Biocompatible hyaluronic acid hydrogels

Abstract

The present disclosure describes biocompatable hydrogels, isocyanate cross-linkers, and methods for preparation and use of the same. Cross-linking of polymers such as hyaluronic acid and other polymers with poly-functional isocyanate cross-linkers form urethane cross-linked polymers which may be hydrated in various aqueous media to form hydrogels. Cross-linkers include isocyanate terminated polycaprolactones, optionally modified with small molecules such as dopamine. Example hydrogels include dermal fillers for use in cosmetic and medical applications such as tissue engineering, wound healing, and controlled drug release. Hydrogels described herein achieve enhanced resistance to enzymatic degradation and hydrolysis, effectively prolonging the in vivo lifespan of the hydrogel, and address challenges in hydrogel longevity, safety, and versatility. Balancing hydrophobic and hydrophilic segments within the hydrogel allows precise control over key hydrogel properties, including elasticity, viscosity, and porosity. In vitro and in vivo studies confirm significant improvements over existing technologies.

Description

BIOCOMPATIBLE HYALURONIC ACID HYDROGELSCROSS REFERENCE

[0001] This PCT application claims the benefit to U.S. Provisional Application No. 63 / 618,119 filed January 5, 2024, the contents of which are incorporated herein by reference.INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BACKGROUND

[0003] Dermal fillers have become integral to aesthetic medicine and therapeutic applications, serving to enhance or restore facial contours, reduce the appearance of wrinkles and lines, and correct deformities or asymmetries. A variety of materials, both natural and synthetic, have been utilized as dermal fillers over the years, with hyaluronic acid-based hydrogels emerging as a possible choice due to their biocompatibility, hydrating properties, and reversible nature. However, the rapid degradation of these hydrogels in the body has led to a quest for materials and methods to extend their in vivo lifespan.

[0004] Hyaluronic acid (HA) is a naturally occurring glycosaminoglycan found in various tissues in the human body. HA's unique ability to retain water and its intrinsic biocompatibility has made it an attractive starting material for creating dermal fillers. Despite its many advantages, HA-based hydrogels have faced challenges in terms of their relatively short in vivo lifespan. This degradation often requires patients to undergo frequent and sometimes uncomfortable reinjections, adding to both the financial and time burdens.

[0005] Cross-linking plays a crucial role in the development and functionality of hyaluronic acid-based hydrogels, dictating several physical properties such as mechanical strength, degradation rate, and overall performance. Traditional methods have employed diepoxide cross-linkers, such as 1,4-butanediol diglycidyl ether (BDDE), to achieve these objectives. BDDE has been widely used as a cross-linking agent due to its highly reactive epoxy groups that enable effective cross-linking. However, its usage has raised significant concerns.

[0006] BDDE is a water-soluble molecule, and its high reactivity makes it difficult to control the cross-linking reaction's selectivity. This difficulty leads to the potential presence of partially reacted or unreacted residual BDDE in the final hydrogel product. Residual BDDE, being a toxic substance, raises alarming health concerns when used in dermal filler applications. Research has demonstrated that even trace amounts of BDDE may result in local inflammation, tissue necrosis, or other adverse reactions.

[0007] The use of BDDE as a cross-linker has also been linked to unwanted immune responses in some cases. Unpredictable interactions between BDDE-cross-linked hydrogels and the host's immune system can cause complications such as prolonged inflammation or foreign body reactions. These responses not only affect the filler's cosmetic appearance but may also lead to longer-term health concerns that necessitate medical intervention.

[0008] Despite its ability as an effective cross-linker, BDDE does not provide sufficient longevity in vivo. The linkages formed by BDDE are susceptible to enzymatic degradation and hydrolysis, leading to the rapid breakdown of the hydrogel. This limited lifespan is one of the significant drawbacks of BDDE-cross-linked hydrogels, necessitating frequent reinjections, adding to the inconvenience and costs for patients.

[0009] Furthermore, the use of BDDE as a cross-linking agent offers a restricted range of properties for customization. While cross-linking density can be controlled to some extent, achieving a finely tuned balance between various desirable hydrogel characteristics like elasticity, viscosity, and porosity has been challenging. This limitation restricts the versatility and potential applications of BDDE-cross-linked hydrogels.

[0010] The conventional method of cross-linking hydrogels using di epoxide crosslinkers like BDDE has revealed substantial limitations and concerns. From toxicity and immune responses to limited longevity and a restricted set of achievable properties, these challenges have driven the need for innovative cross-linking moieties and methods. The persistent issues surrounding BDDE and similar cross-linking agents highlight the urgency for approaches that offer both enhanced performance and safety in dermal filler applications.

[0011] There exists a significant need for innovative approaches to dermal filler hydrogel design that can offer enhanced in vivo longevity without sacrificing other key attributes. The need extends beyond aesthetic applications to medical and therapeutic uses, where consistent, long-lasting hydrogel performance is essential. The burgeoning demand for effective, long-lasting dermal fillers in the cosmetic industry and the increasingrecognition of the potential therapeutic applications of such hydrogels have heightened the urgency of this unmet need.BRIEF SUMMARY OF THE INVENTION

[0012] Hyaluronic acid-based dermal filler hydrogels disclosed herein are specifically designed to address limitations in the existing landscape of dermal fillers and hydrogels. While the current market offers a variety of hydrogel fillers, many suffer from serious limitations such as toxicity, limited longevity, a lack of reversibility and a constrained set of achievable performance characteristics. Hyaluronic acid cross-linking technology described herein allows for the formation of hydrogels addressing these shortcomings, thereby offering advanced solutions in the fields of cosmetic and therapeutic applications.

[0013] Example cross-linking moieties include a defined set of polycaprolactone alcohols and isocyanates that cross-link hyaluronate acid with urethane linkages. Unlike conventional cross-linkers like BDDE, which are prone to toxicity, impair a limited range of properties and suffer from rapid in vivo degradation, the urethane linkages impart improved resistance to enzymatic degradation and hydrolysis. The incorporated polycaprolactone can also slow the rate of degradation and introduce highly desirable benefits such as collagen stimulation. These features enhance the hydrogel's longevity in vivo and provide an inherent safety advantage, significantly minimizing the risks associated with residual cross-linker toxicity and immune responses from unreacted epoxide groups.

[0014] Another innovative aspect is the tunable balance between hydrophobic and hydrophilic segments in the cross-linked structure. The nature and quantity of the crosslinker can be tuned to produce hydrogels with varying elasticity, viscosity, porosity, and degradation rate. The ability to fine-tune these characteristics paves the way for a versatile and customizable hydrogel that can be tailored to specific needs and applications, ranging from aesthetic enhancements to controlled drug delivery, wound healing and tissue engineering.

[0015] An example formulation process of a hydrogel of the present invention includes a precisely defined pathway for synthesis of the cross-linking moiety, followed by a controlled cross-linking method with hyaluronic acid. The synthesis process involves converting hyaluronic acid to an organic soluble form, forming the cross-linker by terminating the polycaprolactone alcohols with isocyanate groups, then cross-linking hyaluronic acid to form the desired product. Additionally, when there are enough crosslinking moieties present, dopamine can be conjugated to the polycaprolactone backbone orHA to form an adhesive hydrogel. This method can also be used to conjugate other groups onto the polycaprolactone backbone or onto HA directly, which highlights the versatility of the platform.

[0016] In addition to its primary application in cosmetic enhancements, the hydrogels described herein present a wide spectrum of therapeutic and medical applications. The unique designs allow leveraging for additional applications in controlled drug release, tissue engineering, and wound healing. The innovation transcends the conventional constraints of hydrogel fillers, showcasing a wide breadth of potential impact.

[0017] The present invention represents a significant advancement in the field of hydrogels and hyaluronic acid-based materials. Cross-linking moieties capable of forming chemically resistant urethane linkages and introducing a balance between hydrophobic and hydrophilic segments provide material solutions over previously described hydrogels with distinct advancements. The multifaceted designs described herein address current challenges faced by the industry, including concerns over toxicity, limited longevity, and constrained properties, thereby ushering in a new era of safe, durable, and versatile dermal fillers.

[0018] The present invention provides a biocompatible hydrogel. In some embodiments, the biocompatible hydrogel comprises a hydrated, cross-linked polymer comprising a plurality of urethane linkages. In some embodiments, the biocompatible hydrogel comprises a hydrated, cross-linked polysaccharide comprising a plurality of urethane linkages.

[0019] In some embodiments, the biocompatible hydrogel comprises a) hyaluronic acid; b) a poly-functional urethane cross-linker reacted with the hyaluronic acid to form a crosslinked polymer with a plurality of urethane linkages; and c) an aqueous fluid configured to swell the cross-linked polymer to form the biocompatible hydrogel material.

[0020] In some embodiments, the hydrogel is configured for cosmetic use, medical use, use as a dermal filler, use in wound care, use in tissue engineering, use in controlled drug release, to encapsulate a therapeutic agent, or any combination thereof.

[0021] The present invention also provides a cross-linked polymer (e.g. a polysaccharide) for formation of a biocompatible hydrogel. In some embodiments, the cross-linked polymer comprises a) a polymer (e.g. a polysaccharide); and b) a polyfunctional urethane cross-linker reacted with the polymer to form a cross-linked polymer with a plurality of urethane linkages; wherein the cross-linked polymer is configured to swell in an aqueous fluid to form a biocompatible hydrogel.

[0022] The present invention also provides an isocyanate cross-linker for formation of a biocompatible hydrogel. In some embodiments, the cross-linker comprises a) a polyfunctional core; b) a plurality of di-functional polymeric linkers; each bound to the polyfunctional core; and c) a plurality of isocyanate substituents, each bound to one of the plurality of di-functional polymeric linkers such that the isocyanate substituents are coupled with the poly-functional core via the di-functional polymeric linkers, thereby forming the cross-linker; wherein the cross-linker is configured to react with a polymer to form a cross-linked polymer configured to swell in an aqueous fluid to form a biocompatible hydrogel.

[0023] The present invention provides a method of preparing a cross-linked polymer (e.g. a cross-linked polysaccharide) for formation of a biocompatible hydrogel. In some embodiments, the present invention provides a method of preparing a cross-linked polymer for formation of a biocompatible hydrogel, comprising a) providing a polyfunctional isocyanate cross-linker; b) reacting a polymer with an organic compound to form an organic polymer salt, wherein the organic polymer salt is soluble in an organic solvent; and c) reacting the organic polymer salt with the poly-functional isocyanate crosslinker in the organic solvent to form a plurality of urethane cross-links, thereby forming the cross-linked polymer; wherein the cross-linked polymer is configured to swell in an aqueous fluid to form a biocompatible hydrogel. In some embodiments, the present invention provides a method of preparing a cross-linked polysaccharide for formation of a biocompatible hydrogel, comprising a) providing a poly-functional isocyanate crosslinker; b) reacting a polysaccharide with an organic compound to form an organic polysaccharide salt, wherein the organic polysaccharide salt is soluble in an organic solvent; and c) reacting the organic polysaccharide salt with the poly-functional isocyanate cross-linker in the organic solvent to form a plurality of urethane cross-links, thereby forming the cross-linked polysaccharide; wherein the cross-linked polysaccharide is configured to swell in an aqueous fluid to form a biocompatible hydrogel.

[0024] The present invention provides a method of preparing a biocompatible hydrogel. In some embodiments, the method comprises a) providing a cross-linked polymer of any one of the preceding claims; and b) hydrating the cross-linked polymer with an aqueous fluid to form a biocompatible hydrogel. In some embodiments, the method comprises a) providing a cross-linked polysaccharide of any one of the preceding claims; and b) hydrating the cross-linked polysaccharide with an aqueous fluid to form a biocompatible hydrogel.

[0025] The present invention provides a cosmetic skin treatment method. In some embodiments, the method comprises a) providing a hydrogel material of any one of the preceding claims; and b) performing subdermal injection a volume of the hydrogel material via a needle.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and accompanying drawings, that set forth illustrative embodiments in which the principles of the present disclosure are utilized.

[0027] FIG. 1 depicts sodium hyaluronate, an example polymer for cross-linking to form a biocompatible hydrogel, n is an integer.

