Crosslinked ha-collagen hydrogels as skin fillers

Abstract

The present disclosure relates to a cross-linked HA-collagen hydrogel as a skin filler, in particular to a cross-linked macromolecular matrix comprising: lysine; hyaluronic acid; and collagen; wherein the hyaluronic acid is crosslinked with the collagen via at least one endogenous amino group on the collagen and / or via at least one amino group present on the lysine.

Description

[0001] This patent application is a divisional application of the patent application with application number 2020800974820, filing date December 28, 2020, and invention title "Crosslinked HA-Collagen Hydrogel as a Dermal Filler".

[0002] Cross-reference to Related Applications

[0003] This application claims the benefit of the priority of U.S. Patent Application No. 62 / 953,910, filed on December 26, 2019, the entire content of which is incorporated herein by reference. Technical Field

[0004] The present disclosure relates to crosslinked macromolecular matrices comprising hyaluronic acid, collagen, and lysine. Such compositions can be used as tissue fillers with enhanced tissue integration. Background Art

[0005] Aging is a natural process that occurs over time and can be influenced by genetic and lifestyle factors (alcohol abuse, tobacco, UVA / UVB exposure, diet). For example, the characteristics of facial skin aging include muscle and fat atrophy, skin laxity, age spots, sagging, and jowling. Relaxation of the subcutaneous tissue can lead to excess skin and sagging, which may cause drooping of the cheeks and eyelids. Jowling refers to the increase in excess weight through swelling at the bottom of the face and neck. These changes may be associated with dryness, loss of elasticity, and rough texture.

[0006] 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 defects such as wrinkles and volume loss due to the effects of aging. Initially, dermal filler compositions containing bovine collagen entered the market in the 1970s. Human-derived collagen was approved by the FDA in 2003 and is superior to bovine-derived collagen, which may cause allergic reactions in patients. However, due to enzymes within the skin tissue, human-derived collagen compositions rapidly degrade within 3 to 6 months. Therefore, patients using these early compositions required frequent surgical procedures to maintain their desired corrective aesthetic appearance.

[0007] As an alternative to collagen-based dermal fillers, hyaluronan (also known as hyaluronic acid, abbreviated HA)-based fillers were introduced in the 1990s. HA is a naturally occurring water-soluble polysaccharide, specifically a glycosaminoglycan, which 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. In addition, HA has the ability to bind a large amount 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 duration or physical properties to act as a wrinkle filler, so crosslinked HA has been used to maximize their longevity in dermal tissues. Thus, there is a need for improved dermal fillers. SUMMARY OF THE INVENTION

[0008] Embodiments herein encompass methods and compositions (e.g., hydrogels or dermal fillers) that include a crosslinked macromolecular matrix comprising hyaluronic acid, collagen, and lysine, wherein the hyaluronic acid is crosslinked to the collagen via at least one endogenous amino group on the collagen and / or via at least one amino group present on the lysine.

[0009] In some embodiments of any one or any combination of the above or below embodiments, the crosslinked macromolecular matrix further comprises lidocaine. In some embodiments of any one or any combination of the above or below embodiments, the concentration of lidocaine in the matrix ranges from about 0.15% (w / w) to about 0.45% (w / w). In some embodiments of any one or any combination of the above or below embodiments, the concentration of lidocaine in the matrix ranges from about 0.27% (w / w) to about 0.33% (w / w). In some embodiments of any one or any combination of the above or below embodiments, the concentration of lidocaine is about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w), or about 0.45% (w / w) of the matrix, or any concentration between any two of the above values. In some embodiments of any one or any combination of the above or below embodiments, the concentration of lidocaine in the matrix is about 0.3% (w / w).

[0010] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix further comprises uncrosslinked HA. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the matrix is up to about 5% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the matrix is 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), or about 5% (w / w) or any concentration between the ranges defined by any two of the above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the matrix is about 1% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the matrix is about 2% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the matrix is about 5% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the uncrosslinked HA improves the extrudability of the macromolecular matrix.

[0011] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix is stable for at least about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months or for any amount of time between the ranges defined by any two of the above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix is stable at temperatures between about 4°C and about 25°C. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix is stable at about 4°C. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix is stable at about 25°C. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix is stable for about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36 months or for any time between the ranges defined by any two of the above values.

[0012] In any one of the above or below-described embodiments or in some embodiments of any embodiment, the crosslinked macromolecular matrix has minimal degradation at about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months or at any amount of time within the range defined by any two of the above values.

[0013] In any one of the above or below-described embodiments or in some embodiments of any embodiment, the elastic modulus (G') of the crosslinked macromolecular matrix is from about 30 Pa to about 10,000 Pa. In any one of the above or below-described embodiments or in some embodiments of any embodiment, the elastic modulus (G') of 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 2400 Pa, about 2500 Pa, about 2600 Pa, about 2700 Pa, about 2800 Pa, about 2900 Pa, about 3000 Pa, about 3100 Pa, about 3200 Pa, about 3300 Pa, about 3400 Pa, about 3500 Pa, about 3600 Pa, about 3700 Pa, about 3800 Pa, about 3900 Pa, about 4000 Pa, about 4100 Pa, about 4200 Pa, about 4300 Pa, about 4400 Pa, about 4500 Pa, about 4600 Pa, about 4700 Pa, about 4800 Pa, about 4900 Pa, about 5000 Pa, about 5100 Pa, about 5200 Pa, about 5300 Pa, about 5400 Pa, about 5500 Pa, about 5600 Pa, about 5700 Pa, about 5800 Pa, about 5900 Pa, about 6000 Pa, about 6100 Pa, about 6200 Pa, about 6300 Pa, about 6400 Pa, about 6500 Pa, about 6600 Pa, about 6700 Pa, about 6800 Pa, about 6900 Pa, about 7000 Pa, about 7100 Pa, about 7200 Pa, about 7300 Pa, about 7400 Pa, about 7500 Pa, about 7600 Pa, about 7700 Pa, 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 10,000 Pa or any elastic modulus between the ranges defined by any two of the above values.

[0014] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the compression force value of the crosslinked macromolecular 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 180 gmf, about 190 gmf, about 200 gmf, about 210 gmf, about 220 gmf, about 230 gmf, about 240 gmf, about 250 gmf, about 260 gmf, about 270 gmf, about 280 gmf, about 290 gmf, about 300 gmf, about 310 gmf, about 320 gmf, about 330 gmf, about 340 gmf, about 350 gmf, about 360 gmf, about 370 gmf, about 380 gmf, about 390 gmf, about 400 gmf, about 410 gmf, about 420 gmf, about 430 gmf, about 440 gmf, about 450 gmf, about 460 gmf, about 470 gmf, about 480 gmf, about 490 gmf, about 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 compression force value between the ranges defined by any two of the above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the compression force value of the crosslinked macromolecular matrix is about 100 gmf, about 200 gmf, about 300 gmf, about 400 gmf, about 500 gmf or about 600 gmf or any compression force value between the ranges defined by any two of the above values.

[0015] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of hyaluronic acid is 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 above values.

[0016] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the collagen comprises type I collagen. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the collagen comprises type II collagen. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the collagen comprises type III collagen. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the collagen contains from about 1% to 3% of type I or type III collagen. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the collagen contains from about 0% to about 3% of type II collagen. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the collagen contains from about 97% to about 99% of type I collagen. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the collagen comprises a mixture of type I and type III collagen. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the matrix contains from about 0% to about 3% of type III collagen.

[0017] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix is formulated for injection or for use with a needle and / or cannula.

[0018] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of the collagen is 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 above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of the collagen is about 3 mg / mL. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of the collagen is about 6 mg / mL. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of the collagen is about 10 mg / mL. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of the collagen is about 12 mg / mL.

[0019] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix further comprises a salt. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix comprises NaCl in the range of about 50 mM to about 400 mM. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix comprises NaCl, wherein the concentration of NaCl is about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, 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 above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix comprises about 150 mM of NaCl. In certain embodiments, the crosslinked macromolecular matrix does not contain salt.

[0020] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the crosslinked macromolecular matrix comprises about 0.01 M phosphate buffer, about 137 mM of NaCl and KCl at a concentration of about 2.7 mM.

[0021] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the average molecular weight of hyaluronic acid is from about 20,000 Daltons to about 10,000,000 Daltons. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the average molecular weight of hyaluronic acid is 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 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 or about 1,000,000 Daltons, or an average molecular weight between the ranges defined by any two of the above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the average molecular weight of hyaluronic acid is from about 20,000 Daltons to about 10,000,000 Daltons.In any one of the above or below embodiments or in some embodiments of any embodiment, wherein the hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, and the mixture comprises hyaluronic acid having the following molecular weights: about 20,000, 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 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 the range between any two of the above values.

[0022] The present invention also provides a composition, which comprises: hyaluronic acid, collagen, lysine and a buffer; and wherein the composition is an aqueous hydrogel.

[0023] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, hyaluronic acid is crosslinked with collagen through at least one endogenous amino group on the collagen and / or at least one amino group present on lysine. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the composition further comprises lidocaine. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of lidocaine in the matrix ranges between about 0.15% (w / w) and about 0.45% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of lidocaine in the composition ranges between about 0.27% (w / w) and about 0.33% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of lidocaine is about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w) of the composition or any concentration between the ranges defined by any two of the above values.

[0024] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the composition further comprises uncrosslinked HA. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the composition is up to about 5% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the composition is 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 any two of the above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the composition is about 1% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the composition is about 2% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the composition is about 5% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the uncrosslinked HA improves the extrudability of the composition. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the buffer is phosphate buffered saline.

[0025] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the average molecular weight of the hyaluronic acid in the composition is from about 20,000 Daltons to about 10,000,000 Daltons.

[0026] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, wherein the mixture comprises hyaluronic acid having the following molecular weights: 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 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 the range between any two of the above values.

[0027] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the collagen of the composition comprises type I collagen. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the collagen comprises type II collagen. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the collagen comprises type III collagen.

[0028] In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the composition is stable for about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or for any amount of time between any two of the above values. In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the composition is stable at about 4°C. In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the composition is stable at about 25°C. In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the composition has minimal degradation for about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or for any amount of time within the range defined by any two of the above values.

[0029] In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the composition further comprises uncrosslinked HA. In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the concentration of uncrosslinked HA in the composition is up to about 5% (w / w). In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the uncrosslinked HA improves the extrudability of the composition. In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the composition is stable for about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or for any amount of time between any two of the above values. In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the composition is stable at 4°C. In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the composition is stable at 25°C. In some embodiments of any one of the above or below-described embodiments, or of any embodiment, the composition has minimal degradation for 6 months, 12 months, 18 months, 24 months, 30 months, or 36 months or for any amount of time within the range defined by any two of the above values.

[0030] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the viscosity of the composition is 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, about 6900 Pa S, about 7000 Pa S, about 7100 Pa S, about 7200 Pa S, about 7300 Pa S, about 7400 Pa S, about 7500 Pa S, about 7600 Pa S, about 7700 Pa S, about 7800 Pa S, about 7900 Pa S, about 8000 Pa S, about 8100 Pa S, about 8200 Pa S, about 8300 Pa S, about 8400 Pa S, about 8500 Pa S, about 8600 Pa S, about 8700 Pa S, about 8800 Pa S, about 8900 Pa S, about 9000 Pa S, about 9100 Pa, about 9200 Pa S, about 9300 Pa S, about 9400 Pa S, about 9500 Pa S, about 9600 Pa S, about 9700 Pa S, about 9800 Pa S, about 9900 Pa S or about 10,000 Pa S or any viscosity between the ranges defined by any two of the above values.

[0031] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the parameter of the tangent value of the dielectric loss angle (G" / G') of the composition is from about 0.01 to about 0.5. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the parameter of the tangent value of the dielectric loss angle (G" / G') of the composition is 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 parameter of the tangent value of the dielectric loss angle between the ranges defined by any two of the above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the buffer solution includes phosphate buffered saline.

[0032] The present invention also provides a method for crosslinking hyaluronic acid and collagen. The method includes dissolving collagen, hyaluronic acid, and lysine in an aqueous solution to form a pre-reaction aqueous solution, wherein the pH of the pre-reaction aqueous solution is between 4 and 6, and preparing a second solution that includes: a water-soluble carbodiimide; and N-hydroxysuccinimide or N-hydroxy sulfosuccinimide; adding the second solution to the pre-reaction aqueous solution to form a crosslinking reaction mixture, reacting the crosslinking reaction mixture by crosslinking hyaluronic acid and collagen with lysine, wherein hyaluronic acid is crosslinked to collagen through at least one endogenous amino group on the collagen and / or through at least one amino group present on the lysine, wherein the degradation of HA and collagen is minimal and the structures of HA and collagen remain intact, thereby forming a crosslinked macromolecular matrix. In some embodiments of any one or any of the above or below-described embodiments, the pH of the pre-reaction aqueous solution 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 above values. In some embodiments of any one or any of the above or below-described embodiments, the method further includes providing an activator that includes triazole, fluorinated phenol, succinimide, or sulfosuccinimide.

[0033] In some embodiments of any one or any of the above or below-described embodiments, the method further includes adding lidocaine to the crosslinked macromolecular matrix. In some embodiments of any one or any of the above or below-described embodiments, the concentration of lidocaine in the matrix ranges from about 0.15% (w / w) to about 0.45% (w / w). In some embodiments of any one or any of the above or below-described embodiments, the concentration of lidocaine in the matrix ranges from about 0.27% (w / w) to about 0.33% (w / w). In some embodiments of any one or any of the above or below-described embodiments, wherein the concentration of lidocaine is about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w), or about 0.45% (w / w) of the matrix, or any concentration between the ranges defined by any two of the above values. In some embodiments of any one or any of the above or below-described embodiments, the concentration of lidocaine in the matrix is about 0.3% (w / w).

[0034] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the method further includes adding uncrosslinked HA to the crosslinked macromolecular matrix. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the crosslinked macromolecular matrix is up to about 5% w / w. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA in the added matrix is 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 any two of the above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA added to the matrix is about 1% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA added to the matrix is about 3% (w / w). In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the concentration of uncrosslinked HA added to the matrix is about 5% (w / w).

[0035] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the reaction step is carried out between about 4°C and about 35°C. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the reaction 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 at any temperature between any two of the above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the reaction step is carried out at about 4°C or about 22°C.

[0036] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the method further includes purifying the crosslinked macromolecular matrix, wherein the purification step is performed using dialysis purification. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, dialysis is carried out between about 2°C and about 30°C. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, 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 at any temperature between the ranges defined by any two of the above values. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the purification step is carried out between about 2°C and about 8°C. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the purification step is carried out at about 2°C, about 4°C, about 6°C, about 8°C or at any temperature between the ranges defined by any two of the above values.

[0037] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the method is carried out at a temperature below room temperature. In any one of the above or below-described embodiments or in some embodiments of any of the 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 at a temperature between the ranges defined by any two of the above values.

[0038] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the pH of the crosslinking reaction mixture is between about 4 and about 6.0. In any one of the above or below-described embodiments or in some embodiments of any of the 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 above values.

[0039] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the solution before the reaction contains a salt, wherein the sodium chloride concentration in the crosslinking reaction mixture is about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, 325 mM, about 350 mM, about 375 mM or about 400 mM, or any concentration between any two of the above values.

[0040] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the crosslinking reaction mixture is about 20 mM to about 200 mM. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is about 20 mM, about 40 mM, about 60 mM, about 80 mM, about 100 mM, about 120 mM, about 140 mM, about 160 mM, about 180 mM or about 200 mM, or any concentration between any two of the above values.

