Biological mixtures comprising biologically crosslinked

Abstract

The invention provides a biological mixture containing a biological cross-linking material. The biological cross-linking material comprises collagen, collagen polypeptide, first hyaluronic acid and second hyaluronic acid; wherein the collagen, the collagen polypeptide, the first hyaluronic acid and the second hyaluronic acid are cross-linked with one another. The biological cross-linking material has high cross-linking degree, low collagen degradation rate and low hyaluronic acid degradation rate, and can prolong the retention time of the biological cross-linking material in a human body. The invention also provides an application and a preparation method of the biological mixture.

Description

Technical Field

[0001] The present invention relates to a biological mixture, particularly a biological mixture containing a biological crosslinking material. The present invention further relates to the use and preparation method of the biological mixture. Background Art

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

[0003] Currently, there are various medical aesthetic products on the market, such as hyaluronic acid injectables and collagen injectables. These injectables belong to filler injections. When there are depressions, wrinkles or looseness on the superficial skin due to aging, hyaluronic acid or collagen is subcutaneously injected to adjust the skin appearance or shape. After hyaluronic acid and collagen are injected into the human body, they are easily decomposed by the human body and need to be administered regularly. Therefore, how to effectively increase the residence time of such materials in the human body is an important issue. Summary of the Invention

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

[0005] According to the present invention, the biological crosslinking material or the biological crosslinking material product has excellent structural strength, that is, a high crosslinking degree, a low collagen degradation rate and a low hyaluronic acid degradation rate, so as to increase the residence time of the biological crosslinking material containing hyaluronic acid and collagen in the human body, thereby effectively reducing the frequency of an individual's need to supplement hyaluronic acid and collagen.

[0006] In one embodiment, the biological mixture contains water. The biological mixture can be freeze-dried 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 rehydrated before use, for example, adding water or a pharmaceutically acceptable carrier, such as phosphate buffered saline (PBS).

[0007] Specifically, the structure of the collagen mainly consists of an amino acid sequence of glycine - hydroxylysine - hydroxyproline, but is not limited thereto. In addition, the components of the collagen polypeptide are also rich in glycine, hydroxylysine, and hydroxyproline, but are not limited thereto. Since collagen, collagen polypeptide, the first hyaluronic acid, and the second hyaluronic acid are not single - component or single - molecular - weight compounds, the biological mixture contains the biological cross - linked material formed by cross - linking the four types of components in various bonding ways.

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

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

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

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

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

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

[0014] Preferably, the average cross - linking degree of the biological cross - linked material is from 66% to 96%, for example: 66%, 70%, 75%, 80%, 85%, 90%, 95%, or 96%. More preferably, the average cross - linking degree of the biological cross - linked material is from 72% to 78%.

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

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

[0017] In one embodiment, based on the total volume of the biological cross-linked material, the total content of collagen and collagen polypeptides is 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 biological cross-linked material, the total content of collagen and collagen polypeptides is 35 mg / ml to 45 mg / ml.

[0018] In one embodiment, based on the total volume of the biological cross-linked material, the total content of the first hyaluronic acid and the second hyaluronic acid is 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 biological cross-linked material, the total content of the first hyaluronic acid and the second hyaluronic acid is 4.5 mg / ml to 5.5 mg / ml.

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

[0020] In one embodiment, based on the total volume of the biological mixture, the concentration of the exosomes is 1×10 5 particles to 1×10 11 particles, for example: 1×10 5 particles / ml, 5×10 5 particles / ml, 1×10 6 particles / ml, 5×10 6 particles / ml, 1×10 7 particles / ml, 5×10 7 particles / ml, 1×10 8 particles / ml, 5×10 8 particles / ml, 1×10 9 particles / ml, 5×10 9particles / mL, 1×10 10 particles / mL, 5×10 10 particles / mL or 1×10 11 particles / mL. Preferably, based on the total volume of the biological mixture, the concentration of the exosomes is 8×10 6 particles / mL to 1.2×10 7 particles / mL.

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

[0022] In one embodiment, the dosage form of the skin tissue filling product includes an injection. In one embodiment, the injection is a filling injection for microplastic surgery.

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

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

[0025] In one embodiment, the product includes a drug, a cosmetic, or a medical device. Preferably, the product includes a beauty drug, a medical cosmetic, or a beauty medical device.

[0026] The present invention further provides a method for preparing the biological mixture, comprising:

[0027] First cross-linking step: Mix collagen, the first hyaluronic acid, a first cross-linking agent, and a first carrier to obtain a first mixed solution, and react for 20 hours to 28 hours to obtain a first cross-linked complex; wherein, based on the total volume of the first mixed solution, the content of collagen is 10 weight / volume percentage (w / v%) to 40 weight / volume percentage, and the content of the first hyaluronic acid is 0.1 weight / volume percentage to 9 weight / volume percentage;

[0028] Second cross-linking step: Mix the first cross-linked complex, a second hyaluronic acid, a second cross-linking agent, and a second carrier to obtain a second mixed solution, and react for 20 hours to 28 hours to obtain a second cross-linked complex; wherein, based on the total volume of the second mixed solution, the content of the first cross-linked complex is 10 weight / volume percentage to 40 weight / volume percentage, and the content of the second hyaluronic acid is 0.1 weight / volume percentage to 9 weight / volume percentage; and

[0029] Third crosslinking step: After mixing the second crosslinked complex, collagen polypeptide, third crosslinking agent and third carrier, a third mixed solution is obtained, and reacted for 20 to 28 hours to obtain the biological crosslinked material; wherein, based on the total volume of the third mixed solution, the content of the second crosslinked complex is 10 wt% to 40 wt%, and the content of the collagen polypeptide is 10 wt% to 40 wt%; wherein,

[0030] The first crosslinking agent, the second crosslinking agent and the third crosslinking agent are different crosslinking agents, and the first carrier, the second carrier and the third carrier all contain water.

[0031] Preferably, any one or a combination of the first carrier, the second carrier and the third carrier contains phosphate buffered saline (PBS).

[0032] In one embodiment, the collagen is from a collagen solution, and the collagen concentration in the collagen solution is 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 10.5 mg / ml to 11.5 mg / ml.

[0033] In one embodiment, the collagen polypeptide is from a collagen polypeptide solution, and the concentration of the collagen polypeptide in the collagen polypeptide solution is 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 polypeptide in the collagen polypeptide solution is 26 mg / ml to 30 mg / ml.

[0034] Preferably, based on the total volume of the first mixture, the content of the collagen is 15 wt% to 35 wt%, such as: 15 wt%, 19 wt%, 23 wt%, 27 wt%, 31 wt% or 35 wt%, and the content of the first hyaluronic acid is 0.2 wt% to 8 wt%, such as: 0.2 wt%, 0.5 wt%, 1 wt%, 3 wt%, 6 wt% or 8 wt%. Preferably, based on the total volume of the first mixture, the content of the collagen is 18 wt% to 22 wt%, and the content of the first hyaluronic acid is 0.8 wt% to 1.2 wt%. More preferably, the rest is phosphate buffered saline and the first crosslinking agent.