[0028] FIG. 2 depicts cetrimonium hyaluronate, an organic -soluble form of hyaluronic acid.

[0029] FIG. 3 depicts ethyl lysine diisocyanate (eLDI), an example di-functional isocyanate cross-linker.

[0030] FIG. 4 depicts dopamine.

[0031] FIG. 5a depicts a an example tetrol core, 2,2-bis(((6-((6- hydroxyhexanoyl)oxy)hexanoyl)oxy)methyl)propane- 1,3 -diyl bis(6-((6- hy droxy hexanoy l)oxy)hexanoate) .

[0032] FIG. 5b depicts a tetrol core with extended polycapralactone linkages, n is an integer.

[0033] FIG 6. depicts a an example tetrol cross-linker, formed by reation of a tetrol with eLDI to yield a tetra-functional isocyanate. R represents a tetrol core, which could include extended polycapralactone linkages.

[0034] FIG. 7 depicts a cross-linked tetrol with isocyanate terminated groups. This example can be formed by linking two tetrol cores via eLDI to form a hexafunctional isocyanate. R represents a tetrol core, which could include extended polycapralactone linkages.

[0035] FIG. 8A depicts hyaluronic acid cross-linked with an example tetrol cross-linker. R represents a tetrol core, which could include extended polycapralactone linkages, n is an integer.

[0036] FIG. 8B depicts hyaluronic acid cross-linked with an example tetrol cross-linker. R represents a tetrol core, which could include extended polycapralactone linkages. HA represents hyaluronic acid.

[0037] FIG. 9 depicts an example dopamine-modified tetrol cross-linker. The tetrafunctional isocyanate cross-linker is reacted with two molecules of dopamine to yield a functionalized difunctional isocyanate cross-linker. R represents a tetrol core, which could include extended polycapralactone linkages.

[0038] FIG. 10A depicts hyaluronic acid cross-linked with the dopamine-modified tetrol cross-linker. R represents a tetrol core, which could include extended polycapralactone linkages, n is an integer.

[0039] FIG. 10B depicts hyaluronic acid cross-linked with the dopamine-modified tetrol cross-linker. R represents a tetrol core, which could include extended polycapralactone linkages. HA represents hyaluronic acid.

[0040] FIG. 11 A depicts an example triol.

[0041] FIG. 1 IB depicts an example triol with extended polycapralactone linkages, n is an integer.

[0042] FIG. 12A depicts an example trifunctional isocyanate cross-linker formed by the reaction of a triol with eLDI.

[0043] FIG. 12B depicts an example trifunctional isocyanate cross-linker formed by the reaction of a triol with eLDI. R represents a trifunctional core which could include extended polycapralactone linkages.

[0044] FIG 13A. depicts hyaluronic acid cross-linked with an example trifunctional isocyanate cross-linker. R represents a trifunctional core which could include extended polycapralactone linkages, n is an integer.

[0045] FIG 13B. depicts hyaluronic acid cross-linked with an example trifunctional isocyanate cross-linker. R represents a trifunctional core which could include extended polycapralactone linkages. HA represents hyaluronic acid.

[0046] FIG. 14A depicts a dopamine-modified difunctional isocyanate cross-linker, formed from a triol core.

[0047] FIG. 14B depicts a dopamine-modified difunctional isocyanate cross-linker, formed from a triol core. R represents a trifunctional core which could include extended polycapralactone linkages.

[0048] FIG. 15A depicts hyaluronic acid cross-linked with an example dopamine- modified cross-linker formed from a triol. R represents a trifunctional core which could include extended polycapralactone linkages, n is an integer.

[0049] FIG. 15B depicts hyaluronic acid cross-linked with an example dopamine- modified cross-linker formed from a triol. R represents a trifunctional core which could include extended polycapralactone linkages. HA represents hyaluronic acid.

[0050] FIG. 16 depicts a diol having extended polycapralactone linkages, n is an integer.

[0051] FIG. 17A depicts a difunctional isocyanate cross-linker formed by reaction of a diol with eLDI. n is an integer.

[0052] FIG. 17B depicts a difunctional isocyanate cross-linker formed by reaction of a diol with eLDI. R represents a difunctional core, optionally having extended polymer linkages.

[0053] FIG. 18A depicts hyaluronic acid cross-linked with an example difunctional isocyanate cross-linker, n is an integer.

[0054] FIG. 18B depicts hyaluronic acid cross-linked with an example difunctional isocyanate cross-linker. R represents a difunctional core, optionally having extended polymer linkages. HA represents hyaluronic acid.

[0055] FIG. 19 depicts the degree of swelling for Commercial Gel 3 and example hydrogels DFF10, DFF4, and DFF5 as a percentage of the initial gel mass. Error bars indicate the standard deviation of 3 samples.

[0056] FIG. 20 depicts a hyaluronidase-based degradation assay of three DF example hydrogel formulations, DFF4, DFF5, DFF10, and Commercial Gel 3 for comparison.

[0057] FIG. 21 depicts the viability of DF example hydrogel formulations DFF4, DFF5, DFF9, DFF10 and CCF3 (Commercial Gel 3). NC: Negative control, PC: Positive control (DMSO)

[0058] FIG. 22 depicts a graph of the number of inflammatory cells for all three groups (PT-J, PT-S, and PT-T) at three, seven, thirty, and sixty day timepoints.

[0059] FIG. 23 depicts images depicting inflammatory response at different timepoints represented by dermal filler PT-T of the present invention.

[0060] FIG. 24 depicts a graph of filler area for both groups at three, seven, thirty, and sixty day timepoints.

[0061] FIG. 25 depicts images depicting collagen production for both test and commercial fillers at early and late timepoints.DETAILED DESCRIPTIONI. GENERAL

[0062] This disclosure provides a hyaluronic acid-based hydrogel that offers significant improvements over existing technologies. A unique cross-linking moiety allows for a harmonious balance between hydrophobic and hydrophilic segments to create a variety of hydrogels. Cross-linking moieties of the present invention can also be used to introduce other components into the hydrogel to extend the benefits of the material.

[0063] Polycaprolactone (PCL) is a biodegradable polyester suitable for use in dermal fillers. Its biodegradability ensures safety and natural metabolization over time, making it a preferable alternative to permanent synthetic fillers. PCL fillers are also long-lasting, reducing the need for frequent repeat treatments, which can be cost-effective. Known for its strong safety profile and biocompatibility, PCL poses a low risk of allergic reactions. Its versatility allows for customization, making it suitable for treating both deep wrinkles and fine lines. Additionally, PCL stimulates collagen production, leading to naturallooking and gradual cosmetic improvements.

[0064] The family of cross-linking moieties include examples having isocyanate groups and polycaprolactones terminated with these isocyanate groups, which would cross-link polymers such as hyaluronic acid to form chemically resistant urethane linkages. These linkages can impart enhanced resistance to enzymatic degradation and hydrolysis, thereby extending the in vivo lifespan of the hydrogel. These urethane linkages can be synergistic with an added polycaprolactone backbone, which can provide even slower degradation rates and can promote collagen formation. By carefully controlling the isocyanate groups, polycaprolactone backbone and the cross-linking density, the hydrophobic and hydrophilic segments of the gel can be gently balanced to allow precise control over key hydrogel properties, including elasticity, viscosity, and porosity, without sacrificing biocompatibility. This enables the tailoring of the hydrogel to specific applications, ensuring optimal performance.

[0065] The formulation process for these hydrogels is also important. The synthesized products can be hydrated easily in various media to unlock further benefits. These media can include water, PBS, hyaluronic acid dissolved in PBS, bacterial cellulose suspended in PBS, etc. By changing the media, the benefits from the cross-linked product can be easily translated to other applications without compromising other desired properties.

[0066] Method of Cross-linking

[0067] Polymers such as hyaluronic acid (HA) may be cross-linked through the strategic formation of urethane linkages. This is accomplished by reacting HA with an isocyanate, or a caprolactone-based polyol (polycaprolactone polyol) or other polyols that have been terminated with isocyanate (NCO) groups. Initially, the polycaprolactone with terminal hydroxyl (OH) groups is reacted with an isocyanate containing two or more NCO groups (such as eLDl). This reaction is performed in specified ratios to yield products that possess terminal NCO groups. This initial stage produces a multifaceted mixture comprising two primary categories: first, the foundational polycaprolactone that has reacted solely with the isocyanate; and second, the more complex cross-linked products formed by interconnecting multiple PCL units via the isocyanates. This resultant mixture avails one or multiple NCO reactive sites for subsequent chemical interactions. Additional unreacted isocyanates can also be contained in this mixture to vary the hydrophilic and hydrophobic segments.

[0068] This cross-linking platform allows for additional molecules to be attached to the cross-linking moiety, which significantly extends the versatility of the platform. As an example, dopamine can be reacted onto the cross-linking moiety to incorporate adhesive properties.

[0069] This cross-linking moiety is then introduced to a polymer such as hyaluronic acid or a derivative of hyaluronic acid. During this phase, the primary OH groups within the hyaluronic acid or other polymer readily react with the available NCO groups, facilitating cross-linking. Secondary OH groups may also react to a lesser extent. Moreover, the formation of pendant chains is also feasible when compounds with just a single available NCO group are part of the mixture. This reaction can be catalyzed by numerous catalysts to enhance the rate of the reaction. In another example of this platform’s utility, biomolecules could be entangled or entrapped within the cross-linked matrix, delaying or controlling release based on either diffability or degradation of the cross-linked matrix.II. DEFINITIONS

[0070] As used herein, the term “hydrogel” refers to a water-insoluble, three- dimensional polymer network capable of holding at least 10% by weight or volume of interstitial fluid.

[0071] As used herein, the term “polysaccharide” refers to a carbohydrate comprising two or more monosaccharides, bound together via one or more glycosidic linkages.

[0072] As used herein, the term “linker,” “linked,” “linking” or “linkage” refers to a chemical moiety that links two or more other chemical moieties.

[0073] As used herein, the term “polymer chain” refers to a polymeric molecule or a segment of a polymeric molecule. Polymer chains may be linear or branched.III. HYDROGEL

[0074] The present invention provides a biocompatible hydrogel. In some embodiments, the biocompatible hydrogel comprises a hydrated, cross-linked polymer comprising a plurality of urethane linkages. In some embodiments, the biocompatible hydrogel comprises a hydrated, cross-linked polysaccharide comprising a plurality of urethane linkages. In some embodiments, the biocompatible hydrogel comprises a hydrated, crosslinked hyaluronic acid comprising a plurality of urethane linkages.

[0075] In some embodiments, the polymer comprises a polysaccharide, a natural or synthetic glycosaminoglycan, a modified glycosaminoglycan, hyaluronic acid, a modified hyaluronic acid, cellulose, a polypeptide, gelatin, collagen, or a combination thereof.

[0076] In some embodiments, the biocompatible hydrogel comprises a) hyaluronic acid; b) a poly-functional urethane cross-linker reacted with the hyaluronic acid to form a crosslinked polymer with a plurality of urethane linkages; and c) an aqueous fluid configured to swell the cross-linked polymer to form the biocompatible hydrogel material.

[0077] In some embodiments, the hydrogel is configured for cosmetic use, medical use, use as a dermal filler, use in wound care, use in tissue engineering, use in controlled drug release, to encapsulate a therapeutic agent, or any combination thereof.

[0078] In some embodiments, the hydrogel is sufficiently firm or cohesive for use as a dermal filler.

[0079] In some embodiments, the hydrogel is configured to flow through a 12-gauge, a 14-gauge, a 16-gauge, a 18-gauge, a 19-gauge, a 20-gauge, a 21 -gauge, a 22-gauge, a 23- gauge, a 24-gauge, a 25-gauge, a 26-gauge, a 27-gauge, a 28-gauge, a 29-gauge, a 30- gauge, or a 32-gauge needle.

[0080] In some embodiments, the hydrogel is configured to retain sufficient rheological properties for use as a dermal filler after autoclaving.

[0081] In some embodiments, the hydrogel comprises an in vivo half-life greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, or 30 days.

[0082] In some embodiments, the hydrogel comprises an in vivo degradation rate of less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, lessthan about 10%, less than about 12%, less than about 14%, less than about 16%, less than about 18%, less than about 20%, less than about 22%, less than about 24%, less than about 26%, less than about 28%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, or less than about 50% per day.