[0041] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the molar ratio of the water-soluble carbodiimide to the hyaluronic acid in the water-soluble carbodiimide: hyaluronic acid repeating unit is about 0.5 to about 2.0. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the molar ratio of the water-soluble carbodiimide to the hyaluronic acid in the water-soluble carbodiimide: hyaluronic acid repeating unit 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.

[0042] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the molar:molar (lysine:HA repeating unit) ratio of lysine and hyaluronic acid is between about 0.01 and about 0.6. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the molar:molar (lysine:HA repeating 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, 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.

[0043] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the method further comprises sterilizing the crosslinked macromolecular matrix, the method comprising: transferring the crosslinked macromolecular matrix to a container and performing steam sterilization; and sterilizing the hydrogel by steam sterilization. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the container is a syringe.

[0044] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the method further comprises dialyzing the crosslinked macromolecular matrix, wherein the dialysis is performed through a membrane having a molecular weight cut-off of about 1000 Daltons to about 100,000 Daltons, and wherein the dialysis is performed before sterilization. In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the dialysis is performed in phosphate buffered saline.

[0045] In any one of the above or below-described embodiments or in some embodiments of any of the embodiments, the hyaluronic acid in the pre-reaction solution is hydrated for at least 60 minutes before adding the second solution.

[0046] In some embodiments of any one or any of the above or below-described embodiments, the reaction mixture is crosslinked for about 16 hours to about 24 hours. In some embodiments of any one or any of the above or below-described embodiments, the reaction mixture is crosslinked for about 16 hours, about 18 hours, about 20 hours, about 22 hours or about 24 hours, or for an amount of time within the range defined by any two of the above values.

[0047] In some embodiments of any one or any of the above or below-described embodiments, the crosslinking reaction is carried out at a temperature of about 2°C to about 35°C. In some embodiments of any one or any of the above or below-described 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 at any temperature within the range defined by any two of the above values.

[0048] In some embodiments of any one or any of the above or below-described embodiments, the crosslinking reaction is carried out at a temperature of about 2°C to about 8°C. In some embodiments of any one or any of the above or below-described embodiments, the crosslinking reaction is carried out at about 2°C, about 4°C, about 6°C or about 8°C, or at any temperature within the range defined by any two of the above values.

[0049] The present invention also provides a crosslinked macromolecular matrix prepared by the method of any one of the above or below-described embodiments.

[0050] In addition, the present disclosure provides a method for improving the aesthetic quality of human anatomical features. The method includes: injecting a composition into human tissue so as to improve the aesthetic quality of the anatomical features; wherein the composition comprises a crosslinked macromolecular matrix, the matrix comprising: hyaluronic acid; lysine; and collagen; wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amino group on the collagen and / or through at least one amino group present on the lysine.

[0051] The present disclosure also provides a method of improving the appearance of an individual. The method includes injecting a composition into the tissue of the individual at an injection site to improve the aesthetic quality of an anatomical feature, wherein infiltrating cells from the tissue integrate into the composition within the injection site and deposit new collagen in the composition; wherein the composition includes a crosslinked macromolecular matrix that includes: hyaluronic acid; lysine; and collagen; wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amino group on the collagen and / or through at least one amino group present on the lysine; and wherein the tissue injected with the composition exhibits tissue integration, collagen deposition, and angiogenesis. In some embodiments of any one or any combination of the above or below-described embodiments, the composition is injected into the nasolabial fold. In some embodiments of any one or any combination of the above or below-described embodiments, the method improves the symmetry between facial features. In some embodiments of any one or any combination of the above or below-described embodiments, the method enhances and restores the volume of facial features. In some embodiments of any one or any combination of the above or below-described embodiments, the method restores the volume of the cheeks and / or temples. In some embodiments, the method increases, corrects, restores, or creates volume in the chin, jawline, or nasolabial fold. In some embodiments of any one or any combination of the above or below-described embodiments, the composition is injected into the tear trough of the individual. In some embodiments of any one or any combination of the above or below-described embodiments, the composition is injected into an area including skin atrophy and / or fat pad atrophy. In some embodiments of any one or any combination of the above or below-described embodiments, the method provides a natural appearance, feel, and movement in the tissue receiving the injection, wherein the composition causes an increase in collagen infiltration of the tissue surrounding the injection site. In some embodiments of any one or any combination of the above or below-described embodiments, the duration of the composition is increased due to tissue integration into the injection site. In some embodiments of any one or any combination of the above or below-described embodiments, the method improves the hydration and elasticity of the skin surrounding the injection site.

[0052] The present disclosure also provides a method for increasing tissue infiltration and depositing collagen in a dermal filler implant. The method includes injecting a composition into the tissue of an individual to create a dermal filler reservoir containing the composition, wherein the composition includes a crosslinked macromolecular matrix that includes: hyaluronic acid; lysine; and collagen; wherein the hyaluronic acid is crosslinked to the collagen via at least one endogenous amino group on the collagen and / or via at least one amino group present on the lysine; and wherein cells from the tissue surrounding the dermal filler reservoir infiltrate the dermal filler reservoir containing the composition, wherein the cells integrate into the composition and deposit new collagen into the composition, thereby creating infiltrated tissue in the composition, and wherein blood vessels connect the infiltrated tissue in the composition to the blood supply of the individual's body.

[0053] In some embodiments of any one or any combination of the above or below-described embodiments, the collagen includes type I collagen and / or type III collagen.

[0054] In some embodiments of any one or any combination of the above or below-described embodiments, the composition includes 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 any two of the above values. In some embodiments of any one or any combination of the above or below-described embodiments, the composition includes about 13 mg / mL hyaluronic acid.

[0055] In some embodiments of any one or any of the above or below embodiments of the method, the composition or macromolecular matrix further comprises lidocaine. In some embodiments of any one or any of the above or below embodiments of the method, the concentration of lidocaine in the matrix ranges between 0.15% (w / w) and 0.45% (w / w). In some embodiments of any one or any of the above or below embodiments of the method, the concentration of lidocaine in the matrix ranges between 0.27% (w / w) and 0.33% (w / w). In some embodiments of any one or any of the above or below embodiments of the method, the concentration of lidocaine in the matrix is about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration between the ranges defined by any two of the above values. In some embodiments of any one or any of the above or below embodiments of the method, the concentration of lidocaine in the matrix is about 0.3% (w / w).

[0056] In some embodiments of any one or any of the above or below embodiments of the method, the composition or macromolecular matrix further comprises uncrosslinked HA. In some embodiments of any one or any of the above or below embodiments of the method, the concentration of uncrosslinked HA in the composition or matrix is up to about 5% (w / w). In some embodiments of any one or any of the above or below embodiments of the method, the concentration of uncrosslinked HA in the composition or matrix is 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 above values. In some embodiments of any one or any of the above or below embodiments of the method, the concentration of uncrosslinked HA in the composition or matrix is about 1% (w / w). In some embodiments of any one or any of the above or below embodiments of the method, the concentration of uncrosslinked HA in the composition or matrix is about 2% (w / w). In some embodiments of any one or any of the above or below embodiments of the method, the concentration of uncrosslinked HA in the composition or matrix is about 5% (w / w). Description of the Drawings

[0057] Figure 1 Shows in vitro cell viability cells in close contact with the HA / collagen crosslinked hydrogel formulation.

[0058] Figure 2 shows the actin filament alignment index (2A), cell aspect ratio (2B), and convex hull to cell area ratio (2C) of fibroblasts cultured on HA-only hydrogels or HA / collagen crosslinked hydrogels as depicted. The HA-collagen hydrogel (24:6 HA:collagen) (Formulation X) formulated at a hydration temperature of 5 °C exhibited significantly higher actin filament alignment index, cell aspect ratio, and convex hull to cell area ratio compared to a similar hydrogel (Formulation VI) formulated at a hydration temperature of 22 °C. *p<0.05, ANOVA with Tukey's post hoc analysis. The HA-collagen hydrogel with a HA:collagen ratio of 20:6 formulated at a hydration temperature of 5 °C exhibited particularly high actin filament alignment index, cell aspect ratio, and convex hull to cell area ratio compared to the HA-only gel (Formulation XIX). *p<0.05, ANOVA with Tukey's post hoc analysis. Figure 2D Shows the ranking of the HA-collagen hydrogel as a function of the Euclidean distance (3-dimensional space encompassing the actin filament alignment index, cell aspect ratio, and convex hull to cell area ratio) from the HA-only hydrogel. An increase in the Euclidean distance indicates improved cell spreading and adhesion compared to the low-adhesion HA-only gel. Overall, the hydrogels formulated at a hydration temperature of 5 °C had a greater Euclidean distance compared to the HA-only gels.

[0059] Figure 3 Shows the lift curves (mean + / - SEM) of Formulation I vs. Formulation II vs. Formulation III.

[0060] Figure 4 Shows the lift curves (mean + / - SEM) of Formulation XV vs. Formulation III.

[0061] Figure 5 Shows the lift curves (mean + / - SEM) of Formulation II vs. Formulation XV vs. Formulation XVI.

[0062] Figure 6 shows the tissue integration of hydrogels with 4 mg / mL collagen and increasing HA concentrations. (6A) H&E, (6B) collagen 1a, (6C) vimentin, (6D) procollagen 1, (6E) CD31. H&E staining showed a decrease in tissue ingrowth with increasing HA concentration. As shown, dense collagen 1a staining was observed in the 13 mg / mL HA formulation (Formulation I). The 20 mg / mL HA formulation showed reduced collagen 1a filling, while the 25 mg / mL HA formulation showed large areas without collagen 1a deposition. Vimentin-positive fibroblast / fibrocyte infiltration was observed in all formulations, and the degree of infiltration decreased with increasing HA concentration. Procollagen I 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. As shown by CD31-positive staining, angiogenesis was observed within the hydrogel boluses in the 20 mg / mL and 25 mg / mL HA formulations. CD31 staining was not performed on the 13 mg / mL HA formulation.

[0063] Figure 7 Shows the tissue integration of hydrogels prepared with a higher proportion of low molecular weight HA relative to high molecular weight HA. (A) Colloidal iron, (B) collagen 1a, (C) vimentin, (D) procollagen 1, (E) CD31. Colloidal iron staining showed tissue integration at the edge of the Formulation XV hydrogel and strong tissue integration throughout the Formulation XVI gel bolus. Dense collagen 1a deposition was observed on the back of the Formulation XV bolus, but the deposit did not completely fill the gel. Fine chains of collagen 1a-positive tissue were observed throughout the Formulation XVI hydrogel. Vimentin-positive fibroblast / fibrocyte infiltration was observed in all formulations. Most of the Formulation XVI bolus was infiltrated with vimentin-positive cells. Procollagen I staining was present in both the Formulation XV and Formulation XVI gels. The presence of procollagen I staining may indicate continued collagen deposition over time. Angiogenesis was observed within the hydrogel boluses in both formulations (arrows). The Formulation XVI formulation showed the most robust angiogenesis throughout the bolus.

[0064] Figure 8Shows the tissue integration of hydrogels containing 24 mg / mL HA and 6 mg / mL collagen prepared at room temperature (Formulation VI) and at a hydration temperature of 5 °C (Formulation X). Collagen 1a staining shows a fine collagen distribution around the Formulation VI hydrogel, with limited deposition around the hydrogel particles. Collagen 1a staining of the Formulation X gel shows strong collagen deposition around the hydrogel and dense collagen deposition around the hydrogel particles.

[0065] Figure 9 Shows hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining of hydrogel implants after 12 weeks of subcutaneous injection in rats. H&E staining shows tissue deposition closely associated with the hydrogel particles in Formulation XIX, while sparse tissue deposition is observed around the large hydrogel deposits in the HA-only hydrogel. Vimentin staining shows a more extensive fibrocyte / fibroblast infiltration into the Formulation XIX hydrogel bolus compared to the HA-only gel. As shown by the extensive CD31 positive labeling, the Formulation XIX bolus is also more vascularized than the HA-only bolus. The enhanced cell infiltration and vascularization of the Formulation XIX bolus result in a denser and more uniform tissue deposition within the bolus, as shown by collagen I labeling.

[0066] Figure 10 Shows the immunohistochemical (IHC) quantification of the positive staining area, which shows an increase in the levels of vimentin (fibroblasts), collagen I, and CD31 (blood vessels) in the Formulation XIX hydrogel bolus after 12 weeks of subcutaneous implantation in rats compared to the HA-only hydrogel.

[0067] Figure 11 Shows the lifting ability in a rat subcutaneous injection model. Formulation XIX exhibits a lifting ability similar to that of the 24 mg / mL HMW HA-only gel from 4 to 12 weeks. As shown, this formulation exhibits enhanced tissue integration while maintaining a lifting ability similar to that of the HA-only gel.

[0068] Figure 12 Shows the 28-week lifting ability data of crosslinked HA-collagen gels. The lifting ability of the HA-only gel steadily declines over time. The lifting ability of the HA-collagen gel remains stable from 12 to 28 weeks. Without limiting the present 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, particularly Formulation XIX has significantly better tissue ingrowth than the HA-only gel.

[0069] Figure 13Shows the differences observed in the 24:6 HA:collagen gel before and after autoclaving. The 24:6 HA:collagen gel (repeated runs for Sample 1 and Sample 2) generally had lower cell viability. However, both autoclaved and non-autoclaved preparations showed higher cell viability than the HA-only gel. As shown, experiments (Sample 1 and Sample 2) with the gel containing 24:6 HA:collagen were repeated before (B) and after autoclaving (BA). A small but significant difference in cell viability was observed in Sample 1, while no significant difference was observed in Sample 2 after autoclaving.

[0070] Figure 14 Shows the H&E staining of the gel implants in the subcutaneous tissue of a rat model after 4 weeks of implantation. Preparation XXII (A; 20 mg HA: 4 mg collagen, hydrated at 5°C) showed similar or better tissue integration compared to Preparation XIX (B; 20 mg HA: 6 mg collagen, hydrated at 5°C). Blind review scores by pathologists also demonstrated enhanced integration for Preparation XXII, with a score of 2.33, while Preparation XIX had a score of 1.83. Higher scores indicate better tissue integration. In contrast, Preparation XX (C; 20 mg HA: 10 mg collagen, synthesized at 25°C) showed worse tissue integration than Preparation XXII and Preparation XIX. The tissue integration score for Preparation XX was 1.13. The results also indicate that tissue integration does not follow a linear trend with collagen concentration. Instead, there are optimal synthesis conditions and collagen concentrations for achieving enhanced tissue response.

[0071] Figure 15 Shows the in vitro cell viability of human dermal fibroblasts cultured with HA-only and HA-collagen (Preparations XXII and XXIII) gels.

[0072] Figure 16 Shows the image analysis of the aspect ratio of human dermal fibroblasts cultured with HA-only or HA-collagen gels (Preparations XXII and XXIII).

[0073] Figure 17 Shows the tissue integration scores of the gel implants in rats subcutaneously implanted with Preparations XXII and XXIII or the HA-only control after 4 weeks.

[0074] Figure 18 Shows the collagen 1a staining of the tissue integration of Preparations XXII and XXIII compared to the HA-only gel.

[0075] Figure 19 Shows the quantification of the percentage of the area positive for collagen 1a staining within the hydrogel implant after subcutaneous implantation of Preparation XXII in rats for 4 weeks.

[0076] Figure 20 Shows confocal micrographs of human dermal fibroblasts cultured for 48 hours on gels containing only HA, formulation XXII, or formulation XXIII. The samples were stained for HA-binding protein, Hoechst, and wheat germ agglutinin (cell membrane).

[0077] Figure 21 Shows immunohistochemical analysis of the tissue response to gels containing only HA and HA-collagen hydrogels (formulations XXII and XXIII) after 4 weeks of subcutaneous implantation in rats.

[0078] Figure 22 Shows data on the lifting ability of formulation XXII at 52 weeks compared to gels containing only HA.

