comprises: a delivery device; a plurality of spherical biodegradable microgel particles having a diameter in the range of 30 μm to 1000 μm contained in an aqueous solution and stored in the delivery device; and an annealing agent or precursor to the annealing agent stored in the delivery device.
[0025]
25. Use of a plurality of spherical microgel particles and an annealing agent, as defined in any one of claims 1 to 24, characterized by the fact that it is for the manufacture of a wound healing medicament, wherein the wound healing agent Annealing pairs the spherical microgel particles to form a covalently stabilized scavenger having interstitial spaces interconnected in it where the mean pore diameter is at least 12 μm.
[0026]
26. Use of layers of covalently stabilized sequestrants of spherical microgel particles, as defined in any one of claims 1 to 24, having a diameter in the range of 30 μm to 1000 μm, in which at least one covalently stabilized sequester has interstitial spaces interconnected in it defined by an average pore diameter of at least 12 μm, characterized by the fact that it is for the manufacture of a medicine for wound healing.
[0027]
27. Use of a covalently stabilized scavenger of spherical microgel particles having a diameter in the range of 30 μm to 1000 μm having interconnected interstitial spaces therein defined by an average pore diameter of at least 12 μm, as defined in any of claims 1 to 24, characterized by the fact that it is for the manufacture of a medicine for wound healing.
[0028]
28. Method for the preparation of spherical microgel particles, as defined in any one of claims 1 to 24, characterized by the fact that it comprises: providing a microfluidic device that generates droplets of water in oil that has a plurality of channels of inlets leading to a common channel and a pair of oil compression channels that intersect the common channel at a downstream location; flowing a first prepolymer solution containing a polymeric backbone modified with oligopeptides in a first inlet channel; flowing a second solution containing a biodegradable crosslinking agent to a second inlet channel; an oil and a surfactant flow into the pair of oil compression channels to form droplets containing the first prepolymer solution and the second solution; and collecting spherical microgel particles formed by cross-linking the droplets.

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Ndreu-Halili0|Cell/Tissue Microenvironment Engineering and Monitoring in Tissue Engineering, Regenerative Medicine, and In Vitro Tissue Models

同族专利:

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公开号 | 公开日

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US20200085859A1|2020-03-19|

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IL250092A|2021-05-31|

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EP3169372A1|2017-05-24|

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IL250092D0|2017-03-30|

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BR112017000813A2|2017-12-05|

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US20180078671A1|2018-03-22|

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KR20210072133A|2021-06-16|

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AU2015289474B2|2019-12-05|

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CN106714854B|2020-09-04|

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JP6651500B2|2020-02-19|

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KR20170031741A|2017-03-21|

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CN111939316A|2020-11-17|

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KR102264607B1|2021-06-14|

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US20190151497A1|2019-05-23|

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CA2955357A1|2016-01-21|

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WO2016011387A1|2016-01-21|

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IL282559D0|2021-06-30|

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US10912860B2|2021-02-09|

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US20170368224A1|2017-12-28|

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AU2015289474A1|2017-02-02|

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JP2017522113A|2017-08-10|

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US20160279283A1|2016-09-29|

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CN106714854A|2017-05-24|

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US20210138105A1|2021-05-13|

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EP3169372A4|2018-03-21|

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JP2020075150A|2020-05-21|

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法律状态:
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2020-09-29| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-01-12| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-16| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 17/07/2015, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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