[0035] Preferably, based on the total volume of the second mixture, the content of the first crosslinked complex is 15 wt% to 35 wt%, such as: 15 wt%, 19 wt%, 23 wt%, 27 wt%, 31 wt% or 35 wt%, and the content of the second hyaluronic acid is 0.2 wt% to 8 wt%, such as: 0.2 wt%, 0.5 wt%, 1 wt%, 3 wt%, 6 wt% or 8 wt%. Preferably, based on the total volume of the second mixture, the content of the first crosslinked complex is 18 wt% to 22 wt%, and the content of the second hyaluronic acid is 0.8 wt% to 1.2 wt%. More preferably, the rest is phosphate buffered saline and the second crosslinking agent.

[0036] Preferably, based on the total volume of the third mixture, the content of the second crosslinked complex is 15 wt% to 35 wt%, such as: 15 wt%, 19 wt%, 23 wt%, 27 wt%, 31 wt% or 35 wt%, and the content of the collagen polypeptide is 15 wt% to 35 wt%, such as: 15 wt%, 19 wt%, 23 wt%, 27 wt%, 31 wt% or 35 wt%. Preferably, based on the total volume of the third mixture, the content of the second crosslinked complex is 18 wt% to 22 wt%, and the content of the collagen polypeptide is 18 wt% to 22 wt%. More preferably, the rest is phosphate buffered saline and the third crosslinking agent.

[0037] In one embodiment, the raw material source of any one or a combination of the collagen solution and the collagen polypeptide solution includes any one or a combination of pigskin, cowhide, and sheepskin. Preferably, the raw material source includes cowhide.

[0038] In one embodiment, the collagen in the collagen solution is any one or a combination of porcine collagen, bovine collagen, and ovine collagen. Preferably, the collagen in the collagen solution is bovine collagen.

[0039] In one embodiment, the first crosslinking agent includes 1,4-butanediol diglycidyl ether (BDDE).

[0040] In one embodiment, based on the total volume of the first mixture, the content of the first crosslinking agent is 0.5 wt% to 5 wt%, for example: 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, or 5 wt%. Preferably, based on the total volume of the first mixture, the content of the first crosslinking agent is 1.8 wt% to 2.2 wt%.

[0041] In one embodiment, the second crosslinking agent includes glutaraldehyde (GA).

[0042] In one embodiment, based on the total volume of the second mixture, the content of the second crosslinking agent is 0.005 wt% to 0.1 wt%, for example: 0.005 wt%, 0.01 wt%, 0.04 wt%, 0.06 wt%, 0.08 wt%, or 0.1 wt%. Preferably, based on the total volume of the second mixture, the content of the second crosslinking agent is 0.009 wt% to 0.011 wt%.

[0043] In one embodiment, the third crosslinking agent includes 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).

[0044] In one embodiment, based on the total volume of the third mixed solution, the content of the third crosslinking agent is 0.5 wt% to 5 wt%, for example: 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt% or 5 wt%. Preferably, based on the total volume of the third mixed solution, the content of the third crosslinking agent is 2.7 wt% to 3.3 wt%.

[0045] In one embodiment, any one or a combination of the first crosslinking step and the third crosslinking step includes adding an alkali agent. Preferably, the alkali agent includes sodium hydroxide.

[0046] In one embodiment, the alkali agent is a pH adjuster.

[0047] In one embodiment, any one or a combination of the first mixed solution in the first crosslinking step and the third mixed solution in the third crosslinking step is alkaline. Preferably, the pH value of any one or a combination of the first mixed solution in the first crosslinking step and the third mixed solution in the third crosslinking step is 8 to 12, for example: 8, 9, 10, 11 or 12. More preferably, the pH value of any one or a combination of the first mixed solution in the first crosslinking step and the third mixed solution in the third crosslinking step is 9.8 to 10.2.

[0048] 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, after the first precipitation step of the first crosslinking step, a first precipitate is obtained; after the second precipitation step of the second crosslinking step, a second precipitate is obtained; and after the third precipitation step of the third crosslinking step, a third precipitate is obtained. Preferably, the precipitation step includes adding an aqueous sodium chloride solution and centrifuging at 10000×g to 15000×g for 5 minutes 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 minutes to 12 minutes. The precipitate mainly contains the crosslinked complex generated in the crosslinking step.

[0049] 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, after the first precipitate is subjected to the first washing step of the first crosslinking step, the first crosslinked complex is obtained; after the second precipitate is subjected to the second washing step of the second crosslinking step, the second crosslinked complex is obtained; and after the third precipitate is subjected to the third washing step of the third crosslinking step, the biocompatible crosslinked material is obtained. Preferably, the washing step includes adding an aqueous ethanol solution and centrifuging at 10,000×g to 15,000×g for 5 minutes to 20 minutes. More preferably, the washing step includes adding an aqueous ethanol solution and centrifuging at 12,000×g to 13,000×g for 8 minutes to 12 minutes.

[0050] In one embodiment, the method for preparing the biocompatible mixture further includes a crosslinking neutralization step: mixing the biocompatible crosslinked material and an aqueous glycine solution to obtain a fourth mixture, reacting for 20 hours to 28 hours to obtain a biocompatible crosslinked material product. According to the present invention, the crosslinking neutralization step can ensure that the biocompatible crosslinked material no longer continues to crosslink, so as to improve the stability of the product, and there is no substantial difference in weight between the biocompatible crosslinked material and the biocompatible crosslinked material product, that is, the weight difference is within 1%.

[0051] Preferably, the concentration of the aqueous glycine solution is 0.05 M to 1 M. Preferably, the concentration of the aqueous glycine solution is 0.08 M to 0.12 M.

[0052] In one embodiment, the crosslinking neutralization step includes a precipitation step to obtain a precipitate, that is, after the crosslinking neutralization step, a fourth precipitate is obtained. Preferably, the precipitation step includes centrifuging at 10,000×g to 15,000×g for 1 minute to 10 minutes. More preferably, the precipitation step includes centrifuging at 12,000×g to 13,000×g for 2.5 minutes to 3.5 minutes.

[0053] In one embodiment, the crosslinking neutralization step includes a washing step to obtain a washed precipitate, that is, after the fourth precipitate is subjected to the fourth washing step of the crosslinking neutralization step, the biocompatible crosslinked material product is obtained. Preferably, the washing step includes adding phosphate buffered saline and centrifuging at 10,000×g to 15,000×g for 1 minute to 10 minutes. More preferably, the washing step includes adding phosphate buffered saline and centrifuging at 12,000×g to 13,000×g for 2.5 minutes to 3.5 minutes.

[0054] In summary, the biological mixture containing the biological cross-linking material of the present invention is a bionic tissue repair material. In addition to being applicable to subcutaneous tissue filling and wrinkle reduction, it is also applicable to medical applications such as tissue repair, scar repair, and tissue reconstruction, and has good biocompatibility, which can reduce allergic or inflammatory reactions. In addition, the biological cross-linking material can be used in combination with exosomes to accelerate tissue repair and further promote human tissue health. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is an aspect of the preparation method of the biological mixture of the present invention.

[0056] Figure 2 This is another aspect of the preparation method of the biological mixture of the present invention.

[0057] Figure 3 This is a photo of the biological mixture of the present invention made into an injection. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0058] The following provides several embodiments to illustrate the implementation manners 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 make various modifications and changes without departing from the spirit of the present invention to implement or apply the content of the present invention.