[0079] Figure 23 Shows data on the lifting ability of formulation XXIII at 26 weeks compared to gels containing only HA.

[0080] Figure 24 Shows confocal micrographs of human dermal fibroblasts cultured for 48 hours on gels containing only HA, formulation XXVI, or formulation XXV. The samples were stained for HA-binding protein, Hoechst, and wheat germ agglutinin (cell membrane).

[0081] Figure 25 Shows two-photon imaging of second harmonic generation signals (white) and tissue autofluorescence (green) in rats treated with subcutaneous bolus injections of gels containing only HA, formulation XXV, or formulation XXIII after 12 weeks.

[0082] Figure 26 Shows immunohistochemical analysis of the tissue response to formulation XXV after 4 weeks of subcutaneous implantation in rats.

[0083] Figure 27 Shows immunohistochemical analysis of the tissue response to formulation XXVI after 4 weeks of subcutaneous implantation in rats.

[0084] Figure 28 Shows data on the lifting ability of formulations XXV and XXVI at 30 weeks compared to gels containing only HA. Detailed Description

[0085] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0086] In cases where the definition of a term used in the specification departs from the common meaning of that term, unless specifically indicated otherwise, the applicant intends to use the definitions provided herein.

[0087] The present disclosure relates to crosslinked macromolecular matrices, compositions comprising crosslinked macromolecular matrices, methods for preparing crosslinked macromolecular matrices, and methods for improving the appearance of an individual. Fillers comprising the crosslinked macromolecular matrices described in the embodiments have immediate filling and lifting capabilities after injection and are subsequently integrated into the injection site through tissue, which can produce long-term and natural effects.

[0088] Advantageously, the crosslinking method provides HA / collagen materials with adjustable physical properties, which produce a range of filling and lifting characteristics, thus allowing such materials to be injected into a range of tissue depths, facial regions, and for different purposes (volumization, severe wrinkles, fine lines, etc.). In addition, the synthesis method allows control of cell infiltration from the surrounding tissue into the injected bolus by covalently binding collagen to the crosslinked hydrogel. In addition, crosslinking can also protect collagen from denaturation. The combination of lifting and tissue integration characteristics is expected to provide excellent facial aesthetic enhancement with a natural feel, appearance, and movement. As described herein, methods for improving the quality of fillers, which result in hybrid materials with superior performance compared to previous collagen fillers and current HA fillers.

[0089] 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.

[0090] The terms "a," "an," "the," and similar references used in the context of describing the present invention (especially in the context of the following claims) should be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context. As used herein, when referring to a measurable value, "about" means including variations of +20% or +10%, more preferably +5%, even more preferably +1%, still more preferably +0.1% compared to the specified value.

[0091] As used herein, unless the context requires otherwise, the term "comprising" and variations of this term, such as "comprising," "comprises," and "comprised," are not intended to exclude further additives, components, integers, or steps.

[0092] "Crosslinked macromolecular matrix" refers to a matrix formed by crosslinking HA and collagen. HA and collagen can be crosslinked by activating the natural carboxylic acid moieties on HA and collagen so that such moieties can react with the endogenous amino groups present on collagen. In addition, lysine can be added as a carboxylic acid / diamine crosslinking agent to further enhance the crosslinking of HA and collagen. The addition of such lysine allows adjustment of the physical properties of the resulting hydrogel. The crosslinked macromolecular matrix can be used in medical aesthetic compositions or formulations (e.g., as a cosmetic or skin filler).

[0093] "Hyaluronic acid (or Hyaluronan)" as described herein refers to a non-sulfated glycosaminoglycan that is widely distributed in connective tissue, epithelial tissue, and nerve tissue throughout the human body. Hyaluronic acid is abundant in different layers of the skin and has multiple functions, such as ensuring good hydration, assisting in the organization of the extracellular matrix, acting as a filler material; and participating in tissue repair mechanisms.

[0094] "Collagen" as described herein is the main structural protein in the extracellular space of various connective tissues in the body. Collagen forms fibrils and sheets that withstand tensile loads. Collagen also has specific integrin-binding sites for cell adhesion and is known to promote cell adhesion, migration, and proliferation. Collagen may be positively charged because it contains a high content of basic amino acid residues, such as arginine, lysine, and hydroxylysine. More than 90% of the collagen in the human body is type I collagen. Type III collagen is the main component of reticular fibers and is usually found together with type I collagen. Those skilled in the art will understand that collagen can be provided by commercial sources. In some embodiments of any one or any of the above or below-described embodiments, the provided collagen material can be a mixture comprising about 97% to about 99% type I collagen and the balance being about 1% to 3% type III collagen.

[0095] In some embodiments of any one or any of the above or below-described embodiments, the collagen is cross-linked collagen. In some embodiments of any one or any of the above or below-described embodiments, the collagen is non-cross-linked collagen.

[0096] In some embodiments of any one or any of the above or below-described embodiments, HA is cross-linked with an amine and can have multiple cross-links through lysine on collagen or HA or through another amine group.

[0097] "Elastic modulus" is also known as the modulus of elasticity and refers to a quantity that measures the ability of an object or material to resist elastic deformation (i.e., non-permanent) when a stress is applied to it.

[0098] As described herein, "compressive force" refers to applying a force, pressure, or effort to an object so that it is squeezed, flattened, or compacted.

[0099] As described herein, "sterilization" refers to subjecting a material to a sterilization process that may cause the death of microorganisms in the material. The methods of disinfection and sterilization can be physical, chemical, and physicochemical means.

[0100] For materials such as hydrogels, for example, disinfection can be accomplished under less aggressive conditions, such as shorter disinfection times, lower temperatures, and lower exposure doses.

[0101] Non - limitingly, sterilization can include steam heat, dry heat, and / or ionizing radiation

[0102] As a result of undergoing a sterilization process, a sterile product, such as a hydrogel, can be formed. Such sterilization processes can be found in Chitre et al. (US2014 / 0011980A1), Chitre et al. (US2018 / 0147307A1), and Chitre et al. (US2016 / 0101200A1).

[0103] In one embodiment, the composition or matrix contains an anesthetic. Non - limiting anesthetics include benzocaine, chlorprocaine, procaine, proparacaine, tetracaine, amirocaine, oxybuprocaine, articaine, bupivacaine, dibucaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, solacaine, tonicaine, cocaine.

[0104] Method

[0105] Hyaluronic acid and collagen can be cross - linked using 1 - ethyl - 3-(N,N′ - dimethylaminopropyl)carbodiimide (EDC) and N - hydroxysuccinimide (NHS) to activate the reaction of the native carboxylic acid moieties present on HA and collagen with the endogenous amine groups present on collagen. In one embodiment, lysine is added as an additional diamine cross - linker to further enhance the chemical modification of HA and collagen and to regulate the physical properties of the resulting hydrogel.

[0106] The addition of lysine allows for independent regulation of cross - linking, such that the physical properties of the hydrogel can be adjusted without changing the amount of the HA:collagen composition or the activating reagent. In one embodiment, the cross - linking reaction is carried out under mild pH and temperature conditions (pH 5.5 and 4°C to 25°C), rather than at the higher pH and temperature required for BDDE cross - linking of HA skin fillers. Surprisingly, using these methods, the HA and the sensitive collagen components exhibit minimal degradation during the cross - linking process, and their structures remain substantially intact, as shown in the examples.

[0107] In one embodiment, hyaluronic acid is hydrated for at least 60 minutes prior to the step of cross-linking with collagen. In a further embodiment, hyaluronic acid is hydrated at a temperature below room temperature. In yet another embodiment, hyaluronic acid is hydrated at a temperature of about 2°C, about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 20°C, about 22°C, about 24°C or at any temperature between the ranges defined by any two of the above values. In one embodiment, hyaluronic acid is hydrated at a temperature above room temperature. Thus, the method of making the hydrogel can be adjusted to adjust the properties of the hydrogel, such as the loss tangent parameter (G" / G').

[0108] In one embodiment, collagen can be provided in a solution, where the solution has an acidic pH and the collagen is soluble therein.

[0109] In one embodiment, collagen is provided as pre-fibrillar collagen, where the collagen is processed prior to cross-linking. In one embodiment, the pre-fibrillar collagen is in an alkaline solution. In one embodiment, collagen is provided as soluble collagen, where the collagen is in an acidic solution. In one embodiment, the pre-fibrillar collagen is in a solution, where the solution has a neutral pH.

[0110] 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 at any pH between the ranges defined by any two of the above values.

[0111] The physical properties of the hydrogel can depend on the HA and collagen concentrations, HA molecular weight, EDC concentration, EDC / NHS ratio, temperature, pH, salt / buffer concentration and lysine concentration. In one embodiment, an elastic modulus (G′) value in the range of 30 Pa to almost 10,000 Pa is obtained. In one embodiment, the elastic modulus depends on the formulation and synthesis parameters. The formulation and synthesis parameters can be adjusted. In one embodiment, the compression force value ranges from 20 gmf to greater than 500 gmf and the hydrogel swelling ranges from 1.5 times to 5 times the original gel volume. Based on the wide range of physical properties obtained, different HA / collagen configurations may be used as skin fillers for different facial applications.

[0112] In one embodiment of the cross-linking reaction, the method includes stopping the cross-linking step.

[0113] As described in the embodiments herein, formulations with low G' and compression force values and minimal swelling can be used for very superficially placed fine line fillers or injectable skin quality enhancers, while more robust configurations with higher G' and compression force and more swelling can be used for moderate to severe wrinkle correction and facial augmentation / plasticity.

[0114] In one embodiment, a method of filling fine lines includes the step of providing a composition to a patient in need thereof, the composition having a low G' and compressive force value. In one embodiment, a method of treating moderate to severe wrinkle correction and facial augmentation / plasticity is provided, the method including providing a composition having a higher G' and compressive force to a patient in need thereof.

[0115] Consider increasing the HA concentration of the crosslinked macromolecular matrix. For example, increasing the HA concentration may result in a hydrogel having a higher G', a higher compressive force value, and a higher opacity. The increase in opacity may also reduce the likelihood of blue discoloration at the injection site due to the Tyndall effect. The strongly crosslinked macromolecular matrix can provide the forces required to lift the tissue and resist subsequent deformation, which can produce 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.

[0116] The elastic modulus can be a function of the hyaluronic acid concentration. In one embodiment, the HA concentration ranges between 13 mg / mL and 28 mg / mL. In one embodiment, the G' of the composition 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 2400 Pa, about 2500 Pa, about 2600 Pa, about 2700 Pa, about 2800 Pa, about 2900 Pa, about 3000 Pa, about 3100 Pa, about 3200 Pa, about 3300 Pa, about 3400 Pa, about 3500 Pa, about 3600 Pa, about 3700 Pa, about 3800 Pa, about 3900 Pa, about 4000 Pa, about 4100 Pa, about 4200 Pa, about 4300 Pa, about 4400 Pa, about 4500 Pa, about 4600 Pa, about 4700 Pa, about 4800 Pa, about 4900 Pa, about 5000 Pa, about 5100 Pa, about 5200 Pa, about 5300 Pa, about 5400 Pa, about 5500 Pa, about 5600 Pa, about 5700 Pa, about 5800 Pa, about 5900 Pa, about 6000 Pa, about 6100 Pa, about 6200 Pa, about 6300 Pa, about 6400 Pa, about 6500 Pa, about 6600 Pa, about 6700 Pa, about 6800 Pa, about 6900 Pa, about 7000 Pa, about 7100 Pa, about 7200 Pa, about 7300 Pa, about 7400 Pa, about 7500 Pa, about 7600 Pa, about 7700 Pa, 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 10,000 Pa or any elastic modulus between the ranges defined by any two of the above values.

[0117] In one embodiment, a higher G' value is desired. Higher G' values can also be obtained by increasing the EDC:HA ratio or the EDC:NHS ratio. Mixtures containing hyaluronic acid components of different molecular weights were considered, which may affect the G' and compressive force values. For example, in an HA:collagen formulation, a decrease in the hydration temperature may result in an increase in the G' value, a decrease in swelling, and a decrease in opacity (increase in translucency). Using these synthesis parameters and results, HA-collagen formulations with desired physical properties can be synthesized in a targeted manner.

[0118] The collagen concentration can also affect the physical properties. For a given hydration temperature, an increased collagen concentration may result in an increase in opacity, an increase in G′, and a decrease in swelling. The increased opacity may result in a decrease in the Tyndall effect of the filler. The physical and optical properties of the HA-collagen hydrogel depend on the degree of dissolution of collagen during the synthesis step. Synthesis parameters such as temperature, pH, and salt concentration affect the solubility of collagen. The solubility of collagen may decrease with an increase in temperature, pH, and salt concentration, and a decrease in the solubility of collagen during the synthesis process may result in a decrease in the G' of the gel, an increase in the degree of swelling, and an increase in the extrusion force. HA can also interact with collagen to reduce the solubility of collagen, as described by Taguchi and his colleagues (Taguchi et al., Journal of Biomedical Materials Research, 2002, 61(2), 330-336; incorporated by reference herein). Adjusting the salt concentration can change the interaction between HA and collagen, thereby regulating the solubility of collagen and changing its physical properties. In one embodiment, the composition contains a salt at a concentration between 50 mM and 400 mM. In one embodiment, the composition contains an NaCl concentration of approximately 150 mM. Thus, during the synthesis process, by decreasing the hydration temperature and optimizing the salt / buffer concentration, for a given HA concentration, the maximum G′ and minimum swelling values and the maximum collagen solubility can be obtained.

[0119] In one embodiment, the composition is transparent. In one embodiment, the composition is translucent. In one embodiment, the concentration of HA, the hydration temperature, the salt concentration, and / or the collagen concentration affect the opacity of the composition. The increased opacity may reduce the Tyndall effect, i.e., the blue discoloration that may occur at the injection site. Those skilled in the art can understand the method for determining the opacity of the composition.

[0120] In one embodiment, the biological properties and tissue responses of these materials and compositions have been characterized. In one embodiment, the formulations show enhanced cell activity compared to HA-only materials. The level of activity depends on the collagen concentration, and also on the HA concentration and the synthesis process. In one embodiment, for a given collagen concentration, formulations with a lower HA concentration (13 mg / mL) were found to produce an enhanced in vitro response compared to formulations with a higher HA concentration (20 mg / mL to 28 mg / mL). In one embodiment, compositions with similar HA concentrations, such as those hydrogels with a higher collagen concentration, show a stronger in vitro response. Additionally, in one embodiment, formulations hydrated at temperatures below room temperature stimulate higher cell activity than those similar formulations hydrated at room temperature. For a particular filler indication, a particular level of cell activity may be required, and by selecting the formulation and synthesis parameters, a preferred level of activity can be achieved.

[0121] In one embodiment, the tissue responses of the HA / collagen formulations were also evaluated in a tissue integration model. Tissue sections of the HA-collagen material implants showed cell infiltration from the surrounding tissue and new collagen deposition and angiogenesis within the injected filler bolus. The degree of infiltration and tissue integration varied for different formulations. In some embodiments of the formulations described herein, the collagen structure and crosslinking were unexpectedly important in the degree of infiltration and tissue integration. In one embodiment, the collagen structure and crosslinking may be important in tissue integration and infiltration (see, for example, Figure 14 ). In some embodiments of the formulations described herein, tissue integration decreased with increasing HA concentration. In some embodiments of the formulations described herein, formulations with a low HA concentration (13 mg / mL) injected into the tissue were found to have the surrounding tissue infiltrate the entire gel bolus at 4 weeks. In these embodiments, cell nuclei and newly deposited collagen were found to be dispersed throughout the gel. This can be seen in Example 7 using Formulation I ( Figure 6B and 6C ). Thus, these formulations achieved surprising results of tissue infiltration into the gel bolus.

