Biological mixtures containing biological cross-linking materials, their uses and preparation methods

Abstract

This invention provides a biological mixture comprising a biological cross-linking material, wherein the biological cross-linking material comprises collagen, collagen peptides, a first hyaluronic acid, and a second hyaluronic acid; wherein the collagen, the collagen peptides, the first hyaluronic acid, and the second hyaluronic acid are cross-linked with each other. The biological cross-linking material has a high degree of cross-linking, a low collagen degradation rate, and a low hyaluronic acid degradation rate, which can increase its residence time in the human body. This invention also provides the uses and methods for preparing the biological mixture.

Description

[Technical Field]

[0001] This invention relates to a biological mixture, particularly a biological mixture comprising a biological crosslinking material. The invention also relates to the use and preparation method of said biological mixture. [Previous Technology]

[0002] Medical aesthetics has gradually emerged with frequent social activities and includes facial care, body shaping and hair care; among them, the face is the primary aspect of an individual's appearance, so how to effectively maintain facial youth and health is the focus of medical aesthetics.

[0003] Currently, there are various medical aesthetic products on the market, such as hyaluronic acid injections and collagen injections. These injections are filler-type injections, used to adjust or reshape the skin's appearance by subcutaneous injection when superficial skin shows depressions, wrinkles, or sagging due to aging. However, hyaluronic acid and collagen are easily broken down by the body after injection, requiring regular injections. Therefore, how to effectively extend the time these materials remain in the body is an important issue. [Summary of the Invention]

[0004] To solve the above problems, the present invention provides a biological mixture comprising a biological crosslinking material, wherein the biological crosslinking material comprises collagen, collagen peptides, a first hyaluronic acid, and a second hyaluronic acid; wherein the molecular weight of the first hyaluronic acid is from 80 kilodaltons (kDa) to 500 kilodaltons, and the molecular weight of the second hyaluronic acid is from 1000 kilodaltons to 3000 kilodaltons; and the collagen, the collagen peptides, the first hyaluronic acid, and the second hyaluronic acid are crosslinked with each other, and the average degree of crosslinking of the biological crosslinking material is from 65% to 97%.

[0005] According to the present invention, the bio-crosslinked material or bio-crosslinked material product has excellent structural strength, namely, high crosslinking degree, low collagen degradation rate and low hyaluronic acid degradation rate, so as to increase the residence time of the bio-crosslinked material containing hyaluronic acid and collagen in the human body, thereby effectively reducing the frequency of personal supplementation of hyaluronic acid and collagen.

[0006] In one embodiment, the biological mixture comprises water. The biological mixture can be lyophilized to obtain the biological crosslinking material. The biological crosslinking material of the present invention can be made into a lyophilized powder for long-term storage and can be rehydrated before use, for example, by adding water or a pharmaceutically acceptable carrier, such as phosphate-buffered saline (PBS).

[0007] It should be specifically noted that the structure of the collagen is mainly composed of the amino acid sequence of glycine-hydroxylysine-hydroxyproline, but is not limited thereto. Furthermore, the collagen polypeptide is also rich in glycine, hydroxylysine, and hydroxyproline, but is not limited thereto. Since collagen, collagen polypeptide, first hyaluronic acid, and second hyaluronic acid are not single components or compounds of a single molecular weight, the biological mixture comprises the biological cross-linked material formed by cross-linking the above four types of components in multiple bonding modes.

[0008] In one embodiment, the collagen contains a repeating sequence of glycine-hydroxylysine-hydroxyproline.

[0009] In one embodiment, the collagen comprises type I collagen.

[0010] In one embodiment, the collagen polypeptide comprises any one or a combination of glycine, hydroxylysine, and hydroxyproline.

[0011] In one embodiment, the collagen polypeptide is a product obtained by digesting it with pepsin. Preferably, the collagen polypeptide is a product obtained by digesting type I collagen with pepsin.

[0012] Preferably, the molecular weight of the first hyaluronic acid is from 85 kilodaltons to 450 kilodaltons, for example: 85 kilodaltons, 100 kilodaltons, 150 kilodaltons, 200 kilodaltons, 250 kilodaltons, 300 kilodaltons, 350 kilodaltons, 400 kilodaltons, or 450 kilodaltons. More preferably, the molecular weight of the first hyaluronic acid is from 90 kilodaltons to 110 kilodaltons.

[0013] Preferably, the molecular weight of the second hyaluronic acid is from 1100 kilodaltons to 2900 kilodaltons, for example: 1100 kilodaltons, 1400 kilodaltons, 1700 kilodaltons, 2000 kilodaltons, 2300 kilodaltons, 2600 kilodaltons, or 2900 kilodaltons. More preferably, the molecular weight of the first hyaluronic acid is from 1350 kilodaltons to 1650 kilodaltons.

[0014] Preferably, the average degree of crosslinking of the bio-crosslinked material is 66% to 96%, for example: 66%, 70%, 75%, 80%, 85%, 90%, 95% or 96%. Preferably, the average degree of crosslinking of the bio-crosslinked material is 72% to 78%.

[0015] In one embodiment, the average collagen degradation rate of the bio-crosslinked material is less than 20%. Preferably, the average collagen degradation rate of the bio-crosslinked material is less than 15%. More preferably, the average collagen degradation rate of the bio-crosslinked material is 3% to 10%.

[0016] In one embodiment, the average hyaluronic acid degradation rate of the bio-crosslinked material is less than 17%. Preferably, the average hyaluronic acid degradation rate of the bio-crosslinked material is less than 15%. More preferably, the average hyaluronic acid degradation rate of the bio-crosslinked material is 9% to 14%.

[0017] In one embodiment, based on the total volume of the bio-crosslinked material, the total content of collagen and collagen peptides is from 30 mg / ml to 80 mg / ml, for example: 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, or 80 mg / ml. Preferably, based on the total volume of the bio-crosslinked material, the total content of collagen and collagen peptides is from 35 mg / ml to 45 mg / ml.

[0018] In one embodiment, based on the total volume of the bio-crosslinked material, the total content of the first hyaluronic acid and the second hyaluronic acid is from 1 mg / ml to 18 mg / ml, for example: 1 mg / ml, 3 mg / ml, 6 mg / ml, 9 mg / ml, 12 mg / ml, 15 mg / ml, or 18 mg / ml. Preferably, based on the total volume of the bio-crosslinked material, the total content of the first hyaluronic acid and the second hyaluronic acid is from 4.5 mg / ml to 5.5 mg / ml.

[0019] In one embodiment, the biological mixture further comprises exosomes. Preferably, the exosomes are adipose-derived stem cell exosomes.

[0020] In one embodiment, based on the total volume of the biological mixture, the concentration of the exosomes is from 1×10⁵ to 1×10¹¹ per milliliter, for example: 1×10⁵ exosomes / mL, 5×10⁵ exosomes / mL, 1×10⁶ exosomes / mL, 5×10⁶ exosomes / mL, 1×10⁷ exosomes / mL, 5×10⁷ exosomes / mL, 1×10⁸ exosomes / mL, 5×10⁸ exosomes / mL, 1×10⁹ exosomes / mL, 5×10⁹ exosomes / mL, 1×10¹⁰ exosomes / mL, 5×10¹⁰ exosomes / mL, or 1×10¹¹ exosomes / mL. Preferably, based on the total volume of the biological mixture, the concentration of the exosomes is from 8×10⁶ exosomes / mL to 1.2×10⁷ exosomes / mL.

[0021] The present invention further provides the use of the biological mixture for preparing skin tissue filling products.

[0022] In one embodiment, the dosage form of the skin tissue filler product includes an injectable form. In one embodiment, the injectable form is a filler injectable for minimally invasive cosmetic procedures.

[0023] The skin tissue filling product of the present invention can provide the biological cross-linked material through subcutaneous injection.

[0024] Preferably, the skin tissue filling product is a skin tissue filling product for use on the face.

[0025] In one embodiment, the product comprises a pharmaceutical, cosmetic, or medical device. Preferably, the product comprises a cosmetic pharmaceutical, a medical cosmetic, or a cosmetic medical device.