[0059] I. Raw materials:

[0060] (I) Collagen solution

[0061] 1. Steps: Add 6 grams (g) of porcine epidermal fragments (size: 1 cm × 1 cm × 0.04 cm) and 600 milligrams (mg) of pepsin to 400 milliliters (ml) of 0.01N hydrochloric acid aqueous solution, and then stir at room temperature for 12 hours with a magnetic stirrer (rotation speed: 250 rpm) to carry out a decomposition reaction. Filter using a square filter cloth of a steamer cloth (mesh number: about 1800 to 1900) to obtain the collagen solution.

[0062] 2. Analysis: This analysis measures the concentration of the collagen solution. Among them, hydroxyproline (Hypro) mainly exists in connective tissue collagens, collagen, and elastin, and the primary structure sequence of collagen is mostly a repeating sequence of three amino acid fragments of "glycine-hydroxylysine-hydroxyproline". Therefore, the generally recognized method for collagen quantification is to measure the content of "hydroxyproline". This invention refers to the method described in the journal "A.L. 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 as follows:

[0063] (1) Standard: This invention uses hydroxyproline standards to prepare hydroxyproline solutions with different concentrations, and uses a spectrophotometer to measure the absorbance at a wavelength of 555 nanometers (nm) to plot a standard curve of absorbance against hydroxyproline concentration.

[0064] (2) Collagen solution: After freeze-drying the collagen solution, a lyophilized powder is obtained, and hydrolysis reaction is carried out at 100 °C for 16 hours with concentrated hydrochloric acid to obtain a hydrolysate. The hydrolysate is diluted 50 times with distilled water and then further diluted with isopropanol, and the added amount of isopropanol is 2 times that of distilled water to obtain a dilution. The dilution is further added with Chloramine T reagent, citrate buffer, distilled water, Ehrlich’s reagent, perchloric acid, and isopropanol and then homogenized, and reacted at 70 °C for 10 minutes. The absorbance at a wavelength of 555 nanometers is measured with a spectrophotometer, and combined with the standard curve to obtain the concentration of hydroxyproline.

[0065] Furthermore, the journal "N.Yu. Ignat’eva et al., 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 in different collagens, the proportion of hydroxyproline is generally 12.8% to 14.7%. Therefore, 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. Thus, after the concentration of the hydroxyproline is corrected by the dilution factor, another conversion is performed: Collagen concentration (mg / ml) = (Hydroxyproline concentration (mg / ml) × 7.69).

[0066] 3. Results: The collagen concentration in the collagen solution is approximately 11.1 mg / ml.

[0067] (II) Collagen polypeptide solution

[0068] 1. Steps: Similar to the steps for obtaining the collagen solution, the only difference is that the decomposition reaction time is 72 hours instead of 12 hours. In addition, due to the longer decomposition reaction time, the main product of the collagen polypeptide solution is collagen polypeptide further decomposed from collagen.

[0069] 2. Analysis: The measurement method and calculation method for the collagen polypeptide concentration in the collagen polypeptide solution are the same as those for the collagen solution, which are also obtained by measuring the concentration of hydroxyproline and then performing further conversion.

[0070] 3. Results: The collagen polypeptide concentration in the collagen polypeptide solution is approximately 28.3 mg / ml.

[0071] (III) First hyaluronic acid

[0072] Purchased from Pei Shi Biotechnology in Taiwan, China, with the product name HA - LM100 and an average molecular weight of approximately 100 kDa.

[0073] (IV) Second hyaluronic acid

[0074] Purchased from Pei Shi Biotechnology in Taiwan, China, with the product name HA - LM2000 and an average molecular weight of approximately 1500 kDa.

[0075] II. Example 1: Biological mixture containing a biological cross - linking material

[0076] As Figure 1As shown, the preparation method of the biological cross-linked material includes: Step S1: the first cross-linking step, Step S2: the second cross-linking step, and Step S3: the third cross-linking step to obtain the biological cross-linked material of the present invention, which belongs to a biological mixture. In addition, as Figure 2 shown, the biological cross-linked material of the present invention also undergoes Step S4: the cross-linking neutralization step to obtain a biological cross-linked material product, which is described as follows.

[0077] (I) The first cross-linking step:

[0078] 1. The first mixing step: Add a collagen solution, the first hyaluronic acid, the first cross-linking agent: 1,4-butanediol diglycidyl ether (BDDE), and sodium hydroxide (NaOH) to phosphate buffered saline (PBS) to obtain a first mixed solution; wherein, the collagen solution contains collagen, the pH of the first mixed solution is 10, and based on the total volume of the first mixed solution, 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%, and the rest is phosphate buffered saline. Specifically, the content of collagen being 20 w / v% (i.e., 20 g / 100 ml) means that in every 100 milliliters of the first mixed solution, it is equivalent to containing 20 grams of collagen (excluding the weight of water, that is, equivalent to the weight of the freeze-dried powder), and the content of the first hyaluronic acid is also not counted as the weight of water.

[0079] 2. The first reaction step: Stir the first mixed solution at room temperature (about 28°C) (rotation speed of 250 rpm) for 24 hours to obtain a first cross-linked solution.

[0080] 3. The first precipitation step: Add an aqueous sodium chloride (NaCl) solution with the same volume and a concentration of 2 M (i.e., 2 mol / L) as the first cross-linked solution to the first cross-linked solution, and remove the supernatant after centrifugation at 12,500×g for 10 minutes to obtain a first precipitate.

[0081] 4. The first washing step: Add an appropriate volume of 60% aqueous ethanol solution to the first precipitate, and remove the supernatant after centrifugation at 12,500×g for 10 minutes, for a total of 5 times, to obtain a first cross-linked complex; wherein, the first cross-linked complex is a thick aqueous mixture. In addition, the appropriate volume of 60% aqueous ethanol solution means that about 1 ml of 60% aqueous ethanol solution is added to 1 gram of the first precipitate.

[0082] (II) The second cross-linking step:

[0083] 1. Second mixing step: Add the first cross-linked complex, the second hyaluronic acid, and the second cross-linking agent: glutaraldehyde (GA) into PBS to obtain a second mixed solution; wherein, the pH of the second mixed solution is 7.2, and based on the total volume of the second mixed solution, the content of the first cross-linked 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%, and the rest is phosphate buffered saline.

[0084] 2. Second reaction step: After stirring the second mixed solution at room temperature (rotation speed: 250 rpm) for 24 hours, a second cross-linked solution is obtained.

[0085] 3. Second precipitation step: Similar to the first precipitation step, the only difference is that the precipitation target is the second cross-linked solution, not the first cross-linked solution. In other words, add an aqueous sodium chloride solution with the same volume and a concentration of 2 M as the second cross-linked solution into the second cross-linked solution, and after centrifuging at 12,500×g for 10 minutes, remove the supernatant to obtain a second precipitate.

[0086] 4. Second washing step: Similar to the first washing step, the only difference is that the washing target is the second precipitate, not the first precipitate, and the number of washing times is 3 times, not 5 times. In other words, add an appropriate volume of 60% aqueous ethanol solution into the second precipitate, and after centrifuging at 12,500×g for 10 minutes, remove the supernatant, and perform this operation 3 times to obtain a second cross-linked complex; wherein, the second cross-linked complex is a thick aqueous mixture.

[0087] (III) Third cross-linking step:

[0088] 1. Third mixing step: Add the second cross-linked complex, the collagen polypeptide solution, the third cross-linking agent: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and sodium hydroxide into PBS to obtain a third mixed solution; wherein, the pH of the third mixed solution is 10, and based on the total volume of the third mixed solution, the content of the second cross-linked complex is 20 w / v%, the content of the collagen polypeptide solution is 20 w / v%, and the content of the third cross-linking agent (EDC) is 3 w / v%, and the rest is phosphate buffered saline.

