Physically mixed HA-collagen skin filler

Abstract

The present disclosure relates to a physically mixed HA-collagen skin filler, in particular to a composition comprising cross-linked hyaluronic acid physically mixed with collagen. Methods of improving the aesthetic quality of anatomical features of the human body are also contemplated.

Description

[0001] This patent application is a divisional application of the patent application with application number 2020800966345, application date December 28, 2020, and invention name “Physically mixed HA-collagen skin filler”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Patent Application No. 62 / 953,925, filed on December 26, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0004] The present disclosure relates to compositions and methods of making compositions comprising hyaluronic acid mixed with collagen for use as a filler.Other methods include methods of treating volume deficiencies within tissues. Background Art

[0005] Embodiments described herein relate to dermal filler compositions, and more particularly to dermal filler compositions comprising hyaluronic acid physically mixed with collagen.

[0006] Skin aging occurs over time and is exacerbated by a variety of factors, such as sun exposure (UVA and UVB rays), genetics, and negative personal habits (such as smoking, alcohol, and a poor diet). As your skin ages, it may feel and look rougher, and your skin may also begin to thin and become less elastic.

[0007] Hyaluronan (also known as hyaluronic acid, abbreviated HA) is a non-sulfated glycosaminoglycan that is widely distributed in the connective tissue, epithelial tissue, and neural tissue of the human body. Hyaluronan is abundant in different layers of the skin, and its role is to ensure good hydration, assist in the organization of the extracellular matrix, act as a space filler; and participate in tissue repair mechanisms. However, with age, the amount of hyaluronan, collagen, elastin, and other matrix polymers present in the skin decreases. For example, repeated exposure to ultraviolet rays from the sun can cause dermal cells to reduce their production of hyaluronan and increase its degradation rate. This loss of hyaluronan can lead to various skin conditions, such as dry skin, wrinkles, blemishes, imperfections, and reduced skin thickness.

[0008] The body also has endogenous collagen found in tendons, muscles, bones, skin and ligaments. However, over time, collagen content in the body can be lost due to exposure to UV rays, tobacco and alcohol, and the aging process. Reduced collagen in the skin can lead to loss of elasticity, reduced skin thickness, wrinkle formation and sagging.

[0009] As a result, there is growing interest in both surgical and nonsurgical treatments to address aging issues. Nonsurgical treatments include botulinum toxin injections and soft tissue filler injections, which are the most commonly performed procedures for facial rejuvenation.

[0010] Dermal fillers have been used to treat volume loss, smooth the appearance of wrinkles, correct facial asymmetry, and contour the face and other areas of the body. Results may last from a few months to two years, depending on where the filler was injected and the patient; however, the effects of fillers are temporary and require follow-up treatments to maintain the desired aesthetic results.

[0011] Fillers based on hyaluronic acid (HA) were developed approximately 20 years ago, and treatment with such fillers is one of the most frequently performed procedures in cosmetic dermatology practice. A major advantage of HA fillers is that HA produced by biotechnological methods is chemically identical to HA produced naturally by the human body and therefore rarely causes allergic reactions. HA is susceptible to enzymatic degradation in tissues due to the body's endogenous enzymes and clearance, and therefore, HA can be cross-linked or otherwise modified to extend the residence time of the filler. Furthermore, because HA is susceptible to enzymatic degradation by hyaluronidase, HA fillers can degrade rapidly and, in the event of an adverse event, the filler effect can be reversed by treatment with exogenous hyaluronidase. Therefore, HA fillers are an attractive option for many patients because the corrective effects of HA treatment are long-lasting and HA fillers have a good safety profile.

[0012] Along with their many advantages, HA fillers also have several disadvantages. One such disadvantage is the Tyndall effect, a bluish tint that can be seen at the site of injection of certain surface HA fillers. Since HA is hydrophilic and will absorb water, swelling can also occur at the injection site. It should also be noted that the higher the degree of cross-linking, the higher the risk of inflammation and granuloma formation that HA may have.

[0013] Before HA fillers appeared on the market, collagen fillers were introduced to the market in the 1980s. Collagen has several favorable properties as a filler material. For example, collagen fillers are opaque, so injecting these materials under the skin does not result in the Tyndall effect as seen with HA fillers. Additionally, collagen can act as a scaffold and support tissue ingrowth into the filler material. However, collagen fillers are not without their disadvantages. High collagen concentrations are required in filler formulations to achieve the desired filling effect, and despite these high collagen concentrations, the volumizing and wrinkle-filling abilities of the material are lower than those of HA fillers. Additionally, the duration of wrinkle correction has been shown to be only 3 to 6 months, and these fillers are irreversible as no enzymatic treatments exist that can be used to degrade the collagen material.

[0014] Therefore, there is a need to provide dermal fillers that have the advantages of both HA and collagen fillers. As described in embodiments herein, the present invention provides compositions that provide lift and volume upon injection while providing the benefits of both collagen and HA fillers. Summary of the invention

[0015] Thus, new dermal filler compositions are provided, as well as methods for making the same. Some embodiments include homogenous hydrogel compositions prepared from hyaluronic acid and collagen. Some embodiments also include hydrogel compositions containing completely uniformly mixed HA and / or collagen particles. Some embodiments may also include partially homogenous or heterogeneous products containing particles of HA or collagen (e.g., collagen fibrils). These compositions may be prepared by including cross-linked hyaluronic acid to produce cross-linked hyaluronic acid and then mixing the cross-linked hyaluronic acid with collagen.

[0016] In a first aspect, a method for preparing a macromolecular matrix is ​​provided. The method comprises providing cross-linked hyaluronic acid, providing collagen, and physically mixing the collagen into the cross-linked hyaluronic acid, wherein the collagen is homogeneously mixed throughout the cross-linked hyaluronic acid, thereby forming a macromolecular matrix, wherein the macromolecular matrix comprises cross-linked hyaluronic acid and is physically mixed in the collagen.

[0017] In some embodiments of any one or any of each of the foregoing or following embodiments, the collagen is provided in a soluble state as a solution.

[0018] In any one of the above or following embodiments respectively or in some embodiments of any one, collagen is provided in the form of a solution, wherein the pH of the collagen solution is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, or any pH between the ranges defined by any two of the above values, and wherein collagen is soluble at acidic pH. In any one of the above or following embodiments respectively or in some embodiments of any one, collagen is provided in the form of an acidic pH solution, wherein the pH of the collagen solution is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, or any pH between the ranges defined by any two of the above values, and wherein collagen is soluble at acidic pH. In any one of the above or following embodiments respectively or in some embodiments of any one, collagen is provided in the form of a neutral pH solution.

[0019] In some embodiments of any one or any of the foregoing or following embodiments, the collagen and cross-linked hyaluronic acid are physically mixed with a buffer. In some embodiments of any one or any of the foregoing or following embodiments, the buffer comprises PBS.

[0020] In some embodiments of any of the foregoing or following embodiments, the method further comprises neutralizing the macromolecular matrix to a pH of about 7 after thoroughly homogenizing the collagen in the cross-linked hyaluronic acid.

[0021] In some embodiments of any one or any of each of the foregoing or following embodiments, the method further comprises neutralizing the collagen to a pH of about 7 prior to physically mixing the collagen with the cross-linked hyaluronic acid. In some embodiments of any one or any of each of the foregoing or following embodiments, neutralizing the collagen prior to physically mixing the collagen into the cross-linked hyaluronic acid causes the collagen to precipitate into collagen fibrils or particles, wherein the collagen fibrils or particles are further mixed into the cross-linked hyaluronic acid, wherein the fibrils or particles are completely homogenously mixed in the cross-linked hyaluronic acid.

[0022] In the above-mentioned or following embodiments respectively any one or in some embodiments of any embodiment, collagen is provided in the form of fibrillated collagen or collagen fiber. In the above-mentioned or following embodiments respectively any one or in some embodiments of any embodiment, wherein collagen is provided in the form of fibrillated collagen, and wherein collagen is prepared at neutral or alkaline pH, thereby producing the fibrillated collagen of collagen fiber.

[0023] In any one of the above or following embodiments or some embodiments of any embodiment, collagen is prepared with at least one salt to obtain fibrillated collagen or collagen fibers. In any one of the above or following embodiments or some embodiments of any embodiment, the concentration of at least one salt is: about 20mM, about 50mM, about 100mM, about 150mM, about 200mM, about 250mM, about 300mM, about 350mM, about 400mM, about 450mM or about 500mM, or in any concentration between the ranges defined by any two of the above values. In any one of the above or following embodiments or some embodiments of any embodiment, at least one salt comprises anions, wherein the anions comprise H2PO 4- 、SO4 2- , Cl - or SCN -In some embodiments of any of the foregoing or following embodiments, at least one salt comprises NaCl, Na2SO4, or Li2SO4.

[0024] In some embodiments of any of the foregoing or following embodiments, the collagen is homogeneously mixed throughout the macromolecular matrix.

[0025] In some embodiments of any of the foregoing or following embodiments, the cross-linked hyaluronic acid comprises a hyaluronic acid component having an average molecular weight of about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 40,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 90,000 Daltons, about 100,000 Daltons, about 200,000 Daltons, about 300,000 Daltons, about 400,000 Daltons, about 500,000 Daltons, about 600,000 Daltons, about 700,000 Daltons, about 800,000 Daltons, about 90,000 Daltons, about 100,000 Daltons, about 200,000 Daltons, about 300,000 Daltons, about 400,000 Daltons, about 500,000 Daltons, about 600,000 Daltons, about 700,000 Daltons, about 800,000 Daltons, about Daltons, about 900,000 Daltons, about 1,000,000 Daltons, about 1,100,000 Daltons, about 1,200,000 Daltons, about 1,300,000 Daltons, about 1,400,000 Daltons, about 1,500,000 Daltons, about 1,600,000 Daltons, about 1,700,000 Daltons, about 1,800,000 Daltons, about 1,900,000 Daltons, about 2,000,000 Daltons, about 2,100,000 Daltons, about 2,200,000 Daltons, about 2,300,000 Daltons, about 2,400,000 Daltons, about 2,500,000 Daltons, about 2,600,000 Daltons Daltons, about 2,700,000 Daltons, about 2,800,000 Daltons, about 2,900,000 Daltons, about 3,000,000 Daltons, about 3,100,000 Daltons, about 3,200,000 Daltons, about 3,300,000 Daltons, about 3,400,000 Daltons, about 3,500,000 Daltons, about 3,600,000 Daltons, about 3,700,000 Daltons, about 3,800,000 Daltons, about 3,900,000 Daltons, about 4,000,000 Daltons, about 4,100,000 Daltons, about 4,200,000 Daltons, about 4,300,000 Daltons, about 4,400,000 Daltons. ,000 Daltons, about 4,500,000 Daltons, about 4,600,000 Daltons, about 4,700,000 Daltons, about 4,800,000 Daltons, about 4,900,000 Daltons, about 5,000,000 Daltons, about 5,100,000 Daltons, about 5,200,000 Daltons, about 5,300,000 Daltons, about 5400,000 Daltons, about 5500,000 Daltons, about 5600,000 Daltons, about 5700,000 Daltons, about 5800,000 Daltons, about 5900,000 Daltons, about 6000,000 Daltons, about 6100,000 Daltons, about 6200,000 Daltons, about000 Daltons, about 6,300,000 Daltons, about 6,400,000 Daltons, about 6,500,000 Daltons, about 6,600,000 Daltons, about 6,700,000 Daltons, about 6,800,000 Daltons, about 6,900,000 Daltons, about 7,000,000 Daltons, about 7,100,000 Daltons, about 7,200,000 Daltons 00 Daltons, about 7,300,000 Daltons, about 7,400,000 Daltons, about 7,500,000 Daltons, about 7,600,000 Daltons, about 7,700,000 Daltons, about 7,800,000 Daltons, about 7,900,000 Daltons, about 8,000,000 Daltons, about 8,100,000 Daltons, about 8,200,000 Daltons 0 Daltons, about 8,300,000 Daltons, about 8,400,000 Daltons, about 8,500,000 Daltons, about 8,600,000 Daltons, about 8,700,000 Daltons, about 8,800,000 Daltons, about 8,900,000 Daltons, about 9,000,000 Daltons, about 9,100,000 Daltons, about 9,200,000 Daltons Daltons, about 9,300,000 Daltons, about 9,400,000 Daltons, about 9,500,000 Daltons, about 9,600,000 Daltons, about 9,700,000 Daltons, about 9,800,000 Daltons, about 9,900,000 Daltons, or about 10,000,000 Daltons, or any molecular weight between the ranges defined by any two of the above values. In some embodiments of any of the foregoing or following embodiments, the cross-linked hyaluronic acid comprises a mixture of hyaluronic acid components, the hyaluronic acid components comprising at least two different molecular weights.

[0026] In some embodiments of any one or any embodiment of the above or following embodiments, the method further comprises adding lidocaine to the macromolecular matrix. In some embodiments of any one or any embodiment of the above or following embodiments, the concentration of lidocaine in the matrix is ​​between 0.15% (w / w) and 0.45% (w / w). In some embodiments of any one or any embodiment of the above or following embodiments, the concentration of lidocaine in the matrix is ​​between 0.27% (w / w) and 0.33% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of lidocaine in the matrix is ​​about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration in the range defined by any two of the above values. In some embodiments of any one or any of the above or following embodiments, the concentration of lidocaine in the matrix is ​​about 0.3% (w / w).

[0027] In some embodiments of any or any of the above or following embodiments, the method further comprises adding uncrosslinked HA to the matrix. In some embodiments of any or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​up to about 5% (w / w). In some embodiments of any or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), or any concentration between the ranges defined by any two of the above values. In some embodiments of any or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 1% (w / w). In some embodiments of any or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​2% (w / w). In some embodiments of any or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 5% (w / w). In some embodiments of any of the foregoing or following embodiments, the uncrosslinked HA improves the extrudability of the matrix.

[0028] In some embodiments of any of the foregoing or following embodiments, the macromolecular matrix has enhanced extrudability through a needle, wherein the needle comprises a gauge size of 27G, 28G, 29G, 30G, 31G, or 32G.

[0029] In some embodiments of any or any of the above or following embodiments, the macromolecular matrix is ​​stable for at least 6 months. In some embodiments of any or any of the above or following embodiments, the macromolecular matrix is ​​stable for about 6, about 9, about 12, about 15, about 18, about 21, about 24, about 27, about 30, about 33 or about 36 months, or any amount of time between the ranges defined by any two of the above values. In some embodiments of any or any of the above or following embodiments, the macromolecular matrix is ​​stable at a temperature between about 4°C and about 25°C. In some embodiments of any or any of the above or following embodiments, the macromolecular matrix is ​​stable at about 4°C, about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, or any temperature between the ranges defined by any two of the above values. In some embodiments of any or any of the above or following embodiments, the macromolecular matrix is ​​stable at 4°C. In some embodiments of any or any of the above or following embodiments, the macromolecular matrix is ​​stable at about 25°C.

[0030] In some embodiments of any of the foregoing or following embodiments, the macromolecular matrix has minimal degradation in about 3, about 6, about 9, about 12, about 15, about 18, about 21, about 24, about 27, about 30, about 33, about 36 months, or any amount of time between the ranges defined by any two of the foregoing values.

[0031] In the second aspect, a method for preparing a macromolecular matrix is ​​provided. The method includes dissolving hyaluronic acid in an aqueous solution to form a pre-reaction aqueous solution and preparing a second solution, wherein the second solution includes a water-soluble carbodiimide; and N-hydroxysuccinimide or N-hydroxysulfosuccinimide; or 1,4-butanediol diglycidyl ether (BDDE) in a sodium hyaluronate solution in the presence of sodium hydroxide; the second solution is added to the pre-reaction aqueous solution to form a cross-linked reaction mixture; cross-linked hyaluronic acid, wherein the cross-linked reaction mixture reacts by cross-linking hyaluronic acid, thereby forming a cross-linked hyaluronic acid; and collagen is provided, and collagen is physically mixed into the cross-linked hyaluronic acid, thereby producing a macromolecular matrix comprising cross-linked hyaluronic acid and collagen, wherein collagen is homogeneously mixed throughout the cross-linked hyaluronic acid. In any one of the above or following embodiments or some embodiments of any embodiment, the second solution includes a water-soluble carbodiimide and N-hydroxysuccinimide or N-hydroxysulfosuccinimide, wherein hyaluronic acid is cross-linked with a naturally occurring amine, thereby forming a cross-linked hyaluronic acid. In any one or some embodiments of any embodiment of the above-mentioned or following embodiments respectively, the second solution comprises a water-soluble carbodiimide and N-hydroxysuccinimide or N-hydroxysulfosuccinimide, and wherein cross-linking is carried out in the presence of MES. In any one or some embodiments of any embodiment of the above-mentioned or following embodiments respectively, the second solution comprises 1,4-butanediol diglycidyl ether (BDDE) in a sodium hyaluronate solution in the presence of sodium hydroxide, and wherein BDDE is used to cross-link hyaluronic acid using epoxide chemistry. In any one or some embodiments of any embodiment of the above-mentioned or following embodiments respectively, the physical mixing step is carried out in the presence of a buffer. In any one or some embodiments of any embodiment of the above-mentioned or following embodiments respectively, the buffer comprises PBS. In any one of the above or following embodiments or some embodiments of any embodiment, collagen is provided in the form of a collagen solution, wherein the pH of the collagen solution is: about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5 or about 7.0, or any pH between the ranges defined by any two of the above values, and wherein the collagen is soluble at acidic pH. In any one of the above or following embodiments or some embodiments of any embodiment, collagen is provided in the form of an acidic pH solution, wherein the pH of the collagen solution is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5 or any pH between the ranges defined by any two of the above values, and wherein the collagen is soluble at acidic pH.In some embodiments of any one of the foregoing or following embodiments or any of the embodiments, the collagen is provided in the form of a collagen solution, wherein the pH of the collagen solution is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5 or about 7.0 or any pH in the range defined by any two of the foregoing values, and wherein the collagen is soluble at acidic pH.

[0032] In some embodiments of any of the foregoing or following embodiments, the collagen is provided at an acidic to neutral pH.

[0033] In some embodiments of any one of the foregoing or following embodiments or any of the embodiments, the method further comprises neutralizing the macromolecular matrix to a pH of about 7 after homogenizing the collagen throughout the cross-linked hyaluronic acid.

[0034] In some embodiments of any one of the foregoing or following embodiments or any of the embodiments, the method further comprises neutralizing the macromolecular matrix to a pH of about 7 after homogenizing the collagen throughout the cross-linked hyaluronic acid.

[0035] In some embodiments of any one or any of the foregoing or following embodiments, the collagen is provided for prefibrosis in a solution at about pH 5 to pH 7. In some embodiments of any one or any of the foregoing or following embodiments, for prefibrosis, the collagen is provided in the form of a solution having a pH of about 5.0, about 5.5, about 6.0, about 6.5, or about 7, or any pH in the range defined by any two of the foregoing values.

