Cross-linked HA-collagen hydrogel as a dermal filler

Abstract

To provide an improved dermal filler.SOLUTION: A dermal filler for improving the aesthetic quality of an anatomic feature of a human face by injection into the anatomic feature, contains a crosslinked macromolecular matrix containing lysine, hyaluronic acid, and collagen. The hyaluronic acid is crosslinked to the collagen by at least one endogenous amine group on the collagen and by at least one amine group present on the lysine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Patent Application No. 62 / 953,910, filed December 26, 2019, which is incorporated herein by reference in its entirety.

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

[0003] Aging is a natural process that occurs over time and can be influenced by genetics and lifestyle factors (recreational drugs, alcohol abuse, tobacco, UVA / UVB exposure, diet). Characteristics of facial skin aging include, for example, muscle and fat atrophy, skin laxity, age spots, sagging, and weight gain. Loosening of the subcutaneous tissue can lead to excess skin and ptosis, resulting in the appearance of drooping cheeks and eyelids. Weight gain refers to excess weight gain due to swelling of the lower face and neck. These changes can be associated with dryness, loss of elasticity, and a rough texture.

[0004] Dermal fillers have been used to improve the appearance of aging skin. Various types of dermal fillers have been developed and used to treat or improve / correct physical imperfections, such as wrinkles and volume loss due to the effects of aging. The first dermal filler compositions containing bovine collagen entered the market in 1970. Human-derived collagen was approved by the FDA in 2003, which offered advantages over bovine-derived collagen, which has the potential for allergic reactions in patients. However, human-derived collagen compositions rapidly degrade within three to six months due to enzymes in skin tissue. As a result, patients using these early compositions required frequent procedures to maintain the desired corrective aesthetic appearance.

[0005] Hyaluronan, or hyaluronic acid (HA), based fillers were introduced in 1990 as an alternative to collagen-based dermal fillers. HA is a naturally occurring water-soluble polysaccharide, specifically a glycosaminoglycan. It is a major component of the extracellular matrix and is widely distributed in animal tissues. HA has excellent biocompatibility and does not cause allergic reactions when implanted in patients. Furthermore, HA has the ability to bind large amounts of water, making it an excellent volumizer for soft tissues. HA is similar to collagen in that it can also be degraded by endogenous enzymes in the skin. For example, uncrosslinked HA does not have sufficient longevity or physical properties to function as a wrinkle filler; therefore, crosslinked HA has been used to maximize its longevity in dermal tissue. Therefore, improved dermal fillers are needed. Summary of the Invention

[0006] Embodiments herein include methods and compositions (e.g., hydrogels or dermal fillers) comprising a crosslinked polymeric matrix comprising hyaluronic acid, collagen, and lysine, wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine.

[0007] In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix further comprises lidocaine. In some embodiments of each or any of the above or below embodiments, the lidocaine is present in the matrix at a concentration ranging from about 0.15% (w / w) to about 0.45% (w / w). In some embodiments of each or any of the above or below embodiments, the lidocaine is present in the matrix at a concentration ranging from about 0.27% (w / w) to about 0.33% (w / w). In some embodiments of each or any of the above or below embodiments, lidocaine is at 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 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w), or about 0.45% (w / w) of the matrix, or any concentration between the ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the lidocaine is at a concentration of about 0.3% (w / w) in the matrix.

[0008] In some embodiments of each or any of the above or below embodiments, the cross-linked polymer matrix further comprises non-cross-linked HA. In some embodiments of each or any of the above or below embodiments, the non-cross-linked HA has a concentration of up to about 5% (w / w) in the matrix. In some embodiments of each or any of the above or below embodiments, the non-cross-linked 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 ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the non-cross-linked HA has a concentration of about 1% (w / w) in the matrix. In some embodiments of each or any of the above or below embodiments, the non-cross-linked HA has a concentration of about 2% (w / w) in the matrix. In some embodiments of each or any of the above or below embodiments, the non-cross-linked HA has a concentration of about 5% (w / w) in the matrix. In some embodiments of each or any of the above or below embodiments, the non-crosslinked HA improves the extrudability of the polymeric matrix.

[0009] In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric 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 any time period between ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix is ​​stable at a temperature of about 4°C to about 25°C. In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix is ​​stable at about 4°C. In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix is ​​stable at about 25°C. In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric 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 any time between the ranges defined by any two of the foregoing values.

[0010] In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix undergoes negligible degradation in about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or any time between the ranges defined by any two of the foregoing values.

[0011] In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix has a modulus of elasticity (G') of from about 30 Pa to about 10,000 Pa.In some embodiments of each or any of the above or below embodiments, the matrix has a viscosity of about 30 Pa, about 40 Pa, about 50 Pa, about 60 Pa, about 70 Pa, about 80 Pa, about 90 Pa, about 100 Pa, about 200 Pa, about 300 Pa, about 400 Pa, about 500 Pa, about 600 Pa, about 700 Pa, about 800 Pa, about 900 Pa, about 1000 Pa, about 1100 Pa, about 1200 Pa, about 1300 Pa, about 1400 Pa, about 1500 Pa, about 1600 Pa, about 1700 Pa, about 1800 Pa, about 1900 Pa, about 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 has a modulus of elasticity (G') of 7800 Pa, about 7900 Pa, about 8000 Pa, about 8100 Pa, about 8200 Pa, about 8300 Pa, about 8400 Pa, about 8500 Pa, about 8600 Pa, about 8700 Pa, about 8800 Pa, about 8900 Pa, about 9000 Pa, about 9100 Pa, about 9200 Pa, about 9300 Pa, about 9400 Pa, about 9500 Pa, about 9600 Pa, about 9700 Pa, about 9800 Pa, about 9900 Pa or about 10000 Pa, or any modulus of elasticity between a range defined by any two of the foregoing values.

[0012] In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix has a viscosity of about 10 gmf, about 20 gmf, about 30 gmf, about 40 gmf, about 50 gmf, about 60 gmf, about 70 gmf, about 80 gmf, about 90 gmf, about 100 gmf, about 110 gmf, about 120 gmf, about 130 gmf, 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, In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix has a compressive force value of about 100 gmf, about 200 gmf, about 300 gmf, about 400 gmf, about 500 gmf, about 600 gmf, or any compressive force value between the ranges defined by any two of the above values.

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

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

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

[0016] In some embodiments of each or any of the above or below embodiments, 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 ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the collagen has a concentration of about 3 mg / ml. In some embodiments of each or any of the above or below embodiments, the collagen has a concentration of about 6 mg / ml. In some embodiments of each or any of the above or below embodiments, the collagen has a concentration of about 10 mg / ml. In some embodiments of each or any of the above or below embodiments, the collagen has a concentration of about 12 mg / ml.

[0017] In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix further comprises a salt. In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix comprises NaCl in the range of about 50 mM to about 400 mM. In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix comprises NaCl, wherein the NaCl has 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 ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the crosslinked polymeric matrix comprises about 150 mM NaCl. In certain embodiments, the crosslinked polymeric matrix does not comprise a salt.

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

[0019] In some embodiments of each or any of the above or below embodiments, the hyaluronic acid has an average molecular weight of from about 20,000 daltons to about 10,000,000 daltons. In some embodiments of each or any of the above or below embodiments, the hyaluronic acid is selected from the group consisting of about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,500,000 daltons, about 2,000,000 daltons, about 2,500,000 daltons, and the like. Daltons, about 3,000,000 Daltons, about 3,500,000 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 1,000,000 Daltons, or an average molecular weight between the ranges defined by any two of the foregoing values. In some embodiments of the compositions of each or any of the above or below embodiments, the hyaluronic acid has an average molecular weight of from about 20,000 daltons to about 10,000,000 daltons.

[0033] In some embodiments of each or any of the above or below embodiments, the hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, the mixture comprising: about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,500,000 daltons, about 2,000,000 daltons, Hyaluronic acid having a molecular weight of about 2,500,000 daltons, about 3,000,000 daltons, about 3,500,000 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 a range between any two of the foregoing values.

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

[0021] In some embodiments of each or any of the above or below embodiments, the hyaluronic acid is crosslinked to the collagen via at least one endogenous amine group on the collagen and / or at least one amine group present on lysine. In some embodiments of each or any of the above or below embodiments, the composition further comprises lidocaine. In some embodiments of each or any of the above or below embodiments, the lidocaine is at a concentration in the matrix ranging from about 0.15% (w / w) to about 0.45% (w / w). In some embodiments of each or any of the above or below embodiments, the lidocaine is at a concentration in the composition ranging from about 0.27% (w / w) to about 0.33% (w / w). In some embodiments of each or any of the above or below embodiments, lidocaine is at 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 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w), or about 0.45% (w / w) of the composition, or any concentration between the ranges defined by any two of the foregoing values.

[0022] In some embodiments of each or any of the above or below embodiments, the composition further comprises non-crosslinked HA. In some embodiments of each or 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 some embodiments of each or any of the above or below embodiments, the non-crosslinked HA has 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) in the composition, or any concentration between the ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the non-crosslinked HA has a concentration of about 1% (w / w) in the composition. In some embodiments of each or any of the above or below embodiments, the non-crosslinked HA has a concentration of about 2% (w / w) in the composition. In some embodiments of each or any of the above or below embodiments, the non-crosslinked HA has a concentration of about 5% (w / w) in the composition. In some embodiments of each or any of the above or below embodiments, the non-crosslinked HA improves the extrudability of the composition. In some embodiments of each or any of the above or below embodiments, the buffer is phosphate buffered saline.

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

[0024]

[0033] In some embodiments of each or any of the above or below embodiments, the hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, the mixture comprising: about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,500,000 daltons, about 2,000,000 daltons, Hyaluronic acid having a molecular weight of about 2,500,000 daltons, about 3,000,000 daltons, about 3,500,000 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 a range between any two of the foregoing values.

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

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

[0027] In some embodiments of each or any of the above or below embodiments, the composition further comprises non-crosslinked HA. In some embodiments of each or 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 some embodiments of each or any of the above or below embodiments, the non-crosslinked HA improves the extrudability of the composition. In some embodiments of each or 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 any time between the ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the composition is stable at 4°C. In some embodiments of each or any of the above or below embodiments, the composition is stable at 25°C. In some embodiments of each or any of the above or below embodiments, the composition shows negligible degradation for 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months, or any time between the ranges defined by any two of the foregoing values.

[0028]

[0033] In some embodiments of each or any of the above or below embodiments, the composition comprises at least one of: about 4,000 Pa S, about 4100 Pa S, about 4200 Pa S, about 4300 Pa S, about 4400 Pa S, about 4500 Pa S, about 4600 Pa S, about 4700 Pa S, about 4800 Pa S, about 4900 Pa S, about 5000 Pa S, about 5100 Pa S, about 5200 Pa S, about 5300 Pa S, about 5400 Pa S, about 5500 Pa S, about 5600 Pa S, about 5700 Pa S, about 5800 Pa S, about 5900 Pa S, about 6000 Pa S, about 6100 Pa S, about 6200 Pa S, about 6300 Pa S, about 6400 Pa S, about 6500 Pa S, about 6600 Pa S, about 6700 Pa S, about 6800 Pa S 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 5, about 9900 Pa S, or about 10,000 Pa S, or any viscosity between the range defined by any two of the foregoing values.

[0029] In some embodiments of each or any of the above or below embodiments, the composition has a tan delta parameter (G" / G') of about 0.01 to about 0.5. In some embodiments of each or any 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 the ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the composition comprises a phosphate buffered saline solution.