[0089] 2. Third reaction step: After stirring the third mixed solution at 40°C (rotation speed: 250 rpm) for 24 hours, a third cross-linked solution is obtained.

[0090] 3. Third precipitation step: Similar to the first precipitation step, with the only difference being that the precipitation target is the third crosslinked solution instead of the first crosslinked solution. The third precipitation step can obtain a third precipitate.

[0091] 4. Third washing step: Similar to the first washing step, with the only differences being that the washing target is the third precipitate instead of the first precipitate, and the number of washings is 3 times instead of 5 times. The third washing step can obtain the biocrosslinked material, which is a biological mixture; wherein, the biocrosslinked material is a thick aqueous mixture.

[0092] (IV) Crosslinking neutralization step:

[0093] 1. Fourth mixing step: Add the biocrosslinked material to an aqueous glycine solution with a volume 10 times that of the biocrosslinked material and a concentration of 0.1 M to obtain a fourth mixture.

[0094] 2. Fourth reaction step: After gently stirring the fourth mixture at room temperature (rotation speed of 250 rpm) for 24 hours, a fourth crosslinked solution is obtained.

[0095] 3. Fourth precipitation step: Centrifuge the fourth crosslinked solution at 12500×g for 3 minutes and then remove the supernatant to obtain a fourth precipitate.

[0096] 4. Fourth washing step: Add the fourth precipitate to PBS with the same volume and containing sodium hydroxide (pH = 9), centrifuge at 12500×g for 3 minutes and then remove the supernatant, for a total of 3 times, to obtain the biocrosslinked material product; wherein, the biocrosslinked material product is a thick aqueous mixture, and its wet weight has no substantial difference from the wet weight of the biocrosslinked material. The crosslinking neutralization step is not a necessary step for manufacturing the biocrosslinked material of the present invention.

[0097] Furthermore, add the biological mixture containing the biocrosslinked material of Example 1 to PBS containing 0.35 w / v% lidocaine hydrochloride and fill it into a syringe to make an injection; wherein, based on the total volume of the injection content, the total concentration of collagen and collagen polypeptide is about 40 mg / ml, and a photo of the injection is as Figure 3 shown.

[0098] III. Analysis of the monomer ratio of the biocrosslinked material of Example 1

[0099] The monomers of the biological cross-linked material of Example 1 include collagen from the collagen solution, collagen polypeptides from the collagen polypeptide solution (the components are from collagen), the first hyaluronic acid, and the second hyaluronic acid. Regarding the part of analyzing the collagen and collagen polypeptide content, the measuring method and calculation method of the concentration of collagen and collagen polypeptides contained in the biological cross-linked material of Example 1 are the same as those of the "collagen solution" (that is, first measure the concentration of "hydroxyproline" and then convert it to the collagen concentration). The calculation result is 40 mg / ml, that is, the total concentration of collagen and collagen polypeptides in the biological cross-linked material of Example 1 is 40 mg / ml.

[0100] Regarding the part of analyzing the content of the first hyaluronic acid and the second hyaluronic acid, after freeze-drying 1 ml of the biological cross-linked material of Example 1, a freeze-dried powder is obtained. After weighing the freeze-dried powder and subtracting the content of collagen and collagen polypeptides obtained by the above conversion, the weight of hyaluronic acid per milliliter can be obtained to obtain the hyaluronic acid concentration. The calculation result is 4.8 mg / ml, that is, the total concentration of the first hyaluronic acid and the second hyaluronic acid in the biological cross-linked material of Example 1 is 4.8 mg / ml.

[0101] It should be specifically noted that the "total concentration of collagen and collagen polypeptides (40 mg / ml)" and the "total concentration of the first hyaluronic acid and the second hyaluronic acid (4.8 mg / ml)" in the biological cross-linked material of Example 1 are both calculation results without taking into account the water content. Therefore, the numerator in the sum of the two concentrations (40 + 4.8 = 44.8 mg / ml) is not the wet weight of each milliliter of the biological cross-linked material (a thick water-containing mixture).

[0102] IV. Example 2: Combined use of biological cross-linked material and exosomes

[0103] The biological cross-linked material of Example 1 and the exosome solution (a concentrated adipose-derived stem cell exosome solution, named Exo-ASC, purchased from Pei Shi Biotechnology in Taiwan, China) are 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 biological cross-linked material is 4 w / v%, and in the fifth mixture, the concentration of the exosomes (i.e., adipose-derived stem cell exosomes) is about 1×10 7 particles (EV) / ml.

[0104] V. Examples 3 to 5: Biological cross-linked materials

[0105] Examples 3 to 5 are similar to Example 1, except for the different sources of collagen, which are described as follows: The raw materials of the collagen solution and the collagen polypeptide solution in Example 1 are all pigskins. The source of collagen in Example 3 includes pigskin and cowhide, and the raw material weights of pigskin and cowhide are 1:1; the source of collagen in Example 4 includes pigskin and sheepskin (after defleecing), and the raw material weights of pigskin and sheepskin are 1:1; and the source of collagen in Example 5 includes cowhide and sheepskin, and the raw material weights of cowhide and sheepskin are 1:1. In other words, the differences in the sources of collagen in each group are shown in Table 1.

[0106] Table 1: Sources of Collagen in Each Group

[0107] Group Collagen source Example 1 Pig skin Example 2 Pig skin Example 3 The raw material weights of pig skin and cowhide are 1:1 Example 4 The raw material weights of pig skin and sheepskin are 1:1 Example 5 The raw material weights of cowhide and sheepskin are 1:1

[0108] VI. Comparative Examples 1 to 11: Biologically Crosslinked Material Products (or in combination with exosomes)

[0109] The key differences in the preparation methods of each group are shown in Table 2. The preparation methods of each group are described in sequence as follows.

[0110] Table 2: Preparation Methods of Each Group

[0111]

[0112]

[0113] (I) Comparative Example 1: Four raw materials (collagen solution, collagen polypeptide solution, first hyaluronic acid, and second hyaluronic acid) are crosslinked together once, and three crosslinking agents (BDDE, GA, and EDC) are used simultaneously, which are described as follows:

[0114] 1. Crosslinking step:

[0115] (1) Mixing step: Add the collagen solution, collagen polypeptide solution, first hyaluronic acid, second hyaluronic acid, first crosslinking agent (BDDE), second crosslinking agent (GA), third crosslinking agent (EDC), and sodium hydroxide to PBS to obtain a mixed solution; wherein, the pH of the mixed solution is 10, and based on the total volume of the mixed solution, the content of the collagen solution is 20 w / v%, the content of the collagen polypeptide solution is 20 w / v%, the content of the first hyaluronic acid is 1 w / v%, the content of the second hyaluronic acid is 1 w / v%, the content of the first crosslinking agent (BDDE) is 2 w / v%, the content of the second crosslinking agent (GA) is 0.01 w / v%, and the content of the third crosslinking agent (EDC) is 3 w / v% to obtain the mixed solution of Comparative Example 1.

[0116] (2) Reaction step: After stirring the mixture of Comparative Example 1 at room temperature (rotation speed: 250 rpm) for 24 × 3 = 72 hours, a cross-linked solution of Comparative Example 1 was obtained.

