Cross-linked HA-collagen hydrogel as a skin filler

Abstract

The present invention provides a skin filler and a method for treating wrinkles in a patient's skin. [Solution] A polymer matrix is ​​provided which contains lysine, hyaluronic acid, and collagen, wherein the hyaluronic acid and collagen form a hydrogel, and the polymer matrix does not contain chitosan.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Patent Application No. 62 / 953,910, filed on December 26, 2019, which is hereby incorporated by reference in its entirety.

[0002] This disclosure relates to a cross - linked polymer matrix comprising hyaluronic acid, collagen, and lysine. Such a composition can be used as a tissue filler with enhanced tissue integration.

Background Art

[0003] Aging is a natural process that occurs over time and can be influenced by genetic characteristics and lifestyle factors (recreational drugs, alcohol abuse, tobacco, UVA / UVB exposure, diet). Characteristics of facial skin aging include, for example, atrophy of muscles and fat, skin laxity, spots, sagging, and swelling. The relaxation of the subcutaneous tissue can lead to excessive skin and drooping, resulting in the appearance of sagging cheeks and eyelids. Swelling refers to an increase in excessive body weight due to the expansion of the face and the lower part of the neck. These changes can be related to dryness, loss of elasticity, and a rough texture.

[0004] Skin fillers have been used to improve the appearance of aging skin. Various types of skin fillers have been developed and used for the treatment or improvement / correction of physical imperfections, such as wrinkles and volume loss due to the effects of aging. Initially, skin filler compositions containing bovine collagen entered the market in 1970. Human - derived collagen was approved by the FDA in 2003, which was more advantageous than bovine - derived collagen that had the potential for allergic reactions in patients. However, human - derived collagen compositions rapidly degraded within 3 to 6 months due to enzymes in the skin tissue. As a result, patients using these initial compositions required frequent procedures to maintain the desired corrected aesthetic appearance.

[0005] Hyaluronan or hyaluronic acid (HA) fillers were introduced in 1990 as an alternative to collagen-based dermal fillers. HA is a naturally occurring water-soluble polysaccharide, specifically a glycosaminoglycan, a major component of the extracellular matrix and widely distributed in animal tissues. HA possesses excellent biocompatibility and does not cause allergic reactions when implanted in patients. Furthermore, HA has the ability to bind to large amounts of water, making it an excellent volume-forming agent for soft tissues. HA is similar to collagen in that it can also be broken down by endogenous enzymes in the skin. For example, non-crosslinked HA does not possess sufficient duration or physical properties to act as a wrinkle filler; therefore, crosslinked HA has been used to maximize its lifespan in skin tissue. Consequently, improved dermal fillers are needed. [Overview of the project]

[0006] Embodiments of this specification encompass methods and compositions (e.g., hydrogels or dermal fillers) comprising a crosslinked polymer matrix containing hyaluronic acid, collagen, and lysine, wherein the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine.

[0007] In each or any of the embodiments described above or below, the crosslinked polymer matrix further comprises lidocaine. In each or any of the embodiments described above or below, the concentration of lidocaine in the matrix ranges from about 0.15% (w / w) to about 0.45% (w / w). In each or any of the embodiments described above or below, the concentration of lidocaine in the matrix ranges from about 0.27% (w / w) to about 0.33% (w / w). In each or any of the embodiments described above or below, lidocaine is present in a concentration of approximately 0.15% (w / w), approximately 0.17% (w / w), approximately 0.19% (w / w), approximately 0.21% (w / w), approximately 0.23% (w / w), approximately 0.25% (w / w), approximately 0.27% (w / w), approximately 0.29% (w / w), approximately 0.31% (w / w), approximately 0.33% (w / w), approximately 0.35% (w / w), approximately 0.37% (w / w), approximately 0.39% (w / w), approximately 0.41% (w / w), approximately 0.43% (w / w), or approximately 0.45% (w / w) of the matrix, or any concentration between the ranges defined by any two of the aforementioned values. In each or any of the embodiments described above or below, lidocaine is present in the matrix at a concentration of approximately 0.3% (w / w).

[0008] In each or any of the embodiments described above or below, the crosslinked polymer matrix further comprises uncrosslinked HA. In each or any of the embodiments described above or below, the uncrosslinked HA has a concentration of up to about 5% (w / w) in the matrix. In each or any of the embodiments described above or below, the uncrosslinked HA has a concentration of 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), or about 5% (w / w) in the matrix, or any concentration between the range defined by any two of the aforementioned values. In each or any of the embodiments described above or below, the uncrosslinked HA has a concentration of about 1% (w / w) in the matrix. In each or any of the embodiments described above or below, the uncrosslinked HA has a concentration of about 2% (w / w) in the matrix. In each or any of the embodiments described above or below, the uncrosslinked HA has a concentration of about 5% (w / w) in the matrix. In each or any of the embodiments described above or below, the non-crosslinked HA improves the extrudeability of the polymer matrix.

[0009] In each or any of the embodiments described above or below, the crosslinked polymer matrix is ​​stable for at least about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or for any time within the range defined by any two of the aforementioned values. In each or any of the embodiments described above or below, the crosslinked polymer matrix is ​​stable at a temperature of about 4°C to about 25°C. In each or any of the embodiments described above or below, the crosslinked polymer matrix is ​​stable at about 4°C. In each or any of the embodiments described above or below, the crosslinked polymer matrix is ​​stable at about 25°C. In each or any of the embodiments described above or below, the crosslinked polymer matrix is ​​stable for about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22, about 23 months, about 24 months, about 25 months, about 26 months, about 27 months, about 28 months, about 29 months, about 30 months, about 31 months, about 32 months, about 33 months, about 34 months, about 35 months, about 36 months, or at any point in time between the range defined by any two of the aforementioned values.

[0010] In each or any of the embodiments described above or below, the crosslinked polymer matrix undergoes minimal degradation over a period of about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or any time within the range defined by any two of the aforementioned values.

[0011] In each or any of the embodiments described above or below, the crosslinked polymer matrix has an elastic modulus (G') ranging from about 30 Pa to about 10,000 Pa.In each or any of the embodiments described above or below, the matrix is ​​approximately 30 Pa, approximately 40 Pa, approximately 50 Pa, approximately 60 Pa, approximately 70 Pa, approximately 80 Pa, approximately 90 Pa, approximately 100 Pa, approximately 200 Pa, approximately 300 Pa, approximately 400 Pa, approximately 500 Pa, approximately 600 Pa, approximately 700 Pa, approximately 800 Pa, approximately 900 Pa, approximately 1000 Pa, approximately 1100 Pa, approximately 1200 Pa, approximately 1300 Pa, approximately 1400 Pa, approximately 1500 Pa, approximately 1600 Pa, approximately 1700 Pa, approximately 1800 Pa, approximately 1900 Pa, approximately 20 00Pa, about 2100Pa, about 2200Pa, about 2300Pa, about 2400Pa, about 2500Pa, about 2600Pa, about 2700Pa, about 2800Pa, about 2900Pa, about 3000Pa, about 3100Pa, about 3200Pa, about 3300Pa, about 3400P a, about 3500Pa, about 3600Pa, about 3700Pa, about 3800Pa, about 3900Pa, about 4000Pa, about 4100Pa, about 4200Pa, about 4300Pa, about 4400Pa, about 4500Pa, about 4600Pa, about 4700Pa, about 4800Pa, about 4 900Pa, approximately 5000Pa, approximately 5100Pa, approximately 5200Pa, approximately 5300Pa, approximately 5400Pa, approximately 5500Pa, approximately 5600Pa, approximately 5700Pa, approximately 5800Pa, approximately 5900Pa, approximately 6000Pa, approximately 6100Pa, approximately 6200Pa, approximately 6300Pa, approximately 6400Pa, approximately 6500Pa, approximately 6600Pa, approximately 6700Pa, approximately 6800Pa, approximately 6900Pa, approximately 7000Pa, approximately 7100Pa, approximately 7200Pa, approximately 7300Pa, approximately 7400Pa, approximately 7500Pa, approximately 7600Pa, approximately 7700Pa, approximately It has an elastic modulus (G') of 7800 Pa, approximately 7900 Pa, approximately 8000 Pa, approximately 8100 Pa, approximately 8200 Pa, approximately 8300 Pa, approximately 8400 Pa, approximately 8500 Pa, approximately 8600 Pa, approximately 8700 Pa, approximately 8800 Pa, approximately 8900 Pa, approximately 9000 Pa, approximately 9100 Pa, approximately 9200 Pa, approximately 9300 Pa, approximately 9400 Pa, approximately 9500 Pa, approximately 9600 Pa, approximately 9700 Pa, approximately 9800 Pa, approximately 9900 Pa, or approximately 10000 Pa, or any elastic modulus between the range defined by any two of the aforementioned values.

[0012] In each or any of the embodiments described above or below, the crosslinked polymer matrix is ​​approximately 10 gmf, approximately 20 gmf, approximately 30 gmf, approximately 40 gmf, approximately 50 gmf, approximately 60 gmf, approximately 70 gmf, approximately 80 gmf, approximately 90 gmf, approximately 100 gmf, approximately 110 gmf, approximately 120 gmf, and approximately 130 gmf. f, about 140gmf, about 150gmf, about 160gmf, about 170gmf, about 180gmf, about 190gmf, about 200gmf, about 210gmf, 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 31 0gmf, approx. 320gmf, approx. 330gmf, approx. 340gmf, approx. 350gmf, approx. 360gmf, approx. 370gmf, approx. 380gmf, approx. 390gmf, approx. 400gmf, about 410gmf, about 420gmf, about 430gmf, about 440gmf, about 450gmf, about 460gmf, about 470gmf, about 480gmf, The compressive force values ​​are approximately 490 gmf, 500 gmf, 510 gmf, 520 gmf, 530 gmf, 540 gmf, 550 gmf, 560 gmf, 570 gmf, 580 gmf, 590 gmf, or 600 gmf, or any compressive force value within the range defined by any two of the aforementioned values. In each or any of the embodiments described above or below, the crosslinked polymer matrix has compressive force values ​​of approximately 100 gmf, 200 gmf, 300 gmf, 400 gmf, 500 gmf, or 600 gmf, or any compressive force value within the range defined by any two of the aforementioned values.

[0013] In each or any of the embodiments described above or below, the hyaluronic acid is at a concentration of approximately 5 mg / ml, approximately 6 mg / ml, approximately 8 mg / ml, approximately 10 mg / ml, approximately 12 mg / ml, approximately 14 mg / ml, approximately 16 mg / ml, approximately 18 mg / ml, approximately 20 mg / ml, approximately 22 mg / ml, approximately 24 mg / ml, approximately 26 mg / ml, approximately 28 mg / ml, approximately 30 mg / ml, approximately 32 mg / ml, approximately 34 mg / ml, or approximately 36 mg / ml, or any concentration between the range defined by any two of the aforementioned values.

[0014] In each or any of the embodiments described above or below, the collagen comprises type I collagen. In each or any of the embodiments described above or below, the collagen comprises type II collagen. In each or any of the embodiments described above or below, the collagen comprises type III collagen. In each or any of the embodiments described above or below, the collagen comprises about 1-3% type I or type III collagen. In each or any of the embodiments described above or below, the collagen comprises about 0% to about 3% type II collagen. In each or any of the embodiments described above or below, the collagen comprises about 97% to about 99% type I collagen. In each or any of the embodiments described above or below, the collagen comprises a mixture of both type I and type III collagen. In each or any of the embodiments described above or below, the matrix comprises about 0% to about 3% type III collagen.

[0015] In each or any of the embodiments described above or below, the crosslinked polymer matrix is ​​formulated for injection or application using a needle and / or cannula.

[0016] In each or any of the embodiments described above or below, the collagen has a concentration of about 1 mg / ml, 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, or any concentration between the range defined by any two of the aforementioned values. In each or any of the embodiments described above or below, the collagen has a concentration of about 3 mg / ml. In each or any of the embodiments described above or below, the collagen has a concentration of about 6 mg / ml. In each or any of the embodiments described above or below, the collagen has a concentration of about 10 mg / ml. In each or any of the embodiments described above or below, the collagen has a concentration of about 12 mg / ml.

[0017] In each or any of the embodiments described above or below, the crosslinked polymer matrix further comprises salts. In each or any of the embodiments described above or below, the crosslinked polymer matrix comprises NaCl in the range of about 50 mM to about 400 mM. In each or any of the embodiments described above or below, the crosslinked polymer matrix comprises NaCl, the NaCl having concentrations of 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, about 325 mM, about 350 mM, about 375 mM or about 400 mM, or any concentration between the range defined by any two of the aforementioned values. In each or any of the embodiments described above or below, the crosslinked polymer matrix comprises about 150 mM NaCl. In certain embodiments, the crosslinked polymer matrix does not contain salts.

[0018] In each or any of the embodiments described above or below, the crosslinked polymer matrix comprises about 0.01 M phosphate buffer, about 137 mM NaCl, and about 2.7 mM KCl.

[0019] In each or any of the embodiments described above or below, the hyaluronic acid has an average molecular weight ranging from about 20,000 daltons to about 10,000,000 daltons. In each or any of the embodiments described above or below, hyaluronic acid is approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,500,000 Daltons, approximately 2,000,000 Daltons, and approximately 2,500,000 The average molecular weight is approximately 3,000,000 daltons, approximately 3,500,000 daltons, approximately 4,000,000 daltons, approximately 4,500,000 daltons, approximately 5,000,000 daltons, approximately 5,500,000 daltons, approximately 6,000,000 daltons, approximately 6,500,000 daltons, approximately 7,500,000 daltons, approximately 8,000,000 daltons, approximately 8,500,000 daltons, approximately 9,000,000 daltons, approximately 9,500,000 daltons and / or approximately 1,000,000 daltons, or the average molecular weight is between any two of the aforementioned values. In some embodiments of each or any of the compositions of the embodiments described above or below, the hyaluronic acid has an average molecular weight of about 20,000 daltons to about 10,000,000 daltons.In each or any of the embodiments described above or below, the hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, the mixture being approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,500,000 Daltons, approximately 2,000,000 Daltons, This includes hyaluronic acid having a molecular weight of approximately 2,500,000 Daltons, approximately 3,000,000 Daltons, approximately 3,500,000 Daltons, approximately 4,000,000 Daltons, approximately 4,500,000 Daltons, approximately 5,000,000 Daltons, approximately 5,500,000 Daltons, approximately 6,000,000 Daltons, approximately 6,500,000 Daltons, approximately 7,500,000 Daltons, approximately 8,000,000 Daltons, approximately 8,500,000 Daltons, approximately 9,000,000 Daltons, approximately 9,500,000 Daltons and / or approximately 10,000,000 Daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

[0020] This disclosure also provides a composition comprising hyaluronic acid, collagen, lysine, and a buffer, the composition being an aqueous hydrogel.

[0021] In each or any of the embodiments described above or below, hyaluronic acid is crosslinked to collagen by at least one endogenous amine group on collagen and / or at least one amine group present on lysine. In each or any of the embodiments described above or below, the composition further comprises lidocaine. In each or any of the embodiments described above or below, the concentration of lidocaine in the matrix ranges from about 0.15% (w / w) to about 0.45% (w / w). In each or any of the embodiments described above or below, the concentration of lidocaine in the composition ranges from about 0.27% (w / w) to about 0.33% (w / w). In each or any of the embodiments described above or below, lidocaine is present in the composition at a concentration of approximately 0.15% (w / w), approximately 0.17% (w / w), approximately 0.19% (w / w), approximately 0.21% (w / w), approximately 0.23% (w / w), approximately 0.25% (w / w), approximately 0.27% (w / w), approximately 0.29% (w / w), approximately 0.31% (w / w), approximately 0.33% (w / w), approximately 0.35% (w / w), approximately 0.37% (w / w), approximately 0.39% (w / w), approximately 0.41% (w / w), approximately 0.43% (w / w), or approximately 0.45% (w / w), or any concentration between the ranges defined by any two of the aforementioned values.

[0022] In each or any of the embodiments described above or below, the composition further comprises non-crosslinked HA. In each or any of the embodiments described above or below, the non-crosslinked HA is present in the composition at a maximum concentration of about 5% (w / w). In each or any of the embodiments described above or below, the non-crosslinked HA is present in the composition at a concentration of about 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 range defined by any two of the aforementioned values. In each or any of the embodiments described above or below, the non-crosslinked HA is present at a concentration of about 1% (w / w) of the composition. In each or any of the embodiments described above or below, the non-crosslinked HA is present at a concentration of about 2% (w / w) of the composition. In each or any of the embodiments described above or below, the non-crosslinked HA is present at a concentration of about 5% (w / w) of the composition. In each or any of the embodiments described above or below, the non-crosslinked HA improves the extrudeability of the composition. In each or any of the embodiments described above or below, the buffer is phosphate-buffered saline.

[0023] In each or any of the embodiments described above or below, the hyaluronic acid in the composition has an average molecular weight of about 20,000 daltons to about 10,000,000 daltons.

[0024] In each or any of the embodiments described above or below, the hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, the mixture being approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,500,000 Daltons, approximately 2,000,000 Daltons, This includes hyaluronic acid having a molecular weight of approximately 2,500,000 Daltons, approximately 3,000,000 Daltons, approximately 3,500,000 Daltons, approximately 4,000,000 Daltons, approximately 4,500,000 Daltons, approximately 5,000,000 Daltons, approximately 5,500,000 Daltons, approximately 6,000,000 Daltons, approximately 6,500,000 Daltons, approximately 7,500,000 Daltons, approximately 8,000,000 Daltons, approximately 8,500,000 Daltons, approximately 9,000,000 Daltons, approximately 9,500,000 Daltons and / or approximately 10,000,000 Daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

[0025] In each or any of the embodiments described above or below, the collagen of the composition includes type I collagen. In each or any of the embodiments described above or below, the collagen includes type II collagen. In each or any of the embodiments described above or below, the collagen includes type III collagen.

[0026] In each or some of any of the above or below embodiments, the composition is stable for about 6 months, about 12 months, about 18 months, about 24 months, about 30 months or about 36 months, or for any time between ranges defined by any two of the foregoing values. In each or some of any of the above or below embodiments, the composition is stable at about 4°C. In each or some of any of the above or below embodiments, the composition is stable at about 25°C. In each or some of any of the above or below embodiments, the composition degrades very slightly at about 6 months, about 12 months, about 18 months, about 24 months, about 30 months or about 36 months, or for any time between ranges defined by any two of the foregoing values.

[0027] In each or some of any of the above or below embodiments, the composition further comprises non-crosslinked HA. In each or some of any of the above or below embodiments, the non-crosslinked HA has a concentration of up to about 5% (w / w) in the composition. In each or some of any of the above or below embodiments, the non-crosslinked HA improves the extrudability of the composition. In each or some of any of the above or below embodiments, the composition is stable for about 6 months, about 12 months, about 18 months, about 24 months, about 30 months or about 36 months, or for any time between ranges defined by any two of the foregoing values. In each or some of any of the above or below embodiments, the composition is stable at 4°C. In each or some of any of the above or below embodiments, the composition is stable at 25°C. In each or some of any of the above or below embodiments, the composition degrades very slightly at 6 months, 12 months, 18 months, 24 months, 30 months or 36 months, or for any time between ranges defined by any two of the foregoing values.

[0028] In each or any of the embodiments described above or below, the composition is used in approximately 4,000 Pa S, approximately 4,100 Pa S, approximately 4,200 Pa S, approximately 4,300 Pa S, approximately 4,400 Pa S, approximately 4,500 Pa S, approximately 4,600 Pa S, approximately 4,700 Pa S, approximately 4,800 Pa S, approximately 4,900 Pa S, approximately 5,000 Pa S, approximately 5,100 Pa S, approximately 5,200 Pa S, approximately 5,300 Pa S, approximately 5,400 Pa S, approximately 5,500 Pa S, approximately 5,600 Pa S, approximately 5,700 Pa S, approximately 5,800 Pa S, approximately 5,900 Pa S, approximately 6,000 Pa S, approximately 6,100 Pa S, approximately 6,200 Pa S, approximately 6,300 Pa S, approximately 6,400 Pa S, approximately 6,500 Pa S, approximately 6,600 Pa S, approximately 6,700 Pa S, and approximately 6,800 Pa S, approximately 6900Pa S, approximately 7000Pa S, approximately 7100Pa S, approximately 7200Pa S, approximately 7300Pa S, approximately 7400Pa S, approximately 7500Pa S, approximately 7600Pa S, approximately 7700Pa S, approximately 7800Pa S, approximately 7900Pa S, approximately 8000Pa S, approximately 8100Pa S, approximately 8200Pa S, about 8300Pa S, about 8400Pa S, about 8500Pa S, about 8600Pa S, about 8700Pa S, about 8800Pa S, about 8900Pa S, about 9000Pa S, about 9100Pa, about 9200Pa S, about 9300Pa S, about 9400Pa S, about 9500Pa S, about 9600Pa S, approx. 9700Pa S, approx. 9800Pa The viscosity is S, approximately 9900 Pa S, or approximately 10,000 Pa S, or any viscosity between the range defined by any two of the aforementioned values.

[0029] In each or some embodiments of the above or below embodiments, the composition has a tan delta parameter (G’’ / G’) of from about 0.01 to about 0.5. In each or some embodiments of the above or below embodiments, the composition has a tan delta parameter (G’’ / G’) of about 0.01, about 0.05, about 0.10, about 0.15, about 0.20, about 0.25, about 0.30, about 0.35, about 0.40, about 0.45 or about 0.50, or any tan delta parameter between ranges defined by any two of the foregoing values. In some embodiments of each or some compositions of the above or below embodiments, the buffer solution contains phosphate buffered saline.

[0030] The present disclosure also provides a method for crosslinking hyaluronic acid and collagen. The method includes dissolving collagen, hyaluronic acid and lysine in an aqueous solution to form a pre-reaction aqueous solution, wherein the pre-reaction aqueous solution has a pH of 4 - 6, forming the pre-reaction aqueous solution, and preparing a second solution containing a water-soluble carbodiimide and N-hydroxysuccinimide or N-hydroxysulfosuccinimide, adding the second solution to the pre-reaction aqueous solution to form a crosslinking reaction mixture, and reacting the crosslinking reaction mixture by crosslinking hyaluronic acid and collagen with lysine, wherein hyaluronic acid is crosslinked to collagen by at least one endogenous amine group on collagen and / or at least one amine group on lysine, HA and collagen are slightly decomposed, and the structures of HA and collagen remain intact, thereby forming a crosslinked polymer matrix. In each or some embodiments of the above or below embodiments, the pre-reaction aqueous solution includes a pH of about 4.0, about 4.5, about 5.0, about 5.5 or about 6, or any pH between ranges defined by any two of the foregoing values. In each or some embodiments of the above or below embodiments, the method further includes applying an activator including triazole, fluorinated phenol, succinimide or sulfosuccinimide.

[0031] In each or any of the embodiments described above or below, the method further comprises adding lidocaine to a crosslinked polymer matrix. In each or any of the embodiments described above or below, the concentration of lidocaine in the matrix ranges from about 0.15% (w / w) to about 0.45% (w / w). In each or any of the embodiments described above or below, the concentration of lidocaine in the matrix ranges from about 0.27% (w / w) to about 0.33% (w / w). In each or any of the embodiments described above or below, lidocaine is present in a concentration of approximately 0.15% (w / w), approximately 0.17% (w / w), approximately 0.19% (w / w), approximately 0.21% (w / w), approximately 0.23% (w / w), approximately 0.25% (w / w), approximately 0.27% (w / w), approximately 0.29% (w / w), approximately 0.31% (w / w), approximately 0.33% (w / w), approximately 0.35% (w / w), approximately 0.37% (w / w), approximately 0.37% (w / w), approximately 0.39% (w / w), approximately 0.41% (w / w), approximately 0.43% (w / w), or approximately 0.45% (w / w) of the matrix, or any concentration between the ranges defined by any two of the aforementioned values. In each or any of the embodiments described above or below, lidocaine is present in the matrix at a concentration of approximately 0.3% (w / w).