[0083] In some embodiments, the hydrogel is shelf-stable at room temperature for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, or at least 30 days.

[0084] In some embodiments, the hydrogel comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% water by weight.

[0085] In some embodiments, the hydrogel comprises a cross-linking density of at least about 0.1%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

[0086] In some embodiments, the hydrogel comprises covalently-bound dopamine.

[0087] In some embodiments, the hydrogel comprises a plurality of adhesive substituents.

[0088] In some embodiments, the hydrogel has a degree of swelling of less than about 100%, less than about 200%, less than about 300%, less than about 400%, less than about 500%, or less than about 600% by mass.

[0089] In some embodiments, the hydrogel is substantially free of leachable toxins.

[0090] In some embodiments, the hydrogel comprises water, a buffer, a PBS buffer, a gelatin medium, a collagen medium, a cellulose medium, or a combination thereof.

[0091] In some embodiments, the hydrogel is reversibly cross-linked.

[0092] In some embodiments, the hydrogel is not water-soluble.

[0093] In some embodiments, the hydrogel has a water solubility at 25 °C of less than 30 mg / L, 25 mg / L, 20 mg / L, or 15 mg / L.

[0094] In some embodiments, the hydrogel is substantially free of unreacted isocyanate groups.IV. CROSS-LINKED POLYMER FOR FORMATION OF BIOCOMPATIBLE HYDROGEL

[0095] The present invention also provides a cross-linked polymer for formation of a biocompatible hydrogel. In some embodiments, the cross-linked polymer comprises a) a polymer; and b) a poly-functional urethane cross-linker reacted with the polymer to form a cross-linked polymer with a plurality of urethane linkages; wherein the cross-linked polymer is configured to swell in an aqueous fluid to form a biocompatible hydrogel.

[0096] The present invention also provides a cross-linked polysaccharide for formation of a biocompatible hydrogel. In some embodiments, the cross-linked polysaccharide comprises a) a polysaccharide; and b) a poly-functional urethane cross-linker reacted with the polysaccharide to form a cross-linked polysaccharide with a plurality of urethane linkages; wherein the cross-linked polysaccharide is configured to swell in an aqueous fluid to form a biocompatible hydrogel.

[0097] In some embodiments, the polymer or polysaccharide has a number average molecular weight of about 50-60, about 60-70, about 70-80, about 80-90, about 90-100, about 100-120, about 120-140, about 140-160, about 160-180, about 180-200, about 200- 220, about 220-240, about 240-260, about 260-280, about 280-300, about 300-350, about 350-400, about 400-450, about 450-500, about 500-550, about 550-600, about 600-650, about 650-700, about 700-750, about 750-800, about 800-850, about 850-900, about 900- 950, about 950-1000, about 1000-1100, about 1100-1200, about 1200-1300, about 1300- 1400, about 1400-1500, about 1500-1600, about 1600-1700, about 1700-1800, about 1800-1900, about 1900-2000, or greater than about 2000 kDa prior to cross-linking.V. ISOCYANATE CROSS-LINKER FOR FORMATION OF BIOCOMPATIBLE HYDROGEL

[0098] The present invention also provides an isocyanate cross-linker for formation of a biocompatible hydrogel. In some embodiments, the cross-linker comprises a) a polyfunctional core; b) a plurality of di-functional polymeric linkers; each bound to the polyfunctional core; and c) a plurality of isocyanate substituents, each bound to one of the plurality of di-functional polymeric linkers such that the isocyanate substituents are coupled with the poly-functional core via the di-functional polymeric linkers, thereby forming the cross-linker; wherein the cross-linker is configured to react with a polymer(e.g. a polysaccharide) to form a cross-linked polymer (e.g. a cross-linked polysaccharide) configured to swell in an aqueous fluid to form a biocompatible hydrogel.

[0099] In some embodiments, the cross-linker is bi-functional, tri-functional, tetrafunctional, penta-functional, or hexafunctional.

[0100] In some embodiments, the cross-linker is formed via reaction of a polyfunctional alcohol with a di-functional isocyanate. In some embodiments, the cross-linker is formed via reaction of a poly-functional alcohol with a multi-functional isocyanate, wherein the multi-functional isocyanate comprises a compound having two or more isocyanate groups, or a compound having one or more isocyanate groups and one or more non-isocyanate groups.

[0101] In some embodiments, the cross-linker comprises a modified polycaprolactone (PCL) polymer.

[0102] In some embodiments, the cross-linker comprises a poly-functional polycaprolactone (PCL) polymer functionalized with a plurality of isocyanate substituents.

[0103] In some embodiments, the cross-linker comprises one or more polymer linkers.

[0104] In some embodiments, multiple poly-functional alcohols are linked together via reaction with a poly-functional isocyanate to form a poly-functional isocyanate having a greater functionality than that of the unreacted poly-functional alcohol.

[0105] In some embodiments, the cross-linker is formed via the reaction of a polycaprolactone (PCL) polymer with terminal hydroxyl groups with ethyl lysine diisocyanate (eLDl) or another poly-functional isocyanate.

[0106] In some embodiments, the cross-linker is functionalized with dopamine.VI. METHOD OF PREPARING CROSS-LINKED POLYMER FOR FORMATION OF BIOCOMPATIBLE HYDROGEL

[0107] The present invention provides a method of preparing a cross-linked polymer for formation of a biocompatible hydrogel. In some embodiments, the present invention provides a method of preparing a cross-linked polymer for formation of a biocompatible hydrogel, comprising a) providing a poly-functional isocyanate cross-linker; b) reacting a polymer with an organic base or organic salt to form an organic polymer salt, wherein the organic polymer salt is soluble in an organic solvent; and c) reacting the organic polymer salt with the poly-functional isocyanate cross-linker in the organic solvent to form a plurality of urethane cross-links, thereby forming the cross-linked polymer; wherein the cross-linked polymer is configured to swell in an aqueous fluid to form a biocompatible hydrogel.

[0108] The present invention provides a method of preparing a cross-linked polysaccharide for formation of a biocompatible hydrogel. In some embodiments, the present invention provides a method of preparing a cross-linked polysaccharide for formation of a biocompatible hydrogel, comprising a) providing a poly-functional isocyanate cross-linker; b) reacting a polysaccharide with an organic base or organic salt to form an organic polysaccharide salt, wherein the organic polysaccharide salt is soluble in an organic solvent; and c) reacting the organic polysaccharide salt with the polyfunctional isocyanate cross-linker in the organic solvent to form a plurality of urethane cross-links, thereby forming the cross-linked polysaccharide; wherein the cross-linked polysaccharide is configured to swell in an aqueous fluid to form a biocompatible hydrogel.

[0109] In some embodiments, a weight ratio of the polymer and the cross-linker is about 1 :0.01-1 :0.1, about 1 :0.1-l:0.2, about 1 :0.2-1:0.3, about 1:03-1:0.4, about 1 :0.4-1:0.5, about 1:0.5-1 :0.6, about 1:0.6-1:0.7, about 1:0.7-1:0.8, about 1 :0.8-1 :0.9, about 1 :0.9-1: 1, about 1:1-1: 1.1, about 1:1.1-1 : 1.2, about 1 :1.2-1:13, about 1:13-1 :1.4, about 1 :1.4-1: 1.5, about 1: 1.5-1 : 1.6, about 1:1.6- 1:1.7, about 1:1.7-1: 1.8, about 1 :1.8-1 : 1.9, about 1 :1.9-1:2, about 1:2-1 :2.2, about 1:2.2-1 :2.4, about 1 :2.4-1:2.6, about 1:2.6-1 :2.8, about 1 :2.8-1 , about 1 -1 .5, about 1 .5-1 :4, about 1:4-1 :4.5, or about 1:4.5-1:5.

[0110] In some embodiments, the poly-functional isocyanate cross-linker is ethyl lysine diisocyanate (eLDl) or another bi-functional cross-linker.

[0111] In some embodiments, the method additionally comprises one or more workup, isolation, autoclaving, or hydrating steps.

[0112] In some embodiments, the method additionally comprises a reaction with dopamine.

[0113] In some embodiments, the polymer is reacted with cetrimonium bromide, tetrabutylammonium bromide or tetrabutylammonium hydroxide to form the organic polymer salt.VII. METHOD OF PREPARING BIOCOMPATIBLE HYDROGEL

[0114] The present invention provides a method of preparing a biocompatible hydrogel. In some embodiments, the method comprises a) providing a cross-linked polymer of any one of the preceding claims; and b) hydrating the cross-linked polymer with an aqueous fluid to form a biocompatible hydrogel.

[0115] The present invention provides a method of preparing a biocompatible hydrogel. In some embodiments, the method comprises a) providing a cross-linked polysaccharideof any one of the preceding claims; and b) hydrating the cross-linked polysaccharide with an aqueous fluid to form a biocompatible hydrogel.

[0116] In some embodiments, the method of preparing a biocompatible hydrogel additionally comprises autoclaving the cross-linked polymer or polysaccharide.VIII. COSMETIC SKIN TREATMENT METHOD

[0117] The present invention provides a cosmetic skin treatment method. In some embodiments, the method comprises a) providing a hydrogel material of any one of the preceding claims; and b) performing subdermal injection a volume of the hydrogel material via a needle.IX. EXAMPLES

[0118] The cross-linking approach of this example involves a series of key steps to form the optimal product. The sequence involves creating an organic soluble form of hyaluronic acid, fabrication of the cross-linking moiety, cross-linking hyaluronic acid with the crosslinking moiety and finally one or more workup steps to obtain the final product.

[0119] Creating an organic soluble form of hyaluronic acid from a water soluble form, such as sodium hyaluronate (FIG. 1), is an important step in this example process as the organic solvent facilitates the reaction. Examples of these organic soluble species include cetrimonium hyaluronate (FIG. 2) or tetrabutylammonium hyaluronate. These species can be dissolved in organic solvents such as dimethyl sulfoxide. The hyaluronic acid used can have multiple forms. The starting material used in this synthesis is sodium hyaluronate of different molecular weight categories. These categories are low (10,000Da-200,000Da), medium (200,000Da-l,600,000Da), and high (>l,600,000Da) molecular weights, which are designated as LMW, MMW and HMW, respectively.

[0120] The synthesis of the cross-linking moiety starts by combining the desired polycaprolactone polyol and isocyanate to form the aforementioned NCO terminated molecules. Examples of the polycaprolactone polyol include tetrol (FIG. 5), triol (FIG. 11) and diol (FIG. 16) of various molecular weights. Examples of the isocyanates include lysine diisocyanate, ethyl lysine diisocyanate (FIG. 3) etc. The ratio of these molecules can be changed to tailor the characteristics of the final product. These reactions can be done in dimethyl sulphoxide (DMSO), dimethylacetamide (DMAC), etc. Various catalysts can be used to catalyze the reaction such as 72-toluenesulphonic acid, dibutyltin dilaurate, etc.

[0121] After the formation of the initial cross-linker, additional biomolecules that can react with isocyanate groups can be added directly to the reaction mixture to be appendedto the cross-linker. For instance, dopamine (FIG. 4) can react to form a urea group and become bound to the cross-linker to modify its properties and the properties of the final product.

[0122] The resulting cross-linker (examples include FIG. 6, FIG. 7, FIG. 9, FIG. 12, FIG. 14 and FIG. 17) is then transferred into a hyaluronic acid solution. The ratio of crosslinker and hyaluronic acid can be changed to obtain the desired cross-linking density. The catalyst for the reaction can be transferred with the cross-linker or one of the catalysts could be added at this stage of the reaction.

[0123] Finally, the product (examples include FIG. 8, FIG. 10, FIG. 13, FIG. 15 and FIG. 18) is precipitated from the reaction and thoroughly washed and dried to remove the organic solvents. The product is then used to create a variety of formulations or final products.