[0036] In some embodiments of any one of the foregoing or following embodiments or any of the embodiments, the method further comprises neutralizing the macromolecular matrix to a pH of about 7 after homogenizing the collagen throughout the cross-linked hyaluronic acid.

[0037] In some embodiments of any one of the above or below embodiments or any of the embodiments, the method further includes neutralizing the collagen to a pH of about 7 before physically mixing the collagen with the cross-linked hyaluronic acid, wherein neutralizing the collagen before physically mixing the collagen into the cross-linked hyaluronic acid causes the collagen to precipitate into collagen fibrils or particles, wherein the collagen fibrils or particles are further mixed into the cross-linked hyaluronic acid, and wherein the fibrils or particles are homogenously mixed throughout the cross-linked hyaluronic acid.

[0038] In some embodiments of any of the foregoing or following embodiments, the collagen is provided in the form of fibrillated collagen or collagen fibers.

[0039] In some embodiments of any of the foregoing or following embodiments, the collagen is prepared at an alkaline pH, thereby producing fibrillated collagen or collagen fibers.

[0040] In some embodiments of any of the foregoing or following embodiments, the collagen is prepared with at least one salt to obtain fibrillated collagen or collagen fibers.

[0041] In some embodiments of any of the foregoing or following embodiments, the collagen is prepared with at least one salt to obtain fibrillated collagen or collagen fibers.

[0042] In some embodiments of any of the foregoing or following embodiments, the concentration of at least one salt is about 20 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM or about 500 mM, or any concentration in the range defined by any two of the foregoing values.

[0043] In some embodiments of any of the foregoing or following embodiments, at least one salt comprises an anion, wherein the anion comprises H2PO 4- 、SO4 2- , Cl - or SCN - In some embodiments of any of the foregoing or following embodiments, at least one salt comprises NaCl, Na2SO4, or Li2SO4.

[0044] In some embodiments of any of the foregoing or following embodiments, the hyaluronic acid comprises a mixture of hyaluronic acid components comprising at least two different molecular weights.

[0045] In some embodiments of any one of the above or below embodiments or any of the embodiments, the dissolving step also includes dissolving both hyaluronic acid and collagen in an aqueous solution to form a pre-reacted aqueous solution comprising hyaluronic acid and collagen, wherein the cross-linking reaction reacts by cross-linking both hyaluronic acid and collagen into an HA-collagen coupled gel, and wherein the mixing step includes physically mixing the HA-collagen coupled gel with additional collagen, wherein the collagen is in a soluble state and wherein the collagen is in a solution, wherein the solution is at an acidic pH, thereby producing a macromolecular matrix comprising the HA-collagen coupled gel physically mixed in the collagen.

[0046] In some embodiments of any one of the foregoing or following embodiments, collagen fibrils or collagen precipitates are formed after a mixing step with collagen, and wherein the collagen fibrils or collagen precipitates are homogeneously mixed in the cross-linked hyaluronic acid. In some embodiments of any one of the foregoing or following embodiments, collagen fibrils or collagen precipitates are formed during a mixing step with collagen, and wherein the collagen fibrils or collagen precipitates are homogeneously mixed in the cross-linked hyaluronic acid.

[0047] In some embodiments of any one of the foregoing or following embodiments or any of the embodiments, the method further comprises adjusting the osmotic pressure of the macromolecular matrix after mixing the collagen throughout the cross-linked hyaluronic acid.

[0048] In some embodiments of any or any of the foregoing or following embodiments, the mixing step is performed at room temperature. In some embodiments of any or any of the foregoing or following embodiments, the mixing step is performed at about 4°C. In some embodiments of any or any of the foregoing or following embodiments, the method is performed at about 4°C.

[0049] In some embodiments of any one or any embodiment of the above or following embodiments, the method also includes purifying cross-linked hyaluronic acid or HA-collagen coupling gel, wherein the mixing step of mixing with collagen is carried out before purification. In some embodiments of any one or any embodiment of the above or following embodiments, purification is carried out using dialysis purification. In some embodiments of any one or any embodiment of the above or following embodiments, the pH of the cross-linked reaction mixture is adjusted after cross-linking is completed, wherein the pH is adjusted before the purification step, and wherein the pH is adjusted to about 7.0, about 7.2, about 7.4, about 7.6, or any pH between the ranges defined by any two of the above values. In some embodiments of any one or any embodiment of the above or following embodiments, purification is carried out between the ranges between about 2 ° C to about 8 ° C. In some embodiments of any one or any embodiment of the above or following embodiments, purification is carried out at about 2 ° C, about 4 ° C, about 6 ° C or about 8 ° C, or at any temperature between the ranges defined by any two of the above values.

[0050] In some embodiments of any one or any embodiment of the above or following embodiments, the method also includes sterilizing the cross-linked hyaluronic acid or HA-collagen coupling gel before mixing with collagen, wherein the sterilization step is performed after the purification step. In some embodiments of any one or any embodiment of the above or following embodiments, the sterilization step includes transferring the cross-linked hyaluronic acid or HA-collagen coupling gel to a container for sterilization; and sterilizing the cross-linked hyaluronic acid or HA-collagen coupling gel. In some embodiments of any one or any embodiment of the above or following embodiments, the container is a syringe. In some embodiments of any one or any embodiment of the above or following embodiments, sterilization is by heat (dry heat, steam heat, moist heat sterilization), radiation (i.e. non-ionizing, UV), ionization (particles (e.g., beta rays, gamma rays, x-rays), electromagnetic (e.g., electron beam), filtration) or terminal sterilization.

[0051] In the above-mentioned or following embodiments each in any one or some embodiments of any embodiment, the method also includes dialyzing cross-linked macromolecular matrix or HA-collagen coupling gel, wherein dialysis is carried out by having a molecular weight cutoff value in the range between about 1000 daltons to about 100,000 daltons, and wherein dialysis is carried out before sterilization. In the above-mentioned or following embodiments each in any one or some embodiments of any embodiment, dialysis is carried out in buffer. In the above-mentioned or following embodiments each in any one or some embodiments of any embodiment, dialysis is carried out in phosphate buffered saline or sodium phosphate buffer. In the above-mentioned or following embodiments each in any one or some embodiments of any embodiment, buffer also includes NaCl and / or KCl.

[0052] In some embodiments of any one or any embodiment of the above or following embodiments, the method further comprises raising the pH of the cross-linked hyaluronic acid or HA-collagen coupling gel to a neutral pH after the cross-linking reaction is completed, wherein the pH increase is performed before the sterilization step. In some embodiments of any one or any embodiment of the above or following embodiments, the method further comprises raising the pH of the cross-linked hyaluronic acid or HA-collagen coupling gel to about 7.0, about 7.2, or about 7.4, or any pH between the ranges defined by any two of the above values ​​after the cross-linking reaction is completed, wherein the pH increase is performed before the sterilization step.

[0053] In some embodiments of any one or any of the foregoing or following embodiments, the second solution comprises a water-soluble carbodiimide and N-hydroxysuccinimide or N-hydroxysulfosuccinimide, and wherein the adding and cross-linking steps are performed at a temperature between about 2° C. and about 22° C. In some embodiments of any one or any of the foregoing or following embodiments, the adding and cross-linking steps are performed at a temperature of about 2° C., about 4° C., about 6° C., about 8° C., about 10° C., about 12° C., about 14° C., about 16° C., about 18° C., about 20° C., about 22° C., or at any temperature between the ranges defined by any two of the foregoing values.

[0054] In some embodiments of any one or any of the foregoing or following embodiments, crosslinking is performed at about 22°C. In some embodiments of any one or any of the foregoing or following embodiments, crosslinking is performed at about 4°C.

[0055] In some embodiments of any one or any of the above or following embodiments, the second solution comprises 1,4-butanediol diglycidyl ether (BDDE) in a sodium hyaluronate solution in the presence of sodium hydroxide, and wherein the adding and cross-linking steps are performed between about 45°C and about 75°C. In some embodiments of any one or any of the above or following embodiments, the second solution comprises 1,4-butanediol diglycidyl ether (BDDE) in a sodium hyaluronate solution in the presence of sodium hydroxide, and wherein the adding and cross-linking steps are performed at a temperature of about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, or any temperature between the ranges defined by any two of the above values. In some embodiments of any one or any of the above or following embodiments, the second solution comprises 1,4-butanediol diglycidyl ether (BDDE) in a sodium hyaluronate solution in the presence of sodium hydroxide, and wherein the adding and cross-linking steps are performed at a temperature of about 50°C.

[0056] In some embodiments of any one of the foregoing or following embodiments or any of the embodiments, the method further comprises a stopping step of cross-linking, wherein the stopping step comprises adding a base to the cross-linking reaction mixture to a pH of about 8 to about 10 for at least about 10 minutes, and then adding an acidic solution to the cross-linking mixture until a pH of about 7 is reached.

[0057] In some embodiments of any one or any of the foregoing or following embodiments, the cross-linking reaction mixture comprises a pH between about 4.0 and about 10.0. In some embodiments of any one or any of the foregoing or following embodiments, the pH of the cross-linking reaction mixture is between about 4.0 and about 6.0.

[0058] In some embodiments of any of the foregoing or following embodiments, the second solution comprises 1,4-butanediol diglycidyl ether (BDDE) in a sodium hyaluronate solution in the presence of sodium hydroxide, and wherein the crosslinking is performed under alkaline conditions.

[0059] In some embodiments of any of the foregoing or following embodiments, the pre-reaction solution comprises a salt, wherein the concentration of the salt is about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, about 500 mM, or any concentration in the range defined by any two of the foregoing values.

[0060] In some embodiments of any one or any of the above or following embodiments, the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and wherein the concentration of the water-soluble carbodiimide in the cross-linking reaction mixture is about 20 mM to about 300 mM. In some embodiments of any one or any of the above or following embodiments, the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration of about 20 mM, about 40 mM, about 60 mM, about 80 mM, about 100 mM, about 120 mM, about 140 mM, about 160 mM, about 180 mM, about 200 mM, about 220 mM, about 240 mM, about 260 mM, about 280 mM, about 300 mM, or any concentration between the ranges defined by any two of the above values. In some embodiments of any of the foregoing or following embodiments, the molar ratio of water-soluble carbodiimide to hyaluronic acid water-soluble carbodiimide:hyaluronic acid repeating units is from about 0.3 to about 3.0. In some embodiments of any of the foregoing or following embodiments, the molar ratio of water-soluble carbodiimide to hyaluronic acid water-soluble carbodiimide:hyaluronic acid repeating units is about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9 or about 3.0, or any molar ratio between the ranges defined by any two of the foregoing values.

[0061] In some embodiments of any one of the above or below embodiments, the mole: mole (lysine: HA repeating unit) ratio of lysine to hyaluronic acid is between about 0.01 and about 0.6. In some embodiments of any one of the above or below embodiments, the mole: mole (lysine: HA repeating unit) ratio of lysine to hyaluronic acid is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.2, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49, about 10. about 0.54, about 0.55, about 0.56, about 0.57, about 0.58, about 0.59, about 0.6, or any molar ratio in the range defined by any two of the above values.

[0062] In any one or some embodiments of any of the above or following embodiments respectively, before adding the second solution, the hyaluronic acid in the pre-reaction solution is hydrated for at least 60 minutes. In any one or some embodiments of any of the above or following embodiments respectively, the cross-linking of the reaction mixture is carried out for about 4 hours to about 24 hours.

[0063] In some embodiments of any one or any embodiment of the above or following embodiments, the method further comprises adding lidocaine to the macromolecular matrix. In some embodiments of any one or any embodiment of the above or following embodiments, the concentration of lidocaine in the matrix is ​​between about 0.15% (w / w) to about 0.45% (w / w). In some embodiments of any one or any embodiment of the above or following embodiments, the concentration of lidocaine in the matrix is ​​between about 0.27% (w / w) to about 0.33% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of lidocaine in the matrix is ​​about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration in the range defined by any two of the above values. In some embodiments of any one or any of the above or following embodiments, the concentration of lidocaine in the matrix is ​​about 0.3% (w / w).

[0064] In some embodiments of any or any of the above or following embodiments, the method further comprises adding uncrosslinked HA to the matrix. In some embodiments of any or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​up to about 5% (w / w). In some embodiments of any or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), or any concentration between the ranges defined by any two of the above values. In some embodiments of any or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 1% (w / w). In some embodiments of any or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 2% (w / w). In some embodiments of any or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 5% (w / w). In some embodiments of any of the foregoing or following embodiments, the uncrosslinked HA improves the extrudability of the matrix.

[0065] In some embodiments of any one of the foregoing or following embodiments or any of the embodiments, the method includes a neutralization step performed after forming a macromolecular matrix comprising cross-linked hyaluronic acid and collagen or after forming an HA-collagen coupled gel with physically mixed collagen, wherein the neutralization step includes adjusting the macromolecular matrix to physiological pH and physiological salt concentration.

[0066] In any one or some embodiments of any of the above or following embodiments, neutralization includes adding an alkaline solution or a buffer solution to adjust the pH after the mixing step under sterile conditions. In any one or some embodiments of any of the above or following embodiments, adjusting the pH and physiological salt concentration causes the collagen to precipitate into fibrils or particles, wherein the collagen fibrils or particles are homogeneously distributed in the macromolecular matrix.

[0067] In some embodiments of any of the foregoing or following embodiments, the macromolecular matrix has enhanced extrudability through a needle, wherein the needle comprises a gauge size of 27G, 28G, 29G, 30G, 31G, or 32G.

[0068] In a third aspect, a macromolecular matrix is ​​provided, wherein the macromolecular matrix is ​​prepared by any one of the embodiments described herein.

[0069] In some embodiments of any one or any embodiment of the above or following embodiments, the macromolecular matrix includes cross-linked hyaluronic acid, wherein collagen is physically mixed with cross-linked hyaluronic acid, and wherein collagen is homogeneous throughout the macromolecular complex. In some embodiments of any one or any embodiment of the above or following embodiments, collagen is uniformly mixed throughout the macromolecular complex. In some embodiments of any one or any embodiment of the above or following embodiments, the macromolecular matrix includes HA-collagen coupling gel, wherein HA-collagen coupling gel also includes physically mixed collagen that is not cross-linked to HA-collagen coupling gel. In some embodiments of any one or any embodiment of the above or following embodiments, the macromolecular matrix also includes lidocaine. In some embodiments of any one or any embodiment of the above or following embodiments, the concentration of lidocaine in the matrix is ​​in the range of about 0.15% (w / w) to about 0.45% (w / w). In some embodiments of any of the foregoing or following embodiments, the concentration of lidocaine in the matrix is ​​in the range of about 0.27% (w / w) to about 0.33% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of lidocaine in the matrix is ​​about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration in the range defined by any two of the above values. In some embodiments of any one or any of the above or following embodiments, the concentration of lidocaine in the matrix is ​​about 0.3% (w / w).

[0070] In some embodiments of any one or any of the above or following embodiments, the matrix further comprises uncrosslinked HA. In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​up to 5% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), or any concentration between the ranges defined by any two of the above values. In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 1% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 2% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 5% (w / w). In some embodiments of any of the foregoing or following embodiments, the uncrosslinked HA improves the extrudability of the matrix.

[0071] In a fourth aspect, a macromolecular matrix is ​​provided, wherein the macromolecular matrix is ​​prepared by the method described in any one or some embodiments of any of the above or below embodiments. In some embodiments of any one or some embodiments of any of the above or below embodiments, the method further comprises concentrating the collagen before adding the collagen to the cross-linked HA.

[0072] In a fifth aspect, a macromolecular matrix is ​​provided, wherein the macromolecular matrix comprises: hyaluronic acid, wherein the hyaluronic acid is cross-linked; and collagen; wherein the collagen is physically mixed with the cross-linked hyaluronic acid. In any one of the above or following embodiments or some embodiments of any embodiment, the macromolecular matrix also comprises lidocaine. In any one of the above or following embodiments or some embodiments of any embodiment, the concentration of lidocaine in the matrix is ​​between about 0.15% (w / w) and about 0.45% (w / w). In any one of the above or following embodiments or some embodiments of any embodiment, the concentration of lidocaine in the matrix is ​​between about 0.27% (w / w) and about 0.33% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of lidocaine in the matrix is ​​about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration in the range defined by any two of the above values. In some embodiments of any one or any of the above or following embodiments, the concentration of lidocaine in the matrix is ​​0.3% (w / w).

[0073] In some embodiments of any one or any of the above or following embodiments, the matrix further comprises uncrosslinked HA. In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​up to about 5% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), or any concentration between the ranges defined by any two of the above values. In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 1% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 2% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 5% (w / w). In some embodiments of any of the foregoing or following embodiments, the uncrosslinked HA improves the extrudability of the matrix.

[0074] In the sixth aspect, a macromolecular matrix is ​​provided, wherein the macromolecular matrix includes: HA-collagen coupling gel, wherein the HA-collagen coupling gel includes hyaluronic acid cross-linked with collagen; and physically mixed collagen, wherein the physically mixed collagen is not cross-linked to the HA-collagen coupling gel, and wherein the physically mixed collagen is uniformly mixed in the HA-collagen coupling gel. In any one of the above or below embodiments or some embodiments of any embodiment, the macromolecular matrix also includes lidocaine. In any one of the above or below embodiments or some embodiments of any embodiment, the concentration of lidocaine in the matrix is ​​between about 0.15% (w / w) and about 0.45% (w / w). In any one of the above or below embodiments or some embodiments of any embodiment, the concentration of lidocaine in the matrix is ​​between about 0.27% (w / w) and about 0.33% (w / w). In some embodiments of any of the foregoing or following embodiments, the concentration of lidocaine in the matrix is ​​about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w)), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration in the range defined by any two of the foregoing values. In some embodiments of any of the foregoing or following embodiments, the concentration of lidocaine in the matrix is ​​about 0.3% (w / w).

[0075] In some embodiments of any one or any of the above or following embodiments, the matrix further comprises uncrosslinked HA. In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​up to about 5% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), or any concentration between the ranges defined by any two of the above values. In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 1% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 2% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 5% (w / w). In some embodiments of any of the foregoing or following embodiments, the uncrosslinked HA improves the extrudability of the matrix.

[0076] In the above-mentioned or following embodiments each in any one or some embodiments of any embodiment, hyaluronic acid is cross-linked with naturally occurring amines. In the above-mentioned or following embodiments each in any one or some embodiments of any embodiment, naturally occurring amines are from lysine. In the above-mentioned or following embodiments each in any one or some embodiments of any embodiment, collagen is from bovine collagen, marine collagen, human collagen or porcine collagen. In the above-mentioned or following embodiments each in any one or some embodiments of any embodiment, collagen is recombinant human collagen.