[0026] The present invention further provides a method for preparing the biological mixture, comprising: a first crosslinking step: mixing collagen, the first hyaluronic acid, the first crosslinking agent and the first carrier to obtain a first mixture, and reacting for 20 hours to 28 hours to obtain a first crosslinking complex; wherein, based on the total volume of the first mixture, the content of collagen is 10% to 40% by weight (w / v%), and the content of the first hyaluronic acid is 0.1% to 9% by weight (w / v%). The second crosslinking step involves mixing the first crosslinking complex, the second hyaluronic acid, the second crosslinking agent, and the second carrier to obtain a second mixture. This mixture is then reacted for 20 to 28 hours to obtain a second crosslinking complex. The first crosslinking complex comprises 10 to 40% by weight (W / V) and the second hyaluronic acid comprises 0.1 to 9% by weight (W / V). The third crosslinking step involves mixing the second crosslinking complex, collagen peptides, the third crosslinking agent, and the third carrier to obtain a third mixture. This mixture is then reacted for 20 to 28 hours to obtain the bio-crosslinked material. The second crosslinking complex comprises 10 to 40% by weight (W / V) and the collagen peptide comprises 10 to 40% by weight (W / V) based on the total volume of the third mixture. The first, second, and third crosslinking agents are different crosslinking agents, and all three carriers contain water.

[0027] Preferably, any one or a combination of the first carrier, the second carrier and the third carrier comprises phosphate buffered saline (PBS).

[0028] In one embodiment, the collagen is derived from a collagen solution, and the collagen concentration in the collagen solution is from 10 mg / ml to 16 mg / ml, for example: 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, or 16 mg / ml. Preferably, the collagen concentration in the collagen solution is from 10.5 mg / ml to 11.5 mg / ml.

[0029] In one embodiment, the collagen peptide is derived from a collagen peptide solution, and the concentration of the collagen peptide in the collagen peptide solution is from 25 mg / ml to 50 mg / ml, for example: 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml. Preferably, the concentration of the collagen peptide in the collagen peptide solution is from 26 mg / ml to 30 mg / ml.

[0030] Preferably, based on the total volume of the first mixture, the collagen content is from 15% to 35% by weight, for example: 15%, 19%, 23%, 27%, 31%, or 35% by weight, and the first hyaluronic acid content is from 0.2% to 8% by weight, for example: 0.2%, 0.5%, 1%, 3%, 6%, or 8% by weight. Preferably, based on the total volume of the first mixture, the collagen content is from 18% to 22% by weight, and the first hyaluronic acid content is from 0.8% to 1.2% by weight.

[0031] Preferably, based on the total volume of the second mixture, the content of the first crosslinking complex is from 15 to 35% by weight, for example: 15%, 19%, 23%, 27%, 31%, or 35% by weight, and the content of the second hyaluronic acid is from 0.2 to 8% by weight, for example: 0.2%, 0.5%, 1%, 3%, 6%, or 8%. Preferably, based on the total volume of the second mixture, the content of the first crosslinking complex is from 18 to 22% by weight, and the content of the second hyaluronic acid is from 0.8 to 1.2% by weight.

[0032] Preferably, based on the total volume of the third mixture, the content of the second crosslinking complex is from 15 to 35% by weight, for example: 15%, 19%, 23%, 27%, 31%, or 35% by weight, and the content of the collagen peptide is from 15 to 35% by weight, for example: 15%, 19%, 23%, 27%, 31%, or 35% by weight. Preferably, based on the total volume of the third mixture, the content of the second crosslinking complex is from 18 to 22% by weight, and the content of the collagen peptide is from 18 to 22% by weight.

[0033] In one embodiment, the raw material source for either or both of the collagen solution and the collagen polypeptide solution comprises any or both of pigskin, cowhide, and sheepskin. Preferably, the raw material source comprises cowhide.

[0034] In one embodiment, the collagen in the collagen solution is any one or a combination of porcine collagen, bovine collagen, and sheep collagen. Preferably, the collagen in the collagen solution is bovine collagen.

[0035] In one embodiment, the first crosslinking agent comprises 1,4-butanediol diglycidal ether (BDDE).

[0036] In one embodiment, based on the total volume of the first mixture, the content of the first crosslinking agent is from 0.5 to 5% by weight, for example: 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%. Preferably, based on the total volume of the first mixture, the content of the first crosslinking agent is from 1.8 to 2.2% by weight.

[0037] In one embodiment, the second crosslinking agent comprises glutaraldehyde (GA).

[0038] In one embodiment, based on the total volume of the second mixture, the content of the second crosslinking agent is from 0.005 weight / volume percentage to 0.1 weight / volume percentage, for example: 0.005 weight / volume percentage, 0.01 weight / volume percentage, 0.04 weight / volume percentage, 0.06 weight / volume percentage, 0.08 weight / volume percentage, or 0.1 weight / volume percentage. Preferably, based on the total volume of the second mixture, the content of the second crosslinking agent is from 0.009 weight / volume percentage to 0.011 weight / volume percentage.

[0039] In one embodiment, the third crosslinking agent comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0040] In one embodiment, based on the total volume of the third mixture, the content of the third crosslinking agent is from 0.5 to 5% by weight, for example: 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%. Preferably, based on the total volume of the third mixture, the content of the third crosslinking agent is from 2.7 to 3.3% by weight.

[0041] In one embodiment, either or a combination of the first crosslinking step and the third crosslinking step comprises adding an alkali agent. Preferably, the alkali agent comprises sodium hydroxide.

[0042] In one embodiment, the alkali is a pH adjuster.

[0043] In one embodiment, either or a combination of the first mixture in the first crosslinking step and the third mixture in the third crosslinking step is alkaline. Preferably, the pH value of either or a combination of the first mixture in the first crosslinking step and the third mixture in the third crosslinking step is 8 to 12, for example: 8, 9, 10, 11 or 12. More preferably, the pH value of either or a combination of the first mixture in the first crosslinking step and the third mixture in the third crosslinking step is 9.8 to 10.2.

[0044] In one embodiment, any one or a combination of the first crosslinking step, the second crosslinking step, and the third crosslinking step includes a precipitation step to obtain a precipitate; for example, a first precipitate is obtained after the first precipitation step of the first crosslinking step; a second precipitate is obtained after the second precipitation step of the second crosslinking step; and a third precipitate is obtained after the third precipitation step of the third crosslinking step. Preferably, the precipitation step includes adding an aqueous sodium chloride solution and centrifuging at 10000×g to 15000×g for 5 to 20 minutes. More preferably, the precipitation step includes adding an aqueous sodium chloride solution and centrifuging at 12000×g to 13000×g for 8 to 12 minutes. The precipitate mainly comprises the crosslinked complex generated in the crosslinking step.

[0045] In one embodiment, any one or a combination of the first crosslinking step, the first crosslinking step, and the third crosslinking step includes a washing step to obtain a washed precipitate; for example, the first precipitate is subjected to a first washing step of the first crosslinking step to obtain the first crosslinked composite; the second precipitate is subjected to a second washing step of the second crosslinking step to obtain the second crosslinked composite; and the third precipitate is subjected to a third washing step of the third crosslinking step to obtain the bio-crosslinked material. Preferably, the washing step includes adding an aqueous ethanol solution and centrifuging at 10000×g to 15000×g for 5 to 20 minutes. More preferably, the washing step includes adding an aqueous ethanol solution and centrifuging at 12000×g to 13000×g for 8 to 12 minutes.

[0046] In one embodiment, the method for preparing the bio-mixture further includes a crosslinking neutralization step: mixing the bio-crosslinking material and an aqueous glycine solution to obtain a fourth mixture, and reacting for 20 to 28 hours to obtain a bio-crosslinking material product. According to the present invention, the crosslinking neutralization step ensures that the bio-crosslinking material no longer continues to crosslink, thereby improving the stability of the product, and the weight of the bio-crosslinking material and the bio-crosslinking material product is not substantially different, that is, the weight difference is within 1%.

[0047] Preferably, the concentration of the glycine aqueous solution is 0.05 M to 1 M. More preferably, the concentration of the glycine aqueous solution is 0.08 M to 0.12 M.

[0048] In one embodiment, the crosslinking neutralization step includes a precipitation step to obtain a precipitate, i.e., a fourth precipitate is obtained after the crosslinking neutralization step. Preferably, the precipitation step includes centrifugation at 10000×g to 15000×g for 1 to 10 minutes. More preferably, the precipitation step includes centrifugation at 12000×g to 13000×g for 2.5 to 3.5 minutes.

[0049] In one embodiment, the crosslinking neutralization step includes a washing step to obtain a washed precipitate, i.e., the fourth precipitate is subjected to a fourth washing step in the crosslinking neutralization step to obtain the bio-crosslinked material product. Preferably, the washing step includes adding phosphate-buffered saline and centrifuging at 10000×g to 15000×g for 1 to 10 minutes. More preferably, the washing step includes adding phosphate-buffered saline and centrifuging at 12000×g to 13000×g for 2.5 to 3.5 minutes.