[0032] In each or any of the embodiments described above or below, the method further includes adding non-crosslinked HA to a crosslinked polymer matrix. In each or any of the embodiments described above or below, the non-crosslinked HA is present in the crosslinked polymer matrix at a maximum concentration of about 5% w / w. In each or any of the embodiments described above or below, the non-crosslinked HA is added to the matrix to a concentration of about 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 range defined by any two of the aforementioned values. In each or any of the embodiments described above or below, the non-crosslinked HA is added to the matrix to a concentration of about 1% (w / w). In each or any of the embodiments described above or below, the non-crosslinked HA is added to the matrix to a concentration of about 3% (w / w). In each or any of the embodiments described above or below, non-crosslinked HA is added to the matrix to a concentration of about 5% (w / w).

[0033] In each or any of the embodiments described above or below, the reaction step takes place at a temperature of about 4°C to about 35°C. In each or any of the embodiments described above or below, the reaction step takes place at a temperature of about 4°C, about 5°C, about 7°C, about 9°C, about 11°C, about 13°C, about 15°C, about 17°C, about 19°C, about 21°C, about 23°C, about 25°C, about 27°C, about 29°C, about 31°C, about 33°C, about 35°C, or any temperature between the range defined by any two of the aforementioned values. In each or any of the embodiments described above or below, the reaction step takes place at about 4°C or about 22°C.

[0034] In each or any of the embodiments described above or below, the method further comprises purifying the crosslinked polymer matrix, the purification step being carried out by dialysis. In each or any of the embodiments described above or below, the dialysis is carried out at a temperature of about 2°C to about 30°C. In each or any of the embodiments described above or below, the dialysis is carried out at a temperature of about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C, or any temperature between the range defined by any two of the aforementioned values. In each or any of the embodiments described above or below, the purification process is carried out at a temperature of about 2°C to about 8°C. In each or any of the embodiments described above or below, the purification process is carried out at a temperature of about 2°C, about 4°C, about 6°C, about 8°C, or any temperature within the range defined by any two of the aforementioned values.

[0035] In each or any of the embodiments described above or below, the method is carried out below room temperature. In each or any of the embodiments described above or below, the method is carried out 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, about 24°C, about 26°C, about 28°C, about 30°C, about 32°C, about 34°C, or about 36°C, or at a temperature between the range defined by any two of the aforementioned values.

[0036] In each or any of the embodiments described above or below, the pH of the crosslinking reaction mixture is about 4 to about 6.0. In each or any of the embodiments described above or below, the pH of the crosslinking reaction mixture is about 4.0, about 4.5, about 5.0, about 5.5, or about 6.0, or any pH between the range defined by any two of the aforementioned values.

[0037] In each or any of the embodiments described above or below, the pre-reaction solution contains a salt containing sodium chloride at a concentration of 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, or about 400 mM, or any concentration between the range defined by any two of the aforementioned values ​​in the crosslinking reaction mixture.

[0038] In each or any of the embodiments described above or below, the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the crosslinking reaction mixture at a concentration of about 20 mM to about 200 mM. In each or any of the embodiments described above or below, 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 or about 200 mM, or any concentration between the ranges defined by any of the aforementioned values.

[0039] In some embodiments of each or any of the embodiments described above or below, the molar-to-molar ratio of repeating units of water-soluble carbodiimide to repeating units of hyaluronic acid is about 0.5 to about 2.0. In some embodiments of each or any of the embodiments described above or below, the molar-to-molar ratio of repeating units of water-soluble carbodiimide to repeating units of hyaluronic acid is 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, or about 2.0.

[0040] In each or any of the embodiments described above or below, the molar-to-molar (lysine repeating unit:HA repeating unit) ratio of lysine to hyaluronic acid is approximately 0.01 to approximately 0.6. In each or any of the embodiments described above or below, the molar-to-molar (lysine repeating unit:HA repeating unit) ratio of lysine to hyaluronic acid is approximately 0.01, approximately 0.02, approximately 0.03, approximately 0.04, approximately 0.05, approximately 0.06, approximately 0.07, approximately 0.08, approximately 0.09, approximately 0.10, approximately 0.11, approximately 0.12, approximately 0.13, approximately 0.14, approximately 0.15, approximately 0.16, approximately 0.17, approximately 0.18, approximately 0.19, approximately 0.2, approximately 0.21, approximately 0.22, approximately 0.23, approximately 0.2 4. Approximately 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, or 0.6.

[0041] In each or any of the embodiments described above or below, the method further comprises sterilizing the crosslinked polymer matrix, which includes transferring the crosslinked polymer matrix to a container for steam sterilization and sterilizing the hydrogel by steam sterilization. In each or any of the embodiments described above or below, the container is a syringe.

[0042] In each or any of the embodiments described above or below, the method further comprises dialysis of a crosslinked polymer matrix, wherein the dialysis is performed through a membrane having a molecular weight cutoff of about 1,000 daltons to about 100,000 daltons, and the dialysis is performed before sterilization. In each or any of the embodiments described above or below, the dialysis is performed in phosphate-buffered saline.

[0043] In each or any of the embodiments described above or below, the hyaluronic acid in the pre-reaction solution is hydrated for at least 60 minutes before the second solution is added.

[0044] In each or any of the embodiments described above or below, the crosslinking reaction mixture is carried out for about 16 to about 24 hours. In each or any of the embodiments described above or below, the crosslinking reaction mixture is carried out for about 16 hours, about 18 hours, about 20 hours, about 22 hours, or about 24 hours, or any time between the range defined by any two of the aforementioned values.

[0045] In each or any of the embodiments described above or below, the crosslinking reaction takes place at a temperature between approximately 2°C and approximately 35°C. In each or any of the embodiments described above or below, the crosslinking reaction takes place at approximately 2°C, approximately 3°C, approximately 4°C, approximately 5°C, approximately 7°C, approximately 9°C, approximately 11°C, approximately 13°C, approximately 15°C, approximately 17°C, approximately 19°C, approximately 21°C, approximately 23°C, approximately 25°C, approximately 27°C, approximately 29°C, approximately 31°C, approximately 33°C, approximately 35°C, or any temperature within the range defined by any two of the aforementioned values.

[0046] In each or any of the embodiments described above or below, the crosslinking reaction takes place at a temperature between approximately 2°C and approximately 8°C. In each or any of the embodiments described above or below, the crosslinking reaction takes place at a temperature of approximately 2°C, approximately 4°C, approximately 6°C, or approximately 8°C, or any temperature within the range defined by any two of the aforementioned values.

[0047] This disclosure also provides a crosslinked polymer matrix prepared by any one of the embodiments described above or below.

[0048] Furthermore, the present disclosure provides a method for improving the aesthetics of human anatomical features. The method comprises injecting a composition into human tissue to improve the aesthetics of the anatomical features, the composition comprising a crosslinked polymer matrix comprising hyaluronic acid, lysine, and collagen, wherein the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine.

[0049] This disclosure also provides a method for improving the appearance of an individual. The method involves injecting a composition into the tissue of an individual at an injection site to improve the aesthetics of anatomical features, wherein infiltrating cells from the tissue are integrated into the composition at the injection site, and depositing new collagen within the composition, wherein the composition comprises a cross-linked polymer matrix comprising hyaluronic acid, lysine, and collagen, and the hyaluronic acid is cross-linked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine, and depositing new collagen, wherein the tissue into which the composition has been injected has tissue integration, collagen deposition, and angiogenesis. In each or any of the embodiments described above or below, the composition is injected into the nasolabial folds. In each or any of the embodiments described above or below, the method improves the symmetry between facial features. In each or any of the embodiments described above or below, the method increases and restores volume to facial features. In each or any of the embodiments described above or below, the method restores volume to the cheeks / or temples. In some embodiments, the method increases, corrects, restores, or imparts volume to the chin, jawline, or nasolabial folds. In each or any of the embodiments described above or below, the composition is injected into the tear trough of a solid. In each or any of the embodiments described above or below, the composition is injected into an area including skin atrophy and / or fat body atrophy. In each or any of the embodiments described above or below, the method imparts a natural look, feel, and movement to the injected tissue, and the composition results in increased collagen infiltration from the surrounding tissue at the injection site. In each or any of the embodiments described above or below, the duration of the composition is extended as a result of tissue integration into the injection site. In each or any of the embodiments described above or below, the method improves the hydration and elasticity of the skin surrounding the injection site.

[0050] The disclosure also provides a method for increasing tissue infiltration in a dermal filler graft by collagen deposition. The method involves injecting a composition into the tissue of an individual to create a dermal filler depot containing the composition, wherein the composition comprises a crosslinked polymer matrix comprising hyaluronic acid, lysine, and collagen, and the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine, and the method involves injecting a composition into the tissue of an individual, wherein cells from the surrounding tissue infiltrate the dermal filler depot containing the composition, the cells integrate with the composition and deposit new collagen into the composition, thereby creating infiltrated tissue within the composition, and blood vessels connect the infiltrated tissue within the composition to the blood supply of the individual's body.

[0051] In each or any of the embodiments described above or below, the collagen includes type I collagen and / or type III collagen.

[0052] In each or any of the embodiments described above or below, the composition comprises about 18 mg / ml of hyaluronic acid, about 20 mg / ml of hyaluronic acid, about 22 mg / ml of hyaluronic acid, about 24 mg / ml of hyaluronic acid, about 26 mg / ml of hyaluronic acid, about 28 mg / ml of hyaluronic acid, or about 30 mg / ml of hyaluronic acid, or has any concentration between the range defined by any two of the aforementioned values. In each or any of the embodiments described above or below, the composition comprises about 13 mg / ml of hyaluronic acid.

[0053] In each or any of the embodiments of the above or below embodiments of the method, the composition or polymer matrix further comprises lidocaine. In each or any of the embodiments of the above or below embodiments, the concentration of lidocaine in the matrix is ​​in the range of 0.15% (w / w) to 0.45% (w / w). In each or any of the embodiments of the above or below embodiments, the concentration of lidocaine in the matrix is ​​in the range of 0.27% (w / w) to 0.33% (w / w). In each or any of the embodiments described above or below, lidocaine is present in a concentration of approximately 0.15% (w / w), approximately 0.17% (w / w), approximately 0.19% (w / w), approximately 0.21% (w / w), approximately 0.23% (w / w), approximately 0.25% (w / w), approximately 0.27% (w / w), approximately 0.29% (w / w), approximately 0.31% (w / w), approximately 0.33% (w / w), approximately 0.35% (w / w), approximately 0.37% (w / w), approximately 0.37% (w / w), approximately 0.39% (w / w), approximately 0.41% (w / w), approximately 0.43% (w / w), or approximately 0.45% (w / w) of the matrix, or any concentration between the ranges defined by any two of the aforementioned values. In each or any of the embodiments described above or below, lidocaine is present in the matrix at a concentration of approximately 0.3% (w / w).

[0054] In each or any of the embodiments of the above or below embodiments of this method, the composition or polymer matrix further comprises non-crosslinked HA. In each or any of the embodiments of the above or below embodiments, the non-crosslinked HA is present in the composition or matrix at a maximum concentration of about 5% (w / w). In each or any of the embodiments of the above or below embodiments of this method, the non-crosslinked HA is present in the composition or matrix at a concentration of about 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 range defined by any two of the aforementioned values. In each or any of the embodiments of the above or below embodiments of this method, the non-crosslinked HA is present in the composition or matrix at a concentration of about 1% (w / w). In each or any of the embodiments of the above or below embodiments of this method, the non-crosslinked HA is present in the composition or matrix at a concentration of about 2% (w / w). In each or any of the embodiments of the above or below embodiments of this method, the non-crosslinked HA is present in the composition or matrix at a concentration of about 5% (w / w). [Brief explanation of the drawing]

[0055] [Figure 1] Figure 1 shows in vitro activated cells in close contact with an HA / collagen cross-linked hydrogel formulation.

[0056] [Figure 2A-2D]Figure 2 shows the actin filament alignment index (2A), cell length-to-width ratio (2B), and convex-to-cell area ratio (2C) of fibroblasts cultured on HA-only hydrogels or HA / collagen cross-linked hydrogels. HA-collagen hydrogel (24:6 HA:collagen) (Formulation X), formulated at a hydration temperature of 5°C, exhibits significantly higher actin filament alignment index, cell length-to-width ratio, and convex-to-cell area ratio compared to a similar hydrogel formulated at a hydration temperature of 22°C (Formulation VI). *p<0.05 was observed by ANOVA using Tukey post-hoc analysis. HA-collagen hydrogel containing 20:6 HA:collagen, formulated at a hydration temperature of 5°C, exhibits particularly high actin filament alignment index, cell length-to-width ratio, and convex-to-cell area ratio compared to the HA-only gel (Formulation XIX). *p<0.05 was observed by ANOVA using Tukey post-hoc analysis. Figure 2D shows the ranking of HA-collagen hydrogels as a function of Euclidean distance (in three-dimensional space including actin filament alignment index, cell length-to-width ratio, and convex hull-to-cell area ratio) obtained from HA-only hydrogels. An increase in Euclidean distance indicates improved cell spreadability and adhesion compared to low-adhesion HA-only gels. Overall, hydrogels formulated at a hydration temperature of 5°C exhibit greater Euclidean distances than HA-only gels.

[0057] [Figure 3] Figure 3 shows the lift profiles of Formulation I, Formulation II, and Formulation III (mean + / - SEM).

[0058] [Figure 4] Figure 4 shows the lift profiles of formulation XV versus formulation III (mean + / - SEM).

[0059] [Figure 5] Figure 5 shows the lift profiles of Formulation II vs. Formulation XV vs. Formulation XVI (mean + / - SEM).

[0060] [Figure 6]Figure 6 shows the tissue integration of hydrogels as HA concentration increases with 4 mg / mL collagen. (6A) H&E, (6B) Collagen 1a, (6C) Vimentin, (6D) Procollagen 1, (6E) CD31. H&E staining demonstrates the decrease in tissue proliferation with increasing HA concentration. As shown, dark staining of collagen 1a is observed with the 13 mg / mL HA formulation (Formulation I). The 20 mg / mL HA formulation shows a decrease in collagen 1a fillers, and the 25 mg / mL HA formulation shows broad areas without collagen 1a deposition. Vimentin-positive fibroblast / fibrocystic infiltration was observed with all formulations, and the degree of infiltration decreased with increasing HA concentration. Staining of procollagen 1 appeared to be reduced in the low HA formulation (Formulation I) compared with the 20 mg / mL and 25 mg / mL HA formulations. The presence of procollagen I staining in the 20 mg / mL and 25 mg / mL HA preparations may indicate that collagen deposition continues over time. Vascular angiogenesis within the hydrogel bolus was observed in the 20 mg / mL and 25 mg / mL HA preparations, as indicated by positive CD31 staining. CD31 staining was not performed in the 13 mg / mL HA preparation.

[0061] [Figure 7]Figure 7 shows tissue integration in hydrogels prepared with a higher proportion of low molecular weight HA than high molecular weight HA. (A) Colloidal iron, (B) Collagen 1a, (C) Vimentin, (D) Procollagen 1, (E) CD31. Colloidal iron staining demonstrates tissue integration at the periphery of the Formulation XV hydrogel and robust tissue integration throughout the Formulation XVI gel bolus. High-density collagen 1a deposition was observed on the back of the Formulation XV bolus, but this deposition prevented the gel from being completely filled. Fine chains of collagen 1a-positive tissue were observed throughout the Formulation XVI hydrogel. Vimentin-positive fibroblast / fibrous cell infiltration was observed in all formulations. Vimentin-positive cells infiltrated the majority of the Formulation XVI bolus. Procollagen 1 staining was present in both Formulation XV and Formulation XVI gels. The presence of procollagen 1 staining may indicate that collagen deposition continued over time. Angiogenesis within the hydrogel bolus was observed in both formulations (arrowhead). Formulation XVI showed the most robust angiogenesis throughout the entire bolus.

[0062] [Figure 8] Figure 8 shows the tissue integration of hydrogels containing 24 mg / mL HA and 6 mg / mL collagen, prepared at room temperature (Formulation VI) and 5°C (Formulation X) hydration temperatures. Staining of collagen 1a shows that fine collagen is distributed around the Formulation VI hydrogel, and deposition around the hydrogel particles is limited. Staining of collagen 1a in Formulation X gel shows that robust collagen is deposited around the hydrogel, and high-density collagen is deposited around the hydrogel particles.

[0063] [Figure 9]Figure 9 shows hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining of hydrogel explants 12 weeks after subcutaneous injection into rats. H&E staining shows tissue deposition closely associated with hydrogel particles in Formulation XIX, while sparse tissue deposition is observed around large hydrogel deposits in HA-only hydrogels. Vimentin staining shows more extensive fibrocytic / fibroblast infiltration into Formulation XIX hydrogel boluses than into HA-only gels. Formulation XIX boluses are also more highly angiogenic than HA-only boluses, as indicated by extensive CD31-positive labeling. The enhanced cellular infiltration and angiogenesis in Formulation XIX boluses allow for denser and more uniform tissue deposition within the bolus, as indicated by collagen I labeling.

[0064] [Figure 10] Figure 10 shows that immunohistochemical (IHC) quantification of the positively stained area demonstrates increased levels of vimentin (fibroblasts), collagen I, and CD31 (vascular tissue) in the bolus of formulation XIX hydrogel after 12 weeks of subcutaneous implantation in rats, compared to hydrogel with HA alone.

[0065] [Figure 11] Figure 11 shows the lifting capacity in a rat subcutaneous injection model. Formulation XIX exhibits similar lifting capacity to a 24 mg / mL HMW HA-only gel for 4 to 12 weeks. As shown, this formulation exhibits improved tissue integration while maintaining similar lifting capacity to the HA-only gel.

[0066] [Figure 12]Figure 12 shows the lifting capacity data of the cross-linked HA-collagen gel at 28 weeks. The lifting capacity of the HA-only gel steadily decreased over time. The lifting capacity of the HA-collagen gel remained stable from 12 to 28 weeks. Without limiting this disclosure, this may indicate that the HA-collagen gel has a longer duration of therapeutic effect than the HA-only gel. The extended duration may be a result of better integration and tissue proliferation. As shown, formulation XIX has significantly better tissue proliferation than the HA-only gel, in particular.

[0067] [Figure 13] Figure 13 shows the differences observed before and after autoclaving of 24:6 HA vs. collagen gels. The 24:6 HA vs. collagen gels (run repeatedly as Sample 1 and Sample 2) generally showed lower cell viability. However, both the autoclaved and non-autoclaved formulations showed higher cell viability than the HA-only gel. As shown, the experiment was performed on replicas of gels containing 24:6 HA vs. collagen (Sample 1 and Sample 2) before (B) and after (BA) autoclaving. A slight but significant difference in cell viability was observed in Sample 1, but no significant difference was observed in Sample 2 after autoclaving.

[0068] [Figure 14]Figure 14 shows H&E staining of gel filler boluses after 4 weeks of subcutaneous transplantation in a rat model. Formulation XXII (A; 20 mg HA: 4 mg collagen, hydrated at 5°C) shows similar or better tissue integration than Formulation XIX (B; 20 mg HA: 6 mg collagen, hydrated at 5°C). Blinded scoring by pathologists further demonstrates improved integration of Formulation XXII, with a score of 2.33 compared to Formulation XIX's score of 1.83. Higher scores indicate better tissue integration. In contrast, Formulation XX (C; 20 mg HA: 10 mg collagen, synthesized at 25°C) shows inferior tissue integration compared to Formulation XXII and Formulation XIX. The tissue integration score for Formulation XX is 1.13. This result further demonstrates that tissue integration does not follow a linear trend in collagen concentration. Instead, there are optimal synthesis conditions and collagen concentrations that achieve enhanced tissue response.

[0069] [Figure 15] Figure 15 shows the in vitro cell viability of human dermal fibroblasts cultured with HA-only collagen and HA-collagen gels (formulations XXII and XXIII).

[0070] [Figure 16] Figure 16 shows an image analysis of the length-to-width ratio of human dermal fibroblasts cultured with HA-only collagen or HA-collagen gel (formulations XXII and XXIII).

[0071] [Figure 17] Figure 17 shows the scoring of tissue integration of gelbolus after 4 weeks of subcutaneous transplantation in rats, comparing formulations XXII and XXIII or HA alone as controls.

[0072] [Figure 18] Figure 18 shows the staining of collagen 1a in integrated tissues of formulations XXII and XXIII compared to gels containing only HA.

[0073] [Figure 19] Figure 19 shows the quantification of the positive area percentage of collagen 1a staining within hydrogel bolus after subcutaneous transplantation of formulation XXII into rats for 4 weeks.

[0074] [Figure 20] Figure 20 shows confocal micrographs of human dermal fibroblasts cultured for 48 hours on gels containing HA only, formulation XXII, or formulation XXIII. The samples were stained for HA-binding protein, Hoechst, and wheat germ agglutinin (cell membrane).

[0075] [Figure 21] Figure 21 shows immunohistochemical analyses of tissue responses to HA alone and HA-collagen hydrogel (formulations XXII and XXIII) after 4 weeks of subcutaneous transplantation in rats.

[0076] [Figure 22] Figure 22 shows the 52-week lift capacity data for formulation XXII compared to a gel containing only HA.

[0077] [Figure 23] Figure 23 shows the 26-week lift capacity data for formulation XXIII compared to a gel containing only HA.

[0078] [Figure 24] Figure 24 shows confocal micrographs of human dermal fibroblasts cultured for 48 hours on gels containing HA only, formulation XXVI, or formulation XXV. The samples were stained for HA-binding protein, Hoechst, and wheat germ agglutinin (cell membrane).

[0079] [Figure 25] Figure 25 shows two-photon imaging of second-harmonic generation signals (white) and tissue autofluorescence (green) in rats treated with HA alone, or with subcutaneous bolus injection of formulation XXV or formulation XXIII, after 12 weeks.

[0080] [Figure 26]Figure 26 shows the immunohistochemical analysis of the tissue response to formulation XXV after 4 weeks of subcutaneous transplantation in rats.

[0081] [Figure 27] Figure 27 shows the immunohistochemical analysis of the tissue response to formulation XXVI in rats after 4 weeks of subcutaneous transplantation.

[0082] [Figure 28] Figure 28 shows the 30-week lift capacity data for formulations XXV and XXVI compared to a gel containing only HA. [Modes for carrying out the invention]

[0083] The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0084] If the definitions of terms used herein deviate from the commonly used meanings of those terms, the applicant intends to use the definitions presented herein unless otherwise specifically indicated.

[0085] This specification discloses a crosslinked polymer matrix, a composition containing the crosslinked polymer matrix, a method for producing the crosslinked polymer matrix, and a method for improving the appearance of a solid. The filler containing the crosslinked polymer matrix described in this embodiment has immediate filling and lifting properties after injection, followed by tissue integration at the injection site, which can result in a long-term and natural effect.

[0086] Advantageously, the crosslinking method provides an HA / collagen material with adjustable physical properties that produce a variety of filling and lifting characteristics, thereby enabling the injection of such material into various tissue depths, various areas of the face, and for various purposes (volume enhancement, severe wrinkles, fine wrinkles, etc.). Furthermore, this synthesis method allows for control of cell infiltration from surrounding tissue into the injected bolus through the covalent incorporation of collagen into the crosslinked hydrogel. Moreover, this crosslinking can also protect the collagen from denaturation. The combination of lifting and tissue integration properties is expected to result in superior facial aesthetic enhancement through a natural feel, appearance, and movement. As described herein, this is a method for improving the quality of fillers, resulting in a superior hybrid material that can surpass previous collagen fillers and current HA fillers.