[0077] In some embodiments of any of the foregoing or following embodiments, the elastic modulus (G') of the macromolecular matrix is ​​about 100 Pa, about 200 Pa, about 300 Pa, about 400 Pa, about 500 Pa, about 600 Pa, about 700 Pa, about 800 Pa, about 900 Pa, about 1000 Pa, about 1100 Pa, about 1200 Pa, about 1300 Pa, about 1400 Pa, about 1500 Pa, about 1600 Pa, about 1700 Pa, about 1800 Pa, about 1900 Pa, about 2000 Pa, about 2100 Pa, about 2200 Pa, about 2300 Pa, about 2400 Pa, about 2500 Pa, about 2600 Pa, about 2700 Pa, about 2800 Pa, about 2900 Pa or about 3000 Pa, or any value between the ranges defined by any two of the foregoing values.

[0078] In some embodiments of any of the foregoing or following embodiments, the macromolecular matrix has a compressive force value of about 10 gmf, about 20 gmf, about 30 gmf, about 40 gmf, about 50 gmf, about 60 gmf, about 70 gmf, about 80 gmf, about 90 gmf, about 100 gmf, about 110 gmf, about 120 gmf, about 130 gmf, about 140 gmf, about 150 gmf, about 160 gmf, about 170 gmf, about 180 gmf, about 190 gmf, about 200 gmf, about 210 gmf, about 220 gmf, about 230 gmf, about 240 gmf, about 250 gmf, about 260 gmf, about 270 gmf, about 280 gmf, about 290 gmf, about 300 gmf, about 310 gmf, about 320 gmf, about 330 gmf, about 340 gmf 0gmf, about 140gmf, about 150gmf, about 160gmf, about 170gmf, about 180gmf, about 190gmf, about 200gmf, about 210gmf, about 220gmf, about 230gmf, about 240gmf, about 250gmf, about 260gmf, about 270gmf, about 280gmf, about 290gmf, about 3 00gmf, about 310gmf, about 320gmf, about 330gmf, about 340gmf, about 350gmf, about 360gmf, about 370gmf, about 380gmf, about 390gmf, about 400gmf, about 410gmf, about 420gmf, about 430gmf, about 440gmf, about 450gmf, about 460gmf, about about 590 gmf or about 600 gmf, or any compressive force value between the range defined by any two of the above values.

[0079] In some embodiments of any of the foregoing or following embodiments, the concentration of hyaluronic acid is about 2 mg / mL, about 4 mg / mL, about 6 mg / mL, about 8 mg / mL, about 10 mg / mL, about 12 mg / mL, about 14 mg / mL, about 16 mg / mL, about 18 mg / mL, about 20 mg / mL, about 22 mg / mL, about 24 mg / ml, about 26 mg / mL, about 28 mg / mL, about 30 mg / mL, 32 mg / mL, about 34 mg / mL or about 36 mg / mL, or any concentration between the ranges defined by any two of the foregoing values.

[0080] In some embodiments of any of the foregoing or following embodiments, the weight ratio of hyaluronic acid to collagen is about 20:3, about 24:2.3, about 24:10, about 24:12, about 24:4, about 24:6, about 28:6, or about 28:11.

[0081] In some embodiments of any of the foregoing or following embodiments, the hyaluronic acid comprises a mixture of hyaluronic acids, wherein the mixture comprises a 50:50 blend of HA having two different molecular weights.

[0082] In some embodiments of any of the foregoing or following embodiments, the collagen comprises type I collagen and / or type III collagen.

[0083] In some embodiments of any of the foregoing or following embodiments, the concentration of collagen is about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, or about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, or any concentration in the range defined by any two of the foregoing values.

[0084] In some embodiments of any of the foregoing or following embodiments, the osmotic pressure of the macromolecular matrix is ​​between 250 mOsm / kg and 350 mOsm / kg.

[0085] In some embodiments of any of the foregoing or following embodiments, the osmotic pressure of the macromolecular matrix is ​​about 250 mOsm / kg, about 275 mOsm / kg, about 300 mOsm / kg, about 325 mOsm / kg, or about 350 mOsm / kg, or any osmotic pressure in the range defined by any two of the foregoing values.

[0086] In some embodiments of any of the foregoing or following embodiments, the hyaluronic acid is linear.

[0087] In some embodiments of any of the foregoing or following embodiments, the viscosity modulus (G") of the macromolecular matrix is ​​about 10Pa, about 20Pa, about 30Pa, about 40Pa, about 50Pa, about 60Pa, about 70Pa, about 80Pa, about 90Pa, about 100Pa, about 200Pa, about 300Pa, about 400Pa, about 500Pa, about 600Pa, about 700Pa, about 800Pa, about 900Pa or about 1000Pa, or any viscosity modulus (G") between the ranges defined by any two of the above values.

[0088] In some embodiments of any of the foregoing or following embodiments, the dielectric loss tangent value (G" / G') of the macromolecular matrix is ​​about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.12, about 0.14, about 0.16, about 0.18, about 0.20, about 0.22, about 0.24, about 0.26, about 0.28, about 0.30, about 0.32, about 0.34, about 0.36, about 0.38, about 0.40, about 0.42, about 0.44, about 0.46, about 0.48, about 0.50, or any dielectric loss tangent value (G" / G') between the ranges defined by any two of the foregoing values.

[0089] In some embodiments of any of the foregoing or following embodiments, the pH of the macromolecular matrix is ​​about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, or any pH in the range defined by any two of the above values.

[0090] In a seventh aspect, a method for improving the aesthetic quality of human anatomical features is provided. The method comprises injecting a composition into human tissue, thereby improving the aesthetic quality of the anatomical features; wherein the composition comprises a macromolecular matrix prepared by a method of any embodiment described herein or a macromolecular matrix of any embodiment described herein.

[0091] In an eighth aspect, a method for improving the aesthetic quality of human anatomical features is provided, the method comprising: injecting a composition into human tissue, thereby improving the aesthetic quality of the anatomical features; wherein the composition comprises a macromolecular matrix, the macromolecular matrix comprising: cross-linked hyaluronic acid; lysine; and collagen; wherein the collagen is physically mixed into the cross-linked hyaluronic acid.

[0092] In some embodiments of any of the foregoing or following embodiments, the collagen comprises type I collagen and / or type III collagen.

[0093] In some embodiments of any of the foregoing or following embodiments, the method does not cause a blue discoloration at the injection site.

[0094] In the ninth aspect, a method for improving the appearance of a human body by injecting a composition into human tissue is provided, whereby the composition promotes cell infiltration and collagen deposition from surrounding tissue into the injected composition. The method comprises injecting a composition into human tissue, thereby improving the aesthetic quality of anatomical features; wherein the composition comprises a cross-linked macromolecular matrix, the cross-linked macromolecular matrix comprising: hyaluronic acid, lysine and collagen; wherein the hyaluronic acid comprises cross-linked hyaluronic acid physically mixed with collagen; and wherein the tissue injected with the composition exhibits tissue integration with the composition. In some embodiments of any one or any of the above or below embodiments, the macromolecular matrix further comprises lidocaine. In some embodiments of any one or any of the above or below embodiments, the concentration of lidocaine in the matrix is ​​between about 0.15% (w / w) and about 0.45% (w / w). In some embodiments of any one or any of the above or below embodiments, the concentration of lidocaine in the matrix is ​​between about 0.27% (w / w) and about 0.33% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of lidocaine in the matrix is ​​about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration in the range defined by any two of the above values. In some embodiments of any one or any of the above or following embodiments, the concentration of lidocaine in the matrix is ​​0.3% (w / w).

[0095] In some embodiments of any one or any of the above or following embodiments, the matrix further comprises uncrosslinked HA. In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​up to about 5% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w) or about 5% (w / w), or any concentration between the ranges defined by any two of the above values. In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 1% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 2% (w / w). In some embodiments of any one or any of the above or following embodiments, the concentration of uncrosslinked HA in the matrix is ​​about 5% (w / w). In some embodiments of any of the foregoing or following embodiments, the uncrosslinked HA improves the extrudability of the matrix.

[0096] In some embodiments of any of the foregoing or following embodiments, the hyaluronic acid component has an average molecular weight of about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 40,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 100,000 Daltons, about 200,000 Daltons, about 300,000 Daltons, about 400,000 Daltons, about 500,000 Daltons, about 600,000 Daltons, about 700,000 Daltons, about 800,000 Daltons, about 900,000 Daltons, about 100,000 Daltons, about 150,000 Daltons, about 160,000 Daltons, about 170,000 Daltons, about 180,000 Daltons, about 190,000 Daltons, about 200,000 Daltons, about 250,000 Daltons, about 260,000 Daltons, about 270,000 Daltons, about 280,000 Daltons, about 290,000 Daltons, about 300,000 Daltons, about 310,000 Daltons, about 320,000 Daltons, about 330,000 Daltons, about 340,000 Daltons, about 350,000 Daltons, about 360,000 Daltons, about 370,000 Daltons, about 380,000 Da ,000,000 Daltons, about 1,100,000 Daltons, about 1,200,000 Daltons, about 1,300,000 Daltons, about 1,400,000 Daltons, about 1,500,000 Daltons, about 1,600,000 Daltons, about 1,700,000 Daltons, about 1,800,000 Daltons, about 1,900,000 Daltons, about 2,000,000 Daltons, about 2,100,000 Daltons, about 2,200,000 Daltons, about 2,300,000 Daltons, about 2,400,000 Daltons, about 2,500,000 Daltons, about 2,600,000 Daltons, about 2,700,000 Daltons about 2,800,000 Daltons, about 2,900,000 Daltons, about 3,000,000 Daltons, about 3,100,000 Daltons, about 3,200,000 Daltons, about 3,300,000 Daltons, about 3,400,000 Daltons, about 3,500,000 Daltons, about 3,600,000 Daltons, about 3,700,000 Daltons, about 3,800,000 Daltons, about 3,900,000 Daltons, about 4,000,000 Daltons, about 4,100,000 Daltons, about 4,200,000 Daltons, about 4,300,000 Daltons, about 4,400,000 Daltons, about 4,500,000 Daltons Daltons, about 4,600,000 Daltons, about 4,700,000 Daltons, about 4,800,000 Daltons, about 4,900,000 Daltons, about 5,000,000 Daltons, about 5,100,000 Daltons, about 5,200,000 Daltons, about 5,300,000 Daltons, about 5,400,000 Daltons, about 5,500,000 Daltons, about 5,600,000 Daltons, about 5,700,000 Daltons, about 5,800,000 Daltons, about 5,900,000 Daltons, about 6,000,000 Daltons, about 6,100,000 Daltons, about 6,200,000 Daltons, about 6,300,000 Daltons, about 6,400,000 Daltons, about 6,500,000 Daltons, about 6,600,000 Daltons, about 6,700,000 Daltons, about 6,800,000 Daltons, about 6,900,000 Daltons, about 7,000,000 Daltons, about 7,100,000 Daltons, about 7,200,000 Daltons, about 7,3 00,000 Daltons, about 7,400,000 Daltons, about 7,500,000 Daltons, about 7,600,000 Daltons, about 7,700,000 Daltons, about 7,800,000 Daltons, about 7,900,000 Daltons, about 8,000,000 Daltons, about 8,100,000 Daltons, about 8,200,000 Daltons, about 8,300,000 Daltons, about 8,400,000 Daltons, about 8,500,000 Daltons, about 8,600,000 Daltons, about 8,700,000 Daltons, about 8,800,000 Daltons, about 8,900,000 Daltons, about 9,000,000 Daltons, about 9,100,000 Daltons, about 9,200,000 Daltons Daltons, about 9,300,000 Daltons, about 9,400,000 Daltons, about 9,500,000 Daltons, about 9,600,000 Daltons, about 9,700,000 Daltons, about 9,800,000 Daltons, about 9,900,000 Daltons, or about 10,000,000 Daltons, or any weight in the range defined by any two of the above values. In some embodiments of any of the above or below embodiments, the hyaluronic acid of the cross-linked hyaluronic acid comprises a mixture of hyaluronic acid components, and the hyaluronic acid components comprise at least two different molecular weights.

[0097] In some embodiments of any of the foregoing or following embodiments, the collagen comprises type I collagen and / or type III collagen.

[0098] In some embodiments of any of the foregoing or following embodiments, the tissue into which the composition is injected exhibits integration with the composition, wherein collagen is deposited in the composition by infiltration of cells from surrounding tissues.

[0099] In some embodiments of any one of the foregoing or following embodiments or any of the embodiments, the tissue into which the composition is injected exhibits integration with the composition and, after injection of the composition, has collagen deposition in the injected composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1Several examples of different compositions of hydrogels with different amounts of collagen and hyaluronic acid are shown. For gels containing about 6.5 mg / mL HA and gels containing about 12.7 mg / mL hyaluronic acid, the opacity of the hydrogels increases with increasing collagen concentration.

[0101] Figure 2 Shown are the G', G", and dielectric loss tangent values ​​of HA-collagen hydrogel at 25°C, 5 Hz frequency, and 0.8% strain between two parallel plates with a diameter of 25 mm and a gap length of 1 mm.

[0102] Figure 3 Swelling of the hydrogel formulation is shown.

[0103] Figure 4 The change in compressive force from 2.5 mm to 0.9 mm gap for the HA-collagen formulation is shown.

[0104] Figure 5 shows the use of a 1 mL BD tuberculin syringe via a 27G- 1 Average extrusion force of HA-collagen hydrogel with a 2" needle.

[0105] Figure 6 Shown are the average heights over time for BDDE-crosslinked and naturally occurring amine (NOA)-crosslinked HA hydrogel samples implanted in a rat model, showing increases in NOA-crosslinked formulations with increasing HA concentrations.

[0106] Figure 7 Shown are the average heights over time of BDDE-crosslinked and NOA-crosslinked HA-collagen hydrogel samples implanted in a rat model, showing the effect of collagen admixture (<3 mg / mL) on lifting capacity.

[0107] Figure 8 Shown are the average heights over time of BDDE-crosslinked and NOA-crosslinked HA-collagen hydrogel samples implanted in a rat model, showing the effect of collagen admixture (up to 11 mg / mL) on lifting capacity.

[0108] Fig. 9 Cell viability of human dermal fibroblasts cultured on BDDE-crosslinked and NOA-crosslinked HA-collagen hydrogels is shown. *p<0.05, Tukey post hoc ANOVA.

[0109] Fig.10 Cell morphology analysis of human dermal fibroblasts cultured on NOA hydrogels or NOA cross-linked HA-collagen hydrogels (24 mg / mL HA, 6 mg / mL collagen) is shown. *p<0.05, Student's t-test.

[0110] Fig.11 Immunohistochemistry for rat type I collagen is shown, demonstrating increased collagen deposition in a 20 mg / mL HA hydrogel bolus containing 3 mg / mL collagen.

[0111] Fig.12 Immunohistochemistry for rat type I collagen is shown, demonstrating encapsulation in the center of a hydrogel bolus containing 28 mg / mL HA. Addition of up to 11 mg / mL collagen modestly improved collagen deposition along the periphery of the hydrogel bolus.

[0112] Fig.13 Immunohistochemistry for rat type I collagen is shown, showing increased collagen deposition in 24 mg / mL HA hydrogel bolus injections with increasing collagen concentration (2.3 mg / mL to 6 mg / mL). The formulation containing 24 mg / mL HA and 6 mg / mL collagen (24NOA6CN) exhibited the strongest tissue integration.

[0113] Fig.14 Immunohistochemistry for rat type I collagen is shown, demonstrating collagen deposition / tissue integration within BDDE-crosslinked HA and NOA-crosslinked HA-collagen hydrogels at 4 to 12 weeks following subcutaneous implantation in rats. The formulation containing 24 mg / mL HA and 6 mg / mL collagen (24NOA6CN) demonstrated greater tissue integration at both 4 and 12 weeks compared to the BDDE-crosslinked HA gel alone. In the 24NOA6CN formulation, tissue integration appeared to improve from 4 to 12 weeks.

[0114] Fig.15 Semi-quantitative histopathological scoring of tissue integration into BDDE-only cross-linked HA hydrogels and 24NOA6CN hydrogels is shown. Scoring system: Best integration

[0115] =10, worst integration = -5. (A) Colloidal iron staining shows enhanced tissue integration and associated histopathological scores in 24NOA6CN hydrogel bolus injections. (B) Bar graph showing histopathological scores of BDDE-only cross-linked HA hydrogels and 24NOA6CN after 4 and 12 weeks of subcutaneous implantation in rats. 24NOA6CN gel showed the highest tissue integration score after 12 weeks. DETAILED DESCRIPTION

[0116] Factors such as sun exposure, genetics, disease, injury and lifestyle can damage our underlying tissues and cause undesirable changes to our skin, resulting in wrinkles, folds and sagging of tissue, leading to visible effects of aging. These changes can be largely attributed to the loss of adipose tissue and extracellular matrix proteins such as collagen and elastin. The loss of these components leads to a thinning of the skin layers, which, combined with changes in bone and muscle structure, can lead to volume loss and wrinkle formation. In order to restore the skin to its previous youthful appearance, current methods are aimed at restoring the skin's volume and increasing its thickness.

[0117] Dermal fillers are one of many treatments available for removing wrinkles and restoring volume. Once injected, dermal fillers are used to replace lost collagen and elastin, thereby thickening the skin, lifting tissue and ultimately removing wrinkles. The effects of dermal fillers are temporary and tend to wear off over time. Therefore, follow-up injections are needed to maintain a youthful appearance.

[0118] Hyaluronic acid (HA) dermal fillers are very effective in removing wrinkles and dominate the market for several reasons. Dermal fillers can be improved to affect the duration of the filler in the tissue. Without limitation, improvements such as changing the cross-linking of the dermal filler can affect, for example, the duration of the filler in the tissue.

[0119] In addition to the long-lasting benefits of HA dermal fillers, they are also reversible. Reversibility is a key advantage, especially if the filler is not placed correctly or an adverse event (AE) occurs, such as occluding a blood vessel. For example, in the case of an AE, the dermal filler can be quickly degraded by injecting hyaluronidase into the affected area. Hyaluronidase is an enzyme that breaks down HA and converts the gel filler into a liquid solution that can diffuse away from the affected area. These benefits make HA dermal fillers one of the most popular fillers on the market today.

[0120] Although HA dermal fillers have many advantages, their performance can still be improved. For example, HA fillers do not supplement and / or significantly stimulate the synthesis of extracellular matrix (ECM) proteins associated with rejuvenation, such as collagen. In addition, if injected superficially, HA dermal fillers may cause AE, which manifests as a blue discoloration at the surface of the skin, known as the Tyndall effect (Cohen et al. Understanding, avoiding, and managing dermal filler complications. Dermatol Surg, 2008. 34 Suppl 1: p. S92-9; incorporated herein by reference). This is because the transparent particles in the dermal filler scatter light in a certain way, resulting in a blue hue. The injected HA product and the anatomical area of ​​injection both affect the likelihood of the Tyndall effect (Bailey et al., Etiology, prevention, and treatment of dermal filler complications. Aesthet Surg J, 2011. 31 (1): p. 110-21; incorporated herein by reference). Swelling can also be a concern with HA fillers because HA is extremely hydrophilic and strongly absorbs water. Therefore, as the HA concentration in the hydrogel increases or the degree of cross-linking decreases, water absorption increases. For example, some HA fillers can absorb more than 6 times their starting weight when exposed to saline. Water absorption can cause swelling, especially when the filler is injected superficially or into areas where the skin is thin. Additionally, available fillers on the market that contain only HA have not been shown to allow tissue integration. Despite these issues, HA dermal fillers have become the most popular fillers on the market due to their performance.