[0124] Various examples of these reactions are given in the following sections.Example 1: Synthesis of hyaluronic acid hydrogels

[0125] This example pertains to dermal fillers used in cosmetic and medical applications. More specifically, this example relates to hyaluronic acid-based hydrogels, designed with a polycaprolactone-based cross-linking moiety, to extend the in vivo longevity of the dermal filler. In conventional dermal filler technology, the frequency of reinjections is often a limiting factor due to the natural degradation of the hydrogel over time. This example addresses the need for prolonged effectiveness by slowing the degradation rate of the hydrogel, thereby offering a valuable solution for both patients and practitioners. This example includes a unique cross-linking agent, enabling the creation of a hyaluronic acid-based hydrogel that exhibits enhanced stability and durability in vivo. This extended longevity reduces the need for frequent reinjections, offering significant benefits in cosmetic enhancements, reconstructive surgeries, and other dermal filler applications. The technology is applicable not only to aesthetic medicine but also has potential applications in various therapeutic fields where long-lasting biocompatible hydrogels are desired. It may find utility in wound care, tissue engineering, controlled drug release, and other biomedical applications where the longevity of the hydrogel plays an important role.

[0126] Cetrimonium hyaluronate. The reaction begins by dissolving 5 g of sodium hyaluronate (FIG. 1) in 400 mL of water. Separately, 4.8 g of cetrimonium bromide is dissolved in 50 mL of water at 40°C. This cetrimonium bromide solution is then slowly added to the sodium hyaluronate solution while stirring. Upon mixing, cetrimoniumhyaluronate (FIG. 2) precipitates instantaneously. The precipitate is then isolated by filtration and rinsed with warm water (~50°C) until to remove excess cetrimonium bromide. Finally, it is lyophilized for use in subsequent reactions.Table 1: Caprolactone-based polyols used for synthesis

[0127] Reaction of hyaluronic acid and isocyanate (Reaction pathway 1).Cetrimonium hyaluronate is dissolved in DMSO and kept under inert atmosphere. Ethyl lysine diisocyanate is then added directly to the solution in the desired ratio (Table 2). The solution is heated to 60°C and maintained at that temperature for 3 h. The reaction is then cooled and allowed to proceed for 72 h. This solution is then ready for the workup step. Toluene sulphonic acid can be used as a catalyst (1 mol%) at this step to reduce the reaction time to 24 h. The reaction workup step is then done.

[0128] PCL hyaluronic acid based hydrogels (Reaction pathway 2)

[0129] Polycaprolactone and ethyl lysine diisocyanate reaction. The specific polycaprolactones used in these reactions are described in Table 1 and examples of reactant quantities are given in Table 2. Prior to the reaction, the PCL is dried under vacuum at 80°C for 4 h. It is then dissolved in DMAC to approximately 20 wt%. Ethyl lysine diisocyanate is introduced at a predetermined ratio under a nitrogen atmosphere to ensure inert conditions. The reaction mixture is then maintained at 60°C for 3 h before proceeding to the next phase. The ratio of PCL to eLDI is carefully selected based on the availability of terminal OH groups and the desired cross-linking density, as detailed in Table 2.

[0130] Hyaluronic acid and polycaprolactone cross-linking reaction. For the crosslinking stage, cetrimonium hyaluronate is dissolved in DMSO to ~1 wt%. Under an inert atmosphere, the pre-reacted mixture from the preceding step is added. For uncatalyzed reactions, the mixture is allowed to react at 60°C for 3 h before being maintained at 25°C for an additional 72 h. For catalyzed reactions, a 1 mol% toluene sulfonic acid is introduced. The reaction conditions remain the same but the duration is shortened to 24hours at 25°C. Specific formulations for these reactions are found in Table 2. The reaction workup step is then done.

[0131] Dopamine modified PCL hyaluronic acid hydrogel (Reaction pathway 3).To graft dopamine onto the structure, dopamine is added into the PCL diisocyanate reaction before it is added to hyaluronic acid. It is reacted onto the structure and then transferred to hyaluronic acid for the final reaction. An example recipe for the reactants is given in Table 2.Table 2: Reactant quantities for the hydrogel products normalized to 1.00 g of cetrimonium hyaluronate

[0132] Dopamine modified PCL-diisocyanate reaction. The polycaprolactone polyol with at least 3 OH groups is first dried at 80°C for 4 h and dissolved with DMAC to 20 wt%. Ethyl lysine diisocyanate is added under nitrogen and the reaction is heated to 60°C and maintained for 3 h. The reaction mixture is then cooled to 25°C.

[0133] Dopamine hydrochloride is dissolved separately in DMAC to ~ 5 wt% and cooled to 4°C under an inert atmosphere. An equimolar amount of triethylamine is then added to the solution and stirred. Triethylamine hydrochloride is precipitated leaving dopamine in solution. The dopamine solution is then added to the PCL-isocyanate reaction and stirred for 4 h.

[0134] Dopamine modified hyaluronic acid reaction. The reaction proceeds exactly as Pathway 2. The reaction workup step is then done.

[0135] Reaction workup. The final workup of all preceding reactions involves replacing the organic soluble salt with its sodium variant. A sevenfold molar excess of sodium chloride is introduced in a 20 wt% aqueous solution. This mixture is then poured into methanol, which is maintained at twice the original volume of the reaction mixture. The precipitated product is washed with a mixture of methanol and isopropyl alcohol toremove residual DMSO, rehydrated with water, and lyophilized to obtain the final solid hydrogel product, which is subsequently utilized in dermal filler formulations.

[0136] Hydrogel formulations. Hydrogel formulations are made by hydrating the cross-linked HA product in aqueous media including but not limited to, phosphate buffered saline (PBS), water, growth media, bacterial cellulose, among others. The products can be formulated in concentrations ranging from 0.5 to 5.0 wt%. The medium and concentration are carefully chosen based on the application and required characteristics (degree of swelling, degradation time, pH, strength, viscosity) of the hydrogel. Depending on the molecular weight of HA and the cross-linking density of the product, the hydration time for this process can vary from medium to medium. Some examples for formulations in PBS are given in Table 3 and formulations in bacterial cellulose (in PBS) are given in Table 4. The pH of the hydrating media ranged from 5.5 to 8.5.Table 3: Description of hydrogel formulations made in PBSTable 4: Description of hydrogel formulations made in a 0.1 wt% bacterial cellulose-PBS mediumExample 2: Characterization

[0137] Sterilizability. The suitability of hydrogels to undergo a sterilization process without sustaining significant damage or alteration to its intended properties was investigated as sterilization is an essential step for any implantable / injectable medical device. First, the hydrogels were prepared, and their initial properties were qualitatively assessed prior to autoclaving, as outlined in the Physical and Mechanical Properties section. Then, the hydrogels underwent sterilization via a steam autoclave using a predefined cycle adhering to ISO 17665 guidelines. This autoclave cycle encompassed a temperature of 121 °C for a duration of 15 minutes. Next, the hydrogels were allowed to cool, following which a re-evaluation of their gel properties was conducted to verify the persistence of gel characteristics. Any hydrogel products that no longer retained their gel characteristics following the autoclaving process were not subjected to further quantitative analysis. The properties are reported in Table 5.

[0138] Physical and Mechanical Properties. The evaluation of the physical and mechanical properties, and the overall performance of the example cross-linked hyaluronic acid-based hydrogel is crucial to ensure its suitability for cosmetic and therapeutic applications. The formulations from Table 3 were assessed to compare their injectability, cohesivity and firmness. These assessments were done pre- and post-sterilization, and the results are shown in Table 5. Hydrogels that exhibited suitable properties after the sterilization process were subjected to comprehensive analysis including rheological analysis, longevity and cytotoxicity. These analyses aimed to provide a comprehensive understanding of the hydrogel's behavior and potential within the context of cosmetic and therapeutic applications. Two commercial dermal fillers were utilized as benchmarks for comparison.Example 3: Screening Analysis

[0139] Injectability. To determine injectability, the formulation was transferred into a 1 mL syringe and administered onto a glass slide through a 27 / 30G needle, as dictated by the specific formulation. A rating scale of 0 to 5 was employed for evaluation, wherein ascore of 0 means non-injectability, a score of 5 denotes effortless injectability with no additional force required, and a score of 2.5 means injectability with some degree of effort. The results are reported in Table 5.

[0140] Cohesivity. After injecting the sample, a spatula was used to spread the formulation. The cohesiveness was evaluated as a binary assessment (yes / no). If fragments remain behind, the sample is deemed non-cohesive. The results are shown in Table 5.

[0141] Firmness. Post- injection, the gel's firmness was evaluated through tactile perception. A rating scale of 0 to 5 was used for scoring, with a score of 0 representing zero resistance to applied pressure, a score of 2.5 indicating slight resistance, and a score of 5 signifying complete firmness. The results are shown in Table 5.Table 2: Hydrogels’ qualitative properties prior and post autoclaving in comparison to commercial autoclaved gels.Example 4: Rheology

[0142] Rheology analyses were conducted using a TA-lnstruments AR-1500ex Rheometer at a temperature of 25°C, employing a sandblasted parallel plates geometry, a sample volume of 0.9 mL, and a 500 pm gap between the parallel plate and the rheometer. The viscoelasticity parameters were determined by frequency sweep curves, which were performed under a constant stress determined by the amplitude curve for each sample separately. The results of this experiment (Table 6) established the viscosity (G” and r]*), elasticity or firmness (G’ and tan delta) and total resistance to deformation (G*) of each product.Table 6: Rheological properties of hydrogels in comparison with commercial formulationsExample 5: Longevity studies (Hyaluronidase assay)

[0143] To accelerate the rate of degradation and thereby assess the longevity of the hydrogels, the enzyme hyaluronidase was used. Approximately 100 pL of hyaluronic acidbased dermal filler gel is extruded from a syringe into a pre-massed cell strainer. Every such sample is massed in its respective cell strainer, then pre-swelled by incubating in 10.0 mL Dulbecco’s phosphate buffered saline (DPBS) for more than 12 h at room temperature. Each sample is re-massed to account for solvent uptake and then transferred to 10.0 mL fresh DPBS with 100 U / mL hyaluronidase (nominal) and incubated, covered, at 37°C and pH 7.4. Intermittently (initially 1 hour intervals), the cell strainers are removed from the hyaluronidase solution, blotted dry, and massed in order to track the degradation of the samples. The hyaluronidase solution can be replaced as necessary to replenish the solution volume. The time course for each sample is complete when the mass nears that of the empty cell strainer.

[0144] Within a single experiment, degradation profiles of different samples can be directly compared in order to evaluate the relative longevity and resistance to enzymatic breakdown. By first comparing the degree of swelling, FIG. 19Error! Reference sourcenot found, shows that the experimental dermal fillers do not swell to any appreciable degree as opposed to Commercial Gel 3, which absorbed ~4x its mass in water. For these swollen samples, FIG. 20 demonstrates that Commercial Gel 3 degraded within about 4 h whereas the experimental dermal fillers (i.e. DFF4, DFF5 and DFF10) took ~10 days to be substantially degraded.Example 6: Biocompatibility studies

[0145] Hydrogels were assessed for cytotoxicity using Neutral Red (NR) assay as per IS010993-5(2009) guidelines. Following hydration and sterilization 1 ml of Dulbecco's Modified Eagle Medium (DMEM) was added per 0.2ml of hydrogel. Hydrogels were incubated at 37°C for 24 h and then 200ul of DMEM extract was added in per well of 96- well plate seeded with 100 000 A10 cells / well in triplicate. After incubation of extracts with cells at 37°C for 24 h NR reagent was added at a concentration of 40ug / ml. Plates were developed and read at 540nm. <70% cell viability compared to control is considered a cytotoxic effect.

[0146] Formulations DFF4, DFF5, DFF9 and DFF10 were tested with this method along with a commercial gel, commercial gel 3 (CCF3). All proprietary formulations showed viability over the 70% cut off compared to control, while the commercial gel failed, as shown in FIG. 21Example 7: In vivo assessment (small animal study)

[0147] An in vivo study was conducted to evaluate the tissue histopathological response to the implantation of the novel dermal filler formulations, in comparison with formulations available in the market. The study included 50 male rats (Rattus Norvegicus, albino variation, Holtzman), approximately 90 days old and weighing 350 grams. The animals were identified and randomly divided into 4 experimental implantation time points: 3, 7, 30, and 60 days (n=5). Each animal received 3 different materials on its dorsal side, consisting of: saline solution (PT-S, negative control), novel gel crosslinked with Cohesys system DFF5 (PT-T, experimental), and commercial gel (PT-J, positive control).