[0122] In some embodiments, formulations with a higher HA concentration (20 mg / mL to 25 mg / mL) also exhibited strong tissue integration, but as with those formulations with a lower HA concentration, the tissue did not infiltrate the entire bolus.

[0123] In some embodiments, the HA molecular weight and / or gel particle characteristics also affect the surrounding tissue integration.

[0124] However, another surprising result suggests that structure / crosslinking may be more important than the collagen concentration in the composition. An example of this surprising finding is that a gel containing 20:6 HA:collagen (where HA is hydrated at 5 °C) (fraction = 2.0) exhibited a better tissue integration fraction than a gel containing 20:10 HA:collagen (hydrated at room temperature) (fraction = 0.5). The tissue integration scores were performed by blinded histopathologists and were normalized against an internal study control (HA-only gel). Higher scores indicate better tissue integration.

[0125] Surprisingly, differences in the results were shown in compositions with different mixed gel structures. Compositions in which collagen was mixed had a different response compared to compositions in which the gel had collagen crosslinked with HA at 5 °C.

[0126] As shown in the embodiments herein, formulation XIX synthesized with HA hydrated at 5 °C demonstrated improved in vitro and in vivo performance and optimal tissue integration.

[0127] In addition to collagen concentration, the level or crosslinking and structure of the composition were shown to be equally significant. For example, compositions produced at reduced temperatures such as gel formulations produced improved in vitro and in vivo performance, such as improved tissue integration. This can be seen in formulation XIX (having a 20:6 HA:collagen ratio and a hydration temperature of about 5 °C). The preparation of the formulation also led to surprising results, such as improved in vitro and in vivo performance. In some embodiments, preparing the gel formulation at a low temperature (e.g., 5 °C) for hydration results in the gel formulation having improved in vitro and in vivo performance. In some embodiments, the gel formulation demonstrated tissue integration into the site of the injection formulation.

[0128] Formulations having HA:collagen ratios of 20:6 and 20:4 and a hydration temperature of about 5 °C also produced surprising results, such as improved in vitro and in vivo performance.

[0129] In some embodiments, a formulation is provided, wherein the formulation increases the penetration of collagen into tissue. The formulation contains 13 mg / mL hyaluronic acid. In some embodiments, the formulation is injected into tissue, thereby creating a depot containing the formulation, wherein cells from the tissue surrounding the depot deposit into the depot. In one embodiment, the tissue injected with the formulation was shown to have tissue integration and collagen deposition as well as angiogenesis. In one embodiment, the formulation has a 20:6 HA:collagen ratio and a hydration temperature of about 5 °C. In one embodiment, the formulation has a 20:4 HA:collagen ratio and a hydration temperature of about 5 °C.

[0130] The primary function of a dermal filler is to fill in wrinkles and support the overlying tissue into which the filler is injected. The amount of lift required depends on the specific facial indication. Articles designed to plump an indication and placed deeper under the skin will need to exhibit more structure and more lift. Preparations for superficial fine lines do not need to exhibit such a large lift, but should be smoother and blend into the existing tissue. Accordingly, HA / collagen preparations are evaluated in an animal lift-ability model to determine lift-ability. For preparations crosslinked in a similar manner, lift-ability depends on the HA concentration, with higher HA concentrations providing increased lift.

[0131] In some embodiments, crosslinked HA:collagen preparations to which lysine has been added exhibit increasing lift as the HA concentration increases from 13 mg / mL to 20 mg / mL to 25 mg / mL. In some embodiments, the HA molecular weight also affects lift. In some embodiments, a preparation containing 25 mg / mL high molecular weight HA exhibits greater lift than a preparation composed of a mixture of 25 mg / mL low and high molecular weight HA. Accordingly, the desired lift can be achieved by selecting optimal synthesis parameters, HA concentration, and HA molecular weight ratio. In some embodiments of any one or any combination of the above or below embodiments, the preparation comprises a mixture of hyaluronic acid components having different molecular weights, wherein the mixture comprises hyaluronic acid having 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 1,500,000 daltons, about 2,000,000 daltons, 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 defined by any two of the above values.

[0132] Method for synthesizing lysine-crosslinked HA-collagen hydrogel

[0133] The methods provided by the present disclosure include 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 comprises a weight ratio of hyaluronic acid to collagen 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 comprises a collagen concentration of about 6 mg / mL. In some embodiments, the hydrogel is stirred for homogenization. In some embodiments, the hydrogel is hydrated at a temperature below room temperature. In one embodiment, the HA is hydrated at a temperature between 2 °C and 35 °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, 19 °C, 21 °C, 23 °C, 25 °C, 27 °C, 29 °C, 31 °C, 33 °C, 35 °C, or at any temperature between the ranges defined by any two of the above 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 at any temperature between the ranges defined by any two of the above 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 above room temperature. In one embodiment, the HA is hydrated at a temperature of at least 35 °C. In some embodiments, another hydration step is performed for at least 60 minutes. In some embodiments, the hydration is performed at a pH of about 5.5 in MES buffer. The mixture can be contained within a syringe and passed between two syringes at least fifty times. An EDC / NHS solution can be added to the mixture. Mixing can be performed by passing the solution between two syringes. After adding the EDC / NHS solution, the mixture is reacted at a temperature between 2 °C and 8 °C for at least 16 hours. 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 several parameters of the composition, such as compressive force, swelling characteristics, and extrusion force.

[0134] In one embodiment, the macromolecular matrix further comprises uncrosslinked HA, which can be used to facilitate injection and reduce extrusion force.

[0135] In one embodiment, the lysine:HA ratio is optimized to maximize crosslinking efficiency. In one embodiment, the lysine:HA ratio is between about 0.0 and 0.5 and may allow for more efficient crosslinking. Crosslinking without lysine may rely on collagen to provide amines for crosslinking and may allow for more water-labile ester crosslinks between HA chains. Crosslinking with a high lysine:HA ratio may saturate the activated carboxylic acids on the HA chains and may result in pendant lysine molecules being attached to the HA chains on only one side rather than crosslinking between the chains. By selecting the optimal lysine:HA ratio for a given indication, the physical properties of the resulting hydrogel can be adjusted and desired characteristics obtained. In some embodiments, the optimal lysine:HA ratio may depend on the composition.

[0136] Sterilization of the composition

[0137] Before administering the composition by injection or implantation into a human patient, the developed biomaterial may need to be sterilized, or unwanted biomaterials, such as pathogens, bacterial microorganisms, destroyed. These compositions include the embodiments described herein, such as materials like crosslinked macromolecular matrices. The proteins, polysaccharides, and carbohydrates in these materials may be prone to molecular decomposition when exposed to conventional heat sterilization procedures (such as autoclaving) or subjected to ionizing radiation (such as gamma radiation). Traditionally, many of these energy-sensitive biomaterials are batch sterilized by microfiltration processes designed to physically remove microorganisms from the composition. The filtered composition must then be packaged in syringes and / or vials for use by a physician.

[0138] In one embodiment, the crosslinked macromolecular matrix is sterile. In one embodiment, the method of preparing the crosslinked macromolecular matrix further includes the step of sterilizing the crosslinked macromolecular matrix.

[0139] In one embodiment, the method further includes the step of subjecting the composition or crosslinked macromolecular matrix to a dose of broadband spectral radiation to effectively inactivate pathogens, microorganisms, and other microbes.

[0140] In one embodiment, the method further includes the step of subjecting the composition or crosslinked macromolecular matrix to pulsed radiation (sometimes hereinafter pulsed light, including broadband spectral radiation). The broadband spectral radiation may have a band within the wavelength range of about 100 nm to about 1100 nm. The broadband spectral radiation includes wavelengths in the ultraviolet range, visible light range, and infrared range. In some embodiments, it has a wavelength distribution of about 54% UV wavelengths, 26% visible light wavelengths, and about 20% infrared wavelengths. This form of radiation can be provided by a xenon lamp.

[0141] In one embodiment, pulsed light inactivates microorganisms (microorganisms and microbes) in the composition throughout the composition without causing significant deterioration of the composition and without causing significant changes in the rheology of the composition.

[0142] In one embodiment, the pulsed light has an energy defined by a UV flux at 254 nm between about 100 mJ / sqcm and about 2000 mJ / sqcm. In one embodiment, the pulsed light has an energy defined by a UV flux at 254 nm between about 300 mJ / sqcm and about 1800 mJ / sqcm.

[0143] In one embodiment, the pulsed light has an energy between about 700 mJ / sqcm and about 800 mJ / sqcm defined by a UV flux at 254 nm. In one embodiment, the pulsed light has an energy defined by a UV flux at 254 nm between about 1400 mJ / sqcm and about 1600 mJ / sqcm.

[0144] In one embodiment, the pulsed light has a pulse frequency between about 1 pulse per second and about 10 pulses per second, for example, about 3 pulses per second.

[0145] In one embodiment, the composition is subjected to pulsed light for a period of no more than 240 seconds. In one embodiment, the composition is subjected to pulsed light for a period of no more than 120 seconds. In one embodiment, the composition is subjected to pulsed light for a period of no more than 40 seconds. In one embodiment, the composition is subjected to pulsed light for a period of no more than 30 seconds. In one embodiment, the composition is subjected to pulsed light for a period of no more than 20 seconds. In one embodiment, the composition is subjected to pulsed light for a period of 10 seconds.

[0146] In one embodiment, the composition is subjected to pulsed light for a period of 5 seconds. In one embodiment, the composition is subjected to pulsed light for a period of no more than one second.

[0147] In one embodiment, the pulsed light effectively sterilizes the composition without increasing the temperature of the composition by more than 90 °C. In one embodiment, the pulsed light can effectively sterilize the composition without increasing the temperature of the composition by more than 20 °C. In one embodiment, the dose effectively sterilizes the composition without increasing the temperature of the composition by more than 15 °C, for example, more than 10 °C, for example, more than 5 °C.

[0148] In one embodiment, the pulsed light effectively sterilizes the composition with a rheological loss (G′ / G") of less than about 10%, or less than about 8%, or less than about 5%.

[0149] In one embodiment, pulsed light effectively sterilizes the composition, i.e., inactivates pathogens, microbes, and other microorganisms in the composition without causing significant degradation, e.g., without causing a significant change in the rheological properties of the composition.

[0150] In one embodiment, the effective sterilization dose of radiation preserves the rheology of the hydrogel. In one embodiment, the method effectively sterilizes the hydrogel with a rheological loss (G′ / G") of less than about 10%, or less than about 8%, or less than about 5%.

[0151] Examples

[0152] The following examples, including the experiments conducted and the results obtained, are for illustrative purposes only and should not be construed as limiting the disclosure.

[0153] Example 1 – Synthesis of lysine-crosslinked HA-collagen hydrogel

[0154] A 4.96 mg / mL collagen solution in 0.01 M HCl was added to a 30 mL HSW Norm-Ject syringe together with lysine·HCl, HMW HA, MES buffer / NaCl solid, and 1 M NaOH. The concentrations were adjusted accordingly to prepare hydrogels with HA:collagen ratios of, for example, 13:4 mg / mL (Formulation I), 20:4 mg / mL (Formulation II), and 25:4 (Formulation III). The mixture was stirred to homogenize the solution and the HA was allowed to hydrate at room temperature for about 60 minutes. After about 60 minutes to about 90 minutes, the mixture was passed between syringes and hydrated again for about 30 minutes to about 60 minutes. After the second hydration, the mixture was passed between syringes several times. An EDC / NHS solution was prepared in a third 30 mL syringe by adding water, NHS, and EDC and mixed by shaking. The EDC / NHS solution was added to the HA / collagen mixture and passed between two syringes and then transferred to a glass vial allowing reaction at 2 °C to 8 °C. In some embodiments, the reaction time was about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, or any time between the ranges defined by any two of the above values. After this time, the gel was transferred to a syringe and passed between two syringes again. The pH of the gel was adjusted to about 7.40 using 2 M NaOH and the final volume was adjusted using PBS. The gel formulation was dialyzed against PBS at 2 °C to 8 °C for about 70 hours, during which the buffer was changed several times to remove EDC / NHS. The gel was then transferred from the dialysis membrane to a syringe and passed through a stainless steel mesh (60 μm pore to 104 μm pore) and between two syringes. The gel was transferred to a 1 mL syringe and the syringe was steam sterilized. The resulting sterile hydrogel was characterized using rheology, compression force measurement, extrusion force measurement, and swelling.

[0155] For Formulation XXVI, NaCl was omitted during the crosslinking process.

[0156] For Formulations XXV and XXVI, uncrosslinked HMW HA (2% (w / w) relative to the total composition) and lidocaine hydrochloride (0.3% (w / w) relative to the total composition) were added before syringe filling and sterilization.

[0157] Example 2 – Synthesis of lysine-crosslinked HA-collagen hydrogel with a final collagen concentration of 6 mg / mL

[0158] Add a 7.16 mg / mL collagen solution in 0.01 M HCl together with lysine·HCl, HMW and / or LMW HA, and MES buffer / NaCl solid to a 30 mL HSW Norm-Ject syringe. Adjust the pH with 1 M NaOH. Stir the mixture to homogenize it and allow the HA to hydrate at the specified temperature for about 60 minutes. After about 60 to 90 minutes, pass the mixture multiple times between two syringes and hydrate again for at least 30 minutes. After the second hydration step, pass the mixture multiple times between the two syringes again. Prepare an EDC / NHS solution in a third 30 mL syringe by adding water, NHS, and EDC and shake to mix. Hydration can be carried out at about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, or at any temperature between the ranges defined by any two of the above. Add the EDC / NHS solution to the HA / collagen mixture and pass it multiple times between two syringes, then transfer it to a Thinky Mixer reaction vessel and allow it to react at 2 °C to 8 °C for at least 16 hours. After this period, homogenize the gel using the Thinky Mixer. Adjust the pH of the gel to about 7.40 with 2 M NaOH and adjust the final volume with PBS. Dialyze the gel formulation against PBS at 2 °C to 8 °C for about 70 hours, during which the buffer is changed multiple times. Then transfer the gel from the dialysis membrane to a syringe, pass it through a stainless steel mesh (104 μm pores), and homogenize it using the Thinky Mixer. Transfer the gel to a 1 mL syringe and autoclave the syringe. Characterize the resulting sterile hydrogel as described in the above examples.

[0159] 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 preparing the gel, the hyaluronic acid is hydrated at a temperature of 5 °C.

[0160] Example 3 – Synthesis of lysine-crosslinked HA-collagen hydrogel (Formulation XVI) with an HA:collagen concentration of 28:2 mg / mL Example 4 – Synthesis of lysine-crosslinked HA-collagen hydrogel (Formulation XV) with an HA:collagen concentration of 25:4 mg / mL prepared at 1.25x the final concentration

[0161] A 3.20 mg / mL collagen solution in 0.01 M HCl was added to a 30 mL HSW Norm-Ject syringe together with 0.01 M HCl, lysine·HCl, HMW HA, LMW HA, and MES buffer / NaCl solid. The pH was adjusted using NaOH. The mixture was stirred to homogenize and the HA was hydrated in water at room temperature for about 90 minutes. After 90 minutes, the mixture was passed several times between syringes and hydrated again for about 30 minutes. After the second hydration step, the mixture was passed several times between syringes. An EDC / NHS solution was prepared in a third 30 mL syringe by adding water, NHS, and EDC and mixed by shaking. The EDC / NHS solution was added to the HA / collagen mixture and passed several times between syringes and then transferred to a glass vial and allowed to react at 2 °C to 8 °C for at least 16 hours. Thereafter, the gel was transferred to a syringe and passed between syringes. The pH of the gel was adjusted to about 7.40 using 2 M NaOH and the final volume was adjusted using PBS. The gel formulation was dialyzed against PBS at 2 °C to 8 °C for about 70 hours during which the buffer was changed several times. The gel was then transferred from the dialysis membrane to a syringe and passed through a stainless steel mesh (104 μm pores) and passed between syringes for homogenization. The gel was transferred to a 1 mL syringe and the syringe was steam sterilized. The resulting sterile hydrogel was characterized as described in the above example.