[0121] Collagen dermal fillers were introduced to the market in 1981. Collagen is an ideal material for this application because it is a natural component of the skin, giving it strength, fullness, and texture. Once injected, these fillers add collagen back into the skin, replenishing ECM proteins associated with rejuvenation. In addition, the gels do not cause the Tyndall effect. For example, 1 and Collagen dermal fillers such as HA are injected into the papillary dermis (Gold et al., Use of hyaluronic acid fillers for the treatment of the aging face. Clinical Interventions in Aging, 2007. 2 (3): p. 369-376; incorporated herein by reference). Although they are superficially injected, the opaque appearance of the material prevents observation of the Tyndall effect. Therefore, collagen fillers have some advantages over HA-based fillers.

[0122] Despite these advantages, collagen fillers have some disadvantages that make HA fillers preferable. For example, collagen fillers cannot be reversed using injectable, commercially available enzyme solutions like HA fillers. To achieve the desired effect, collagen dermal fillers have a high concentration of collagen (35mg / mL to 65mg / mL collagen), and this high concentration of collagen can be difficult to remove if an adverse reaction occurs. In addition, collagen dermal fillers have been shown to have a limited duration, with an average correction time of 3 to 6 months. Cross-linking of collagen can produce a stronger hydrogel that can be placed deeper in the skin and can last longer. However, cross-linked collagen gels are less similar to the collagen present in the body than uncross-linked collagen fillers and are therefore less natural.

[0123] In the following embodiments, the preparation and characterization of a hybrid material made of a HA filler containing physically mixed collagen is described. The material takes advantage of the advantages provided by collagen and HA fillers while eliminating the problems of each material alone.

[0124] The advantages of HA dermal fillers are retained due to the hybrid nature of the material, while the inclusion of collagen actively addresses other issues. For example, since the addition of collagen to HA gels results in an opaque formulation (see Examples 2 and Figure 1), so this can solve the problem of the Tyndall effect. In addition, the addition of collagen enables the production of high gel stiffness without changing the swelling properties. Using HA fillers alone, G' (i.e. stiffness) can be increased by increasing the HA concentration or increasing the crosslinking density of the hydrogel network. However, increasing the HA concentration will lead to increased swelling, and the higher the crosslinking degree, the lower the swelling of the gel. Therefore, the uniqueness of physically mixed HA collagen hydrogels is that G' / stiffness increases with increasing collagen concentration without affecting the swelling properties (Examples 3 and 4). Finally, since collagen is associated with young skin, injection of HA filler compositions mixed with collagen can supplement ECM proteins associated with young skin. HA fillers mixed with collagen can also be used as a scaffold for tissue ingrowth and new collagen deposition after injection. This enhanced tissue integration may translate into a more natural and lasting effect. Therefore, when collagen is incorporated into HA skin fillers, it surprisingly improves the physical properties and biological performance of the final product.

[0125] Likewise, the shortcomings of collagen dermal fillers can be addressed by including HA dermal fillers in the formulation. For example, the short duration of collagen fillers can be improved by using a longer-lasting HA dermal filler mixed with collagen. In addition, formulations of HA fillers mixed with collagen require significantly less collagen to achieve the desired biological effect, and therefore, the reversibility of the formulation is enhanced compared to collagen-only dermal fillers because the HA portion of the composition can be degraded using injected hyaluronidase. With HA-collagen gels, the filling effect is produced by the HA dermal filler. If an AE is encountered, HA can be degraded using hyaluronidase, thereby turning the material into a liquid and ensuring the safety of the patient. Therefore, mixed HA dermal fillers with collagen formulations have significant advantages over using HA or collagen alone.

[0126] As described in the embodiments herein, the mixed HA and collagen compositions produce hydrogels with surprising beneficial properties. It was found that different combined concentrations of HA and collagen enable hydrogels to achieve lifting and tissue integration. In some embodiments, compositions comprising 20mg / mL HA or 24mg / mL HA produce good lifting in tissues. However, some compositions wherein the concentration is increased to at least 28mg / mL produce improved lifting, but do not improve the tissue integration of tissue integration to the injection site. In some embodiments, the composition comprises 24mg / mL HA and 6mg / mL collagen. In some embodiments, the composition comprises 20mg / mL HA and 10mg / mL collagen. In some embodiments, the composition produces tissue lifting and enhanced tissue integration to the tissue integration of the composition injection site. In some embodiments, the composition has good lifting and higher tissue integration compared to gels with only HA.

[0127] Ease of injectability is also a key property of dermal fillers and can be quantified using extrusion force (EF) measurement. Essentially, EF is the force required to apply to the plunger to extrude the gel through a needle of a specific metering. Easily injectable dermal fillers are characterized by having a small extrusion force and a smooth and uniform extrusion profile over the length of the syringe. A force of <40N is considered acceptable and easy to inject, control and precision during injection. The average extrusion force of HA collagen preparations is substantially less than 40N (see Example 6). In some embodiments herein, the macromolecular matrix has an extrusion force of less than 40N. In addition, the extrusion force curve looks smooth, indicating that the material is homogeneous. Therefore, HA collagen dermal fillers are easy to inject through a fine needle. In any one of the above or below embodiments or some embodiments of any embodiment, the macromolecular complex also includes uncrosslinked HA. In any one of the above or below embodiments or some embodiments of any embodiment, the concentration of uncrosslinked HA in the macromolecular complex is 1%, 2% or 5%. In any one of the above or below embodiments or some embodiments of any embodiment, the uncrosslinked HA makes it easy to extrude and reduces the extrusion force of the composition.

[0128] These embodiments herein specifically describe HA dermal fillers that are physically mixed with collagen. Alternatively, collagen can be chemically cross-linked with HA to form different types of HA collagen gels. However, compared to chemically cross-linked gels, physically mixed gels contain collagen that is more similar to collagen in natural tissue.

[0129] In some embodiments of any of the foregoing or following embodiments, the composition provides improved skin quality, such as skin hydration, improved elasticity, and reduced skin wrinkles.

[0130] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0131] As used herein, "a" or "an" may mean one or more than one. As used herein, when referring to a measurable value, "about" means including a change of +20% or +10%, more preferably +5%, even more preferably +1%, and still more preferably +0.1% compared to the specified value.

[0132] As used herein, the term "comprise" and variations of the term such as "comprising, comprises, comprised" are not intended to exclude further additives, components, integers or steps, unless the context requires otherwise.

[0133] The term "hyaluronic acid" or "HA" is a non-sulfated glycosaminoglycan that is widely distributed in connective tissue, epithelial tissue, and neural tissue throughout the human body. Without limitation, this sodium hyaluronate may also be used. Hyaluronic acid is abundant in different layers of the skin and has multiple functions, such as ensuring good hydration, assisting in the organization of the extracellular matrix, acting as a filler material; and participating in tissue repair mechanisms. However, with age, the amount of hyaluronic acid, collagen, elastin, and other matrix polymers present in the skin decreases. For example, repeated exposure to ultraviolet light (such as from the sun) causes dermal cells to reduce their production of hyaluronic acid and increase its degradation rate. This loss of hyaluronic acid leads to various skin conditions, such as imperfections, defects, diseases and / or disorders. For example, there is a strong correlation between the water content in the skin and the level of hyaluronic acid in the dermal tissue. As the skin ages, both the amount and quality of hyaluronic acid in the skin decrease. These changes can cause dryness and wrinkling of the skin.

[0134] HA can include hyaluronic acid and any hyaluronate thereof, including, for example, sodium hyaluronate (sodium salt), potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate. Hyaluronic acid from a variety of sources can be used herein. For example, hyaluronic acid can be extracted from animal tissues, harvested as a product of bacterial fermentation, or produced in commercial quantities by bioprocess technology. In embodiments described herein, cross-linked hyaluronic acid comprises a molecular weight of about 10,000 daltons to about 10,000,000 daltons.

[0135] "Macromolecular matrix" refers to a matrix formed by cross-linked HA physically mixed with collagen. In some embodiments, the macromolecular matrix also includes collagen cross-linked with HA.

[0136] "Collagen" as described herein is a structural protein that is present in various connective tissues in the body. Any type of collagen can be used in the methods and compositions described herein. In some embodiments, type I collagen, type II collagen, type III collagen, type IV collagen, type VI collagen, or a combination thereof can be used. For example, collagen can be derived from cell culture, animal tissue, or recombinant means, and can be derived from human, porcine, recombinant, or bovine sources. Some embodiments include collagen derived from human fibroblast cultures. Some embodiments include collagen that has been denatured into gelatin. In some embodiments of the macromolecular complex, the macromolecular complex comprises type I and / or type III collagen.

[0137] Those skilled in the art will appreciate the considerations for collagen from different sources. Histopathologically, bovine collagen fibers may be thicker than human collagen, have a homogeneous appearance with little space between them, fewer fibroblasts, and cannot refract polarized light. Skin testing may be required before injecting bovine or other animal collagen products. Rare allergic reactions have been reported, including foreign body granulomas and palisade granulomas of bovine collagen. Rare systemic complications include flu-like symptoms, paresthesias, or dyspnea, and severe anaphylactic shock has been reported after injection of bovine collagen. Therefore, skin testing may be required before injection to identify patients at risk of allergic reactions and their short duration of action. Those skilled in the art will consider the type of collagen used in the embodiments herein and will understand the differences between collagen sources.

[0138] In some embodiments of any of the foregoing or following embodiments, the composition further comprises HA particles or particles or fibrils of collagen.

[0139] As used herein, "local delivery" refers to administering a composition to or near a tissue in need of the therapeutic composition. In some embodiments of any of the foregoing or following embodiments, the macromolecular matrix is ​​delivered locally to an area of ​​the patient's body where there is a volume deficit.

[0140] Treatment

[0141] In some embodiments of any of the above or below embodiments, the composition is administered as an injection into facial soft tissue to increase the size, shape, and / or contour of facial features, such as increasing the size, shape, and / or contour of the lips, cheeks, or eye areas; change the size, shape, and / or contour of facial features, such as changing the size, shape, and / or contour of the lips, cheeks, or eye areas; reduce or eliminate wrinkles, folds, or wrinkles in the skin; resist wrinkles, folds, or wrinkles in the skin; hydrate the skin; increase skin elasticity; reduce or eliminate skin roughness; increase and / or improve skin firmness. Without limitation, these areas may include, for example, the eye area, cheek area, and / or neck area.

[0142] Those skilled in the art will appreciate that prior to administration, sterile techniques may be used, such as appropriate disinfection of the skin with 2% to 4% chlorhexidine or 70% isopropyl alcohol solution, and avoiding contamination of the treatment area after cleaning the patient's skin. An injection method should be used that minimizes the number of skin punctures and uses the smallest gauge available needle for injection. It is also important to avoid injection into inflamed or infected skin.

[0143] In some embodiments of any one or any of the foregoing or following embodiments, the compositions described herein provide instant lift and volume upon injection while delivering and sequestering collagen. In embodiments described herein, the methods provide a natural-looking effect to the patient receiving the injection.

[0144] In some embodiments of any of the foregoing or following embodiments, the collagen is not chemically bound to the cross-linked HA network.

[0145] In some embodiments of any or any of the foregoing or following embodiments, the HA component is linear HA. In some embodiments of any or any of the foregoing or following embodiments, the HA is cross-linked to naturally occurring amines using EDC and NHS chemistry or BDDE cross-linked HA. In some embodiments of any or any of the foregoing or following embodiments, the naturally occurring amine is lysine.

[0146] In any one or some embodiments of any of the above or below embodiments, the composition stimulates the synthesis of essential structural units (e.g., ECM proteins) of the skin. The production of proteins (e.g., collagen) can be stimulated from the tissue into which the composition is injected, but is not limited thereto.

[0147] In some embodiments of any of the foregoing or following embodiments, the method of treatment is reversible after hyaluronidase treatment. During an adverse event or if the outcome is unfavorable, hyaluronidase treatment can be provided.

[0148] In some embodiments of any of the foregoing or following embodiments, the composition can be used superficially to eliminate fine lines and deep (subcutaneous and / or supraperiosteal) for cheek augmentation to correct age-related volume loss in the mid-face.

[0149] In some embodiments of any of the foregoing or following embodiments, the method of treatment does not result in a Tyndall effect at the injection site.

[0150] One skilled in the art will appreciate the different ways in which the macromolecular matrix can be administered to a patient in need thereof.In some embodiments of any one of the foregoing or following embodiments, the macromolecular matrix is ​​administered with a needle or cannula.

[0151] The collagen used in the embodiments herein can be provided from any source. Without limitation, for example, porcine, bovine, recombinant and human collagen can be used. In some embodiments, the composition does not require allergy testing.

[0152] As described herein, HA collagen dermal fillers can provide immediate lift and volume after injection while delivering and capturing collagen. In some embodiments of any one or any of the foregoing or following embodiments, the composition can stimulate the synthesis of necessary building blocks, thereby obtaining skin that looks young and healthy. In some embodiments of any one or any of the foregoing or following embodiments, the method is reversible after hyaluronidase treatment. In some embodiments of any one or any of the foregoing or following embodiments, the composition allows for an increase in modulus (stiffness) without significantly affecting the swelling properties of the filler.

[0153] In some embodiments of any of the foregoing or following embodiments, the composition can be used to superficially eliminate fine and deep lines (subcutaneous and / or supraperiosteal) for cheek augmentation to correct age-related volume loss in the mid-face.

[0154] In some embodiments of any of the foregoing or following embodiments, the composition produces little or no granulomas over the life of the filler. Thus, the filler does not cause a "pebble-stone" appearance in the skin or injected area.

[0155] In some embodiments of any of the foregoing or following embodiments, the composition provides benefits such as hydration and rejuvenation of the skin.

[0156] Method for producing hydrogel

[0157] In some embodiments of any one of the above or below embodiments or any of the embodiments, the method of making a hydrogel includes providing: HA, collagen, water and PBS, and mixing at an acidic pH. The HA used in the method can be cross-linked. The collagen can be provided by any source. In a non-limiting manner, the collagen can be porcine collagen, human collagen, or a recombinant form of collagen.

[0158] There are different methods of mixing collagen into hyaluronic acid or the above mixture. Those skilled in the art will appreciate the various methods of preparing hydrogels. In a non-limiting manner, collagen can be mixed into cross-linked hyaluronic acid using, for example, a Thinky mixer, an acoustic mixer, a reactor, a syringe-syringe mixing, a stainless steel cylinder.

[0159] In some embodiments of any of the above or following embodiments, the HA concentration of the mixture is about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 24 mg / mL, about 28 mg / mL, or any concentration in a range between any two of the above values. In some embodiments of any of the above or following embodiments, the HA concentration of the mixture is about 5 mg / mL. In some embodiments of any of the above or following embodiments, the HA concentration of the mixture is about 25 mg / mL.

[0160] In some embodiments, wherein the HA concentration is about 20 mg / mL, the composition provides both lifting and tissue integration.

[0161] In some embodiments of any of the foregoing or following embodiments, the composition comprises 24 mg / mL HA and about 6 mg / mL collagen.

[0162] In some embodiments of any of the foregoing or following embodiments, the composition comprises 20 mg / mL HA and about 10 mg / mL collagen.

[0163] In some embodiments of any of the above or following embodiments, the method further comprises sterilizing the composition. There are many methods for sterilizing the hydrogel, and these methods can be understood by those skilled in the art. Without limitation, the composition can be sterilized, for example, by heat (dry heat, steam heat), radiation (non-ionizing, ionizing (particles (such as gamma rays), electromagnetic (such as electron beam), filtration) or terminal sterilization (such as sterilization of the product in its final container, such as a syringe). In some embodiments of any of the above or following embodiments, the composition can be sterilized by heat (dry heat, steam heat), radiation (non-ionizing, ionizing (particles (such as gamma rays), electromagnetic (such as electron beam), filtration or terminal sterilization (such as sterilization of the product in its final container, such as a syringe).

[0164] For example, steam heat can be used to sterilize the product by exposing the composition to saturated steam under pressure. For example, this can be done at a temperature of 121° C. to 124° C. for 15 minutes. For example, in some alternatives, different amounts of time and higher temperatures can be used.

[0165] Dry heat may be used and may require higher temperatures and longer exposure times (Galante et al., 2017). In some embodiments of any of the foregoing or following embodiments, a method of preparing a macromolecular matrix is ​​provided, wherein a cross-linked HA hydrogel is provided and collagen is provided, wherein the collagen is in a soluble state in solution, and wherein the collagen is physically mixed into the cross-linked HA hydrogel.

[0166] In some embodiments of any embodiment in the above or following embodiments, collagen is provided in the form of a solution, wherein the solution is acidic. In some embodiments of any embodiment in the above or following embodiments, collagen is provided in a solution state. In some embodiments of any embodiment in the above or following embodiments, collagen is in a soluble state. In some embodiments of any embodiment in the above or following embodiments, the method for manufacturing a macromolecular matrix also includes neutralizing a mixture of collagen comprising cross-linked hyaluronic acid and mixed. In some embodiments of any embodiment in the above or following embodiments, collagen fibrils or collagen precipitates are formed after mixing. However, collagen fibrils or collagen precipitates are homogenously mixed into HA hydrogel. In some embodiments of any embodiment in the above or following embodiments, the method also includes adjusting the osmotic pressure of the hydrogel after mixing soluble collagen with cross-linked HA. In some embodiments, the adjustment step may cause collagen to form a precipitate or fibrils, but the collagen precipitate or fibrils are homogenously mixed around the cross-linked hyaluronic acid.

[0167] In the embodiments described herein, it is important that the HA is cross-linked and the collagen is added as a solution. The mixture should be mixed so that the mixture is well mixed and homogenous. The mixture is then neutralized and the osmotic pressure is adjusted so that the macromolecular matrix is ​​at the correct pH / osmotic pressure. In essence, it is important that the composition is uniform and homogenous so that the functions of the filler can be exerted, such as lifting ability and properties that allow tissue integration.

[0168] In some embodiments, before being mixed into the cross-linked hyaluronic acid, the collagen is neutralized (in an acidic solution). In some embodiments, neutralization to a pH of about 7 results in different collagen structures, such as fibrillation of collagen. In some embodiments, these fibrils are homogeneously mixed into the cross-linked hyaluronic acid.

