Injectable collagen material and method for preparing same

Abstract

The present invention relates to the field of biomaterials, and in particular to an injectable collagen material and a method for preparing the same. The injectable collagen material of the present invention has properties such as neutral pH, homogeneity, and fluidity, and satisfies the requirements for direct injection of an aqueous collagen solution into the body, and can form a collagen hydrogel with a predetermined mechanical strength in the body.

Description

[Technical Field]

[0001] The present invention relates to a biomaterial and a method for preparing the same, and in particular to an injectable collagen material and a method for preparing the same. [Background technology]

[0002] Collagen is the major structural protein of connective tissue and is widely distributed in tissues and organs such as bone, skin, blood vessels, ligaments, cartilage, muscles, and tendons, accounting for 25% to 30% of all proteins. To date, more than 20 types of collagen have been identified, of which type I collagen is the most common, accounting for approximately 90%. Due to its excellent biocompatibility, biodegradability, and biological activity, collagen is widely used as a conventional biomaterial in fields such as tissue engineering and regenerative medicine.

[0003] Injectable collagen materials, one of the applications of collagen, are typically obtained by dissolving collagen extracted by salt precipitation or solid collagen in an acidic aqueous solution followed by dialysis. During the dialysis process, the pH of the acidic collagen solution gradually changes to neutral. Furthermore, because collagen has an isoelectric point of 7.5 to 7.8, collagen molecules dissolved in the aqueous solution tend to gradually self-assemble into collagen fibers. Therefore, conventional injectable collagen materials are essentially dispersions of concentrated, phase-separated collagen fibers in an aqueous solution. This means that they are heterogeneous collagen fiber suspensions rather than homogeneous aqueous solutions with adequate collagen molecules. Such collagen fiber suspensions typically require high-speed shearing and filtration to meet injection requirements. Furthermore, because they are essentially heterogeneous, they do not form collagen hydrogels with the required mechanical strength in vivo after injection.

[0004] In view of the above problems with the preparation and use of injectable collagen materials, there is a need to develop an injectable collagen material that has a neutral pH and homogeneous fluidity, which can form a collagen hydrogel with a predetermined mechanical strength in the body, and a method for preparing such an injectable collagen material. Summary of the Invention

[0005] One object of the present invention is to provide an injectable collagen material that has pH-neutral, homogeneous fluidity, and is capable of forming a collagen hydrogel with a predetermined mechanical strength in vivo. Another object of the present invention is to provide a method for preparing the injectable collagen material.

[0006] The present invention provides a method for preparing an injectable collagen material, comprising the steps of:

[0007] (1) a step of sufficiently dissolving a solid collagen material in water, centrifuging the resulting solution, and collecting the supernatant, wherein the solid collagen material is selected from one or a combination of two or more types of collagen selected from type I, type II, type III, and type V, and the total concentration of the various collagens in the supernatant is 0.1 wt.% or more and 5 wt.% or less;

[0008] (2) A simulated body fluid component is added to the supernatant obtained in step (1), and the simulated body fluid component contains Na + , K. + , Mg 2+ , Ca 2+ , Cl - , HCO3 - , HPO4 2- , SO4 2- , CO3 2- , PO4 3- , H2PO4 - a step including one or a combination of two or more of the following:

[0009] (3) collecting the solution and supernatant obtained in step (2);

[0010] (4) a final step of adjusting the pH value of the supernatant obtained in step (3) to a range of 6.0 to 8.0 using an alkaline solution and an acidic solution;

[0011] (5) centrifuging the solution obtained in step (4) and collecting the supernatant;

[0012] (6) adding a protective molecule to the supernatant obtained in step (5), the protective molecule comprising at least one of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, Tween, Triton, ethylene glycol, phenylalanine, proline, lecithin, glutamic acid, lysine, cysteine, mangiferin, glycine, aspartic acid, n-butanol, propylene glycol, ethylenediaminetetraacetic acid sodium salt, stearic acid, span, glycerin, and gelatin;

[0013] (7) subjecting the solution obtained in step (6) to low-temperature irradiation treatment;

[0014] (8) centrifuging the solution obtained in step (7) and collecting the supernatant; The method for preparing an injectable collagen material comprises the steps of:

[0015] In the method for preparing an injectable collagen material of the present invention, the centrifugation rotation speed in the above steps (3), (5), and (8) is 1,000 to 10,000 rpm. Because the self-assembly of collagen molecules into collagen fibers during the preparation process is unavoidable, insoluble matter in the collagen aqueous solution can be removed by centrifugation to meet the requirements for an injectable material. An appropriate centrifugation rotation speed can be selected depending on the size of the injection needle. For example, a centrifugation rotation speed of 1,000 rpm can be selected for an 8G injection needle, and a centrifugation rotation speed of 3,000 rpm can be selected for a 21G injection needle, which will meet the requirements for injection.