[0050] In summary, the bio-mixture containing bio-crosslinking materials of the present invention is a biomimetic tissue repair material. Besides being suitable for subcutaneous tissue filling and wrinkle reduction, it is also suitable for medical applications such as tissue repair, scar repair, and tissue reconstruction, and has good biocompatibility, reducing allergic or inflammatory reactions. Furthermore, the bio-crosslinking material can be used in combination with exosomes to accelerate tissue repair, thereby further promoting human tissue health.

Implementation Method

[0051] The following provides several embodiments to illustrate the implementation of the present invention; those skilled in the art can easily understand the advantages and effects that the present invention can achieve through the content of this specification, and can make various modifications and changes without departing from the spirit of the present invention to implement or apply the content of the present invention.

[0052] I. Raw materials:

[0053] (a) Collagen solution

[0054] 1. Steps: Add 6 g of porcine skin fragments (1 cm × 1 cm × 0.04 cm) and 600 mg of pepsin to 400 ml of 0.01 N hydrochloric acid aqueous solution, and stir with a magnetic stir bar (250 rpm) at room temperature for 12 hours to carry out the decomposition reaction. Filter the solution using a square filter cloth (approximately 1800 to 1900 mesh) to obtain the collagen solution.

[0055] 2. Analysis: This analysis measures the concentration of the collagen solution. Hydroxyproline (Hypro) is mainly found only in connective tissue sclerein, collagen, and elastin. Furthermore, the primary structural sequence of collagen is mostly a repeating sequence of the three amino acid segments "glycine-hydroxylysine-hydroxyproline." Therefore, the generally accepted method for quantifying collagen is to measure the content of "hydroxyproline." This invention references the method described in the journal "AL Helling et al., In Vitro Enzymatic Degradation of Tissue Grafts and Collagen Biomaterials by Matrix Metalloproteinases: Improving the Collagenase Assay, ACS Biomaterials Science & Engineering Vol 3 / Issue 9, 2016" to measure the concentration of hydroxyproline, and the steps are briefly described below:

[0056] (1) Standard: In this invention, hydroxyproline standard is used to prepare hydroxyproline solutions of different concentrations, and the absorbance at a wavelength of 555 nanometers (nm) is measured by spectrophotometer to draw a standard curve of absorbance versus hydroxyproline concentration.

[0057] (2) Collagen solution: The collagen solution was freeze-dried to obtain a freeze-dried powder, and then hydrolyzed with concentrated hydrochloric acid at 100°C for 16 hours to obtain a hydrolysate. The hydrolysate was diluted 50 times with distilled water, and then further diluted with isopropanol, with the amount of isopropanol added being twice that of distilled water, to obtain a diluted solution. The diluted solution was further homogenized by adding chloramine T reagent, citrate buffer, distilled water, Ehrlich's reagent, perchloric acid and isopropanol, and reacted at 70°C for 10 minutes. The absorbance at a wavelength of 555 nm was measured using a spectrophotometer, and the concentration of hydroxyproline was obtained by referring to the standard curve.

[0058] Furthermore, the journal article "N. Yu. Ignat'eva et al., I Determination of Hydroxyproline in Tissues and the Evaluation of the Collagen Content of the Tissues, Journal of Analytical Chemistry 62(1):51-57, 2007" reveals that the proportion of hydroxyproline in different collagens is generally 12.8% to 14.7%, so it is uniformly assumed that the proportion of hydroxyproline is 13%. In other words, the collagen content is 1 / 0.13 = 7.69 times that of hydroxyproline. Therefore, the concentration of hydroxyproline is further converted after being corrected for dilution ratio: collagen concentration (mg / ml) = (hydroxyproline concentration (mg / ml) × 7.69).

[0059] 3. Results: The collagen concentration in the collagen solution was approximately 11.1 mg / ml.

[0060] (II) Collagen polypeptide solution

[0061] 1. Procedure: Similar to the procedure for obtaining collagen solution, the only difference being that the decomposition reaction time is 72 hours instead of 12 hours. Furthermore, due to the longer decomposition reaction time, the product obtained from the collagen peptide solution is mainly collagen peptides resulting from further decomposition of collagen.

[0062] 2. Analysis: The measurement and calculation methods for the concentration of collagen peptides in collagen peptide solution are the same as those for collagen protein solution. It is also obtained by further conversion after measuring the concentration of hydroxyproline.

[0063] 3. Results: The concentration of collagen peptides in the collagen peptide solution was approximately 28.3 mg / ml.

[0064] (III) First Hyaluronic Acid

[0065] Purchased from Pei-Chan Biomedical, product name HA-LM100, average molecular weight approximately 100 kDa.

[0066] (IV) Second Hyaluronic Acid

[0067] Purchased from Pei-Chan Biomedical, product name HA-LM2000, average molecular weight is approximately 1500 kDa.

[0068] II. Example 1: Bio-mixture containing bio-crosslinking materials

[0069] As shown in Figure 1, the method for preparing the bio-crosslinked material includes: step S1: a first crosslinking step, step S2: a second crosslinking step, and step S3: a third crosslinking step, to obtain the bio-crosslinked material of the present invention, which is a biological mixture. Furthermore, as shown in Figure 2, the bio-crosslinked material of the present invention undergoes a further step S4: a crosslinking neutralization step to obtain a bio-crosslinked material product, as explained below.

[0070] (a) First crosslinking step:

[0071] 1. First mixing step: Collagen solution, first hyaluronic acid, first cross-linking agent: 1,4-butanediol diglycidal ether (BDDE) and sodium hydroxide (NaOH) are added to phosphate buffered saline (PBS) to obtain a first mixture; wherein, the collagen solution contains collagen, the pH of the first mixture is 10, and based on the total volume of the first mixture, the content of collagen is 20 w / v% (i.e., 20 g / 100 ml), the content of the first hyaluronic acid is 1 w / v%, and the content of the first cross-linking agent (BDDE) is 2 w / v%, with the remainder being phosphate buffered saline. It should be noted that the collagen content of 20 w / v% (i.e., 20 g / 100 ml) means that each 100 ml of the first mixture contains 20 grams of collagen (excluding the weight of water, i.e., the weight of the lyophilized powder), and the hyaluronic acid content also excludes the weight of water.

[0072] 2. First reaction step: After stirring the first mixture at room temperature (about 28°C) (at 250 rpm) for 24 hours, a first crosslinking solution is obtained.

[0073] 3. First precipitation step: Add the same volume of sodium chloride (NaCl) aqueous solution with a concentration of 2M (i.e. 2 mol / L) as the first crosslinking solution to the first crosslinking solution, centrifuge at 12500×g for 10 minutes and remove the supernatant to obtain the first precipitate.

[0074] 4. First washing step: Add an appropriate volume of 60% ethanol aqueous solution to the first precipitate, centrifuge at 12500×g for 10 minutes, and remove the supernatant. Repeat this process 5 times to obtain the first cross-linking complex; wherein, the first cross-linking complex is a viscous aqueous mixture. Furthermore, the appropriate volume of 60% ethanol aqueous solution refers to approximately 1 ml of 60% ethanol aqueous solution added to 1 gram of the first precipitate.

[0075] (II) Second crosslinking step:

[0076] 1. Second mixing step: The first cross-linking complex, the second hyaluronic acid, and the second cross-linking agent: glutaraldehyde (GA) are added to PBS to obtain a second mixture; wherein the pH of the second mixture is 7.2, and based on the total volume of the second mixture, the content of the first cross-linking complex is 20 w / v%, the content of the second hyaluronic acid is 1 w / v%, and the content of the second cross-linking agent (GA) is 0.01 w / v, with the remainder being phosphate-buffered saline.

[0077] 2. Second reaction step: After stirring the second mixture at room temperature (250 rpm) for 24 hours, a second crosslinking solution is obtained.

[0078] 3. Second precipitation step: Similar to the first precipitation step, except that the precipitate is the second crosslinking solution, not the first crosslinking solution. In other words, add the same volume of 2M sodium chloride aqueous solution as the second crosslinking solution to the second crosslinking solution, centrifuge at 12500×g for 10 minutes, and then remove the supernatant to obtain the second precipitate.