[0169] In some embodiments, cross-linked hyaluronic acid and collagen are mixed together at an acidic pH. After mixing, the pH of the solution can be raised to between 7.0 and 7.5 by adding a base and mixing a second time. In some embodiments, the increase in pH can trigger the self-assembly of the collagen network. In some embodiments, the mixture can be autoclaved after mixing.

[0170] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0171] As used herein, "a" or "an" may mean one or more than one. As used herein, when referring to a measurable value, "about" means including a change of +20% or +10%, more preferably +5%, even more preferably +1%, and still more preferably +0.1% compared to the specified value.

[0172] As used herein, the term "comprise" and variations of the term such as "comprising, comprises and comprised" are not intended to exclude further additives, components, integers or steps, unless the context requires otherwise.

[0173] The term "hyaluronic acid" or "HA" is a non-sulfated glycosaminoglycan that is widely distributed in connective, epithelial, and neural tissues throughout the human body. Hyaluronic acid is abundant in different layers of the skin and has a variety of functions, such as ensuring good hydration, assisting in the organization of the extracellular matrix, acting as a filler material; and participating in tissue repair mechanisms. However, with age, the amount of hyaluronic acid, collagen, elastin, and other matrix polymers present in the skin decreases. For example, repeated exposure to ultraviolet light (such as from the sun) causes dermal cells to reduce their production of hyaluronic acid and increase its degradation rate. This loss of hyaluronic acid leads to various skin conditions, such as imperfections, defects, diseases and / or disorders. For example, there is a strong correlation between the amount of water in the skin and the level of hyaluronic acid in the dermal tissue. As the skin ages, both the amount and quality of hyaluronic acid in the skin decreases. These changes can cause the skin to become dry and wrinkled.

[0174] HA can include hyaluronic acid and any hyaluronate thereof, including, for example, sodium hyaluronate (sodium salt), potassium hyaluronate, magnesium hyaluronate and calcium hyaluronate. Hyaluronic acid from a variety of sources can be used herein. For example, hyaluronic acid can be extracted from animal tissues, harvested as a product of bacterial fermentation, or produced in commercial quantities by bioprocess technology.

[0175] As used herein, "collagen" is a structural protein that is present in various connective tissues in the body. The collagen provided may be from any source. Without limitation, for example, the collagen may be marine collagen, bovine collagen, porcine collagen, human collagen, human collagen from cell culture, or recombinant collagen.

[0176] As used herein, "local delivery" refers to administering a composition to or near a tissue in need of the therapeutic composition. In some embodiments of any of the foregoing or following embodiments, the macromolecular matrix is ​​delivered locally to an area of ​​the patient's body having a volume deficit.

[0177] "Physical mixing" is the addition or introduction of another substance for mixing. Physical mixing can be performed by, for example, stirring, a stir bar, an acoustic mixer, an orbital mixer, but is not limited thereto.

[0178] Subject Technology as a Description of the Terms

[0179] For convenience, various embodiments of aspects of the present disclosure are described as numbered clauses (1, 2, 3, etc.). These are provided as examples and do not limit the subject technology. The drawings and reference numerals provided below are for example and illustrative purposes only, and the clauses are not limited by these identifiers.

[0180] Item 1. A method for preparing a macromolecular matrix, the method comprising: providing cross-linked hyaluronic acid, providing collagen; physically mixing the collagen into the cross-linked hyaluronic acid, wherein the collagen is homogeneously mixed throughout the cross-linked hyaluronic acid, thereby forming a macromolecular matrix, wherein the macromolecular matrix comprises cross-linked hyaluronic acid and physically mixed collagen.

[0181] Item 2. The method of Item 1, wherein the collagen is provided in a soluble state as a solution.

[0182] Item 3. The method of Item 1 or 2, wherein the collagen is provided in the form of an acidic pH solution, wherein the pH of the collagen solution is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, or any pH in the range between any two of the above values, and wherein the collagen is soluble at acidic pH.

[0183] Item 4. The method of any one of items 1 to 3, wherein the collagen is provided in the form of an acidic pH solution, wherein the pH of the collagen solution is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, or any pH in the range between any two of the above values, and wherein the collagen is soluble at acidic pH.

[0184] Clause 5. The method of any one of clauses 1 to 3, wherein the collagen is provided in the form of a neutral pH solution.

[0185] Item 6. The method of any one of Items 1 to 5, wherein the collagen and cross-linked hyaluronic acid are physically mixed with a buffer.

[0186] Clause 7. The method of Clause 6, wherein the buffer solution comprises PBS.

[0187] Clause 8. The method of any one of Clauses 1 to 7, wherein the method further comprises neutralizing the macromolecular matrix to a pH of about 7 after homogenizing the collagen throughout the cross-linked hyaluronic acid.

[0188] Clause 9. The method of any one of Clauses 1 to 8, wherein the method further comprises neutralizing the collagen to a pH of about 7 prior to physically mixing the collagen with the cross-linked hyaluronic acid.

[0189] Item 10. The method of Item 9, wherein the collagen is neutralized prior to being physically mixed into the cross-linked hyaluronic acid, thereby causing the collagen to precipitate into collagen fibrils or particles, wherein the collagen fibrils or particles are further mixed into the cross-linked hyaluronic acid, wherein the fibrils or particles are homogeneously mixed throughout the cross-linked hyaluronic acid.

[0190] Item 11. The method of Item 1, wherein the collagen is provided as fibrillated collagen or collagen fibers.

[0191] Item 12. The method of Item 11, wherein the collagen is prepared at an alkaline pH, thereby producing fibrillated collagen or collagen fibers.

[0192] Item 13. The method of Item 11 or 12, wherein the collagen is prepared with at least one salt to obtain fibrillated collagen or collagen fibers.

[0193] Clause 14. The method of Clause 13, wherein the concentration of at least one salt is about 20 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM or about 500 mM, or any concentration in a range defined by any two of the above values.

[0194] Clause 15. The method of clause 13 or 14, wherein at least one salt comprises an anion, wherein the anion comprises H2PO 4- 、SO4 2- , Cl - or SCN - .

[0195] Clause 16. The method of any one of clauses 13 to 15, wherein the at least one salt comprises NaCl, Na2SO4, or Li2SO4.

[0196] Clause 17. The method of any one of Clauses 1 to 16, wherein the collagen is homogenously mixed throughout the macromolecular matrix.

[0197] Clause 18. The method of any one of Clauses 1 to 16, wherein the collagen is uniformly mixed throughout the macromolecular matrix.

[0198] Clause 19. The method of any one of clauses 1 to 18, wherein the cross-linked hyaluronic acid comprises a hyaluronic acid component having an average molecular weight of about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 40,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 90,000 Daltons, about 100,000 Daltons, about 200,000 Daltons, about 300,000 Daltons, about 400,000 Daltons, about 500,000 Daltons, about 600,000 Daltons, about 700,000 Daltons, about 800,000 Daltons, about 900,000 Daltons Daltons, about 1,000,000 Daltons, about 1,100,000 Daltons, about 1,200,000 Daltons, about 1,300,000 Daltons, about 1,400,000 Daltons, about 1,500,000 Daltons, about 1,600,000 Daltons, about 1,700,000 Daltons, about 1,800,000 Daltons, about 1,900,000 Daltons, about 2,000,000 Daltons, about 2,100,000 Daltons, about 2,200,000 Daltons, about 2,300,000 Daltons, about 2,400,000 Daltons, about 2,500,000 Daltons, about 2,600,000 Daltons, about 2,700,000 Daltons 00 Daltons, about 2,800,000 Daltons, about 2,900,000 Daltons, about 3,000,000 Daltons, about 3,100,000 Daltons, about 3,200,000 Daltons, about 3,300,000 Daltons, about 3,400,000 Daltons, about 3,500,000 Daltons, about 3,600,000 Daltons, about 3,700,000 Daltons, about 3,800,000 Daltons, about 3,900,000 Daltons, about 4,000,000 Daltons, about 4,100,000 Daltons, about 4,200,000 Daltons, about 4,300,000 Daltons, about 4,400,000 Daltons, about 4,500,000 Daltons, about about 4,600,000 Daltons, about 4,700,000 Daltons, about 4,800,000 Daltons, about 4,900,000 Daltons, about 5,000,000 Daltons, about 5,100,000 Daltons, about 5,200,000 Daltons, about 5,300,000 Daltons, about 5400,000 Daltons, about 5500,000 Daltons, about 5600,000 Daltons, about 5700,000 Daltons, about 5800,000 Daltons, about 5900,000 Daltons, about 6000,000 Daltons, about 6,100,000 Daltons, about 6,200,000 Daltons, about 6,300,000 Daltons, about 6,400,000 Daltons, about 6,500,000 Daltons, about 6,600,000 Daltons, about 6,700,000 Daltons, about 6,800,000 Daltons, about 6,900,000 Daltons, about 7,000,000 Daltons, about 7,100,000 Daltons, about 7,200,000 Daltons, about 7,300,000 Daltons, about 7,400,000 Daltons, about 7,500,000 Daltons, about 7,600,000 Daltons, about 7,700,000 Daltons, about 7,800,000 Daltons, about 7,900,000 Daltons, about 8,000,000 Daltons, about 8,100,000 Daltons, about 8,200,000 Daltons, about 8,300,000 Daltons, about 8,400,000 Daltons, about 8,500,000 Daltons, about 8,600,000 Daltons, about 8,700,000 Daltons, about 8,800,000 Daltons, about 8,900,000 Daltons, about 9,000,000 Daltons, about 9,100,000 Daltons, about 9,200,000 Daltons Daltons, about 9,300,000 Daltons, about 9,400,000 Daltons, about 9,500,000 Daltons, about 9,600,000 Daltons, about 9,700,000 Daltons, about 9,800,000 Daltons, about 9,900,000 Daltons, or about 10,000,000 Daltons, or any molecular weight between any two of the above values.

[0199] Clause 20. The method of any one of Clauses 1 to 19, wherein the cross-linked hyaluronic acid comprises a mixture of hyaluronic acid components having at least two different molecular weights.

[0200] Clause 21. The method of any one of Clauses 1 to 20, wherein the method further comprises adding lidocaine to the macromolecular matrix.

[0201] Clause 22. The method of any one of Clauses 1 to 21, wherein the concentration of lidocaine in the matrix ranges from about 0.15% (w / w) to about 0.45% (w / w).

[0202] Clause 23. The method of clause 21 or 22, wherein the concentration of lidocaine in the matrix is ​​about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration between the range defined by any two of the above values.

[0203] Clause 24. The method of any one of Clauses 21 to 23, wherein the concentration of lidocaine in the matrix ranges from about 0.27% (w / w) to about 0.33% (w / w).

[0204] Clause 25. The method of any one of Clauses 1 to 24, wherein the matrix further comprises non-cross-linked HA.

[0205] Clause 26. The method of Clause 25, wherein the concentration of uncrosslinked HA within the matrix is ​​up to about 5% (w / w).

[0206] Item 27. The method of Item 25 or 26, wherein the concentration of uncross-linked HA in the matrix is ​​about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), or any concentration between the ranges defined by any two of the above values.

[0207] Clause 28. The method of any one of Clauses 25 to 27, wherein the concentration of uncrosslinked HA in the matrix is ​​about 1% (w / w).

[0208] Clause 29. The method of any one of Clauses 25 to 27, wherein the concentration of uncrosslinked HA in the matrix is ​​about 2% (w / w).

[0209] Clause 30. The method of any one of Clauses 25 to 27, wherein the concentration of uncrosslinked HA in the matrix is ​​about 5% (w / w).

[0210] Clause 31. The method of any one of clauses 1 to 30, wherein the macromolecular matrix has enhanced extrudability through a needle, wherein the needle comprises a gauge size of 27G, 28G, 29G, 30G, 31G, or 32G.

[0211] Clause 32. The method of any one of clauses 1 to 31, wherein the macromolecular matrix is ​​stable for at least 6 months.

[0212] Clause 33. The method of any of clauses 1 to 32, wherein the macromolecular matrix is ​​stable for about 6, about 9, about 12, about 15, about 18, about 21, about 24, about 27, about 30, about 33 months, or about 36 months, or any amount of time between the ranges defined by any two of the above values.

[0213] Clause 34. The method of any one of clauses 1 to 33, wherein the macromolecular matrix is ​​stable at about 4°C, about 5°C, about 10°C, about 15°C, about 20°C, about 25°C, or any temperature between a range defined by any two of the above values.

[0214] Clause 35. The method of any one of clauses 1 to 34, wherein the macromolecular matrix is ​​stable at about 4°C.

[0215] Clause 36. The method of any one of Clauses 1 to 35, wherein the macromolecular matrix is ​​stable at about 25°C.

[0216] Clause 37. The method of any of clauses 1 to 36, wherein the macromolecular matrix has minimal degradation at about 3, about 6, about 9, about 12, about 15, about 18, about 21, about 24, about 27, about 30, about 33 or about 36 months, or any amount of time between the range defined by any two of the above values.

[0217] Item 38. A method for preparing a macromolecular matrix, the method comprising: dissolving hyaluronic acid in an aqueous solution to form a pre-reacted aqueous solution and preparing a second solution, wherein the second solution comprises: a) a water-soluble carbodiimide; and N-hydroxysuccinimide or N-hydroxysulfosuccinimide; or b) 1,4-butanediol diglycidyl ether (BDDE) in a sodium hyaluronate solution in the presence of sodium hydroxide; and adding the second solution to the pre-reacted aqueous solution to form a cross-linked reaction mixture; cross-linking the hyaluronic acid, wherein the cross-linking reaction mixture reacts by cross-linking the hyaluronic acid to form a cross-linked hyaluronic acid; and

[0218] providing collagen; and physically mixing the collagen into the cross-linked hyaluronic acid, thereby producing a macromolecular matrix comprising the cross-linked hyaluronic acid and the collagen, wherein the collagen is homogeneously mixed throughout the cross-linked hyaluronic acid.

[0219] Clause 39. The method of Clause 38, wherein the second solution comprises a water-soluble carbodiimide and N-hydroxysuccinimide or N-hydroxysulfosuccinimide, wherein the hyaluronic acid is cross-linked with the naturally occurring amines to form cross-linked hyaluronic acid.

[0220] Clause 40. The method of clause 38 or 39, wherein the second solution comprises a water-soluble carbodiimide and N-hydroxysuccinimide or N-hydroxysulfosuccinimide, and wherein the cross-linking is performed in the presence of MES.

[0221] Clause 41. The method of Clause 38, wherein the second solution comprises 1,4-butanediol diglycidyl ether (BDDE) in a sodium hyaluronate solution in the presence of sodium hydroxide, and wherein the BDDE is used to crosslink the hyaluronic acid using epoxide chemistry.

[0222] Clause 42. The method of any one of clauses 38 to 41, wherein the collagen is provided in a soluble state as a solution.

[0223] Clause 43. The method of any one of clauses 38 to 42, wherein the physical mixing step is performed in the presence of a buffer.

[0224] Item 44. The method according to Item 43, wherein the buffer comprises PBS.

[0225] Clause 45. The method of any one of clauses 38 to 44, wherein the collagen is provided in the form of a collagen solution, wherein the pH of the collagen solution is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, or any pH in the range bounded by any two of the above values, and wherein the collagen is soluble at acidic pH.

[0226] Clause 46. A method as described in any of clauses 38 to 45, wherein the collagen is provided in the form of an acidic pH solution, wherein the pH of the collagen solution is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, or any pH in the range defined by any two of the above values, and wherein the collagen is soluble at acidic pH.

[0227] Clause 47. The method of any one of clauses 38 to 46, wherein the collagen is provided at an acidic to neutral pH.

[0228] Clause 48. The method of any one of Clauses 38 to 46, further comprising neutralizing the macromolecular matrix to a pH of about 7 after homogenizing the collagen throughout the cross-linked hyaluronic acid.

[0229] Item 49. The method of any of Items 38 to 48, further comprising neutralizing the collagen to a pH of about 7 prior to physically mixing the collagen with the cross-linked hyaluronic acid, wherein neutralizing the collagen prior to physically mixing the collagen into the cross-linked hyaluronic acid causes the collagen to precipitate into collagen fibrils or particles, wherein the collagen fibrils or particles are further mixed into the cross-linked hyaluronic acid, wherein the fibrils or particles are homogeneously mixed throughout the cross-linked hyaluronic acid.

[0230] Clause 50. The method of Clause 49, wherein the collagen is provided in the form of fibrillated collagen or collagen fibers.

[0231] Clause 51. The method of clause 50, wherein the collagen is prepared at an alkaline pH, thereby producing fibrillated collagen or collagen fibers.

[0232] Clause 52. The method of clause 50 or 51, wherein the collagen is prepared with at least one salt to obtain fibrillated collagen or collagen fibers.

[0233] Clause 53. The method of clause 52, wherein the concentration of at least one salt is about 20 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM or about 500 mM, or any concentration in the range defined by any two of the above values.

[0234] Clause 54. The method of clause 52 or 53, wherein at least one salt comprises an anion, wherein the anion comprises H2PO 4- 、SO4 2- , Cl - or SCN - .

[0235] Clause 55. The method of any of clauses 52-54, wherein the at least one salt comprises NaCl, Na2SO4, or Li2SO4.

[0236] Clause 56. The method of any one of clauses 38 to 55, wherein the hyaluronic acid comprises an average molecular weight of about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, 40,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 90,000 Daltons, about 100,000 Daltons, about 200,000 Daltons, about 300,000 Daltons, about 400,000 Daltons, about 500,000 Daltons, about 600,000 Daltons, about 700,000 Daltons, about 800,000 Daltons, about 900,000 Daltons, about 100 0,000 Daltons, about 1,100,000 Daltons, about 1,200,000 Daltons, about 1,300,000 Daltons, about 1,400,000 Daltons, about 1,500,000 Daltons, about 1,600,000 Daltons, about 1,700,000 Daltons, about 1,800,000 Daltons, about 1,900,000 Daltons, about 2,000,000 Daltons, about 2,100,000 Daltons, about 2,200,000 Daltons, about 2,300,000 Daltons, about 2,400,000 Daltons, about 2,500,000 Daltons, about 2,600,000 Daltons, about 2,700,000 Daltons, about 2,800,000 Daltons, about 2,900,000 Daltons, about 3,000,000 Daltons, about 3,100,000 Daltons, about 3,200,000 Daltons, about 3,300,000 Daltons, about 3,400,000 Daltons, about 3,500,000 Daltons, about 3,600,000 Daltons, about 3,700,000 Daltons, about 3,800,000 Daltons, about 3,900,000 Daltons, about 4,000,000 Daltons, about 4,100,000 Daltons, about 4,200,000 Daltons, about 4,300,000 Daltons, about 4,400,000 Daltons, about 4,500,000 Daltons Daltons, about 4,600,000 Daltons, about 4,700,000 Daltons, about 4,800,000 Daltons, about 4,900,000 Daltons, about 5,000,000 Daltons, about 5,100,000 Daltons, about 5,200,000 Daltons, about 5,300,000 Daltons, about 5,400,000 Daltons, about 5,500,000 Daltons, about 5,600,000 Daltons, about 5,700,000 Daltons, about 5,800,000 Daltons, about 5,900,000 Daltons, about 6,000,000 Daltons, about 6,100,000 Daltons, about 6,200,000 Daltons, about 6,300,000 Daltons,000 Daltons, about 6,400,000 Daltons, about 6,500,000 Daltons, about 6,600,000 Daltons, about 6,700,000 Daltons, about 6,800,000 Daltons, about 6,900,000 Daltons, about 7,000,000 Daltons, about 7,100,000 Daltons, about 7,200,000 Daltons, about 7,3 00,000 Daltons, about 7,400,000 Daltons, about 7,500,000 Daltons, about 7,600,000 Daltons, about 7,700,000 Daltons, about 7,800,000 Daltons, about 7,900,000 Daltons, about 8,000,000 Daltons, about 8,100,000 Daltons, about 8,200,000 Daltons, about 8 ,300,000 Daltons, about 8,400,000 Daltons, about 8,500,000 Daltons, about 8,600,000 Daltons, about 8,700,000 Daltons, about 8,800,000 Daltons, about 8,900,000 Daltons, about 9,000,000 Daltons, about 9,100,000 Daltons, about 9,200,000 Daltons , about 9,300,000 Daltons, about 9,400,000 Daltons, about 9,500,000 Daltons, about 9,600,000 Daltons, about 9,700,000 Daltons, about 9,800,000 Daltons, about 9,900,000 Daltons, or about 10,000,000 Daltons, or any molecular weight between any two of the above values.