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains.

[0088] In the context describing the present invention (particularly in the context of the following claims), the terms “a,” “an,” “the,” and similar references should be interpreted as encompassing both singular and plural unless otherwise specifically indicated herein or unless otherwise clearly contradicted by the context. As used herein when referring to measurable values, “about” means encompassing a variation of +20% or +10%, more preferably +5%, even more preferably +1%, and even more preferably +0.1% from the specified value.

[0089] As used herein, unless the context requires otherwise, the term “comprise,” and variations such as “comprising,” “comprises,” and “comprised,” are not intended to exclude further additives, components, integers, or processes.

[0090] A "crosslinked polymer matrix" refers to a matrix formed by crosslinking HA and collagen. HA and collagen can be crosslinked by activating the natural carboxylic acid portions of HA and collagen so that these portions can react with endogenous amine groups present on the collagen. Furthermore, lysine may be added as a carboxylic acid / diamine crosslinking agent to further promote crosslinking between HA and collagen. Adding lysine in this way allows for adjustment of the physical properties of the resulting hydrogel. The crosslinked polymer matrix can be used in compositions or formulations for medical aesthetics (e.g., as aesthetic fillers or dermal fillers).

[0091] As used herein, "hyaluronic acid" or "hyaluronan" refers to a non-sulfated glycosaminoglycan widely distributed throughout the human body in connective tissue, epithelial tissue, and nerve tissue. Hyaluronan is abundant in various layers of the skin and has multiple functions, such as ensuring good hydration, assisting in the organization of the extracellular matrix, and acting as a filler material, and is involved in tissue repair mechanisms.

[0092] As used herein, “collagen” is the primary structural protein of the extracellular space in various connective tissues in the body. Collagen forms fibrils and sheets that support tensile loads. Collagen also has specific integrin binding sites for cell adhesion and is known to promote cell adhesion, migration, and proliferation. Collagen may be positively charged due to its high content of basic amino acid residues such as arginine, lysine, and hydroxylysine. More than 90% of the collagen in the human body is type I collagen. Type III collagen is the main component of reticular fibers and can generally be found together with type I collagen. Those skilled in the art will understand that collagen can be supplied from commercial sources. In each or any of the embodiments described above or below, the collagen material provided may have a mixture of collagen that is about 97% to about 99% type I collagen and the remaining collagen that is about 1% to 3% type III collagen.

[0093] In each or any of the embodiments described above or below, the collagen is cross-linked collagen. In each or any of the embodiments described above or below, the collagen is uncross-linked collagen.

[0094] In each or any of the embodiments described above or below, HA is crosslinked with an amine and may have two or more crosslinks via lysine on either collagen or HA, or by another amine group.

[0095] "Elastic modulus," also known as the elastic coefficient, is a quantity that measures the resistance of an object or substance that deforms elastically (i.e., not permanently) when stress is applied.

[0096] As used herein, “compressive force” refers to the application of force, pressure, or exertion to an object that causes the object to be compressed, crushed, or compressed.

[0097] As used herein, "sterilization" refers to submitting materials to a sterilization process that can result in the death of microorganisms in the materials. Methods for disinfection and sterilization may be physical, chemical, and physicochemical means.

[0098] For materials such as hydrogels, sterilization can be achieved under less aggression conditions, such as shorter sterilization times, lower temperatures, and lower dose exposures.

[0099] Sterilization may include, but is not limited to, steam heat, dry heat, and / or ionizing radiation.

[0100] As a result of being subjected to a sterilization process, sterilized products such as hydrogels may be formed. Such sterilization processes can be found in Chitre et al. (U.S. Patent Application Publication 2014 / 0011980), Chitre et al. (U.S. Patent Application Publication 2018 / 0147307), and Chitre et al. (U.S. Patent Application Publication 2016 / 0101200).

[0101] In one embodiment, the composition or matrix contains an anesthetic. Examples of anesthetics, but not limited to, include benzocaine, chloroprocaine, procaine, propalacaine, tetracaine, amylocaine, oxybuprocaine, articaine, bupivacaine, dibucaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, sameridine, tonicaine, and cincocaine.

[0102] method Hyaluronic acid and collagen can be co-crosslinked with 1-ethyl-3-(N,N'-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the naturally occurring carboxylic acid moieties present on HA and collagen for reaction with endogenous amine groups present on collagen. In one embodiment, lysine is added as an additional diamine crosslinking agent to further enhance the chemical modification of HA and collagen and to adjust the physical properties of the resulting hydrogel.

[0103] By adding lysine, independent adjustment of crosslinking can be made, allowing for the modification of the physical properties of the hydrogel without changing the HA:collagen composition or the amount of activating reagent. In one embodiment, the crosslinking reaction is carried out under mild pH and temperature conditions (pH 5.5 and 4-25°C) rather than the higher pH and temperature required for BDDE crosslinking, which is standard in HA dermal fillers. Using this method, it has been shown that, surprisingly, the HA and fragile collagen protein components are only slightly degraded during crosslinking, as shown in the example, while their structures remain almost intact.

[0104] In one embodiment, hyaluronic acid is hydrated for at least 60 minutes before the crosslinking step with collagen. In a further embodiment, hyaluronic acid is hydrated at a temperature lower than room temperature. In yet another embodiment, hyaluronic acid is hydrated 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 20°C, about 22°C, about 24°C, or any temperature between the range defined by any two of the aforementioned values. In one embodiment, hyaluronic acid is hydrated at a temperature higher than room temperature. Thus, the method for preparing the hydrogel can be adjusted to control hydrogel properties, such as the tan del parameter (G'' / G').

[0105] In one embodiment, the collagen may be provided in the form of a solution having an acidic pH, and the collagen is soluble.

[0106] In one embodiment, collagen is provided as pre-fibrotic collagen, and the collagen is treated before crosslinking. In one embodiment, the pre-fibrotic collagen is present in a basic solution. In one embodiment, collagen is provided as soluble collagen, and the collagen is present in an acidic solution. In one embodiment, the pre-fibrotic collagen is in a solution having a neutral pH.

[0107] In one embodiment, the crosslinking reaction is carried out at a pH of 4.0, 5.0, 5.5, 6.0, 6.5, or 7.0, or at any pH within the range defined by any two of the aforementioned values.

[0108] The physical properties of the hydrogel may depend on the concentrations of HA and collagen, the molecular weight of HA, the concentration of EDC, the EDC / NHS ratio, temperature, pH, salt / buffer concentration, and lysine concentration. In one embodiment, elastic modulus (G') values ​​ranging from 30 Pa to approximately 10,000 Pa can be obtained. In one embodiment, the elastic modulus depends on formulation and synthesis parameters, which can be adjusted. In one embodiment, the compressive force values ​​range from 20 gmf to over 500 gmf, and the swelling of the hydrogel may range from 1.5 to 5 times the original gel volume. Based on the broad spectrum of physical properties obtained, various HA / collagen compositions can find applications as skin fillers for various facial applications.

[0109] In one embodiment of the crosslinking reaction, this method includes stopping the crosslinking process.

[0110] As described in the embodiments herein, formulations with low G' and compressive force values ​​and minimal swelling can be applied as fillers for very surface wrinkles or as injectable skin texture improvers, while more robust formulations with high G' and compressive force and greater swelling can be used for correcting moderate to severe wrinkles and for facial volume and contouring.

[0111] In one embodiment, a method for filling fine wrinkles includes the step of supplying a composition having a low G' and compressive force value to a patient. In another embodiment, a method is provided for correcting moderate to severe wrinkles and performing facial volume-forming / contouring treatment, and this method includes providing a composition to a patient who requires it, having a higher G' and compressive force.

[0112] The aim is to increase the HA concentration of the crosslinked polymer matrix. Increasing the HA concentration can yield hydrogels with, for example, higher G', higher compressive force values, and higher opacity. Increased opacity can also reduce the possibility of blue discoloration at the injection site due to the Tyndall effect. A strong crosslinked polymer matrix can provide the necessary force to lift the tissue and resist subsequent deformation, which can result in the desired straightening and appearance. Thus, high lifting ability may require high matrix strength. The modulus of elasticity (G') can represent the stiffness of the matrix and the ease of extrusion of the matrix.

[0113] The modulus of elasticity may be a function of the hyaluronic acid concentration. In one embodiment, the HA concentration is in the range of 13 mg / ml to 28 mg / ml. In one embodiment, the composition is approximately 30 Pa, approximately 40 Pa, approximately 50 Pa, approximately 60 Pa, approximately 70 Pa, approximately 80 Pa, approximately 90 Pa, approximately 100 Pa, approximately 200 Pa, approximately 300 Pa, approximately 400 Pa, approximately 500 Pa, approximately 600 Pa, approximately 700 Pa, approximately 800 Pa, approximately 900 Pa, approximately 1000 Pa, approximately 1100 Pa, approximately 1200 Pa, approximately 1300 Pa, approximately 1400 Pa, approximately 1500 Pa, approximately 1600 Pa, approximately 1700 Pa, approximately 1800 Pa, approximately 1900 Pa, approximately 2000 Pa, approximately 2100 Pa, and approximately 2200 Pa. , about 2300Pa, about 2400Pa, about 2500Pa, about 2600Pa, about 2700Pa, about 2800Pa, about 2900Pa, about 3000Pa, about 3100Pa, about 3200Pa, about 3300Pa, about 3400Pa, about 3500Pa, about 3600P a, about 3700Pa, about 3800Pa, about 3900Pa, about 4000Pa, about 4100Pa, about 4200Pa, about 4300Pa, about 4400Pa, about 4500Pa, about 4600Pa, about 4700Pa, about 4800Pa, about 4900Pa, about 5000P a, about 5100Pa, about 5200Pa, about 5300Pa, about 5400Pa, about 5500Pa, about 5600Pa, about 5700Pa, about 5800Pa, about 5900Pa, about 6000Pa, about 6100Pa, about 6200Pa, about 6300Pa, about 6400 Pa, about 6500Pa, about 6600Pa, about 6700Pa, about 6800Pa, about 6900Pa, about 7000Pa, about 7100Pa, about 7200Pa, about 7300Pa, about 7400Pa, about 7500Pa, about 7600Pa, about 7700Pa, about 7800 It has an elastic modulus of Pa, G' of approximately 7900 Pa, approximately 8000 Pa, approximately 8100 Pa, approximately 8200 Pa, approximately 8300 Pa, approximately 8400 Pa, approximately 8500 Pa, approximately 8600 Pa, approximately 8700 Pa, approximately 8800 Pa, approximately 8900 Pa, approximately 9000 Pa, approximately 9100 Pa, approximately 9200 Pa, approximately 9300 Pa, approximately 9400 Pa, approximately 9500 Pa, approximately 9600 Pa, approximately 9700 Pa, approximately 9800 Pa, approximately 9900 Pa, or approximately 10000 Pa, or any modulus of elasticity between the range defined by any two of the aforementioned values.

[0114] In one embodiment, a higher G' value is desirable. A higher G' value can also be obtained by increasing the EDC:HA ratio or the EDC:NHS ratio. Mixtures of hyaluronic acid components having different molecular weights are intended, which may affect the G' and compressive force values. For example, in the formulation of HA:collagen, a lower hydration temperature may result in a higher G' value, lower swelling, and decreased opacity (increased translucency). Using these synthesis parameters and results, formulations of HA-collagen with desired physical properties can be synthesized.

[0115] Collagen concentration can also affect the physical properties. At a given hydration temperature, increasing collagen concentration can lead to increased opacity, a rise in G', and decreased swelling. Increased opacity can lead to a decrease in the Tyndall effect of the filler. The physical and optical properties of HA-collagen hydrogels depend on the degree to which collagen is soluble during the synthesis process. Synthesis parameters such as temperature, pH, and salt concentration affect the solubility of collagen. Collagen solubility can decrease with increasing temperature, pH, and salt concentration, and decreased collagen solubility during synthesis results in a gel with a lower G', higher swelling, and increased extruder force. HA can also interact with collagen to decrease its solubility, as described by Taguchi et al. Journal of Biomedical Materials Research, 2002, 61(2), 330-336, incorporated herein by reference. By adjusting the salt concentration, the interaction between HA and collagen can be altered, thereby adjusting collagen solubility and changing the physical properties. In one embodiment, the composition contains a salt having a concentration of 50 mM to 400 mM. In another embodiment, the composition has an NaCl concentration of approximately 150 mM. Therefore, by lowering the hydration temperature and optimizing the salt / buffer concentration, it is possible to obtain the maximum G' and minimum swelling values, along with the maximum collagen solubility for a given HA concentration during synthesis.

[0116] In one embodiment, the composition is transparent. In another embodiment, the composition is semi-transparent. In one embodiment, the HA concentration, hydration temperature, salt concentration, and / or collagen concentration affect the opacity of the composition. Increased opacity can reduce the blue discoloration that may be observed at the injection site, which is the Tyndall effect. Those skilled in the art will understand how to measure the opacity of the composition.

[0117] In one embodiment, the biological properties and tissue responses to these materials and compositions are characterized. In one embodiment, this formulation exhibits enhanced cellular activity compared to HA-only materials. The activity level depends on the collagen concentration, but also on the HA concentration and synthesis procedure. In one embodiment, formulations with lower HA concentrations (13 mg / mL) were found to give a more enhanced in vitro response than formulations with higher HA concentrations (20-28 mg / mL) for a given collagen concentration. In one embodiment, compositions with similar HA concentrations, such as hydrogels with higher collagen concentrations, showed a greater in vitro response. Furthermore, in one embodiment, formulations hydrated at temperatures below room temperature stimulated higher cellular activity than similar formulations hydrated at room temperature. Specific levels of cellular activity may be required to adapt certain fillers, and desirable activity levels could be achieved by selecting formulation and synthesis parameters.

[0118] In one embodiment, the HA / collagen formulation was also evaluated for its tissue response in a tissue integration model. Tissue sections obtained from grafts of the HA-collagen material showed cell infiltration from surrounding tissue, as well as deposition of new collagen and angiogenesis within the injected filler bolus. The degree of infiltration and tissue integration varied between different formulations. In some embodiments of the formulations described herein, collagen structure and crosslinking are surprisingly important in the degree of infiltration and tissue integration. In one embodiment, collagen structure and crosslinking may be important in tissue integration and infiltration (see, for example, Figure 14). In some embodiments of the formulations described herein, tissue integration decreases with increasing HA concentration. In some embodiments of the formulations described herein, with low HA concentration (13 mg / ml) injected into tissue, surrounding tissue was found to infiltrate the gel bolus within 4 weeks. In these embodiments, cell nuclei and newly deposited collagen were found to be scattered within the gel. This can be seen in Example 7 (Figures 6B and 6C) using Formulation I. Thus, these formulations yielded remarkable results regarding tissue infiltration into the gelbolus.

[0119] In some embodiments, formulations with higher HA concentrations (20–25 mg / mL) also showed strong tissue integration, but the tissue did not infiltrate the entire bolus as it did with formulations with lower HA concentrations.

[0120] In some embodiments, the molecular weight of HA and / or the properties of the gel particles also affect the integration of the surrounding tissue.

[0121] However, another surprising result showed that structure / crosslinking may be more important than collagen concentration in the composition. An example of this surprising finding is that a gel with 20:6 HA:collagen hydrated at 5°C showed a better tissue integration score (score = 2.0) than a gel with 20:10 HA:collagen hydrated at room temperature (score = 0.5). Tissue integration scoring was performed by blinded histopathologists and normalized against an internal control (HA-only gel). Higher scores indicate better tissue integration.

[0122] Surprisingly, differences in results were observed in compositions with different mixed gel structures. Compositions containing collagen showed a different response than compositions with collagen crosslinked to HA at 5°C.

[0123] Formulation XIX, synthesized using HA hydrated at 5°C, demonstrated improved in vitro and in vivo performance, as well as the best tissue integration shown in the embodiments herein.

[0124] Aside from collagen concentration, the levels of composition, crosslinking, and structure showed equal importance. For example, compositions such as gel formulations prepared at low temperatures resulted in improved in vitro and in vivo performance, such as improved tissue integration. This can be seen for formulation XIX, which has an HA:collagen ratio of 20:6 and a hydration temperature of approximately 5°C. The preparation of the formulation also yielded remarkable results, such as improved in vitro and in vivo performance. In some embodiments, the preparation of gel formulations at low temperatures for hydration, such as 5°C, resulted in gel formulations with improved in vitro and in vivo performance. In some embodiments, the gel formulations demonstrated tissue integration into the site of the injected formulation.

[0125] Formulations with HA:collagen ratios of 20:6 and 20:4 and a hydration temperature of approximately 5°C also yielded remarkable results, including improved in vitro and in vivo performance.

[0126] In some embodiments, formulations are provided that increase collagen infiltration into tissue. The formulation contains 13 mg / ml of hyaluronic acid. In some embodiments, the formulation is injected into tissue, resulting in the creation of a depot containing the formulation, and cells from the surrounding tissue deposit into the depot. In one embodiment, the tissue injected with the formulation is shown to have tissue integration, collagen deposition, and angiogenesis. In one embodiment, the formulation has an HA:collagen ratio of 20:6 and a hydration temperature of about 5°C. In another embodiment, the formulation has an HA:collagen ratio of 20:4 and a hydration temperature of about 5°C.

[0127] The primary function of dermal fillers is to fill wrinkles and support the tissue covering the filler injected beneath. The required amount of lift depends on the specific facial indication. Products placed deeper beneath the skin to achieve volume formation in the indication need to exhibit more structure and greater lifting force. Formulations for fine wrinkles with superficial placement do not need to exhibit as much lifting force, but need to be smoother and integrate with existing tissue. Therefore, the lifting capacity of HA / collagen formulations was evaluated in an animal lifting capacity model to determine their effectiveness. For similarly cross-linked formulations, the lifting capacity depended on the HA concentration, with higher HA concentrations resulting in increased lift.

[0128] In some embodiments, cross-linked HA:collagen formulations with added lysine showed increased lifting force as the HA concentration increased from 13 mg / mL to 20 mg / mL and then to 25 mg / mL. In some embodiments, the molecular weight of HA also affected the lifting force. In some embodiments, formulations containing 25 mg / mL of high molecular weight HA showed greater lifting force than formulations composed of a mixture of 25 mg / mL of low molecular weight HA and high molecular weight HA. Thus, by selecting optimal synthesis parameters, HA concentration, and HA molecular weight ratio, the desired lifting force can be achieved. In each or any of the embodiments described above or below, the formulation comprises a mixture of hyaluronic acid components having different molecular weights, the mixture being approximately 20,000 daltons, approximately 40,000 daltons, approximately 60,000 daltons, approximately 80,000 daltons, approximately 100,000 daltons, approximately 200,000 daltons, approximately 300,000 daltons, approximately 400,000 daltons, approximately 500,000 daltons, approximately 600,000 daltons, approximately 700,000 daltons, approximately 800,000 daltons, approximately 900,000 daltons, approximately 1,000,000 daltons, approximately 1,500,000 daltons, approximately 2,000,000 daltons, and approximately 2 This includes hyaluronic acid having an average molecular weight of 500,000 Daltons, approximately 3,000,000 Daltons, approximately 3,500,000 Daltons, approximately 4,000,000 Daltons, approximately 4,500,000 Daltons, approximately 5,000,000 Daltons, approximately 5,500,000 Daltons, approximately 6,000,000 Daltons, approximately 6,500,000 Daltons, approximately 7,500,000 Daltons, approximately 8,000,000 Daltons, approximately 8,500,000 Daltons, approximately 9,000,000 Daltons, approximately 9,500,000 Daltons and / or approximately 10,000,000 Daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

[0129] Method for synthesizing lysine-crosslinked HA-collagen hydrogel This disclosure provides a method comprising providing a collagen solution and adding the collagen solution to a second solution comprising lysine-HCl, high molecular weight HA, MES buffer, NaCl, and NaOH. In some embodiments, the hydrogel has a hyaluronic acid-to-collagen weight ratio of about 24:12, about 28:2, about 20:4, about 25:4, about 22:6, about 22:4, about 24:6, about 20:6, or about 13:4. In some embodiments, the hydrogel has a collagen concentration of about 6 mg / ml. In some embodiments, the hydrogel is stirred for homogenization. In some embodiments, the hydrogel is hydrated below room temperature. In one embodiment, the HA is hydrated at a temperature of 2°C to 35°C. In one embodiment, HA is hydrated at 2°C, 3°C, 5°C, 7°C, 9°C, 11°C, 13°C, 15°C, 17°C, 19°C, 21°C, 23°C, 25°C, 27°C, 29°C, 31°C, 33°C, 35°C, or at any temperature within the range defined by any two of the aforementioned values. In one embodiment, HA is hydrated at a temperature of 2°C to 19°C. In one embodiment, HA is hydrated at a temperature of 2°C, 3°C, 5°C, 7°C, 9°C, 11°C, 13°C, 15°C, 17°C, or 19°C, or at any temperature within the range defined by any two of the aforementioned values. In one embodiment, HA is hydrated at room temperature for at least 60 minutes. In one embodiment, HA is hydrated at a temperature higher than room temperature. In one embodiment, HA is hydrated at a temperature of at least 35°C. In some embodiments, another hydration step is performed for at least 60 minutes. In some embodiments, hydration is carried out in MES buffer at approximately pH 5.5. The mixture may be placed in a single syringe or passed between two syringes at least 50 times. An EDC / NHS solution may be added to the mixture. Mixing can be done by passing the solution between two syringes. After adding the EDC / NHS solution, the mixture is reacted at a temperature of 2–8°C for at least 16 hours. In some embodiments, the pH of the solution is adjusted to 7.4 using NaOH and purified by dialysis. The properties of the formed hydrogel can be obtained using a rheometer. Those skilled in the art can measure the composition for several parameters, such as compressive force, swelling properties, and extrusion force.

[0130] In one embodiment, the polymer matrix further comprises non-crosslinked HA which can be used to facilitate injection and reduce extrusion force.

[0131] In one embodiment, the lysine:HA ratio is optimized to maximize crosslinking efficiency. In one embodiment, the lysine:HA ratio is approximately 0.0 to 0.5, which may allow for more efficient crosslinking. Crosslinking without lysine may rely on collagen to supply amines for crosslinking, making ester crosslinking between HA chains more water-insensitive. Crosslinking at a high lysine:HA ratio may saturate the activated carboxylic acid on the HA chain, resulting in pendant lysine molecules bound only to one side of the HA chain rather than crosslinking between chains. By selecting the optimal lysine:HA ratio for a given indication, the physical properties of the resulting hydrogel can be adjusted to achieve the desired properties. In some embodiments, the optimal lysine:HA ratio may be composition-dependent.

[0132] Sterilization of composition The developed biomaterials may require sterilization or destruction of undesirable biological materials, such as pathogens and bacterial microorganisms, before administration of the composition by injection or implantation to human patients. These compositions include the embodiments described herein and include, for example, materials such as cross-linked polymer matrices. Proteins, polysaccharides, and carbohydrates in these materials may be susceptible to molecular destruction when exposed to conventional heat sterilization procedures such as autoclaving, or to ionizing radiation such as gamma rays. Conventionally, many of these energy-sensitive biomaterials are sterilized in bulk by a microfiltration process aimed at physically removing microorganisms from the composition. The filtered composition then has to be packaged in syringes and / or vials for use by physicians.

[0133] In one embodiment, the crosslinked polymer matrix is ​​sterile. In one embodiment, the method for preparing the crosslinked polymer matrix further includes a step of sterilizing the crosslinked polymer matrix.

[0134] In one embodiment, the method further includes exposing the composition or a crosslinked polymer matrix to broadband spectral radiation in a dose effective in inactivating pathogens, microorganisms, and other microorganisms.