[0162] Embodiment 5 – Physical properties of the hydrogel Example 6 – In vitro testing of the hydrogel 。

[0163] A 5.67 mg / mL collagen solution in 0.01 M HCl was added to a 30 mL HSW Norm-Ject syringe together with lysine·HCl, HMW HA, LMW HA, and MES buffer / NaCl solid. The pH was adjusted with 1 M NaOH. The mixture was stirred to homogenize and the HA was hydrated in water at room temperature for about 90 minutes. Then the mixture was passed between syringes several times and hydrated again for 30 minutes. After the second hydration step, the mixture was passed between syringes again. An EDC / NHS solution was prepared in a third 30 mL syringe by adding water, NHS, and EDC and mixed by shaking. The EDC / NHS solution was added to the HA / collagen mixture and passed between syringes several times, then transferred to a glass vial and allowed to react at 2 °C to 8 °C for at least 16 hours. Thereafter, the gel was transferred to a syringe and passed between syringes. The pH of the gel was adjusted to about 7.40 with 2 M NaOH and the final volume was adjusted with PBS. The gel formulation was dialyzed against PBS at 2 °C to 8 °C for about 70 hours, during which the buffer was changed several times. Then the gel was transferred from the dialysis membrane to a syringe, passed through a stainless steel mesh (104 μm pores) and passed between syringes for homogenization. The gel was transferred to a 1 mL syringe and the syringe was steam sterilized. The resulting sterile hydrogel was characterized as described in the above example.

[0164] In vitro cell proliferation and viability 。

[0165] Rheological properties were obtained using an Anton-Paar MCR301 / 302 rheometer with a 25 mm parallel plate geometry measuring tool. Samples were analyzed at a 1 mm gap height by frequency sweep (10 Hz to 0.1 Hz, 1% strain) and amplitude sweep (0.3% to 300% strain, 5 Hz frequency). Compression force was measured using the same instrument with a 2.5 mm gap height and vertical compression. The gap height was set to 2.5 mm and held for 5 minutes, 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 volume of the gel after equilibrium. The swollen gel volume was compared to the original gel volume added before adding the buffer. Swelling was expressed as the percentage of additional liquid absorption relative to the original gel volume. Gel extrusion force of the gel formulation was measured using a texture analyzer set at a speed of 50 mm / min in a 1 mL COC syringe equipped with a 1 / 2” 27G TSK needle (unless otherwise stated).

[0166]

[0167]

[0168] Table 1: Synthesis parameters and physical properties of hydrogel formulations. 1 equivalent (Eq) corresponds to a 0.1 M MES buffer concentration with 0.9% NaCl.

[0169] As shown in Table 1, at a constant collagen concentration, as the HA concentration increased from 13 mg / mL to 20 mg / mL to 25 mg / mL (Formulation I vs. Formulation II vs. Formulation III), the G′ values (380 → 645 → 1370 Pa), as well as the compressive force (47 → 180 → 310 gmf) and extrusion force (13.8 (30G) → 28.0 → 51.7 N) increased, while the G″ / G′ ratio decreased with increasing HA concentration (0.123 → 0.103 → 0.056).

[0170] At the same HA and collagen concentrations, as the HMW / LMW HA ratio decreased from 100 / 0 to 65 / 35 to 35 / 65 (Formulation VI vs. Formulation VII vs. Formulation VIII), the G′ values (1360 → 1180 → 932 Pa), as well as the compressive force (292 → 226 → 163 gmf) and extrusion force (63.6 → 29.2 → 20.0 N) decreased, while the G″ / G′ ratio increased with decreasing HMW / LMW ratio (0.055 → 0.068 → 0.085).

[0171] At constant HA and collagen concentrations, as the synthesis hydration temperature decreased from 35 °C to 22 °C to 15 °C to 5 °C (Formulation XI vs. Formulation VI vs. Formulation IX vs. Formulation X), the G′ values increased (876 → 1360 → 3145 → 4750 Pa), while the hydrogel swelling degree (257% → 238% → 159% → 127%) and extrusion force (56.1–63.6 → 33.8 → 20.1 N) decreased. The compressive force was not affected by the change in hydration temperature, except at higher hydration temperatures (35 °C vs. other temperatures). Adjusting the hydration temperature during synthesis changes the solubility of collagen and thus results in changes in the physical properties of the resulting hydrogel.

[0172] By reducing the salt / buffer concentration by two-thirds, similar G' values (5470 Pa vs. 4750 Pa), degrees of swelling (108% vs. 127%), and extrusion forces (20.5 N vs. 20.1 N) were obtained for the formulation hydrated at 22 °C with the reduced salt / buffer concentration and the formulation hydrated at 5 °C with the full concentration of salt / buffer (Formulation XIII vs. Formulation X). Additionally, the synthesis using the reduced concentration of salt / buffer was less sensitive to the hydration temperature of 22 °C vs. 5 °C than the synthesis using the full concentration of salt / buffer. For the formulations synthesized using the reduced concentration of salt / buffer, with hydration temperatures of 22 °C and 5 °C (Formulation XIII vs. Formulation XII), the G′, degree of swelling, and extrusion force values did not differ significantly, while for similar formulations synthesized using the full concentration of salt / buffer, these physical properties were significantly different (Formulation VI vs. Formulation X).

[0173] The effect of added lysine was observed by comparing similar formulations synthesized at a HA:collagen concentration of 28:2 mg / mL (Formulation XVIII vs. Formulation XVI vs. Formulation XVII). The optimal lysine:HA ratio was used to maximize the crosslinking efficiency, and the exact ratio depended on the HA molecular weight, the concentration of the activating reagent, and the synthesis conditions. For example, for a series of formulations synthesized at a HA:collagen concentration of 28:2 mg / mL (Formulation XVIII vs. Formulation XVI vs. Formulation XVII), the lysine:HA ratio increased (0 → 0.333 → 0.5). For the formulation prepared with a lysine:HA ratio of 0.333, the physical properties (such as G' and compressive force) were maximized, while for the same formulation, the swelling and G" / G' values were minimized. 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. Crosslinking without lysine may rely on collagen to provide the amines for crosslinking and may allow for more water-labile ester crosslinks between HA chains. Crosslinking with a high lysine:HA ratio may saturate the activated carboxylic acids on the HA chains and may result in pendant lysine molecules being attached to the HA chains on only one side rather than crosslinking between the chains. These cases with very low or high lysine:HA ratios may lead to 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 adjusted and the desired properties obtained.

[0174] Figure 1 。

[0175] Figure 1 。

[0176] The viability and proliferation of fibroblasts in close contact with HA-collagen hydrogels were quantified using the XTT assay. 100 μL of hydrogel (n = 3) was spread on the bottom of a 24-well cell culture plate with a low-adhesion surface coating and placed in a humidified incubator at 37 °C for 30 minutes. 50,000 adult dermal fibroblasts in 500 μL of cell culture medium were added on top of the hydrogel bed and incubated at 37 °C. After 48 hours of incubation, 250 μL of XTT reagent was added to each well and incubated at 37 °C for 4 hours. The plate was then spun at 300 x g for 5 minutes, and 200 μL of the supernatant from each well was transferred to the wells of a 96-well filter plate with a 20 μm pore size. The filter plate containing the XTT supernatant was spun at 300 x g for 5 minutes. 100 μL of the filtered supernatant from each well was transferred to a clean 96-well plate (black wall, clear bottom), and the absorbance of the supernatant was read on a microplate reader (450 nm, background corrected at 630 nm). The data were normalized to the XTT cell activity of fibroblasts cultured on positive control tissue culture polystyrene (TCPS).

[0177] It was found that the cell viability and proliferation of formulations with lower HA concentrations were higher than those of similarly cross-linked 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 concentration, 13 mg / mL (formulation I), 20 mg / mL (formulation II), and 25 mg / mL (formulation III) showed proliferation values relative to the TCPS positive control of 53%, 30%, and 20% ( Figure 1 ). The negative control gel containing only HA showed a proliferation value of 12% relative to the TCPS control.

[0178] Furthermore, for similarly cross-linked formulations with the same HA and collagen concentrations, the hydration temperature during synthesis showed an effect on cell viability and proliferation. For hydrogels with an HA:collagen concentration of 24:6 mg / mL, those formulated at 5 °C (formulation X) showed higher cell proliferation capacity than those formulated at 22 °C (formulation VI) or 35 °C (formulation XI), with proliferation values of 39%, 27%, and 19% ( Figure 1 ).

[0179] Cell viability and proliferation are also affected by the salt and buffer concentrations during hydrogel synthesis. For synthesis carried out at 5 °C (formulation X, 1 equivalent, compared to formulation XII, 0.33 equivalents), there was no significant change in the cellular response upon decreasing the salt / buffer concentration. However, for formulations hydrated at 22 °C or 35 °C, a significant increase in cell viability and proliferation was observed for those formulations prepared with a decreased salt / buffer concentration (formulation XIII, 22 °C vs. formulation XIV, 35 °C) compared to those prepared with one equivalent of salt / buffer (formulation VI, 22 °C vs. formulation XI, 35 °C). Figure 15 )

[0180] Formulation XIX was prepared at 5 °C with 20 mg / mL HA and 6 mg / mL collagen. This formulation exhibited higher cell viability and proliferation compared to other gels with HA concentrations of 20 mg / mL or higher. In vitro cell morphology )

[0181] Formulations containing an HA:collagen ratio of 20:4 also showed enhanced in vitro cellular responses Figure 13 , formulation XXII).

[0182] In addition, formulation XIX demonstrated consistent stability and performance after autoclaving.

[0183] Figures 2A to 2D .

[0184] Analyze cell morphology to evaluate the effects of the hydrogel formulation on cell size, shape, and cytoskeletal organization. Image and quantify the actin filament alignment index and morphology of fibroblasts cultured on HA-only or HA / collagen crosslinked hydrogels. Increased actin filament alignment may be associated with increased cell adhesion to its substrate. An increased aspect ratio is associated with increased cell spreading on the substrate. The convex hull to cell area ratio is a measure of cell shape, where 1.0 indicates a cell with a uniform shape and values greater than 1 indicate a cell with a more irregular shape. Cells that make multiple contacts and extend / migrate with the matrix exhibit a more irregular cell shape and a higher convex hull to cell area ratio. The actin filament alignment index, aspect ratio, and convex hull to cell area ratio can be analyzed together in 3-dimensional Euclidean space. Based on the Euclidean distance of the hydrogel from a negative control (in this case, a non-adhesive HA-only gel), the overall cellular response to the filler can be ranked. A greater Euclidean distance from the HA-only control indicates enhanced cell adhesion and spreading on the hydrogel. Hydrogels that support greater cell adhesion and spreading are expected to induce more cell infiltration into the gel, and these cells will deposit ECM in the gel matrix. Increased cell infiltration and ECM deposition may facilitate tissue integration into the hydrogel depot in vivo. In contrast, those formulations with lower cell adhesion and spreading values will behave more inertly and allow less tissue infiltration and integration. In some embodiments, the method of preparing the hydrogel further includes an autoclaving step, where autoclaving does not alter the properties of the hydrogel (see Figure 16 ).

[0185] In a typical procedure, hydrogels (n = 3) in cell culture medium and human dermal fibroblasts are added to a 96-well cell culture plate with a low-adhesion surface coating. After incubation for 48 hours, the cells are fixed in formalin and stained with Hoechst, WGA-488, and AlexaFluor-Phalloidin. The wells are imaged with a confocal microscope, and the actin filament alignment (phalloidin) and cell morphology (WGA-488) are analyzed using image analysis software.

[0186] For similar cross-linked formulations with the same HA and collagen concentrations, the hydration temperature during synthesis showed an effect on cell adhesion and spreading. For hydrogels with an HA:collagen concentration of 24:6, those formulated at 5 °C (formulation X) showed increased cell adhesion (actin filament alignment index) compared to those formulated at 22 °C (formulation VI), with cell adhesion values of 0.054 and 0.015, respectively (Figure 2). Formulations hydrated at 5 °C (formulation X) exhibited increased cell spreading (cell aspect ratio) compared to those hydrated at 22 °C (formulation VI), with cell spreading values of 2.52 and 1.40, respectively. Formulations hydrated at 5 °C (325_B) also showed an increased ratio of the convex hull to cell area compared to those hydrated at 22 °C (formulation VI), with area ratios of 1.34 and 1.07, respectively. The actin filament alignment index, cell aspect ratio, and ratio of the convex hull to cell area of formulations hydrated at 22 °C (formulation VI) were similar to those of the HA-only control. The optimized formulation (20:6 HA:collagen, hydrated at 5 °C; formulation XIX) showed significantly higher actin filament alignment index, aspect ratio, and ratio of the convex hull to cell area compared to the HA-only gel. Ranking the hydrogels using the Euclidean distance from the HA-only gel showed that the optimized formulation ranked higher than the other HA-collagen hydrogels.

[0187] Cell morphology analysis was highly relevant to the XTT cell viability assay because formulation X, which demonstrated higher viability than formulation VI in the viability assay, also showed evidence of enhanced cell adhesion and spreading in the morphology assay. Formulation XIX also showed higher viability than the other HA-collagen formulations and the HA-only gel. Cell spreading and adhesion are associated with higher cell viability, and thus, the results of each assay were highly consistent with each other ( Example 7 – In vivo testing of the hydrogel ).

[0188] Fibroblasts cultured with formulations XXII and XXIII showed significantly greater cell aspect ratios compared to fibroblasts cultured with the HA-only gel ( Lifting ability ).

[0189] Figure 3 .

[0190] Figure 4

[0191] The ability of the hydrogel to support tissue projection (elevation) was evaluated in vivo using a rat subcutaneous implantation model. 125 μL of the hydrogel (n = 10) was injected as a subcutaneous bolus onto the top of the skull. A clinical 3-D imaging system (Canfield Vectra) was used to generate 3-D reconstructions of the bolus over a 12-week period. Medical imaging software (Canfield Mirror) was used to analyze the average height of the bolus.

[0192] The in vivo elevation ability of a series of HA-collagen formulations with the same collagen concentration (4 mg / mL) and HMW / LMW HA ratio (100 / 0) measured between 4 and 12 weeks showed a positive correlation with HA concentration. The formulation with 25 mg / mL HA (formulation III) showed more projection than formulations with 20 mg / mL (formulation II) or 13 mg / mL (formulation I), see Figure 5 . Since the compressive force of these formulations increased with increasing HA concentration, in vivo projection from 4 to 12 weeks showed a positive correlation with compressive force. In vivo elevation also depends on gel synthesis conditions. Two formulations contained the same 25:4 mg / mL HA:collagen concentration (formulation III vs. formulation XV), but were synthesized at different HMW / LMW HA ratios and different crosslinking conditions, and produced different elevation curves over 4 to 12 weeks. The formulation synthesized with high MW HA at 1x synthesis concentration showed better elevation than the formulation prepared with 10 / 90 HMW / LMW HA at 1.25x synthesis concentration ( Figure 11 ). In addition, hydrogel formulations (formulation II, formulation XV, formulation XVI) with different HA / collagen concentrations and synthesis conditions but similar compressive force values (166 to 180) showed similar in vivo elevation curves between 4 and 12 weeks ( Figure 22 ). Thus, by selecting the optimal composition and synthesis conditions, the elevation curve required for a given application can be obtained.