[0237] Clause 57. The method of any one of Clauses 38 to 56, wherein the hyaluronic acid comprises a mixture of hyaluronic acid components having at least two different molecular weights.

[0238] Clause 58. The method of any one of clauses 38 to 57, wherein the dissolving step further comprises dissolving both hyaluronic acid and collagen in an aqueous solution to form a pre-reacted aqueous solution comprising both hyaluronic acid and collagen, and wherein the cross-linking reaction is carried out by cross-linking the hyaluronic acid and collagen into an HA-collagen coupled gel, wherein the mixing step comprises physically mixing the HA-collagen coupled gel with additional collagen, wherein the collagen is in a soluble state and wherein the collagen is in a solution, wherein the solution is at an acidic pH, thereby producing a macromolecular matrix comprising the HA-collagen coupled gel with physically mixed collagen.

[0239] Clause 59. The method of any one of clauses 38 to 58, wherein collagen fibrils or collagen precipitates are formed after the mixing step with the collagen, and wherein the collagen fibrils or collagen precipitates are homogeneously mixed within the cross-linked hyaluronic acid.

[0240] Clause 60. The method of any one of clauses 38 to 58, wherein collagen fibrils or collagen precipitates are formed during the mixing step of mixing with the collagen, and wherein the collagen fibrils or collagen precipitates are homogenously mixed within the cross-linked hyaluronic acid.

[0241] Clause 61. The method of any one of Clauses 1 to 60, wherein the method further comprises adjusting the osmotic pressure of the macromolecular matrix after mixing the collagen throughout the cross-linked hyaluronic acid.

[0242] Clause 62. The method of any one of clauses 1 to 61, wherein the mixing step is performed at room temperature.

[0243] Clause 63. The method of any one of clauses 1 to 61, wherein the mixing step is performed at about 4°C.

[0244] Clause 64. The method of any one of clauses 1 to 63, wherein the method is carried out at about 4°C.

[0245] Clause 65. The method of any one of clauses 1 to 64, wherein the method further comprises purifying the cross-linked hyaluronic acid or HA-collagen coupled gel, wherein the purification is performed prior to the step of mixing with the collagen.

[0246] Clause 66. The method of Clause 65, wherein the purification is performed using dialysis purification.

[0247] Clause 67. The method of clause 65 or 66, wherein the pH of the cross-linking reaction mixture is adjusted after cross-linking is completed, wherein the pH adjustment is performed prior to the purification step, and wherein the pH is adjusted to about 7.0, about 7.2, about 7.4, about 7.6, or any pH between the range defined by any two of the above values.

[0248] Clause 68. The method of any one of clauses 65 to 67, wherein the purification is performed at a temperature in the range of about 2°C to about 8°C.

[0249] Clause 69. The method of any one of clauses 65 to 68, wherein the purification is carried out at about 2°C, about 4°C, about 6°C or about 8°C, or any temperature between a range defined by any two of the foregoing values.

[0250] Clause 70. The method of any one of clauses 65 to 69, wherein the method further comprises a sterilization step of sterilizing the cross-linked hyaluronic acid or HA-collagen coupling gel prior to mixing with the collagen, wherein the sterilization step is performed after the purification step.

[0251] Clause 71. The method of Clause 70, wherein the sterilization step comprises transferring the cross-linked hyaluronic acid or HA-collagen coupled gel to a container for sterilization; and sterilizing the cross-linked hyaluronic acid or HA-collagen coupled gel.

[0252] Clause 72. The method of Clause 71, wherein the container is a syringe.

[0253] Clause 73. The method of any one of clauses 70 to 72, wherein sterilization is performed by heat (dry heat, steam heat, moist heat sterilization), radiation (non-ionizing, UV), ionization (particles (beta rays, gamma rays, x-rays), electromagnetic (electron beam), filtration or terminal sterilization.

[0254] Clause 74. The method of any one of clauses 1 to 73, wherein the method further comprises dialyzing the cross-linked macromolecular matrix or the HA-collagen coupled gel, wherein the dialysis is performed through a membrane having a molecular weight cutoff in the range of between 1000 Daltons to about 100,000 Daltons, and wherein the dialysis is performed prior to sterilization.

[0255] Clause 75. The method of Clause 74, wherein the dialysis is performed in a buffer.

[0256] Clause 76. The method of Clause 75, wherein the buffer comprises phosphate buffered saline or sodium phosphate buffer.

[0257] Clause 77. The method of any of Clauses 75 or 76, wherein the buffer further comprises NaCl and / or KCl.

[0258] Clause 78. The method of any one of clauses 38 to 75, wherein the method further comprises raising the pH of the cross-linked hyaluronic acid or HA-collagen coupled gel to a neutral pH after the cross-linking reaction is complete, wherein raising the pH is performed prior to the sterilization step.

[0259] Item 79. The method of any one of items 38 to 75, wherein the method further comprises raising the pH of the cross-linked hyaluronic acid or HA-collagen coupled gel to about 7.0, about 7.2 or about 7.4, or any pH between the ranges defined by any two of the above values ​​after the cross-linking reaction is completed, wherein raising the pH is performed prior to the sterilization step.

[0260] Clause 80. The method of any one of clauses 38 to 78, wherein the second solution comprises a water-soluble carbodiimide and N-hydroxysuccinimide or N-hydroxysulfosuccinimide, and wherein the adding and cross-linking steps are performed at a temperature between about 4°C and about 22°C.

[0261] Clause 81. The method of Clause 80, wherein the crosslinking is performed at about 22°C.

[0262] Clause 82. The method of Clause 80, wherein the cross-linking is performed at about 4°C.

[0263] Clause 83. The method of any one of clauses 38 to 78, wherein the second solution comprises 1,4-butanediol diglycidyl ether (BDDE) in a sodium hyaluronate solution in the presence of sodium hydroxide, and wherein the adding and cross-linking steps are performed between about 45°C and about 75°C.

[0264] Clause 84. The method of any of clauses 38 to 83, wherein the second solution comprises 1,4-butanediol diglycidyl ether (BDDE) in a solution of sodium hyaluronate in the presence of sodium hydroxide, and wherein the adding and cross-linking steps are performed at a temperature of about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, or at any temperature in the range defined by any two of the foregoing values.

[0265] Clause 85. The method of any one of clauses 38 to 84, wherein the second solution comprises 1,4-butanediol diglycidyl ether (BDDE) in a solution of sodium hyaluronate in the presence of sodium hydroxide, and wherein the adding and cross-linking steps are performed at a temperature of about 50°C.

[0266] Item 86. The method of any one of items 1 to 85, wherein the method comprises a neutralization step performed after forming a macromolecular matrix comprising cross-linked hyaluronic acid and collagen or after forming an HA-collagen coupled gel with physically mixed collagen, wherein the neutralization step comprises adjusting the macromolecular matrix to physiological pH and physiological salt concentration.

[0267] Clause 87. The method of Clause 86, wherein the neutralizing comprises adding an alkaline solution or a buffer solution under sterile conditions to adjust the pH after the mixing step.

[0268] Clause 88. The method of clause 86 or 87, wherein the pH and physiological salt concentration are adjusted to cause the collagen to precipitate into fibrils or particles, wherein the collagen fibrils or particles are homogeneously distributed in the macromolecular matrix.

[0269] Clause 89. The method of any one of clauses 38 to 88, wherein the method further comprises a stopping cross-linking step, wherein the stopping step comprises adding a base to the cross-linking reaction mixture until the pH is between about 8 and about 10 for at least 10 minutes, and then adding an acidic solution to the cross-linking mixture until the pH reaches about 7.

[0270] Clause 90. The method of any one of clauses 38 to 89, wherein the naturally occurring amine is derived from lysine.

[0271] Clause 91. The method of any of Clauses 38 to 90, wherein the pH of the cross-linking reaction mixture is between about 4.0 and about 10.0.

[0272] Clause 92. The method of Clause 91, wherein the pH of the cross-linking reaction mixture is between about 4.0 and about 6.0.

[0273] Clause 93. The method of any one of clauses 38 to 78 or 83 to 92, wherein the second solution comprises 1,4-butanediol diglycidyl ether (BDDE) in a sodium hyaluronate solution in the presence of sodium hydroxide, and wherein cross-linking is performed under alkaline conditions.

[0274] Clause 94. The method of any one of clauses 38 to 93, wherein the pre-reaction solution comprises a salt, wherein the concentration of the salt is about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, about 500 mM, or any concentration in the range defined by any two of the above values.

[0275] Clause 95. The method of any one of clauses 38 to 81, 86 to 92 or 94, wherein the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and wherein the concentration of the water-soluble carbodiimide in the cross-linking reaction mixture is about 20 mM to about 300 mM.

[0276] Clause 96. The method of clause 95, wherein the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and its concentration is about 20 mM, about 40 mM, about 60 mM, about 80 mM, about 100 mM, about 120 mM, about 140 mM, about 160 mM, about 180 mM, about 200 mM, about 220 mM, about 240 mM, about 260 mM, about 280 mM, about 300 mM, or any concentration in the range bounded by any two of the above values.

[0277] Clause 97. The method of any one of clauses 38 to 81, 86 to 92, or 94 to 96, wherein the molar ratio of water-soluble carbodiimide to hyaluronic acid water-soluble carbodiimide:hyaluronic acid repeating units is from about 0.3 to about 3.0.

[0278] Clause 98. The method of clause 97, wherein the molar ratio of the water-soluble carbodiimide and the water-soluble carbodiimide:hyaluronic acid repeating units of hyaluronic acid is about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9 or about 3.0, or any molar ratio between the ranges defined by any two of the above values.

[0279] Clause 99. The method of any one of clauses 38 to 98, wherein the mole:mole (lysine:HA repeating unit) ratio of lysine to hyaluronic acid is from about 0.01 to about 0.6.

[0280] Clause 100. The method of clause 99, wherein the mole:mole (lysine:HA repeating unit) ratio of lysine to hyaluronic acid is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.2, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.51, about 0.52, about 0.53, about 0.54, about 0.55, about 0.56, about 0.57, about 0.58, about 0.59, about 0.6, or any molar ratio between the ranges defined by any two of the above values.

[0281] Clause 101. The method of any one of clauses 38 to 100, wherein the hyaluronic acid in the pre-reaction solution is hydrated for at least about 60 minutes prior to adding the second solution.

[0282] Clause 102. The method of any one of Clauses 38 to 101, wherein crosslinking of the reaction mixture is performed for about 4 hours to about 24 hours.

[0283] Clause 103. The method of any one of clauses 38 to 102, wherein the method further comprises adding lidocaine to the macromolecular matrix.

[0284] Clause 104. The method of Clause 103, wherein the concentration of lidocaine in the matrix ranges from about 0.15% (w / w) to about 0.45% (w / w).

[0285] Clause 105. The method of clause 103 or 104, wherein the concentration of lidocaine in the matrix is ​​about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration between the range bounded by any two of the above values.

[0286] Clause 106. The method of any one of clauses 103 to 105, wherein the concentration of lidocaine in the matrix ranges from about 0.27% (w / w) to about 0.33% (w / w).

[0287] Clause 107. The method of any one of Clauses 38 to 106, wherein the matrix further comprises uncrosslinked HA.

[0288] Clause 108. The method of Clause 107, wherein the concentration of uncrosslinked HA in the matrix is ​​up to about 5% (w / w).

[0289] Clause 109. The method of clauses 107 to 108, wherein the concentration of uncross-linked HA in the matrix is ​​about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), or any concentration between the range bounded by any two of the above values.

[0290] Clause 110. The method of any one of clauses 107 to 109, wherein the concentration of uncrosslinked HA in the matrix is ​​about 1% (w / w).

[0291] Clause 111. The method of any one of clauses 107 to 109, wherein the concentration of uncrosslinked HA in the matrix is ​​about 2% (w / w).

[0292] Clause 112. The method of any one of clauses 107 to 109, wherein the concentration of uncrosslinked HA in the matrix is ​​about 5% (w / w).

[0293] Clause 113. The method of any one of clauses 38 to 112, wherein the macromolecular matrix has enhanced extrudability through a needle, wherein the needle comprises a gauge size of 27G, 28G, 29G, 30G, 31G, or 32G.

[0294] Clause 114. The method of any one of clauses 1 to 113, wherein the method further comprises concentrating the collagen prior to adding the collagen to the cross-linked HA.

[0295] Clause 115. A macromolecular matrix prepared by the method of any one of clauses 1 to 114.

[0296] Item 116. The macromolecular matrix of Item 115, wherein the macromolecular matrix comprises cross-linked hyaluronic acid, wherein the collagen is physically mixed with the cross-linked hyaluronic acid, and wherein the collagen is homogeneous throughout the macromolecular complex.

[0297] Item 117. The macromolecular matrix of Item 115, wherein the macromolecular matrix comprises an HA-collagen coupled gel, wherein the HA-collagen coupled gel further comprises physically mixed collagen that is not cross-linked in the HA-collagen coupled gel.

[0298] Item 118. A macromolecular matrix comprising: hyaluronic acid, wherein the hyaluronic acid is cross-linked; and collagen; wherein the collagen is physically mixed with the cross-linked hyaluronic acid.

[0299] Item 119. A macromolecular matrix comprising: an HA-collagen coupled gel, wherein the HA-collagen coupled gel contains hyaluronic acid cross-linked with collagen; and physically mixed collagen, wherein the physically mixed collagen is not cross-linked with the HA-collagen coupled gel, and wherein the physically mixed collagen is homogeneously mixed within the HA-collagen coupled gel.

[0300] Item 120. The macromolecular matrix of any one of items 115 to 119, wherein the hyaluronic acid is cross-linked with naturally occurring amines.

[0301] Item 121. The macromolecular matrix of Item 120, wherein the naturally occurring amine is derived from lysine.

[0302] Clause 122. The macromolecular matrix of any one of clauses 115 to 121, wherein the collagen is derived from bovine collagen, marine collagen, human collagen or porcine collagen.

[0303] Clause 123. The macromolecular matrix of any one of clauses 115 to 122, wherein the collagen is recombinant human collagen.

[0304] Clause 124. The macromolecular matrix of any one of clauses 115 to 123, wherein the elastic modulus (G') of the macromolecular matrix is ​​about 100 Pa, about 200 Pa, about 300 Pa, about 400 Pa, about 500 Pa, about 600 Pa, about 700 Pa, about 800 Pa, about 900 Pa, about 1000 Pa, about 1100 Pa, about 1200 Pa, about 1300 Pa, about 1400 Pa, about 1500 Pa, about 1600 Pa, about 1700 Pa, about 1800 Pa, about 1900 Pa, about 2000 Pa, about 2100 Pa, about 2200 Pa, about 2300 Pa, about 2400 Pa, about 2500 Pa, about 2600 Pa, about 2700 Pa, about 2800 Pa, about 2900 Pa, about 3000 Pa, about 3100 Pa, about 3200 Pa, about 3300 Pa, about 3400 Pa, about 3500 Pa, about 3600 Pa, about 3700 Pa, about 3800 Pa, about 3900 Pa, about 4000 Pa, about 4100 Pa, about 4200 Pa, about 4300 Pa, about 4400 Pa, about 4500 Pa, about 4600 Pa, about 4700 Pa, about 4800 Pa, about 4900 Pa, about 5000 Pa, about 5100 Pa, about 5200 Pa, about 5300 Pa, about 5400 Pa, about 5500 Pa, about 5600 Pa, about 5700 Pa, about 580 Pa, about 500Pa, about 1600Pa, about 1700Pa, about 1800Pa, about 1900Pa, about 2000Pa, about 2100Pa, about 2200Pa, about 2300Pa, about 2400Pa, about 2500Pa, about 2600Pa, about 2700Pa, about 2800Pa, about 2900Pa, or about 3000Pa, or any value in the range defined by any two of the above values.

[0305] Clause 125. The macromolecular matrix of any one of clauses 115 to 124, wherein the macromolecular matrix has a compressive force value of about 10 gmf, about 20 gmf, about 30 gmf, about 40 gmf, about 50 gmf, about 60 gmf, about 70 gmf, about 80 gmf, about 90 gmf, about 100 gmf, about 110 gmf, about 120 gmf, about 130 gmf , about 140gmf, about 150gmf, about 160gmf, about 170gmf, about 180gmf, about 190gmf, about 200gmf, about 210gmf, about 220gmf, about 230gmf, about 240gmf, about 250gmf, about 260gmf, about 270gmf, about 280gmf, about 290gmf, about 300g gmf, about 310 gmf, about 320 gmf, about 330 gmf, about 340 gmf, about 350 gmf, about 360 gmf, about 370 gmf, about 380 gmf, about 390 gmf, about 400 gmf, about 410 gmf, about 420 gmf, about 430 gmf, about 440 gmf, about 450 gmf, about 460 gmf, about 470 gmf gmf, about 580 gmf, about 590 gmf or about 600 gmf, or any compressive force value between a range defined by any two of the above values.

[0306] Clause 126. The macromolecular matrix of any one of clauses 115 to 125, wherein the concentration of hyaluronic acid is about 2 mg / mL, about 4 mg / mL, about 6 mg / mL, about 8 mg / mL, about 10 mg / mL, about 12 mg / mL, about 14 mg / mL, about 16 mg / mL, about 18 mg / mL, about 20 mg / mL, about 22 mg / mL, about 24 mg / mL, about 26 mg / mL, about 28 mg / mL, about 30 mg / mL, 32 mg / mL, about 34 mg / mL or about 36 mg / mL, or any concentration between the range defined by any two of the above values.