[0030] The present disclosure also provides 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, the pre-reaction aqueous solution having a pH of 4 to 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 mixture; and reacting the crosslinking reaction mixture by crosslinking the hyaluronic acid and collagen with the lysine, wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine, and the HA and collagen are negligibly degraded, while the structures of the HA and collagen remain intact, thereby forming a crosslinked polymeric matrix. In some embodiments of each or any of the above or below embodiments, the pre-reaction aqueous solution comprises a pH of about 4.0, about 4.5, about 5.0, about 5.5, or about 6, or any pH between the ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the method further comprises providing an activator comprising a triazole, a fluorinated phenol, a succinimide, or a sulfosuccinimide.

[0031] In some embodiments of each or any of the above or below embodiments, the method further comprises adding lidocaine to the crosslinked polymer matrix. In some embodiments of each or any of the above or below embodiments, the lidocaine is at a concentration in the matrix ranging from about 0.15% (w / w) to about 0.45% (w / w). In some embodiments of each or any of the above or below embodiments, the lidocaine is at a concentration in the matrix ranging from about 0.27% (w / w) to about 0.33% (w / w). In some embodiments of each or any of the above or below embodiments, lidocaine is at 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) of the matrix, or any concentration between the ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the lidocaine is at a concentration of about 0.3% (w / w) in the matrix.

[0032] In some embodiments of each or any of the above or below embodiments, the method further comprises adding non-crosslinked HA to the crosslinked polymer matrix. In some embodiments of each or any of the above or below embodiments, the non-crosslinked HA has a concentration of up to about 5% w / w in the crosslinked polymer matrix. In some embodiments of each or any of the above or below embodiments, the non-crosslinked HA is added 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) in the matrix, or any concentration between the ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the non-crosslinked HA is added to a concentration of about 1% (w / w) in the matrix. In some embodiments of each or any of the above or below embodiments, the non-crosslinked HA is added to a concentration of about 3% (w / w) in the matrix. In some embodiments of each or any of the above or below embodiments, non-crosslinked HA is added to a concentration of about 5% (w / w) in the matrix.

[0033] In some embodiments of each or any of the above or below embodiments, the reacting step is carried out at about 4° C. to about 35° C. In some embodiments of each or any of the above or below embodiments, the reacting step is carried out at 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 ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the reacting step is carried out at about 4° C. or about 22° C.

[0034] In some embodiments of each or any of the above or below embodiments, the method further comprises purifying the crosslinked polymeric matrix, wherein the purification step is carried out using dialysis purification. In some embodiments of each or any of the above or below embodiments, the dialysis is carried out at about 2°C to about 30°C. In some embodiments of each or any of the above or below embodiments, the dialysis is carried out at 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 ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the purification step is carried out at about 2° C. to about 8° C. In some embodiments of each or any of the above or below embodiments, the purification step is carried out at about 2° C., about 4° C., about 6° C., about 8° C., or any temperature between the ranges defined by any two of the foregoing values.

[0035] In some embodiments of each or any of the above or below embodiments, the method is carried out below room temperature. In some embodiments of each or any of the above or below embodiments, 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 a temperature between the ranges defined by any two of the foregoing values.

[0036] In some embodiments of each or any of the above or below embodiments, the pH of the crosslinking reaction mixture is about 4 to about 6.0. In some embodiments of each or any of the above or below embodiments, 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 ranges defined by any two of the foregoing values.

[0037] In some embodiments of each or any of the above or below embodiments, the pre-reaction solution comprises a salt comprising 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 ranges defined by any two of the foregoing values ​​in the crosslinking reaction mixture.

[0038] In some embodiments of each or any of the above or below embodiments, the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration of about 20 mM to about 200 mM in the crosslinking reaction mixture. In some embodiments of each or any of the above or below embodiments, the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a concentration of about 20 mM, about 40 mM, about 60 mM, about 80 mM, about 100 mM, about 120 mM, about 140 mM, about 160 mM, about 180 mM, or about 200 mM, or any concentration between the ranges defined by any of the foregoing values.

[0039] In some embodiments of each or any of the methods above or below, the water-soluble carbodiimide and hyaluronic acid have a molar to molar ratio of repeating units of the water-soluble carbodiimide to repeating units of the hyaluronic acid of about 0.5 to about 2.0. In some embodiments of each or any of the above or below, the water-soluble carbodiimide and hyaluronic acid have a molar to molar ratio of repeating units of the water-soluble carbodiimide to repeating units of the hyaluronic acid of 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 some embodiments of each or any of the above or below embodiments, the mole:mol (lysine repeat units:HA repeat units) ratio of lysine to hyaluronic acid is about 0.01 to about 0.6. In some embodiments of each or any of the above or below embodiments, the mole:mol (lysine repeat units:HA repeat units) ratio of lysine to hyaluronic acid is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.2, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 0.4, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.3, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.4, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49, about 0.5, about 0.51, about 0.52, about 0.53, about 0.54, about 0.55, about 0.56, about 0.57, about 0.58, about 0.59 or about 0.6.

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

[0042] In some embodiments of each or any of the above or below embodiments, the method further comprises dialyzing the crosslinked polymeric matrix, wherein the dialysis is performed through a membrane having a molecular weight cutoff of about 1000 daltons to about 100,000 daltons, and wherein the dialysis is performed prior to sterilization. In some embodiments of each or any of the above or below embodiments, the dialysis is performed with phosphate buffered saline.

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

[0044] In some embodiments of each or any of the above or below embodiments, the crosslinking reaction mixture is carried out for about 16 to about 24 hours. In some embodiments of each or any of the above or below embodiments, 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 period between the ranges defined by any two of the foregoing values.

[0045] In some embodiments of each or any of the above or below embodiments, the crosslinking reaction is carried out at about 2° C. to about 35° C. In some embodiments of each or any of the above or below embodiments, the crosslinking reaction is carried out at about 2° C., about 3° C., 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 within a range defined by any two of the foregoing values.

[0046] In some embodiments of each or any of the above or below embodiments, the crosslinking reaction is carried out at about 2° C. to about 8° C. In some embodiments of each or any of the above or below embodiments, the crosslinking reaction is carried out at about 2° C., about 4° C., about 6° C., or about 8° C., or any temperature within the range defined by any two of the foregoing values.

[0047] The present disclosure also provides a crosslinked polymeric matrix prepared by the method of any one of the above or below embodiments.

[0048] Additionally, the present disclosure provides a method for improving the aesthetics of a human anatomical feature, comprising injecting a composition into human tissue, thereby improving the aesthetics of the anatomical feature, the composition comprising a crosslinked polymeric matrix comprising hyaluronic acid, lysine, and collagen, wherein the hyaluronic acid is crosslinked to the collagen via at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine.

[0049] The present disclosure also provides a method for improving the appearance of an individual. The method includes injecting a composition into tissue of an individual at an injection site, thereby improving the aesthetics of an anatomical feature, wherein infiltrating cells from the tissue are integrated into the composition within the injection site; depositing new collagen within the composition, wherein the composition comprises a crosslinked polymeric 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; and depositing new collagen, wherein the tissue into which the composition is injected exhibits tissue integration and collagen deposition and angiogenesis. In some embodiments of each or any of the above or below embodiments, the composition is injected into the nasolabial folds. In some embodiments of each or any of the above or below embodiments, the method improves symmetry between facial features. In some embodiments of each or any of the above or below embodiments, the method increases and restores volume to facial features. In some embodiments of each or any of the above or below embodiments, the method restores volume to the cheeks and / or temples. In some embodiments, the method increases, corrects, restores, or provides volume to the chin, jawline, or nasolabial folds. In some embodiments of each or any of the above or below embodiments, the composition is injected into the individual's tear trough. In some embodiments of each or any of the above or below embodiments, the composition is injected into an area containing skin atrophy and / or fat pad atrophy. In some embodiments of each or any of the above or below embodiments, the method provides a natural look, feel, and movement to the tissue receiving the injection, and the composition results in increased collagen infiltration from tissue surrounding the injection site. In some embodiments of each or any of the above or below embodiments, the duration of the composition is extended as a result of tissue integration into the injection site. In some embodiments of each or any of the above or below embodiments, the method improves hydration and elasticity of the skin surrounding the injection site.

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

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

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

[0053] In some embodiments of each or any of the above or below method embodiments, the composition or polymeric matrix further comprises lidocaine. In some embodiments of each or any of the above or below embodiments, the lidocaine is at a concentration in the matrix ranging from 0.15% (w / w) to 0.45% (w / w). In some embodiments of each or any of the above or below embodiments, the lidocaine is at a concentration in the matrix ranging from 0.27% (w / w) to 0.33% (w / w). In some embodiments of each or any of the above or below embodiments, lidocaine is at 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) of the matrix, or any concentration between the ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments, the lidocaine is at a concentration of about 0.3% (w / w) in the matrix.

[0054] In some embodiments of each or any of the above or below embodiments of the method, the composition or polymeric matrix further comprises non-crosslinked HA. In some embodiments of each or any of the above or below embodiments, the non-crosslinked HA has a concentration of up to about 5% (w / w) in the composition or matrix. In some embodiments of each or any of the above or below embodiments of the method, the non-crosslinked HA has 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 ranges defined by any two of the foregoing values. In some embodiments of each or any of the above or below embodiments of the method, the non-crosslinked HA has a concentration of about 1% (w / w) in the composition or matrix. In some embodiments of each or any of the above or below embodiments of the method, the non-crosslinked HA has a concentration of about 2% (w / w) in the composition or matrix. In some embodiments of each or any of the above or below embodiments of the method, the non-crosslinked HA has a concentration of about 5% (w / w) in the composition or matrix. [Brief explanation of the drawings]

[0055] [Figure 1] Figure 1 shows in vitro cytoactive cells in intimate contact with the HA / collagen crosslinked hydrogel formulation.

[0056] [Figures 2A-2D]Figure 2 shows the actin filament alignment index (2A), cell length-to-width ratio (2B), and convex hull-to-cell area ratio (2C) of fibroblasts cultured on HA-only hydrogels or HA / collagen crosslinked hydrogels. The HA-collagen hydrogel (24:6 HA:collagen) formulated at a hydration temperature of 5°C (Formulation X) exhibits significantly higher actin filament alignment index, cell length-to-width ratio, and convex hull-to-cell area ratio than a similar hydrogel formulated at a hydration temperature of 22°C (Formulation VI). *p<0.05 by ANOVA with Tukey post-hoc analysis. The HA-collagen hydrogel with 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 hull-to-cell area ratio when compared to the HA-only gel (Formulation XIX). *p<0.05 by ANOVA with Tukey post-hoc analysis. Figure 2D shows the ranking of HA-collagen hydrogels as a function of Euclidean distance (a 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 spreading and attachment compared to the low-adhesion HA-only gels. Overall, hydrogels formulated at a hydration temperature of 5°C exhibit greater Euclidean distance than HA-only gels.

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

[0058] [Figure 4] FIG. 4 shows the lift profile of Formulation XV vs. Formulation III (mean + / - SEM).

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

[0060] [Figure 6]Figure 6 shows the tissue integration of hydrogels with increasing HA concentrations at 4 mg / mL collagen. (6A) H&E, (6B) collagen 1a, (6C) vimentin, (6D) procollagen 1, and (6E) CD31. H&E staining demonstrates a decrease in ingrowth with increasing HA concentration. As shown, intense collagen 1a staining is observed in the 13 mg / mL HA formulation (Formulation I). The 20 mg / mL HA formulation shows decreased collagen 1a packing, and the 25 mg / mL HA formulation shows extensive areas without collagen 1a deposition. Vimentin-positive fibroblast / fibrocyte infiltration was observed in all formulations, and the extent of infiltration decreased with increasing HA concentration. Procollagen 1 staining appeared reduced in the low HA formulation (Formulation I) compared to 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 formulations may indicate continued collagen deposition over time. Vascularization within the hydrogel bolus was observed in the 20 mg / mL and 25 mg / mL HA formulations, as indicated by positive CD31 staining. CD31 staining was absent in the 13 mg / mL HA formulation.