[0117] (3) Precipitation step: Similar to the first precipitation step of Example 1, except that the precipitation target was the cross-linked solution of Comparative Example 1 to obtain the precipitate of Comparative Example 1.

[0118] (4) Washing step: Similar to the first washing step of Example 1, except that the washing target was the precipitate of Comparative Example 1 to obtain the cross-linked complex of Comparative Example 1.

[0119] 2. Cross-linking neutralization step: Similar to the cross-linking neutralization step of Example 1, except that the cross-linking neutralization target was the cross-linked complex of Comparative Example 1 to obtain the cross-linked product of Comparative Example 1.

[0120] (2) Comparative Example 2: Four raw materials (collagen solution, collagen polypeptide solution, first hyaluronic acid, and second hyaluronic acid) were cross-linked in three times, but the cross-linking agents for the three cross-linkings were the same, all being BDDE.

[0121] 1. First cross-linking step: The same as the first cross-linking step of Example 1.

[0122] 2. Second cross-linking step: Similar to the second cross-linking step of Example 1, except that the second cross-linking agent was BDDE instead of GA, and based on the total volume of the second mixture, the content of the second cross-linking agent (BDDE) was 2 w / v% to obtain the second cross-linked complex of Comparative Example 2.

[0123] 3. Third cross-linking step: Similar to the third cross-linking step of Example 1, except that (1) the third cross-linking agent was BDDE instead of EDC, and based on the total volume of the third mixture, the content of the third cross-linking agent (BDDE) was 2 w / v%; and (2) the target of the third cross-linking step was the second cross-linked complex of Comparative Example 2 to obtain the cross-linked material of Comparative Example 2.

[0124] 4. Cross-linking neutralization step: Similar to the cross-linking neutralization step of Example 1, except that the cross-linking neutralization target was the cross-linked complex of Comparative Example 2 to obtain the cross-linked product of Comparative Example 2.

[0125] (3) Comparative Example 3: Four raw materials (collagen solution, collagen polypeptide solution, first hyaluronic acid, and second hyaluronic acid) were cross-linked in three times, but the cross-linking agents for the three cross-linkings were the same, all being GA.

[0126] 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 based on the total volume of the first mixed solution, the content of the first crosslinking agent (GA) is 0.01 w / v%, to obtain the first crosslinked complex of Comparative Example 3.

[0127] 2. Second crosslinking step: Similar to the second crosslinking step of Example 1, except that the second crosslinking target is the first crosslinked complex of Comparative Example 3, to obtain the second crosslinked complex of Comparative Example 3.

[0128] 3. Third crosslinking step: Similar to the third crosslinking step of Example 1, except that (1) the third crosslinking agent is GA instead of EDC, and based on the total volume of the third mixed solution, the content of the third crosslinking agent (BDDE) is 0.01 w / v%; and (2) the target of the third crosslinking step is the second crosslinked complex of Comparative Example 3, to obtain the crosslinked material of Comparative Example 3.

[0129] 4. Crosslinking neutralization step: Similar to the crosslinking neutralization step of Example 1, except that the crosslinking neutralization target is the crosslinked complex of Comparative Example 3, to obtain the crosslinked product of Comparative Example 3.

[0130] (IV) Comparative Example 4: Four raw materials (collagen solution, collagen polypeptide solution, the first hyaluronic acid, and the second hyaluronic acid) are crosslinked in three times, but the crosslinking agents for the three crosslinkings are the same, all being EDC.

[0131] 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 based on the total volume of the first mixed solution, the content of the first crosslinking agent (EDC) is 3 w / v%, to obtain the first crosslinked complex of Comparative Example 4.

[0132] 2. Second crosslinking step: Similar to the second crosslinking step of Example 1, except that (1) the second crosslinking agent is EDC instead of GA, and based on the total volume of the second mixed solution, the content of the second crosslinking agent (EDC) is 3 w / v%; and (2) the target of the third crosslinking step is the first crosslinked complex of Comparative Example 4, to obtain the second crosslinked complex of Comparative Example 4.

[0133] 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 crosslinked complex of Comparative Example 4, to obtain the crosslinked material of Comparative Example 4.

[0134] 4. Crosslinking neutralization step: Similar to the crosslinking neutralization step of Example 1, except that the crosslinking neutralization target is the crosslinked complex of Comparative Example 4, to obtain the crosslinked product of Comparative Example 4.

[0135] (5) Comparative Example 5: Two raw materials (collagen solution and collagen polypeptide solution) are crosslinked together once, and the crosslinking agent is GA, which is described as follows:

[0136] 1. Crosslinking step:

[0137] (1) Mixing step: Add the collagen solution, collagen polypeptide solution, crosslinking agent (GA), and sodium hydroxide into PBS to obtain a mixed solution; wherein, the pH of the mixed solution is 10, and based on the total volume of the mixed solution, the content of the collagen solution is 20 w / v%, the content of the collagen polypeptide solution is 20 w / v%, and the content of the crosslinking agent (GA) is 0.01 w / v% to obtain the mixed solution of Comparative Example 5.

[0138] (2) Reaction step: Stir the mixed solution of Comparative Example 5 at room temperature (rotation speed is 250 rpm) for 24×3 = 72 hours to obtain the crosslinked solution of Comparative Example 5.

[0139] (3) Precipitation step: Similar to the first precipitation step of Example 1, the difference is only that the precipitation target is the crosslinked solution of Comparative Example 5 to obtain the precipitate of Comparative Example 5.

[0140] (4) Washing step: Similar to the first washing step of Example 1, the difference is only that the washing target is the precipitate of Comparative Example 5 to obtain the crosslinked complex of Comparative Example 5.

[0141] 2. Crosslinking neutralization step: Similar to the crosslinking neutralization step of Example 1, the difference is only that the crosslinking neutralization target is the crosslinked complex of Comparative Example 5 to obtain the crosslinked product of Comparative Example 5.

[0142] (6) Comparative Example 6: Two raw materials (the first hyaluronic acid and the second hyaluronic acid) are crosslinked together once, and the crosslinking agent is BDDE, which is described as follows:

[0143] 1. Crosslinking step:

[0144] (1) Mixing step: Add the first hyaluronic acid, the second hyaluronic acid, the crosslinking agent (BDDE), and sodium hydroxide into PBS to obtain a mixed solution; wherein, the pH of the mixed solution is 10, and based on the total volume of the mixed solution, 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 mixed solution of Comparative Example 6.

[0145] (2) Reaction step: Stir the mixed solution of Comparative Example 6 at room temperature (rotation speed is 250 rpm) for 24×3 = 72 hours to obtain the crosslinked solution of Comparative Example 6.

[0146] (3) Precipitation step: Similar to the first precipitation step of Example 1, with the only difference being that the precipitation target is the crosslinked solution of Comparative Example 6 to obtain the precipitate of Comparative Example 6.

[0147] (4) Washing step: Similar to the first washing step of Example 1, with the only difference being that the washing target is the precipitate of Comparative Example 6 to obtain the crosslinked complex of Comparative Example 6.

[0148] 2. Crosslinking and neutralization step: Similar to the crosslinking and neutralization step of Example 1, with the only difference being that the crosslinking and neutralization target is the crosslinked complex of Comparative Example 6 to obtain the crosslinked product of Comparative Example 6.