[0016] In the method for preparing an injectable collagen material of the present invention, the acidic solution in step (4) contains at least one of salicylic acid, lactic acid, sulfuric acid, tartaric acid, citric acid, phosphoric acid, oxalic acid, acetic acid, ethanedioic acid, succinic acid, hydrochloric acid, maleic acid, benzoic acid, nitric acid, malic acid, nicotinic acid, sodium dihydrogen phosphate, and formic acid, and the alkaline solution contains at least one of tetramethylethylenediamine, sodium hydroxide, triethylamine, sodium carbonate, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, disodium hydrogen phosphate, and sodium hydrogen carbonate. The main purpose of selecting an appropriate acidic or alkaline solution is to adjust the pH value of the collagen aqueous solution to a range acceptable for intracorporeal injection. Furthermore, because the normal pH of the human body is 7.35 to 7.45, collagen molecules can self-assemble within this range to form insoluble matter that can clog injection needles. Therefore, by preferentially selecting a weak acid or weak alkali as the acidic or alkaline solution, the pseudo-body fluid components and protective molecules added to the collagen aqueous solution act synergistically to weaken the self-assembly behavior of the collagen molecules, making it easier to use the collagen aqueous solution by injection.

[0017] In the method for preparing an injectable collagen material of the present invention, the pH value of the supernatant in step (4) is ultimately in the range of 6.8 to 7.8, and the pH value of the collagen aqueous solution is adjusted to be close to the normal pH range of the human body, ensuring excellent tissue compatibility of the material injected into the body.

[0018] Compared to conventional techniques, the method for preparing an injectable collagen material of the present invention first selects an appropriate type of collagen and controls the total collagen concentration, then adds a simulated body fluid component, adjusts the pH with an alkaline or acidic solution, and adds protective molecules. In addition to the synergistic effects of these steps, the insoluble matter in the collagen aqueous solution is removed by centrifugation several times during this process, ensuring uniform fluidity of the collagen aqueous solution. The collagen molecules do not aggregate and clog injection needles, and even after irradiation, they do not crosslink and clog injection needles. The preparation method of the present invention allows for the production of an injectable collagen material with neutral pH and uniform fluidity, which can form a collagen hydrogel with a predetermined mechanical strength in the body after injection.

[0019] The injectable collagen material of the present invention is prepared using the above-mentioned method for preparing an injectable collagen material.

[0020] The injectable collagen material of the present invention is injected into the body using an 8-34G needle, preferably a 21-32G needle, preferably a 25-30G needle. Depending on the application scenario, different needle specifications must be selected when using the injectable collagen material of the present invention, such as a 10G needle with an inner diameter of approximately 2.69 mm for filling internal cavities and a 32G needle with an inner diameter of approximately 0.11 mm for minor facial cosmetic injections. Therefore, the main parameters of the preparation method must be adjusted to ensure uniform fluidity of the collagen aqueous solution and prevent needle clogging.

[0021] The injectable collagen material of the present invention can form a collagen hydrogel with a compressive modulus in the range of 0.1 to 20 kPa after injection into the body. Because the collagen molecules of the injectable collagen material of the present invention do not self-assemble to form collagen fibers before injection and no crosslinking reaction occurs after irradiation, a collagen hydrogel with a predetermined mechanical strength can be formed after injection through interactions such as aggregation of collagen molecules under body temperature conditions. This is different from the inability to form a collagen hydrogel with a predetermined mechanical strength after injection into the body, as occurs with heterogeneous collagen fiber suspensions in which collagen molecules have already aggregated before injection. The mechanical strength of the formed collagen hydrogel can be adjusted by adjusting the collagen concentration in the injectable collagen material.