[0061] [Figure 7]Figure 7 shows tissue integration in hydrogels prepared with a higher percentage 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. Dense collagen 1a deposition was observed on the back of the Formulation XV bolus, but this deposition did not completely fill the gel. Fine strands of collagen 1a-positive tissue were observed throughout the Formulation XVI hydrogel. Vimentin-positive fibroblast / fibrocyte 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 continued collagen deposition over time. Vascularization within the hydrogel bolus was observed in both formulations (arrowheads). Formulation XVI formulation demonstrated the most robust angiogenesis throughout the bolus.

[0062] [Figure 8] Figure 8 shows tissue integration for hydrogels containing 24 mg / mL HA and 6 mg / mL collagen prepared at room temperature (Formulation VI) and 5°C (Formulation X) hydration temperatures. Collagen 1a staining shows a fine collagen distribution around the hydrogel particles in the Formulation VI hydrogel, with limited deposition around the hydrogel particles. Collagen 1a staining for the Formulation X gel shows a robust collagen deposition around the hydrogel, with dense collagen deposition around the hydrogel particles.

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

[0064] [Figure 10] Figure 10 shows that immunohistochemical (IHC) quantification of positively stained areas demonstrated increased levels of vimentin (fibroblasts), collagen I, and CD31 (vascular) in boluses of Formulation XIX hydrogels after 12 weeks of subcutaneous implantation in rats compared to HA-only hydrogels.

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

[0066] [Figure 12]Figure 12 shows 28-week lifting capacity data for crosslinked HA-collagen gels. 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. While not limiting the 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 the result of better integration and tissue ingrowth. As shown, Formulation XIX, in particular, has significantly better tissue ingrowth than the HA-only gel.

[0067] [Figure 13] Figure 13 shows the differences observed in 24:6 HA to collagen gels before and after autoclaving. The 24:6 HA to collagen gels (run in duplicate as Sample 1 and Sample 2) had lower cell viability overall. However, both the autoclaved and non-autoclaved formulations all show higher cell viability than the HA-only gel. As shown, experiments were performed on replicates (Sample 1 and Sample 2) of gels containing 24:6 HA to collagen before (B) and after (A-A) autoclaving. A small 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 implantation in a rat model. Formulation XXII (A; 20 mg HA: 4 mg collagen, hydrated at 5°C) demonstrates similar or better tissue integration than Formulation XIX (B; 20 mg HA: 6 mg collagen, hydrated at 5°C). Blinded scoring by a pathologist further demonstrates the 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, compounded at 25°C) demonstrates inferior tissue integration compared to Formulations XXII and XIX. Formulation XX's tissue integration score is 1.13. This result further demonstrates that tissue integration does not follow a linear trend with collagen concentration. Instead, there are optimal compounding conditions and collagen concentrations that achieve an enhanced tissue response.

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

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

[0071] [Figure 17] FIG. 17 shows the scoring of tissue integration of gel boluses after 4 weeks of subcutaneous implantation of Formulation XXII and Formulation XXIII or HA only control in rats.

[0072] [Figure 18] FIG. 18 shows collagen 1a staining of tissue integration for Formulation XXII and Formulation XXIII compared to HA-only gels.

[0073] [Figure 19] FIG. 19 shows quantification of the percent positive area of ​​collagen 1a staining within the hydrogel bolus after 4 weeks of subcutaneous implantation of Formulation XXII in rats.

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

[0075] [Figure 21] FIG. 21 shows immunohistochemical analysis of tissue responses to HA alone and HA-collagen hydrogels (Formulations XXII and XXIII) after 4 weeks of subcutaneous implantation in rats.

[0076] [Figure 22] Figure 22 shows the 52-week lifting capacity data for Formulation XXII compared to the HA-only gel.

[0077] [Figure 23] Figure 23 shows the 26-week lifting capacity data for Formulation XXIII compared to the HA-only gel.

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

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

[0080] [Figure 26]FIG. 26 shows immunohistochemical analysis of tissue responses to Formulation XXV after 4 weeks of subcutaneous implantation in rats.

[0081] [Figure 27] FIG. 27 shows immunohistochemical analysis of tissue responses to Formulation XXVI after 4 weeks of subcutaneous implantation in rats.

[0082] [Figure 28] Figure 28 shows the 30-week lifting capacity data for Formulations XXV and XXVI compared to the HA-only gel. DETAILED DESCRIPTION OF THE INVENTION

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

[0084] Where the definition of a term used herein deviates from the commonly used meaning of that term, applicants intend to utilize the definition provided herein unless specifically indicated otherwise.

[0085] Disclosed herein are cross-linked polymeric matrices, compositions comprising the cross-linked polymeric matrices, methods for making the cross-linked polymeric matrices, and methods for improving an individual's appearance. The fillers comprising the cross-linked polymeric matrices described herein have immediate filling and lifting properties after injection, which may be followed by tissue integration into the injection site, which may result in long-term, natural-looking results.

[0086] Advantageously, the crosslinking method provides HA / collagen materials with tunable physical properties that result in a variety of filling and lifting properties, allowing such materials to be injected at various tissue depths, into various areas of the face, and for various purposes (volume creation, severe wrinkles, fine wrinkles, etc.). Furthermore, this synthesis method allows for controlled cellular infiltration from surrounding tissue into the injected bolus through the covalent incorporation of collagen into the crosslinked hydrogel. Furthermore, 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 with a natural feel, appearance, and movement. As described herein, methods for improving filler quality result in superior hybrid materials that may surpass previous collagen fillers and current HA fillers.

[0087] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0088] As used in the context of describing the present invention (particularly in the context of the claims below), the terms "a," "an," "the," and similar referents should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. As used herein when referring to a measurable value, "about" is meant to encompass 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 of the term such as "comprising," "comprises," and "comprised" are not intended to exclude additional additives, components, integers, or steps.

[0090] "Crosslinked polymeric matrix" refers to a matrix formed by crosslinking HA and collagen. HA and collagen can be crosslinked by activating the natural carboxylic acid moieties of the HA and collagen so that they can react with endogenous amine groups present on the collagen. Furthermore, to further promote crosslinking of the HA and collagen, lysine may be added as a carboxylic acid / diamine crosslinker. The addition of lysine in this manner allows for tailoring of the physical properties of the resulting hydrogel. The crosslinked polymeric matrix can be used in medical aesthetic compositions or formulations (e.g., as aesthetic or dermal fillers).

[0091] As used herein, "hyaluronic acid" or "hyaluronan" refers to a non-sulfated glycosaminoglycan that is widely distributed throughout the human body in connective, epithelial, and nervous tissues. 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 a major structural protein in the extracellular space of 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 attachment, migration, and proliferation. Collagen can be positively charged due to its high content of basic amino acid residues, such as arginine, lysine, and hydroxylysine. Over 90% of the collagen in the human body is type I collagen. Type III collagen is the major component of reticular fibers and is commonly found together with type I collagen. Those skilled in the art will understand that collagen can be provided from commercial sources. In some embodiments of each or any of the above or below embodiments, the provided collagen material may have a mixture of about 97% to about 99% type I collagen, with the remaining collagen being about 1% to 3% type III collagen.

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

[0094] In some embodiments of each or any of the above or below embodiments, the HA is cross-linked to an amine and may have two or more cross-links through lysines on either the collagen or the HA, or through separate amine groups.

[0095] "Modulus of elasticity," also known as the modulus of elasticity, refers to a quantity that measures the resistance of an object or substance to elastically (i.e., non-permanently) deform when stress is applied.

[0096] As used herein, "compressive force" refers to the application of force, pressure, or effort to an object that compresses, crushes, or compacts the object.

[0097] "Sterilization," as used herein, refers to the submission of a material to a sterilization process that can result in the death of microorganisms in the material. Methods for disinfection and sterilization can be by physical, chemical, and physicochemical means.

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

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

[0100] When subjected to a sterilization process, a sterile product such as a hydrogel can be formed. Such sterilization processes can be found in Chitre et al. (U.S. Patent Application Publication No. 2014 / 0011980), Chitre et al. (U.S. Patent Application Publication No. 2018 / 0147307), and Chitre et al. (U.S. Patent Application Publication No. 2016 / 0101200).

[0101] In one embodiment, the composition or matrix comprises an anesthetic, including but not limited to, benzocaine, chloroprocaine, procaine, proparacaine, tetracaine, amylocaine, oxybuprocaine, articaine, bupivacaine, dibucaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, sameridine, tonicaine, and cinchocaine.

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

[0103] The addition of lysine may allow for independent adjustment of crosslinking, modulating the physical properties of the hydrogel without changing the HA:collagen composition or the amount of activation 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, surprisingly, the HA and sensitive collagen protein components have been shown to degrade only slightly during crosslinking, maintaining their structure largely intact, as shown in the examples.

[0104] In one embodiment, the hyaluronic acid is hydrated for at least 60 minutes before the cross-linking step with collagen. In a further embodiment, the hyaluronic acid is hydrated at a temperature below room temperature. In yet a further embodiment, the 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 ranges defined by any two of the foregoing values. In one embodiment, the hyaluronic acid is hydrated at a temperature above room temperature. Thus, the method of making the hydrogel can be adjusted to tailor the hydrogel properties, such as, for example, the tan del parameter (G" / G').

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

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

[0107] In one embodiment, the cross-linking reaction is carried out at a pH of 4.0, 5.0, 5.5, 6.0, 6.5, or 7.0, or any pH between the range defined by any two of the foregoing values.

[0108] The physical properties of the hydrogel may depend on the concentrations of HA and collagen, the molecular weight of HA, the EDC concentration, the EDC / NHS ratio, the temperature, the pH, the salt / buffer concentration, and the lysine concentration. In one embodiment, elastic modulus (G') values ​​ranging from 30 Pa to approximately 10,000 Pa are obtained. In one embodiment, the elastic modulus depends on the 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 wide spectrum of physical properties obtained, various HA / collagen configurations may find use as dermal fillers for various facial applications.

[0109] In one embodiment of the cross-linking reaction, the method includes terminating the cross-linking process.

[0110] As described in embodiments herein, formulations with low G' and compressive force values ​​and minimal swelling can be applied as very superficial wrinkle fillers or injectable skin enhancement agents, while more robust configurations with high G' and compressive force and increased swelling can be used for moderate to severe wrinkle correction and facial volumization / contouring.

[0111] In one embodiment, a method of filling fine wrinkles comprises providing a composition having low G' and compressive force values ​​to a patient in need thereof. In one embodiment, a method of performing moderate to severe wrinkle correction and facial volume / contouring procedures is provided, the method comprising providing a composition to a patient in need thereof, the composition having higher G' and compressive force.

[0112] It is contemplated to increase the HA concentration of the crosslinked polymer matrix. Increasing the HA concentration can result in a hydrogel with, for example, a higher G', a higher compressive force value, and higher opacity. Increasing 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 correction and appearance. Thus, a high lifting capacity may require a high strength matrix. The elastic modulus (G') can represent the stiffness of the matrix and the ease of extrusion of the matrix.