[0149] (VII) Comparative Example 7: Comparative Example 7 is obtained by further adding exosomes to Comparative Example 5, and the description is as follows: The preparation method is similar to that of Example 2, with the only difference being that the crosslinked product of Comparative Example 5 is used instead of the biocompatible crosslinking 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-derived stem cell exosomes) is also approximately 1×10 7 particles (EV) / ml.

[0150] (VIII) Comparative Example 8: Comparative Example 8 is obtained by further adding exosomes to Comparative Example 6, and the description is as follows: The preparation method is similar to that of Example 2, with the only difference being that the crosslinked product of Comparative Example 6 is used instead of the biocompatible crosslinking 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-derived stem cell exosomes) is also approximately 1×10 7 particles (EV) / ml.

[0151] (IX) Comparative Example 9: The preparation method of Comparative Example 9 is similar to that of Comparative Example 3, with the only difference being that the collagen source is pigskin and cowhide, and the raw material weights of the two are 1:1.

[0152] (IX) Comparative Example 10: The preparation method of Comparative Example 10 is similar to that of Comparative Example 3, with the only difference being that the collagen source is pigskin and sheepskin, and the raw material weights of the two are 1:1.

[0153] (IX) Comparative Example 11: The preparation method of Comparative Example 11 is similar to that of Comparative Example 3, with the only difference being that the collagen source is cowhide and sheepskin, and the raw material weights of the two are 1:1.

[0154] Analysis 1: Crosslinking degree, collagen degradation rate, and hyaluronic acid degradation rate

[0155] This analysis aims to understand the structural strength of the biocompatible crosslinking material product. Therefore, the crosslinking degree, collagen degradation rate, and hyaluronic acid degradation rate of Example 1, Examples 3 to 5, Comparative Examples 1 to 6, and Comparative Examples 9 to 11 are compared, and the description is as follows.

[0156] (1) Crosslinking degree analysis

[0157] Trinitrobenzenesulfonic acid (TNBS) can bind to the free amino groups in proteins to form a yellow substance, and the absorbance value at a wavelength of 345 nm is measured using a spectrophotometer for quantification. Therefore, the TNBS method is used in this invention to detect free amino groups and calculate the crosslinking degree accordingly. The details are as follows:

[0158] 1. Sample preparation:

[0159] (1) Test sample: The lyophilized powder obtained by lyophilizing the biological crosslinked material products of each group is used as the sample of the test product.

[0160] (2) Control sample: The lyophilized powder of the raw materials of each group itself is used as the control sample. Taking Example 1 as an example, 4 raw materials are mixed: collagen solution, collagen polypeptide solution, the first hyaluronic acid, and the second hyaluronic acid, with a weight ratio of 20:20:1:1, to obtain a raw material mixture, which is then lyophilized to obtain the lyophilized powder of the raw materials as the sample. Additionally, taking Comparative Example 5 as an example, 2 raw materials are mixed: collagen solution and collagen polypeptide solution, with a weight ratio of 20:20, to obtain a raw material mixture, which is then lyophilized to obtain the lyophilized powder of the raw materials as the sample.

[0161] 2. Analysis steps:

[0162] (1) TNBS method: Take 1 mg of the sample and add it to 100 μl of a 0.1% TNBS aqueous solution, place it in a dry bath, and set the reaction temperature at 50 °C for 1 hour. Then, add 300 μl of a 6N hydrochloric acid aqueous solution, set the reaction temperature at 60 °C for 1.5 hours, and then cool it to room temperature to obtain the test solution. Take 200 μl of the test solution into a 96-well plate, and measure the absorbance value at a wavelength of 345 nm using a spectrophotometer for quantification.

[0163] (2) Standard glycine solution: Since glycine has only 1 free amino group, glycine is used as the standard solution. Since the relationship between the absorbance value and the free amino group concentration is not necessarily linear, standard glycine solutions with different concentrations are prepared and reacted according to the above TNBS method, and the absorbance value at a wavelength of 345 nm is measured using a spectrophotometer to plot a standard curve of the absorbance value against the glycine concentration (i.e., the free amino group concentration), so as to calculate the free amino group concentration of each test product and control sample.

[0164] (3) Calculation formula: Crosslinking degree (%) = [1 - (free amino group concentration of the test product / free amino group concentration of the control sample)] × 100%; where the free amino group concentration of the test product is the uncrosslinked part, so it is deducted to obtain the concentration of the crosslinked part, that is, the crosslinking degree.

[0165] (2) Analysis of collagen degradation rate:

[0166] Ninhydrin can bind to the free amino groups in proteins to form a blue-violet substance, and the absorbance value at a wavelength of 570 nm is measured by a spectrophotometer for quantification. Therefore, the ninhydrin method is used in this invention to detect the concentration of free amino groups after collagen degradation, and the collagen degradation rate is calculated therefrom. The description is as follows:

[0167] 1. Sample preparation:

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

[0169] (2) Control samples: The raw material mixtures (non-freeze-dried powder) of each group itself are used as the samples of the control articles. Taking Example 1 as an example, 4 raw materials are mixed: collagen solution, collagen polypeptide solution, the first hyaluronic acid, and the second hyaluronic acid, and the weight ratio is 20:20:1:1 to obtain the raw material mixture. Additionally, taking Comparative Example 5 as an example, 2 raw materials are mixed: collagen solution and collagen polypeptide solution, and the weight ratio is 20:20 to obtain the raw material mixture.

[0170] 2. Analysis steps:

[0171] (1) Degrade collagen: Take 1 g of the sample and add 0.1 ml of an aqueous collagenase solution, and react at 37 °C for 24 hours; wherein, the aqueous collagenase solution contains 20 mM of NaH 2 PO 4 .H 2 O and 0.36 mM of CaCl 2 .2H 2 O, the pH value is 7.4, and its collagenase concentration is 0.5 unit / ml. Then centrifuge at 10000 rpm for 10 minutes to obtain the supernatant.

[0172] (2) Ninhydrin method: Take 0.2 ml of the supernatant and add 25 μl of ninhydrin (concentration: 20 mg / ml, product number: 151173, purchased from Sigma-Aldrich), react at 100 °C for 20 minutes, wait for it to cool to room temperature, and measure the absorbance value at a wavelength of 570 nm by a spectrophotometer for quantification.

[0173] (3) Standard glycine solution: Prepare standard glycine solutions with different concentrations and react according to the above ninhydrin method, and measure the absorbance value at a wavelength of 570 nm by a spectrophotometer to plot a standard curve of the absorbance value against the glycine concentration (i.e., the free amino group concentration) to calculate the free amino group concentrations of the test articles and control articles of each group after being degraded by collagenase respectively.

[0174] (4) Calculation formula: Collagen degradation rate (%) = (Free amino group concentration of the test sample / Free amino group concentration of the control sample) × 100%.

[0175] (III) Analysis of hyaluronic acid degradation rate:

[0176] D-Glucurono-δ-lactone (D-GUL) is one of the main products after the degradation of hyaluronic acid and can be used to quantify the degradation rate of hyaluronic acid. Further, D-GUL will be decomposed into D-Glucuronic Acid (D-GlcA) in an acidic environment, which has a carboxyl group (-COOH) and can undergo a condensation reaction with carbazole to form a purple-red substance, and the absorbance value at a wavelength of 530 nm is measured using a spectrophotometer for quantification. The description is as follows.

[0177] 1. Sample preparation:

[0178] (1) Test sample: The biological cross-linked material products (non-freeze-dried powder) of each group are used as the samples of the test sample.