[0022] Unlike conventional injectable collagen materials, which are heterogeneous collagen fiber suspensions, the injectable collagen material of the present invention is prepared using the above-mentioned method for preparing an injectable collagen material, and has a neutral pH, is capable of homogeneous flow, and is capable of forming a collagen hydrogel with a predetermined mechanical strength in the body. [Brief explanation of the drawings]

[0023] [Figure 1] 1A and 1B are graphs showing turbidity tests of injectable collagen materials (FIG. 1A is a sample of Example 1, FIG. 1B is a sample of Example 2, FIG. 1C is a sample of Example 3, FIG. 1D is a sample of Comparative Example 1, and FIG. 1E is a sample of Comparative Example 2). [Figure 2] These are injection needles of different specifications used in testing injectable collagen materials. [Figure 3] 1 shows a subcutaneous injection test of injectable collagen material in nude mice. DETAILED DESCRIPTION OF THE INVENTION

[0024] Example 1 1. A method for preparing an injectable collagen material, comprising:

[0025] (1) thoroughly dissolving 1 g of type I collagen in 1000 g of water, centrifuging the solution at 2000 rpm, and collecting the supernatant;

[0026] (2) adding 32 g of KHCO, 0.01 g of CaCl, and 0.5 g of NaHPO to the supernatant obtained in step (1);

[0027] (3) centrifuging the solution obtained in step (2) at 1000 rpm and collecting the supernatant;

[0028] (4) adjusting the supernatant obtained in step (3) to pH 7.2 with 1 M succinic acid, 0.5 M acetic acid, and 2 M sodium carbonate;

[0029] (5) centrifuging the solution obtained in step (4) at a rotation speed of 3000 rpm and collecting the supernatant;

[0030] (6) adding 1% Triton, 0.5% ethylenediaminetetraacetic acid sodium salt, and 0.2% glycine to the supernatant obtained in step (5);

[0031] (7) The solution obtained in step (6) was heated at 0°C with Co 60 A step of performing an irradiation treatment;

[0032] (8) centrifuging the solution obtained in step (7) at a rotation speed of 3000 rpm and collecting the supernatant; The method for preparing an injectable collagen material comprises the steps of:

[0033] The injectable collagen material prepared in this example can be formed into an injectable collagen material with homogeneous fluidity, in which the collagen molecules are sufficiently soluble in aqueous solution in their molecular state, making it possible to inject it using a 32G needle with an inner diameter of 0.11 mm, and forming a collagen hydrogel with a compressive modulus of 0.25±0.08 KPa after injection into the body.

[0034] Example 2 1. A method for preparing an injectable collagen material, comprising:

[0035] (1) thoroughly dissolving 2 g of type II collagen and 0.1 g of type III collagen in 200 g of water, centrifuging the solution at 3000 rpm, and collecting the supernatant;

[0036] (2) adding 0.1 g of NaCl, 0.1 g of Na2CO3, and 0.1 g of K3PO4 to the supernatant obtained in step (1);

[0037] (3) centrifuging the solution obtained in step (2) at a rotation speed of 2000 rpm and collecting the supernatant;

[0038] (4) adjusting the supernatant obtained in step (3) to pH 6.0 with 0.5 M phosphoric acid and 0.1 M potassium hydroxide;

[0039] (5) centrifuging the solution obtained in step (4) at a rotation speed of 4000 rpm and collecting the supernatant;

[0040] (6) adding 0.4% n-butanol, 1% aspartic acid, and 2% glycerin to the supernatant obtained in step (5);

[0041] (7) subjecting the solution obtained in step (6) to electron beam irradiation treatment at 4°C;

[0042] (8) centrifuging the solution obtained in step (7) at a rotation speed of 5000 rpm and collecting the supernatant; The method for preparing an injectable collagen material comprises the steps of:

[0043] The injectable collagen material prepared in this example can be formed into an injectable collagen material with homogeneous fluidity, in which the collagen molecules are sufficiently soluble in aqueous solution in their molecular state, allowing injection using a 30G needle with an inner diameter of 0.16 mm. After injection into the body, a collagen hydrogel with a compressive modulus of 5.92±0.43 KPa can be formed.