[0113] The elastic modulus can be a function of the concentration of hyaluronic acid. In one embodiment, the HA concentration is between 13 mg / ml and 28 mg / ml. In one embodiment, the composition has a modulus of elasticity of about 30 Pa, about 40 Pa, about 50 Pa, about 60 Pa, about 70 Pa, about 80 Pa, about 90 Pa, about 100 Pa, about 200 Pa, about 300 Pa, about 400 Pa, about 500 Pa, about 600 Pa, about 700 Pa, about 800 Pa, about 900 Pa, about 1000 Pa, about 1100 Pa, about 1200 Pa, about 1300 Pa, about 1400 Pa, about 1500 Pa, about 1600 Pa, about 1700 Pa, about 1800 Pa, about 1900 Pa, about 2000 Pa, about 2100 Pa, or about 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 Pa, about 7900 Pa, about 8000 Pa, about 8100 Pa, about 8200 Pa, about 8300 Pa, about 8400 Pa, about 8500 Pa, about 8600 Pa, about 8700 Pa, about 8800 Pa, about 8900 Pa, about 9000 Pa, about 9100 Pa, about 9200 Pa, about 9300 Pa, about 9400 Pa, about 9500 Pa, about 9600 Pa, about 9700 Pa, about 9800 Pa, about 9900 Pa or about 10000 Pa, or any modulus between a range defined by any two of the foregoing 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 with different molecular weights are contemplated, which can affect the G' and compressive force values. For example, in HA:collagen formulations, lowering the hydration temperature can result in higher G' values, lower swelling, and decreased opacity (increased translucency). These synthesis parameters and results can be used to synthesize HA-collagen formulations with desired physical properties.

[0115] Collagen concentration can also affect physical properties. At a given hydration temperature, increasing collagen concentration can result in increased opacity, higher G', and decreased swelling. Increased opacity can result in 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 collagen solubility. Collagen solubility can be reduced by increasing temperature, pH, and salt concentration, and reduced collagen solubility during synthesis results in gels with lower G', higher swelling, and increased extrusion force. HA can also interact with collagen and reduce its solubility, as described by Taguchi and coworkers (Taguchi et al., Journal of Biomedical Materials Research, 2002, 61(2), 330-336, incorporated herein by reference). Adjusting the salt concentration can change the interaction between HA and collagen, thereby adjusting collagen solubility and changing physical properties. In one embodiment, the composition contains a salt having a concentration of 50 mM to 400 mM. In one embodiment, the composition has an NaCl concentration of about 150 mM. Thus, by lowering the hydration temperature and optimizing the salt / buffer concentration, maximum collagen solubility for a given HA concentration during synthesis can be achieved along with maximum G' and minimum swelling values.

[0116] In one embodiment, the composition is transparent. In one embodiment, the composition is translucent. In one embodiment, the HA concentration, hydration temperature, salt concentration, and / or collagen concentration affect the opacity of the composition. Increasing the opacity can reduce the Tyndall effect, a blue discoloration that may be observed at the injection site. 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 have been characterized. In one embodiment, this formulation exhibits enhanced cellular activity compared to HA-only materials. Activity levels depend not only on collagen concentration but also on HA concentration and synthesis procedure. In one embodiment, formulations with lower HA concentrations (13 mg / mL) were found to provide enhanced in vitro responses compared to 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, demonstrated greater in vitro responses. Furthermore, in one embodiment, formulations hydrated below room temperature stimulated higher cellular activity than similar formulations hydrated at room temperature. A specific level of cellular activity may be required for a particular filler application, and the desired activity level can be achieved by selecting formulation and synthesis parameters.

[0118] In one embodiment, HA / collagen formulations were also evaluated for tissue response in a tissue integration model. Histological sections obtained from implants of HA-collagen material showed cellular infiltration from surrounding tissue, as well as new collagen deposition and vascularization within the injected filler bolus. The extent of infiltration and tissue integration varied among different formulations. In some embodiments of the formulations described herein, collagen structure and crosslinking surprisingly play a role in the extent of infiltration and tissue integration. In one embodiment, collagen structure and crosslinking may be important in tissue integration and infiltration (see, e.g., Figure 14). In some embodiments of the formulations described herein, tissue integration decreased with increasing HA concentration. In some embodiments of the formulations described herein, including those with low HA concentrations (13 mg / ml) injected into tissue, the surrounding tissue was found to infiltrate the gel bolus by 4 weeks. In these embodiments, cell nuclei and newly deposited collagen were found interspersed throughout the gel. This can be seen in Example 7 (Figures 6B and 6C), where Formulation I was used. Thus, these formulations provided surprising results regarding tissue infiltration into the gel bolus.

[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 with formulations with lower HA concentrations.

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

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

[0122] Surprisingly, differences in results were observed for compositions with different mixed gel structures: compositions with mixed collagen responded differently than compositions in which the gel had collagen cross-linked to HA at 5°C.

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

[0124] With the exception of collagen concentration, the level or crosslinking and structure of the composition were of equal importance. For example, compositions such as gel formulations produced at lower 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. Preparation of the formulation also resulted in surprising results, such as improved in vitro and in vivo performance. In some embodiments, preparation of gel formulations at lower 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 at the site of injection.

[0125] Formulations with 20:6 and 20:4 HA:collagen ratios and hydration temperatures of about 5°C also provided surprising results, including improved in vitro and in vivo performance.

[0126] In some embodiments, a formulation is provided that increases collagen infiltration into tissue. The formulation comprises 13 mg / ml 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 tissue surrounding the depot are deposited in the depot. In one embodiment, tissue injected with the formulation exhibits tissue integration and 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 one 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 underlying tissue that covers the injected dermal filler. The amount of lift required depends on the specific facial indication. Products placed deeper under the skin to create volume for the indication need to exhibit more structure and greater lifting power. Formulations for fine wrinkles with superficial placement do not need to exhibit as much lifting power, but they should be smoother and integrate with existing tissue. Therefore, HA / collagen formulations were evaluated in an animal lifting capacity model to determine lifting capacity. For formulations crosslinked in a similar manner, lifting capacity was dependent on HA concentration, with higher HA concentrations resulting in increased lift.

[0128] In some embodiments, cross-linked HA:collagen formulations with added lysine exhibited increasing lifting force as the HA concentration increased from 13 mg / mL to 20 mg / mL to 25 mg / mL. In some embodiments, the molecular weight of the HA also affects lifting force. In some embodiments, a formulation containing 25 mg / mL of high molecular weight HA exhibited greater lifting force than a formulation consisting of a 25 mg / mL mixture of low and high molecular weight HA. Thus, by selecting optimal synthesis parameters, HA concentration, and HA molecular weight ratio, a desired lifting force can be achieved. In some embodiments of each or any of the above or below embodiments, the formulation comprises a mixture of hyaluronic acid components having different molecular weights, the mixture comprising: about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,500,000 daltons, about 2,0 ... Hyaluronic acid having an average molecular weight of about 1,500,000 daltons, about 3,000,000 daltons, about 3,500,000 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 a range between any two of the aforementioned values.

[0129] Method for synthesizing lysine-crosslinked HA-collagen hydrogels The present 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 to homogenize. In some embodiments, the hydrogel hydrates below room temperature. In one embodiment, the HA hydrates at a temperature between 2°C and 35°C. In one embodiment, the 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 any temperature in between the range defined by any two of the foregoing values. In one embodiment, the HA is hydrated at a temperature between 2°C and 19°C. In one embodiment, the 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 any temperature in between the range defined by any two of the foregoing values. In one embodiment, the HA is hydrated at room temperature for at least 60 minutes. In one embodiment, the HA is hydrated at a temperature higher than room temperature. In one embodiment, the HA is hydrated at a temperature of at least 35°C. In some embodiments, a separate hydration step is performed for at least 60 minutes. In some embodiments, the hydration is performed in MES buffer at about pH 5.5. The mixture may be placed in one syringe or passed between two syringes at least 50 times. A solution of EDC / NHS may be added to the mixture. Mixing may be performed by passing the solution between the two syringes. After adding the EDC / NHS solution, the mixture is allowed to react for at least 16 hours at a temperature of 2-8°C. In some embodiments, the pH of the solution is adjusted to 7.4 using NaOH and purified using 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 compression force, swelling characteristics, and extrusion force.

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

[0131] In one embodiment, the lysine:HA ratio is optimized to maximize crosslinking efficiency. In one embodiment, the lysine:HA ratio is approximately 0.0-0.5, which may allow for more efficient crosslinking. Lysine-free crosslinking may rely on collagen to provide amines for crosslinking, making ester crosslinks between HA chains more water-labile. Crosslinking at a high lysine:HA ratio may saturate activated carboxylic acids on the HA chains, resulting in pendant lysine molecules attached to only one side of the HA chains 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 tailored to achieve desired properties. In some embodiments, the optimal lysine:HA ratio may be composition-dependent.

[0132] Sterilization of the composition Developed biomaterials may require sterilization or destruction of unwanted biological materials, such as pathogens and bacterial microorganisms, prior to administration of the composition by injection or implantation into a human patient. These compositions include embodiments described herein and include materials such as cross-linked polymeric matrices. Proteins, polysaccharides, and carbohydrates in these materials may be susceptible to molecular breakdown when exposed to conventional heat sterilization procedures, such as autoclaving, or to ionizing radiation, such as gamma radiation. Traditionally, many of these energy-sensitive biomaterials are sterilized in bulk by microfiltration processes designed to physically remove microorganisms from the composition. The filtered composition must then be packaged into syringes and / or vials for use by physicians.

[0133] In one embodiment, the cross-linked polymeric matrix is ​​sterile. In one embodiment, the method of making a cross-linked polymeric matrix further comprises sterilizing the cross-linked polymeric matrix.

[0134] In one embodiment, the method further comprises exposing the composition or crosslinked polymeric matrix to a dose of broad-spectrum radiation effective to inactivate pathogens, microorganisms, and other microorganisms.

[0135] In one embodiment, the method further comprises exposing the composition or crosslinked polymer matrix to pulsed radiation (hereinafter sometimes referred to as pulsed light) comprising broadband spectrum radiation. The broadband spectrum radiation may have a wavelength range of about 100 nm to about 1100 nm. The broadband spectrum 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 provided by a xenon lamp.

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

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

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

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

[0142] In one embodiment, the pulsed light is effective to sterilize the composition without raising the temperature of the composition above 90° C. In one embodiment, the pulsed light is effective to sterilize the composition without raising the temperature of the composition above 20° C. In one embodiment, the dose is effective to sterilize the composition without raising the temperature of the composition above 15° C., such as above 10° C., for example above 5° C.

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

[0144] In one embodiment, the pulsed light is effective to sterilize the composition, i.e., inactivate pathogens, microorganisms, and other microorganisms in the composition, without causing significant degradation, e.g., without causing significant changes in the rheological properties of the composition.

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

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

[0147] Example 1 - Synthesis of lysine cross-linked HA-collagen hydrogel A solution of 4.96 mg / mL collagen 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 concentrations were adjusted accordingly to create hydrogels with HA:collagen ratios of 13:4 mg / mL (Formulation I), 20:4 mg / mL (Formulation II), and 25:4 mg / mL (Formulation III). The mixture was stirred to homogenize the solution, and the HA was allowed to hydrate for approximately 60 minutes at room temperature. After approximately 60 to 90 minutes, the mixture was passed between the syringes and allowed to hydrate again for approximately 30 to 60 minutes. After the second hydration, the mixture was passed between the syringes several times. An EDC / NHS solution was prepared in a third 30 mL syringe by adding water, NHS, and EDC and shaking to mix. The EDC / NHS solution was added to the HA / collagen mixture, passed through two syringes, and then transferred to a glass vial, which was allowed to react at 2-8°C. In some embodiments, the reaction time is approximately 16 hours, approximately 18 hours, approximately 20 hours, approximately 22 hours, approximately 24 hours, or any time between the ranges defined by any two of the aforementioned values. After this, the gel was transferred to a syringe and passed through the two syringes again. The pH of the gel was adjusted to approximately 7.40 using 2 M 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 time the buffer was exchanged several times to remove the EDC / NHS. The gel was then transferred from the dialysis membrane to a syringe, passed through a stainless steel mesh (60 μm pore size - 104 μm pore size), and passed through the two syringes. The gel was then transferred to a 1 mL syringe, which was then steam sterilized. The resulting sterile hydrogels were characterized using rheology, compression force measurements, extrusion force measurements, and swelling.