[0148] After anesthesia and preparation of the animals, 100 pL of each formulation were injected into the dermis using a 22-gauge cannula attached to a 1 mL syringe. The animals were euthanized at different time points (3, 7, 30, and 60 days) with an overdose of intraperitoneal anesthetic Thiopental (0.16 mL / 100 g body weight; Cristalia). The skin on the animals' backs was removed with a safety margin and immediately fixed in buffered formalin solution (Lillie's solution, pH 7.4) for subsequent histopathological processing. The samples were stretched on filter paper and immersed in buffered formalinfor 96 hours. Subsequently, the specimens were washed in water for 24 hours, dehydrated by immersion in ethanol solutions of increasing concentration (70%, 80%, 90%, and absolute ethanol), cleared in alcohol / xylene baths, and finally embedded in paraffin blocks.

[0149] Each piece was individually and alphanumerically coded to identify each product / time to be analyzed. The obtained sections were embedded in paraffin and cut into 6 pm thick semi-serial sections. The slides obtained from each piece were subjected to either hematoxylin and eosin (H&E) staining or Masson's Trichrome staining, for histological and histometric analyses. Each slide was photomicrographed using a light microscope (Carl Zeiss - Jena) equipped with a digital camera. An experienced pathologist analyzed the slides using ImageJ Software (National Institutes of Health - USA), considering 10 fields of interest in each.Inflammatory response

[0150] The inflammatory response to the dermal filler formulations was quantified across the three experimental groups: PT-J (commercial filler), PT-S (control filler), and PT-T (test group filler). At 3 days post-injection, the PT-J group exhibited a higher number of inflammatory cells compared to both PT-T and PT-S groups. By 7 days, the PT-J group showed a significant increase in presence of inflammatory cells, while the PT- T group maintained similar cellular load. In contrast, the PT-S group consistently showed a low inflammatory profile throughout the observed time points. At 30 days post-injection, both PT-J and PT-T groups experienced a decrease in inflammatory cell counts, aligning with the PT-S group's consistently low inflammatory profile. By 60 days, the cellular profiles remained stable across all groups, indicating a diminishing inflammatory response over time. Overall, the test group PT-T exhibited a milder and controlled inflammatory reduction compared to the commercial group PT-J, particularly notable at the 7-day time point where PT-J maintained a significantly higher level of inflammatory cells. This suggests that the PT-T formulation promotes a more favorable inflammatory environment, enhancing biocompatibility and facilitating smoother integration within the tissue matrix. FIG. 22 illustrates the inflammatory response across the different groups and time points, highlighting the observed differences.

[0151] Qualitative assessment indicated that throughout all the time points the dermal filler formulation is present along the subepithelial area where the gel was injected. At 3 days post injection, the filler is surrounded by immature fibroblasts. By 7 days, fibroblasts are maturing and there is deposition of collagen fibers around the filler. At these earlytimepoints, 3 and 7 days, there is mild inflammatory response marked by the presence of giant cells. At the later timepoints, 30 and 60 days, there is noticeable reduction in the volume of filler, and no evidence of giant cells. By 60 days post injection the remaining filler is surrounded by mature fibroblasts and organized collagen fibers. When compared to the commercial filler, the immune responses were comparable, however they differed in the time for collagen deposition. For the commercial filler, it took 30 days to observe collagen deposition whereas collagen deposition was evident in the novel filler at 7 days post injection. FIG. 23 summarizes results for the novel formulation at different timepoints.Filler Area

[0152] FIG. 24 presents the changes in filler area over time across the different groups. Both PT-J and PT-T groups showed a reduction in filler volume from 3 to 60 days postinjection. The PT-J group experienced a progressive decrease in the amount of remaining material, consistently reducing the filler area from 3 days onward, with notable reductions observed up to 60 days. Similarly, the PT-T group demonstrated a steady decrease in filler volume, particularly between the early time points of 3 days to 30 days and from 3 days to 60 days. When comparing the two groups, PT-T exhibited a smaller amount of remaining filler material at both 3 and 7 days compared to PT-J. However, by 30 and 60 days, the filler volumes between PT-J and PT-T were comparable, indicating that both formulations achieved similar levels of filler reduction over the longer term. This suggests that while PT-T may integrate and remodel more efficiently in the early stages, both fillers ultimately reach a similar state of filler volume reduction as time progresses. The reduction in filler volume over time in both groups indicates effective integration and remodeling within the tissue matrix, with PT-T offering a more pronounced decrease in the initial phases postinjection. PT-J demonstrated a more abrupt reduction in filler area between the timepoints of 7 and 30 days.Collagen Stimulation

[0153] FIG. 25 shows collagen deposition over time across the test and commercial groups. At the early time points of 3- and 7-days post-injection, collagen production was similar for both PT-T and PT-J groups, indicating comparable initial tissue responses. However, at the longer time points of 30 and 60 days, the test group PT-T demonstrated an increased amount of collagen compared to the commercial group PT-J. This enhanced collagen deposition in the PT-T group suggests a more robust stimulation of the tissue matrix, contributing to improved structural support and integration within the tissue.

[0154] The novel fillers achieved mature and organized collagen fiber formation earlier and to a greater extent at later time points compared to the commercial filler. This accelerated collagen stimulation underscores the potential of the PT-T formulation in promoting effective tissue regeneration and enhancing the longevity of the filler effect.

[0155] The similarities in collagen production during the early stages (3 and 7 days) between PT-J and PT-T groups likely reflect the universal inflammatory response triggered by the injection process itself, a common physiological reaction that facilitates tissue repair and filler integration. The subsequent increase in collagen production observed in the PT-T group at later time points (30 and 60 days) may be attributed to the presence of the polycaprolactone (PCL) based crosslinker in the test dermal filler. PCL is recognized for its biostimulatory properties, which enhance collagen synthesis and promote sustained tissue regeneration. This biostimulatory effect of the PCL crosslinker likely contributes to the higher collagen levels observed in the PT-T group, suggesting that PCL plays a significant role in optimizing the performance of the dermal filler by managing inflammation and stimulating collagen production. Consequently, the PT-T formulation not only supports a controlled inflammatory response but also facilitates a more efficient and robust collagen network, improving the structural integrity and longevity of the dermal filler within the tissue matrix.Example 7: Applications of the Hydrogels

[0156] One application of the hydrogels described herein is in the field of dermal fillers where the unique balance between the hydrophilic and hydrophobic groups allows for tailoring specific formulations for different parts of the body. Beyond aesthetic enhancements, the hydrogels of the present invention offer a wide range of therapeutic and medical applications, such as tissue engineering, wound healing, and controlled drug release.

[0157] For cosmetic applications, the hydrogel formulations can be used as dermal fillers in the face. Since specific formulations do not swell when equilibrated in water, post injection swelling would not be observed so that the clinician and patient would see the result immediately rather than waiting to see the effects. This is ideal for observing the anti-aging effects of the dermal filler on the face to show reduced wrinkles and fine lines. Moreover, these hydrogels could be used for tissue augmentation to add volume with the same benefits and longevity.

[0158] These hydrogels can also act as efficient carriers for the controlled release of drugs. Due to the versatility of the platform, fast or slow degrading formulations can easilybe obtained with an embedded therapeutic agent to efficiently control the release kinetics of drugs into the body.

[0159] In combination with drug release, the hydrogels can also be used for wound healing applications. They can help retain moisture at the site of the wound to facilitate healing. Moreover, the hydrogel can act as a scaffold structure to allow cells to grow and move easily, which will assist tissue regeneration. This tissue regeneration can help to reduce the appearance of scars. Additionally, the unique nature of the platform allows for the incorporation of antimicrobial agents, such as dopamine in the incorporated dopamine- PCL-HA hydrogel created in this platform. These formulations can help to reduce infection and inflammation.

[0160] These properties also make the hydrogels ideal for tissue engineering as they can mimic the extracellular matrix, providing cells with an environment for adhesion and proliferation. As they can be used as a scaffold, they are excellent candidates for cell seeding and 3D culturing. The platform can be used to fine tune the mechanical properties of the gel to obtain soft hydrogels for neural tissue engineering and stiffer hydrogels for bone. Example hydrogels are fully biocompatible and biodegradable so that new tissue can eventually replace the scaffold.Example 8: Reproducibility

[0161] Reproducibility was improved by conducting reactions at lower concentrations (0.25%) compared to standard (1%) and scaled-up (3.5%) conditions. Lyophilization was identified as a critical step affecting product consistency due to 'fractionation.' This issue was addressed by shearing the gel prior to lyophilization, improving reproducibility significantly.

[0162] Reproducibility of the dermal filler formulations was significantly enhanced by optimizing reaction conditions and addressing critical processing steps. Experiments demonstrated that conducting reactions at lower concentrations (0.25%) improved reproducibility compared to the standard conditions (1%) and scaled-up reactions (3.5%). These findings suggest that reduced reaction concentrations allow for more controlled cross-linking and homogeneity in the final product.

[0163] Lyophilization was identified as a critical step influencing product consistency. During this process, "fractionation" of the solid dermal filler product was observed, leading to variations in reproducibility. To mitigate this issue, the gel was subjected to shearing by syringe extrusion prior to lyophilization. This additional step effectively reduced inconsistencies and enhanced the uniformity of the final product. Alternativeshearing methods include mechanical stirring, mesh extrusion, and roller milling. Modifications to the work-up method, including the introduction of shearing and refinements in reaction handling, further contributed to the observed reproducibility improvements.

[0164] New dermal filler formulations, outlined in Table 7, were developed and tested to explore the effects of these process optimizations. These formulations included variations with and without lidocaine, providing a basis for evaluating the influence of lidocaine on material properties. The shear sensitivity of the formulations was further investigated and is detailed in Table 8. Notably, the formulations DFF 19.1 -Control and DFF 19.1 -Control 27 G Needle revealed distinct differences in rheological behavior when exposed to varying shear conditions. For instance, using a thinner needle gauge (27 G) resulted in reduced elastic and viscous moduli while increasing the damping factor. This indicates a thinner flow profile and altered viscoelastic properties due to the higher shear forces experienced during injection.

[0165] The findings demonstrate the importance of reaction concentration, process control during lyophilization, and the impact of shear forces on the performance of dermal fillers. The developed formulations, featuring both high molecular weight (HMW) and medium molecular weight (MMW) hyaluronic acid cross-linked with the tetrol crosslinker(F!G 6), are detailed in Table 7. These formulations represent a significant advancement in achieving reproducible, high-quality dermal fillers tailored for clinical application.Table 7: New dermal filler formulations made using DF2 in Table 2. Lidocaine hydrochloride monohydrate was added at 0.3 wt%Example 9: Scale Up

[0166] Scaling up the reaction linearly was challenging due to low reaction concentrations (~1%). To overcome this, the reaction was modified to use 3.5% concentration with continuous heating (~60°C) to dissolve and react the crosslinker effectively. A revised work-up procedure incorporating methanol dilution followed by aqueous sodium chloride precipitation ensured consistent yields, whether processed at room temperature or with heating.

[0167] Scaling up the reaction posed significant challenges due to the inherently low reaction concentrations (~1%) used in standard processes. These low concentrations resulted in difficulties achieving high yields when attempting a linear scale-up. To address this, a modified approach was developed wherein the reaction concentration was increased to 3.5%, coupled with continuous heating at approximately 60°C. This adjustment facilitated the efficient dissolution of the reactants and enabled effective cross-linking of the materials, ensuring scalability without compromising the quality of the product.

[0168] The work-up procedure was also extensively revised to accommodate the scaled- up reaction. Methanol was introduced as the first addition to dilute the reaction mixture, followed by the gradual addition of aqueous sodium chloride to precipitate the product. This sequential work-up strategy allowed for precise control over the precipitation process, which was critical to maintaining consistency and yield. Notably, the work-up step was performed successfully under both heated and room temperature conditions, with comparable yields observed across both approaches.