[0179] (2) Control sample: The raw material mixtures (non-freeze-dried powder) of each group itself are used as the samples of the control sample.

[0180] 2. Analysis steps:

[0181] (1) Degrade hyaluronic acid: Add 300 units of hyaluronidase to 1 g of the 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 absolute ethanol to terminate the enzymatic hydrolysis reaction. The mixture is centrifuged at 12,000 revolutions per minute for 20 minutes to obtain the supernatant.

[0182] (2) Carbazole method: The experimental procedures refer to 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 microliters (μl) of the supernatant was added to 200 microliters of a carbazole solution with a concentration of 0.1% by volume (the solvent is absolute ethanol), and after boiling at 100 °C for 10 minutes, color development occurred, and the absorbance value at a wavelength of 530 nanometers was measured using a spectrophotometer for quantification.

[0183] (3) Standard D-glucuronic acid lactone solution: Standard D-glucuronic acid lactone solutions with different concentrations were reacted according to the above carbazole method, and the absorbance values at a wavelength of 530 nanometers were measured using a spectrophotometer to plot a standard curve of absorbance value against D-glucuronic acid lactone concentration, so as to calculate the D-glucuronic acid lactone concentration of each group of test samples and reference samples after being degraded by hyaluronidase.

[0184] (4) Calculation formula: Hyaluronic acid degradation rate (%) = (D-glucuronic acid lactone concentration of the test sample / D-glucuronic acid lactone concentration of the reference sample) × 100%.

[0185] (IV) Results

[0186] Three replicate tests were performed for each group, and the results of the crosslinking degree, collagen degradation rate, and hyaluronic acid degradation rate of each group are shown in Table 3.

[0187] Table 3: Crosslinking degree, collagen degradation rate, and hyaluronic acid degradation rate of each group (including measured values and their averages of three replicate tests)

[0188]

[0189]

[0190]

[0191] As can be seen from Table 3, first, in Example 1 and Examples 3 to 5, the degree of crosslinking is greater than 74%, with a high degree of crosslinking; the collagen degradation rate is lower than 10%, with a low collagen degradation rate; and the hyaluronic acid degradation rate is lower than 14%, with a low hyaluronic acid degradation rate.

[0192] In contrast, in Comparative Examples 1 to 6 and Comparative Examples 9 to 11, the degree of crosslinking is lower than 50%, significantly lower than that in Example 1 and Examples 3 to 5 (at least 74%); the collagen degradation rate is greater than 20%, significantly higher than that in Example 1 and Examples 3 to 5 (less than 10%); and the hyaluronic acid degradation rate is greater than 20%, significantly higher than that in Example 1 and Examples 3 to 5 (less than 14%). It can be seen that compared with Comparative Examples 1 to 6 and Comparative Examples 9 to 11, Example 1 and Examples 3 to 5 are less likely to be degraded by collagenase and hyaluronidase in the human body, and the residence time of the bio-crosslinked material in the human body can be greatly increased, so as to effectively reduce the frequency of supplementing the bio-crosslinked material.

[0193] Second, from the comparison between Example 1 and Examples 3 to 5, it can be seen that the collagen degradation rates of Examples 3 and 5 (both collagen sources include cowhide) are lower than those of Example 1 and Example 4. It can be known that using cowhide as the collagen source can reduce the collagen degradation rate, so as to increase the residence time of the bio-crosslinked material in the human body and effectively reduce the frequency of supplementing the bio-crosslinked material.

[0194] Third, Example 1 uses three crosslinking steps. Compared with Comparative Example 1 using a single crosslinking step, the degree of crosslinking of Example 1 (75.09%) is significantly higher than that of Comparative Example 1 (49.41%); the collagen degradation rate of Example 1 (9.31%) is significantly lower than that of Comparative Example 1 (21.85%); and the hyaluronic acid degradation rate of Example 1 (13.04%) is significantly lower than that of Comparative Example 1 (20.14%). Therefore, the three crosslinking steps adopted in the present invention can effectively increase the degree of crosslinking, reduce the collagen degradation rate and the hyaluronic acid degradation rate compared with the single crosslinking step, so as to increase the residence time of the bio-crosslinked material in the human body, thereby effectively reducing the frequency of supplementing the bio-crosslinked material.

[0195] Fourth, the three cross-linking steps of Example 1 sequentially use BDDE, GA, and EDC. Compared with Comparative Examples 2 to 4 that use a single cross-linking agent, the cross-linking degree of Example 1 (75.09%) is significantly higher than that of Comparative Examples 2 to 4 (the highest is 34.92%); the collagen degradation rate of Example 1 (9.31%) is significantly lower than that of Comparative Examples 2 to 4 (the lowest is 31.95%); and the hyaluronic acid degradation rate of Example 1 (13.04%) is significantly lower than that of Comparative Examples 2 to 4 (the lowest is 31.63%). Therefore, in the three cross-linking steps of the present invention, BDDE, GA, and EDC are sequentially used. Compared with a single cross-linking agent, the cross-linking degree can be effectively improved, and the collagen degradation rate and hyaluronic acid degradation rate can be reduced, so as to increase the residence time of the biological cross-linked material in the human body and effectively reduce the frequency of supplementing the biological cross-linked material.

[0196] Fifth, Example 1 uses 4 raw materials (both collagen raw material and hyaluronic acid raw material). Compared with Comparative Example 5 that only uses collagen raw material, the collagen degradation rate of Example 1 (9.31%) is significantly lower than that of Comparative Example 5 (46.48%); and compared with Comparative Example 6 that only uses hyaluronic acid raw material, 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 4 raw materials. Compared with a single collagen raw material or hyaluronic acid raw material, the collagen degradation rate and hyaluronic acid degradation rate can be effectively reduced, so as to increase the residence time of the biological cross-linked material in the human body and effectively reduce the frequency of supplementing the biological cross-linked material.

[0197] Analysis 2: Yield

[0198] This analysis aims to understand the yield of the biological cross-linked material product and is divided into 5 groups: Example 1 and Comparative Examples 1 to 4, which are described as follows.

[0199] (1) Yield analysis

[0200] Calculation formula: Yield (%) = (wet weight of the biological cross-linked material product / wet weight of the raw materials) * 100%; where the weight of the cross-linking agent is not included in the raw materials, that is, it includes the total addition amount (wet weight) of the collagen solution, collagen polypeptide solution, first hyaluronic acid, and second hyaluronic acid.

[0201] (2) Results: The yields of each group are shown in Table 4.

[0202] Table 4: Yields of each group (unit: %)

[0203]

[0204] As can be seen from Table 4, the yields of the other groups are similar, except that the yield of Comparative Example 2 is significantly lower; among them, although the yield of Comparative Example 3 is higher than that of Example 1, that is, more raw materials are crosslinked and remain in the biocrosslinked material product, it can be seen from Table 3 that the structural strength of Comparative Example 3 is significantly lower than that of Example 1, that is, the crosslinking quality of Example 1 is significantly better than that of Comparative Example 3.

[0205] Analysis 3: Cell viability analysis

[0206] This analysis aims to understand the effect of the combination of the biocrosslinked material product and exosomes on cell viability, and is divided into a blank group, a control group and an experimental group. The experimental group includes Example 2, Example 2 after long-term storage, Comparative Example 7 and Comparative Example 8; among them, the "Example 2 after long-term storage" is to carry out this analysis after Example 2 is left standing in a refrigerated environment at 4°C for 3 months, and the other groups carry out this analysis within 3 days after preparation, as described below.