[0148] For formulation XXVI, NaCl was omitted during cross-linking.

[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 prior to 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 collagen 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 solids. The pH was adjusted with 1 M NaOH. The mixture was stirred to homogenize, and the HA was allowed to hydrate for approximately 60 minutes at the specified temperature. After approximately 60–90 minutes, the mixture was passed between the two syringes several times and allowed to hydrate again for at least 30 minutes. After the second hydration step, the mixture was again passed between the two syringes several times. An EDC / NHS solution was prepared in a third 30 mL syringe by adding water, NHS, and EDC and shaking to mix. Hydration can be performed at temperatures of approximately 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C, or at 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 between two syringes several times, and then transferred to a Thinky Mixer reaction vessel, which was allowed to react for at least 16 hours at 2-8°C. The gel was then homogenized using the Thinky Mixer. The pH of the gel was adjusted to approximately 7.40 using 2 M 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 several buffer changes during this time. The gel was then transferred from the dialysis membrane into a syringe, passed through a stainless steel mesh (104 μm pores), and homogenized using the Thinky Mixer. The gel was transferred into a 1 mL syringe, which was then steam-sterilized. The resulting sterilized hydrogel was characterized as described in the previous example.

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

[0152] Example 3-28: Synthesis of lysine cross-linked HA-collagen hydrogel with a HA:collagen concentration of 2 mg / mL (Formulation XVI). A solution of 3.20 mg / mL collagen 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 solids. The pH was adjusted with NaOH. The mixture was stirred to homogenize, and the HA was allowed to hydrate for approximately 90 minutes at room temperature. After 90 minutes, the mixture was passed through the syringes several times and allowed to hydrate again for approximately 30 minutes. After the second hydration step, the mixture was passed through the syringes several times. An EDC / NHS solution was prepared in a third 30 mL syringe by adding water, NHS, and EDC, and the mixture was shaken to mix. The EDC / NHS solution was added to the HA / collagen mixture, passed through the syringes several times, and then transferred to a glass vial, which was then allowed to react for at least 16 hours at 2–8°C. The gel was then transferred to the syringe and passed through the syringes. The pH of the gel was adjusted to approximately 7.40 using 2 M 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 several buffer changes during this time. The gel was then transferred from the dialysis membrane into a syringe, passed through a stainless steel mesh (104 μm pores), and homogenized by passing it between syringes. The gel was transferred into a 1 mL syringe, and the syringe was steam-sterilized. The resulting sterilized hydrogel was characterized as described in the previous example.

[0153] Example 4 - Synthesis of lysine cross-linked HA-collagen hydrogel with HA:collagen concentration of 25:4 mg / mL prepared at 1.25x the final concentration (-Formulation XV) A solution of 5.67 mg / mL collagen 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 solids. The pH was adjusted with 1 M NaOH. The mixture was stirred to homogenize, and the HA was allowed to hydrate at room temperature for approximately 90 minutes. The mixture was then passed through the syringes several times and allowed to hydrate again for 30 minutes. After the second hydration step, the mixture was again passed through the syringes. An EDC / NHS solution was prepared in a third 30 mL syringe by adding water, NHS, and EDC and shaking to mix. The EDC / NHS solution was added to the HA / collagen mixture, passed through the syringes several times, and then transferred to a glass vial, which was allowed to react for at least 16 hours at 2–8°C. The gel was then transferred to a syringe and passed through the syringes. The pH of the gel was adjusted to approximately 7.40 using 2 M 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 several buffer changes during this time. The gel was then transferred from the dialysis membrane into a syringe, passed through a stainless steel mesh (104 μm pores), and homogenized by passing it between syringes. The gel was transferred into a 1 mL syringe, and the syringe was steam-sterilized. The resulting sterilized hydrogel was characterized as described in the previous example.

[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 profilometer. Samples were analyzed at a 1 mm gap height using both frequency sweep (10 Hz to 0.1 Hz, 1% strain) and amplitude sweep (0.3% to 300% strain, 5 Hz frequency) measurements. Compression force was measured using the same instrument with a 2.5 mm gap height and normal compression. The gap height was established at 2.5 mm and held there for 5 min, then compressed from 2.5 mm to 0.89 mm at a rate of 13.33 μm / s. Hydrogel swelling was measured by mixing the gel sample with an excess of phosphate buffer and determining the gel volume after equilibration. The volume of the swollen gel was compared to the original gel volume before buffer addition. Swelling is expressed as the uptake of additional fluid as a percentage of the original gel volume. Gel extrusion force was measured for gel formulations in 1 mL COC syringes fitted with ½ inch 27G TSK needles (unless otherwise stated) using a texture analyzer set at a speed of 50 mm / min. [Table 1]

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

[0156] As shown in Table 1, as the HA concentration increases from 13 mg / mL to 20 mg / mL to 25 mg / mL (Formulation I vs. Formulation II vs. Formulation III) at a constant collagen concentration, the G' value increases (380 Pa → 645 Pa → 1370 Pa) along with the compression force (47 gmf → 180 gmf → 310 gmf) and extrusion 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] As the HMW / LMW HA ratio decreased from 100 / 0 to 65 / 35 to 35 / 65 at the same HA and collagen concentrations (Formulation VI vs. Formulation VII vs. Formulation VIII), the G' value decreased (1360 Pa → 1180 Pa → 932 Pa) along with the compression force (292 → 226 → 163 gmf) and extrusion force (63.6 → 29.2 → 20.0 N), while the G'' / G' ratio increased (0.055 → 0.068 → 0.085) as the HMW / LMW ratio decreased.

[0158] Decreasing the synthesis hydration temperature 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) increases the G' value (876 → 1360 → 3145 → 4750 Pa), but decreases the hydrogel swelling (257% → 238% → 159% → 127%) along with the extrusion force (56.1-63.6 → 33.8 → 20.1 N). Compression force is unaffected by changes in hydration temperature, except at higher hydration temperatures (35°C vs. others). Adjusting the hydration temperature during synthesis alters the solubility of collagen and, consequently, the physical properties of the resulting hydrogel.

[0159] By reducing the salt / buffer concentration by two-thirds, similar G' values ​​(5470 Pa vs. 4750 Pa), swelling (108% vs. 127%), and extrusion force (20.5 N vs. 20.1 N) are obtained for formulations hydrated at 22°C with the lower salt / buffer concentration and at 5°C with the original salt / buffer concentration (Formulation XIII vs. Formulation X). Furthermore, syntheses using lower salt / buffer concentrations are less sensitive to hydration temperatures of 22°C vs. 5°C than syntheses using the original salt / buffer concentrations. While the values ​​of G', swelling force, and extrusion force are not significantly different for formulations synthesized using lower salt / buffer concentrations with hydration temperatures of 22°C and 5°C (Formulation XIII vs. Formulation XII), these physical properties are significantly different for similar formulations synthesized using the original salt / buffer concentrations (Formulation VI vs. Formulation X).

[0160] The effect of added lysine can be seen by comparing similar formulations synthesized at a 28:2 mg / mL HA:collagen concentration (Formulation XVIII vs. Formulation XVI vs. Formulation XVII). The optimal lysine:HA ratio helps maximize crosslinking efficiency, and the exact ratio depends on the molecular weight of HA, the concentration of the activating reagent, and the synthesis conditions. For example, the lysine:HA ratio was increased (0 → 0.333 → 0.5) for a series of formulations synthesized at a 28:2 mg / mL HA:collagen concentration (Formulation XVIII vs. Formulation XVI vs. Formulation XVII). Physical properties such as G' and compressive force were maximized for formulations prepared with a lysine:HA ratio of 0.333, while swelling and G'' / G' values ​​were minimized for those same formulations. Higher G' and lower swelling are generally associated with more highly crosslinked hydrogels. A lysine:HA ratio between 0 and 0.5 allows for more efficient crosslinking. Lysine-free crosslinking may rely on collagen to provide amines for crosslinking, making the ester crosslinks between HA chains more water-labile. Crosslinking at high lysine:HA ratios can saturate activated carboxylic acids on the HA chains, resulting in pendant lysine molecules attached to only one side of the HA chains rather than crosslinking between chains. These scenarios, with very low or high lysine:HA ratios, can 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 tailored to achieve the desired properties.

[0161] Example 6 - In vitro testing of hydrogels In vitro cell proliferation and viability The viability and proliferation of fibroblasts in intimate contact with HA-collagen hydrogels were quantified using the XTT assay. 100 μL of hydrogel (n=3) was layered 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 48 hours of incubation, 250 μL of XTT reagent was added to each well and incubated at 37°C for 4 hours. The plate was then spun at 300 x g for 5 minutes, and 200 μL of supernatant from each well was transferred to a well of a 96-well filter plate with a 20 μm mesh. The filter plate containing the XTT supernatant was spun at 300 x g for 5 minutes. 100 μL of filtered supernatant from each well was transferred to a clean 96-well plate (black walls, clear bottom), and the absorbance of the supernatant was read using a microplate reader (450 nm with 630 nm background correction). Data were normalized to the XTT cell viability of fibroblasts cultured on tissue culture polystyrene (TCPS) as a positive control.

[0162] Cell viability and proliferation were found to be higher in formulations with lower HA concentrations than in similarly crosslinked formulations with the same collagen concentration and higher HA concentrations. For example, hydrogels synthesized with 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) demonstrated proliferation values ​​of 53%, 30%, and 20% compared to the TCPS positive control (Figure 1). The HA-only negative control gel demonstrated proliferation values ​​of 12% compared to the TCPS control.

[0163] Furthermore, the effect of hydration temperature during the synthesis procedure on cell viability and proliferation of similarly crosslinked formulations with the same HA and collagen concentrations was observed. For hydrogels with a 24:6 mg / mL HA:collagen concentration, those 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 salt and buffer concentrations during hydrogel synthesis. For syntheses performed at 5°C, no change in cellular response was evident when the salt / buffer concentration was reduced (Formulation X (1 equivalent) vs. Formulation XII (0.33 equivalents)). However, for formulations hydrated at 22°C or 35°C, significantly increased cell viability and proliferation were observed for formulations prepared with reduced salt / buffer concentrations (Formulation XIII, 22°C, and Formulation XIV, 35°C) compared with formulations prepared with 1 equivalent of salt / buffer (Formulation VI, 22°C, and Formulation XI, 35°C) (Figure 1).

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

[0166] A formulation with a 20:4 HA:collagen ratio was also shown to enhance cellular responses in vitro (Figure 15, Formulation XXII).

[0167] Additionally, Formulation XIX was shown to have consistent stability and performance after autoclaving.