[0193] The elevation ability was also tested with formulation XIX and a formulation containing only HA ( Figure 23 and 12 ). As shown, formulation XIX showed similar elevation ability to a 24 mg / mL HMW HA-only gel over 4 to 28 weeks.

[0194] The extended (52-week) elevation ability data of formulation XXII were tested. The elevation ability of the HA-only control decreased steadily over time. In contrast, the elevation ability of the HA-collagen gel (formulation XXII) remained stable between 30 and 52 weeks ( Figure 28)。This surprising result indicates that these HA - collagen gel formulations can have a longer lifting duration than gels containing only HA. This is associated with better tissue ingrowth compared to gels containing only HA (see below).

[0195] Data on the extended (26 - week) lifting ability of formulation XXII were tested. Formulation XXIII and a comparative example containing only HA showed similar lifting ability over the course of 26 weeks( Figure 28 )。The enhanced tissue integration of formulation XXIII surprisingly led to an increase in the duration of the lifting ability and other benefits to the overall effect. For example, the newly created tissue can maintain skin quality, lifting ability, and correct wrinkles.

[0196] Data on the extended (30 - week) lifting ability of formulation XXV were tested. The lifting ability of the gel containing only HA steadily declined over time( In vivo tissue integration )。In contrast, the lifting ability of the HA - collagen gels (formulations XXV and XXVI) remained stable between 18 and 30 weeks( Figures 6A to 6E )。This surprising result indicates that these HA - collagen gel formulations can have a longer lifting duration than gels containing only HA. This is associated with better tissue ingrowth compared to gels containing only HA (see below).

[0197] Figure 7

[0198] The in - vivo tissue integration of a series of formulations was evaluated using a rat subcutaneous implantation model. In a typical procedure, 125 μL of the hydrogel was delivered as a subcutaneous bolus to the dorsal side of the rats. Four weeks later, the bolus was removed, fixed in formalin, and embedded in paraffin for histological examination. The tissue sections were stained with hematoxylin and eosin (H&E) and colloidal iron. Immunohistochemical staining for type I collagen, vimentin, CD31, and procollagen type I was also performed.

[0199] For formulations prepared similarly with HMW HA and 4 mg / mL collagen, tissue integration showed a negative correlation with HA concentration. The collagen density deposited in the surrounding tissue of the formulation with 13 mg / mL HA (formulation I) was higher than those prepared with 20 mg / mL HA (formulation II) or 25 mg / mL (formulation III), and was related to the HA concentration( Figure 8)。In addition, compared to those materials with 20 mg / mL or 25 mg / mL HA, the 13 mg / mL HA formulation had fewer areas without tissue in the injection bolus. In addition to HA concentration, integration is expected to depend mainly on collagen concentration. However, it seems that other factors may 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) showed collagen deposition throughout the bolus and few areas without tissue.

[0200] Formulation XVI was mainly prepared with LMW HA, which is different from the formulations mentioned earlier prepared with HMW HA. However, the molecular weight of HA is not the only influencing factor, as a second formulation (Formulation XV) with an HA:collagen concentration of 25:4 mg / mL, which was mainly prepared with LMW HA, did not show the same strong integration throughout the bolus ( Figure 17 ). The hydration temperature during the above synthesis process has been shown to affect in vitro cell responses and, through subsequent studies, also in vivo cell infiltration and tissue integration. Two similar formulations prepared with an HA:collagen concentration of 24:6 mg / mL but with different hydration temperatures of 5 °C (Formulation X) and 22 °C (Formulation VI) showed different collagen deposition densities around the periphery of the bolus, with a higher response compared to the formulation prepared at 5 °C ( Figure 18 ). The degree of tissue integration is affected by the combination of factors such as HA concentration, HA molecular weight ratio, collagen concentration, and synthesis conditions rather than a single parameter. A range of tissue responses have been achieved using these materials, and thus, this tissue integration and infiltration can be customized to suit specific filler applications by optimizing the above synthesis parameters.

[0201] Formulations XXII and XXIII showed enhanced tissue integration compared to gels containing only HA ( Figure 19 ). Collagen 1a staining showed a fine collagen distribution around the gel particles in the HA-collagen formulations, while there was limited deposition of collagen 1a in the gels containing only HA ( Figure 20 ). Quantification of the percentage of positive area of collagen 1a staining in the hydrogel bolus after 4 weeks of subcutaneous implantation in rats showed that Formulation XXII produced more collagen 1a-positive tissue than the hydrogel containing only HA ( Figure 9 ).

[0202] Figure 21Confocal micrographs of human dermal fibroblasts cultured for 48 hours on gels containing only HA, formulation XXII, or formulation XXIII are shown. Samples were stained for HA-binding protein, Hoechst, and cell membrane. The resulting gels showed a significant improvement in cell adhesion compared to the crosslinked products containing only HA, demonstrating the potential of the gels as scaffolds for tissue integration and collagen deposition.

[0203] The ability of formulation XIX to increase the levels of vimentin (fibroblasts), collagen I, and CD31 was also tested. As Figure 24 and 10 shown, compared to the HA-only hydrogel, after 12 weeks of subcutaneous implantation in rats, formulation XIX was able to increase the levels of vimentin (fibroblasts), collagen I, and CD31 (blood vessels) in the formulation XIX hydrogel bolus. This confirmed that formulation XIX had improved cell spreading and adhesion. Similarly, compared to the HA-only control, formulations XXII and XXIII promoted greater fibroblast infiltration (vimentin staining) and angiogenesis (CD31 staining, arrows) ( Figure 25 ). This indicated tissue regeneration in the hydrogel bolus with a morphology consistent with endogenous tissue.

[0204] Figure 26 Confocal micrographs of human dermal fibroblasts cultured for 48 hours on gels containing only HA, formulation XXVI, or formulation XXV are shown. Samples were stained for HA-binding protein, Hoechst, and cell membrane. The gels of formulations XXVI and XXV showed a surprising improvement in cell adhesion compared to the crosslinked products containing only HA, demonstrating the potential of the gels as scaffolds for tissue integration and collagen deposition.

[0205] Figure 27 Two-photon imaging of second harmonic generation signal (white) and tissue autofluorescence (green) in rats treated with subcutaneous bolus injections of HA-only, formulation XXV, or formulation XXIII after 12 weeks is shown. 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 was observed in the HA-only gels.

[0206] Description of the subject technology as a clauseShows immunohistochemical analysis of the tissue response to formulation XXV after 4 weeks of subcutaneous implantation in rats. Formulation XXV promotes tissue integration (H&E staining), fibroblast infiltration (vimentin), limited macrophage response (CD68), deposition of collagen I, limited collagen III, and vascularization (CD31). This may indicate natural tissue regeneration in the hydrogel bolus. Additionally, formulation XXV received a higher histological score for tissue integration (4.67) compared to the HA-only control (0.67).

[0207] ​ Shows immunohistochemical analysis of the tissue response to formulation XXVI after 4 weeks of subcutaneous implantation in rats. Formulation XXVI promotes tissue integration (H&E staining), fibroblast infiltration (vimentin), limited macrophage response (CD68), deposition of collagen I, limited collagen III, and vascularization (CD31). This may indicate natural tissue regeneration in the hydrogel bolus. Additionally, formulation XXVI received a higher histological score for tissue integration (4.17) compared to the HA-only control (0.67).

[0208] ​

[0209] For convenience, various embodiments of aspects of the present disclosure are described as numbered clauses (1, 2, 3, etc.). These are provided as examples and do not limit the subject technology. The identification of the figures and reference numerals provided below are for illustrative and example purposes only, and the clauses are not limited by these identifications.

[0210] Clause 1. A crosslinked macromolecular matrix comprising: lysine; hyaluronic acid; and collagen; wherein the hyaluronic acid is crosslinked to the collagen via at least one endogenous amino group on the collagen and / or via at least one amino group present on the lysine.

[0211] Clause 2. The crosslinked macromolecular matrix of any one of the above or following clauses, wherein the crosslinked macromolecular matrix further comprises lidocaine.

[0212] Clause 3. The crosslinked macromolecular matrix of any one of the above or following clauses, wherein the concentration of lidocaine in the matrix ranges from about 0.15% (w / w) to about 0.45% (w / w).

[0213] Clause 4. A crosslinked macromolecular matrix according to any one of the above or following clauses, wherein the concentration of lidocaine in the matrix is about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration between the ranges defined by any two of the above values.

[0214] Clause 5. A crosslinked macromolecular matrix according to any one of the above or following clauses, wherein the concentration of lidocaine in the matrix is between about 0.27% (w / w) and about 0.33% (w / w).

[0215] Clause 6. A crosslinked macromolecular matrix according to any one of the above or following clauses, wherein the matrix further comprises uncrosslinked HA.

[0216] Clause 7. A crosslinked macromolecular matrix according to any one of the above or following clauses, wherein the concentration of uncrosslinked HA in the matrix is up to about 5% (w / w).

[0217] Clause 8. A crosslinked macromolecular matrix according to any one of the above or following clauses, wherein the concentration of uncrosslinked HA in the matrix is about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w) or about 5% (w / w), or any concentration between the ranges defined by any two of the above values.

[0218] Clause 9. A crosslinked macromolecular matrix according to any one of the above or following clauses, wherein the concentration of uncrosslinked HA in the matrix is about 1% (w / w).

[0219] Clause 10. A crosslinked macromolecular matrix according to any one of the above or following clauses, wherein the concentration of uncrosslinked HA in the matrix is about 2% (w / w).

[0220] Clause 11. A crosslinked macromolecular matrix according to any one of the above or following clauses, wherein the concentration of uncrosslinked HA in the matrix is about 5% (w / w).

[0221] Clause 12. A crosslinked macromolecular matrix according to any one of the above or following clauses, wherein the uncrosslinked HA improves the extrudability of the macromolecular matrix.

[0222] Clause 13. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the crosslinked macromolecular matrix is stable at about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months or at any amount of time between ranges defined by any two of the above values.

[0223] Clause 14. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the crosslinked macromolecular matrix is stable at temperatures between about 4 °C and about 25 °C.

[0224] Clause 15. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the crosslinked macromolecular matrix is stable at about 4 °C.

[0225] Clause 16. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the crosslinked macromolecular matrix is stable at about 25 °C.

[0226] Clause 17. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the crosslinked macromolecular matrix is stable at about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36 months or at any time between ranges defined by any two of the above values.

[0227] Clause 18. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the crosslinked macromolecular matrix has minimal degradation at about 6 months, about 12 months, about 18 months, about 24 months, about 30 months or about 36 months or at any amount of time within the ranges defined by any two of the above values.

[0228] Clause 19. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the elastic modulus (G') of the matrix is from about 30 Pa to about 10,000 Pa, or any elastic modulus between ranges defined by any two of the above values.

[0229] Crosslinked macromolecular matrix of any one of the above or following clauses, wherein the elastic modulus (G') of 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 2400 Pa, about 2500 Pa, about 2600 Pa, about 2700 Pa, about 2800 Pa, about 2900 Pa, about 3000 Pa, about 3100 Pa, about 3200 Pa, about 3300 Pa, about 3400 Pa, about 3500 Pa, about 3600 Pa, about 3700 Pa, about 3800 Pa, about 3900 Pa, about 4000 Pa, about 4100 Pa, about 4200 Pa, about 4300 Pa, about 4400 Pa, about 4500 Pa, about 4600 Pa, about 4700 Pa, about 4800 Pa, about 4900 Pa, about 5000 Pa, about 5100 Pa, about 5200 Pa, about 5300 Pa, about 5400 Pa, about 5500 Pa, about 5600 Pa, about 5700 Pa, about 5800 Pa, about 5900 Pa, about 6000 Pa, about 6100 Pa, about 6200 Pa, about 6300 Pa, about 6400 Pa, about 6500 Pa, about 6600 Pa, about 6700 Pa, about 6800 Pa, about 6900 Pa, about 7000 Pa, about 7100 Pa, about 7200 Pa, about 7300 Pa, about 7400 Pa, about 7500 Pa, about 7600 Pa, about 7700 Pa, 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 10,000 Pa or any elastic modulus within the range defined by any two of the above values.

[0230] Clause 21. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the compression force value of 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 180 gmf, about 190 gmf, about 200 gmf, about 210 gmf, about 220 gmf, about 230 gmf, about 240 gmf, about 250 gmf, about 260 gmf, about 270 gmf, about 280 gmf, about 290 gmf, about 300 gmf, about 310 gmf, about 320 gmf, about 330 gmf, about 340 gmf, about 350 gmf, about 360 gmf, about 370 gmf, about 380 gmf, about 390 gmf, about 400 gmf, about 410 gmf, about 420 gmf, about 430 gmf, about 440 gmf, about 450 gmf, about 460 gmf, about 470 gmf, about 480 gmf, about 490 gmf, about 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 compression force value between the ranges defined by any two of the above values.

[0231] Clause 22. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the compression force value of the matrix is about 100 gmf, about 200 gmf, about 300 gmf, about 400 gmf, about 500 gmf or about 600 gmf, or any compression force value between the ranges defined by any two of the above values.

[0232] Clause 23. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the concentration of hyaluronic acid is 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 above values.

[0233] Clause 24. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the collagen includes type I collagen.

[0234] Clause 25. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the collagen comprises type II collagen.

[0235] Clause 26. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the collagen comprises type III collagen.

[0236] Clause 27. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the collagen comprises 0% to 3% of type II collagen.

[0237] Clause 28. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the collagen comprises 1% to 3% of type I collagen.

[0238] Clause 29. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the matrix comprises approximately 0% to approximately 3% of type III collagen.

[0239] Clause 30. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the collagen comprises approximately 97% to approximately 99% of type I collagen.

[0240] Clause 31. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the collagen comprises a mixture of type I and type III collagen.

[0241] Clause 32. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the concentration of the collagen is approximately 1 mg / mL, approximately 2 mg / mL, approximately 4 mg / mL, approximately 6 mg / mL, approximately 8 mg / mL, approximately 10 mg / mL, approximately 12 mg / mL, approximately 14 mg / mL, or any concentration between the ranges defined by any two of the above values.

[0242] Clause 33. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the crosslinked macromolecular matrix further comprises salt.

[0243] Clause 34. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the crosslinked macromolecular matrix comprises NaCl in the range of approximately 50 mM to approximately 400 mM.

[0244] Clause 35. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the crosslinked macromolecular matrix comprises NaCl, and the concentration of NaCl is approximately 50 mM, approximately 75 mM, approximately 100 mM, approximately 125 mM, approximately 150 mM, approximately 175 mM, approximately 200 mM, approximately 225 mM, approximately 250 mM, approximately 275 mM, approximately 300 mM, approximately 325 mM, approximately 350 mM, approximately 375 mM, or approximately 400 mM, or any concentration between the ranges defined by any two of the above values.

[0245] Clause 36. A crosslinked macromolecular matrix according to any one of the foregoing or following clauses, wherein the crosslinked macromolecular matrix comprises NaCl, and wherein the concentration of NaCl is about 150 mM.

[0246] Clause 37. A crosslinked macromolecular matrix according to any one of the foregoing or following clauses, wherein the crosslinked macromolecular matrix comprises about 0.01 M phosphate buffer, about 137 mM NaCl, and KCl at a concentration of about 2.7 mM.

[0247] Clause 38. A crosslinked macromolecular matrix according to any one of the foregoing or following clauses, wherein the crosslinked macromolecular matrix is formulated for injection or for use with a needle and / or cannula.

[0248] Clause 39. A crosslinked macromolecular matrix according to any one of the foregoing or following clauses, wherein the hyaluronic acid component has an average molecular weight of from about 20,000 daltons to about 10,000,000 daltons.