[0044] Example 3 1. A method for preparing an injectable collagen material, comprising: (1) thoroughly dissolving 40 g of type I collagen and 0.1 g of type V collagen in 500 g of water, centrifuging the solution at 6,000 rpm, and collecting the supernatant;

[0045] (2) adding 0.5 g of KSO, 1 g of KHPO, and 2 g of KCO to the supernatant obtained in step (1);

[0046] (3) centrifuging the solution obtained in step (2) at a rotation speed of 4000 rpm and collecting the supernatant;

[0047] (4) adjusting the supernatant obtained in step (3) to pH 8.0 with 2 M salicylic acid, 1 M triethylamine, and 1 M aqueous ammonia;

[0048] (5) centrifuging the solution obtained in step (4) at a rotation speed of 6000 rpm and collecting the supernatant;

[0049] (6) adding 0.01% gelatin, 0.2% ethylene glycol, and 1% proline to the supernatant obtained in step (5);

[0050] (7) subjecting the solution obtained in step (6) to gamma irradiation treatment at 4°C;

[0051] (8) centrifuging the solution obtained in step (7) at a rotation speed of 10,000 rpm and collecting the supernatant; The method for preparing an injectable collagen material comprises the steps of:

[0052] The injectable collagen material prepared in this example can be formed into an injectable collagen material with homogeneous fluidity, in which the collagen molecules are sufficiently soluble in aqueous solution in their molecular state, making it possible to inject it using a 22G needle with an inner diameter of 0.41 mm, and after injection into the body, it can form a collagen hydrogel with a compressive elastic modulus of 17.86±0.15 KPa.

[0053] In the method for preparing an injectable collagen material of the present invention, the solid collagen material used in step (1) is selected from one or a combination of two or more types of type I, II, III, and V collagen. However, the solid collagen material is not limited to the types disclosed in Examples 1 to 3; other types can also be used, such as type II, III, or V collagen used alone, or a combination of four types: type I, II, III, and V. The mass of the solid collagen material can be adjusted so long as the total concentration of the various collagens in the supernatant in step (1) is between 0.1 wt.% and 5 wt.%. This is because a collagen concentration below 0.1 wt.% will not allow the formation of a collagen hydrogel with a required mechanical strength in the body after injection, and a collagen concentration above 5 wt.% will result in an aqueous collagen solution with too high a viscosity to permit injection. Therefore, selecting this range of total collagen concentration is a prerequisite for ensuring that the prepared injectable collagen material meets the requirements for injection and use.

[0054] The simulated body fluid component in step (2) of the present invention is Na + , K. + , Mg 2+ , Ca 2+ , Cl - , HCO3 - , HPO4 2- , SO4 2- , CO3 2- , PO4 3- , H2PO4 - Furthermore, because the isoelectric point of collagen is 7.5 to 7.8, collagen molecules in an aqueous collagen solution tend to self-assemble during the subsequent pH adjustment process to form collagen fibers, clogging the needle and making injection impossible. On the other hand, the addition of simulated body fluid components balances the ionic strength of the aqueous solution, weakening the interactions between collagen molecules and simulating the in vivo environment, thereby improving the tissue compatibility of the injectable collagen material.

[0055] The acidic solution in step (4) of the present invention contains at least one of salicylic acid, lactic acid, sulfuric acid, tartaric acid, citric acid, phosphoric acid, oxalic acid, acetic acid, ethanedioic acid, succinic acid, hydrochloric acid, maleic acid, benzoic acid, nitric acid, malic acid, nicotinic acid, sodium dihydrogen phosphate, and formic acid. The alkaline solution contains at least one of tetramethylethylenediamine, sodium hydroxide, triethylamine, sodium carbonate, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, disodium hydrogen phosphate, and sodium hydrogen carbonate. The primary purpose of selecting an alkaline or acidic solution is to adjust the pH of the collagen solution to a range acceptable for intracorporeal injection. Furthermore, because the normal pH of the human body is 7.35 to 7.45, collagen molecules can self-assemble within this range, forming insoluble matter that clogs injection needles. Therefore, preferentially selecting a weak acid or alkaline solution can act synergistically with the simulated body fluid components and protective molecules added to the collagen solution to weaken the self-assembly behavior of the collagen molecules, facilitating the use of the collagen solution by injection. The final pH of the supernatant in step (4) is in the range of 6.0 to 8.0, preferably 6.8 to 7.8. Furthermore, the pH of the collagen solution is adjusted to a value close to the normal pH range of the human body, ensuring excellent tissue compatibility of the material injected into the body.

[0056] The protective molecules used in step (6) of the present invention include at least one of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, Tween, Triton, ethylene glycol, phenylalanine, proline, lecithin, glutamic acid, lysine, cysteine, mangiferin, glycine, aspartic acid, n-butanol, propylene glycol, sodium ethylenediaminetetraacetic acid, stearic acid, spanish, glycerin, and gelatin. The main purpose of adding an appropriate protective molecule is to create weak interactions, such as hydrogen bonds, with collagen molecules in aqueous solution, reducing the possibility of needle clogging due to crosslinking of collagen molecules during irradiation. At the same time, the protective molecules also weaken the interactions between collagen molecules in aqueous solution, reducing the possibility of collagen molecules self-aggregating to form insoluble matter and clogging the injection needle.