[0079] 4. Second cleaning step: Similar to the first cleaning step, except that the cleaning target is the second precipitate, not the first precipitate, and the cleaning is performed 3 times, not 5 times. In other words, an appropriate volume of 60% ethanol aqueous solution is added to the second precipitate, and after centrifugation at 12500×g for 10 minutes, the supernatant is removed. This process is repeated 3 times to obtain the second cross-linked complex; wherein the second cross-linked complex is a viscous aqueous mixture.

[0080] (III) Third crosslinking step:

[0081] 1. Third mixing step: The second cross-linking complex, collagen peptide solution, third cross-linking agent: 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and sodium hydroxide are added to PBS to obtain a third mixture; wherein the pH of the third mixture is 10, and based on the total volume of the third mixture, the content of the second cross-linking complex is 20 w / v%, the content of the collagen peptide solution is 20 w / v%, and the content of the third cross-linking agent (EDC) is 3 w / v, with the remainder being phosphate-buffered saline.

[0082] 2. Third reaction step: After stirring the third mixture at 40°C (250 rpm) for 24 hours, a third crosslinking solution is obtained.

[0083] 3. Third precipitation step: Similar to the first precipitation step, except that the precipitate is a third cross-linking solution, not a first cross-linking solution. The third precipitation step yields a third precipitate.

[0084] 4. Third cleaning step: Similar to the first cleaning step, except that the cleaning target is the third precipitate, not the first precipitate, and the cleaning is performed 3 times, not 5 times. The third cleaning step can obtain the biological cross-linking material, which is a biological mixture; wherein, the biological cross-linking material is a thick aqueous mixture.

[0085] (IV) Crosslinking neutralization step:

[0086] 1. Fourth mixing step: Add the bio-crosslinking material to 10 times its volume of a 0.1 M glycine aqueous solution to obtain a fourth mixture.

[0087] 2. Fourth reaction step: After stirring the fourth mixture at room temperature (250 rpm) for 24 hours, a fourth crosslinking solution is obtained.

[0088] 3. Fourth precipitation step: After centrifuging the fourth crosslinking solution at 12500×g for 3 minutes, remove the supernatant to obtain the fourth precipitate.

[0089] 4. Fourth washing step: Add the fourth precipitate to the same volume of PBS (pH=9) containing sodium hydroxide, centrifuge at 12500×g for 3 minutes, and remove the supernatant. Repeat this process 3 times to obtain the bio-crosslinked material product; wherein the bio-crosslinked material product is a viscous aqueous mixture, and its wet weight is not substantially different from that of the bio-crosslinked material. The crosslinking neutralization step is not a necessary step in manufacturing the bio-crosslinked material of the present invention.

[0090] Furthermore, the biological mixture containing the biological cross-linking material of Example 1 was added to PBS containing 0.35 w / v % lidocaine hydrochloride and filled into a syringe to prepare an injection; wherein, based on the total volume of the injection contents, the total concentration of collagen and collagen peptides was approximately 40 mg / ml, and a photograph of the injection is shown in Figure 3.

[0091] III. Monomer Ratio Analysis of the Bio-crosslinked Material in Example 1

[0092] The monomers of the bio-crosslinking material of Example 1 include collagen from a collagen solution, collagen peptides (derived from collagen) from a collagen peptide solution, first hyaluronic acid, and second hyaluronic acid. Regarding the analysis of the collagen and collagen peptide content, the measurement and calculation methods for the concentration of collagen and collagen peptides in the bio-crosslinking material of Example 1 are the same as those for the "collagen solution" (i.e., the concentration of "hydroxyproline" is measured first and then converted to the collagen concentration). The calculation result is 40 mg / ml, that is, the total concentration of collagen and collagen peptides in the bio-crosslinking material of Example 1 is 40 mg / ml.

[0093] Regarding the analysis of the content of the first and second hyaluronic acid, 1 ml of the bio-crosslinking material of Example 1 was freeze-dried to obtain a freeze-dried powder. The freeze-dried powder was weighed, and the content of collagen and collagen peptides obtained above was subtracted to obtain the weight of hyaluronic acid per milliliter, thus obtaining the hyaluronic acid concentration. The calculated result was 4.8 mg / ml, that is, the total concentration of the first and second hyaluronic acid in the bio-crosslinking material of Example 1 was 4.8 mg / ml.

[0094] It should be noted that the "total concentration of collagen and collagen peptides (40 mg / ml)" and the "total concentration of first hyaluronic acid and second hyaluronic acid (4.8 mg / ml)" in the bio-crosslinked material of Example 1 are calculation results without taking into account the water content. Therefore, the molecules in the sum of the two concentrations (40+4.8=44.8 mg / ml) are not the wet weight of each milliliter of bio-crosslinked material (thick aqueous mixture).

[0095] IV. Example 2: Combination of biological cross-linking materials with exosomes

[0096] The bio-crosslinking material of Example 1 and the exosome solution (a concentrated adipose stem cell exosome solution, brand name Exo-ASC, purchased from Pei-Chan Biomedical) were added to PBS containing 0.35 w / v% lidocaine hydrochloride to obtain a fifth mixture; wherein, based on the total volume of the fifth mixture, the content of the bio-crosslinking material was 4 w / v%, and the concentration of the exosomes (i.e., adipose stem cell exosomes) in the fifth mixture was approximately 1 × 10⁷ particles (EV) / ml.

[0097] V. Examples 3 to 5: Bio-crosslinked materials

[0098] Examples 3 to 5 are similar to Example 1, except that the collagen source differs, as described below: The collagen solution and collagen peptide solution in Example 1 were all made from pigskin. The collagen source in Example 3 included both pigskin and cowhide, with a weight ratio of 1:1; the collagen source in Example 4 included both pigskin and sheepskin (after hair removal), with a weight ratio of 1:1; and the collagen source in Example 5 included both cowhide and sheepskin, with a weight ratio of 1:1. In other words, the differences in the collagen source among the groups are shown in Table 1.

[0099] Table 1: Collagen Sources in Each Group Group Collagen sources Example 1 pigskin Example 2 pigskin Example 3 The raw materials of pigskin and cowhide are in a 1:1 weight ratio. Example 4 The raw materials of pigskin and sheepskin are in a 1:1 weight ratio. Example 5 The raw materials of cowhide and sheepskin are in a 1:1 weight ratio.

[0100] VI. Comparative Examples 1 to 11: Bio-crosslinking materials (or used in combination with exosomes)

[0101] The key differences in the preparation methods of each group are shown in Table 2. The preparation methods of each group are explained in order below.

[0102] Table 2: Preparation methods for each group Group Manufacturing method Comparative Example 1 Four raw materials (collagen solution, collagen peptide solution, primary hyaluronic acid, and secondary hyaluronic acid) are cross-linked together in a single process, and three cross-linking agents (BDDE, GA, and EDC) are used simultaneously; collagen source: pig skin. Comparative Example 2 The four raw materials were cross-linked in three stages (same as in Example 1), but the cross-linking agent used in all three stages was the same, namely BDDE; the collagen source was pig skin. Comparative Example 3 The four raw materials were cross-linked in three stages (same as in Example 1), but the cross-linking agent for all three stages was the same, namely GA; the collagen source was pig skin. Comparative Example 4 The four raw materials were cross-linked in three stages (same as in Example 1), but the cross-linking agent used in all three stages was the same, namely EDC; the collagen source was pig skin. Comparative Example 5 Two raw materials (collagen solution and collagen peptide solution) are cross-linked together in a single process, and the cross-linking agent is GA; the collagen source is pig skin. Comparative Example 6 Two raw materials (first hyaluronic acid and second hyaluronic acid) are cross-linked together in a single process, and the cross-linking agent is BDDE. Comparative Example 7 Comparative Example 5: Exosomes were added; Collagen source: pig skin. Comparative Example 8 Comparative Example 6 was supplemented with exosomes. Comparative Example 9 Preparation method is the same as Comparative Example 3; Collagen source: Raw materials from pigskin and cowhide in a 1:1 weight ratio. Comparative Example 10 Preparation method is the same as Comparative Example 3; Collagen source: Raw materials from pigskin and sheepskin in a 1:1 weight ratio. Comparative Example 11 Preparation method is the same as Comparative Example 3; Collagen source: Raw materials from cowhide and sheepskin in a 1:1 weight ratio.