[0169] These modifications represent a key advancement in the patent, enabling reliable scale-up of the dermal filler production process. By addressing the limitations associated with low reaction concentrations and introducing optimized reaction and work-up methods, the new process achieves consistent and reproducible yields at larger scales. Thisinnovation ensures that the high-performance characteristics of the dermal fillers are preserved, even in scaled-up manufacturing settings, making it a robust and scalable solution for commercial production.Example 10: Lidocaine Addition

[0170] Formulations containing 0.3 wt% lidocaine hydrochloride monohydrate were developed to align with the standard practices observed in commercial dermal fillers, which commonly include this concentration of lidocaine to enhance patient comfort during injection. Lidocaine acts as a local anesthetic, minimizing injection discomfort and improving the overall patient experience. Its inclusion in the formulations also influenced the physical and functional properties of the dermal fillers.

[0171] One key observation was a reduction in water uptake at higher gel concentrations (2%) when lidocaine was included. This effect likely contributes to maintaining the filler’s structural integrity and performance after injection. To optimize this benefit while ensuring injectability and handling properties, the gel concentration was adjusted to 1%. Rheological testing confirmed that lidocaine inclusion significantly influenced the viscoelastic properties of the formulations. These changes were reflected in parameters such as the elastic modulus (G'), viscous modulus (G"), and damping factor (Tan 5), as detailed in Table 8. Such modifications suggest that lidocaine not only improves patient comfort but also allows for fine-tuning of the gel’s mechanical properties to achieve an ideal balance between injectability, cohesivity, and durability.

[0172] By incorporating lidocaine, the developed formulations mirror the anesthetic benefits and physical performance characteristics of existing commercial fillers, while potentially offering improved material properties and patient outcomes. This strategic approach positions the lidocaine-containing formulations as competitive and innovative additions to the dermal filler market.Table 8: Rheological properties of dermal filler formulations at 1 Hz and using a standard 21 G needle

[0173] AnalysisG' (Elastic Modulus): o Dermal fillers with lidocaine (DFF19.1, DFF19.2, and DFF19.3 Lidocaine) generally show higher elastic modulus values compared to those without lidocaine, indicating a more pronounced elastic behavior.G" (Viscous Modulus): o The viscous modulus is highest in DFF19.3-Lidocaine, indicating a strong viscous component for this sample. o Samples without lidocaine (DFF 19.1 -Control, DFF19.2-Control, DFF19.3- Control) exhibit lower G" compared to most lidocaine samples, except DFF 19.1 -Lidocaine.|^*| (Complex Viscosity):o The samples with lidocaine (DFF19.2 and DFF19.3 Lidocaine) show higher complex viscosities, suggesting a thicker or more resistant material flow under deformation.Tan 5 (Damping Factor): o The inclusion of lidocaine appears to modulate the damping factor, with some formulations (like DFF19.2-Lidocaine) exhibiting a more elastic nature, while others (like DFF19.3-Lidocaine) demonstrates higher viscosity due to altered cross-linking interactions at lower gel concentrations. This suggests that the specific formulation can significantly impact the energy dissipation and viscoelastic balance of the filler.Screening AnalysisFormulation Optimization: Lidocaine-Containing Gels

[0174] To comprehensively evaluate the impact of lidocaine inclusion on gel formulations, the series of new formulations given in Table 7 were analyzed. These included DFF 19.1 -Control, DFF 19.1 -Lidocaine, DFF19.2-Control, DFF19.2-Lidocaine, DFF19.3-Control, and DFF19.3-Lidocaine. In this naming convention, "Control" denotes gels prepared without lidocaine, while "Lidocaine" indicates gels containing 0.3 wt% lidocaine hydrochloride monohydrate, consistent across all lidocaine -containing formulations.