[0057] The centrifugation speed in steps (3), (5), and (8) of the present invention is 1,000 to 10,000 rpm. Because the self-assembly of collagen molecules to form collagen fibers is unavoidable during the preparation process, insoluble matter in the collagen aqueous solution can be removed by centrifugation to ensure the material is injectable. The appropriate centrifugation speed can be selected depending on the size of the injection needle. For example, a centrifugation speed of 1,000 rpm can be selected for an 8G injection needle, and a centrifugation speed of 3,000 rpm can be selected for a 21G injection needle, which will meet the injection requirements.

[0058] The injectable collagen material of the present invention is prepared using the above-mentioned method for preparing an injectable collagen material.

[0059] The injectable collagen material of the present invention is injected into the body using an 8-34G needle, preferably a 21-32G needle, preferably a 25-30G needle. Depending on the application scenario, different needle specifications must be selected when using the injectable collagen material of the present invention, such as a 10G needle with an inner diameter of approximately 2.69 mm for filling internal cavities and a 32G needle with an inner diameter of approximately 0.11 mm for minor facial cosmetic injections. Therefore, the main parameters of the preparation method must be adjusted to ensure uniform fluidity of the collagen aqueous solution and prevent needle clogging.

[0060] The injectable collagen material of the present invention can form a collagen hydrogel with a compressive modulus in the range of 0.1-20 kPa after injection into the body. Because the collagen molecules of the injectable collagen material of the present invention do not self-assemble to form collagen fibers before injection and no crosslinking reaction occurs after irradiation, a collagen hydrogel with a desired mechanical strength can be formed after injection through interactions such as aggregation of collagen molecules under body temperature conditions. This is different from the inability to form a collagen hydrogel with a desired mechanical strength after injection into the body, as is the case with heterogeneous collagen fiber suspensions in which collagen molecules have already aggregated before injection. The mechanical strength of the formed collagen hydrogel can be adjusted by adjusting the collagen concentration in the injectable collagen material.

[0061] Unlike conventional injectable collagen materials, which are heterogeneous collagen fiber suspensions, the injectable collagen material of the present invention has a neutral pH, is capable of homogeneous flow, and is capable of forming a collagen hydrogel with a predetermined mechanical strength in vivo. In the preparation method of the present invention, the synergistic effects of steps such as adding a simulated body fluid component, adjusting the pH with an alkaline or acidic solution, and adding protective molecules ensure homogeneous fluidity of the collagen aqueous solution, and the collagen molecules do not aggregate and clog injection needles, nor do they crosslink when irradiated, causing needle clogging. The injectable collagen material of the present invention can form a collagen hydrogel with a predetermined mechanical strength when injected into the body in a homogeneous flow state.

[0062] The effects of the present invention will be explained below by comparing Examples 1 to 3 with Comparative Examples 1 and 2.

[0063] Comparative study of the effectiveness of injectable collagen materials 1. Preparation of Comparative Collagen Materials Comparative Example 1: A method for preparing a collagen material, comprising:

[0064] (1) a step of thoroughly dissolving 1 g of type I collagen in 1000 g of water; (2) adding 32 g of KHCO, 0.01 g of CaCl, and 0.5 g of NaHPO to the supernatant obtained in step (1); (3) adding 1% Triton and 0.2% glycine to the supernatant obtained in step (2); (4) The solution obtained in step (3) is cooled to room temperature. 60 A step of performing irradiation treatment; a method for preparing a collagen material, the method comprising the steps of:

[0065] Comparative Example 2: A method for preparing a collagen material, comprising: (1) thoroughly dissolving 1 g of type I collagen in 1000 g of water, centrifuging the solution at 2000 rpm, and collecting the supernatant; (2) adjusting the supernatant obtained in step (1) to pH 7.2 with 1 M succinic acid and 2 M sodium carbonate; (3) centrifuging the solution obtained in step (2) at a rotation speed of 3000 rpm and collecting the supernatant; (4) The solution obtained in step (3) was heated at 0°C with Co 60 A step of performing an irradiation treatment; (5) centrifuging the solution obtained in step (4) at a rotation speed of 3000 rpm and collecting the supernatant; a method for preparing a collagen material, the method comprising the steps of:

[0066] 2. Comparison of efficacy studies

[0067] (1) Turbidity test of injectable collagen material: The turbidity of the injectable collagen material was observed at a wavelength of 625 nm using a turbidity meter (TZD-BZ-905, Suzhou DiagVita Technology Co., Ltd.). The test results are shown in Table 1. The turbidity test diagram of the injectable collagen material is shown in Figure 1, where Figure 1A is the sample of Example 1, Figure 1B is the sample of Example 2, Figure 1C is the sample of Example 3, Figure 1D is the sample of Comparative Example 1, and Figure 1E is the sample of Comparative Example 2.

[0068] Table 1. Turbidity test of injectable collagen materials JPEG2025540977000002.jpg12170

[0069] As can be seen from Table 1, in the injectable collagen materials of Examples 1 to 3, the collagen molecules do not aggregate to form collagen fibers, but are fully dissolved in a molecular state in an aqueous solution with homogeneous fluidity and low turbidity. In Figures 1A, 1B, and 1C, the injectable collagen materials of Examples 1, 2, and 3 are transparent, and the scale on the syringe can be clearly observed. In Comparative Examples 1 and 2, the collagen molecules have already aggregated to form collagen fibers, forming a collagen fiber suspension similar to that of conventional injectable collagen materials, and the aqueous solution is highly turbid. In Figures 1D and 1E, the aqueous solutions of Comparative Examples 1 and 2 are cloudy, and the scale on the syringe cannot be clearly observed.

[0070] (2) Injection test of injectable collagen material using different needle specifications: An injection test was conducted on the injectable collagen material prepared in Example 1 using the different needle specifications shown in Figure 2. From left to right, the needles shown in the figure were a 22G needle with an inner diameter of 0.41 mm, a 27G needle with an inner diameter of 0.21 mm, a 30G needle with an inner diameter of 0.16 mm, a 32G needle with an inner diameter of 0.11 mm, and a 34G needle with an inner diameter of 0.06 mm. As can be seen from Figure 2, the injectable collagen material prepared in Example 1 has homogeneous fluidity, and the collagen molecules do not aggregate to form collagen fibers. Therefore, injection using the above-mentioned needle specifications is possible and the needle does not clog. However, in Comparative Example 1, the collagen fiber suspension is heterogeneous and is prone to aggregation and clogging the needle, making injection using the above-mentioned needle specifications impossible.

[0071] (3) Subcutaneous injection test of injectable collagen material in nude mice: The injectable collagen material of Example 1 was subjected to a subcutaneous injection test in nude mice using an 8G injection needle. As can be seen from Figure 3, the material was easy to inject, meeting the requirements for injection.

[0072] (4) Hydrogel compression modulus test of injectable collagen materials formed in vivo: Samples from Examples 1 to 3 and Comparative Examples 1 and 2 were subcutaneously injected into mice. After 24 hours, the injected materials were separated from the mouse tissue and subjected to a compression test. A uniaxial mechanical testing machine (E10000, Instron) was used, with a load of 1 KN and a compression speed of 0.4 mm min -1 The test was carried out at 25°C. The specimens were kept wet during the test. The specimen size was 5mm x 5mm x 5mm, and the compression was 30%. The compressive modulus was calculated from the slope of the stress-strain curve. The test results are shown in Table 2.

[0073] Table 2. Compressive elastic modulus test of hydrogels formed from injectable collagen materials JPEG2025540977000003.jpg13170

[0074] As can be seen from Table 2, the injectable collagen materials of Examples 1 to 3 were in an aqueous solution state with homogeneous fluidity and were able to form collagen hydrogels with the required mechanical strength in mice, whereas the collagen aqueous solutions of Comparative Examples 1 and 2 were actually collagen fiber suspensions, which were unable to form collagen hydrogels in mice and were in an amorphous state, making it impossible to test the compressive modulus.

[0075] As can be seen from the above tests, compared to Comparative Examples 1 and 2, Examples 1 to 3 are able to form injectable collagen materials with homogeneous fluidity, in which the collagen molecules are sufficiently dissolved in an aqueous solution in a molecular state, allowing injection using a needle with a smaller inner diameter, and are able to form a collagen hydrogel with a predetermined mechanical strength after injection into the body. On the other hand, Comparative Examples 1 and 2 formed collagen fiber suspensions in which the collagen molecules had already aggregated to form collagen fibers, lacking homogeneity, and unable to form a collagen hydrogel after injection into the body.