[0168] In vitro cell morphology Cell morphology was analyzed to assess the effect of hydrogel formulations on cell size, shape, and cytoskeletal organization. The actin filament alignment index and morphology of fibroblasts cultured on HA-only or HA / collagen cross-linked hydrogels were imaged and quantified. Increased actin filament alignment can be correlated with increased cell adhesion to the substrate. An increase in the length-to-width ratio correlates with increased cell spreading on the substrate. The ratio of the convex hull to the cell area is a measure of cell shape, with 1.0 indicating uniformly shaped cells and values ​​above 1 indicating more irregularly shaped cells. Cells that make multiple contacts with the matrix and are elongating / migrating exhibit more irregular cell shapes and higher convex hull to cell area ratio values. The actin filament alignment index, length-to-width ratio, and convex hull to cell area ratio can be analyzed together in 3D Euclidean space. The Euclidean distance obtained from the negative control hydrogel (in this case, the non-adherent HA-only gel) allows for the overall ranking of cellular responses to the filler. A larger Euclidean distance obtained from the HA-only control indicates enhanced cell attachment and spreading on the hydrogel. Hydrogels that support greater cell attachment and spreading are expected to induce more cell infiltration into the gel, which then deposits ECM within the gel matrix. Increased cell infiltration and ECM deposition may be beneficial for in vivo tissue integration into the hydrogel depot. Conversely, formulations that result in lower cell attachment and spreading values ​​behave more inertly, resulting in less tissue infiltration and integration. In some embodiments, the method of making the hydrogel further includes an autoclaving step, which does not alter the hydrogel's properties. (See Figure 13.)

[0169] In a typical procedure, hydrogels (n = 3) and human dermal fibroblasts in cell culture medium were added to a 96-well cell culture plate with a low-adhesion surface coating. After 48 hours of incubation, 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 (phalloidin) and cell morphology (WGA-488) were analyzed using image analysis software.

[0170] The effect of hydration temperature during the synthesis procedure on cell attachment and spreading of similarly crosslinked formulations with the same HA and collagen concentrations was observed. For hydrogels with a 24:6 HA:collagen concentration, the formulation hydrated at 5°C (Formulation X) exhibited higher cell attachment (actin filament alignment index) than the formulation hydrated at 22°C (Formulation VI), with values ​​of 0.054 and 0.015, respectively (Figure 2). The formulation hydrated at 5°C (Formulation X) also exhibited increased cell spreading (cell length-to-width ratio) compared to the formulation hydrated at 22°C (Formulation VI), with values ​​of 2.52 and 1.40, respectively. The formulation hydrated at 5°C (325_B) also exhibited a higher convex hull-to-cell area ratio than the 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 the HA-only control. The optimized formulation (20:6 HA:collagen, hydrated at 5°C, Formulation XIX) exhibits 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 reveals that the optimized formulation outperforms the other HA-collagen hydrogels.

[0171] The cell morphology analysis correlates well with the XTT cell activity assay in that Formulation X, which showed greater activity than Formulation VI in the activity assay, also showed evidence of increased cell attachment and spreading in the morphology assay. Formulation XIX also exhibits greater activity than other HA-collagen formulations and HA-only gels. Cell spreading and attachment are associated with greater cell activity, and therefore, the results of each assay are in good agreement with each other (Figures 2A-D).

[0172] Fibroblasts cultured with Formulation XXII and Formulation XXIII exhibit significantly greater cell length-to-width ratios than fibroblasts cultured with HA-only gels (Figure 16).

[0173] Example 7 - In vivo testing of hydrogels Lifting Capacity The hydrogel's ability to support tissue elevation (lifting force) was evaluated in vivo using a subcutaneous implantation model in rats. 125 μL of hydrogel (n=10) was injected as a subcutaneous bolus above the skull. A clinical 3D imaging system (Canfield Vectra) was used to generate 3D reconstructions of the bolus over a 12-week period. The average bolus height 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 over 4 to 12 weeks showed a positive correlation with the HA concentration. The formulation containing 25 mg / mL HA (Formulation III) showed more lift than the 20 mg / mL (Formulation II) or 13 mg / mL (Formulation I) formulations (Figure 3). Because compressive force increases with HA concentration in these formulations, in vivo lifting over 4 to 12 weeks correlates positively with compressive force. In vivo lifting force also depends on the gel synthesis conditions. Two formulations (Formulation III vs. Formulation XV) contained the same HA:collagen concentration of 25:4 mg / mL but were synthesized under different HMW / LMW HA ratios and different crosslinking conditions, resulting in different lifting profiles over 4 to 12 weeks. The formulation synthesized using high-molecular-weight HA at a 1x synthesis concentration exhibited superior lifting force compared to the formulation prepared using 10 / 90 HMW / LMW HA at a 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) exhibited similar in vivo lifting profiles between 4 and 12 weeks (Figure 5). Thus, by selecting the optimal composition and synthesis conditions, the desired lifting profile for a given application can be obtained.

[0175] Lifting ability was also tested using Formulation XIX and an HA-only formulation (Figures 11 and 12). As shown, Formulation XIX demonstrated similar lifting ability to the 24 mg / ml HMW HA-only gel from 4 to 28 weeks.

[0176] Long-term (52-week) lifting capacity data for Formulation XXII was examined. 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 surprising result indicates that these HA-collagen gel formulations are capable of longer lift duration than HA-only gels. This correlates with better tissue ingrowth than HA-only gels (see below).

[0177] Long-term (26-week) lifting performance data for Formulation XXII was tested. Formulation XXIII and the HA-only comparison demonstrate similar lifting performance over 26 weeks (Figure 23). The enhanced tissue integration of Formulation XXIII surprisingly results in longer duration of lifting performance and other benefits to the overall effect. For example, the newly formed tissue maintains skin quality, lifting performance, and wrinkle correction.

[0178] Long-term (30-week) lifting capacity data for Formulation XXV was examined. 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 surprising result indicates that these HA-collagen gel formulations are capable of longer lift duration than HA-only gels, which correlates with better tissue ingrowth than HA-only gels (see below).

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

[0180] Tissue integration correlates negatively with HA concentration for similarly prepared formulations using HMW HA and 4 mg / mL collagen. The density of collagen deposited near the surrounding tissue is higher for the formulation containing 13 mg / mL HA (Formulation I) than for those prepared with 20 mg / mL HA (Formulation II) or 25 mg / mL HA (Formulation III), and is dependent on HA concentration (Figures 6A-6E). Furthermore, the 13 mg / mL HA formulation exhibits fewer areas of the injected bolus devoid of tissue than the materials containing 20 mg / mL or 25 mg / mL HA. In addition to HA concentration, integration is expected to depend primarily on collagen concentration. However, it is possible that other factors strongly influence tissue infiltration into the bolus. For example, a formulation with a high HA concentration (28 mg / mL) and a low collagen concentration (2 mg / mL) (Formulation XVI) demonstrates collagen deposition throughout the bolus with few areas devoid of tissue.

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

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

[0183] Figure 20 shows confocal micrographs of human dermal fibroblasts cultured for 48 hours on HA-only, Formulation XXII, or Formulation XXIII gels. Samples were stained for HA-binding proteins, Hoechst, and cell membranes. The resulting gels showed marked improvement in cell attachment when compared to the HA-only crosslinked product, thus indicating the gels' potential to function as a scaffold for tissue integration and collagen deposition.

[0184] Formulation XIX was also tested for its ability to increase the levels of vimentin (fibroblasts), collagen I, and CD31. As shown in Figures 9 and 10, Formulation XIX was able to increase the levels of vimentin (fibroblasts), collagen I, and CD31 (blood vessels) in the Formulation XIX hydrogel bolus compared to the HA-only hydrogel after 12 weeks of subcutaneous implantation in rats. This is supported by the improved cell spreading 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 tissue regeneration in the hydrogel bolus with a morphology consistent with endogenous tissue.

[0185] Figure 24 shows confocal micrographs of human dermal fibroblasts cultured for 48 hours on HA-only, Formulation XXVI, or Formulation XXV gels. Samples were stained for HA-binding proteins, Hoechst, and cell membranes. Formulation XXVI and Formulation XXV gels showed a surprising improvement in cell attachment when compared to the HA-only crosslinked product, thus demonstrating the potential of the gels to function as a scaffold 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 subcutaneous bolus injections of HA alone, Formulation XXV, or Formulation XXIII after 12 weeks. The presence of second harmonic generation (white) indicates the formation of fully assembled fibrillar collagen in the HA-collagen treated implants. Limited second harmonic generation can be seen in the HA-only gel.

[0187] Figure 26 shows immunohistochemical analysis of the tissue response to Formulation XXV after 4 weeks of subcutaneous implantation in rats. Formulation XXV promoted 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 hydrogel bolus. Furthermore, Formulation XXV resulted in a higher tissue integration pathology score (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 implantation in rats. Formulation XXVI promoted 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 hydrogel bolus. Furthermore, Formulation XXVI resulted in a higher tissue integration pathology score (4.17) compared to the HA-only control (0.67).

[0189] Description of the subject technology in the form of clauses Various examples of aspects of the present disclosure are described for convenience in the form of numbered clauses (1, 2, 3, etc.). These are offered by way of example and not as limitations on the subject technology. Identification of figures and reference numbers is provided below merely by way of example and for illustrative purposes, and the clauses are not limited by those identifications.

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

[0191] Item 2. A crosslinked polymeric matrix according to any one of the above or below items, further comprising lidocaine.

[0192] Item 3. A crosslinked polymer matrix according to any one of the preceding or following items, wherein the lidocaine is present in the matrix at a concentration ranging from about 0.15% (w / w) to about 0.45% (w / w).

[0193] Item 4. The crosslinked polymeric matrix of any one of the preceding or following items, wherein the lidocaine is at 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) of the matrix, or any concentration between the ranges defined by any two of the foregoing values.

[0194] Item 5. A crosslinked polymer matrix according to any one of the preceding or following items, wherein the lidocaine is present in the matrix at a concentration ranging from about 0.27% (w / w) to about 0.33% (w / w).

[0195] Item 6. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the matrix further comprises non-crosslinked HA.

[0196] Item 7. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the non-crosslinked HA has a concentration in the matrix of up to about 5% (w / w).

[0197] Section 8. A crosslinked polymeric matrix according to any one of the preceding or following sections, wherein the non-crosslinked HA has a concentration in the matrix 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 ranges defined by any two of the foregoing values.

[0198] Item 9. A crosslinked polymer matrix according to any one of the preceding or following items, wherein the non-crosslinked HA has a concentration of about 1% (w / w) in the matrix.

[0199] Item 10. A crosslinked polymer matrix according to any one of the preceding or following items, wherein the non-crosslinked HA has a concentration of about 2% (w / w) in the matrix.

[0200] Item 11. A crosslinked polymer matrix according to any one of the preceding or following items, wherein the non-crosslinked HA has a concentration of about 5% (w / w) in the matrix.

[0201] Item 12. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the non-crosslinked HA improves the extrudability of the polymeric matrix.

[0202] Item 13. A crosslinked polymeric matrix described in any one of the above or below items, which is stable for about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or any time between the ranges defined by any two of the foregoing values.

[0203] Item 14. A crosslinked polymer matrix according to any one of the preceding or following items, which is stable at temperatures between about 4°C and about 25°C.

[0204] Item 15. A crosslinked polymer matrix according to any one of the preceding or following items, which is stable at about 4°C.

[0205] Item 16. A crosslinked polymeric matrix according to any one of the preceding or following items, which is stable at about 25°C.

[0206] Item 17. The crosslinked polymeric matrix of any one of the preceding or following items, which 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 months, 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 any time point between the ranges defined by any two of the foregoing values.

[0207] Item 18. A crosslinked polymeric matrix according to any one of the preceding or following items, which exhibits negligible degradation within about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or any time between the ranges defined by any two of the foregoing values.

[0208] Item 19. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the matrix has an elastic modulus (G') of from about 30 Pa to about 10,000 Pa, or any modulus between the ranges defined by any two of the foregoing values.