[0175] Qualitative assessments of these formulations focused on key properties such as injectability, cohesivity, and firmness. The results revealed no significant differences in injectability or cohesivity between the gels with and without lidocaine. However, slight variations in firmness were observed among the DFF19.1, DFF19.2, and DFF19.3 formulations, irrespective of lidocaine inclusion. These differences in firmness may reflect intrinsic variations due to the specific formulation parameters, such as molecular weight of hyaluronic acid or cross-linking density, rather than the presence of lidocaine itself. The detailed results of these assessments are summarized in Table 9.Table 9: Qualitative properties of hydrogels containing lidocaine in comparison to control gels without lidocaine.Enumerated embodiments:Enumerated Embodiment 1. Enumerated embodiment 1. A hydrogel comprising: a. a plurality of polymer chains; b. a plurality of poly-functional urethane cross-linkers reacted with the plurality of polymer chains to form a cross-linked polymer with a plurality of urethane linkages; and c. an aqueous fluid dispersed within the cross-linked polymer to form the hydrogel.Enumerated Embodiment 2. A hydrogel comprising a hydrated cross-linked polymer comprising a plurality of polymer chains crosslinked together with one or more poly-functional urethane cross-linkers.Enumerated Embodiment 3. The hydrogel of any one of the preceding embodiments, wherein one or more of the polymer chains comprises hyaluronic acid, a modified hyaluronic acid, a derivative of hyaluronic acid, or any combination thereof.Enumerated Embodiment 4. The hydrogel of any one of the preceding embodiments, wherein one or more of the polymer chains comprises a polysaccharide, a natural or synthetic glycosaminoglycan, a modified glycosaminoglycan, hyaluronic acid, a modified hyaluronic acid, a derivative of hyaluronic acid cellulose, a polypeptide, gelatin, or collagen.Enumerated Embodiment 5. A hydrogel comprising a hydrated, cross-linked polymer comprising a plurality of urethane linkages.Enumerated Embodiment 6. The hydrogel of any one of the preceding embodiments, wherein the plurality of urethane linkages were formed via reaction of a plurality of isocyanates.Enumerated Embodiment 7. The hydrogel of any one of the preceding embodiments, additionally comprising lidocaine or lidocaine hydrochloride monohydrate.Enumerated Embodiment 8. The hydrogel of any one of the preceding embodiments, additionally comprising about 0.3 weight percent of lidocaine or lidocaine hydrochloride mo no hydrate.Enumerated Embodiment 9. The hydrogel of any one of the preceding embodiments, additionally comprising about 0.01-0.5 weight percent of lidocaine or lidocaine hydrochloride monohydrate.Enumerated Embodiment 10. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is configured to stimulate collagen formation.Enumerated Embodiment 11. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is configured for cosmetic use, medical use, use as a dermal filler, use in wound care, use in tissue engineering, use in controlled drug release, to encapsulate a therapeutic agent, or any combination thereof.Enumerated Embodiment 12. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is sufficiently firm for use as a dermal filler.Enumerated Embodiment 13. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is sufficiently cohesive for use as a dermal filler.Enumerated Embodiment 14. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is configured to flow through a 12-gauge, a 14-gauge, a 16-gauge, a 18-gauge, a 19-gauge, a 20-gauge, a 21 -gauge, a 22-gauge, a 23-gauge, a 24-gauge, a 25-gauge, a 26-gauge, a 27-gauge, a 28-gauge, a 29-gauge, a 30-gauge, or a 32-gauge needle.Enumerated Embodiment 15. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is configured to retain sufficient rheological properties for use as a dermal filler after autoclaving.Enumerated Embodiment 16. The hydrogel of any one of the preceding embodiments, comprising an in vivo half-life greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, or 30 days.Enumerated Embodiment 17. The hydrogel of any one of the preceding embodiments, comprising an in vivo degradation rate of less than about 1 %, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 12%, less than about 14%, less than about 16%, less than about 18%, less than about 20%, less than about 22%, less than about 24%, less than about 26%, less than about 28%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, or less than about 50% per day.Enumerated Embodiment 18. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is shelf-stable at room temperature for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, or at least 30 days.Enumerated Embodiment 19. The hydrogel of any one of the preceding embodiments, comprising at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% water by weight.Enumerated Embodiment 20. The hydrogel of any one of the preceding embodiments, comprising a cross-linking density of at least about 0.1%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.Enumerated Embodiment 21. The hydrogel of any one of the preceding embodiments, additionally comprising a plurality of adhesive substituents.Enumerated Embodiment 22. The hydrogel of any one of the preceding embodiments, additionally comprising dopamine.Enumerated Embodiment 23. The hydrogel of any one of the preceding embodiments, additionally comprising covalently-bound dopamine.Enumerated Embodiment 24. The hydrogel of any one of the preceding embodiments, wherein the hydrogel has a degree of swelling of less than about 100%, less than about 200%, less than about 300%, less than about 400%, less than about 500%, or less than about 600% by mass.Enumerated Embodiment 25. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is substantially free of leachable toxins.Enumerated Embodiment 26. The hydrogel of any one of the preceding embodiments, comprising water, a buffer, a PBS buffer, a gelatin medium, a collagen medium, a cellulose medium, or a combination thereof.Enumerated Embodiment 27. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is reversibly cross-linked.Enumerated Embodiment 28. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is not water-soluble.Enumerated Embodiment 29. The hydrogel of any one of the preceding embodiments, wherein the hydrogel has a water solubility at 25 °C of less than 30 mg / L, 25 mg / L, 20 mg / L, or 15 mg / L.Enumerated Embodiment 30. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is substantially free of unreacted isocyanate groups.Enumerated Embodiment 31. The hydrogel of any one of the preceding embodiments, wherein the hydrogel is biocompatible.Enumerated Embodiment 32. A cross-linked polymer comprising a plurality of polymer chains crosslinked together with one or more poly-functional urethane cross-linkers.Enumerated Embodiment 33. The cross-linked polymer of any one of the preceding embodiments wherein the cross-linked polymer swells in an aqueous fluid to form a biocompatible hydrogel.Enumerated Embodiment 34. The cross-linked polymer of any one of the preceding embodiments, wherein the one or more of the cross-linkers is bifunctional, tri-functional, tetra-functional, penta-functional, or hexa-functional.Enumerated Embodiment 35. The cross-linked polymer of any one of the preceding embodiments, wherein the one or more of the cross-linkers is formed via reaction of a poly-functional alcohol with a multi-functional isocyanate, wherein the multi-functional isocyanate comprises a compound having two or more isocyanate groups, or a compound having one or more isocyanate groups and one or more non-isocyanate groups.Enumerated Embodiment 36. The cross-linked polymer of any one of the preceding embodiments, wherein the one or more of the cross-linkers comprises a polycaprolactone (PCL), polyethylene glycol (PEG), or polyvinyl alcohol (PVA) polymeric linker.Enumerated Embodiment 37. The cross-linked polymer of any one of the preceding embodiments, wherein the one or more of the cross-linkers comprises a poly-functional polycaprolactone (PCL) polymer functionalized with a plurality of isocyanate substituents.Enumerated Embodiment 38. The cross-linked polymer of any one of the preceding embodiments, wherein the plurality of polymer chains has a number average molecular weight of about 50-60, about 60-70, about 70-80, about 80-90, about 90-100, about 100-120, about 120-140, about 140-160, about 160-180, about 180-200, about 200-220, about 220-240, about 240-260, about 260-280, about 280- 300, about 300-350, about 350-400, about 400-450, about 450-500, about 500-550, about 550-600, about 600-650, about 650-700, about 700-750, about 750-800, about 800-850, about 850-900, about 900-950, about 950-1000, about 1000-1100, about 1100-1200, about 1200-1300, about 1300-1400, about 1400-1500, about 1500-1600, about 1600-1700, about 1700-1800, about 1800-1900, about 1900- 2000, or greater than about 2000 kDa prior to cross-linking.Enumerated Embodiment 39. An isocyanate cross-linker for formation of a crosslinked polymer, the cross-linker comprising: a. a poly-functional core;b. a plurality of di-functional polymeric linkers; each covalently bound to the polyfunctional core; and c. a plurality of isocyanate substituents, each covalently bound to one of the plurality of di-functional polymeric linkers such that the isocyanate substituents are coupled with the poly-functional core via the di-functional polymeric linkers, thereby forming the cross-linker.Enumerated Embodiment 40. The cross-linker of any one of the preceding embodiments, wherein the cross-linker is configured to react with a polymer to form the cross-linked polymer.Enumerated Embodiment 41. The cross-linker of any one of the preceding embodiments, wherein the cross-linked polymer is configured to swell in an aqueous fluid to form a hydrogel.Enumerated Embodiment 42. The cross-linker of any one of the preceding embodiments, wherein the cross-linker is bi-functional, tri-functional, tetrafunctional, penta-functional, or hexa-functional.Enumerated Embodiment 43. The cross-linker of any one of the preceding embodiments, wherein the cross-linker is formed via reaction of a poly-functional alcohol with a multi-functional isocyanate, wherein the multi-functional isocyanate comprises a compound having two or more isocyanate groups, or a compound having one or more isocyanate groups and one or more non-isocyanate groups.Enumerated Embodiment 44. The cross-linker of any one of the preceding embodiments, wherein the cross-linker comprises a plurality of polycaprolactone (PCL) polyethylene glycol (PEG) or polyvinyl alcohol (PVA) polymeric linkers.Enumerated Embodiment 45. The cross-linker of any one of the preceding embodiments, wherein the cross-linker comprises a poly-functional polycaprolactone (PCL) polymer functionalized with a plurality of isocyanate substituents.Enumerated Embodiment 46. The cross-linker of any one of the preceding embodiments, wherein the cross-linker comprises one or more polymer linkers.Enumerated Embodiment 47. The cross-linker of any one of the preceding embodiments, wherein multiple poly-functional alcohols are linked together via reaction with a poly-functional isocyanate to form a poly-functional isocyanate having a greater functionality than that of the unreacted poly-functional alcohol.Enumerated Embodiment 48. The cross-linker of any one of the preceding embodiments, wherein the cross-linker is formed via the reaction of a polycaprolactone (PCL) polymer with terminal hydroxyl groups with ethyl lysine diisocyanate (eLDl) or another poly-functional isocyanate.Enumerated Embodiment 49. The cross-linker of any one of the preceding embodiments, wherein the cross-linker is functionalized with dopamine.Enumerated Embodiment 50. A method of preparing a cross-linked polymer for formation of a hydrogel, the method comprising: a. providing a poly-functional isocyanate cross-linker; b. reacting a polymer with an organic base or organic salt to form an organic polymer salt, wherein the organic polymer salt is soluble in an organic solvent; and c. reacting the organic polymer salt with the poly-functional isocyanate crosslinker in the organic solvent to form a plurality of urethane cross-links, thereby forming the cross-linked polymer.Enumerated Embodiment 51. The method of any one of the preceding embodiments, wherein the cross-linked polymer is configured to swell in an aqueous fluid to form a hydrogel.Enumerated Embodiment 52. The method of any one of the preceding embodiments, wherein the reaction between the organic polymer salt and the polyfunctional isocyanate cross-linker is at a concentration of about 3.5 weight percent.Enumerated Embodiment 53. The method of any one of the preceding embodiments, wherein the reaction between the organic polymer salt and the polyfunctional isocyanate cross-linker is at a concentration between about 0.1 to about 3.5 weight percent.Enumerated Embodiment 54. The method of any one of the preceding embodiments, wherein the reaction between the organic polymer salt and the polyfunctional isocyanate cross-linker is at about 60 °C.Enumerated Embodiment 55. The method of any one of the preceding embodiments, wherein the reaction between the organic polymer salt and the polyfunctional isocyanate cross-linker between about 25 °C and about 80 °C.Enumerated Embodiment 56. The method of any one of the preceding embodiments, wherein the reaction between the organic polymer salt and the polyfunctional isocyanate cross-linker between about 25 °C and about 90 °C.Enumerated Embodiment 57. The method of any one of the preceding embodiments, wherein the product is purified by: a. diluting the reaction mixture of the organic polymer salt and the poly-functional isocyanate cross-linker with methanol; and b. adding an aqueous salt solution to precipitate the cross-linked polymer.Enumerated Embodiment 58. The method of claim 53, wherein the aqueous salt solution is a sodium chloride solution.Enumerated Embodiment 59. The method of any one of the preceding embodiments, wherein a weight ratio of the polymer and the cross-linker is about 1:0.01-1 :0.1, about 1 :0.1-l:0.2, about 1 :0.2-l :0.3, about 1:03-1:0.4, about 1 :0.4- 1:0.5, about 1 :0.5-1:0.6, about 1:0.6-1 :0.7, about 1 :0.7-1:0.8, about 1 :0.8-1:0.9, about 1 :0.9-1 :1, about 1 : 1-1 :1.1, about 1 :1.1-1: 1.2, about 1:1.2-1:13, about 1:1.3- 1:1.4, about 1 :1.4- 1:1.5, about 1: 1.5-1 : 1.6, about 1 : 1.6-1: 1.7, about 1 :1.7-1: 1.8, about 1 :1.8-1 : 1.9, about 1 :1.9-1 :2, about 1:2-1 :2.2, about 1 :2.2-1 :2.4, about 1 :2.4- 1:2.6, about 1 :2.6-1:2.8, about 1:2.8-1 , about 1 -13.5, about 1 .5-1:4, about 1:4-1 :4.5, or about 1 :4.5-1:5.Enumerated Embodiment 60. The method of any one of the preceding embodiments, wherein the poly-functional isocyanate cross-linker is ethyl lysine diisocyanate (eLDl) or another bi-functional cross-linker.Enumerated Embodiment 61. The method of any one of the preceding embodiments, additionally comprising one or more workup, isolation, autoclaving, or hydrating steps.Enumerated Embodiment 62. The method of any one of the preceding embodiments, additionally comprising reaction with dopamine.Enumerated Embodiment 63. The method of any one of the preceding embodiments, wherein the polymer is reacted with cetrimonium bromide, tetrabutylammonium bromide or tetrabutylammonium hydroxide to form the organic polymer salt.Enumerated Embodiment 64. A method of preparing a hydrogel, the method comprising: a. providing a cross-linked polymer of any one of the preceding embodiments; and b. hydrating the cross-linked polymer with an aqueous fluid to form a hydrogel.Enumerated Embodiment 65. The method of any one of the preceding embodiments additionally comprising autoclaving the cross-linked polymer.Enumerated Embodiment 66. The method of any one of the preceding embodiments additionally comprising shearing the hydrogel.Enumerated Embodiment 67. The method of any one of the preceding embodiments additionally comprising lyophilizing the hydrogel.Enumerated Embodiment 68. The method of any one of the preceding embodiments additionally comprising shearing and then lyophilizing the hydrogel.Enumerated Embodiment 69. A method for cosmetic skin treatment of a subject, the method comprising: a. providing a hydrogel comprising: i. a plurality of polymer chains; ii. a plurality of poly-functional urethane cross-linkers reacted with the plurality of polymer chains to form a cross-linked polymer with a plurality of urethane linkages; and iii. an aqueous fluid dispersed within the cross-linked polymer to form the hydrogel; and b. sub-dermally injecting a volume of the hydrogel to the subject.Enumerated Embodiment 70. A method for stimulating subdermal collogen deposition of a subject, the method comprising: a. providing a hydrogel comprising: i. a plurality of polymer chains; ii. a plurality of poly-functional urethane cross-linkers reacted with the plurality of polymer chains to form a cross-linked polymer with a plurality of urethane linkages; and iii. an aqueous fluid dispersed within the cross-linked polymer to form the hydrogel; and b. sub-dermally injecting a volume of the hydrogel to the subject thereby stimulating collogen deposition.Enumerated Embodiment 71. The method of any one of the preceding embodiments, wherein the hydrogel additionally comprises lidocaine.Enumerated Embodiment 72. The method of any one of the preceding embodiments, wherein subdermal injection of the hydrogel stimulates subdermal collagen deposition.Enumerated Embodiment 73. The method of any one of the preceding embodiments, wherein the hydrogel is injected via a needle.Enumerated Embodiment 74. The method of any one of the preceding embodiments, wherein the hydrogel is injected via a 12-gauge, a 14-gauge, a 16- gauge, a 18-gauge, a 19-gauge, a 20-gauge, a 21 -gauge, a 22-gauge, a 23-gauge, a 24-gauge, a 25-gauge, a 26-gauge, a 27-gauge, a 28-gauge, a 29-gauge, a 30- gauge, or a 32-gauge needle.Enumerated Embodiment 75. A kit for preparation of a hydrogel, the kit comprising: a. a cross linked polymer comprising a plurality of polymer chains reacted with a plurality of polymer chains to form a cross-linked polymer with a plurality of urethane linkages, the cross-linked polymer configured to swell in an aqueous fluid to form a hydrogel; and b. an aqueous fluid.Enumerated Embodiment 76. The kit of embodiment 75, additionally comprising instructions for swelling the cross-linked polymer with the aqueous fluid to form the hydrogel.Enumerated Embodiment 77. A kit for preparation of a hydrogel, the kit comprising: a. a cross-linked polymer comprising a plurality of polymer chains reacted with a plurality of polymer chains to form a cross-linked polymer with a plurality of urethane linkages; and b. instructions for adding an aqueous fluid to the cross-linked polymer to form a hydrogel.Enumerated Embodiment 78. The kit of embodiment 77, wherein the cross-linked polymer is configured to swell in an aqueous fluid to form a biocompatible hydrogel.

Claims

WHAT IS CLAIMED IS:

1. A hydrogel comprising: a. a plurality of polymer chains; b. a plurality of poly-functional urethane cross-linkers reacted with the plurality of polymer chains to form a cross-linked polymer with a plurality of urethane linkages; and c. an aqueous fluid dispersed within the cross-linked polymer to form the hydrogel.

2. A hydrogel comprising a hydrated cross-linked polymer comprising a plurality of polymer chains crosslinked together with one or more poly-functional urethane cross-linkers.

3. The hydrogel of claim 1 or claim 2, wherein one or more of the polymer chains comprises hyaluronic acid, a modified hyaluronic acid, a derivative of hyaluronic acid, or any combination thereof.

4. The hydrogel of any one of claims 1 -3, wherein one or more of the polymer chains comprises a polysaccharide, a natural or synthetic glycosaminoglycan, a modified glycosaminoglycan, hyaluronic acid, a modified hyaluronic acid, a derivative of hyaluronic acid cellulose, a polypeptide, gelatin, or collagen.

5. A hydrogel comprising a hydrated, cross-linked polymer comprising a plurality of urethane linkages.

6. The hydrogel of any one of claims 1-5, wherein the plurality of urethane linkages were formed via reaction of a plurality of isocyanates.

7. The hydrogel of any one of claims 1-6, additionally comprising lidocaine or lidocaine hydrochloride mo no hydrate.

8. The hydrogel of any one of claims 1-7, additionally comprising about 0.3 weight percent of lidocaine or lidocaine hydrochloride monohydrate.

9. The hydrogel of any one of claims 1-8, additionally comprising about 0.01-0.5 weight percent of lidocaine or lidocaine hydrochloride mono hydrate.

10. The hydrogel of any one of claims 1-9, wherein the hydrogel is configured to stimulate collagen formation.

11. The hydrogel of any one of claims 1-10, wherein the hydrogel is configured for cosmetic use, medical use, use as a dermal filler, use in wound care, use in tissue engineering, use in controlled drug release, to encapsulate a therapeutic agent, or any combination thereof.

12. The hydrogel of any one of claims 1-11, wherein the hydrogel is sufficiently firm for use as a dermal filler.

13. The hydrogel of any one of claims 1-12, wherein the hydrogel is sufficiently cohesive for use as a dermal filler.

14. The hydrogel of any one of claims 1-13, wherein the hydrogel is configured to flow through a 12-gauge, a 14-gauge, a 16-gauge, a 18-gauge, a 19-gauge, a 20- gauge, a 21 -gauge, a 22-gauge, a 23-gauge, a 24-gauge, a 25-gauge, a 26-gauge, a 27-gauge, a 28-gauge, a 29-gauge, a 30-gauge, or a 32-gauge needle.

15. The hydrogel of any one of claims 1-14, wherein the hydrogel is configured to retain sufficient rheological properties for use as a dermal filler after autoclaving.

16. The hydrogel of any one of claims 1-15, comprising an in vivo half-life greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, or 30 days.

17. The hydrogel of any one of claims 1-16, comprising an in vivo degradation rate of less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 12%, less than about 14%, less than about 16%, less than about 18%, less than about 20%, less than about 22%, less than about 24%, less than about 26%, less than about 28%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, or less than about 50% per day.