[0135] In one embodiment, the method further includes the step of exposing a composition or crosslinked polymer matrix to pulsed radiation (which may hereafter be referred to as pulsed light) containing broadband spectral radiation. The broadband spectral radiation may have a wavelength band range of about 100 nm to about 1100 nm. The broadband spectral radiation includes wavelengths in the ultraviolet, visible, and infrared ranges. In some embodiments, the wavelength distribution is about 54% UV wavelengths, 26% visible wavelengths, and about 20% infrared wavelengths. This form of radiation can be supplied by a xenon lamp.

[0136] In one embodiment, pulsed light inactivates microorganisms and other microorganisms in the composition without causing significant degradation of the composition or significant changes in its rheology.

[0137] In one embodiment, the pulsed light has an energy of approximately 100 mJ / sqcm to approximately 2000 mJ / sqcm, defined by a UV fluence of 254 nm. In another embodiment, the pulsed light has an energy of approximately 300 mJ / sqcm to approximately 1800 mJ / sqcm, defined by a UV fluence of 254 nm.

[0138] In one embodiment, the pulsed light has an energy of approximately 700 mJ / sqcm to approximately 800 mJ / sqcm, defined by a UV fluence at 254 nm. In another embodiment, the pulsed light has an energy of approximately 1400 mJ / sqcm to approximately 1600 mJ / sqcm, defined by a UV fluence at 254 nm.

[0139] In one embodiment, the pulsed light has a pulse frequency of about 1 pulse / second to about 10 pulses / second, for example, about 3 pulses / second.

[0140] In one embodiment, the composition is exposed to pulsed light for a period of 240 seconds or less. In one embodiment, the composition is exposed to pulsed light for a period of 120 seconds or less. In one embodiment, the composition is exposed to pulsed light for a period of 40 seconds or less. In one embodiment, the composition is exposed to pulsed light for a period of 30 seconds or less. In one embodiment, the composition is exposed to pulsed light for a period of 20 seconds or less. In one embodiment, the composition is exposed to pulsed light for a period of 10 seconds.

[0141] In one embodiment, the composition is exposed to pulsed light for a period of 5 seconds. In another embodiment, the composition is exposed to pulsed light for a period of 1 second or less.

[0142] In one embodiment, pulsed light is effective in sterilizing the composition without raising its temperature above 90°C. In one embodiment, pulsed light is effective in sterilizing the composition without raising its temperature above 20°C. In one embodiment, the dose is effective in sterilizing the composition without raising its temperature above 15°C, for example above 10°C, for example above 5°C.

[0143] In one embodiment, pulsed light is effective in sterilizing the composition with a rheological loss (G' / G'') of less than about 10%, less than about 8%, or less than about 5%.

[0144] In one embodiment, pulsed light is effective in sterilizing the composition, that is, inactivating pathogens, microorganisms, and other microorganisms in the composition, without causing significant degradation, for example, without causing significant changes in the rheological properties of the composition.

[0145] In one embodiment, the effective sterilization dose of radiation preserves the rheology of the hydrogel. In one embodiment, the method is effective for sterilizing the hydrogel with a rheological loss (G' / G'') of less than about 10%, less than about 8%, or less than about 5%.

[0146] example The following examples, including experiments conducted and results achieved, are provided for illustrative purposes only and should not be construed as limiting this disclosure.

[0147] Example 1: Synthesis of Lysine-Crosslinked HA-Collagen Hydrogel A solution of 4.96 mg / mL of collagen dissolved in 0.01 M HCl was added to a 30 mL HSW Norm-Ject syringe along with lysine-HCl, HMW HA, MES buffer / NaCl solid, and 1 M NaOH. The concentration was adjusted accordingly to prepare hydrogels with HA:collagen ratios of, for example, 13:4 mg / ml (Formulation I), 20:4 mg / ml (Formulation II), and 25:4 (Formulation III). The mixture was stirred to homogenize the solution, and the HA was hydrated at room temperature for approximately 60 minutes. After approximately 60 to 90 minutes, the mixture was passed between syringes and hydrated again for approximately 30 to 60 minutes. After the second hydration, the mixture was passed between syringes several times. An EDC / NHS solution was prepared in a third 30 mL syringe by adding water, NHS, and EDC, and mixed by shaking. The EDC / NHS solution was added to the HA / collagen mixture, passed between two syringes, and then transferred to a glass vial, which was reacted at 2–8°C. In some embodiments, the reaction time was approximately 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, or any time between the range defined by any two of the aforementioned values. After this, the gel was transferred to a syringe and again passed between two syringes. The pH of the gel was adjusted to approximately 7.40 using 2M NaOH, and the final volume was adjusted using PBS. The gel formulation was dialyzed against PBS at 2–8°C for approximately 70 hours, during which the buffer was changed several times to remove the EDC / NHS. The gel was then transferred from the dialyzing membrane to a syringe, passed through a stainless steel mesh (60 μm pores–104 μm pores), and passed between two syringes. This gel was transferred to a 1 mL syringe, and the syringe was steam sterilized. The obtained sterile hydrogels were characterized using rheology, compressive force measurements, extrusion force measurements, and swelling.

[0148] For formulation XXVI, NaCl was omitted during the crosslinking process.

[0149] For formulations XXV and XXVI, non-crosslinked HMW HA (2% w / w of the total composition) and lidocaine HCl (0.3% w / w of the total composition) are added before syringe filling and sterilization.

[0150] Example 2 - Synthesis of lysine-crosslinked HA-collagen hydrogel with a final collagen concentration of 6 mg / mL A solution of 7.16 mg / mL of collagen dissolved in 0.01 M HCl was added to a 30 mL HSW Norm-Ject syringe along with lysine-HCl, HMW and / or LMW HA and MES buffer / NaCl solid. The pH was adjusted with 1 M NaOH. The mixture was stirred to homogenize, and the HA was hydrated at the specified temperature for approximately 60 minutes. After approximately 60-90 minutes, the mixture was passed through two syringes several times and hydrated again for at least 30 minutes. After the second hydration step, the mixture was again passed through two syringes several times. A third 30 mL EDC / NHS solution was prepared in a 30 mL syringe by adding water, NHS, and EDC, and mixed by shaking. Hydration can be carried out at temperatures of approximately 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, or any temperature within the range defined by any two of the aforementioned values. The EDC / NHS solution was added to the HA / collagen mixture, passed through two syringes several times, and then transferred to a Thinky Mixer reaction vessel, where it was reacted at 2–8°C for at least 16 hours. After this, the gel was homogenized using the Thinky Mixer. The pH of the gel was adjusted to approximately 7.40 using 2M NaOH, and the final volume was adjusted using PBS. The gel formulation was dialyzed against PBS at 2–8°C for approximately 70 hours, with the buffer being changed several times during this period. The gel was then transferred from the dialyzing membrane to a syringe, passed through a stainless steel mesh (104 μm pores), and homogenized using the Thinky Mixer. This gel was transferred to a 1 mL syringe, and the syringe was steam sterilized. The resulting sterile hydrogel was characterized as described in the example above.

[0151] In some embodiments, the gel contains 20 mg / ml of hyaluronic acid. In some embodiments, the gel contains 6 mg / ml of collagen. In some embodiments of the method for preparing the gel, the hyaluronic acid is hydrated at a temperature of 5°C.

[0152] Example 3-28: Synthesis of lysine-crosslinked HA-collagen hydrogel with an HA:collagen concentration of 2 mg / mL (Formulation XVI). A solution of 3.20 mg / mL of collagen dissolved in 0.01 M HCl was added to a 30 mL HSW Norm-Ject syringe along with 0.01 M HCl, lysine-HCl, HMW HA, LMW HA, and MES buffer / NaCl solid. The pH was adjusted with NaOH. The mixture was stirred to homogenize, and the HA was hydrated at room temperature for approximately 90 minutes. After 90 minutes, the mixture was passed through the syringe several times and hydrated again for approximately 30 minutes. After the second hydration step, the mixture was passed through the syringe several times. An EDC / NHS solution was prepared in a third 30 mL syringe by adding water, NHS, and EDC, and mixed by shaking. The EDC / NHS solution was added to the HA / collagen mixture, passed through the syringe several times, and then transferred to a glass vial, which was reacted at 2-8°C for at least 16 hours. After this, the gel was transferred to a syringe and passed through the syringe. The pH of the gel was adjusted to approximately 7.40 using 2M NaOH, and the final volume was adjusted using PBS. The gel formulation was dialyzed in PBS at 2-8°C for approximately 70 hours, during which the buffer solution was changed several times. The gel was then transferred from the dialyzing membrane to a syringe, passed through a stainless steel mesh (104 μm pores), and homogenized by passing it between syringes. This gel was then transferred to a 1 mL syringe, and the syringe was steam sterilized. The resulting sterile hydrogel was characterized as described in the example above.

[0153] Example 4 - Synthesis of lysine-crosslinked HA-collagen hydrogel with an HA:collagen concentration of 25:4 mg / mL, prepared at 1.25 times the final concentration (-Formulation XV) A solution of 5.67 mg / mL of collagen dissolved in 0.01 M HCl was added to a 30 mL HSW Norm-Ject syringe along with lysine-HCl, HMW HA, LMW HA, and MES buffer / NaCl solid. The pH was adjusted with 1 M NaOH. The mixture was stirred to homogenize, and the HA was hydrated at room temperature for approximately 90 minutes. The mixture was then passed through the syringe several times and hydrated again for 30 minutes. After the second hydration step, the mixture was passed through the syringe again. An EDC / NHS solution was prepared in a third 30 mL syringe by adding water, NHS, and EDC, and mixed by shaking. The EDC / NHS solution was added to the HA / collagen mixture, passed through the syringe several times, and then transferred to a glass vial, which was reacted at 2–8°C for at least 16 hours. After this, the gel was transferred to a syringe and passed through the syringe. The pH of the gel was adjusted to approximately 7.40 using 2M NaOH, and the final volume was adjusted using PBS. The gel formulation was dialyzed in PBS at 2-8°C for approximately 70 hours, during which the buffer solution was changed several times. The gel was then transferred from the dialyzing membrane to a syringe, passed through a stainless steel mesh (104 μm pores), and homogenized by passing it between syringes. This gel was then transferred to a 1 mL syringe, and the syringe was steam sterilized. The resulting sterile hydrogel was characterized as described in the example above.

[0154] Example 5 - Physical properties of hydrogels Rheological properties were obtained using an Anton-Paar MCR301 / 302 rheometer equipped with a 25 mm parallel plate shape measuring tool. Samples were analyzed at a gap height of 1 mm using both frequency sweep (10 Hz to 0.1 Hz, 1% strain) and amplitude sweep (0.3% to 300% strain, 5 Hz frequency) measurements. Compressive force was measured using the same instrument at a gap height of 2.5 mm and for vertical compression. The gap height was established at 2.5 mm, remained there for 5 minutes, and then compressed from 2.5 mm to 0.89 mm at a speed of 13.33 μm / s. Hydrogel swelling was measured by mixing the gel sample with an excess of phosphate buffer and determining the volume of the gel after equilibrium. The volume of the swollen gel was compared to the volume of the original gel added before the buffer was added. Swelling is expressed as a percentage of the original gel volume, as an incorporation of additional fluid. The gel extrusion force was measured for gel formulations in 1 mL COC syringes fitted with a 1 / 2-inch 27G TSK needle (unless otherwise specified) using a texture analyzer set to a speed of 50 mm / min. [Table 1]

[0155] Table 1: Synthesis parameters and physical properties of hydrogel formulations. One equivalent corresponds to a concentration of 0.1 M MES buffer containing 0.9% NaCl.

[0156] As shown in Table 1, at a constant collagen concentration, as the HA concentration increases from 13 mg / mL to 20 mg / mL and 25 mg / mL (Formulation I vs. Formulation II vs. Formulation III), the G' value increases (380 Pa → 645 Pa → 1370 Pa) along with the compressive force (47 gmf → 180 gmf → 310 gmf) and the pressing force (13.8 N (30 G) → 28.0 N → 51.7 N), while the G'' / G' ratio decreases with increasing HA concentration (0.123 → 0.103 → 0.056).

[0157] With the same HA and collagen concentrations (Formulation VI vs. Formulation VII vs. Formulation VIII), as the HMW / LMW HA ratio decreased from 100 / 0 to 65 / 35 and then to 35 / 65, the G' value decreased along with the compressive force (292→226→163 gmf) and the pressing force (63.6→29.2→20.0 N) (1360 Pa→1180 Pa→932 Pa). On the other hand, as the HMW / LMW ratio decreased, the G'' / G' ratio increased (0.055→0.068→0.085).

[0158] When the synthesis hydration temperature is reduced from 35°C to 22°C to 15°C to 5°C at constant HA and collagen concentrations (Formulation XI vs. Formulation VI vs. Formulation IX vs. Formulation X), the G' value increases (876 → 1360 → 3145 → 4750 Pa), but the hydrogel swelling decreases along with the compressive force (56.1~63.6 → 33.8 → 20.1 N) (257% → 238% → 159% → 127%). The compressive force is not affected by changes in hydration temperature, except at higher hydration temperatures (35°C vs. others). Adjusting the hydration temperature during synthesis changes the solubility of collagen, and consequently, the physical properties of the resulting hydrogel change.

[0159] By reducing the salt / buffer concentration to 2 / 3, similar G' values ​​(5470 Pa vs. 4750 Pa), swelling (108% vs. 127%), and extrusion force (20.5 N vs. 20.1 N) were obtained for formulations hydrated at 22°C with a lower salt / buffer concentration and formulations hydrated at 5°C with the original salt / buffer concentration (Formulation XIII vs. Formulation X). Furthermore, synthesis using the lower salt / buffer concentration is less sensitive to the hydration temperature difference between 22°C and 5°C than synthesis using the original salt / buffer concentration. While the G', swelling force, and extrusion force values ​​did not differ significantly between formulations synthesized using the lower salt / buffer concentrations at 22°C and 5°C (Formulation XIII vs. Formulation XII), these physical properties differed significantly between similar formulations synthesized using the original salt / buffer concentration (Formulation VI vs. Formulation X).

[0160] The effect of added lysine can be observed by comparing similar formulations synthesized with an HA:collagen concentration of 28:2 mg / mL (Formulation XVIII vs. Formulation XVI vs. Formulation XVII). The optimal lysine:HA ratio helps maximize crosslinking efficiency, and its precise ratio depends on the molecular weight of HA, the concentration of the activating reagent, and the synthesis conditions. For example, the lysine:HA ratio increased for a series of formulations synthesized with an HA:collagen concentration of 28:2 mg / mL (Formulation XVIII vs. Formulation XVI vs. Formulation XVII) (0 → 0.333 → 0.5). Physical properties such as G' and compressive force were maximized in the formulation prepared with a lysine:HA ratio of 0.333, while swelling and G'' / G' values ​​were minimized in the same formulation. Higher G' and lower swelling are generally associated with more highly crosslinked hydrogels. A lysine:HA ratio of 0 to 0.5 allows for more efficient crosslinking. Lysine-free crosslinking may rely on collagen to supply amines for crosslinking, making ester crosslinking between HA chains more water-insensitive. Crosslinking at high lysine:HA ratios can saturate the activated carboxylic acid on the HA chains, resulting in penetrating lysine molecules bound only to one side of the HA chain rather than crosslinking between chains. These scenarios with very low or high lysine:HA ratios may result in inefficient crosslinking and suboptimal gel properties. By selecting the optimal lysine:HA ratio for a given indication, the physical properties of the resulting hydrogel can be tuned to achieve the desired properties.

[0161] Example 6 - In vitro testing of hydrogels Cell proliferation and survival rate in vitro The viability and proliferation of fibroblasts in close contact with HA-collagen hydrogel were quantified using the XTT assay. 100 μL of hydrogel (n=3) was stacked 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 human dermal fibroblasts in 500 μL of cell culture medium were added to the hydrogel bed and incubated at 37°C. After incubation for 48 hours, 250 μL of XTT reagent was added to each well and incubated at 37°C for 4 hours. The plate was then rotated at 300xg for 5 minutes, and 200 μL of supernatant obtained from each well was transferred to the wells of a 96-well filter plate with a 20 μm mesh. The filter plate containing the XTT supernatant was rotated at 300xg for 5 minutes. 100 μL of filtered supernatant obtained from each well was transferred to a clean 96-well plate (black wall, clear bottom), and the absorbance of the supernatant was read using a microplate reader (450 nm with 630 nm background correction). The data were normalized against the XTT cell viability of fibroblasts cultured on positive control tissue culture polystyrene (TCPS).

[0162] Cell viability and proliferation were found to be higher with formulations having lower HA concentrations than with similarly cross-linked formulations having the same collagen concentration and higher HA concentration. For example, hydrogels synthesized using HMW HA and 4 mg / mL collagen, but with increasing HA concentrations of 13 mg / mL (Formulation I), 20 mg / mL (Formulation II), and 25 mg / mL (Formulation III), showed proliferation values ​​of 53%, 30%, and 20% compared to the TCPS-positive control. (Figure 1) A negative control gel with HA alone showed a proliferation value of 12% compared to the TCPS control.

[0163] Furthermore, the hydration temperature during the synthesis procedure was found to have an effect on the cell viability and proliferation of similarly cross-linked formulations having the same HA and collagen concentrations. For hydrogels with an HA:collagen concentration of 24:6 mg / mL, formulations hydrated at 5°C (Formulation X) showed higher cell proliferation than those hydrated at 22°C (Formulation VI) or 35°C (Formulation XI), with values ​​of 39%, 27%, and 19%, respectively. (Figure 1)

[0164] Cell viability and proliferation are also affected by the concentrations of salt and buffer during hydrogel synthesis. In synthesis performed at 5°C, there was no clear change in cellular response when the salt / buffer concentration was reduced (Formulation X (1 equivalent) vs. Formulation XII (0.33 equivalents)). However, in formulations hydrated at 22°C or 35°C, formulations prepared with reduced salt / buffer concentrations (Formulation XIII, 22°C, and Formulation XIV, 35°C) showed a significant increase in cell viability and proliferation compared to formulations prepared with 1 equivalent of salt / buffer (Formulation VI, 22°C, and Formulation XI, 35°C). (Figure 1)

[0165] Formulation XIX was prepared at 5°C using 20 mg / mL of HA and 6 mg / mL of collagen. This formulation showed higher cell viability and proliferation compared to other gels with HA concentrations of 20 mg / mL or higher. (Figure 1)

[0166] Formulations with an HA:collagen ratio of 20:4 were also shown to enhance cellular responses in vitro (Figure 15, Formulation XXII).

[0167] Furthermore, formulation XIX was shown to exhibit consistent stability and performance after autoclaving.

[0168] Morphology of in vitro cells Cell morphology was analyzed to evaluate the effects of hydrogel formulations on cell size, shape, and cytoskeletal organization. Actin filament alignment index and morphology of fibroblasts cultured on HA alone or on HA / collagen crosslinked hydrogels were imaged and quantified. Increased actin filament alignment may correlate with increased cell adhesion to the substrate. An increase in the length-to-width ratio correlates with increased cell spreadability on the substrate. The hull-to-cell area ratio is a measure of cell shape, where 1.0 indicates a uniformly shaped cell, and values ​​greater than 1 indicate a more irregularly shaped cell. Cells that have made multiple contacts with the matrix and are elongating / migrating show more irregular cell shapes and higher hull-to-cell area ratio values. Actin filament alignment index, length-to-width ratio, and hull-to-cell area ratio can be analyzed together in three-dimensional Euclidean space. The Euclidean distance of hydrogels obtained from negative controls (in this case, gels with only non-adherent HA) allows for ranking of the overall cellular response to the filler. Larger Euclidean distances obtained from HA-only controls indicate enhanced cell adhesion and spreadability on the hydrogel. Hydrogels that support greater cell adhesion and spreadability are expected to induce more cell infiltration into the gel, where these cells deposit extracellular matrix (ECM) within the gel matrix. Increased cell infiltration and ECM deposition may be beneficial in in vivo tissue integration into the hydrogel depot. Conversely, formulations resulting in lower cell adhesion and spreadability values ​​behave more inactively, leading to less tissue infiltration and integration. In some embodiments, the method for preparing the hydrogel further includes an autoclave step, where the autoclave does not alter the properties of the hydrogel (see Figure 13).

[0169] In a typical procedure, a hydrogel (n=3) in cell culture medium and human dermal fibroblasts were added to a 96-well cell culture plate with a low-adhesion surface coating. After 48 hours of incubation, the cells were fixed with formalin and stained with Hoechst, WGA-488, and Alexa Fluor-Phalloidin. The wells were imaged using a confocal microscope, and actin filament alignment (phaloidin) and cell morphology (WGA-488) were analyzed using image analysis software.

[0170] The hydration temperature during the synthesis procedure had an effect on the cell adhesion and spreadability of similarly crosslinked formulations having the same HA and collagen concentrations. In the case of hydrogels with an HA:collagen concentration of 24:6, formulation hydrated at 5°C (Formulation X) showed higher cell adhesion (actin filament alignment index) than formulation hydrated at 22°C (Formulation VI), with values ​​of 0.054 and 0.015, respectively (Figure 2). Formulation hydrated at 5°C (Formulation X) also showed increased cell spreadability (cell length to width ratio) than formulation hydrated at 22°C (Formulation VI), with values ​​of 2.52 and 1.40, respectively. Formulation hydrated at 5°C (325_B) also showed a higher convex hull to cell area ratio than formulation hydrated at 22°C (Formulation VI), with values ​​of 1.34 and 1.07, respectively. The actin filament alignment index, cell length-to-width ratio, and convex hull-to-cell area ratio of the formulation hydrated at 22°C (Formulation VI) were similar to those of the HA-only control. The optimized formulation (20:6 HA:collagen, hydrated at 5°C, Formulation XIX) showed significantly higher actin filament alignment index, length-to-width ratio, and convex hull-to-cell area ratio than the HA-only gel. Ranking the hydrogels using the Euclidean distance obtained from the HA-only gel revealed that the optimized formulation performed better than the other HA-collagen hydrogels.

[0171] Cell morphology analysis correlates well with the XTT cell activity assay, as formulation X showed higher activity than formulation VI in the activity assay and also provided evidence of increased cell adhesion and spreadability in the morphology assay. Formulation XIX also shows higher activity than other HA-collagen formulations and HA-only gels. Cell spreadability and adhesion are associated with higher cell activity, and therefore the results of each assay agree well with each other (Figures 2A-2D).

[0172] Fibroblasts cultured with formulations XXII and XXIII exhibit a significantly larger cell length-to-width ratio than fibroblasts cultured using HA-only gel. (Figure 16)

[0173] Example 7 - In vivo testing of hydrogels Lifting capacity The ability of hydrogels to support tissue elevation (lifting force) was evaluated in vivo using a rat subcutaneous transplantation model. 125 μL of hydrogel (n=10) was injected as a subcutaneous bolus onto the skull. 3D reconstructions of the bolus were generated over 12 weeks using a clinical 3D imaging system (Canfield Vectra). The mean height of the bolus was analyzed using medical imaging software (Canfield Mirror).

[0174] The in vivo lifting capacity of a series of HA-collagen formulations with the same collagen concentration (4 mg / mL) and HMW / LMW HA ratio (100 / 0), measured between 4 and 12 weeks, showed a positive correlation with HA concentration. The formulation containing 25 mg / mL of HA (Formulation III) showed more elevation than the formulations containing 20 mg / mL (Formulation II) or 13 mg / mL (Formulation I) (Figure 3). Since compressive force increased with HA concentration in these formulations, the in vivo elevation between 4 and 12 weeks showed a positive correlation with compressive force. In vivo lifting force also depended on the gel synthesis conditions. Two formulations (Formulation III vs. Formulation XV), containing the same HA:collagen concentration of 25:4 mg / mL but synthesized under different HMW / LMW HA ratios and different crosslinking conditions, produced different lift profiles between 4 and 12 weeks. Formulations synthesized with high-MW HA at 1x synthesis concentration showed superior lifting power compared to formulations prepared with 10 / 90 HMW / LMW HA at 1.25x synthesis concentration (Figure 4). Furthermore, hydrogel formulations (Formulation II, Formulation XV, Formulation XVI) with different HA / collagen concentrations and synthesis conditions but similar compressive force values ​​(166-180) showed similar in vivo lift profiles over 4-12 weeks (Figure 5). Thus, by selecting the optimal composition and synthesis conditions, the desired lift profile for a given application can be obtained.