[0103] (I) Comparative Example 1: Four raw materials (collagen solution, collagen peptide solution, primary hyaluronic acid and secondary hyaluronic acid) were cross-linked together in a single process, and three cross-linking agents (BDDE, GA and EDC) were used simultaneously, as described below:

[0104] 1. Crosslinking steps:

[0105] (1) Mixing step: Collagen solution, collagen peptide solution, first hyaluronic acid, second hyaluronic acid, first crosslinking agent (BDDE), second crosslinking agent (GA) and third crosslinking agent (EDC) and sodium hydroxide were added to PBS to obtain a mixture; wherein the pH of the mixture was 10, and based on the total volume of the mixture, the content of the collagen solution was 20 w / v%, the content of the collagen peptide solution was 20 w / v%, the content of the first hyaluronic acid was 1 w / v%, the content of the second hyaluronic acid was 1 w / v%, the content of the first crosslinking agent (BDDE) was 2 w / v%, the content of the second crosslinking agent (GA) was 0.01 w / v%, and the content of the third crosslinking agent (EDC) was 3 w / v% to obtain the mixture of Comparative Example 1.

[0106] (2) Reaction steps: The mixture of Comparative Example 1 was stirred at room temperature (250 rpm) for 24×3=72 hours to obtain the crosslinking solution of Comparative Example 1.

[0107] (3) Precipitation step: Similar to the first precipitation step in Example 1, except that the precipitate is the cross-linked solution of Comparative Example 1, so as to obtain the precipitate of Comparative Example 1.

[0108] (4) Cleaning step: Similar to the first cleaning step of Example 1, except that the cleaning target is the precipitate of Comparative Example 1, so as to obtain the crosslinked complex of Comparative Example 1.

[0109] 2. Crosslinking neutralization step: Similar to the crosslinking neutralization step in Example 1, except that the crosslinking neutralization target is the crosslinking complex of Comparative Example 1, so as to obtain the crosslinking product of Comparative Example 1.

[0110] (II) Comparative Example 2: Four raw materials (collagen solution, collagen peptide solution, first hyaluronic acid and second hyaluronic acid) were cross-linked in three stages, but the cross-linking agent used in the three stages was the same, namely BDDE.

[0111] 1. First crosslinking step: Same as the first crosslinking step in Example 1.

[0112] 2. Second crosslinking step: Similar to the second crosslinking step of Example 1, except that the second crosslinking agent is BDDE, not GA, and the content of the second crosslinking agent (BDDE) is 2 w / v% based on the total volume of the second mixture to obtain the second crosslinking complex of Comparative Example 2.

[0113] 3. Third crosslinking step: Similar to the third crosslinking step of Example 1, except that (1) the third crosslinking agent is BDDE, not EDC, and the content of the third crosslinking agent (BDDE) is 2 w / v% based on the total volume of the third mixture; and (2) the target of the third crosslinking step is the second crosslinking complex of Comparative Example 2, so as to obtain the crosslinking material of Comparative Example 2.

[0114] 4. Crosslinking neutralization step: Similar to the crosslinking neutralization step in Example 1, except that the crosslinking neutralization target is the crosslinking complex of Comparative Example 2, so as to obtain the crosslinking product of Comparative Example 2.

[0115] (III) Comparative Example 3: Four raw materials (collagen solution, collagen peptide solution, first hyaluronic acid and second hyaluronic acid) were cross-linked in three stages, but the cross-linking agent used in the three stages was the same, which was GA.

[0116] 1. First crosslinking step: Similar to the first crosslinking step of Example 1, except that the first crosslinking agent is GA instead of BDDE, and the content of the first crosslinking agent (GA) is 0.01 w / v% based on the total volume of the first mixture to obtain the first crosslinking complex of Comparative Example 3.

[0117] 2. Second crosslinking step: Similar to the second crosslinking step of Example 1, except that the target of the second crosslinking is the first crosslinking complex of Comparative Example 3, so as to obtain the second crosslinking complex of Comparative Example 3.

[0118] 3. Third crosslinking step: Similar to the third crosslinking step of Example 1, except that (1) the third crosslinking agent is GA, not EDC, and the content of the third crosslinking agent (BDDE) is 0.01 w / v% based on the total volume of the third mixture; and (2) the target of the third crosslinking step is the second crosslinking complex of Comparative Example 3, so as to obtain the crosslinking material of Comparative Example 3.

[0119] 4. Crosslinking neutralization step: Similar to the crosslinking neutralization step in Example 1, except that the crosslinking neutralization target is the crosslinking complex of Comparative Example 3, so as to obtain the crosslinking product of Comparative Example 3.

[0120] (iv) Comparative Example 4: Four raw materials (collagen solution, collagen peptide solution, first hyaluronic acid and second hyaluronic acid) were cross-linked in three steps, but the cross-linking agent for the three steps was the same, all of which was EDC.

[0121] 1. First crosslinking step: Similar to the first crosslinking step of Example 1, except that the first crosslinking agent is EDC instead of BDDE, and the content of the first crosslinking agent (EDC) is 3 w / v% based on the total volume of the first mixture to obtain the first crosslinking complex of Comparative Example 4.

[0122] 2. Second crosslinking step: Similar to the second crosslinking step of Example 1, except that (1) the second crosslinking agent is EDC, not GA, and the content of the second crosslinking agent (EDC) is 3 w / v based on the total volume of the second mixture; and (2) the target of the third crosslinking step is the first crosslinking complex of Comparative Example 4, so as to obtain the second crosslinking complex of Comparative Example 4.

[0123] 3. Third crosslinking step: Similar to the third crosslinking step of Example 1, except that the target of the third crosslinking step is the second crosslinking complex of Comparative Example 4, so as to obtain the crosslinking material of Comparative Example 4.

[0124] 4. Crosslinking neutralization step: Similar to the crosslinking neutralization step in Example 1, except that the crosslinking neutralization target is the crosslinking complex of Comparative Example 4, so as to obtain the crosslinking product of Comparative Example 4.

[0125] (V) Comparative Example 5: Two raw materials (collagen solution and collagen peptide solution) were cross-linked together in a single step, and the cross-linking agent was GA. The explanation is as follows:

[0126] 1. Crosslinking steps:

[0127] (1) Mixing step: Collagen solution, collagen peptide solution, cross-linking agent (GA) and sodium hydroxide were added to PBS to obtain a mixture; wherein the pH of the mixture was 10, and based on the total volume of the mixture, the content of the collagen solution was 20 w / v%, the content of the collagen peptide solution was 20 w / v%, and the content of the cross-linking agent (GA) was 0.01 w / v% to obtain the mixture of Comparative Example 5.

[0128] (2) Reaction steps: The mixture of Comparative Example 5 was stirred at room temperature (250 rpm) for 24×3=72 hours to obtain the crosslinking solution of Comparative Example 5.

[0129] (3) Precipitation step: Similar to the first precipitation step in Example 1, except that the precipitate is the cross-linked solution of Comparative Example 5, so as to obtain the precipitate of Comparative Example 5.

[0130] (4) Cleaning step: Similar to the first cleaning step of Example 1, except that the cleaning target is the precipitate of Comparative Example 5, so as to obtain the crosslinked complex of Comparative Example 5.

[0131] 2. Crosslinking neutralization step: Similar to the crosslinking neutralization step in Example 1, except that the crosslinking neutralization target is the crosslinking complex of Comparative Example 5, so as to obtain the crosslinking product of Comparative Example 5.

[0132] (vi) Comparative Example 6: Two raw materials (first hyaluronic acid and second hyaluronic acid) were cross-linked together in a single process, and the cross-linking agent was BDDE, as described below:

[0133] 1. Crosslinking steps:

[0134] (1) Mixing step: First hyaluronic acid, second hyaluronic acid, crosslinking agent (BDDE) and sodium hydroxide are added to PBS to obtain a mixture; wherein the pH of the mixture is 10, and based on the total volume of the mixture, the content of the first hyaluronic acid is 1 w / v%, the content of the second hyaluronic acid is 1 w / v%, and the content of the crosslinking agent (BDDE) is 2 w / v%, to obtain the mixture of Comparative Example 6.

[0135] (2) Reaction steps: The mixture of Comparative Example 6 was stirred at room temperature (250 rpm) for 24×3=72 hours to obtain the crosslinking solution of Comparative Example 6.

[0136] (3) Precipitation step: Similar to the first precipitation step in Example 1, except that the precipitate is the cross-linking solution of Comparative Example 6, so as to obtain the precipitate of Comparative Example 6.

[0137] (4) Cleaning step: Similar to the first cleaning step of Example 1, except that the cleaning target is the precipitate of Comparative Example 6, so as to obtain the crosslinked complex of Comparative Example 6.

[0138] 2. Crosslinking neutralization step: Similar to the crosslinking neutralization step in Example 1, except that the crosslinking neutralization target is the crosslinking complex of Comparative Example 6, so as to obtain the crosslinking product of Comparative Example 6.