[0249] Crosslinked macromolecular matrix of any one of the foregoing or following clauses, wherein the average molecular weight of the hyaluronic acid component is 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, about 1,500,000 Daltons, about 1,600,000 Daltons, about 1,700,000 Daltons, about 1,800,000 Daltons, about 1,900,000 Daltons, about 2,000,000 Daltons, about 2,100,000 Daltons, about 2,200,000 Daltons, about 2,300,000 Daltons, about 2,400,000 Daltons, about 2,500,000 Daltons, about 2,600,000 Daltons, about 2,700,000 Daltons, about 2,800,000 Daltons, about 2,900,000 Daltons, about 3,000,000 Daltons, about 3,100,000 Daltons, about 3,200,000 Daltons, about 3,300,000 Daltons, about 3,400,000 Daltons, about 3,500,000 Daltons, about 3,600,000 Daltons, about 3,700,000 Daltons, about 3,800,000 Daltons, about 3,900,000 Daltons, about 4,000,000 Daltons, about 4,100,000 Daltons, about 4,200,000 Daltons, about 4,300,000 Daltons, about 4,400,000 Daltons, about 4,500,000 Daltons, about 4,600,000 Daltons, about 4,700,000 Daltons, about 4,800,000 Daltons, about 4,900,000 Daltons, about 5,000,000 Daltons, about 5,100,000 Daltons, about 5,200,000 Daltons, about 5,300,000 Daltons, about 5,400,000 Daltons, about 5,500,000 Daltons, about 5,600,000 Daltons, about 5,700,000 Daltons, about 5,800,000 Daltons, about 5,900,000 Daltons, about 6,000,000 Daltons, about 6,100,000 Daltons, about 6,200,000 Daltons, about 6,300,000 Daltons, about 6,400,000 Daltons, about 6,500,000 Daltons, about 6,600,000 Daltons, about 6,700,000 Daltons, 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 8,500,000 Daltons, approximately 8,600,000 Daltons, approximately 8,700,000 Daltons, approximately 8,800,000 Daltons, approximately 8,900,000 Daltons, approximately 9,000,000 Daltons, approximately 9,100,000 Daltons, approximately 9,200,000 Daltons, approximately 9,300,000 Daltons, approximately 9,400,000 Daltons, approximately 9,500,000 Daltons, approximately 9,600,000 Daltons, approximately 9,700,000 Daltons, approximately 9,800,000 Daltons, approximately 9,900,000 Daltons or approximately 10,000,000 Daltons or any molecular weight within the range defined by any two of the above values.,

[0250] Clause 41. A crosslinked macromolecular matrix of any one of the above or below clauses, wherein the hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, and the mixture comprises hyaluronic acid having an average molecular weight of about 20,000, 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 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 the range between any two of the above values.

[0251] Clause 42. A composition comprising: hyaluronic acid; collagen; lysine; and a buffer; wherein the composition is an aqueous hydrogel.

[0252] Clause 43. A composition of any one of the above or below clauses, wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amino group on the collagen and / or at least one amino group present on the lysine.

[0253] Clause 44. A composition of any one of the above or below clauses, wherein the composition further comprises lidocaine.

[0254] Clause 45. A composition of any one of the above or below clauses, wherein the concentration of lidocaine in the matrix is between about 0.15% (w / w) and about 0.45% (w / w).

[0255] Clause 46. A composition of any one of the foregoing or following clauses, wherein the concentration of lidocaine is about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w) of the composition or any concentration between the ranges defined by any two of the foregoing values.

[0256] Clause 47. A composition of any one of the foregoing or following clauses, wherein the composition further comprises uncrosslinked HA.

[0257] Clause 48. A composition of any one of the foregoing or following clauses, wherein the concentration of uncrosslinked-crosslinked HA in the composition is up to about 5% (w / w).

[0258] Clause 49. A composition of any one of the foregoing or following clauses, wherein the concentration of uncrosslinked HA is about 0% (w / w), about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w) of the composition or any concentration between the ranges defined by any two of the foregoing values.

[0259] Clause 50. A composition of any one of the foregoing or following clauses, wherein the concentration of uncrosslinked HA in the composition is about 1% (w / w).

[0260] Clause 51. A composition of any one of the foregoing or following clauses, wherein the concentration of uncrosslinked HA in the composition is about 2% (w / w).

[0261] Clause 52. A composition of any one of the foregoing or following clauses, wherein the concentration of uncrosslinked HA in the composition is about 5% (w / w).

[0262] Clause 53. A composition of any one of the foregoing or following clauses, wherein the uncrosslinked HA improves the extrudability of the composition.

[0263] Clause 54. A composition of any one of the foregoing or following clauses, wherein the buffer is phosphate buffered saline.

[0264] Clause 55. A composition of any one of the foregoing or following clauses, wherein the average molecular weight of hyaluronic acid is from about 20,000 daltons to about 10,000,000 daltons.

[0265] Clause 56. A composition according to any one of the above or following clauses, wherein the hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, and the mixture comprises hyaluronic acid having the following molecular weights: 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 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 the range between any two of the above values.

[0266] Clause 57. A composition according to any one of the above or following clauses, wherein the collagen comprises type I collagen.

[0267] Clause 58. A composition according to any one of the above or following clauses, wherein the collagen comprises type II collagen.

[0268] Clause 59. A composition according to any one of the above or following clauses, wherein the collagen comprises type III collagen.

[0269] Clause 60. A composition according to any one of the foregoing or following clauses, wherein the viscosity of the composition is: 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, about 6900 Pa S, about 7000 Pa S, about 7100 Pa S, about 7200 Pa S, about 7300 Pa S, about 7400 Pa S, about 7500 Pa S, about 7600 Pa S, about 7700 Pa S, about 7800 Pa S, about 7900 Pa S, about 8000 Pa S, about 8100 Pa S, about 8200 Pa S, about 8300 Pa S, about 8400 Pa S, about 8500 Pa S, about 8600 Pa S, about 8700 Pa S, about 8800 Pa S, about 8900 Pa S, about 9000 Pa S, about 9100 Pa, about 9200 Pa S, about 9300 Pa S, about 9400 Pa S, about 9500 Pa S, about 9600 Pa S, about 9700 Pa S, about 9800 Pa S, about 9900 Pa S, or about 10,000 Pa S or any viscosity within the range defined by any two of the foregoing values.

[0270] Clause 61. A composition according to any one of the foregoing or following clauses, wherein the parameter of the tangent of the dielectric loss angle (G" / G') of the composition is from about 0.01 to about 0.5.

[0271] Clause 62. A composition according to any one of the foregoing or following clauses, wherein the parameter of the tangent of the dielectric loss angle (G" / G') of the composition is 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 parameter of the tangent of the dielectric loss angle between the ranges defined by any two of the foregoing values.

[0272] Clause 63. A composition according to any one of the above or following clauses, wherein the composition is stable for about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or for any amount of time between ranges defined by any two of the above values.

[0273] Clause 64. A composition according to any one of the above or following clauses, wherein the composition is stable at about 4 °C.

[0274] Clause 65. A composition according to any one of the above or following clauses, wherein the composition is stable at about 25 °C.

[0275] Clause 66. A composition according to any one of the above or following clauses, wherein the composition has minimal degradation at about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, or about 36 months, or for any amount of time within a range defined by any two of the above values.

[0276] Clause 67. A method of 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 pH of the pre-reaction aqueous solution is between about 4 and about 6; and preparing a second solution comprising: a water-soluble carbodiimide; and N-hydroxysuccinimide or N-hydroxy sulfosuccinimide; and adding the second solution to the pre-reaction aqueous solution to form a crosslinking reaction mixture; and reacting the crosslinking reaction mixture by crosslinking hyaluronic acid and collagen with lysine; wherein hyaluronic acid is crosslinked to collagen through at least one endogenous amino group on the collagen and / or through at least one amino group present on lysine; and wherein the degradation of HA and collagen is minimal and the structures of HA and collagen are intact, thereby forming a crosslinked macromolecular matrix.

[0277] Clause 68. A method according to any one of the above or following clauses, wherein the pH of the pre-reaction aqueous solution is about 4.0, about 4.5, about 5.0, about 5.5, or about 6.0, or any pH between ranges defined by any two of the above values.

[0278] Clause 69. A method according to any one of the above or following clauses, wherein the method further comprises adding lidocaine to the crosslinked macromolecular matrix.

[0279] Clause 70. A method according to any one of the above or following clauses, wherein the concentration of lidocaine added to the crosslinked macromolecular matrix is in the range of about 0.15% (w / w) to about 0.45% (w / w).

[0280] Clause 71. The method of any one of the above or following clauses, wherein the concentration in the lidocaine matrix is about 0.15% (w / w), about 0.17% (w / w), about 0.19% (w / w), about 0.21% (w / w), about 0.23% (w / w), about 0.25% (w / w), about 0.27% (w / w), about 0.29% (w / w), about 0.31% (w / w), about 0.33% (w / w), about 0.35% (w / w), about 0.37% (w / w), about 0.39% (w / w), about 0.41% (w / w), about 0.43% (w / w) or about 0.45% (w / w), or any concentration between the ranges defined by any two of the above values.

[0281] Clause 72. The method of any one of the above or following clauses, wherein the method further comprises providing an activator, and the activator comprises triazole, fluorinated phenol, succinimide or sulfosuccinimide.

[0282] Clause 73. The method of any one of the above or following clauses, wherein the method is carried out at a temperature of about 2°C, about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, about 20°C, about 22°C, about 24°C, about 26°C, about 28°C, about 30°C, about 32°C, about 34°C, or about 36°C or at a temperature between the ranges defined by any two of the above values.

[0283] Clause 74. The method of any one of the above or following clauses, wherein the reaction step is carried out between about 4°C and about 35°C.

[0284] Clause 75. The method of any one of the above or following clauses, wherein the reaction step is carried out at about 4°C or about 22°C.

[0285] Clause 76. The method of any one of the above or following clauses, wherein the method further comprises purifying the cross-linked macromolecular matrix, and dialysis is used for the purification step.

[0286] Clause 77. The method of any one of the above or following clauses, wherein the purification step is carried out between 2°C and 30°C.

[0287] Clause 78. The method of any one of the above or following clauses, wherein 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, about 30°C or at any temperature between the ranges defined by any two of the above values.

[0288] Clause 79. A method according to any one of the preceding or following clauses, wherein the purification step is carried out at about 2°C to about 8°C.

[0289] Clause 80. A method according to any one of the preceding or following clauses, wherein the cross-linking reaction is carried out at about 2°C to about 35°C.

[0290] Clause 81. A method according to any one of the preceding or following clauses, wherein the cross-linking reaction is carried out at about 2°C to about 8°C.

[0291] Clause 82. A method according to any one of the preceding or following clauses, wherein the method is carried out at a temperature below room temperature.

[0292] Clause 83. A method according to any one of the preceding or following clauses, wherein the pH of the cross-linking reaction mixture is between about 4.0 and about 6.0.

[0293] Clause 84. A method according to any one of the preceding or following clauses, wherein the solution before the reaction contains salt, and the concentration of sodium chloride in the salt contained in the cross-linking reaction mixture is about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, 325 mM, about 350 mM, about 375 mM, or about 400 mM, or any concentration within the range defined by any two of the above values.

[0294] Clause 85. A method according to any one of the preceding or following clauses, wherein the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the cross-linking reaction mixture is about 20 mM to about 200 mM.

[0295] Clause 86. A method according to any one of the preceding or following clauses, wherein the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is about 20 mM, about 40 mM, about 60 mM, about 80 mM, about 100 mM, about 120 mM, about 140 mM, about 160 mM, about 180 mM or about 200 mM or any concentration within the range defined by any two of the above values.

[0296] Clause 87. A method according to any one of the preceding or following clauses, wherein the molar ratio of the water-soluble carbodiimide to the hyaluronic acid in the water-soluble carbodiimide:hyaluronic acid repeating unit is about 0.5 to about 2.0.

[0297] Clause 88. A method according to any one of the preceding or following clauses, wherein the molar ratio of water-soluble carbodiimide to hyaluronic acid in the water-soluble carbodiimide and 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.

[0298] Clause 89. A method according to any one of the preceding or following clauses, wherein the molar:molar (lysine:HA repeat unit) ratio of lysine to hyaluronic acid is between about 0.01 and about 0.6.

[0299] Clause 90. A method according to any one of the preceding or following clauses, wherein the molar:molar (lysine:HA repeat unit) ratio of lysine to hyaluronic acid is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.2, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.3, 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.

[0300] Clause 91. A method according to any one of the preceding or following clauses, wherein the method further comprises adding uncrosslinked HA to the crosslinked macromolecular matrix.

[0301] Clause 92. A method according to any one of the preceding or following clauses, wherein the concentration of uncrosslinked HA added to the crosslinked macromolecular matrix is up to 5% w / w.

[0302] Clause 93. A method according to any one of the preceding or following clauses, wherein the concentration of uncrosslinked HA added to the matrix is 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 any two of the above values.

[0303] Article 94. A method according to any one of the preceding or following articles, wherein the concentration of uncrosslinked HA added to the matrix is about 1% (w / w).

[0304] Article 95. A method according to any one of the preceding or following articles, wherein the concentration of uncrosslinked HA added to the matrix is about 3% (w / w).

[0305] Article 96. A method according to any one of the preceding or following articles, wherein the concentration of uncrosslinked HA added to the matrix is about 5% (w / w).

[0306] Article 97. A method according to any one of the preceding or following articles, wherein the method further comprises sterilizing the crosslinked macromolecular matrix, the method comprising: transferring the crosslinked macromolecular matrix to a container and performing steam sterilization; and sterilizing the hydrogel by steam sterilization.

[0307] Article 98. A method according to any one of the preceding or following articles, wherein the container is a syringe.

[0308] Article 99. A method according to any one of the preceding or following articles, wherein the method further comprises dialyzing the crosslinked macromolecular matrix, wherein the dialysis is performed through a membrane having a molecular weight cut-off of about 1,000 Daltons to about 100,000 Daltons, and wherein the dialysis is performed before sterilization.

[0309] Article 100. A method according to any one of the preceding or following articles, wherein the dialysis is performed in phosphate buffered saline.

[0310] Article 101. A method according to any one of the preceding or following articles, wherein the hyaluronic acid in the pre-reaction solution is hydrated for at least about 60 minutes before adding the second solution.

[0311] Article 102. A method according to any one of the preceding or following articles, wherein the reaction mixture is crosslinked for about 16 hours to about 24 hours.

[0312] Article 103. A crosslinked macromolecular matrix prepared by a method according to any one of the preceding or following methods.

[0313] Article 104. A method for improving the aesthetic quality of human anatomical features, the method comprising: injecting a composition into human tissue so as to improve the aesthetic quality of the anatomical features; wherein the composition comprises a crosslinked macromolecular matrix, the matrix comprising: hyaluronic acid; lysine; and collagen; wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amino group on the collagen and / or through at least one amino group present on the lysine.

[0314] Article 105. A method according to any one of the preceding or following articles, wherein the crosslinked macromolecular matrix further comprises lidocaine.

[0315] Method of any one of the foregoing or following clauses, wherein the crosslinked macromolecular matrix further comprises uncrosslinked HA.