[0175] Furthermore, the lifting capacity was tested using formulation XIX and a formulation containing only HA (Figures 11 and 12). As shown, formulation XIX demonstrated similar lifting capacity to the 24 mg / ml HMW HA-only gel for 4 to 28 weeks.

[0176] Long-term (52-week) lifting capacity data for Formulation XXII were tested. The lifting capacity of the HA-only control steadily decreased over time. In contrast, the lifting capacity of the HA-collagen gel (Formulation XXII) remained stable from 30 to 52 weeks. (Figure 22) This remarkable result indicates that these HA-collagen gel formulations are capable of a longer lifting duration than HA-only gels. This correlates with better tissue ingrafting than HA-only gels (see below).

[0177] Long-term (26-week) lifting capacity data for Formulation XXII was tested. Formulation XXIII and HA alone showed similar lifting capacity over 26 weeks (Figure 23). Surprisingly, the enhanced tissue integration of Formulation XXIII results in an extended duration of lifting capacity and other benefits to the overall effect. For example, the newly formed tissue maintains skin quality, lifting capacity, and wrinkle correction.

[0178] Long-term (30-week) lifting capacity data for formulation XXV were tested. The lifting capacity of the HA-only gel steadily decreased over time (Figure 28). In contrast, the lifting capacity of the HA-collagen gels (formulations XXV and XXVI) remained stable from 18 to 30 weeks (Figure 28). This remarkable result indicates that these HA-collagen gel formulations are capable of a longer lifting duration than the HA-only gel. This correlates with better tissue infiltration than the HA-only gel (see below).

[0179] Organizational integration in vivo The in vivo tissue integration of a series of formulations was evaluated using a rat subcutaneous transplantation model. In a typical procedure, 125 μL of hydrogel was delivered as a subcutaneous bolus to the dorsal side of rats. After 4 weeks, the bolus was explanted, fixed with formalin, and embedded in paraffin for histology. Tissue sections were stained for hematoxylin & eosin (H&E) and colloidal iron. Immunohistochemical staining for type I collagen, vimentin, CD31, and type I procollagen was also performed.

[0180] Tissue integration shows a negative correlation with HA concentration for formulations similarly prepared using HMW HA and 4 mg / mL collagen. The density of collagen deposited near the surrounding tissue is higher in the formulation containing 13 mg / mL HA (Formulation I) than in the formulations prepared with 20 mg / mL HA (Formulation II) or 25 mg / mL HA (Formulation III), and is HA concentration-dependent (Figures 6A-6E). Furthermore, the 13 mg / mL HA formulation has fewer areas of tissue-deficient injection bolus than the materials containing 20 mg / mL or 25 mg / mL HA. In addition to HA concentration, integration is expected to be primarily dependent on collagen concentration. However, other factors may strongly influence tissue infiltration into the bolus. For example, formulations with high HA concentration (28 mg / mL) and low collagen concentration (2 mg / mL) (Formulation XVI) demonstrate collagen deposition throughout the entire bolus with almost no areas of tissue-deficient.

[0181] Formulation XVI was prepared primarily using LMW HA, unlike the aforementioned formulation which was prepared using HMW HA. However, the molecular weight of HA is not the only contributing factor, as a second formulation (Formulation XV) with an HA:collagen concentration of 25:4 mg / mL was prepared primarily using LMW HA, but did not show the same strong integration throughout the bolus (Figure 7). It has been previously shown that the hydration temperature during synthesis affects the cellular response in vitro, and subsequent studies have shown that it also affects cell invasion and tissue integration in vivo. Two similar formulations prepared with an HA:collagen concentration of 24:6 mg / mL, but at different hydration temperatures of 5°C (Formulation X) and 22°C (Formulation VI), showed different densities of collagen deposition around the bolus, with the formulation prepared at 5°C showing a higher response (Figure 8). It is not a single parameter, but a combination of factors including HA concentration, HA molecular weight ratio, collagen concentration, and synthesis conditions that influences the degree of tissue integration. Various tissue responses have been achieved with these materials, and therefore, the integration and infiltration of this tissue can be tailored to specific filler applications by optimizing the aforementioned synthesis parameters.

[0182] Formulations XXII and XXIII demonstrate enhanced tissue integration compared to HA-only gels (Figure 17). Staining of collagen 1a indicates the distribution of fine collagen around gel particles in the HA-collagen formulations and the limited deposition of collagen 1a in the HA-only gel (Figure 18). Quantification of the positive area percentage for collagen 1a staining within hydrogel bolus after 4 weeks of subcutaneous transplantation in rats demonstrates that Formulation XXII generates more collagen 1a-positive tissue than the HA-only hydrogel (Figure 19).

[0183] Figure 20 shows confocal micrographs of human dermal fibroblasts cultured for 48 hours on gels containing HA alone, formulation XXII, or formulation XXIII. Samples were stained for HA-binding protein, Hoechst, and cell membrane. The prepared gels showed a significant improvement in cell adhesion compared to cross-linked products containing HA alone, suggesting the potential for the gels to function as scaffolds for tissue integration and collagen deposition.

[0184] Formulation XIX was also tested for its ability to increase levels of vimentin (fibroblasts), collagen I, and CD31. As shown in Figures 9 and 10, Formulation XIX was able to increase levels of vimentin (fibroblasts), collagen I, and CD31 (vascular tissue) in bolus Formulation XIX hydrogels after 12 weeks of subcutaneous implantation in rats, compared to hydrogels with HA alone. This is supported by improved cell spreadability and adhesion of Formulation XIX. Similarly, Formulations XXII and XXIII promoted greater fibroblast infiltration (vimentin staining) and angiogenesis (CD31 staining, arrows) compared to the HA-only control (Figure 21). This indicates the regeneration of tissue with morphology consistent with endogenous tissue in the hydrogel bolus.

[0185] Figure 24 shows confocal micrographs of human dermal fibroblasts cultured for 48 hours on gels containing HA alone, formulation XXVI, or formulation XXV. Samples were stained for HA-binding protein, Hoechst, and cell membrane. Formulation XXVI and formulation XXV gels showed a remarkable improvement in cell adhesion compared to cross-linked products containing HA alone, demonstrating the potential of the gels to function as scaffolds for tissue integration and collagen deposition.

[0186] Figure 25 shows two-photon imaging of second harmonic generation signals (white) and tissue autofluorescence (green) in rats treated with HA alone or formulation XXV or formulation XXIII by subcutaneous bolus injection after 12 weeks. The presence of second harmonic generation (white) indicates the formation of fully aggregated fibrillary collagen in grafts treated with HA-collagen. Second harmonic generation is observed to be limited in HA-only gels.

[0187] Figure 26 shows immunohistochemical analysis of the tissue response to formulation XXV after 4 weeks of subcutaneous transplantation in rats. Formulation XXV promotes tissue integration (H&E staining), fibroblast infiltration (vimentin), limited macrophage response (CD68), collagen I deposition, limited collagen III, and angiogenesis (CD31). This may indicate spontaneous tissue regeneration in the hydrogelbolus. Furthermore, formulation XXV resulted in a higher pathological score for tissue integration (4.67) compared to the HA-only control (0.67).

[0188] Figure 27 shows immunohistochemical analysis of the tissue response to formulation XXVI after 4 weeks of subcutaneous transplantation in rats. Formulation XXVI promotes tissue integration (H&E staining), fibroblast infiltration (vimentin), limited macrophage response (CD68), collagen I deposition, limited collagen III, and angiogenesis (CD31). This may indicate spontaneous tissue regeneration in the hydrogelbolus. Furthermore, formulation XXVI resulted in a higher pathological score for tissue integration (4.17) compared to the HA-only control (0.67).

[0189] Description of subject matter in the form of clauses Various examples of the aspects of this disclosure are provided below for convenience in the form of numbered clauses (1, 2, 3, etc.). These are presented as examples and do not limit the subject art. The references to drawings and reference numbers are provided below merely as examples and for illustrative purposes, and the clauses are not limited by their references.

[0190] Article 1. A crosslinked polymer matrix comprising lysine, hyaluronic acid, and collagen, wherein the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine.

[0191] Section 2. A crosslinked polymer matrix according to any one of the above or below sections, further comprising lidocaine.

[0192] Section 3. A crosslinked polymer matrix according to either of the above or below sections, wherein lidocaine is present in the matrix at a concentration ranging from approximately 0.15% (w / w) to approximately 0.45% (w / w).

[0193] Section 4. A crosslinked polymer matrix as described in either of the above or below sections, wherein lidocaine is present in a concentration of approximately 0.15% (w / w), approximately 0.17% (w / w), approximately 0.19% (w / w), approximately 0.21% (w / w), approximately 0.23% (w / w), approximately 0.25% (w / w), approximately 0.27% (w / w), approximately 0.29% (w / w), approximately 0.31% (w / w), approximately 0.33% (w / w), approximately 0.35% (w / w), approximately 0.37% (w / w), approximately 0.37% (w / w), approximately 0.39% (w / w), approximately 0.41% (w / w), approximately 0.43% (w / w), or approximately 0.45% (w / w) of the matrix, or any concentration between the ranges defined by any two of the aforementioned values.

[0194] Section 5. A crosslinked polymer matrix according to either of the above or below sections, wherein lidocaine is present in the matrix at a concentration ranging from approximately 0.27% (w / w) to approximately 0.33% (w / w).

[0195] Section 6. A crosslinked polymer matrix according to any one of the above or below sections, wherein the matrix further comprises non-crosslinked HA.

[0196] Section 7. A crosslinked polymer matrix according to either of the above or below sections, wherein non-crosslinked HA is present in the matrix at a maximum concentration of approximately 5% (w / w).

[0197] Section 8. A crosslinked polymer matrix according to either of the above or below sections, wherein the non-crosslinked HA is present in the matrix at a concentration of about 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 range defined by any two of the aforementioned values.

[0198] Section 9. A crosslinked polymer matrix according to either of the above or below sections, wherein non-crosslinked HA is present in the matrix at a concentration of approximately 1% (w / w).

[0199] Item 10. A crosslinked polymer matrix according to either of the above or below items, wherein non-crosslinked HA is present in the matrix at a concentration of approximately 2% (w / w).

[0200] Item 11. A crosslinked polymer matrix according to either of the above or below items, wherein non-crosslinked HA is present in the matrix at a concentration of approximately 5% (w / w).

[0201] Section 12. A crosslinked polymer matrix according to any one of the above or below sections, wherein the non-crosslinked HA improves the extrudeability of the polymer matrix.

[0202] Clause 13. A crosslinked polymer matrix as described in any one of the above or below clauses, which is stable for approximately 6 months, approximately 12 months, approximately 18 months, approximately 24 months, approximately 30 months, or approximately 36 months, or for any time within the range defined by any two of the aforementioned values.

[0203] Item 14. A crosslinked polymer matrix according to either of the above or below items, which is stable at a temperature of approximately 4°C to approximately 25°C.

[0204] Item 15. A crosslinked polymer matrix according to either of the above or below items, which is stable at approximately 4°C.

[0205] Item 16. A crosslinked polymer matrix according to either of the above or below items, which is stable at approximately 25°C.

[0206] Clause 17. A crosslinked polymer matrix as described in either of the above or below clauses, which is stable at any point in time between approximately 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, or any point in time between any two of the above values.

[0207] Clause 18. A cross-linked polymer matrix as described in any one of the above or below clauses, which exhibits minimal degradation over a period of approximately 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months, or any time within the range defined by any two of the aforementioned values.

[0208] Section 19. A crosslinked polymer matrix according to either of the above or below sections, wherein the matrix has an elastic modulus (G') of about 30 Pa to about 10,000 Pa, or any elastic modulus between the range defined by any two of the aforementioned values.

[0209] Section 20. The matrix is ​​approximately 30 Pa, approximately 40 Pa, approximately 50 Pa, approximately 60 Pa, approximately 70 Pa, approximately 80 Pa, approximately 90 Pa, approximately 100 Pa, approximately 200 Pa, approximately 300 Pa, approximately 400 Pa, approximately 500 Pa, approximately 600 Pa, approximately 700 Pa, approximately 800 Pa, approximately 900 Pa, approximately 1000 Pa, approximately 1100 Pa, approximately 1200 Pa, approximately 1300 Pa, approximately 1400 Pa, approximately 1500 Pa, approximately 1600 Pa, approximately 1700 Pa, approximately 1800 Pa, approximately 1900 Pa, approximately 2000 Pa, approximately 2100 Pa, approximately 2200 Pa, approximately 2300 Pa, approximately 2400Pa, about 2500Pa, about 2600Pa, about 2700Pa, about 2800Pa, about 2900Pa, about 3000Pa, about 3100Pa, about 3200Pa, about 3300Pa, about 3400Pa, about 3500Pa, about 3600Pa, about 3700Pa, about 3800 Pa, approx. 3900Pa, approx. 4000Pa, approx. 4100Pa, approx. 4200Pa, approx. 4300Pa, approx. 4400Pa, approx. 4500Pa, approx. 4600Pa, approx. 4700Pa, approx. 5300Pa, approximately 5400Pa, approximately 5500Pa, approximately 5600Pa, approximately 5700Pa, approximately 5800Pa, approximately 5900Pa, approximately 6000Pa, approximately 6100Pa, approximately 6200Pa, approximately 6300Pa, approximately 6400Pa, approximately 6500Pa, approximately 6600Pa, approximately 6700Pa, approximately 6800Pa, approximately 6900Pa, approximately 7000Pa, approximately 7100Pa, approximately 7200Pa, approximately 7300Pa, approximately 7400Pa, approximately 7500Pa, approximately 7600Pa, approximately 7700Pa, approximately 7800Pa, approximately 7900Pa, approximately 8000Pa, approximately 8100Pa, A crosslinked polymer matrix according to any one of the above or below paragraphs, having an elastic modulus (G') of approximately 8200 Pa, approximately 8300 Pa, approximately 8400 Pa, approximately 8500 Pa, approximately 8600 Pa, approximately 8700 Pa, approximately 8800 Pa, approximately 8900 Pa, approximately 9000 Pa, approximately 9100 Pa, approximately 9200 Pa, approximately 9300 Pa, approximately 9400 Pa, approximately 9500 Pa, approximately 9600 Pa, approximately 9700 Pa, approximately 9800 Pa, approximately 9900 Pa, or approximately 10000 Pa, or any elastic modulus between the range defined by any two of the aforementioned values.

[0210] Section 21. The matrix is ​​approximately 10 gmf, approximately 20 gmf, approximately 30 gmf, approximately 40 gmf, approximately 50 gmf, approximately 60 gmf, approximately 70 gmf, approximately 80 gmf, approximately 90 gmf, approximately 100 gmf, approximately 110 gmf, approximately 120 gmf, approximately 130 gmf, approximately 140 gmf, approximately 150 gmf, approximately 160 gmf, approximately 170 gmf, About 180gmf, about 190gmf, about 200gmf, about 210gmf, about 220gmf, about 230gmf, about 240gmf, about 250gmf, about 260gm f, about 270gmf, about 280gmf, about 290gmf, about 300gmf, about 310gmf, about 320gmf, about 330gmf, about 340gmf, about 350 A crosslinked polymer matrix as described in either of the above or below paragraphs, having a compressive force value of gmf, approximately 360 gmf, approximately 370 gmf, approximately 380 gmf, approximately 390 gmf, approximately 400 gmf, approximately 410 gmf, approximately 420 gmf, approximately 430 gmf, approximately 440 gmf, approximately 450 gmf, approximately 460 gmf, approximately 470 gmf, approximately 480 gmf, approximately 490 gmf, approximately 500 gmf, approximately 510 gmf, approximately 520 gmf, approximately 530 gmf, approximately 540 gmf, approximately 550 gmf, approximately 560 gmf, approximately 570 gmf, approximately 580 gmf, approximately 590 gmf, or approximately 600 gmf, or any compressive force value between the range defined by any two of the aforementioned values.

[0211] Section 22. A crosslinked polymer matrix according to any one of the above or below sections, wherein the matrix has compressive force values ​​of approximately 100 gmf, approximately 200 gmf, approximately 300 gmf, approximately 400 gmf, approximately 500 gmf, or approximately 600 gmf, or any compressive force value between the range defined by any two of the aforementioned values.

[0212] Item 23. A crosslinked polymer matrix according to either of the above or below items, wherein hyaluronic acid is present in a concentration of approximately 5 mg / ml, approximately 6 mg / ml, approximately 8 mg / ml, approximately 10 mg / ml, approximately 12 mg / ml, approximately 14 mg / ml, approximately 16 mg / ml, approximately 18 mg / ml, approximately 20 mg / ml, approximately 22 mg / ml, approximately 24 mg / ml, approximately 26 mg / ml, approximately 28 mg / ml, approximately 30 mg / ml, approximately 32 mg / ml, approximately 34 mg / ml, or approximately 36 mg / ml, or any concentration between the range defined by any two of the aforementioned values.

[0213] Item 24. A cross-linked polymer matrix according to any one of the above or below items, wherein the collagen comprises type I collagen.

[0214] Item 25. A cross-linked polymer matrix according to any one of the above or below items, wherein the collagen comprises type II collagen.

[0215] Item 26. A cross-linked polymer matrix according to any one of the above or below items, wherein the collagen comprises type III collagen.

[0216] Item 27. A cross-linked polymer matrix according to any one of the above or below items, wherein the collagen comprises 0% to 3% type II collagen.

[0217] Item 28. A cross-linked polymer matrix according to either of the above or below items, wherein the collagen comprises 1% to 3% type I collagen.

[0218] Section 29. A cross-linked polymer matrix according to either of the above or below sections, wherein the matrix contains approximately 0% to approximately 3% type III collagen.

[0219] Item 30. A cross-linked polymer matrix according to either of the above or below items, wherein the collagen comprises approximately 97% to approximately 99% type I collagen.

[0220] Section 31. A crosslinked polymer matrix according to any one of the above or following sections, wherein the collagen comprises a mixture of both type I collagen and type III collagen.

[0221] Section 32. A crosslinked polymer matrix according to either of the above or below sections, wherein collagen is present in concentrations of approximately 1 mg / ml, approximately 2 mg / ml, approximately 4 mg / ml, approximately 6 mg / ml, approximately 8 mg / ml, approximately 10 mg / ml, approximately 12 mg / ml, approximately 14 mg / ml, or any concentration between the range defined by any two of the above values.

[0222] Section 33. A crosslinked polymer matrix according to any one of the above or following sections, further comprising a salt.

[0223] Item 34. A crosslinked polymer matrix according to either of the above or below items, containing NaCl in a range of approximately 50 mM to approximately 400 mM.

[0224] Item 35. A crosslinked polymer matrix according to any one of the above or below items, comprising NaCl, wherein NaCl is present in concentrations of approximately 50 mM, approximately 75 mM, approximately 100 mM, approximately 125 mM, approximately 150 mM, approximately 175 mM, approximately 200 mM, approximately 225 mM, approximately 250 mM, approximately 275 mM, approximately 300 mM, approximately 325 mM, approximately 350 mM, approximately 375 mM, or approximately 400 mM, or any concentration between the range defined by any two of the above values.

[0225] Item 36. A crosslinked polymer matrix according to either of the above or below items, comprising NaCl and having a concentration of NaCl of approximately 150 mM.

[0226] Item 37. A crosslinked polymer matrix according to either of the above or below items, comprising approximately 0.01 M phosphate buffer, approximately 137 mM NaCl, and approximately 2.7 mM KCl.

[0227] Section 38. A cross-linked polymer matrix as described in any one of the above or below sections, formulated for injection or use with a needle and / or cannula.

[0228] Item 39. A cross-linked polymer matrix according to either of the above or below items, wherein the hyaluronic acid component has an average molecular weight of approximately 20,000 daltons to approximately 10,000,000 daltons.

[0229] Section 40. Hyaluronic acid component: Approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,100,000 Daltons, approximately 1,200,000 Daltons, approximately 1,300,000 Daltons, approximately 1,400,000 0 Daltons, approximately 1,500,000 Daltons, approximately 1,600,000 Daltons, approximately 1,700,000 Daltons, approximately 1,800,000 Daltons, approximately 1,900,000 Daltons, approximately 2,000,000 Daltons, approximately 2,100,000 Daltons, approximately 2,200,000 Daltons, approximately 2,300,000 Daltons, approximately 2,400,000 Daltons, approximately 2,500,000 Daltons, approximately 2,600,000 Daltons, approximately 2,700,000 Daltons, approximately 2,800,000 Daltons, approximately 2,900,000 Daltons, approximately 3,000,000 Daltons, approximately 3, 100,000 Daltons, approximately 3,200,000 Daltons, approximately 3,300,000 Daltons, approximately 3,400,000 Daltons, approximately 3,500,000 Daltons, approximately 3,600,000 Daltons, approximately 3,700,000 Daltons, approximately 3,800,000 Daltons, approximately 3,900,000 Daltons, approximately 4,000,000 Daltons, approximately 4,100,000 Daltons, approximately 4,200,000 Daltons, approximately 4,300,000 Daltons, approximately 4,400,000 Daltons, approximately 4,500,000 Daltons, approximately 4,600,000 Daltons, approximately 4,700,000 Daltons ton, approximately 4,800,000 Daltons, approximately 4,900,000 Daltons, approximately 5,000,000 Daltons, approximately 5,100,000 Daltons, approximately 5,200,000 Daltons, approximately 5,300,000 Daltons, approximately 5,400,000 Daltons, approximately 5,500,000 Daltons, approximately 5,600,000 Daltons, approximately 5,700,000 Daltons, approximately 5,800,000 Daltons, approximately 5,900,000 Daltons, approximately 6,000,000 Daltons, approximately 6,100,000 Daltons, approximately 6,200,000 Daltons, approximately 6,300,000 Daltons, approximately 6,400,000 Daltons, approximately 6,500,000 Daltons, approximately 6,600,000 Daltons, approximately 6,700,000 Daltons, approximately 6,800,000 Daltons, approximately 6,900,000 Daltons, approximately 7,000,000 Daltons, approximately 7,100,000 Daltons, approximately 7,200,000 Daltons, approximately 7,300,000 Daltons, approximately 7,400,000 Daltons Approximately 7,500,000 Daltons, approximately 7,600,000 Daltons, approximately 7,700,000 Daltons, approximately 7,800,000 Daltons, approximately 7,900,000 Daltons, approximately 8,000,000 Daltons, approximately 8,100,000 Daltons, approximately 8,200,000 Daltons, approximately 8,300,000 Daltons, approximately 8,400,000 Daltons, approximately 8,50 A crosslinked polymer matrix according to any one of the above or below items, having an average molecular weight of 0,000 Daltons, approximately 8,600,000 Daltons, approximately 8,700,000 Daltons, approximately 8,800,000 Daltons, approximately 8,900,000 Daltons, approximately 9,000,000 Daltons, approximately 9,100,000 Daltons, approximately 9,200,000 Daltons, approximately 9,300,000 Daltons, approximately 9,400,000 Daltons, approximately 9,500,000 Daltons, approximately 9,600,000 Daltons, approximately 9,700,000 Daltons, approximately 9,800,000 Daltons, approximately 9,900,000 Daltons, or approximately 10,000,000 Daltons, or any molecular weight between the range defined by any two of the aforementioned values.