[0139] (VII) Comparative Example 7: Comparative Example 7 is a further addition of exosomes to Comparative Example 5, as described below: The preparation method is similar to that of Example 2, except that the cross-linking product of Comparative Example 5 is used instead of the biological cross-linking material of Example 1 to obtain the mixture of Comparative Example 7, and in the mixture of Comparative Example 7, the concentration of the exosomes (i.e., adipose stem cell exosomes) is also about 1×10 7 particles (EV) / ml.

[0140] (VIII) Comparative Example 8: Comparative Example 8 is a further addition of exosomes to Comparative Example 6, as described below: The preparation method is similar to that of Example 2, except that the cross-linking product of Comparative Example 6 is used instead of the biological cross-linking material of Example 1 to obtain the mixture of Comparative Example 8, and in the mixture of Comparative Example 8, the concentration of the exosomes (i.e., adipose stem cell exosomes) is also about 1×10 7 particles (EV) / ml.

[0141] (ix) Comparative Example 9: The preparation method of Comparative Example 9 is similar to that of Comparative Example 3, except that the collagen source is pig skin and cow skin, and the weight ratio of the two raw materials is 1:1.

[0142] (ix) Comparative Example 10: The preparation method of Comparative Example 10 is similar to that of Comparative Example 3, except that the collagen source is pigskin and sheepskin, and the weight ratio of the two raw materials is 1:1.

[0143] (ix) Comparative Example 11: The preparation method of Comparative Example 11 is similar to that of Comparative Example 3, except that the collagen source is cowhide and sheepskin, and the weight ratio of the two raw materials is 1:1.

[0144] Analysis 1: Crosslinking degree, collagen degradation rate and hyaluronic acid degradation rate

[0145] This analysis is intended to understand the structural strength of the bio-crosslinked material product. Therefore, the degree of crosslinking, collagen degradation rate and hyaluronic acid degradation rate of the comparative examples 1, 3 to 5, 1 to 6, and 9 to 11 are described below.

[0146] (I) Crosslinking Degree Analysis

[0147] Trinitrobenzenesulfonic acid (TNBS) can bind to free amino groups in proteins to form a yellow substance, and its quantification is achieved by measuring the absorbance at a wavelength of 345 nanometers using spectrophotometry. Therefore, this invention employs the TNBS method to detect free amino groups and thereby calculate the degree of crosslinking. The explanation is as follows:

[0148] 1. Sample preparation:

[0149] (1) Test sample: The freeze-dried powder obtained by freeze-drying the biological cross-linking material products of each group is used as the test sample.

[0150] (2) Reference standards: The freeze-dried powder of the raw materials of each group was used as a reference standard. Taking Example 1 as an example, four raw materials were mixed: collagen solution, collagen peptide solution, first hyaluronic acid and second hyaluronic acid, with a weight ratio of 20:20:1:1, to obtain a raw material mixture, which was then freeze-dried to obtain freeze-dried powder of the raw materials as a sample. In addition, taking Comparative Example 5 as an example, two raw materials were mixed: collagen solution and collagen peptide solution, with a weight ratio of 20:20, to obtain a raw material mixture, which was then freeze-dried to obtain freeze-dried powder of the raw materials as a sample.

[0151] 2. Analysis steps:

[0152] (1) TNBS method: 1 mg of sample was added to 100 µL of 0.1% TNBS aqueous solution and placed in a dry bath. The reaction was carried out at 50°C for 1 hour. Then, 300 µL of 6N hydrochloric acid aqueous solution was added and the reaction was carried out at 60°C for 1.5 hours. The solution was then cooled to room temperature to obtain the test solution. 200 µL of the test solution was transferred to a 96-well plate and the absorbance at a wavelength of 345 nm was measured using a spectrophotometer for quantification.

[0153] (2) Standard glycine solution: Glycine has only one free amino group, so glycine is used as the standard solution. Since the absorbance value and the free amino group concentration are not necessarily linearly related, standard glycine solutions of different concentrations are prepared and reacted according to the above TNBS method. The absorbance value at a wavelength of 345 nm is measured by spectrophotometer to draw a standard curve of absorbance value versus glycine concentration (i.e., free amino group concentration) to calculate the free amino group concentration of each group of test samples and reference samples.

[0154] (3) Calculation formula: Crosslinking degree (%) = [1 - (concentration of free amino groups of the test sample / concentration of free amino groups of the reference standard)] × 100%; where the concentration of free amino groups of the test sample is the uncrosslinked part, so it is subtracted to obtain the concentration of the crosslinked part, i.e., the crosslinking degree.

[0155] (II) Collagen degradation rate analysis:

[0156] Ninhydrin can bind to free amino groups in proteins to form a blue-violet substance, and its quantification is achieved by measuring the absorbance at a wavelength of 570 nanometers using spectrophotometry. Therefore, this invention employs the ninhydrin method to detect the concentration of free amino groups after collagen degradation, and thereby calculates the collagen degradation rate. The explanation is as follows:

[0157] 1. Sample preparation:

[0158] (1) Test sample: The biological cross-linked material products (non-lyophilized powder) of each group are used as the test sample.

[0159] (2) Reference standard: The raw material mixture (non-lyophilized powder) of each group was used as the reference standard sample. Taking Example 1 as an example, four raw materials were mixed: collagen solution, collagen peptide solution, first hyaluronic acid and second hyaluronic acid, in a weight ratio of 20:20:1:1 to obtain the raw material mixture. In addition, taking Comparative Example 5 as an example, two raw materials were mixed: collagen solution and collagen peptide solution, in a weight ratio of 20:20 to obtain the raw material mixture.

[0160] 2. Analysis steps:

[0161] (1) Degradation of collagen: 1 gram of sample was added to 0.1 ml of collagenase aqueous solution and reacted at 37°C for 24 hours; wherein the collagenase aqueous solution contained 20 mM NaH 2PO 4·H 2O and 0.36 mM CaCl 2·2H 2O, the pH value was 7.4, and the collagenase concentration was 0.5 units / ml. After centrifugation at 10000 rpm for 10 minutes, the supernatant was obtained.

[0162] (2) Ninhydrin method: Take 0.2 mL of supernatant and add 25 µL of ninhydrin (concentration of 20 mg / mL, product number: 151173, purchased from Sigma-Aldrich). After reacting at 100°C for 20 minutes, wait for it to cool to room temperature and use spectrophotometry to measure the absorbance at a wavelength of 570 nm for quantification.

[0163] (3) Standard glycine solution: Prepare standard glycine solutions of different concentrations and react them according to the above ninhydrin method. Use a spectrophotometer to measure the absorbance at a wavelength of 570 nm to plot a standard curve of absorbance versus glycine concentration (i.e., free amino group concentration) to calculate the free amino group concentration of each group of test samples and control samples after collagenase degradation.

[0164] (4) Calculation formula: Collagen degradation rate (%) = (Free amino concentration of test sample / Free amino concentration of reference sample) × 100%.

[0165] (III) Hyaluronic Acid Degradation Rate Analysis:

[0166] D-Glucurono-δ-lactone (D-GUL) is one of the main products of hyaluronic acid degradation and can be used to quantify the degradation rate of hyaluronic acid. Furthermore, D-GUL will decompose into D-Glucuronic Acid (D-GlcA) in an acidic environment. D-GlcA has a carboxyl group (-COOH) and can undergo a condensation reaction with carbazole to generate a purple-red substance. This substance can be quantified by measuring the absorbance at a wavelength of 530 nanometers using spectrophotometry. The explanation is as follows.

[0167] 1. Sample preparation:

[0168] (1) Test sample: The biological cross-linked material products (non-lyophilized powder) of each group are used as the test sample.

[0169] (2) Reference: The raw material mixture (non-lyophilized powder) of each group is used as the reference sample.

[0170] 2. Analysis steps:

[0171] (1) Degradation of hyaluronic acid: Add 300 units of hyaluronidase to 1 gram of sample, and then add deionized water to adjust the volume of the mixture to 2 ml. After reacting at 42°C for 60 minutes, add 3 ml of anhydrous ethanol to terminate the enzymatic reaction. After centrifuging the mixture at 12,000 rpm for 20 minutes, obtain the supernatant.