[0316] The method of any one of the above or following clauses, wherein 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, about 1,500,000 Daltons, about 1,600,000 Daltons, about 1,700,000 Daltons, about 1,800,000 Daltons, about 1,900,000 Daltons, about 2,000,000 Daltons, about 2,100,000 Daltons, about 2,200,000 Daltons, about 2,300,000 Daltons, about 2,400,000 Daltons, about 2,500,000 Daltons, about 2,600,000 Daltons, about 2,700,000 Daltons, about 2,800,000 Daltons, about 2,900,000 Daltons, about 3,000,000 Daltons, about 3,100,000 Daltons, about 3,200,000 Daltons, about 3,300,000 Daltons, about 3,400,000 Daltons, about 3,500,000 Daltons, about 3,600,000 Daltons, about 3,700,000 Daltons, about 3,800,000 Daltons, about 3,900,000 Daltons, about 4,000,000 Daltons, about 4,100,000 Daltons, about 4,200,000 Daltons, about 4,300,000 Daltons, about 4,400,000 Daltons, about 4,500,000 Daltons, about 4,600,000 Daltons, about 4,700,000 Daltons, about 4,800,000 Daltons, about 4,900,000 Daltons, about 5,000,000 Daltons, about 5,100,000 Daltons, about 5,200,000 Daltons, about 5,300,000 Daltons, about 5,400,000 Daltons, about 5,500,000 Daltons, about 5,600,000 Daltons, about 5,700,000 Daltons, about 5,800,000 Daltons, about 5,900,000 Daltons, about 6,000,000 Daltons, about 6,100,000 Daltons, about 6,200,000 Daltons, about 6,300,000 Daltons, about 6,400,000 Daltons, about 6,500,000 Daltons, about 6,600,000 Daltons, about 6,700,000 Daltons, 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 8,500,000 Daltons, approximately 8,600,000 Daltons, approximately 8,700,000 Daltons, approximately 8,800,000 Daltons, approximately 8,900,000 Daltons, approximately 9,000,000 Daltons, approximately 9,100,000 Daltons, approximately 9,200,000 Daltons, approximately 9,300,000 Daltons, approximately 9,400,000 Daltons, approximately 9,500,000 Daltons, approximately 9,600,000 Daltons, approximately 9,700,000 Daltons, approximately 9,800,000 Daltons, approximately 9,900,000 Daltons or approximately 10,000,000 Daltons or any molecular weight between the ranges defined by any two of the above values.,

[0317] Clause 108. A method as in any of the foregoing or following clauses, wherein the hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, and the mixture comprises hyaluronic acid having 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 1,500,000 daltons, about 2,000,000 daltons, 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 1,000,000 daltons and / or any hyaluronic acid having a molecular weight within the range between any two of the foregoing values.

[0318] Clause 109. A method as in any of the foregoing or following clauses, wherein the collagen comprises type I collagen and / or type III collagen.

[0319] Clause 110. A method of improving the appearance of an individual, the method comprising: injecting a composition into the tissue of the individual at an injection site so as to improve the aesthetic quality of an anatomical feature, wherein infiltrating cells from the tissue integrate into the composition at the injection site and deposit new collagen in the composition; wherein the composition comprises a crosslinked macromolecular matrix, the crosslinked macromolecular matrix comprising: hyaluronic acid; lysine; and collagen; wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amino group on the collagen and / or through at least one amino group present on the lysine; and wherein the tissue injected with the composition shows tissue integration, collagen deposition and angiogenesis.

[0320] Clause 111. A method as in any of the foregoing or following clauses, wherein the composition further comprises lidocaine.

[0321] Clause 112. A method as in any of the foregoing or following clauses, wherein the composition further comprises uncrosslinked HA.

[0322] Article 113. A method according to any one of the foregoing or following articles, wherein a composition is injected into the chin, jawline, lips, or nasolabial fold.

[0323] Article 114. A method according to any one of the foregoing or following articles, wherein the method improves the symmetry between facial features.

[0324] Article 115. A method according to any one of the foregoing or following articles, wherein the method enhances and restores the volume of facial features.

[0325] Article 116. A method according to any one of the foregoing or following articles, wherein the method increases, corrects, restores, or creates volume in the chin, lips, jawline, or nasolabial fold.

[0326] Article 117. A method according to any one of the foregoing or following articles, wherein a composition is injected into the tear trough of an individual.

[0327] Article 118. A method according to any one of the foregoing or following articles, wherein a composition is injected into an area including skin atrophy and / or atrophy of the fat pad.

[0328] Article 119. A method according to any one of the foregoing or following articles, wherein the method provides a natural appearance, feel, and movement in the tissue receiving the injection, wherein the composition causes increased collagen infiltration from the tissue surrounding the injection site.

[0329] Article 120. A method according to any one of the foregoing or following articles, wherein the duration of the composition is increased due to tissue integration into the injection site.

[0330] Article 121. A method according to any one of the foregoing or following articles, wherein the method improves the hydration and elasticity of the skin around the injection site.

[0331] Article 122. A method of increasing collagen infiltration into tissue, the method comprising:

[0332] Injecting a composition into the tissue of an individual to create a skin filler reservoir containing the composition, wherein the composition comprises a crosslinked macromolecular matrix comprising: hyaluronic acid; lysine; and collagen; wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amino group on the collagen and / or through at least one amino group present on the lysine; and wherein cells from the tissue surrounding the skin filler reservoir infiltrate the skin filler reservoir containing the composition, wherein the cells integrate into the composition and deposit new collagen into the composition, thereby creating infiltrated tissue in the composition and wherein blood vessels connect the infiltrated tissue in the composition to the blood supply of the individual's body.

[0333] Clause 123. The method of any one of the above or following clauses, wherein the matrix further comprises lidocaine.

[0334] Clause 124. The method of any one of the above or following clauses, wherein the composition further comprises uncrosslinked HA.

[0335] The method of any one of the above or following clauses, wherein the average molecular weight of hyaluronic acid is: 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, about 1,500,000 Daltons, about 1,600,000 Daltons, about 1,700,000 Daltons, about 1,800,000 Daltons, about 1,900,000 Daltons, about 2,000,000 Daltons, about 2,100,000 Daltons, about 2,200,000 Daltons, about 2,300,000 Daltons, about 2,400,000 Daltons, about 2,500,000 Daltons, about 2,600,000 Daltons, about 2,700,000 Daltons, about 2,800,000 Daltons, about 2,900,000 Daltons, about 3,000,000 Daltons, about 3,100,000 Daltons, about 3,200,000 Daltons, about 3,300,000 Daltons, about 3,400,000 Daltons, about 3,500,000 Daltons, about 3,600,000 Daltons, about 3,700,000 Daltons, about 3,800,000 Daltons, about 3,900,000 Daltons, about 4,000,000 Daltons, about 4,100,000 Daltons, about 4,200,000 Daltons, about 4,300,000 Daltons, about 4,400,000 Daltons, about 4,500,000 Daltons, about 4,600,000 Daltons, about 4,700,000 Daltons, about 4,800,000 Daltons, about 4,900,000 Daltons, about 5,000,000 Daltons, about 5,100,000 Daltons, about 5,200,000 Daltons, about 5,300,000 Daltons, about 5,400,000 Daltons, about 5,500,000 Daltons, about 5,600,000 Daltons, about 5,700,000 Daltons, about 5,800,000 Daltons, about 5,900,000 Daltons, about 6,000,000 Daltons, about 6,100,000 Daltons, about 6,200,000 Daltons, about 6,300,000 Daltons, about 6,400,000 Daltons, about 6,500,000 Daltons, about 6,600,000 Daltons, about 6,700,000 Daltons, about 6,800,000 Daltons, about 6,900,000 Daltons, about 7,000,000 Daltons, about 7,100,000 Daltons, about 7,200,000 Daltons, about 7,300,000 Daltons, about 7,400,000 Daltons, about 7,500,000 Daltons, about 7,600,000 Daltons, about 7,700,000 Daltons, about 7,800,000 Daltons, about 7,900,000 Daltons, about 8,000,000 Daltons, about 8,100,000 Daltons, about 8,200,000 Daltons, about 8,300,000 Daltons, about 8,400,000 Daltons, about 8,500,000 Daltons, about 8,600,000 Daltons, about 8,700,000 Daltons, about 8,800,000 Daltons, about 8,900,000 Daltons, about 9,000,000 Daltons, about 9,100,000 Daltons, about 9,200,000 Daltons, about 9,300,000 Daltons, about 9,400,000 Daltons, about 9,500,000 Daltons, about 9,600,000 Daltons, about 9,700,000 Daltons, about 9,800,000 Daltons, about 9,900,000 Daltons or about 10,000,000 Daltons, or any other molecular weight within the range defined by any two of the above values.,

[0336] Method of any one of the above or following clauses, wherein the hyaluronic acid comprises a mixture of hyaluronic acid components having different molecular weights, and the mixture comprises hyaluronic acid having 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 1,500,000 Daltons, about 2,000,000 Daltons, 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 with a molecular weight within the range between any two of the above values.

[0337] Method of any one of the above or following clauses, wherein the collagen comprises type I collagen, type II collagen and / or type III collagen.

[0338] Method of any one of the above or following clauses, wherein the composition comprises about 13 mg / mL of hyaluronic acid.

[0339] Method of any one of the above or following clauses, wherein the composition comprises about 20 mg / mL of hyaluronic acid, about 22 mg / mL of hyaluronic acid, about 24 mg / mL, about 26 mg / mL of hyaluronic acid, about 28 mg / mL of hyaluronic acid or about 30 mg / mL of hyaluronic acid.

[0340] Method of any one of the above or following clauses, wherein the product is injected into the superficial dermis to improve skin quality, fine lines or roughness.

[0341] In some embodiments, any clause herein may depend on any one independent clause or any one dependent clause. In one aspect, any clause (e.g., a dependent or independent clause) may be combined with any other one or more clauses (e.g., a dependent or independent clause). In one aspect, a claim may include some or all of the words (e.g., steps, operations, methods, or components) recited in a clause, sentence, phrase, or paragraph. In one aspect, a claim may include some or all of the words enumerated in one or more clauses, sentences, phrases, or paragraphs. In one aspect, some words in each clause, sentence, phrase, or paragraph may be deleted. In one aspect, additional words or elements may be added to a clause, sentence, phrase, or paragraph. In one aspect, the subject technology may be implemented without utilizing some of the components, elements, functions, or operations described herein. In one aspect, additional components, elements, functions, or operations may be utilized to implement the subject technology.

[0342] The terms “a,” “an,” “the,” and similar references used in the context of describing the present invention (especially in the context of the following claims) should be construed to cover both the singular and the plural unless otherwise specified herein or clearly contradicted by the context. The recitation of numerical ranges herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise specified herein, each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise specified herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention being claimed. Any language in the specification should not be construed as indicating that any non-claimed element is essential for the practice of the invention.

[0343] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as a limitation. Each member of the group may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. For convenience and / or patentability reasons, it is contemplated that one or more members of the group may be included in or deleted from the group. When any such inclusion or deletion occurs, the specification is deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0344] This document describes certain embodiments of the invention, including the best mode known to the inventors for practicing the invention. Of course, variations of the described embodiments will become apparent to those of ordinary skill in the art after reading the above description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the invention includes all modifications and equivalent means of the subject matter recited in the appended claims as permitted by applicable law. In addition, unless otherwise stated herein or clearly contradicted by context, the invention includes any combination of all possible variations of the above-described elements.

[0345] The specific embodiments disclosed herein may be further limited by the language “consisting of” or “consisting essentially of” used in the claims. When used in the claims, whether submitted or added by amendment, the transitional term “consisting of” excludes any element, step, or ingredient not specified in the claim. 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 inherently or explicitly described and enabled herein.

[0346] In addition, numerous patents and printed publications are referenced throughout the specification. Each of the above-referenced references and printed publications is hereby incorporated by reference in its entirety.

[0347] Finally, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Accordingly, by way of example and not limitation, alternative configurations of the invention may be used in accordance with the teachings herein. Therefore, the invention is not limited to that shown and described herein.

Claims

1. A cross-linked macromolecular matrix, comprising: lysine; hyaluronic acid; and collagen; wherein the hyaluronic acid is cross-linked to the collagen through at least one endogenous amino group on the collagen and through at least one amino group present on the lysine; and wherein the cross-linked macromolecular matrix does not contain chitosan.

2. The cross-linked macromolecular matrix according to claim 1, wherein the cross-linked macromolecular matrix further comprises lidocaine.

3. The cross-linked macromolecular matrix according to claim 1 or 2, wherein the matrix further comprises uncross-linked hyaluronic acid, and the concentration of the uncross-linked hyaluronic acid in the matrix is up to 5% (w / w).

4. The cross-linked macromolecular matrix according to any one of claims 1 to 3, wherein the elastic modulus (G') of the matrix is 30 Pa to 10,000 Pa.

5. The cross-linked macromolecular matrix according to any one of claims 1 to 4, wherein the compression force value of the matrix is 100 gmf to 600 gmf.

6. The cross-linked macromolecular matrix according to any one of claims 1 to 5, wherein the parameter of the loss tangent of the medium of the matrix (G" / G') is 0.01 to 0.

5.

7. The cross-linked macromolecular matrix according to any one of claims 1 to 6, wherein the concentration of the hyaluronic acid is 5 mg / mL to 36 mg / mL.

8. The cross-linked macromolecular matrix according to claim 7, wherein the concentration of the hyaluronic acid is 16 mg / mL to 22 mg / mL.

9. The cross-linked macromolecular matrix according to any one of claims 1 to 8, wherein the collagen comprises one or more of type I collagen, type II collagen, and type III collagen.

10. The cross-linked macromolecular matrix according to claim 9, wherein the collagen comprises 97% to 99% of type I collagen.

11. The cross-linked macromolecular matrix according to any one of claims 1 to 10, wherein the concentration of the collagen is 1 mg / mL to 14 mg / mL.

12. The cross-linked macromolecular matrix according to claim 11, wherein the concentration of the collagen is 2 mg / mL to 6 mg / mL.

13. The cross-linked macromolecular matrix according to any one of claims 1 to 12, wherein the molar:molar ratio of the lysine and the hyaluronic acid is between 0.01 and 0.

6.

14. A method for preparing the cross-linked macromolecular matrix according to any one of claims 1 to 13, the method comprising: (i) dissolving collagen, hyaluronic acid, and lysine in an aqueous solution to form a pre-reaction aqueous solution, wherein the pH of the pre-reaction aqueous solution is between 4 and 6; (ii) preparing a second solution, comprising: water-soluble carbodiimide; and N-hydroxysuccinimide or N-hydroxy sulfosuccinimide; (iii) adding the second solution to the pre-reaction aqueous solution to form a cross-linking reaction mixture; and (iv) reacting the cross-linking reaction mixture to produce a cross-linked macromolecular matrix.

15. A method for cross-linking hyaluronic acid and collagen, comprising: Dissolve collagen, hyaluronic acid and lysine in an aqueous solution to form a pre-reaction aqueous solution, wherein the pH of the pre-reaction aqueous solution is between 4 and 6; and Prepare a second solution, comprising: a water-soluble carbodiimide; and N-hydroxysuccinimide or N-hydroxy sulfosuccinimide; and Add the second solution to the pre-reaction aqueous solution to form a crosslinking reaction mixture; and React the crosslinking reaction mixture by crosslinking the hyaluronic acid and the collagen with the lysine; wherein the hyaluronic acid is crosslinked to the collagen through at least one endogenous amino group on the collagen and through at least one amino group present on the lysine, thereby forming a crosslinked macromolecular matrix; and wherein chitosan is not used in the method.

16. The method according to claim 14 or 15, wherein the pH of the pre-reaction aqueous solution is 4.0 to 5.

5.

17. The method according to claim 14 or 15, wherein the method is carried out at a temperature of 2°C to 36°C.

18. The method according to claim 17, wherein the method is carried out at a temperature of 2°C to 8°C.

19. The method according to claim 14 or 15, wherein the method further comprises providing an activator, the activator comprising triazole, fluorinated phenol, succinimide or sulfosuccinimide.

20. The method according to claim 14 or 15, wherein the water-soluble carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the crosslinking reaction mixture is 20 mM to 200 mM.

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