[0207] (1) Blank group (using the biocrosslinked material product to form a coating without adding cells):

[0208] For each blank group corresponding to the biocrosslinked material product used in the experimental group, after adding 0.1 g of the biocrosslinked material product to a 24-well culture plate for 24 hours, 0.5 ml of cell culture medium was added and soaked for 24 hours to make the biocrosslinked material product adhere to the bottom of the well to form a coating; among them, the cell culture medium was Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS).

[0209] After aspirating the cell culture medium, 1 ml of cell culture medium (without cells) was placed in each well, and then transferred to a cell culture incubator and cultured in a constant environment of 37°C and 5% carbon dioxide for 2 hours. Another 0.5 ml of cell culture medium (without cells) was added to each group and cultured for 7 days, then 0.5 ml of fresh cell culture medium (without cells) was replaced. A cell counting kit-8 (CCK-8, purchased from Sigma-Aldrich / Merck) was used to perform the experimental steps according to the instructions, and 100 μl of each group was transferred to a 96-well culture plate, and the absorbance value at a wavelength of 450 nm was measured using a spectrophotometer.

[0210] (2) Control group (without using the biocrosslinked material product to form a coating and adding cells):

[0211] In a 24-well culture plate, 1 milliliter of cell-containing cell culture medium 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 milliliters of fresh cell culture medium was replaced; wherein, the cell-containing cell culture medium contained the above cell culture medium and contained approximately 1×10 4 human dermal fibroblasts (HDF). The CCK-8 kit was used to perform the experimental steps according to the instructions, and 100 microliters were taken and transferred to a 96-well culture plate, and the absorbance value at a wavelength of 450 nanometers was measured using a spectrophotometer.

[0212] (III) Experimental group:

[0213] After adding 0.1 grams of the biological cross-linked material product to the 24-well culture plate for 24 hours in each group, 0.5 milliliters of the above cell culture medium was added and soaked for 24 hours to allow the biological cross-linked material product to adhere to the bottom of the well to form a coating. In the 24-well culture plate, 1 milliliter of the above cell-containing cell culture medium 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 milliliters of fresh cell culture medium was replaced. The CCK-8 kit was used to perform the experimental steps according to the instructions, and 100 microliters were taken and transferred to a 96-well culture plate, and the absorbance value at a wavelength of 450 nanometers was measured using a spectrophotometer.

[0214] (IV) Calculation formula: Cell survival rate (%) = (absorbance value of the experimental group - absorbance value of the blank group) / (absorbance value of the control group - absorbance value of the blank group) × 100%

[0215] (V) Results: The data of each experimental group are shown in Table 5.

[0216] Table 5: Cell survival rate of each experimental group (unit: %)

[0217]

[0218] As can be seen from Table 5, the biological cross-linked material product of Example 2 still has a cell survival rate similar to that of fresh Example 2 even after 3 months of storage, showing excellent stability. In addition, the cell survival rate of Example 2 is significantly better than that of Comparative Example 7 and Comparative Example 8, indicating that the biological cross-linked material product of Example 2 can effectively promote the growth of human dermal fibroblasts. After being injected into the skin, it can effectively promote skin health.

[0219] In summary, the biological cross-linked material or biological cross-linked material product of the present invention has excellent structural strength, namely high cross-linking degree, low collagen degradation rate and low hyaluronic acid degradation rate. In addition, the biological cross-linked material or biological cross-linked material product of the present invention can be used in combination with exosomes to more effectively maintain skin health.

[0220] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments of equivalent changes within the scope of the technical solution of the present invention by using the technical content disclosed above. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A biological mixture comprising a biological cross-linking material, characterized in that: The biological cross-linking material comprises collagen, collagen polypeptide, a first hyaluronic acid and a second hyaluronic acid; wherein, The molecular weight of the first hyaluronic acid is 100 kilodaltons to 500 kilodaltons, and the molecular weight of the second hyaluronic acid is 1500 kilodaltons to 3000 kilodaltons; and The collagen, the collagen polypeptide, the first hyaluronic acid and the second hyaluronic acid are cross-linked with each other, and the average cross-linking degree of the biological cross-linking material is 65% to 97%.

2. The biological mixture according to claim 1, characterized in that Based on the total volume of the biological cross-linking material, the total content of the collagen and collagen polypeptide is 30 mg / ml to 80 mg / ml.

3. The biological mixture according to claim 1, characterized in that Based on the total volume of the biological cross-linking material, the total content of the first hyaluronic acid and the second hyaluronic acid is 1 mg / ml to 18 mg / ml.

4. The biological mixture according to claim 1, characterized in that Further comprising exosomes.

5. The biological mixture according to claim 4, characterized in that Based on the total volume of the biological mixture, the concentration of the exosomes is 1×10 5 Particles up to 1×10 11 Particles.

6. Use of the biological mixture according to any one of claims 1 to 5 for preparing a skin tissue filling product.

7. The use according to claim 6, characterized in that The dosage form of the skin tissue filling product includes an injection.

8. A method for preparing a biological mixture as claimed in any one of claims 1 to 5, characterized in that: Include: A first cross-linking step: mixing collagen, the first hyaluronic acid, a first cross-linking agent and a first carrier to obtain a first mixed solution, and reacting for 20 to 28 hours to obtain a first cross-linked complex; wherein, based on the total volume of the first mixed solution, the content of the collagen is 10 weight volume percent (w / v%) to 40 weight volume percent, and the content of the first hyaluronic acid is 0.1 weight volume percent to 9 weight volume percent; A second cross-linking step: mixing the first cross-linked complex, the second hyaluronic acid, the second cross-linking agent and the second carrier to obtain a second mixed solution, and reacting for 20 hours to 28 hours to obtain a second cross-linked complex; wherein, based on the total volume of the second mixed solution, the content of the first cross-linked complex is 10 weight volume percent to 40 weight volume percent, and the content of the second hyaluronic acid is 0.1 weight volume percent to 9 weight volume percent; and The third cross-linking step: mixing the second cross-linked complex, the collagen polypeptide, the third cross-linking agent and the third carrier to obtain a third mixed solution, and reacting for 20 hours to 28 hours to obtain the biological cross-linked material; wherein, based on the total volume of the third mixed solution, the content of the second cross-linked complex is 10% to 40% by weight volume, and the content of the collagen polypeptide is 10% to 40% by weight volume; wherein, The first cross-linking agent, the second cross-linking agent and the third cross-linking agent are different cross-linking agents, and the first carrier, the second carrier and the third carrier all contain water.

9. The method according to claim 8, characterized in that: The first cross-linking agent comprises 1,4-butanediol glycidyl ether, and the content of the first cross-linking agent is 0.5% by weight to 5% by weight based on the total volume of the first mixed solution; The second cross-linking agent comprises glutaraldehyde, and based on the total volume of the second mixed solution, the content of the second cross-linking agent is 0.005 weight volume percent to 0.1 weight volume percent; and The third cross-linking agent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and based on the total volume of the third mixed solution, the content of the third cross-linking agent is 0.5% by weight to 5% by weight.

10. The method according to claim 8, characterized in that: Either or a combination of the first cross-linking step and the third cross-linking step comprises adding an alkali agent.

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