[0230] Section 41. Hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, where the mixture is approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,500,000 Daltons, approximately 2,000,000 Daltons, approximately 2,500,000 Daltons, approximately 3,000,000 Daltons, approximately 3, A crosslinked polymer matrix according to any one of the above or below clauses, comprising hyaluronic acid having an average molecular weight of 500,000 Daltons, approximately 4,000,000 Daltons, approximately 4,500,000 Daltons, approximately 5,000,000 Daltons, approximately 5,500,000 Daltons, approximately 6,000,000 Daltons, approximately 6,500,000 Daltons, approximately 7,500,000 Daltons, approximately 8,000,000 Daltons, approximately 8,500,000 Daltons, approximately 9,000,000 Daltons, approximately 9,500,000 Daltons, and / or approximately 10,000,000 Daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

[0231] Section 42. A composition comprising hyaluronic acid, collagen, lysine, and a buffer solution, wherein the composition is an aqueous hydrogel.

[0232] Item 43. The composition according to any one of the above or below items, wherein hyaluronic acid is crosslinked to collagen by at least one endogenous amine group on collagen and / or at least one amine group present on lysine.

[0233] Section 44. The composition according to any one of the above or following sections, further comprising lidocaine.

[0234] Item 45. The composition according to any one of the above or below items, wherein lidocaine is present in the matrix at a concentration ranging from about 0.15% (w / w) to about 0.45% (w / w).

[0235] Section 46. The composition according to either of the above or below paragraphs, wherein lidocaine is present in a concentration of 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 0.37% (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 any two of the above values.

[0236] Item 47. The composition according to any one of the above or following items, further comprising non-crosslinked HA.

[0237] Item 48. The composition according to any one of the above or below items, wherein the non-crosslinked HA is present in the composition at a maximum concentration of about 5% (w / w).

[0238] Item 49. The composition according to any one of the above or below items, wherein the non-crosslinked HA is present in the composition at a concentration of 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.

[0239] Item 50. The composition according to any one of the above or below items, wherein non-crosslinked HA is present in the composition at a concentration of about 1% (w / w).

[0240] Item 51. The composition according to any one of the above or below items, wherein non-crosslinked HA is present in the composition at a concentration of about 2% (w / w).

[0241] Item 52. The composition according to any one of the above or below items, wherein non-crosslinked HA is present in the composition at a concentration of about 5% (w / w).

[0242] Item 53. The composition according to any one of the above or following items, wherein the non-crosslinked HA improves the extrudeability of the composition.

[0243] Item 54. The composition according to any one of the above or below items, wherein the buffer solution is phosphate-buffered saline.

[0244] Item 55. The composition according to any one of the above or below items, wherein the hyaluronic acid has an average molecular weight of about 20,000 daltons to about 10,000,000 daltons.

[0245] Paragraph 56. Hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, such mixture is approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,500,000 Daltons, approximately 2,000,000 Daltons, approximately 2,500,000 Daltons, and approximately 3,000,000 Daltons. A composition according to any one of the above or below items, comprising hyaluronic acid having a molecular weight of 7,000,000 daltons, approximately 3,500,000 daltons, approximately 4,000,000 daltons, approximately 4,500,000 daltons, approximately 5,000,000 daltons, approximately 5,500,000 daltons, approximately 6,000,000 daltons, approximately 6,500,000 daltons, approximately 7,500,000 daltons, approximately 8,000,000 daltons, approximately 8,500,000 daltons, approximately 9,000,000 daltons, approximately 9,500,000 daltons and / or approximately 10,000,000 daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

[0246] Item 57. A composition according to either of the above or below items, wherein the collagen comprises type I collagen.

[0247] Item 58. A composition according to any one of the above or following items, wherein the collagen comprises type II collagen.

[0248] Item 59. A composition according to any one of the above or following items, wherein the collagen comprises type III collagen.

[0249] Section 60. Approximately 4,000Pa S, approximately 4100Pa S, approximately 4200Pa S, approximately 4300Pa S, approximately 4400Pa S, approximately 4500Pa S, approximately 4600Pa S, approximately 4700Pa S, approximately 4800Pa S, approximately 4900Pa S, approximately 5000Pa S, approximately 5100Pa S, approximately 5200Pa S, approximately 5300Pa S, approximately 5400Pa S, approximately 5500Pa S, approximately 5600Pa S, approximately 5700Pa S, approximately 5800Pa S, approximately 5900Pa S, approximately 6000Pa S, approximately 6100Pa S, approximately 6200Pa S, approximately 6300Pa S, approximately 6400Pa S, approximately 6500Pa S, approximately 6600Pa S, approximately 6700Pa S, approx. 6800Pa S, approx. 6900Pa S, about 7000Pa S, about 7100Pa S, about 7200Pa S, about 7300Pa S, about 7400Pa S, about 7500Pa S, about 7600Pa S, about 7700Pa S, about 7800Pa S, about 7900Pa S, about 8000Pa S, about 8100Pa S, about 8200Pa S, about 8300Pa S, about 8400Pa S, about 8500Pa S, about 8600Pa S, about 8700Pa S, about 8800Pa S, about 8900Pa S, about 9000Pa S, about 9100Pa, about 9200Pa S, about 9300Pa S, about 9400Pa S, about 9500Pa S, about 9600Pa S, about 9700Pa S, approx. 9800Pa S, approx. 9900Pa The composition according to either of the above or below items, having a viscosity of S or about 10,000 Pa S, or any viscosity between the range defined by any two of the aforementioned values.

[0250] Section 61. A composition according to any one of the above or following sections, having a tan delta parameter (G'' / G') of about 0.01 to about 0.5.

[0251] Article 62. A composition according to any one of the above or below articles, having a tan delta parameter (G'' / G') of about 0.01, about 0.05, about 0.10, about 0.15, about 0.20, about 0.25, about 0.30, about 0.35, about 0.40, about 0.45 or about 0.50, or any tan delta parameter between the range defined by any two of the above values.

[0252] Paragraph 63. A composition according to any one of the above or below paragraphs, which is stable for about six months, about twelve months, about eighteen months, about twenty-four months, about thirty months, or about thirty-six months, or for any time between the range defined by any two of the above values.

[0253] Section 64. A composition according to any one of the above or following sections, which is stable at approximately 4°C.

[0254] Section 65. A composition according to any one of the above or following sections, which is stable at approximately 25°C.

[0255] Paragraph 66. The composition described in any one of the above or below paragraphs, which undergoes minimal decomposition in approximately 6 months, approximately 12 months, approximately 18 months, approximately 24 months, approximately 30 months, or approximately 36 months, or any time between the ranges defined by any two of the above values.

[0256] Section 67. A method for crosslinking hyaluronic acid and collagen, comprising: dissolving collagen, hyaluronic acid and lysine in an aqueous solution to form a pre-reaction aqueous solution having a pH of about 4 to about 6; preparing a second solution containing a water-soluble carbodiimide and N-hydroxysuccinimide or N-hydroxysulfosuccinimide; adding the second solution to the pre-reaction aqueous solution to form a crosslinking reaction; and reacting the crosslinking reaction by crosslinking hyaluronic acid and collagen with lysine, wherein hyaluronic acid is crosslinked to collagen by at least one endogenous amine group on collagen and / or at least one amine group present on lysine, the HA and collagen are only slightly degraded, the structures of HA and collagen remain intact, and thereby form a crosslinked polymer matrix.

[0257] Section 68. The method according to any one of the above or below sections, wherein the aqueous solution before the reaction has a pH of about 4.0, about 4.5, about 5.0, about 5.5, or about 6.0, or any pH within the range defined by any two of the above values.

[0258] Section 69. The method according to any one of the above or below sections, further comprising adding lidocaine to a crosslinked polymer matrix.

[0259] Item 70. The method according to any one of the above or below items, wherein lidocaine is added to a crosslinked polymer matrix to a concentration ranging from about 0.15% (w / w) to about 0.45% (w / w).

[0260] Paragraph 71. The method according to either of the above or below paragraphs, wherein lidocaine is present in a concentration of approximately 0.15% (w / w), approximately 0.17% (w / w), approximately 0.19% (w / w), approximately 0.21% (w / w), approximately 0.23% (w / w), approximately 0.25% (w / w), approximately 0.27% (w / w), approximately 0.29% (w / w), approximately 0.31% (w / w), approximately 0.33% (w / w), approximately 0.35% (w / w), approximately 0.37% (w / w), approximately 0.37% (w / w), approximately 0.39% (w / w), approximately 0.41% (w / w), approximately 0.43% (w / w), or approximately 0.45% (w / w) of the matrix, or any concentration between the range defined by any two of the aforementioned values.

[0261] Item 72. The method according to any one of the above or following items, further comprising imparting an activator comprising a triazole, a fluorinated phenol, succinimide, or sulfosuccinimide.

[0262] Paragraph 73. The method described in any one of the above or below paragraphs, performed at a temperature of approximately 2°C, approximately 4°C, approximately 6°C, approximately 8°C, approximately 10°C, approximately 12°C, approximately 14°C, approximately 16°C, approximately 18°C, approximately 20°C, approximately 22°C, approximately 24°C, approximately 26°C, approximately 28°C, approximately 30°C, approximately 32°C, approximately 34°C, or approximately 36°C, or at a temperature between any two of the above-mentioned values.

[0263] Item 74. The method according to any one of the above or below items, wherein the reaction step is carried out at approximately 4 to approximately 35°C.

[0264] Item 75. The method according to either of the above or below items, wherein the reaction step is carried out at approximately 4°C or approximately 22°C.

[0265] Paragraph 76. The method according to any one of the above or below paragraphs, further comprising purifying a crosslinked polymer matrix, wherein the purification step is performed by dialysis.

[0266] Item 77. The method according to any one of the above or below items, wherein the purification step is carried out at 2°C to 30°C.

[0267] Item 78. The method according to any one of the above or below items, wherein dialysis is performed at any temperature between about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, or any range defined by any two of the foregoing values.

[0268] Item 79. The method according to any one of the above or below items, wherein the purification step is performed at about 2 °C to about 8 °C.

[0269] Item 80. The method according to any one of the above or below items, wherein the cross-linking reaction is performed at about 2 °C to about 35 °C.

[0270] Item 81. The method according to any one of the above or below items, wherein the cross-linking reaction is performed at about 2 °C to about 8 °C.

[0271] Item 82. The method according to any one of the above or below items, which is performed below room temperature.

[0272] Item 83. The method according to any one of the above or below items, wherein the pH of the cross-linking reaction mixture is about 4.0 to about 6.0.

[0273] Item 84. The method according to any one of the above or below items, wherein the pre-reaction solution contains a salt, and the salt contains sodium chloride at a concentration of 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 or about 400 mM in the cross-linking reaction mixture, or any concentration between any range defined by any two of the foregoing values.

[0274] Item 85. The method according to any one of the above or below items, wherein the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration of about 20 mM to about 200 mM in the cross-linking reaction mixture.

[0275] Paragraph 86. The method according to any one of the above or below paragraphs, wherein the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in 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, or about 200 mM, or any concentration within the range defined by any of the above values.

[0276] Paragraph 87. The method according to any one of the above or below paragraphs, wherein the molar-to-molar ratio of repeating units of water-soluble carbodiimide to repeating units of hyaluronic acid is about 0.5 to about 2.0.

[0277] Paragraph 88. The method according to any one of the above or below paragraphs, wherein the molar-to-molar ratio of repeating units of water-soluble carbodiimide and hyaluronic acid, where the molar-to-molar ratio of repeating units of water-soluble carbodiimide to hyaluronic acid is 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, or about 2.0.

[0278] Paragraph 89. The method according to any one of the above or below paragraphs, wherein the molar:molar (lysine repeating unit:HA repeating unit) ratio of lysine to hyaluronic acid is about 0.01 to about 0.6.

[0279] Section 90. The molar:molar (lysine repeating unit:HA repeating unit) ratio of lysine and hyaluronic acid is approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28. The method described in any one of the above or below items, wherein the ratio is approximately 0.29, approximately 0.3, approximately 0.31, approximately 0.32, approximately 0.33, approximately 0.34, approximately 0.35, approximately 0.36, approximately 0.37, approximately 0.38, approximately 0.39, approximately 0.4, approximately 0.41, approximately 0.42, approximately 0.43, approximately 0.44, approximately 0.45, approximately 0.46, approximately 0.47, approximately 0.48, approximately 0.49, approximately 0.5, approximately 0.51, approximately 0.52, approximately 0.53, approximately 0.54, approximately 0.55, approximately 0.56, approximately 0.57, approximately 0.58, approximately 0.59, or approximately 0.6.

[0280] Section 91. The method according to any one of the above or below sections, further comprising adding non-crosslinked HA to a crosslinked polymer matrix.

[0281] Item 92. The method according to any one of the above or below items, wherein non-crosslinked HA is added to a crosslinked polymer matrix to a maximum concentration of 5% w / w.

[0282] Paragraph 93. The method according to either of the above or below paragraphs, wherein non-crosslinked HA is added to the matrix to a concentration of about 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 to any concentration between the range defined by any two of the above values.

[0283] Item 94. The method according to any one of the above or below items, wherein non-crosslinked HA is added to the matrix to a concentration of about 1% (w / w).

[0284] Item 95. The method according to any one of the above or below items, wherein non-crosslinked HA is added to the matrix to a concentration of about 3% (w / w).

[0285] Item 96. The method according to any one of the above or below items, wherein non-crosslinked HA is added to the matrix to a concentration of about 5% (w / w).

[0286] Paragraph 97. The method according to any one of the above or following paragraphs, further comprising sterilizing a crosslinked polymer matrix, transferring the crosslinked polymer matrix to a container for steam sterilization, and sterilizing a hydrogel by steam sterilization.

[0287] paragraph 98. The method according to any one of the above or below paragraphs, wherein the container is a syringe.

[0288] Paragraph 99. The method according to any one of the above or below paragraphs, further comprising dialysis of a crosslinked polymer matrix, wherein the dialysis is performed through a membrane having a molecular weight cutoff of about 1,000 daltons to about 100,000 daltons, and the dialysis is performed before sterilization.

[0289] Paragraph 100. The method according to any one of the above or below paragraphs, wherein dialysis is performed with phosphate-buffered saline.

[0290] Item 101. The method according to any one of the above or below items, wherein the hyaluronic acid in the pre-reaction solution is hydrated for at least about 60 minutes before the second solution is added.

[0291] Section 102. The method according to any one of the above or below sections, wherein the crosslinking reaction is carried out for approximately 16 to approximately 24 hours.

[0292] Section 103. A crosslinked polymer matrix prepared by the process described in any one of the above or below sections.

[0293] Item 104. A method for improving the aesthetics of human anatomical features, comprising injecting a composition into human tissue, thereby improving the aesthetics of the anatomical features, wherein the composition comprises a crosslinked polymer matrix comprising hyaluronic acid, lysine, and collagen, and the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine.

[0294] Item 105. The method according to any one of the above or below items, wherein the crosslinked polymer matrix further comprises lidocaine.

[0295] Item 106. The method according to any one of the above or below items, wherein the crosslinked polymer matrix further comprises non-crosslinked HA.

[0296] Item 107. Hyaluronic acid component is approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,100,000 Daltons, approximately 1,200,000 Daltons, approximately 1,300,000 Daltons, approximately 1,400,000 Daltons 00 Daltons, approximately 1,500,000 Daltons, approximately 1,600,000 Daltons, approximately 1,700,000 Daltons, approximately 1,800,000 Daltons, approximately 1,900,000 Daltons, approximately 2,000,000 Daltons, approximately 2,100,000 Daltons, approximately 2,200,000 Daltons, approximately 2,300,000 Daltons, approximately 2,400,000 Daltons, approximately 2,500,000 Daltons, approximately 2,600,000 Daltons, approximately 2,700,000 Daltons, approximately 2,800,000 Daltons, approximately 2,900,000 Daltons, approximately 3,000,000 Daltons, approximately 3, 100,000 Daltons, approximately 3,200,000 Daltons, approximately 3,300,000 Daltons, approximately 3,400,000 Daltons, approximately 3,500,000 Daltons, approximately 3,600,000 Daltons, approximately 3,700,000 Daltons, approximately 3,800,000 Daltons, approximately 3,900,000 Daltons, approximately 4,000,000 Daltons, approximately 4,100,000 Daltons, approximately 4,200,000 Daltons, approximately 4,300,000 Daltons, approximately 4,400,000 Daltons, approximately 4,500,000 Daltons, approximately 4,600,000 Daltons, approximately 4,700,000 Daltons ton, approximately 4,800,000 Daltons, approximately 4,900,000 Daltons, approximately 5,000,000 Daltons, approximately 5,100,000 Daltons, approximately 5,200,000 Daltons, approximately 5,300,000 Daltons, approximately 5,400,000 Daltons, approximately 5,500,000 Daltons, approximately 5,600,000 Daltons, approximately 5,700,000 Daltons, approximately 5,800,000 Daltons, approximately 5,900,000 Daltons, approximately 6,000,000 Daltons, approximately 6,100,000 Daltons, approximately 6,200,000 Daltons, approximately 6,300,000 Daltons, approximately 6,400,000 Daltons, approximately 6,500,000 Daltons, approximately 6,600,000 Daltons, approximately 6,700,000 Daltons, approximately 6,800,000 Daltons, approximately 6,900,000 Daltons, approximately 7,000,000 Daltons, approximately 7,100,000 Daltons, approximately 7,200,000 Daltons, approximately 7,300,000 Daltons, approximately 7,400,000 Daltons Dalton, approximately 7,500,000 Dalton, approximately 7,600,000 Dalton, approximately 7,700,000 Dalton, approximately 7,800,000 Dalton, approximately 7,900,000 Dalton, approximately 8,000,000 Dalton, approximately 8,100,000 Dalton, approximately 8,200,000 Dalton, approximately 8,300,000 Dalton, approximately 8,400,000 Dalton, The method according to any one of the above or below items, having an average molecular weight of approximately 8,500,000 Daltons, approximately 8,600,000 Daltons, approximately 8,700,000 Daltons, approximately 8,800,000 Daltons, approximately 8,900,000 Daltons, approximately 9,000,000 Daltons, approximately 9,100,000 Daltons, approximately 9,200,000 Daltons, approximately 9,300,000 Daltons, approximately 9,400,000 Daltons, approximately 9,500,000 Daltons, approximately 9,600,000 Daltons, approximately 9,700,000 Daltons, approximately 9,800,000 Daltons, approximately 9,900,000 Daltons, or approximately 10,000,000 Daltons, or any molecular weight between the range defined by any two of the aforementioned values.

[0297] Paragraph 108. Hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, where the mixture is approximately 20,000 daltons, approximately 40,000 daltons, approximately 60,000 daltons, approximately 80,000 daltons, approximately 100,000 daltons, approximately 200,000 daltons, approximately 300,000 daltons, approximately 400,000 daltons, approximately 500,000 daltons, approximately 600,000 daltons, approximately 700,000 daltons, approximately 800,000 daltons, approximately 900,000 daltons, approximately 1,000,000 daltons, approximately 1,500,000 daltons, approximately 2,000,000 daltons, approximately 2,500,000 daltons, and approximately 3,000,000 daltons. The method according to any one of the above or below items, comprising hyaluronic acid having an average molecular weight of 1 ton, approximately 3,500,000 daltons, approximately 4,000,000 daltons, approximately 4,500,000 daltons, approximately 5,000,000 daltons, approximately 5,500,000 daltons, approximately 6,000,000 daltons, approximately 6,500,000 daltons, approximately 7,500,000 daltons, approximately 8,000,000 daltons, approximately 8,500,000 daltons, approximately 9,000,000 daltons, approximately 9,500,000 daltons and / or approximately 1,000,000 daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

[0298] paragraph 109. The method according to any one of the above or below paragraphs, wherein the collagen comprises type I collagen and / or type III collagen.

[0299] Section 110. A method for improving the appearance of an individual, comprising injecting a composition into the tissue of the individual at an injection site to improve the aesthetics of anatomical features, wherein infiltrating cells from the tissue are integrated into the composition at the injection site, and depositing new collagen within the composition, wherein the composition comprises a cross-linked polymer matrix comprising hyaluronic acid, lysine, and collagen, wherein the hyaluronic acid is cross-linked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine, and depositing new collagen, wherein the tissue into which the composition has been injected is shown to have tissue integration, collagen deposition, and angiogenesis.

[0300] paragraph 111. The method according to either of the above or below paragraphs, wherein the composition further comprises lidocaine.

[0301] Item 112. The method according to any one of the above or following items, wherein the composition further comprises non-crosslinked HA.

[0302] Section 113. The method according to any one of the above or below sections, comprising injecting the composition into the chin, jawline, lips, or nasolabial folds.

[0303] Paragraph 114. The method described in any one of the above or below paragraphs for improving symmetry between facial features.

[0304] Article 115. The method described in any one of the above or below paragraphs for increasing and restoring volume to facial features.

[0305] Item 116. The method according to any one of the above or below items for increasing, correcting, restoring, or giving volume to the chin, lips, jawline, or nasolabial folds.

[0306] Item 117. The method according to any one of the above or below items, for injecting a composition into the tear trough of a solid.

[0307] Item 118. The method according to any one of the above or below paragraphs, comprising injecting the composition into an area including atrophy of the skin and / or atrophy of the fat body.

[0308] Section 119. The method according to any one of the above or below sections, wherein the composition provides a natural appearance, feel, and movement to the injected tissue, and the composition results in increased collagen infiltration from the surrounding tissue at the injection site.

[0309] Paragraph 120. The method according to any one of the above or below paragraphs, wherein the duration of the composition is extended as a result of tissue integration into the injection site.

[0310] Paragraph 121. The method according to any one of the above or below paragraphs, for improving the hydration and elasticity of the skin surrounding the injection site.

[0311] Paragraph 122. A method for increasing collagen infiltration into tissue, comprising injecting a composition into the tissue of an individual to create a depot of a dermal filler comprising the composition comprising a crosslinked polymer matrix comprising hyaluronic acid, lysine, and collagen, wherein the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine, wherein cells from the surrounding tissue infiltrate the depot of the dermal filler comprising the composition, the cells integrate with the composition and deposit new collagen into the composition, thereby creating infiltrated tissue within the composition, and blood vessels connect the infiltrated tissue within the composition to the blood supply of the individual's body.

[0312] Paragraph 123. The method according to any one of the above or below paragraphs, wherein the matrix further comprises lidocaine.

[0313] Item 124. The method according to any one of the above or following items, wherein the composition further comprises non-crosslinked HA.