18. The hydrogel of any one of claims 1-17, wherein the hydrogel is shelf-stable at room temperature for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, or at least 30 days.

19. The hydrogel of any one of claims 1-18, comprising at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% water by weight.

20. The hydrogel of any one of claims 1-19, comprising a cross-linking density of at least about 0.1%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%.

21. The hydrogel of any one of claims 1-20, additionally comprising a plurality of adhesive substituents.

22. The hydrogel of any one of claims 1-21, additionally comprising dopamine.

23. The hydrogel of any one of claims 1-22, additionally comprising covalently-bound dopamine.

24. The hydrogel of any one of claims 1-23, wherein the hydrogel has a degree of swelling of less than about 100%, less than about 200%, less than about 300%, less than about 400%, less than about 500%, or less than about 600% by mass.

25. The hydrogel of any one of claims 1-24, wherein the hydrogel is substantially free of leachable toxins.

26. The hydrogel of any one of claims 1-25, comprising water, a buffer, a PBS buffer, a gelatin medium, a collagen medium, a cellulose medium, or a combination thereof.

27. The hydrogel of any one of claims 1-26, wherein the hydrogel is reversibly crosslinked.

28. The hydrogel of any one of claims 1-27, wherein the hydrogel is not water-soluble.

29. The hydrogel of any one of claims 1-28, wherein the hydrogel has a water solubility at 25 °C of less than 30 mg / L, 25 mg / L, 20 mg / L, or 15 mg / L.

30. The hydrogel of any one of claims 1-29, wherein the hydrogel is substantially free of unreacted isocyanate groups.

31. The hydrogel of any one of claims 1-30, wherein the hydrogel is biocompatible.

32. A cross-linked polymer comprising a plurality of polymer chains crosslinked together with one or more poly-functional urethane cross-linkers.

33. The cross-linked polymer of claim 32, wherein the cross-linked polymer swells in an aqueous fluid to form a biocompatible hydrogel.

34. The cross-linked polymer of claim 32 or 33, wherein the one or more of the crosslinkers is bi-functional, tri-functional, tetra-functional, penta-functional, or hexafunctional .

35. The cross-linked polymer of any one of claims 32-34, wherein the one or more of the cross-linkers is formed via reaction of a poly-functional alcohol with a multi-functional isocyanate, wherein the multi-functional isocyanate comprises a compound having two or more isocyanate groups, or a compound having one or more isocyanate groups and one or more non-isocyanate groups.

36. The cross-linked polymer of any one of claims 32-35, wherein the one or more of the cross-linkers comprises a polycaprolactone (PCL), polyethylene glycol (PEG), or polyvinyl alcohol (PVA) polymeric linker.

37. The cross-linked polymer of any one of claims 32-36, wherein the one or more of the cross-linkers comprises a poly-functional polycaprolactone (PCL) polymer functionalized with a plurality of isocyanate substituents.

38. The cross-linked polymer of any one of claims 32-37, wherein the plurality of polymer chains has a number average molecular weight of about 50-60, about 60- 70, about 70-80, about 80-90, about 90-100, about 100-120, about 120-140, about 140-160, about 160-180, about 180-200, about 200-220, about 220-240, about 240- 260, about 260-280, about 280-300, about 300-350, about 350-400, about 400-450, about 450-500, about 500-550, about 550-600, about 600-650, about 650-700, about 700-750, about 750-800, about 800-850, about 850-900, about 900-950, about 950-1000, about 1000-1100, about 1100-1200, about 1200-1300, about 1300-1400, about 1400-1500, about 1500-1600, about 1600-1700, about 1700- 1800, about 1800-1900, about 1900-2000, or greater than about 2000 kDa prior to cross-linking.

39. An isocyanate cross-linker for formation of a cross-linked polymer, the crosslinker comprising: a. a poly-functional core; b. a plurality of di-functional polymeric linkers; each covalently bound to the poly-functional core; and c. a plurality of isocyanate substituents, each covalently bound to one of the plurality of di-functional polymeric linkers such that the isocyanate substituents are coupled with the poly-functional core via the di-functional polymeric linkers, thereby forming the cross-linker.

40. The cross-linker of claim 39, wherein the cross-linker is configured to react with a polymer to form the cross-linked polymer.

41. The cross-linker of claim 39 or claim 40, wherein the cross-linked polymer is configured to swell in an aqueous fluid to form a hydrogel.

42. The cross-linker of any one of claims 39-41, wherein the cross-linker is bifunctional, tri-functional, tetra-functional, penta-functional, or hexa-functional.

43. The cross-linker of any one of claims 39-42, wherein the cross-linker is formed via reaction of a poly-functional alcohol with a multi-functional isocyanate, wherein the multi-functional isocyanate comprises a compound having two or more isocyanate groups, or a compound having one or more isocyanate groups and one or more non-isocyanate groups.

44. The cross-linker of any one of claims 39-43, wherein the cross-linker comprises a plurality of polycaprolactone (PCL) polyethylene glycol (PEG) or polyvinyl alcohol (PVA) polymeric linkers.

45. The cross-linker of any one of claims 39-44, wherein the cross-linker comprises a poly-functional polycaprolactone (PCL) polymer functionalized with a plurality of isocyanate substituents.

46. The cross-linker of any one of claims 39-45, wherein the cross-linker comprises one or more polymer linkers.

47. The cross-linker of any one of claims 39-46, wherein multiple poly- functional alcohols are linked together via reaction with a poly-functional isocyanate to form a poly-functional isocyanate having a greater functionality than that of the unreacted poly-functional alcohol.

48. The cross-linker of any one of claims 39-47, wherein the cross-linker is formed via the reaction of a polycaprolactone (PCL) polymer with terminal hydroxyl groups with ethyl lysine diisocyanate (eLDl) or another poly-functional isocyanate.

49. The cross-linker of any one of claims 39-48, wherein the cross-linker is functionalized with dopamine.

50. A method of preparing a cross-linked polymer for formation of a hydrogel, the method comprising: a. providing a poly-functional isocyanate cross-linker; b. reacting a polymer with an organic base or organic salt to form an organic polymer salt, wherein the organic polymer salt is soluble in an organic solvent; and c. reacting the organic polymer salt with the poly-functional isocyanate crosslinker in the organic solvent to form a plurality of urethane cross-links, thereby forming the cross-linked polymer.

51. The method of claim 50, wherein the cross-linked polymer is configured to swell in an aqueous fluid to form a hydrogel.

52. The method of claim 50 or claim 51, wherein the reaction between the organic polymer salt and the poly-functional isocyanate cross-linker is at a concentration of about 3.5 weight percent.

53. The method of any one of claims 50-52, wherein the reaction between the organic polymer salt and the poly-functional isocyanate cross-linker is at a concentration between about 0.1 to about 3.5 weight percent.

54. The method of any one of claims 50-53, wherein the reaction between the organic polymer salt and the poly-functional isocyanate cross-linker is at about 60 °C.

55. The method of any one of claims 50-54, wherein the reaction between the organic polymer salt and the poly-functional isocyanate cross-linker between about 25 °C and about 80 °C.

56. The method of any one of claims 50-55, wherein the reaction between the organic polymer salt and the poly-functional isocyanate cross-linker between about 25 °C and about 90 °C.

57. The method of any one of claims 50-56, wherein the product is purified by: a. diluting the reaction mixture of the organic polymer salt and the polyfunctional isocyanate cross-linker with methanol; and b. adding an aqueous salt solution to precipitate the cross-linked polymer.

58. The method of claim 57, wherein the aqueous salt solution is a sodium chloride solution.

59. The method of any one of claims 50-58, wherein a weight ratio of the polymer and the cross-linker is about 1 :0.01-1 :0.1, about 1 :0.1-1 :0.2, about 1 :0.2-l :0.3, about 1:0.3-1:0.4, about l:0.4-l :0.5, about 1 :0.5-1:0.6, about 1:0.6-1:0.7, about 1:0.7- 1:0.8, about 1 :0.8- 1:0.9, about 1:0.9-1 :1, about 1 :1-1: 1.1, about 1 :1.1-1: 1.2, about 1: 1.2-1: 1.3, about 1: 1.3-1 : 1.4, about 1 :1.4- 1:1.5, about 1: 1.5-1: 1.6, about 1: 1.6- 1:1.7, about 1 :1.7- 1:1.8, about 1:1.8-1 : 1.9, about 1 : 1.9-1 :2, about 1:2-1 :2.2, about 1:2.2-1:2.4, about 1:2.4-1 :2.6, about 1 :2.6-1:2.8, about 1:2.8-1:3, about 1:3-1 :3.5, about 1 :3.5-1 :4, about 1 :4-l :4.5, or about 1:4.5-1:5.

60. The method of any one of claims 50-59, wherein the poly-functional isocyanate cross-linker is ethyl lysine diisocyanate (eLDl) or another bi-functional crosslinker.

61. The method of any one of claims 50-60, additionally comprising one or more workup, isolation, autoclaving, or hydrating steps.

62. The method of any one of claims 50-61, additionally comprising reaction with dopamine.

63. The method of any one of claims 50-62, wherein the polymer is reacted with cetrimonium bromide, tetrabutylammonium bromide or tetrabutylammonium hydroxide to form the organic polymer salt.

64. A method of preparing a hydrogel, the method comprising: a. providing a cross-linked polymer of any one of the preceding claims; and b. hydrating the cross-linked polymer with an aqueous fluid to form a hydrogel.

65. The method of claim 64, additionally comprising autoclaving the cross-linked polymer.

66. The method of claim 64 or of claim 65, additionally comprising shearing the hydrogel.

67. The method of any one of the claims 64-66, additionally comprising lyophilizing the hydrogel.

68. The method of any one of claims 64-67 additionally comprising shearing and then lyophilizing the hydrogel.

69. A method for cosmetic skin treatment of a subject, the method comprising: a. providing a hydrogel comprising: i. a plurality of polymer chains; ii. a plurality of poly-functional urethane cross-linkers reacted with the plurality of polymer chains to form a cross-linked polymer with a plurality of urethane linkages; and iii. an aqueous fluid dispersed within the cross-linked polymer to form the hydrogel; and b. sub-dermally injecting a volume of the hydrogel to the subject.

70. A method for stimulating subdermal collogen deposition of a subject, the method comprising: a. providing a hydrogel comprising: i. a plurality of polymer chains;ii. a plurality of poly-functional urethane cross-linkers reacted with the plurality of polymer chains to form a cross-linked polymer with a plurality of urethane linkages; and iii. an aqueous fluid dispersed within the cross-linked polymer to form the hydrogel; and b. sub-dermally injecting a volume of the hydrogel to the subject thereby stimulating collogen deposition.

71. The method of claim 69 or claim 70, wherein the hydrogel additionally comprises lidocaine.

72. The method of any one of claims 69-71, wherein subdermal injection of the hydrogel stimulates subdermal collagen deposition.

73. The method of any one of claims 69-72, wherein the hydrogel is injected via a needle.

74. The method of any one of claims 69-73, wherein the hydrogel is injected via a 12- gauge, a 14-gauge, a 16-gauge, a 18-gauge, a 19-gauge, a 20-gauge, a 21 -gauge, a 22-gauge, a 23-gauge, a 24-gauge, a 25-gauge, a 26-gauge, a 27-gauge, a 28- gauge, a 29-gauge, a 30-gauge, or a 32-gauge needle.

75. A kit for preparation of a hydrogel, the kit comprising: a. a cross linked polymer comprising a plurality of polymer chains reacted with a plurality of polymer chains to form a cross-linked polymer with a plurality of urethane linkages, the cross-linked polymer configured to swell in an aqueous fluid to form a hydrogel; and b. an aqueous fluid.

76. The kit of claim 75, additionally comprising instructions for swelling the crosslinked polymer with the aqueous fluid to form the hydrogel.

77. A kit for preparation of a hydrogel, the kit comprising: a. a cross-linked polymer comprising a plurality of polymer chains reacted with a plurality of polymer chains to form a cross-linked polymer with a plurality of urethane linkages; and b. instructions for adding an aqueous fluid to the cross-linked polymer to form a hydrogel.

78. The kit of claim 77, wherein the cross-linked polymer is configured to swell in an aqueous fluid to form a biocompatible hydrogel.

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