Hydroxyapatite cross-linked recombinant collagen gel for injection and preparation method thereof

Abstract

The invention discloses hydroxyapatite cross-linked recombinant collagen gel for injection and a preparation method of the hydroxyapatite cross-linked recombinant collagen gel. The method comprises the following steps: adding a catalyst into a mixed solution of hydroxyapatite and recombinant collagen, carrying out a cross-linking reaction to form solid gel, cutting the solid gel into blocks, cleaning the solid gel, mixing the gel blocks with a dispersion liquid formed by mixing hydroxyapatite and sodium hyaluronate, and homogenizing the gel blocks to prepare the hydroxyapatite cross-linked recombinant collagen gel. The prepared gel is good in stability, hydroxyapatite can be evenly distributed after subcutaneous injection, the immediate filling effect is achieved, meanwhile, hydroxyapatite microspheres can promote regeneration of collagen, a gel degradation product can be metabolized by a human body, good biocompatibility is achieved, immunogenicity and degradability are avoided, and the preparation method is suitable for large-scale popularization and application. Wide application prospects are realized in the field of medical beauty.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to an injectable hydroxyapatite cross-linked recombinant collagen gel and its preparation method. Background Technology

[0002] As people age, facial aging is an inevitable biological process. External stimuli, changes in the skin microenvironment, and alterations in the immune system all contribute to the progressive aging of skin cells, gradually damaging the extracellular matrix of facial skin and hindering the regeneration process. Visible signs of facial aging include thinning skin, decreased elasticity, moisture loss, and the formation of depressions. Specific indicators of facial aging, such as sunken temples and tear troughs, and deepened nasolabial folds, can be corrected through localized filler treatments to restore tissue plumpness and restore its original state and function.

[0003] Hydroxyapatite (HAP), also known as basic calcium phosphate, is a type of calcium apatite (Ca). 10 (PO4) 6 The natural mineralization of (OH2) is caused by hydroxyl groups (OH) - ), phosphate particles (PO3) 4- ) and calcium ions (Ca 2+ Hydroxyapatite is composed of approximately 70% of human bone and exhibits good biocompatibility and bioactivity, as well as good mechanical and chemical stability. Due to its ease of doping and structural adjustability, hydroxyapatite is also used in fillers to enhance their stability. Hydroxyapatite can also promote collagen production, making it an excellent biomaterial. However, because of its low solubility under physiological conditions, hydroxyapatite tends to aggregate and crystallize in the human body.

[0004] Collagen is widely used in tissue filling, hemostatic sponges, and drug delivery systems. Compared to traditional collagen, recombinant collagen offers significant improvements in biocompatibility, biodegradability, controllability, and production cost. Currently, there are no literature reports on the preparation of injectable medical materials using a combination of recombinant collagen and hydroxyapatite. Summary of the Invention

[0005] The purpose of this invention is to provide an injectable hydroxyapatite cross-linked recombinant collagen gel and its preparation method. This invention incorporates hydroxyapatite into the recombinant collagen, allowing it to be evenly distributed during injection without aggregation in the body. Simultaneously, hydroxyapatite itself promotes collagen production in the human body, and its degradation rate ensures the stability of the recombinant collagen, enabling it to remain in the body for a longer period and prolonging the filling effect.

[0006] The technical solution for achieving the objective of this invention is as follows:

[0007] A method for preparing injectable hydroxyapatite cross-linked recombinant collagen gel includes the following steps:

[0008] (1) Hydroxyapatite and recombinant collagen were stirred and mixed in water, and the pH of the mixed solution was adjusted to 4-6 with an acidic solution. Then, 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) catalyst was added. After reacting for 20-60 seconds, the mixture was transferred to a mold and allowed to stand at room temperature to obtain a solid gel. In the solid gel, the mass concentration of recombinant collagen was 15%-20%, the mass concentration of hydroxyapatite was 20%-40%, and the mass concentration of catalyst was 1%-2%. The recombinant collagen was produced by fermentation of Pichia pastoris with accession number CGMCC No. 5021.

[0009] (2) Cut the solid gel into pieces, then wash it first with an acidic solution with pH=3 to 5, and then wash it with physiological saline.

[0010] (3) Hydroxyapatite and sodium hyaluronate are mixed and dissolved in water to form a dispersion. Then, the washed gel block and the dispersion are mixed evenly and finally homogenized to obtain injectable hydroxyapatite cross-linked recombinant collagen gel. In the dispersion, the mass concentration of hydroxyapatite is 20% to 40% and the mass concentration of sodium hyaluronate is 0.3% to 0.5%.

[0011] Furthermore, in step (1) or (3), the hydroxyapatite is in the form of microspheres with a particle size of 10–50 μm.

[0012] Furthermore, in step (1), the stirring speed is 200-500 rpm / min and the stirring time is 10-30 min.

[0013] Furthermore, in step (1), the reaction time is 20 s.

[0014] Furthermore, in step (1), the settling time is 1 to 3 hours, preferably 2 hours.

[0015] Furthermore, in step (2), the volume of the gel block is 0.5. 3 ~1.0 3 cubic centimeters.