[0172] (2) Carbazole method: The experimental procedure is referenced in the journal: Liuyi Chang et al., Comparative Properties of Hyaluronic Acid Hydrogel Cross-linked with 1,4-Butanediol Diglycidyl Ether Assayed Using a Marine Hyaluronidase, IOP Conference Series: Materials Science and Engineering, Volume 493, 2nd International Conference on Frontiers of Materials Synthesis and Processing 10-11 November 2018, Sanya, China; Among them, 100 μL of supernatant was added to 200 μL of carbazole solution (solvent is anhydrous ethanol) with a concentration of 0.1% by volume weight, and then boiled at 100°C for 10 minutes to develop color. The absorbance at a wavelength of 530 nm was measured by spectrophotometry to quantify the result.

[0173] (3) Standard D-glucuronide solution: Prepare standard D-glucuronide solutions of different concentrations and react them according to the above carbazole method. Measure the absorbance at a wavelength of 530 nm using a spectrophotometer to plot a standard curve of absorbance versus D-glucuronide concentration, and calculate the D-glucuronide concentration of each group of test samples and control samples after degradation by hyaluronidase.

[0174] (4) Calculation formula: Hyaluronic acid degradation rate (%) = (D-glucuronide concentration of the test sample / D-glucuronide concentration of the reference standard) × 100%.

[0175] (IV) Results

[0176] Each group was tested three times. The results of cross-linking degree, collagen degradation rate and hyaluronic acid degradation rate of each group are shown in Table 3.

[0177] Table 3: Crosslinking degree, collagen degradation rate and hyaluronic acid degradation rate of each group (including the measurements and average values ​​of three repeated tests) Group crosslinking degree (%) Collagen degradation rate (%) Hyaluronic acid degradation rate (%) Example 1 75.62 9.17 12.52 70.44 10.41 10.82 79.2 8.34 15.77 Average: 75.09 Average: 9.31 Average: 13.04 Example 3 80 8.31 11.53 75 7.26 14.23 71 6.54 15.77 Average: 75.33 Average: 7.37 Average: 13.84 Example 4 65 8.32 16.22 78 9.51 11.36 80 8.24 13.5 Average: 74.33 Average: 8.69 Average: 13.69 Example 5 92 6.32 12.03 65 4.32 12.58 73 5.47 14.39 Average: 76.67 Average: 5.37 Average: 13.00 Comparative Example 1 50.11 twenty two 18.6 45.77 25 22.5 52.34 18.54 19.33 Average: 49.41 Average: 21.85 Average: 20.14 Comparative Example 2 11.02 34.42 38.5 10.12 40.51 42.6 9.52 30.21 33.2 Average: 10.22 Average: 35.05 Average: 38.10 Comparative Example 3 32.88 49.83 52.1 36.51 54.89 57.8 29.43 55.21 55.6 Average: 32.94 Average: 53.31 Average: 55.17 Comparative Example 4 34.44 32.54 30.5 40.2 29.11 28.7 30.12 34.21 35.7 Average: 34.92 Average: 31.95 Average: 31.63 Comparative Example 5 N / A 45.84 N / A 53.21 40.38 Average: 46.48 Comparative Example 6 N / A N / A 52.31 40.33 57.02 Average: 49.89 Comparative Example 9 31.88 44.83 49.23 37.51 50.89 45.39 25.43 55.11 54.23 Average: 31.61 Average: 50.28 Average: 49.62 Comparative Example 10 31.55 49.83 53.11 30.25 45.31 58.21 35.89 40.22 50.22 Average: 32.56 Average: 45.12 Average: 53.85 Comparative Example 11 36.51 40.21 50.14 29.87 54.89 49.25 24.53 45.52 57.23 Average: 30.30 Average: 46.87 Average: 52.21

[0178] As can be seen from Table 3, firstly, in Examples 1 and 3 to 5, the degree of crosslinking of all of them is greater than 74%, which is a high degree of crosslinking; the collagen degradation rate of all of them is less than 10%, which is a low collagen degradation rate; and the hyaluronic acid degradation rate of all of them is less than 14%, which is a low hyaluronic acid degradation rate.

[0179] In contrast, in Comparative Examples 1 to 6 and Comparative Examples 9 to 11, the degree of crosslinking was less than 50%, significantly lower than that of Examples 1 and 3 to 5 (at least 74%); the collagen degradation rate was greater than 20%, significantly higher than that of Examples 1 and 3 to 5 (less than 10%); and the hyaluronic acid degradation rate was greater than 20%, significantly higher than that of Examples 1 and 3 to 5 (less than 14%). It can be seen that, compared to Comparative Examples 1 to 6 and Comparative Examples 9 to 11, Examples 1 and 3 to 5 are less susceptible to degradation by collagenase and hyaluronidase in the human body, thus significantly increasing the residence time of the bio-crosslinked material in the human body and effectively reducing the frequency of supplementation of the bio-crosslinked material.

[0180] Second, from the comparison of Examples 1 and 3 to 5, it can be seen that the collagen degradation rate of Examples 3 and 5 (the collagen source all includes cowhide) is lower than that of Examples 1 and 4. It can be seen that using cowhide as the collagen source can reduce the collagen degradation rate, thereby increasing the residence time of the bio-crosslinked material in the human body and effectively reducing the frequency of supplementing the bio-crosslinked material.

[0181] Third, Example 1 employs a three-stage crosslinking process. Compared to Comparative Example 1, which employs a single-stage crosslinking process, the degree of crosslinking in Example 1 (75.09%) is significantly higher than that in Comparative Example 1 (49.41%); the collagen degradation rate in Example 1 (9.31%) is significantly lower than that in Comparative Example 1 (21.85%); and the hyaluronic acid degradation rate in Example 1 (13.04%) is significantly lower than that in Comparative Example 1 (20.14%). Therefore, the present invention employs a three-stage crosslinking process, which, compared to a single-stage crosslinking process, can effectively increase the degree of crosslinking, reduce the collagen degradation rate and the hyaluronic acid degradation rate, thereby increasing the residence time of the bio-crosslinked material in the human body and effectively reducing the frequency of supplementing the bio-crosslinked material.

[0182] Fourth, in Example 1, the three crosslinking steps sequentially employ BDDE, GA, and EDC. Compared to Comparative Examples 2 to 4, which use a single crosslinking agent, the degree of crosslinking in Example 1 (75.09) is significantly higher than that in Comparative Examples 2 to 4 (maximum 34.92); the collagen degradation rate in Example 1 (9.31) is significantly lower than that in Comparative Examples 2 to 4 (minimum 31.95); and the hyaluronic acid degradation rate in Example 1 (13.04) is significantly lower than that in Comparative Examples 2 to 4 (minimum 31.63). Therefore, the three crosslinking steps of the present invention, sequentially employing BDDE, GA, and EDC, can effectively increase the degree of crosslinking and reduce the collagen and hyaluronic acid degradation rates compared to a single crosslinking agent, thereby increasing the residence time of the bio-crosslinked material in the human body and effectively reducing the frequency of supplementing the bio-crosslinked material.

[0183] Fifth, Example 1 uses four raw materials (combining collagen and hyaluronic acid). Compared to Comparative Example 5, which uses only collagen, the collagen degradation rate of Example 1 (9.31%) is significantly lower than that of Comparative Example 1 (46.48%); and compared to Comparative Example 6, which uses only hyaluronic acid, the hyaluronic acid degradation rate of Example 1 (13.04%) is significantly lower than that of Comparative Example 6 (49.89%). Therefore, the present invention uses four raw materials, which, compared to a single collagen or hyaluronic acid raw material, can effectively reduce the degradation rate of collagen and hyaluronic acid, thereby increasing the residence time of the bio-crosslinked material in the human body and effectively reducing the frequency of supplementing the bio-crosslinked material.

[0184] Analysis 2: Yield

[0185] This analysis aims to understand the yield of bio-crosslinked material products and is divided into 5 groups: Example 1 and Comparative Examples 1 to 4, as described below.

[0186] (I) Yield Analysis

[0187] Calculation formula: Yield (%) = (Wet weight of biological cross-linking material product / Wet weight of raw material) * 100%; wherein, the weight of the cross-linking agent is not included in the raw material, that is, it includes: the total amount (wet weight) of collagen solution, collagen peptide solution, first hyaluronic acid and second hyaluronic acid added.

[0188] (II) Results: The yield of each group is shown in Table 4.