[0307] Clause 127. The macromolecular matrix of any one of clauses 115 to 126, wherein the weight ratio of hyaluronic acid to collagen is about 20:3, about 24:2.3, about 24:10, about 24:12, about 24:4, about 24:6, about 28:6 or about 28:11.

[0308] Clause 128. The macromolecular matrix of any one of clauses 115 to 127, wherein the cross-linked hyaluronic acid comprises a hyaluronic acid component having an average molecular weight of about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, 40,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 100,000 Daltons, about 200,000 Daltons, about 300,000 Daltons, about 400,000 Daltons, about 500,000 Daltons, about 600,000 Daltons, about 700,000 Daltons, about 800,000 Daltons, about 900,000 Daltons, about 1,000,000 Daltons, about 1,100,000 Daltons, about 1,200,000 Daltons, about 1,300,000 Daltons, about 1,400,000 Daltons, about 1,500,000 Daltons, about 1,600,000 Daltons, about 1,700,000 Daltons, about 1,800,000 Daltons, about 1,900,000 Daltons, about 2,000,000 Daltons, about 2,100,000 Daltons, about 2,200,000 Daltons, about 2,300,000 Daltons, about 2,400,000 Daltons, about 2,500,000 Daltons, about 2,600,000 Daltons, about 2,700,000 Daltons Daltons, about 2,800,000 Daltons, about 2,900,000 Daltons, about 3,000,000 Daltons, about 3,100,000 Daltons, about 3,200,000 Daltons, about 3,300,000 Daltons, about 3,400,000 Daltons, about 3,500,000 Daltons, about 3,600,000 Daltons, about 3,700,000 Daltons, about 3,800,000 Daltons, about 3,900,000 Daltons, about 4,000,000 Daltons, about 4,100,000 Daltons, about 4,200,000 Daltons, about 4,300,000 Daltons, about 4,400,000 Daltons, about 4,500,000 Daltons 0 Daltons, about 4,600,000 Daltons, about 4,700,000 Daltons, about 4,800,000 Daltons, about 4,900,000 Daltons, about 5,000,000 Daltons, about 5,100,000 Daltons, about 5,200,000 Daltons, about 5,300,000 Daltons, about 5,400,000 Daltons, about 5,500,000 Daltons, about 5,600,000 Daltons, about 5,700,000 Daltons, about 5,800,000 Daltons, about 5,900,000 Daltons, about 6,000,000 Daltons, about 6,100,000 Daltons, about 6,200,000 Daltons, about 6,300,000 Daltons, about 6,400,000 Daltons, about 6,500,000 Daltons, about 6,600,000 Daltons, about 6,700,000 Daltons, about 6,800,000 Daltons, about 6,900,000 Daltons, about 7,000,000 Daltons, about 7,100,000 Daltons, about 7,200,000 Daltons, about 7,3 00,000 Daltons, about 7,400,000 Daltons, about 7,500,000 Daltons, about 7,600,000 Daltons, about 7,700,000 Daltons, about 7,800,000 Daltons, about 7,900,000 Daltons, about 8,000,000 Daltons, about 8,100,000 Daltons, about 8,200,000 Daltons, about 8,300,000 Daltons, about 8,400,000 Daltons, about 8,500,000 Daltons, about 8,600,000 Daltons, about 8,700,000 Daltons, about 8,800,000 Daltons, about 8,900,000 Daltons, about 9,000,000 Daltons, about 9,100,000 Daltons, about 9,200,000 Daltons Daltons, about 9,300,000 Daltons, about 9,400,000 Daltons, about 9,500,000 Daltons, about 9,600,000 Daltons, about 9,700,000 Daltons, about 9,800,000 Daltons, about 9,900,000 Daltons, or about 10,000,000 Daltons, or any weight between any two of the above values.

[0309] Item 129. The macromolecular matrix of Item 128, wherein the hyaluronic acid component comprises a mixture of hyaluronic acid components comprising at least two different molecular weights.

[0310] Clause 130. The macromolecular matrix of any one of clauses 115 to 129, wherein the hyaluronic acid comprises a mixture of hyaluronic acids, wherein the mixture comprises a 50:50 blend of HA having two different molecular weights.

[0311] Clause 131. The macromolecular matrix of any one of clauses 115 to 130, wherein the collagen comprises type I collagen and / or type III collagen.

[0312] Clause 132. The macromolecular matrix of any of clauses 115 to 131, wherein the concentration of collagen is about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL or about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL or any concentration between the range defined by any two of the above values.

[0313] Clause 133. The macromolecular matrix of any one of clauses 115 to 132, wherein the macromolecular matrix has an osmotic pressure of between about 250 mOsm / kg and about 390 mOsm / kg.

[0314] Clause 134. The macromolecular matrix of any one of clauses 115 to 133, wherein the macromolecular matrix has an osmotic pressure of between about 250 mOsm / kg, about 275 mOsm / kg, about 300 mOsm / kg, about 325 mOsm / kg, or about 390 mOsm, or any osmotic pressure between a range defined by any two of the foregoing values.

[0315] Clause 135. The macromolecular matrix of any one of clauses 115 to 134, wherein the hyaluronic acid is linear.

[0316] Item 136. The macromolecular matrix of any of Items 115 to 135, wherein the viscosity modulus (G") of the macromolecular matrix is ​​about 10Pa, about 20Pa, about 30Pa, about 40Pa, about 50Pa, about 60Pa, about 70Pa, about 80Pa, about 90Pa, about 100Pa, about 200Pa, about 300Pa, about 400Pa, about 500Pa, about 600Pa, about 700Pa, about 800Pa, about 900Pa or about 1000Pa, or any viscosity modulus (G") between the ranges defined by any two of the above values.

[0317] Item 137. The macromolecular matrix of any of Items 115 to 136, wherein the dielectric loss tangent value (G" / G') of the macromolecular matrix is ​​about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.12, about 0.14, about 0.16, about 0.18, about 0.20, about 0.22, about 0.24, about 0.26, about 0.28, about 0.30, about 0.32, about 0.34, about 0.36, about 0.38, about 0.40, about 0.42, about 0.44, about 0.46, about 0.48, about 0.50, or any dielectric loss tangent value (G" / G') between the ranges defined by any two of the above values.

[0318] Clause 138. The macromolecular matrix of any of clauses 115 to 137, wherein the pH of the macromolecular matrix is ​​about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, or any pH in the range defined by any two of the above values.

[0319] Clause 139. The macromolecular matrix of any one of clauses 115 to 138, wherein the macromolecular matrix further comprises lidocaine.

[0320] Item 140. The macromolecular matrix of Item 139, wherein the concentration of lidocaine in the matrix is ​​about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration in the range defined by any two of the above values.

[0321] Clause 141. The macromolecular matrix of any of Clauses 139 or 140, wherein the concentration of lidocaine in the matrix ranges from about 0.27% (w / w) to about 0.33% (w / w).

[0322] Clause 142. The macromolecular matrix of any one of clauses 115 to 141, wherein the matrix further comprises non-cross-linked HA.

[0323] Item 143. The macromolecular matrix of Item 142, wherein the concentration of uncrosslinked HA in the matrix is ​​up to about 5% (w / w).

[0324] Item 144. The macromolecular matrix of items 142 to 143, wherein the concentration of uncross-linked HA in the matrix is ​​about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), or any concentration between the range defined by any two of the above values.

[0325] Clause 145. The macromolecular matrix of any one of clauses 142 to 144, wherein the concentration of non-cross-linked HA in the matrix is ​​about 1% (w / w).

[0326] Clause 146. The macromolecular matrix of any one of clauses 142 to 144, wherein the concentration of non-cross-linked HA in the matrix is ​​about 2% (w / w).

[0327] Clause 147. The macromolecular matrix of any one of clauses 142 to 144, wherein the concentration of non-cross-linked HA in the matrix is ​​about 5% (w / w).

[0328] Clause 148. The macromolecular matrix of any one of clauses 115 to 147, wherein the macromolecular matrix has enhanced extrudability through a needle, wherein the needle comprises a gauge size of 27G, 28G, 29G, 30G, 31G, or 32G.

[0329] Item 149. A method for improving the aesthetic quality of a human anatomical feature, comprising: injecting a composition into human tissue, thereby improving the aesthetic quality of the anatomical feature; wherein the composition comprises a macromolecular matrix prepared by the method of any one of claims 1 to 102 or a macromolecular matrix of any one of claims 115 to 139.

[0330] Item 150. A method for improving the aesthetic quality of an anatomical feature of the human body, comprising: injecting a composition into human tissue, thereby improving the aesthetic quality of the anatomical feature; wherein the composition comprises a macromolecular matrix, the macromolecular matrix comprising: cross-linked hyaluronic acid; lysine; and collagen; wherein the collagen is physically mixed into the cross-linked hyaluronic acid.

[0331] Clause 151. The method of Clause 150, wherein the composition further comprises lidocaine.

[0332] Clause 152. The method of clause 151, wherein the concentration of lidocaine in the matrix ranges from 0.15% (w / w) to 0.45% (w / w).

[0333] Clause 153. The method of clause 151 or 152, wherein the concentration of lidocaine in the matrix is ​​about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration between the range defined by any two of the above values.

[0334] Clause 154. The method of any one of clauses 151 to 153, wherein the concentration of lidocaine in the matrix ranges from about 0.27% (w / w) to about 0.33% (w / w).

[0335] Clause 155. The method of any one of clauses 150 to 154, wherein the composition further comprises non-cross-linked HA.

[0336] Clause 156. The method of Clause 155, wherein the concentration of uncrosslinked HA in the matrix is ​​up to about 5% (w / w).

[0337] Clause 157. The method of clause 155 or 156, wherein the concentration of uncross-linked HA in the matrix is ​​about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), or any concentration between the range bounded by any two of the above values.

[0338] Clause 158. The method of any one of clauses 155 to 157, wherein the concentration of uncrosslinked HA in the matrix is ​​about 1% (w / w).

[0339] Clause 159. The method of any one of clauses 155 to 157, wherein the concentration of uncrosslinked HA in the matrix is ​​about 2% (w / w).

[0340] Clause 160. The method of any one of clauses 155 to 157, wherein the concentration of uncrosslinked HA in the matrix is ​​about 5% (w / w).

[0341] Clause 161. The method of any of clauses 150 to 160, wherein the cross-linked hyaluronic acid comprises a hyaluronic acid component having an average molecular weight of about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, 40,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 100,000 Daltons, about 200,000 Daltons, about 300,000 Daltons, about 400,000 Daltons, about 500,000 Daltons, about 600,000 Daltons, about 700,000 Daltons, about 800,000 Daltons, about 900,000 Daltons, about 10 about 1,000,000 Daltons, about 1,100,000 Daltons, about 1,200,000 Daltons, about 1,300,000 Daltons, about 1,400,000 Daltons, about 1,500,000 Daltons, about 1,600,000 Daltons, about 1,700,000 Daltons, about 1,800,000 Daltons, about 1,900,000 Daltons, about 2,000,000 Daltons, about 2,100,000 Daltons, about 2,200,000 Daltons, about 2,300,000 Daltons, about 2,400,000 Daltons, about 2,500,000 Daltons, about 2,600,000 Daltons, about 2,700,000 Daltons, about 2,800,000 Daltons, about 2,900,000 Daltons, about 3,000,000 Daltons, about 3,100,000 Daltons, about 3,200,000 Daltons, about 3,300,000 Daltons, about 3,400,000 Daltons, about 3,500,000 Daltons, about 3,600,000 Daltons, about 3,700,000 Daltons, about 3,800,000 Daltons, about 3,900,000 Daltons, about 4,000,000 Daltons, about 4,100,000 Daltons, about 4,200,000 Daltons, about 4,300,000 Daltons, about 4,400,000 Daltons, about 4,500,000 Daltons Daltons, about 4,600,000 Daltons, about 4,700,000 Daltons, about 4,800,000 Daltons, about 4,900,000 Daltons, about 5,000,000 Daltons, about 5,100,000 Daltons, about 5,200,000 Daltons, about 5,300,000 Daltons, about 5,400,000 Daltons, about 5,500,000 Daltons, about 5,600,000 Daltons, about 5,700,000 Daltons, about 5,800,000 Daltons, about 5,900,000 Daltons, about 6,000,000 Daltons, about 6,100,000 Daltons, about 6,200,000 Daltons, about 6,300,000 Daltons, about 6,400,000 Daltons, about 6,500,000 Daltons, about 6,600,000 Daltons, about 6,700,000 Daltons, about 6,800,000 Daltons, about 6,900,000 Daltons, about 7,000,000 Daltons, about 7,100,000 Daltons, about 7,200,000 Daltons, about 7,3 00,000 Daltons, about 7,400,000 Daltons, about 7,500,000 Daltons, about 7,600,000 Daltons, about 7,700,000 Daltons, about 7,800,000 Daltons, about 7,900,000 Daltons, about 8,000,000 Daltons, about 8,100,000 Daltons, about 8,200,000 Daltons, about 8,300,000 Daltons, about 8,400,000 Daltons, about 8,500,000 Daltons, about 8,600,000 Daltons, about 8,700,000 Daltons, about 8,800,000 Daltons, about 8,900,000 Daltons, about 9,000,000 Daltons, about 9,100,000 Daltons, about 9,200,000 Daltons Daltons, about 9,300,000 Daltons, about 9,400,000 Daltons, about 9,500,000 Daltons, about 9,600,000 Daltons, about 9,700,000 Daltons, about 9,800,000 Daltons, about 9,900,000 Daltons, or about 10,000,000 Daltons, or any weight between any two of the above values.

[0342] Clause 162. The method of any one of clauses 150 to 161, wherein the hyaluronic acid in the cross-linked hyaluronic acid comprises a mixture of hyaluronic acid components having at least two different molecular weights.

[0343] Clause 163. The method of any one of clauses 150 to 162, wherein the collagen comprises type I collagen and / or type III collagen.

[0344] Clause 164. The method of any one of Clauses 150 to 163, wherein the method does not cause a blue discoloration at the injection site.

[0345] Clause 165. The method of any one of clauses 150 to 164, wherein the composition has enhanced extrudability through a needle, wherein the needle comprises a gauge size of 27G, 28G, 29G, 30G, 31G, or 32G.

[0346] Item 166. A method for improving the appearance of a human body by injecting a composition into human tissue, whereby the composition promotes cellular infiltration and collagen deposition from surrounding tissue into the injected composition, the method comprising: injecting the composition into human tissue, thereby improving the aesthetic quality of the anatomical feature; wherein the composition comprises a cross-linked macromolecular matrix comprising: hyaluronic acid; lysine; and collagen; wherein the hyaluronic acid comprises cross-linked hyaluronic acid physically mixed with collagen; and wherein the tissue injected with the composition exhibits tissue integration.

[0347] Clause 167. The method of Clause 166, wherein the composition further comprises lidocaine.

[0348] Clause 168. The method of clause 166 or 167, wherein the average molecular weight of the hyaluronic acid component is about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, 40,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 100,000 Daltons, about 200,000 Daltons, about 300,000 Daltons, about 400,000 Daltons, about 500,000 Daltons, about 600,000 Daltons, about 700,000 Daltons, about 800,000 Daltons, about 900,000 Daltons, about 1,000,000 Daltons, about 1,100,000 Daltons, about 2,000,000 Daltons, about 3,000,000 Daltons, about 4,000,000 Daltons, about 5,000,000 Daltons, about 6,000,000 Daltons, about 700,000 Daltons, about 800,000 Daltons, about 900,000 Daltons, about 1,0 ... 0,000 Daltons, about 1,200,000 Daltons, about 1,300,000 Daltons, about 1,400,000 Daltons, about 1,500,000 Daltons, about 1,600,000 Daltons, about 1,700,000 Daltons, about 1,800,000 Daltons, about 1,900,000 Daltons, about 2,000,000 Daltons, about 2,100,000 Daltons, about 2,200,000 Daltons, about 2,300,000 Daltons, about 2,400,000 Daltons, about 2,500,000 Daltons, about 2,600,000 Daltons, about 2,700,000 Daltons, about 2,800,000 Daltons, about about 2,900,000 Daltons, about 3,000,000 Daltons, about 3,100,000 Daltons, about 3,200,000 Daltons, about 3,300,000 Daltons, about 3,400,000 Daltons, about 3,500,000 Daltons, about 3,600,000 Daltons, about 3,700,000 Daltons, about 3,800,000 Daltons, about 3,900,000 Daltons, about 4,000,000 Daltons, about 4,100,000 Daltons, about 4,200,000 Daltons, about 4,300,000 Daltons, about 4,400,000 Daltons, about 4,500,000 Daltons, about 4,600,000 Daltons Daltons, about 4,700,000 Daltons, about 4,800,000 Daltons, about 4,900,000 Daltons, about 5,000,000 Daltons, about 5,100,000 Daltons, about 5,200,000 Daltons, about 5,300,000 Daltons, about 5,400,000 Daltons, about 5,500,000 Daltons, about 5,600,000 Daltons, about 5,700,000 Daltons, about 5,800,000 Daltons, about 5,900,000 Daltons, about 6,000,000 Daltons, about 6,100,000 Daltons, about 6,200,000 Daltons, about 6,300,000 Daltons, about 6,400,000 Daltons,000 Daltons, about 6,500,000 Daltons, about 6,600,000 Daltons, about 6,700,000 Daltons, about 6,800,000 Daltons, about 6,900,000 Daltons, about 7,000,000 Daltons, about 7,100,000 Daltons, about 7,200,000 Daltons, about 7,300,000 Daltons, about 7,400,000 Daltons, about 7,500,000 Daltons, about 7,600,000 Daltons, about 7,700,000 Daltons, about 7,800,000 Daltons, about 7,900,000 Daltons, about 8,000,000 Daltons, about 8,100,000 Daltons, about 8,200,000 Daltons, about 8,300,000 Daltons , about 8,400,000 Daltons, about 8,500,000 Daltons, about 8,600,000 Daltons, about 8,700,000 Daltons, about 8,800,000 Daltons, about 8,900,000 Daltons, about 9,000,000 Daltons, about 9,100,000 Daltons, about 9,200,000 Daltons, about 9,300,000 Daltons, about 9,400,000 Daltons, about 9,500,000 Daltons, about 9,600,000 Daltons, about 9,700,000 Daltons, about 9,800,000 Daltons, about 9,900,000 Daltons, or about 10,000,000 Daltons, or any weight in the range defined by any two of the above values.

[0349] Clause 169. The method of any one of clauses 166 to 168, wherein the hyaluronic acid in the cross-linked hyaluronic acid comprises a mixture of hyaluronic acid components having at least two different molecular weights.