[0209] Item 20. The matrix is ​​about 30 Pa, about 40 Pa, about 50 Pa, about 60 Pa, about 70 Pa, about 80 Pa, about 90 Pa, about 100 Pa, about 200 Pa, about 300 Pa, about 400 Pa, about 500 Pa, about 600 Pa, about 700 Pa, about 800 Pa, about 900 Pa, about 1000 Pa, about 1100 Pa, about 1200 Pa, about 1300 Pa, about 1400 Pa, about 1500 Pa, about 1600 Pa, about 1700 Pa, about 1800 Pa, about 1900 Pa, about 2000 Pa, about 2100 Pa, about 2200 Pa, about 2300 Pa, about 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, 9000 Pa, about 9100 Pa, about 9200 Pa, about 9300 Pa, about 9400 Pa, about 9500 Pa, about 9600 Pa, about 9700 Pa, about 9800 Pa, about 9900 Pa, or about 10000 Pa, or any modulus between the ranges defined by any two of the foregoing values.

[0210] Item 21. The matrix is ​​about 10 gmf, about 20 gmf, about 30 gmf, about 40 gmf, about 50 gmf, about 60 gmf, about 70 gmf, about 80 gmf, about 90 gmf, about 100 gmf, about 110 gmf, about 120 gmf, about 130 gmf, about 140 gmf, about 150 gmf, about 160 gmf, about 170 gmf, About 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 350 500 gmf, about 510 gmf, about 520 gmf, about 530 gmf, about 540 gmf, about 550 gmf, about 560 gmf, about 570 gmf, about 580 gmf, about 590 gmf or about 600 gmf, or any compressive force value between the ranges defined by any two of the aforesaid values.

[0211] Item 22. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the matrix has a compressive force value 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 ranges defined by any two of the foregoing values.

[0212] Item 23. The crosslinked polymeric matrix of any one of the preceding or following items, wherein the hyaluronic acid is at a concentration of about 5 mg / ml, about 6 mg / ml, about 8 mg / ml, about 10 mg / ml, about 12 mg / ml, about 14 mg / ml, about 16 mg / ml, about 18 mg / ml, about 20 mg / ml, about 22 mg / ml, about 24 mg / ml, about 26 mg / ml, about 28 mg / ml, about 30 mg / ml, about 32 mg / ml, about 34 mg / ml, or about 36 mg / ml, or any concentration between the ranges defined by any two of the foregoing values.

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

[0214] Item 25. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the collagen comprises type II collagen.

[0215] Item 26. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the collagen comprises type III collagen.

[0216] Item 27. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the collagen comprises 0% to 3% type II collagen.

[0217] Item 28. A crosslinked polymer matrix according to any one of the above or below items, wherein the collagen comprises 1% to 3% type I collagen.

[0218] Item 29. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the matrix comprises from about 0% to about 3% type III collagen.

[0219] Item 30. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the collagen comprises about 97% to about 99% type I collagen.

[0220] Item 31. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the collagen comprises a mixture of both type I and type III collagen.

[0221] Item 32. The crosslinked polymeric matrix of any one of the preceding or following items, 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 ranges defined by any two of the foregoing values.

[0222] Item 33. A crosslinked polymeric matrix according to any one of the preceding or following items, further comprising a salt.

[0223] Item 34. A crosslinked polymeric matrix according to any one of the preceding or following items, comprising NaCl in the range of about 50 mM to about 400 mM.

[0224] Item 35. The crosslinked polymeric matrix of any one of the preceding or following items, comprising NaCl, wherein the NaCl has 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 ranges defined by any two of the foregoing values.

[0225] Item 36. A crosslinked polymeric matrix according to any one of the preceding or following items, comprising NaCl, wherein the NaCl has a concentration of about 150 mM.

[0226] Item 37. A crosslinked polymeric matrix according to any one of the preceding or following items, comprising a phosphate buffer solution of about 0.01 M, NaCl of about 137 mM, and KCl at a concentration of about 2.7 mM.

[0227] Item 38. A crosslinked polymeric matrix according to any one of the preceding or following items, formulated for injection or application using a needle and / or cannula.

[0228] Item 39. A crosslinked polymeric matrix according to any one of the preceding or following items, wherein the hyaluronic acid component has an average molecular weight of from about 20,000 daltons to about 10,000,000 daltons.

[0229] Item 40. The hyaluronic acid component is selected from the group consisting of about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,100,000 daltons, about 1,200,000 daltons, about 1,300,000 daltons, and about 1,400,000 daltons. 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 tons, 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 10. The crosslinked polymeric matrix of claim 1, having an average molecular weight of about 8,000 daltons, about 8,600,000 daltons, about 8,700,000 daltons, about 8,800,000 daltons, about 8,900,000 daltons, about 9,000,000 daltons, about 9,100,000 daltons, about 9,200,000 daltons, about 9,300,000 daltons, about 9,400,000 daltons, about 9,500,000 daltons, about 9,600,000 daltons, about 9,700,000 daltons, about 9,800,000 daltons, about 9,900,000 daltons or about 10,000,000 daltons, or any molecular weight between the ranges defined by any two of the foregoing values.

[0230] Item 41. The hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, the mixture being about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,500,000 daltons, about 2,000,000 daltons, about 2,500,000 daltons, about 3,000,000 daltons, about 4, 10. The crosslinked polymeric matrix of claim 1, further 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 a range between any two of the aforementioned values.

[0231] Item 42. A composition comprising hyaluronic acid, collagen, lysine, and a buffer, which is an aqueous hydrogel.

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

[0233] Item 44. A composition described in any one of the above or below items, further comprising lidocaine.

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

[0235] Item 46. The composition of any one of the preceding or following items, wherein lidocaine is at 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) of the composition, or any concentration between the ranges defined by any two of the foregoing values.

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

[0237] Item 48. A composition described in any one of the above or below items, wherein the non-crosslinked crosslinked HA has a concentration in the composition of up to about 5% (w / w).

[0238] Item 49. The composition of any one of the above or below items, wherein the non-crosslinked HA has a concentration in the composition 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 foregoing values.

[0239] Item 50. A composition described in any one of the above or below items, wherein the non-crosslinked HA has a concentration of about 1% (w / w) in the composition.

[0240] Item 51. A composition described in any one of the above or below items, wherein the non-crosslinked HA has a concentration of about 2% (w / w) in the composition.

[0241] Item 52. A composition described in any one of the above or below items, wherein the non-crosslinked HA has a concentration of about 5% (w / w) in the composition.

[0242] Item 53. A composition described in any one of the above or below items, wherein the non-crosslinked HA improves the extrudability of the composition.

[0243] Item 54. A composition described in any one of the above or below items, wherein the buffer is phosphate buffered saline.

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

[0245] Item 56. Hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, the mixture being about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,500,000 daltons, about 2,000,000 daltons, about 2,500,000 daltons, about 3,000,000 daltons. 10. The composition of claim 1, further comprising a hyaluronic acid having a molecular weight of about 3,500,000 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 a range between any two of the aforementioned values.

[0246] Item 57. A composition described in any one of the above or below items, wherein the collagen comprises type I collagen.

[0247] Item 58. A composition described in any one of the above or below items, wherein the collagen comprises type II collagen.

[0248] Item 59. A composition described in any one of the above or below 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 10,000 Pa S or about 10,000 Pa S, or any viscosity between the range defined by any two of the foregoing values.

[0250] Item 61. A composition described in any one of the above or below items having a tan delta parameter (G'' / G') of about 0.01 to about 0.5.

[0251] Item 62. A composition described in any one of the above or below items, 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 ranges defined by any two of the foregoing values.

[0252] Item 63. The composition of any one of the above or below items, which is stable for about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or any time between the ranges defined by any two of the foregoing values.

[0253] Item 64. A composition described in any one of the above or below items, which is stable at about 4°C.

[0254] Item 65. A composition described in any one of the above or below items, which is stable at about 25°C.

[0255] Item 66. A composition described in any one of the above or below items, which exhibits negligible degradation within about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or any time between the ranges defined by any two of the foregoing values.

[0256] Item 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, wherein the pre-reaction aqueous solution has a pH of about 4 to about 6; preparing a second solution comprising 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 the hyaluronic acid and collagen with the lysine, wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amine group on the collagen and / or at least one amine group present on the lysine, and wherein the HA and collagen degrade negligibly, leaving the structures of the HA and collagen intact, thereby forming a crosslinked polymeric matrix.

[0257] Item 68. The method of any one of the above or below items, wherein the pre-reaction aqueous solution has a pH of about 4.0, about 4.5, about 5.0, about 5.5, or about 6.0, or any pH between the ranges defined by any two of the foregoing values.

[0258] Item 69. The method of any one of the above or below items, further comprising adding lidocaine to the crosslinked polymer matrix.

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

[0260] Item 71. The method of any one of the above or below items, wherein the lidocaine is at 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) of the matrix, or any concentration between the ranges defined by any two of the foregoing values.

[0261] Item 72. The method of any one of the preceding or following items, further comprising applying an activating agent comprising a triazole, a fluorinated phenol, a succinimide, or a sulfosuccinimide.

[0262] Item 73. The method of any one of the above or below items, 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 a temperature between the ranges defined by any two of the foregoing values.

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

[0264] Item 75. The method according to any one of the above or below items, wherein the reacting step is carried out at about 4°C or about 22°C.

[0265] Item 76. The method according to any one of the above or below items, further comprising purifying the cross-linked polymer matrix, wherein the purification step is carried out using 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 of any one of the above or below items, wherein the dialysis is performed at 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 temperature between the ranges defined by any two of the foregoing values.

[0268] Item 79. The method according to any one of the preceding or following items, wherein the purification step is carried out 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 crosslinking reaction is carried out 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 crosslinking reaction is carried out at about 2°C to about 8°C.

[0271] Item 82. A method according to any one of the preceding or following items, carried out below room temperature.

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

[0273] Item 84. The method of any one of the preceding or following items, wherein the pre-reaction solution comprises a salt, and the salt comprises 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 any concentration between the ranges defined by any two of the foregoing values.

[0274] Item 85. The method of 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 crosslinking reaction mixture.

[0275] Item 86. The method of 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, 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 foregoing values.

[0276] Item 87. The method of any one of the above or below items, wherein the water-soluble carbodiimide and hyaluronic acid have a molar to molar ratio of repeat units of the water-soluble carbodiimide to repeat units of the hyaluronic acid of from about 0.5 to about 2.0.

[0277] Item 88. The method of any one of the preceding or following items, wherein the molar to molar ratio of repeat units of the water-soluble carbodiimide to repeat units of the 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.

[0278] Item 89. The method according to any one of the above or below items, wherein the molar:molar (lysine repeat units:HA repeat units) ratio of lysine and hyaluronic acid is from about 0.01 to about 0.6.

[0279] Item 90. The mole:mol (lysine repeat unit:HA repeat unit) ratio of lysine and hyaluronic acid is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.2, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, 3. The method of any one of the preceding or following clauses, wherein the β-amino acid salt content is about 0.29, about 0.3, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.4, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49, about 0.5, about 0.51, about 0.52, about 0.53, about 0.54, about 0.55, about 0.56, about 0.57, about 0.58, about 0.59 or about 0.6.

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

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

[0282] Clause 93. The method of any one of the above or below clauses, 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 any concentration between the ranges defined by any two of the foregoing values.

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

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

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

[0286] Item 97. The method of any one of the preceding or following items, further comprising sterilizing the cross-linked polymeric matrix, comprising transferring the cross-linked polymeric matrix to a container for steam sterilization, and sterilizing the hydrogel by steam sterilization.

[0287] Item 98. The method of any one of the above or below items, wherein the container is a syringe.

[0288] Item 99. The method of any one of the above or below items, further comprising dialyzing the crosslinked polymer matrix, wherein the dialysis is performed through a membrane having a molecular weight cutoff of about 1000 daltons to about 100,000 daltons, and wherein the dialysis is performed prior to sterilization.

[0289] Item 100. A method according to any one of the above or below items, wherein dialysis is carried out with phosphate buffered saline.