[0314] Item 125. Hyaluronic acid is approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,100,000 Daltons, approximately 1,200,000 Daltons, approximately 1,300,000 Daltons, approximately 1,400,000 Dalton, approximately 1,500,000 Dalton, approximately 1,600,000 Dalton, approximately 1,700,000 Dalton, approximately 1,800,000 Dalton, approximately 1,900,000 Dalton, approximately 2,000,000 Dalton, approximately 2,100,000 Dalton, approximately 2,200,000 Dalton, approximately 2,300,000 Dalton, approximately 2,400,000 Dalton, approximately 2,500,000 Dalton, approximately 2,600,000 Dalton, approximately 2,700,000 Dalton, approximately 2,800,000 Dalton, approximately 2,900,000 Dalton, approximately 3,000,000 Dalton, approximately 3,1 00,000 Daltons, approximately 3,200,000 Daltons, approximately 3,300,000 Daltons, approximately 3,400,000 Daltons, approximately 3,500,000 Daltons, approximately 3,600,000 Daltons, approximately 3,700,000 Daltons, approximately 3,800,000 Daltons, approximately 3,900,000 Daltons, approximately 4,000,000 Daltons, approximately 4,100,000 Daltons, approximately 4,200,000 Daltons, approximately 4,300,000 Daltons, approximately 4,400,000 Daltons, approximately 4,500,000 Daltons, approximately 4,600,000 Daltons, approximately 4,700,000 Daltons Approximately 4,800,000 Daltons, approximately 4,900,000 Daltons, approximately 5,000,000 Daltons, approximately 5,100,000 Daltons, approximately 5,200,000 Daltons, approximately 5,300,000 Daltons, approximately 5,400,000 Daltons, approximately 5,500,000 Daltons, approximately 5,600,000 Daltons, approximately 5,700,000 Daltons, approximately 5,800,000 Daltons, approximately 5,900,000 Daltons, approximately 6,000,000 Daltons, approximately 6,100,000 Daltons, approximately 6,200,000 Daltons, approximately 6,300,000 Daltons, approximately 6,400,000 Daltons, approximately 6,500,000 Daltons, approximately 6,600,000 Daltons, approximately 6,700,000 Daltons, approximately 6,800,000 Daltons, approximately 6,900,000 Daltons, approximately 7,000,000 Daltons, approximately 7,100,000 Daltons, approximately 7,200,000 Daltons, approximately 7,300,000 Daltons, approximately 7,400,000 Daltons Dalton, approximately 7,500,000 Dalton, approximately 7,600,000 Dalton, approximately 7,700,000 Dalton, approximately 7,800,000 Dalton, approximately 7,900,000 Dalton, approximately 8,000,000 Dalton, approximately 8,100,000 Dalton, approximately 8,200,000 Dalton, approximately 8,300,000 Dalton, approximately 8,400,000 Dalton, approximately The method according to any one of the above or below items, having an average molecular weight of 8,500,000 Daltons, approximately 8,600,000 Daltons, approximately 8,700,000 Daltons, approximately 8,800,000 Daltons, approximately 8,900,000 Daltons, approximately 9,000,000 Daltons, approximately 9,100,000 Daltons, approximately 9,200,000 Daltons, approximately 9,300,000 Daltons, approximately 9,400,000 Daltons, approximately 9,500,000 Daltons, approximately 9,600,000 Daltons, approximately 9,700,000 Daltons, approximately 9,800,000 Daltons, approximately 9,900,000 Daltons, or approximately 10,000,000 Daltons, or any other molecular weight between the range defined by any two of the aforementioned values.

[0315] Paragraph 126. Hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, the mixture being approximately 20,000 daltons, approximately 40,000 daltons, approximately 60,000 daltons, approximately 80,000 daltons, approximately 100,000 daltons, approximately 200,000 daltons, approximately 300,000 daltons, approximately 400,000 daltons, approximately 500,000 daltons, approximately 600,000 daltons, approximately 700,000 daltons, approximately 800,000 daltons, approximately 900,000 daltons, approximately 1,000,000 daltons, approximately 1,500,000 daltons, approximately 2,000,000 daltons, approximately 2,500,000 daltons, approximately 3,000,000 daltons. The method according to any one of the above or below items, comprising hyaluronic acid having an average molecular weight of 1 ton, approximately 3,500,000 daltons, approximately 4,000,000 daltons, approximately 4,500,000 daltons, approximately 5,000,000 daltons, approximately 5,500,000 daltons, approximately 6,000,000 daltons, approximately 6,500,000 daltons, approximately 7,500,000 daltons, approximately 8,000,000 daltons, approximately 8,500,000 daltons, approximately 9,000,000 daltons, approximately 9,500,000 daltons and / or approximately 10,000,000 daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

[0316] Paragraph 127. The method according to any one of the above or below paragraphs, wherein the collagen comprises type I collagen, type II collagen and / or type III collagen.

[0317] Item 128. The method according to any one of the above or following items, wherein the composition comprises about 13 mg / ml of hyaluronic acid.

[0318] Item 129. The method according to any one of the above or following items, wherein the composition comprises about 20 mg / ml of hyaluronic acid, about 22 mg / ml of hyaluronic acid, about 24 mg / ml, about 26 mg / ml of hyaluronic acid, about 28 mg / ml of hyaluronic acid, or about 30 mg / ml of hyaluronic acid.

[0319] Item 130. The method described in any one of the above or below items, comprising injecting the product into the superficial dermis to improve skin texture, fine wrinkles, or roughness.

[0320] In some embodiments, any of the provisions of this specification may depend on any one of the independent or dependent provisions. In one embodiment, any of the provisions (e.g., dependent or independent) may be combined with any one or more other provisions (e.g., dependent or independent). In one embodiment, a claim may include some or all of the words (e.g., process, operation, means or component) contained in a section, sentence, phrase or paragraph. In one embodiment, a claim may include some or all of the words contained in one or more sections, sentences, phrases or paragraphs. In one embodiment, some of the words in each section, sentence, phrase or paragraph may be deleted. In one embodiment, additional words or elements may be added to a section, sentence, phrase or paragraph. In one embodiment, the subject art may be implemented without utilizing some of the components, elements, functions or operations described herein. In one embodiment, the subject art may be implemented by utilizing additional components, elements, functions or operations.

[0321] The terms “a,” “an,” “the,” and similar references used in the context describing the present invention (particularly in the context of the following claims) should be construed to encompass both singular and plural unless otherwise specifically indicated herein or unless clearly inconsistent with the context. The descriptions of value ranges herein are intended merely as a convenient way to refer individually to each distinct value that falls within that range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually described herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or unless clearly inconsistent with the context. The use of any examples or exemplary language provided herein (e.g., “etc.”) is intended merely to better illustrate the present invention and not to limit the scope of the claimed invention. No language herein should be construed to indicate an unclaimed element essential to the practice of the present invention.

[0322] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limitation. Members of each group may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in or excluded from a group for convenience and / or patentability reasons. In the event of such inclusion or exclusion, this specification shall be deemed to include the modified group and thus satisfy the description of all Markush groups used in the appended claims.

[0323] Specific embodiments of the Invention, including the best mode for carrying out the Invention as known to the inventors, are described herein. Naturally, variations of these described embodiments will be apparent to those skilled in the art by reading the foregoing description. The inventors expect that those skilled in the art will appropriately use such variations, and the inventors intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all modifications and equivalents of the subject matter described in the appended claims, in a manner permitted by applicable law. Furthermore, unless otherwise indicated herein, or unless clearly inconsistent with the context, any combination of the elements described above in all possible variations thereof is incorporated into the Invention.

[0324] The specific embodiments disclosed herein may be further limited in claims that consist of language or take advantage of essentially consisting of language. Where used in claims, the transitional term "consisting of" excludes elements, processes, or components not specified in the claims, whether added at filing or by amendment. The transitional term "consisting essentially of" limits the scope of the claims to specific materials or processes that do not substantially affect the basic and novel features. Embodiments of the invention as claimed in this manner are essentially or expressly described and enabled herein.

[0325] Furthermore, numerous patents and printed publications are referenced throughout this specification. Each of the references and printed publications cited above is incorporated herein by reference in its entirety.

[0326] Finally, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be adopted are within the scope of the invention. Therefore, alternative configurations of the invention can be utilized in accordance with the teachings herein, not as examples but as examples. Accordingly, the present invention is not limited to those illustrated and described in detail.

Claims

1. Lysine and, Hyaluronic acid and, Collagen and A crosslinked polymer matrix containing, A crosslinked polymer matrix in which the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine.

2. The crosslinked polymer matrix according to claim 1, further comprising lidocaine.

3. The crosslinked polymer matrix according to claim 1 or 2, wherein the lidocaine is present in the matrix at a concentration ranging from about 0.15% (w / w) to about 0.45% (w / w).

4. The crosslinked polymer matrix according to any one of claims 1 to 3, wherein the lidocaine is present in a concentration of approximately 0.15% (w / w), approximately 0.17% (w / w), approximately 0.19% (w / w), approximately 0.21% (w / w), approximately 0.23% (w / w), approximately 0.25% (w / w), approximately 0.27% (w / w), approximately 0.29% (w / w), approximately 0.31% (w / w), approximately 0.33% (w / w), approximately 0.35% (w / w), approximately 0.37% (w / w), approximately 0.37% (w / w), approximately 0.39% (w / w), approximately 0.41% (w / w), approximately 0.43% (w / w), or approximately 0.45% (w / w) of the matrix, or any concentration between the ranges defined by any two of the aforementioned values.

5. The crosslinked polymer matrix according to any one of claims 1 to 4, wherein the lidocaine is present in the matrix at a concentration ranging from about 0.27% (w / w) to about 0.33% (w / w).

6. The crosslinked polymer matrix according to any one of claims 1 to 5, wherein the matrix further comprises non-crosslinked HA.

7. The crosslinked polymer matrix according to claim 6, wherein the non-crosslinked HA has a maximum concentration of about 5% (w / w) in the matrix.

8. The crosslinked polymer matrix according to claim 6 or 7, wherein the non-crosslinked HA is present in the matrix at a concentration of about 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 range defined by any two of the aforementioned values.

9. The crosslinked polymer matrix according to any one of claims 6 to 8, wherein the non-crosslinked HA is present in the matrix at a concentration of about 1% (w / w).

10. The crosslinked polymer matrix according to any one of claims 6 to 8, wherein the non-crosslinked HA is present in the matrix at a concentration of about 2% (w / w).

11. The crosslinked polymer matrix according to any one of claims 6 to 8, wherein the non-crosslinked HA is present in the matrix at a concentration of about 5% (w / w).

12. The crosslinked polymer matrix according to any one of claims 6 to 11, wherein the non-crosslinked HA improves the extrudeability of the polymer matrix.

13. A crosslinked polymer matrix according to any one of claims 1 to 12, which is stable for approximately 6 months, approximately 12 months, approximately 18 months, approximately 24 months, approximately 30 months, or approximately 36 months, or for any time within the range defined by any two of the aforementioned values.

14. A crosslinked polymer matrix according to any one of claims 1 to 13, which is stable at a temperature of approximately 4°C to approximately 25°C.

15. A crosslinked polymer matrix according to any one of claims 1 to 14, which is stable at a temperature of approximately 4°C.

16. A crosslinked polymer matrix according to any one of claims 1 to 15, which is stable at a temperature of approximately 25°C.

17. A crosslinked polymer matrix according to any one of claims 1 to 16, which is stable at any point in time between approximately 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, or any point in time between any two of the aforementioned values.

18. A crosslinked polymer matrix according to any one of claims 1 to 17, wherein degradation is minimal for approximately 6 months, approximately 12 months, approximately 18 months, approximately 24 months, approximately 30 months, or approximately 36 months, or for any time within the range defined by any two of the aforementioned values.

19. The crosslinked polymer matrix according to any one of claims 1 to 18, wherein the matrix has an elastic modulus (G') of about 30 Pa to about 10,000 Pa, or any elastic modulus between the range defined by any two of the aforementioned values.

20. The aforementioned matrix has approximately 30 Pa, approximately 40 Pa, approximately 50 Pa, approximately 60 Pa, approximately 70 Pa, approximately 80 Pa, approximately 90 Pa, approximately 100 Pa, approximately 200 Pa, approximately 300 Pa, approximately 400 Pa, approximately 500 Pa, approximately 600 Pa, approximately 700 Pa, approximately 800 Pa, approximately 900 Pa, approximately 1000 Pa, approximately 1100 Pa, approximately 1200 Pa, approximately 1300 Pa, approximately 1400 Pa, approximately 1500 Pa, approximately 1600 Pa, approximately 1700 Pa, approximately 1800 Pa, approximately 1900 Pa, approximately 2000 Pa, approximately 2100 Pa, approximately 2200 Pa, approximately 2300 Pa, and approximately 24 00Pa, about 2500Pa, about 2600Pa, about 2700Pa, about 2800Pa, about 2900Pa, about 3000Pa, about 3100Pa, about 3200Pa, about 3300Pa, about 3400Pa, about 3500Pa, about 3600Pa, about 3700Pa, about 3800P a, about 3900Pa, about 4000Pa, about 4100Pa, about 4200Pa, about 4300Pa, about 4400Pa, about 4500Pa, Approximately 4600Pa, approximately 4700Pa, approximately 4800Pa, approximately 4900Pa, approximately 5000Pa, approximately 5100Pa, approximately 5200Pa, approximately 5 300 Pa, approximately 5400 Pa, approximately 5500 Pa, approximately 5600 Pa, approximately 5700 Pa, approximately 5800 Pa, approximately 5900 Pa, approximately 6000 Pa, approximately 6100 Pa, approximately 6200 Pa, approximately 6300 Pa, approximately 6400 Pa, approximately 6500 Pa, approximately 6600 Pa, approximately 6700 Pa, approximately 6800 Pa, approximately 6900 Pa, approximately 7000 Pa, approximately 7100 Pa, approximately 7200 Pa, approximately 7300 Pa, approximately 7400 Pa, approximately 7500 Pa, approximately 7600 Pa, approximately 7700 Pa, approximately 7800 Pa, approximately 7900 Pa, approximately 8000 Pa, approximately 8100 Pa, A crosslinked polymer matrix according to any one of claims 1 to 19, having an elastic modulus (G') of approximately 8200 Pa, approximately 8300 Pa, approximately 8400 Pa, approximately 8500 Pa, approximately 8600 Pa, approximately 8700 Pa, approximately 8800 Pa, approximately 8900 Pa, approximately 9000 Pa, approximately 9100 Pa, approximately 9200 Pa, approximately 9300 Pa, approximately 9400 Pa, approximately 9500 Pa, approximately 9600 Pa, approximately 9700 Pa, approximately 9800 Pa, approximately 9900 Pa, or approximately 10000 Pa, or any elastic modulus between the range defined by any two of the aforementioned values.

21. The matrix is ​​approximately 10 gmf, approximately 20 gmf, approximately 30 gmf, approximately 40 gmf, approximately 50 gmf, approximately 60 gmf, approximately 70 gmf, approximately 80 gmf, approximately 90 gmf, approximately 100 gmf, approximately 110 gmf, approximately 120 gmf, approximately 130 gmf, approximately 140 gmf, approximately 150 gmf, approximately 160 gmf, approximately 170 gmf, approximately 1 80gmf, about 190gmf, about 200gmf, about 210gmf, about 220gmf, about 230gmf, about 240gmf, about 250gmf, about 260gmf , about 270gmf, about 280gmf, about 290gmf, about 300gmf, about 310gmf, about 320gmf, about 330gmf, about 340gmf, about 350g A crosslinked polymer matrix according to any one of claims 1 to 20, having a compressive force value of mf, approximately 360 gmf, approximately 370 gmf, approximately 380 gmf, approximately 390 gmf, approximately 400 gmf, approximately 410 gmf, approximately 420 gmf, approximately 430 gmf, approximately 440 gmf, approximately 450 gmf, approximately 460 gmf, approximately 470 gmf, approximately 480 gmf, approximately 490 gmf, approximately 500 gmf, approximately 510 gmf, approximately 520 gmf, approximately 530 gmf, approximately 540 gmf, approximately 550 gmf, approximately 560 gmf, approximately 570 gmf, approximately 580 gmf, approximately 590 gmf, or approximately 600 gmf, or any compressive force value between the range defined by any two of the aforementioned values.

22. The crosslinked polymer matrix according to any one of claims 1 to 21, wherein the matrix has compressive force values ​​of about 100 gmf, about 200 gmf, about 300 gmf, about 400 gmf, about 500 gmf, or about 600 gmf, or any compressive force value between the range defined by any two of the aforementioned values.

23. The crosslinked polymer mixture according to any one of claims 1 to 22, wherein the hyaluronic acid is at a concentration of approximately 5 mg / ml, approximately 6 mg / ml, approximately 8 mg / ml, approximately 10 mg / ml, approximately 12 mg / ml, approximately 14 mg / ml, approximately 16 mg / ml, approximately 18 mg / ml, approximately 20 mg / ml, approximately 22 mg / ml, approximately 24 mg / ml, approximately 26 mg / ml, approximately 28 mg / ml, approximately 30 mg / ml, approximately 32 mg / ml, approximately 34 mg / ml, or approximately 36 mg / ml, or any concentration between the range defined by any two of the aforementioned values.

24. The crosslinked polymer matrix according to any one of claims 1 to 23, wherein the collagen includes type I collagen.

25. The crosslinked polymer matrix according to any one of claims 1 to 24, wherein the collagen comprises type II collagen.

26. The crosslinked polymer matrix according to any one of claims 1 to 25, wherein the collagen comprises type III collagen.

27. The crosslinked polymer matrix according to any one of claims 1 to 26, wherein the collagen comprises 0% to 3% type II collagen.

28. The crosslinked polymer matrix according to any one of claims 1 to 27, wherein the collagen comprises 1% to 3% type I collagen.

29. The crosslinked polymer matrix according to any one of claims 1 to 28, wherein the matrix contains about 0% to about 3% type III collagen.

30. The crosslinked polymer matrix according to any one of claims 1 to 29, wherein the collagen comprises about 97% to about 99% type I collagen.

31. The crosslinked polymer matrix according to any one of claims 1 to 30, wherein the collagen comprises a mixture of both type I collagen and type III collagen.

32. The crosslinked polymer matrix according to any one of claims 1 to 31, wherein the collagen has a concentration of about 1 mg / ml, 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, or any concentration between the range defined by any two of the aforementioned values.

33. A crosslinked polymer matrix according to any one of claims 1 to 32, further comprising a salt.

34. The crosslinked polymer matrix according to claim 33, comprising NaCl in a range of approximately 50 mM to approximately 400 mM.

35. A crosslinked polymer matrix according to any one of claims 1 to 34, comprising NaCl, wherein the NaCl is present in a concentration of 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, about 325 mM, about 350 mM, about 375 mM, or about 400 mM, or any concentration between the range defined by any two of the aforementioned values.

36. A crosslinked polymer matrix according to any one of claims 1 to 35, comprising NaCl, wherein the NaCl has a concentration of about 150 mM.

37. A crosslinked polymer matrix according to any one of claims 1 to 35, comprising approximately 0.01 M phosphate buffer, approximately 137 mM NaCl, and approximately 2.7 mM KCl.

38. A crosslinked polymer matrix according to any one of claims 1 to 37, formulated for injection or use with a needle and / or cannula.

39. The crosslinked polymer matrix according to any one of claims 1 to 38, wherein the hyaluronic acid component has an average molecular weight of about 20,000 daltons to about 10,000,000 daltons.

40. The aforementioned hyaluronic acid component is approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,100,000 Daltons, approximately 1,200,000 Daltons, approximately 1,300,000 Daltons, and approximately 1,400,000 Daltons. Alton, approximately 1,500,000 Dalton, approximately 1,600,000 Dalton, approximately 1,700,000 Dalton, approximately 1,800,000 Dalton, approximately 1,900,000 Dalton, approximately 2,000,000 Dalton, approximately 2,100,000 Dalton, approximately 2,200,000 Dalton, approximately 2,300,000 Dalton, approximately 2,400,000 Dalton, approximately 2,500,000 Dalton, approximately 2,600,000 Dalton, approximately 2,700,000 Dalton, approximately 2,800,000 Dalton, approximately 2,900,000 Dalton, approximately 3,000,000 Dalton, approximately 3,10 0,000 Daltons, approximately 3,200,000 Daltons, approximately 3,300,000 Daltons, approximately 3,400,000 Daltons, approximately 3,500,000 Daltons, approximately 3,600,000 Daltons, approximately 3,700,000 Daltons, approximately 3,800,000 Daltons, approximately 3,900,000 Daltons, approximately 4,000,000 Daltons, approximately 4,100,000 Daltons, approximately 4,200,000 Daltons, approximately 4,300,000 Daltons, approximately 4,400,000 Daltons, approximately 4,500,000 Daltons, approximately 4,600,000 Daltons, approximately 4,700,000 Daltons Approximately 4,800,000 Daltons, approximately 4,900,000 Daltons, approximately 5,000,000 Daltons, approximately 5,100,000 Daltons, approximately 5,200,000 Daltons, approximately 5,300,000 Daltons, approximately 5,400,000 Daltons, approximately 5,500,000 Daltons, approximately 5,600,000 Daltons, approximately 5,700,000 Daltons, approximately 5,800,000 Daltons, approximately 5,900,000 Daltons, approximately 6,000,000 Daltons, approximately 6,100,000 Daltons, approximately 6,200,000 Daltons, approximately 6,300,000 Daltons, approximately 6,400000 Daltons, approximately 6,500,000 Daltons, approximately 6,600,000 Daltons, approximately 6,700,000 Daltons, approximately 6,800,000 Daltons, approximately 6,900,000 Daltons, approximately 7,000,000 Daltons, approximately 7,100,000 Daltons, approximately 7,200,000 Daltons, approximately 7,300,000 Daltons, approximately 7,400,000 Daltons Luton, approximately 7,500,000 Dalton, approximately 7,600,000 Dalton, approximately 7,700,000 Dalton, approximately 7,800,000 Dalton, approximately 7,900,000 Dalton, approximately 8,000,000 Dalton, approximately 8,100,000 Dalton, approximately 8,200,000 Dalton, approximately 8,300,000 Dalton, approximately 8,400,000 Dalton, A crosslinked polymer matrix according to claim 39, having an average molecular weight of approximately 8,500,000 Daltons, approximately 8,600,000 Daltons, approximately 8,700,000 Daltons, approximately 8,800,000 Daltons, approximately 8,900,000 Daltons, approximately 9,000,000 Daltons, approximately 9,100,000 Daltons, approximately 9,200,000 Daltons, approximately 9,300,000 Daltons, approximately 9,400,000 Daltons, approximately 9,500,000 Daltons, approximately 9,600,000 Daltons, approximately 9,700,000 Daltons, approximately 9,800,000 Daltons, approximately 9,900,000 Daltons, or approximately 10,000,000 Daltons, or any molecular weight between the range defined by any two of the aforementioned values.

41. The hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, and the mixture comprises approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,500,000 Daltons, approximately 2,000,000 Daltons, approximately 2,500,000 Daltons, approximately 3,000,000 Daltons, approximately 3, A crosslinked polymer matrix according to any one of claims 1 to 40, comprising hyaluronic acid having an average molecular weight of 500,000 Daltons, about 4,000,000 Daltons, about 4,500,000 Daltons, about 5,000,000 Daltons, about 5,500,000 Daltons, about 6,000,000 Daltons, about 6,500,000 Daltons, about 7,500,000 Daltons, about 8,000,000 Daltons, about 8,500,000 Daltons, about 9,000,000 Daltons, about 9,500,000 Daltons, and / or about 10,000,000 Daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

42. Hyaluronic acid and, Collagen and Lysine and, Buffer and It contains and is an aqueous hydrogel, composition.

43. The composition according to claim 42, wherein the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine.

44. The composition according to claim 42 or 43, further comprising lidocaine.

45. The composition according to claim 44, wherein the lidocaine is present in the matrix at a concentration ranging from about 0.15% (w / w) to about 0.45% (w / w).

46. The composition according to claim 44 or 45, wherein the lidocaine is present in a concentration of 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 0.37% (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 ranges defined by any two of the aforementioned values.

47. The composition according to any one of claims 42 to 46, further comprising non-crosslinked HA.

48. The composition according to any one of claims 47, wherein the non-crosslinked HA has a maximum concentration of about 5% (w / w) in the composition.

49. The composition according to any one of claims 47 or 48, wherein the non-crosslinked HA is present in the composition at a concentration of 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 aforementioned values.

50. The composition according to any one of claims 47 to 49, wherein the non-crosslinked HA is present in the composition at a concentration of about 1% (w / w).

51. The composition according to any one of claims 47 to 49, wherein the non-crosslinked HA is present in the composition at a concentration of about 2% (w / w).

52. The composition according to any one of claims 47 to 49, wherein the non-crosslinked HA is present in the composition at a concentration of about 5% (w / w).

53. The composition according to any one of claims 47 to 52, wherein the non-crosslinked HA improves the extrudeability of the composition.