[0350] Clause 170. The method of any one of clauses 166 to 169, wherein the collagen comprises type I collagen and / or type III collagen.

[0351] Clause 171. The method of any one of Clauses 166 to 170, wherein the tissue injected with the composition exhibits tissue integration into the composition and deposits collagen in the composition by infiltration of cells from surrounding tissue.

[0352] Clause 172. The method of any one of Clauses 166 to 171, wherein the tissue injected with the composition exhibits tissue integration into the composition and collagen is deposited in the composition after injection of the composition.

[0353] Clause 173. The method of any one of clauses 166 to 172, wherein the composition has enhanced extrudability through a needle, wherein the needle comprises a gauge size of 27G, 28G, 29G, 30G, 31G, or 32G.

[0354] Further considerations

[0355] In some embodiments, any clause in this article can rely on any independent clause or any dependent clause. In one aspect, any clause (for example, dependent or independent clause) can be combined with any other one or more clauses (for example, dependent or independent clause). In one aspect, the claim may include some or all of the words (for example, steps, operations, methods or components) quoted in a clause, sentence, phrase or paragraph. In one aspect, the claim may include some or all of the words listed in one or more clauses, sentences, phrases or paragraphs. In one aspect, some words in each clause, sentence, phrase or paragraph can be deleted. In one aspect, additional words or elements can be added to a clause, sentence, phrase or paragraph. In one aspect, the subject technology can be implemented without utilizing some components, elements, functions or operations described herein. In one aspect, additional components, elements, functions or operations can be utilized to realize the subject technology.

[0356] Example

[0357] The following examples, including the experiments performed and the results obtained, are for illustrative purposes only and should not be construed as limiting the present disclosure.

[0358] Example 1 - Hydrogel Preparation

[0359] To prepare the hydrogel formulations, HA dermal filler, porcine collagen, water and 10x PBS were first mixed together at an acidic pH. 10x PBS was added to adjust the solution to isotonic and buffered conditions. The pH of the solution was then raised to ~7.4 by adding 9.1 wt% NaOH and mixing again. The increase in pH triggered the self-assembly of the collagen network (Table 1). Some formulations were also autoclaved after mixing.

[0360] Table 1: Chemical composition, osmotic pressure and pH of HA-collagen hydrogels. (NOA: lysine cross-linked HA gel; Type 1: Formulation 124mg / mL HA; Type 2: Formulation 220mg / mL HA; AS – sterile mixing; AC – autoclaved; TC – 11mg / mL collagen;

[0361] CN-collagen; HA-hyaluronic acid; *-autoclaved samples)

[0362]

[0363]

[0364] Example 2 - Opacity

[0365] As collagen was added, the hydrogel turned white in appearance and gradually became more opaque ( Figure 1 ). The self-assembly of collagen results in a network of physically cross-linked fibers that scatter light, giving the gel an opaque appearance. Thus, the opacity of the hydrogel provides an advantage over transparent HA-only hydrogels in that it does not produce a Tyndall effect at the injection site in patients.

[0366] Example 3 - Rheological properties

[0367] The rheological apparatus used to measure the viscoelastic properties (G', G") of HA / collagen hydrogels consisted of parallel plates with a gap height set to 1 mm and a diameter of 25 mm. The measurement temperature was set to 25 °C, the oscillation frequency to 5 Hz, and the strain to 0.8%. The elastic modulus (G') increased with increasing collagen and HA concentrations. The G' values ​​decreased after autoclaving in the presence of collagen but not in its absence ( Figure 2 ).

[0368] Example 4 - Swelling

[0369] The swelling of each hydrogel formulation was quantified. Briefly, a hydrogel sample (1 part) was mixed with phosphate buffered saline (PBS) (7 parts) using two connected syringes. Mixing was continued for at least 1 hour. After 1 hour, the gel and PBS were transferred to one syringe and centrifuged (750 RCF, 10 minutes, room temperature). Polymer beads containing dye were added to the supernatant and the syringe was spun down again (50 RCF, 2 minutes, room temperature) to observe the gel / supernatant interface. The change in gel volume relative to the total volume was used to calculate the swelling of the hydrogel material ( Figure 3 ). Formulations with higher collagen concentrations showed reduced swelling compared to similar gels without collagen.

[0370] Example 5 - Cohesion

[0371] Cohesion was quantified by measuring the compressive force of the HA-collagen material as the difference in normal force when parallel 25 mm diameter plates were lowered from a gap height of 2.5 mm to 0.9 mm at a rate of 0.8 mm / min. Gels with higher compressive force (normal force varied between 2.5 mm and 0.9 mm gap height) were considered more cohesive. Cohesion increased with the addition of collagen and increasing HA concentration ( Figure 4 ). Autoclaved formulations containing collagen produced cohesion values ​​similar to those with similar HA concentrations but without collagen.

[0372] Example 6 - Extrusion Force

[0373] The extrusion force was measured using a 1 mL BD tuberculin syringe equipped with a 27G, 1 / 2" needle at a speed of 100 mm / min. The extrusion force increased with increasing collagen content ( Figure 5 ). After autoclaving, the extrusion force was found to be lower than that of the same formulation before autoclaving.

[0374] Example 7 - In vivo enhancement capability

[0375] The ability of the hydrogel to support tissue projection (lift) was evaluated in vivo using a rat subcutaneous implant model. 125 μL of the hydrogel (n=10) was injected as a subcutaneous bolus into the top of the skull. A clinical 3-D imaging system (Canfield Vectra) was used to generate 3-D reconstructions of the boluses over the course of 12 weeks. The average height of the boluses was analyzed using medical imaging software (Canfield Mirror).

[0376] The in vivo lifting capacity of a series of NOA cross-linked HA formulations (without collagen) and increased [HA] was measured between 4 and 12 weeks and showed a positive correlation with HA concentrations from 20 mg / mL to 24 mg / mL ( Figure 6 ). Formulations with [HA] above 24 mg / mL did not show an increase in lifting capacity. In addition, the lifting capacity of the 24NOA gel was comparable to that of the BDDE cross-linked gel containing only HA.

[0377] The in vivo lifting capacity of a range of NOA cross-linked HA formulations with 20 mg / mL or 24 mg / mL HA and low concentrations of collagen (2.3 mg / mL to 3.0 mg / mL) measured between 4 and 12 weeks showed that mixing collagen into the HA gel did not affect the lifting capacity ( Figure 7 ). In addition, the lifting capacity of 24NOA2.3 gel was comparable to that of BDDE cross-linked gel containing only HA.

[0378] The in vivo lifting capacity of a range of NOA-crosslinked HA formulations with 24 mg / mL or 28 mg / mL HA and high concentrations of collagen (6 mg / mL to 11 mg / mL) measured between 4 and 52 weeks showed that gels crosslinked with NOA and mixed with collagen exhibited the same or better long-term lifting capacity than the HA-only comparison ( Figure 8 ). In addition, the lifting capacity of NOA-crosslinked HA gel or HA-collagen gel was similar to that of BDDE-crosslinked HA gel alone.

[0379] Example 8 - In vitro testing of cellular responses to hydrogels.

[0380] Quantify the viability of fibroblasts in close contact with HA-collagen hydrogels. The assay measures cell activity by monitoring the reduction of tetrazolium salts to colored formazan products by cellular enzymes. Cells with higher viability exhibit greater absorbance at a specific wavelength, while cells with lower viability / proliferation exhibit less absorbance. The absorbance can be measured and compared to a positive control (tissue culture plate) and a negative control (hydrogel containing HA alone) to derive a relative proliferation value. Hydrogels that support higher cell viability are expected to induce more cells to infiltrate the gel, which deposit ECM in the gel matrix and may facilitate in vivo tissue integration into the hydrogel depot. Conversely, formulations that yield lower cell proliferation values ​​will behave more inertly and allow less tissue infiltration and integration.

[0381] 100 μL of hydrogel (n=3) was spread on the bottom of a 24-well cell culture plate with a low-adhesion surface coating and placed in a humidified incubator at 37°C for 30 minutes. 50,000 adult dermal fibroblasts in 500 μL of cell culture medium were added to the top of the hydrogel bed and incubated at 37°C. After 48 hours of incubation, 250 μL of XTT reagent was added to each well and incubated at 37°C for 4 hours. The plate was then spun at 300 x g for 5 minutes, and 200 μL of supernatant in each well was transferred to a well of a 96-well filter plate with a 20 μm mesh. The filter plate containing the XTT supernatant was spun at 300 x g for 5 minutes. 100 μL of filtered supernatant in each well was transferred to a clean 96-well plate (black wall, transparent bottom) and the absorbance of the supernatant was read on a microplate reader (450 nm, background correction was 630 nm). Data were normalized to XTT cell viability of fibroblasts cultured on positive control tissue culture polystyrene (TCPS).

[0382] It was found that in the preparations with collagen mixed into the HA matrix, the cell activity was higher than that in the preparations without collagen ( Fig. 9 ). For example, hydrogels composed of HA alone (BDDE cross-linked or NOA cross-linked) showed cell viability values ​​ranging from 18.9% to 21%. Hydrogels with 6 mg / mL or 11 mg / mL collagen added showed cell viability values ​​of 28.7% (28NOA11CN), 29.2% (24NOA6CN), or 30.4% (28NOA6CN).

[0383] In addition to the cell viability assays described above, cell morphology analyses were performed to better understand the effects of certain formulations on cell size, shape, and cytoskeletal organization. The actin filament alignment index and morphology of fibroblasts cultured on HA-only or HA / collagen cross-linked hydrogels were imaged and quantified. Increased actin filament alignment may be associated with increased adhesion of cells to their substrate. Increased aspect ratio is associated with increased spreading of cells on the substrate. Hydrogels that support better cell adhesion and spreading are expected to induce more cells to infiltrate the gel, which deposit ECM in the gel matrix. Increased cell infiltration and ECM deposition may be beneficial for in vivo tissue integration into the hydrogel reservoir. In contrast, formulations that yield lower cell adhesion and spreading values ​​will behave more inertly and allow less tissue infiltration and integration.

[0384] In a typical procedure, hydrogels (n=3) and human dermal fibroblasts in cell culture medium were added to 96-well cell culture plates with a low-adhesion surface coating. After 48 hours of incubation, cells were fixed in formalin and stained with Hoechst, WGA-488, and AlexaFluor-Phalloidin. Wells were imaged with a confocal microscope, and actin filament arrangement (phalloidin) and cell morphology (WGA-488) were analyzed using image analysis software.

[0385] Hydrogels containing collagen showed improved cell adhesion and spreading on the hydrogel material ( Fig.10 ). The actin filament alignment index tended to be higher for 24NOA6CN gels (0.026) compared to HA alone (0.016). The aspect ratio (cell spreading) of cells on 24NOA6CN hydrogels (1.41) was significantly higher than that on HA alone (1.28).

[0386] The cell morphology analysis correlated well with the XTT cell viability assay, as the 24NOA6CN formulations that exhibited higher activity in the viability assay than the HA gel alone also showed evidence of enhanced cell adhesion and spreading in the morphology assay.

[0387] Example 9 - In vivo tissue integration

[0388] A rat subcutaneous implant model was used to evaluate the in vivo tissue integration of a range of formulations. In a typical procedure, 125 μL of the hydrogel was delivered as a subcutaneous bolus to the dorsal flank of the rat. After 4 or 12 weeks, the bolus was removed, fixed in formalin, and then embedded in paraffin for histological examination. Tissue sections were stained with hematoxylin and eosin (H&E) and colloidal iron. Immunohistochemical staining for rat type I collagen was performed using an antibody that does not react with exogenous porcine collagen.

[0389] The formulation supplemented with 20 mg / mL HA (20 NOA) and 3 mg / mL collagen showed enhanced cellular infiltration and tissue integration into the injected filler after 4 weeks, as judged by new host collagen deposition by fibroblasts found in the injected hydrogel bolus ( Fig.11 ).

[0390] The formulation with 28 mg / mL HA (28 NOA) showed encapsulation inside the hydrogel bolus after 4 weeks ( Fig.12 ). Addition of up to 11 mg / mL of collagen improved tissue integration / collagen deposition around the hydrogel bolus, but did not prevent encapsulation. This may indicate that there is an upper limit to HA concentration that may prevent tissue integration.

[0391] The formulation with 24 mg / mL HA (24 NOA) showed tissue ingrowth and collagen deposition throughout the bulk of the hydrogel bolus after 4 weeks ( Fig.13 The addition of collagen appeared to improve tissue integration in a dose-dependent manner. 24NOA6CN gels showed robust new collagen deposition around the hydrogel particles.

[0392] The formulation with 24 mg / mL HA and 6 mg / mL collagen (24NOA6CN) showed improved tissue integration / collagen deposition ( Fig.14 ). The 24NOA6CN formulation showed significantly improved tissue integration compared to the BDDE cross-linked HA-only gel. Importantly, this hybrid gel formulation (24NOA6CN) also exhibited similar lifting capabilities to the high-lift BDDE cross-linked HA-only gel. The 24NOA6CN formulation demonstrated a combination of lifting capabilities suitable for correcting volume loss and enhancing tissue integration. This may result in a natural look / feel and increased duration of effect after the initial volume correction.

[0393] A semiquantitative histopathological scoring system was developed to evaluate the tissue integration of hydrogel boluses after subcutaneous injection in rats. Briefly, a blinded pathologist scored the density of tissue ingrowth (0 = worst, 5 = best), the thickness of the tissue band (0 = worst, 5 = best), and the tissue filling of the bolus (-5 = no filling, 0 = bolus filled with tissue) based on H&E stained sections. These three scores were combined to produce a total score of 10 for the most integrated preparation and -5 for the least integrated preparation. The tissue integration of 24NOA6CN gels was quantified using this scoring system and compared to HA gels cross-linked with BDDE alone ( Fig.15). The integration of the 24NOA6CN gel was similar to that of the HA-only gel at 4 weeks (2.33 vs. 2.5, respectively). After 12 weeks, the 24NOA6CN gel had a higher integration score than the HA-only gel (5.17 vs. 2.83, respectively). It is also important to note that the integration of the 24NOA6CN gel improved over time, while the HA-only gel remained essentially unchanged. This may indicate an improving effect of using the HA-collagen hybrid gel over time.

[0394] Regarding the use of plural and / or singular terms herein, those skilled in the art may translate from plural to singular and / or from singular to plural as appropriate, depending on the context and / or application. For clarity, various singular / plural arrangements may be explicitly set forth herein.

[0395] Those skilled in the art will understand that, in general, the terms used herein, and particularly in the appended claims (e.g., the bodies of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "including but not limited to," etc.). Those skilled in the art will also understand that if a specific number of an introduced claim is intended to be recited, such intent will be expressly recited in the claim, and that no such intent is present in the absence of such a recitation. For example, to aid understanding, the following appended claims may contain statements that use the introductory phrases "at least one" and "one or more" to introduce claim statements. However, the use of such phrases should not be interpreted as implying that a claim statement introduced by the indefinite article "a, an" limits any particular claim containing such introduced claim statement to embodiments containing only one such statement, even when the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article, such as "a, an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same is true for the use of definite articles to introduce claim statements. Furthermore, even if a specific number of introduced claim statements is explicitly enumerated, one skilled in the art will recognize that such a statement should be interpreted as referring to at least the recited number (e.g., the simple statement of "two statements," without other modifiers, means at least two statements, or two or more statements). Furthermore, where a convention similar to “at least one of A, B, and C, etc.” is used, generally speaking, such construction refers to the conventional meaning that would be understood by a person skilled in the art (e.g., “a system having at least one of A, B, and C” would include, but is not limited to, systems comprising: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Where a convention similar to “at least one of A, B, or C, etc.” is used, generally speaking, such construction refers to the conventional meaning that would be understood by a person skilled in the art (e.g., “a system having at least one of A, B, or C” would include, but is not limited to, systems comprising: A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art would also understand that any transitional words and / or phrases that actually present two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of these terms, either term or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0396] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0397] Any feature of the embodiment of the first to the sixth aspect is applicable to all aspects and embodiments determined herein. In addition, any feature of the embodiment of the first to the sixth aspect can be combined with other embodiments described herein independently, in part or in whole in any way, for example, one, two or three or more embodiments can be combined in whole or in part. In addition, any feature of the embodiment of the first to the ninth aspect is optional for other aspects or embodiments.

Claims

1. A method for preparing a macromolecular matrix, the method include: Provides cross-linked hyaluronic acid, Provide collagen; and The collagen is physically mixed into the cross-linked hyaluronic acid, wherein the collagen is homogeneously mixed throughout the cross-linked hyaluronic acid, thereby forming the macromolecular matrix, wherein the macromolecular matrix comprises the cross-linked hyaluronic acid and the physically mixed collagen.

2. The method according to claim 1, wherein the collagen is provided in a soluble state as a solution.

3. A method according to claim 1 or 2, wherein the collagen is provided in the form of an acidic pH solution, wherein the pH of the collagen solution is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0 or any pH in the range between any two of the above values, and wherein the collagen is soluble at acidic pH.

4. The method of any one of claims 1 to 3, wherein the collagen is provided in the form of an acidic pH solution, wherein the pH of the collagen solution is about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, or any pH in the range between any two of the above values, and wherein the collagen is soluble at acidic pH.

5. The method according to any one of claims 1 to 3, wherein the collagen is provided in the form of a neutral pH solution.

6. The method according to any one of claims 1 to 5, wherein the collagen and the cross-linked hyaluronic acid are physically mixed with a buffer. The method of claim 6 , wherein the buffer solution comprises PBS.

8. The method according to any one of claims 1 to 7, wherein the method further comprises neutralizing the macromolecular matrix to a pH of about 7 after homogenizing the collagen throughout the cross-linked hyaluronic acid.

9. The method according to any one of claims 1 to 8, wherein the method further comprises neutralizing the collagen to a pH of about 7 before physically mixing the collagen with the cross-linked hyaluronic acid.

10. The method of claim 9, wherein the collagen is neutralized prior to being physically mixed into the cross-linked hyaluronic acid, thereby causing the collagen to precipitate into collagen fibrils or particles, wherein the collagen fibrils or particles are further mixed into the cross-linked hyaluronic acid, wherein the fibrils or particles are homogeneously mixed throughout the cross-linked hyaluronic acid.

11. The method of claim 1, wherein the collagen is provided in the form of fibrillated collagen or collagen fibers.

12. The method of claim 11, wherein the collagen is prepared at an alkaline pH, thereby producing the fibrillated collagen or collagen fibers.

13. The method of claim 11 or 12, wherein the collagen is prepared with at least one salt to obtain fibrillated collagen or collagen fibers.

14. The method of claim 13, wherein the concentration of the at least one salt is about 20 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM or about 500 mM, or any concentration in a range defined by any two of the above values.

15. The method of claim 13 or 14, wherein the at least one salt comprises an anion, wherein the anion comprises H 2 PO 4- 、SO 4 2- , Cl - or SCN - .

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