[0189] Table 4: Yield of each group Group Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Yield 83.2 80.1 60.2 91.2 72.1 87.4 81.1 51.3 89.3 80 82.2 75.3 63.31 94.1 65.2 average 84.27 78.83 58.27 91.53 72.43

[0190] As can be seen from Table 4, except for the significantly lower yield of Comparative Example 2, the yields of the other groups are similar; although the yield of Comparative Example 3 is higher than that of Example 1, that is, more raw materials are cross-linked and remain in the bio-crosslinked material product, as can be seen from Table 3, the structural strength of Comparative Example 3 is significantly lower than that of Example 1, that is, the cross-linking quality of Example 1 is significantly better than that of Comparative Example 3.

[0191] Analysis 3: Cell Viability Analysis

[0192] This analysis aims to understand the effect of combining biological cross-linking material products with exosomes on cell survival rate. It is divided into blank group, control group and experimental group. The experimental group includes Example 2, Example 2 after long-term storage, Comparative Example 7 and Comparative Example 8. Among them, "Example 2 after long-term storage" means that Example 2 was placed in a refrigerated environment at 4°C for 3 months before this analysis. The other groups were analyzed within 3 days after the completion of the storage. The explanation is as follows.

[0193] (a) Blank group (coating formed using biological cross-linking material products, without the addition of cells):

[0194] For each blank group, the corresponding experimental group used a biological cross-linking material product. 0.1 g of the biological cross-linking material product was added to a 24-well culture dish for 24 hours, followed by 0.5 ml of cell culture medium for 24 hours to allow the biological cross-linking material product to adhere to the bottom of the well to form a coating. The cell culture medium was Durbecco's Modified Eagle Medium (DMEM) containing 10% by volume of fetal bovine serum (FBS).

[0195] After aspirating the cell culture medium, add 1 ml of cell culture medium (cell-free) to each well and transfer it to a cell culture incubator for 2 hours at a constant temperature of 37°C and 5% carbon dioxide. Add an additional 0.5 ml of cell culture medium (cell-free) to each group and continue culturing for 7 days. Then replace the medium with 0.5 ml of fresh cell culture medium (cell-free). Use a Cell Counting Kit-8 (CCK-8, purchased from Sigma-Aldrich / Merck) and follow the instructions for the experimental procedures. Transfer 100 μl of each sample to a 96-well culture dish and measure the absorbance at a wavelength of 450 nm using a spectrophotometer.

[0196] (II) Control group (no coating formed using bio-crosslinking materials, no cells added):

[0197] In a 24-well culture dish, 1 ml of cell culture medium containing cells was placed in each well and transferred to a cell culture incubator. After 7 days in a constant environment of 37°C and 5% carbon dioxide, 0.5 ml of fresh cell culture medium was replaced. The cell culture medium containing cells contained the above-mentioned cell culture medium and contained approximately 1 × 10⁴ human dermal fibroblasts (HDF). The experimental procedure was performed according to the instructions using the CCK-8 kit, and 100 μL was transferred to a 96-well culture dish. The absorbance at a wavelength of 450 nm was measured using a spectrophotometer.

[0198] (III) Experimental Group:

[0199] In each group, 0.1 g of the bio-crosslinking material product was added to a 24-well culture dish for 24 hours, followed by 0.5 mL of the cell culture medium and soaking for another 24 hours to allow the bio-crosslinking material product to adhere to the bottom of the wells and form a coating. In each well of the 24-well culture dish, 1 mL of the aforementioned cell culture medium containing cells was placed, and the dish was transferred to a cell culture incubator at a constant temperature of 37°C and 5% carbon dioxide for 7 days. Afterward, 0.5 mL of fresh cell culture medium was replaced. The experimental procedures were performed using the CCK-8 kit according to the instructions, and 100 μL was transferred to a 96-well culture dish. The absorbance at a wavelength of 450 nm was measured using a spectrophotometer.

[0200] (IV) Calculation Formula: Cell viability (%) = (Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance) × 100%

[0201] (V) Results: The data for each experimental group are shown in Table 5.

[0202] Table 5: Cell viability of each experimental group Example 2 Example 2 of long-term storage Comparative Example 7 Comparative Example 8 Cell viability 240 248 170 224 251 230 182 180 238 223 160 190 average 243 233.67 170.67 198.00

[0203] As shown in Table 5, even after 3 months of storage, the cell survival rate of the bio-crosslinked material product of Example 2 is still similar to that of the fresh Example 2, demonstrating excellent stability. Furthermore, the cell survival rate of Example 2 is significantly better than that of Comparative Examples 7 and 8, indicating that the bio-crosslinked material product of Example 2 can effectively promote the growth of human skin fibroblasts, and its injection into the skin can effectively promote skin health.

[0204] In summary, the bio-crosslinked material or bio-crosslinked material product of the present invention has excellent structural strength, namely, high crosslinking degree, low collagen degradation rate, and low hyaluronic acid degradation rate. Furthermore, the bio-crosslinked material or bio-crosslinked material product of the present invention, when used in combination with exosomes, can more effectively maintain skin health. [Simplified Explanation of the Diagram]

[0205] Figure 1 shows one embodiment of the preparation method of the biological mixture of the present invention. Figure 2 shows another embodiment of the preparation method of the biological mixture of the present invention. Figure 3 is a photograph of the biological mixture of the present invention prepared into an injection. [Biomaterial Storage]

[0207] None

Claims

1. A biological mixture comprising a biological cross-linking material, wherein the biological cross-linking material comprises collagen, collagen peptides, a first hyaluronic acid, and a second hyaluronic acid; wherein, The first hyaluronic acid has a molecular weight of 100 kilodaltons (kDa) to 500 kilodaltons, and the second hyaluronic acid has a molecular weight of 1,500 kilodaltons to 3,000 kilodaltons; and the collagen, the collagen peptide, the first hyaluronic acid and the second hyaluronic acid are cross-linked with each other, and the average degree of cross-linking of the biological cross-linking material is 65% to 97%.

2. The biological mixture as claimed in claim 1, wherein the total content of the collagen and collagen peptides is from 30 mg / ml to 80 mg / ml, based on the total volume of the biological crosslinking material.

3. The biological mixture as claimed in claim 1, wherein the total content of the first hyaluronic acid and the second hyaluronic acid is from 1 mg / ml to 18 mg / ml, based on the total volume of the biological crosslinking material.

4. The biological mixture as described in claim 1, further comprising exosomes.

5. The biological mixture as claimed in claim 4, wherein the concentration of the exosomes is from 1 × 10⁵ to 1 × 10¹¹ per milliliter, based on the total volume of the biological mixture.

6. Use of a biocrosslinked material as described in any one of claims 1 to 5 for the preparation of a skin tissue filling product.

7. The use as described in claim 6, wherein the dosage form of the skin tissue filling product includes an injection.

8. A method for preparing a biological mixture as described in any one of claims 1 to 5, comprising: a first cross-linking step: mixing collagen, the first hyaluronic acid, a first cross-linking agent, and a first carrier to obtain a first mixture, and reacting the mixture for 20 hours to 28 hours to obtain a first cross-linked complex; wherein, Based on the total volume of the first mixture, the collagen content is from 10% to 40% by weight volume, and the hyaluronic acid content is from 0.1% to 9% by weight volume. The second crosslinking step involves mixing the first crosslinking complex, the second hyaluronic acid, the second crosslinking agent, and the second carrier to obtain a second mixture. This mixture is then reacted for 20 to 28 hours to obtain a second crosslinking complex. The first crosslinking complex comprises 10 to 40% by weight (W / V) and the second hyaluronic acid comprises 0.1 to 9% by weight (W / V). The third crosslinking step involves mixing the second crosslinking complex, collagen peptides, the third crosslinking agent, and the third carrier to obtain a third mixture. This mixture is then reacted for 20 to 28 hours to obtain the bio-crosslinked material. The second crosslinking complex comprises 10 to 40% by weight (W / V) and the collagen peptide comprises 10 to 40% by weight (W / V) based on the total volume of the third mixture. The first, second, and third crosslinking agents are different crosslinking agents, and all three carriers contain water.

9. The method of preparation as claimed in claim 8, wherein the first crosslinking agent comprises 1,4-butanediol glycidyl ether, and the content of the first crosslinking agent is 0.5 to 5% by weight volume based on the total volume of the first mixture; the second crosslinking agent comprises glutaraldehyde, and the content of the second crosslinking agent is 0.005 to 0.1% by weight volume based on the total volume of the second mixture; and the third crosslinking agent comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the content of the third crosslinking agent is 0.5 to 5% by weight volume based on the total volume of the third mixture.

10. The method of preparation as described in claim 8, wherein either or a combination of the first crosslinking step and the third crosslinking step comprises adding an alkali agent.

Images