54. The composition according to any one of claims 42 to 53, wherein the buffer solution is phosphate-buffered saline.

55. The composition according to any one of claims 42 to 54, wherein the hyaluronic acid has an average molecular weight of about 20,000 daltons to about 10,000,000 daltons.

56. The hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, wherein the mixture contains approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,500,000 Daltons, approximately 2,000,000 Daltons, approximately 2,500,000 Daltons, and approximately 3,000,000 Daltons. A composition according to any one of claims 42 to 55, comprising hyaluronic acid having a molecular weight of approximately 3,500,000 Daltons, approximately 4,000,000 Daltons, approximately 4,500,000 Daltons, approximately 5,000,000 Daltons, approximately 5,500,000 Daltons, approximately 6,000,000 Daltons, approximately 6,500,000 Daltons, approximately 7,500,000 Daltons, approximately 8,000,000 Daltons, approximately 8,500,000 Daltons, approximately 9,000,000 Daltons, approximately 9,500,000 Daltons and / or approximately 10,000,000 Daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

57. The composition according to any one of claims 42 to 56, wherein the collagen comprises type I collagen.

58. The composition according to any one of claims 42 to 57, wherein the collagen comprises type II collagen.

59. The composition according to any one of claims 42 to 58, wherein the collagen comprises type III collagen.

60. Approximately 4,000Pa S, approximately 4100Pa S, approximately 4200Pa S, approximately 4300Pa S, approximately 4400Pa S, approximately 4500Pa S, approximately 4600Pa S, approximately 4700Pa S, approximately 4800Pa S, approximately 4900Pa S, approximately 5000Pa S, about 5100Pa S, about 5200Pa S, about 5300Pa S, about 5400Pa S, about 5500Pa S, about 5600Pa S, about 5700Pa S, about 5800Pa S, about 5900Pa S, about 6000Pa S, about 6100Pa S, approx. 6200Pa S, approx. 6300Pa S, about 6400Pa S, about 6500Pa S, about 6600Pa S, about 6700Pa S, about 6800Pa S, about 6900Pa S, about 7000Pa S, about 7100Pa S, about 7200Pa S, about 7300Pa S, about 7400Pa S, about 7500Pa S, about 7600Pa S, about 7700Pa S, about 7800Pa S, about 7900Pa S, about 8000Pa S, about 8100Pa S, about 8200Pa S, about 8300Pa S, about 8400Pa S, about 8500Pa S, approx. 8600Pa S, approx. 8700Pa The composition according to any one of claims 42 to 59, having a viscosity of S, about 8800 Pa S, about 8900 Pa S, about 9000 Pa S, about 9100 Pa S, about 9200 Pa S, about 9300 Pa S, about 9400 Pa S, about 9500 Pa S, about 9600 Pa S, about 9700 Pa S, about 9800 Pa S, about 9900 Pa S, or about 10,000 Pa S, or any viscosity between the range defined by any two of the aforementioned values.

61. The composition according to any one of claims 42 to 60, having a tan delta parameter (G'' / G') of about 0.01 to about 0.

5.

62. The composition according to any one of claims 42 to 61, having a tan delta parameter (G'' / G') of about 0.01, about 0.05, about 0.10, about 0.15, about 0.20, about 0.25, about 0.30, about 0.35, about 0.40, about 0.45, or about 0.50, or any tan delta parameter between the range defined by any two of the aforementioned values.

63. The composition according to any one of claims 42 to 62, which is stable for approximately 6 months, approximately 12 months, approximately 18 months, approximately 24 months, approximately 30 months, or approximately 36 months, or for any time within the range defined by any two of the aforementioned values.

64. The composition according to any one of claims 42 to 63, which is stable at approximately 4°C.

65. The composition according to any one of claims 42 to 64, which is stable at approximately 25°C.

66. The composition according to any one of claims 42 to 65, wherein the decomposition is minimal for approximately 6 months, approximately 12 months, approximately 18 months, approximately 24 months, approximately 30 months, or approximately 36 months, or for any time within the range defined by any two of the aforementioned values.

67. A method for cross-linking hyaluronic acid and collagen, The process involves dissolving collagen, hyaluronic acid, and lysine in an aqueous solution to form a pre-reaction aqueous solution, wherein the pre-reaction aqueous solution has a pH of approximately 4 to approximately 6, and Water-soluble carbodiimide and N-hydroxysuccinimide or N-hydroxysulfosuccinimide Prepare a second solution containing, Adding the second solution to the pre-reaction aqueous solution to form a crosslinking reaction, The crosslinking reaction is carried out by crosslinking the hyaluronic acid and the collagen with lysine. Includes, The hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine, A method wherein the HA and collagen are slightly degraded, while the structures of the HA and collagen remain intact, thereby forming a cross-linked polymer matrix.

68. The method according to claim 67, wherein the aqueous solution before the reaction has a pH of about 4.0, about 4.5, about 5.0, about 5.5, or about 6.0, or any pH within the range defined by any two of the aforementioned values.

69. The method according to claim 67 or 68, further comprising adding lidocaine to the crosslinked polymer matrix.

70. The method according to claim 69, wherein the lidocaine is added to the crosslinked polymer matrix to a concentration ranging from about 0.15% (w / w) to about 0.45% (w / w).

71. The method according to claim 69 or 70, wherein the lidocaine is at a concentration of approximately 0.15% (w / w), approximately 0.17% (w / w), approximately 0.19% (w / w), approximately 0.21% (w / w), approximately 0.23% (w / w), approximately 0.25% (w / w), approximately 0.27% (w / w), approximately 0.29% (w / w), approximately 0.31% (w / w), approximately 0.33% (w / w), approximately 0.35% (w / w), approximately 0.37% (w / w), approximately 0.37% (w / w), approximately 0.39% (w / w), approximately 0.41% (w / w), approximately 0.43% (w / w), or approximately 0.45% (w / w) of the matrix, or any concentration between the ranges defined by any two of the aforementioned values.

72. The method according to any one of claims 67 to 71, further comprising imparting an activator comprising triazole, fluorinated phenol, succinimide, or sulfosuccinimide.

73. The method according to any one of claims 67 to 72, performed at a temperature of approximately 2°C, approximately 4°C, approximately 6°C, approximately 8°C, approximately 10°C, approximately 12°C, approximately 14°C, approximately 16°C, approximately 18°C, approximately 20°C, approximately 22°C, approximately 24°C, approximately 26°C, approximately 28°C, approximately 30°C, approximately 32°C, approximately 34°C, or approximately 36°C, or at a temperature between any two of the aforementioned values.

74. The method according to any one of claims 67 to 73, wherein the reaction step is carried out at approximately 4 to approximately 35°C.

75. The method according to any one of claims 67 to 74, wherein the reaction step is carried out at approximately 4°C or approximately 22°C.

76. The method according to any one of claims 67 to 75, further comprising purifying the crosslinked polymer matrix, wherein the purification step is performed by dialysis.

77. The method according to claim 76, wherein the purification step is carried out at a temperature of 2°C to 30°C.

78. The method according to claim 76 or 77, wherein the dialysis is performed at a temperature of approximately 2°C, approximately 3°C, approximately 4°C, approximately 5°C, approximately 6°C, approximately 7°C, approximately 8°C, approximately 9°C, approximately 10°C, approximately 11°C, approximately 12°C, approximately 13°C, approximately 14°C, approximately 15°C, approximately 16°C, approximately 17°C, approximately 18°C, approximately 19°C, approximately 20°C, approximately 21°C, approximately 22°C, approximately 23°C, approximately 24°C, approximately 25°C, approximately 26°C, approximately 27°C, approximately 28°C, approximately 29°C, approximately 30°C, or any temperature between the range defined by any two of the aforementioned values.

79. The method according to any one of claims 76 to 78, wherein the purification step is carried out at approximately 2°C or approximately 8°C.

80. The method according to any one of claims 67 to 79, wherein the crosslinking reaction is carried out at a temperature of about 2°C to about 35°C.

81. The method according to any one of claims 67 to 80, wherein the crosslinking reaction is carried out at a temperature of about 2°C to about 8°C.

82. The method according to any one of claims 67 to 81, carried out below room temperature.

83. The method according to any one of claims 67 to 82, wherein the pH of the crosslinking reaction mixture is about 4.0 to about 6.

0.

84. The method according to any one of claims 67 to 83, wherein the pre-reaction solution contains a salt, the salt containing sodium chloride in the crosslinking reaction mixture at a concentration of 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, or about 400 mM, or at any concentration between the range defined by any two of the aforementioned values.

85. The method according to any one of claims 67 to 84, wherein the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the crosslinking reaction mixture at a concentration of about 20 mM to about 200 mM.

86. The method according to claim 85, wherein the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in 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, or about 200 mM, or any concentration within the range defined by the aforementioned arbitrary values.

87. The method according to any one of claims 67 to 86, wherein the molar to molar ratio of repeating units of water-soluble carbodiimide to repeating units of hyaluronic acid of the water-soluble carbodiimide and the hyaluronic acid is about 0.5 to about 2.

0.

88. The method according to claim 87, wherein the molar-to-molar ratio of repeating units of water-soluble carbodiimide to repeating units of hyaluronic acid of the water-soluble carbodiimide and the hyaluronic acid is 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, or about 2.

0.

89. The method according to any one of claims 67 to 88, wherein the molar:molar (lysine repeating unit:HA repeating unit) ratio of lysine to hyaluronic acid is about 0.01 to about 0.

6.

90. The molar:molar ratio of lysine to hyaluronic acid (lysine repeating unit:HA repeating unit) is approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0. The method according to claim 89, wherein the ratio is 0.28, approximately 0.29, approximately 0.3, approximately 0.31, approximately 0.32, approximately 0.33, approximately 0.34, approximately 0.35, approximately 0.36, approximately 0.37, approximately 0.38, approximately 0.39, approximately 0.4, approximately 0.41, approximately 0.42, approximately 0.43, approximately 0.44, approximately 0.45, approximately 0.46, approximately 0.47, approximately 0.48, approximately 0.49, approximately 0.5, approximately 0.51, approximately 0.52, approximately 0.53, approximately 0.54, approximately 0.55, approximately 0.56, approximately 0.57, approximately 0.58, approximately 0.59, or approximately 0.

6.

91. The method according to any one of claims 67 to 90, further comprising adding non-crosslinked HA to the crosslinked polymer matrix.

92. The method according to claim 91, wherein the non-crosslinked HA is added to the crosslinked polymer matrix to a maximum concentration of 5% w / w.

93. The method according to claim 91 or 92, wherein the non-crosslinked HA is added to the matrix to a concentration of about 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 range defined by any two of the aforementioned values.

94. The method according to any one of claims 91 to 93, wherein the non-crosslinked HA is added to the matrix to a concentration of about 1% (w / w).

95. The method according to any one of claims 91 to 93, wherein the non-crosslinked HA is added to the matrix to a concentration of about 3% (w / w).

96. The method according to any one of claims 91 to 93, wherein the non-crosslinked HA is added to the matrix to a concentration of about 5% (w / w).

97. The method further includes sterilizing the crosslinked polymer matrix, Transferring the cross-linked polymer matrix to a container for steam sterilization, and The hydrogel is sterilized by steam sterilization. The method according to any one of claims 67 to 96, including the method described in that claim.

98. The method according to claim 97, wherein the container is a syringe.

99. The method according to any one of claims 67 to 98, further comprising dialysis of the crosslinked polymer matrix, wherein the dialysis is performed through a membrane having a molecular weight cutoff of about 1,000 daltons to about 100,000 daltons, and the dialysis is performed before sterilization.

100. The method according to claim 99, wherein the dialysis is performed with phosphate-buffered saline.

101. The method according to any one of claims 67 to 100, wherein the hyaluronic acid in the pre-reaction solution is hydrated for at least about 60 minutes before the second solution is added.

102. The method according to any one of claims 67 to 101, wherein the crosslinking reaction is carried out for about 16 to about 24 hours.

103. A crosslinked polymer matrix prepared by the process described in any one of claims 67 to 102.

104. A method for improving the aesthetics of human anatomical features, Injecting a composition into human tissue to improve the aesthetics of the anatomical features. Includes, The composition is Hyaluronic acid and, Lysine and, Collagen and It contains a crosslinked polymer matrix, A method wherein the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine.

105. The method according to claim 104, wherein the crosslinked polymer matrix further comprises lidocaine.

106. The method according to claim 104 or 105, wherein the crosslinked polymer matrix further comprises non-crosslinked HA.

107. The aforementioned hyaluronic acid component is approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,100,000 Daltons, approximately 1,200,000 Daltons, approximately 1,300,000 Daltons, and approximately 1,400,000 Daltons. Alton, approximately 1,500,000 Dalton, approximately 1,600,000 Dalton, approximately 1,700,000 Dalton, approximately 1,800,000 Dalton, approximately 1,900,000 Dalton, approximately 2,000,000 Dalton, approximately 2,100,000 Dalton, approximately 2,200,000 Dalton, approximately 2,300,000 Dalton, approximately 2,400,000 Dalton, approximately 2,500,000 Dalton, approximately 2,600,000 Dalton, approximately 2,700,000 Dalton, approximately 2,800,000 Dalton, approximately 2,900,000 Dalton, approximately 3,000,000 Dalton, approximately 3,10 0,000 Daltons, approximately 3,200,000 Daltons, approximately 3,300,000 Daltons, approximately 3,400,000 Daltons, approximately 3,500,000 Daltons, approximately 3,600,000 Daltons, approximately 3,700,000 Daltons, approximately 3,800,000 Daltons, approximately 3,900,000 Daltons, approximately 4,000,000 Daltons, approximately 4,100,000 Daltons, approximately 4,200,000 Daltons, approximately 4,300,000 Daltons, approximately 4,400,000 Daltons, approximately 4,500,000 Daltons, approximately 4,600,000 Daltons, approximately 4,700,000 Daltons Approximately 4,800,000 Daltons, approximately 4,900,000 Daltons, approximately 5,000,000 Daltons, approximately 5,100,000 Daltons, approximately 5,200,000 Daltons, approximately 5,300,000 Daltons, approximately 5,400,000 Daltons, approximately 5,500,000 Daltons, approximately 5,600,000 Daltons, approximately 5,700,000 Daltons, approximately 5,800,000 Daltons, approximately 5,900,000 Daltons, approximately 6,000,000 Daltons, approximately 6,100,000 Daltons, approximately 6,200,000 Daltons, approximately 6,300,000 Daltons, approximately 6,400000 Daltons, approximately 6,500,000 Daltons, approximately 6,600,000 Daltons, approximately 6,700,000 Daltons, approximately 6,800,000 Daltons, approximately 6,900,000 Daltons, approximately 7,000,000 Daltons, approximately 7,100,000 Daltons, approximately 7,200,000 Daltons, approximately 7,300,000 Daltons, approximately 7,400,000 Daltons Luton, approximately 7,500,000 Dalton, approximately 7,600,000 Dalton, approximately 7,700,000 Dalton, approximately 7,800,000 Dalton, approximately 7,900,000 Dalton, approximately 8,000,000 Dalton, approximately 8,100,000 Dalton, approximately 8,200,000 Dalton, approximately 8,300,000 Dalton, approximately 8,400,000 Dalton, approximately The method according to any one of claims 104 to 106, having an average molecular weight of 8,500,000 daltons, approximately 8,600,000 daltons, approximately 8,700,000 daltons, approximately 8,800,000 daltons, approximately 8,900,000 daltons, approximately 9,000,000 daltons, approximately 9,100,000 daltons, approximately 9,200,000 daltons, approximately 9,300,000 daltons, approximately 9,400,000 daltons, approximately 9,500,000 daltons, approximately 9,600,000 daltons, approximately 9,700,000 daltons, approximately 9,800,000 daltons, approximately 9,900,000 daltons, or approximately 10,000,000 daltons, or any molecular weight between the range defined by any two of the aforementioned values.

108. The hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, wherein the mixture comprises approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,500,000 Daltons, approximately 2,000,000 Daltons, approximately 2,500,000 Daltons, and approximately 3,000,000 Daltons. The method according to any one of claims 104 to 107, comprising hyaluronic acid having an average molecular weight of approximately 3,500,000 Daltons, approximately 4,000,000 Daltons, approximately 4,500,000 Daltons, approximately 5,000,000 Daltons, approximately 5,500,000 Daltons, approximately 6,000,000 Daltons, approximately 6,500,000 Daltons, approximately 7,500,000 Daltons, approximately 8,000,000 Daltons, approximately 8,500,000 Daltons, approximately 9,000,000 Daltons, approximately 9,500,000 Daltons and / or approximately 1,000,000 Daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

109. The method according to any one of claims 104 to 108, wherein the collagen comprises type I collagen and / or type III collagen.

110. A method for improving the appearance of an individual, The method involves injecting a composition into the tissue of the individual at the injection site to improve the aesthetics of the anatomical features, wherein infiltrating cells from the tissue are integrated into the composition within the injection site, and depositing new collagen within the composition, wherein the composition is Hyaluronic acid and, Lysine and, Collagen and It contains a crosslinked polymer matrix, A method in which the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine, and the tissue into which the composition is injected has tissue integration, collagen deposition and angiogenesis.

111. The method according to claim 110, wherein the composition further comprises lidocaine.

112. The method according to claim 110 or 111, wherein the composition further comprises non-crosslinked HA.

113. The method according to any one of claims 110 to 112, wherein the composition is injected into the chin, jawline, lips, or nasolabial folds.

114. The method according to claims 110 to 113, for improving symmetry between facial features.

115. The method according to any one of claims 110 to 114, for increasing and restoring volume to facial features.

116. The method according to claim 115, for increasing, correcting, restoring, or giving volume to the chin, jawline, or nasolabial folds.

117. The method according to any one of claims 110 to 112, 114, or 115, wherein the composition is injected into the tear trough of the solid.

118. The method according to any one of claims 110 to 117, comprising injecting the composition into an area including atrophy of the skin and / or atrophy of the fat body.

119. The method according to any one of claims 110 to 118, wherein the composition provides the injected tissue with a natural appearance, feel, and movement, and the composition results in increased collagen infiltration from the surrounding tissue at the injection site.

120. The method according to claim 119, wherein the duration of the composition is extended as a result of tissue integration with the injection site.

121. The method according to any one of claims 104 to 120, which improves the hydration and elasticity of the skin surrounding the injection site.

122. A method for increasing collagen infiltration into tissue, The process includes injecting the composition into the tissue of an individual, thereby creating a depot of a skin filler containing the composition, wherein the composition is Hyaluronic acid, lysine, and collagen A method comprising a crosslinked polymer matrix containing the hyaluronic acid, wherein the hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine, wherein cells from the surrounding tissue infiltrate the depot of the skin filler containing the composition, the cells integrate with the composition and deposit new collagen into the composition, thereby creating infiltrated tissue within the composition, and blood vessels connect the infiltrated tissue within the composition to the blood supply of the body of the individual.

123. The method according to claim 122, wherein the matrix further comprises lidocaine.

124. The method according to claim 122 or 123, wherein the composition further comprises non-crosslinked HA.

125. The aforementioned hyaluronic acid is approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,100,000 Daltons, approximately 1,200,000 Daltons, approximately 1,300,000 Daltons, and approximately 1,400,000 Daltons. Approximately 1,500,000 Daltons, approximately 1,600,000 Daltons, approximately 1,700,000 Daltons, approximately 1,800,000 Daltons, approximately 1,900,000 Daltons, approximately 2,000,000 Daltons, approximately 2,100,000 Daltons, approximately 2,200,000 Daltons, approximately 2,300,000 Daltons, approximately 2,400,000 Daltons, approximately 2,500,000 Daltons, approximately 2,600,000 Daltons, approximately 2,700,000 Daltons, approximately 2,800,000 Daltons, approximately 2,900,000 Daltons, approximately 3,000,000 Daltons, approximately 3,100 ,000 Daltons, approximately 3,200,000 Daltons, approximately 3,300,000 Daltons, approximately 3,400,000 Daltons, approximately 3,500,000 Daltons, approximately 3,600,000 Daltons, approximately 3,700,000 Daltons, approximately 3,800,000 Daltons, approximately 3,900,000 Daltons, approximately 4,000,000 Daltons, approximately 4,100,000 Daltons, approximately 4,200,000 Daltons, approximately 4,300,000 Daltons, approximately 4,400,000 Daltons, approximately 4,500,000 Daltons, approximately 4,600,000 Daltons, approximately 4,700,000 Daltons Approximately 4,800,000 Daltons, approximately 4,900,000 Daltons, approximately 5,000,000 Daltons, approximately 5,100,000 Daltons, approximately 5,200,000 Daltons, approximately 5,300,000 Daltons, approximately 5,400,000 Daltons, approximately 5,500,000 Daltons, approximately 5,600,000 Daltons, approximately 5,700,000 Daltons, approximately 5,800,000 Daltons, approximately 5,900,000 Daltons, approximately 6,000,000 Daltons, approximately 6,100,000 Daltons, approximately 6,200,000 Daltons, approximately 6,300,000 Daltons, approximately 6,400000 Daltons, approximately 6,500,000 Daltons, approximately 6,600,000 Daltons, approximately 6,700,000 Daltons, approximately 6,800,000 Daltons, approximately 6,900,000 Daltons, approximately 7,000,000 Daltons, approximately 7,100,000 Daltons, approximately 7,200,000 Daltons, approximately 7,300,000 Daltons, approximately 7,400,000 Daltons ton, approximately 7,500,000 Daltons, approximately 7,600,000 Daltons, approximately 7,700,000 Daltons, approximately 7,800,000 Daltons, approximately 7,900,000 Daltons, approximately 8,000,000 Daltons, approximately 8,100,000 Daltons, approximately 8,200,000 Daltons, approximately 8,300,000 Daltons, approximately 8,400,000 Daltons, approximately 8 The method according to any one of claims 122 to 124, having an average molecular weight of 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 other molecular weight between any two of the aforementioned values.

126. The hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, wherein the mixture comprises approximately 20,000 Daltons, approximately 40,000 Daltons, approximately 60,000 Daltons, approximately 80,000 Daltons, approximately 100,000 Daltons, approximately 200,000 Daltons, approximately 300,000 Daltons, approximately 400,000 Daltons, approximately 500,000 Daltons, approximately 600,000 Daltons, approximately 700,000 Daltons, approximately 800,000 Daltons, approximately 900,000 Daltons, approximately 1,000,000 Daltons, approximately 1,500,000 Daltons, approximately 2,000,000 Daltons, approximately 2,500,000 Daltons, and approximately 3,000,000 Daltons. The method according to any one of claims 122 to 125, comprising hyaluronic acid having an average molecular weight of approximately 3,500,000 Daltons, approximately 4,000,000 Daltons, approximately 4,500,000 Daltons, approximately 5,000,000 Daltons, approximately 5,500,000 Daltons, approximately 6,000,000 Daltons, approximately 6,500,000 Daltons, approximately 7,500,000 Daltons, approximately 8,000,000 Daltons, approximately 8,500,000 Daltons, approximately 9,000,000 Daltons, approximately 9,500,000 Daltons and / or approximately 10,000,000 Daltons, and / or any hyaluronic acid having a molecular weight within the range between any two of the aforementioned values.

127. The method according to any one of claims 122 to 126, wherein the collagen comprises type I collagen, type II collagen and / or type III collagen.

128. The method according to any one of claims 122 to 127, wherein the composition comprises about 13 mg / ml of hyaluronic acid.

129. The method according to any one of claims 122 to 127, wherein the composition comprises about 20 mg / ml of hyaluronic acid, about 22 mg / ml of hyaluronic acid, about 24 mg / ml, about 26 mg / ml of hyaluronic acid, about 28 mg / ml of hyaluronic acid, or about 30 mg / ml of hyaluronic acid.

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