[0076] The above-mentioned examples merely illustrate the preferred embodiments of the present invention, and various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope determined by the claims of the present invention. [Industrial Applicability]

[0077] The method for preparing a collagen material according to the present invention makes it possible to prepare an injectable collagen material. As one of the applications of collagen, the collagen material according to the present invention is an injectable collagen material with a neutral pH and homogeneous fluidity, and can form a collagen hydrogel with a predetermined mechanical strength in the body, making it widely applicable in fields such as tissue engineering and regenerative medicine.

Claims

1. 1. A method for preparing an injectable collagen material, comprising: (1) a step of sufficiently dissolving a solid collagen material in water, centrifuging the resulting solution, and collecting a supernatant, the solid collagen material being selected from one or a combination of two or more types of collagen selected from type I, type II, type III, and type V, and the total concentration of the various collagens in the supernatant being 0.1 wt. % or more and 5 wt. % or less; (2) A simulated body fluid component is added to the supernatant obtained in step (1), and the simulated body fluid component contains Na ＋ , K. ＋ , Mg ２＋ , Ca ２＋ , Cl － , HCO ３ － , H.P.O. ４ ２－ , S.O. ４ ２－ , CO ３ ２－ , P.O. ４ ３－ , H ２ P.O. ４ － a step comprising one or a combination of two or more of the following: (3) centrifuging the solution obtained in step (2) and collecting the supernatant; (4) a step of finally adjusting the pH value of the supernatant obtained in step (3) to a range of 6.0 to 8.0 using an alkaline solution and an acidic solution; (5) centrifuging the solution obtained in step (4) and collecting the supernatant; (6) adding a protective molecule to the supernatant obtained in step (5), wherein the protective molecule comprises at least one of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, Tween, Triton, ethylene glycol, phenylalanine, proline, lecithin, glutamic acid, lysine, cysteine, mangiferin, glycine, aspartic acid, n-butanol, propylene glycol, ethylenediaminetetraacetic acid sodium salt, stearic acid, span, glycerin, and gelatin; (7) subjecting the solution obtained in step (6) to low-temperature irradiation treatment; (8) centrifuging the solution obtained in step (7) and collecting the supernatant; 1. A method for preparing an injectable collagen material, comprising the steps of:

2. 2. The method for preparing an injectable collagen material according to claim 1, wherein the rotation speed of the centrifugation in step (3) is 1,000 to 10,000 rpm.

3. 2. The method for preparing an injectable collagen material according to claim 1, wherein the rotation speed of the centrifugation in step (5) is 1,000 to 10,000 rpm.

4. 2. The method for preparing an injectable collagen material according to claim 1, wherein the rotation speed of the centrifugation in step (8) is 1,000 to 10,000 rpm.

5. 2. The method for preparing an injectable collagen material according to claim 1, wherein the acidic solution in step (4) contains at least one of salicylic acid, lactic acid, sulfuric acid, tartaric acid, citric acid, phosphoric acid, oxalic acid, acetic acid, ethanedioic acid, succinic acid, hydrochloric acid, maleic acid, benzoic acid, nitric acid, malic acid, nicotinic acid, sodium dihydrogen phosphate, and formic acid, and the alkaline solution contains at least one of tetramethylethylenediamine, sodium hydroxide, triethylamine, sodium carbonate, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia water, disodium hydrogen phosphate, and sodium bicarbonate.

6. 6. The method for preparing an injectable collagen material according to claim 1, wherein the final pH value of the supernatant in step (4) is in the range of 6.8 to 7.

8.

7. An injectable collagen material, characterized in that it is prepared using the method for preparing an injectable collagen material according to any one of claims 1 to 6.

8. The injectable collagen material according to claim 7, characterized in that the injectable collagen material can be injected into the body with an 8 to 34 G injection needle, preferably the injectable collagen material can be injected into the body with a 21 to 32 G injection needle, preferably the injectable collagen material can be injected into the body with a 25 to 30 G injection needle.

9. The injectable collagen material according to claim 8, characterized in that the injectable collagen material can form a collagen hydrogel after being injected into the body, and the compressive elastic modulus of the collagen hydrogel is in the range of 0.1 to 20 kPa.

Images