[0290] Item 101. The method of 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 adding the second solution.

[0291] Item 102. The method of any one of the above or below items, wherein the crosslinking reaction mixture is carried out for about 16 hours to about 24 hours.

[0292] Item 103. A crosslinked polymeric matrix prepared by the process of any one of the methods described above or below.

[0293] Item 104. A method for improving the aesthetics of a human anatomical feature, comprising injecting a composition into human tissue, thereby improving the aesthetics of the anatomical feature, the composition comprising a crosslinked polymeric 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.

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

[0295] Clause 106. The method of any one of the above or below clauses, wherein the cross-linked polymer matrix further comprises non-cross-linked HA.

[0296] Item 107. The hyaluronic acid component has an average molecular weight of about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,100,000 daltons, about 1,200,000 daltons, about 1,300,000 daltons, about 1,400,000 daltons, or about 2,500,000 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 tons, 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 100,000 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, The method of any one of the preceding or following clauses, wherein the polymer has an average molecular weight of about 8,500,000 daltons, about 8,600,000 daltons, about 8,700,000 daltons, about 8,800,000 daltons, about 8,900,000 daltons, about 9,000,000 daltons, about 9,100,000 daltons, about 9,200,000 daltons, about 9,300,000 daltons, about 9,400,000 daltons, about 9,500,000 daltons, about 9,600,000 daltons, about 9,700,000 daltons, about 9,800,000 daltons, about 9,900,000 daltons or about 10,000,000 daltons, or any molecular weight between the ranges defined by any two of the foregoing values.

[0297] Item 108. Hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, the mixture having molecular weights of about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,500,000 daltons, about 2,000,000 daltons, about 2,500,000 daltons, about 3,000,000 daltons, or 10. The method of claim 1, wherein the hyaluronic acid has an average molecular weight of about 3,500,000 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 1,000,000 daltons, and / or any hyaluronic acid having a molecular weight within a range between any two of the foregoing values.

[0298] Item 109. The method of any one of the above or below items, wherein the collagen comprises type I collagen and / or type III collagen.

[0299] Item 110. A method for improving the appearance of an individual, comprising injecting a composition into tissue of the individual at an injection site, thereby improving the aesthetics of an anatomical feature, wherein infiltrating cells from the tissue integrate into the composition within the injection site; depositing new collagen within the composition, wherein the composition comprises a crosslinked polymeric 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; and depositing the new collagen, wherein the tissue into which the composition is injected is shown to have tissue integration and collagen deposition and vascularization.

[0300] Item 111. The method of any one of the above or below items, wherein the composition further comprises lidocaine.

[0301] Clause 112. The method of any one of the above or below clauses, wherein the composition further comprises non-crosslinked HA.

[0302] Item 113. A method according to any one of the preceding or following items, wherein the composition is injected into the chin, jawline, lips, or nasolabial folds.

[0303] Item 114. A method according to any one of the above or below items for improving symmetry between facial features.

[0304] Item 115. A method according to any one of the preceding or following items for augmenting and restoring volume to facial features.

[0305] Item 116. A method according to any one of the preceding or following items for increasing, correcting, restoring or providing volume to the chin, lips, jawline or nasolabial folds.

[0306] Item 117. A method according to any one of the above or below items, wherein the composition is injected into the tear trough of the individual.

[0307] Item 118. A method according to any one of the preceding or following items, wherein the composition is injected into an area containing skin atrophy and / or fat pad atrophy.

[0308] Item 119. A method according to any one of the preceding or following items, which imparts a natural look, feel, and movement to the tissue receiving the injection, and wherein the composition results in increased infiltration of collagen from the tissue surrounding the injection site.

[0309] Item 120. A method according to any one of the preceding or following items, wherein the duration of the composition is extended as a result of tissue integration at the injection site.

[0310] Item 121. A method according to any one of the above or below items, which improves the hydration and elasticity of the skin around the injection site.

[0311] Section 122. A method for increasing collagen infiltration into tissue, comprising injecting a composition into the tissue of an individual, thereby creating a dermal filler depot comprising the composition, wherein the composition comprises a crosslinked polymeric 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 tissue surrounding the dermal filler depot infiltrate the dermal filler depot containing the composition, the cells integrate into the composition and deposit new collagen into the composition, thereby creating an infiltrated tissue within the composition, and blood vessels connect the infiltrated tissue within the composition to a blood supply in the individual's body.

[0312] Item 123. The method of any one of the above or below items, wherein the matrix further comprises lidocaine.

[0313] Clause 124. The method of any one of the above or below clauses, wherein the composition further comprises non-crosslinked HA.

[0314] Item 125. Hyaluronic acid is selected from the group consisting of about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,100,000 daltons, about 1,200,000 daltons, about 1,300,000 daltons, and about 1,400,000 daltons. 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 3,1 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 , about 4,800,000 daltons, about 4,900,000 daltons, about 5,000,000 daltons, about 5,100,000 daltons, about 5,200,000 daltons, about 5,300,000 daltons, about 5,400,000 daltons, about 5,500,000 daltons, about 5,600,000 daltons, about 5,700,000 daltons, about 5,800,000 daltons, about 5,900,000 daltons, about 6,000,000 daltons, about 6,100,000 daltons, about 6,200,000 daltons, about 6,300,000 daltons, about 6,400,000 Daltons, 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 100,000 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 The method of any one of the preceding or following clauses, wherein the polymer has an average molecular weight of about 8,500,000 daltons, about 8,600,000 daltons, about 8,700,000 daltons, about 8,800,000 daltons, about 8,900,000 daltons, about 9,000,000 daltons, about 9,100,000 daltons, about 9,200,000 daltons, about 9,300,000 daltons, about 9,400,000 daltons, about 9,500,000 daltons, about 9,600,000 daltons, about 9,700,000 daltons, about 9,800,000 daltons, about 9,900,000 daltons or about 10,000,000 daltons, or any other molecular weight between the ranges defined by any two of the foregoing values.

[0315] Item 126. The hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, the mixture having molecular weights of about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, about 80,000 daltons, about 100,000 daltons, about 200,000 daltons, about 300,000 daltons, about 400,000 daltons, about 500,000 daltons, about 600,000 daltons, about 700,000 daltons, about 800,000 daltons, about 900,000 daltons, about 1,000,000 daltons, about 1,500,000 daltons, about 2,000,000 daltons, about 2,500,000 daltons, about 3,000,000 daltons, or about 4,000,000 daltons. 10. The method of claim 1, wherein the hyaluronic acid has an average molecular weight of about 3,500,000 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 a range between any two of the foregoing values.

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

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

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

[0319] Item 130. A method according to any one of the preceding or following items, in which the product is injected into the superficial dermis to improve skin quality, fine lines, or roughness.

[0320] In some embodiments, any clause herein may depend on any one of the independent claims or any one of the dependent claims. In an aspect, any clause (e.g., a dependent claim or an independent claim) may be combined with any one or more other clauses (e.g., a dependent claim or an independent claim). In an aspect, a claim may include some or all of the words (e.g., steps, actions, means, or components) recited in a clause, sentence, phrase, or paragraph. In an aspect, a claim may include some or all of the words recited in one or more clauses, sentences, phrases, or paragraphs. In an aspect, some of the words in each clause, sentence, phrase, or paragraph may be deleted. In an aspect, additional words or elements may be added to a clause, sentence, phrase, or paragraph. In an aspect, the subject technology may be implemented without utilizing some of the components, elements, functions, or operations described herein. In an aspect, the subject technology may be implemented using additional components, elements, functions, or operations.

[0321] As used in the context of describing the present invention (particularly in the context of the claims below), the terms "a," "an," "the," and similar referents should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if set forth individually herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better clarify the invention and does not limit the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0322] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limiting. Each group member 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 deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to include the modified group and thus fulfill all Markush group descriptions used in the appended claims.

[0323] Certain embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect such variations to be utilized by those skilled in the art, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, this invention includes any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

[0324] Certain embodiments disclosed herein may be further limited in claims utilizing the language "consisting of" or "consisting essentially of." When used in a claim, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim, whether added at the time of filing or by amendment. The transitional term "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics. Embodiments of the invention so claimed are essentially or explicitly described and enabled herein.

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

[0326] Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the present invention. Thus, by way of example, and not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that particularly shown and described.

Claims

1. 1. A dermal filler for improving the aesthetics of an anatomical feature of a human face by injecting the dermal filler into the anatomical feature, wherein the dermal filler comprises a crosslinked polymeric 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 the hyaluronic acid is crosslinked to the lysine by at least one amine group on the lysine, and the crosslinked polymeric matrix does not comprise chitosan.

2. 10. The dermal filler of claim 1, wherein improving the aesthetics of the anatomical feature increases and restores volume to the anatomical feature.

3. 3. The dermal filler according to claim 1 or 2, wherein the anatomical feature is the chin, jawline, lips, or nasolabial folds.

4. 10. The dermal filler of claim 1, wherein improving the aesthetics of the anatomical feature is to augment, correct, restore, or provide volume to the chin, lips, jawline, or nasolabial folds.

5. 1. A dermal filler for improving the hydration and elasticity of human facial skin by injecting a dermal filler into the skin, wherein the dermal filler comprises a crosslinked polymeric 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 the hyaluronic acid is crosslinked to the lysine by at least one amine group on the lysine, and the crosslinked polymeric matrix does not contain chitosan.

6. 1. A dermal filler for increasing collagen infiltration into tissue, comprising a crosslinked polymeric matrix comprising lysine, hyaluronic acid, and collagen, wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amine group on the collagen and the hyaluronic acid is crosslinked to the lysine through at least one amine group on the lysine, and the crosslinked polymeric matrix does not contain chitosan.

7. The dermal filler according to any one of claims 1 to 6, wherein the dermal filler is injected into the tear trough, areas of skin atrophy, areas of fat pad atrophy, or a combination of two or more thereof.

8. The dermal filler according to any one of claims 1 to 7, wherein the hyaluronic acid is in a concentration of 5 mg / ml to 36 mg / ml.

9. 9. The dermal filler according to claim 8, wherein the hyaluronic acid is in a concentration of 16 mg / ml to 22 mg / ml.

10. The dermal filler according to any one of claims 1 to 9, wherein the collagen comprises one or more of type I collagen, type II collagen, and type III collagen.

11. The dermal filler according to any one of claims 1 to 9, wherein the collagen comprises 97% to 99% type I collagen.

12. The dermal filler according to any one of claims 1 to 11, wherein the collagen is in a concentration of 1 mg / ml to 14 mg / ml.

13. 13. The dermal filler according to claim 12, wherein the collagen is at a concentration of 2 mg / ml to 6 mg / ml.

14. The dermal filler according to any one of claims 1 to 13, wherein the lysine and hyaluronic acid are in a molar:molar ratio of 0.01 to 0.

6.

15. The dermal filler according to any one of claims 1 to 14, wherein the crosslinked polymeric matrix further comprises lidocaine.

16. The dermal filler according to any one of claims 1 to 15, wherein the crosslinked polymeric matrix further comprises non-crosslinked hyaluronic acid.

17. 17. The dermal filler according to claim 16, wherein the non-crosslinked hyaluronic acid is at a concentration of up to 5% (w / w) in the matrix.

18. The dermal filler according to any one of claims 1 to 17, wherein the crosslinked polymeric matrix has an elastic modulus (G') of 30 Pa to 10,000 Pa.

19. The dermal filler according to any one of claims 1 to 18, wherein the crosslinked polymeric matrix has a compressive force value of 100 gmf to 600 gmf.

20. The dermal filler according to any one of claims 1 to 19, wherein the crosslinked polymeric matrix has a tan delta parameter (G'' / G') of 0.01 to 0.5.

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