[0016] Furthermore, in step (2), the hydrochloric acid solution is used for rinsing more than 3 times, and the physiological saline solution is used for rinsing more than 2 times, with each rinsing session lasting 2 to 4 hours.

[0017] Further, in step (2), the weight ratio of hydrochloric acid solution or physiological saline to gel block is 10:1 to 20:1.

[0018] Furthermore, in step (3), the molecular weight of sodium hyaluronate is 80-150W, preferably 120W.

[0019] Furthermore, in step (3), the frequency of the homogenizer is 30-40 Hz.

[0020] The present invention provides an injectable hydroxyapatite cross-linked recombinant collagen gel prepared by the above preparation method.

[0021] Furthermore, the present invention provides the application of the above-mentioned injectable hydroxyapatite cross-linked recombinant collagen gel as an injectable filler material.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] (1) The hydroxyapatite cross-linked collagen gel of the present invention is composed of small particles of gel obtained by homogenization of solid gel. It has high cross-linking degree, high strength, and strong encapsulation. After injection, it stays in the tissue for a longer time than similar gels and can play a better filling role.

[0024] (2) In the preparation process of the hydroxyapatite cross-linked collagen gel of the present invention, the reaction conditions are mild, the required environment is friendly, and there are no toxic or side-effect substances, making it suitable for large-scale production. At the same time, the hydroxyapatite cross-linked collagen gel itself has better biocompatibility, is non-immunogenic, and is degradable.

[0025] (2) In the hydroxyapatite cross-linked collagen gel of the present invention, hydroxyapatite is uniformly distributed in the gel, and can also achieve a uniform support effect in the tissue after injection. At the same time, hydroxyapatite degrades slowly and has a long existence time, which better stimulates collagen production and can provide excellent filling effect for a long time.

[0026] (3) In the hydroxyapatite crosslinked collagen gel of the present invention, the uncrosslinked sodium hyaluronate helps to improve the feel of injection and reduce pain during injection, and provides immediate hydration after injection, resulting in better injection effect. Similarly, the production process allows the gel to contain more hydroxyapatite to achieve better injection effect. Attached Figure Description

[0027] Figure 1 The hydroxyapatite crosslinked recombinant collagen gel sample for injection prepared in Example 3;

[0028] Figure 2 The images show the sterilized hydroxyapatite cross-linked recombinant collagen gel samples prepared in Example 3 and Comparative Example 3, with the left image being Example 3 and the right image being Comparative Example 3, where a and b represent the liquid surface and sediment surface, respectively.

[0029] Figure 3 The extrusion force curves of the hydroxyapatite crosslinked recombinant collagen gel samples prepared in Example 3 and Comparative Example 4 are shown, where a is Comparative Example 4 and b is Example 3.

[0030] Figure 4 The image shows an animal tissue section of the hydroxyapatite cross-linked recombinant collagen gel sample prepared in Example 3, wherein the control group was injected with physiological saline and the B group was injected with Example 3. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0032] Example 1

[0033] (1) Dissolve 4.5g of recombinant collagen powder and 6.0g of hydroxyapatite powder in 19.2g of purified water, and adjust the pH to 5.51 with 1mol / L hydrochloric acid solution. At room temperature and 300rpm, add 0.3g of catalyst EDC, react for 20s, quickly transfer to a mold, and let stand at room temperature for 2h to obtain a solid gel.

[0034] (2) Cut the solid gel into gel blocks with a side length of 0.5 cm, and then wash it 3 times with hydrochloric acid solution with a pH of 4.01 and 3 times with physiological saline, each time for 3 hours.

[0035] (3) Dissolve 0.06g sodium hyaluronate (120W) and 4g hydroxyapatite in purified water, with a total mass of 20g, and stir until homogeneous to form a dispersion. Then mix the dispersion with the washed gel block and homogenize with a homogenizer to obtain injectable hydroxyapatite cross-linked recombinant collagen gel.

[0036] Example 2

[0037] (1) Dissolve 6.0g of recombinant collagen powder and 12g of hydroxyapatite powder in 11.4g of purified water, and adjust the pH to 5.45 with 1mol / L hydrochloric acid solution. At room temperature and 300rpm, add 0.6g of catalyst EDC, react for 20s, quickly transfer to a mold, and let stand at room temperature for 2h to obtain a solid gel.

[0038] (2) Cut the solid gel into gel blocks with a side length of 0.5 cm, then wash it 3 times with hydrochloric acid solution with a pH of 4.01 and 2 times with physiological saline, each time for 3 hours.

[0039] (3) Dissolve 0.1g sodium hyaluronate (120W) and 8g hydroxyapatite in purified water, with a total mass of 20g, and stir until homogeneous to form a dispersion. Then mix the dispersion with the washed gel block and homogenize with a homogenizer to obtain injectable hydroxyapatite cross-linked recombinant collagen gel.

[0040] Example 3

[0041] (1) Dissolve 4.8g of recombinant collagen powder and 9.0g of hydroxyapatite powder in 15.9g of purified water, and adjust the pH to 5.51 with 1mol / L hydrochloric acid solution. At room temperature and 300rpm, add 0.3g of catalyst EDC, react for 20s, quickly transfer to a mold, and let stand at room temperature for 2h to obtain a solid gel.

[0042] (2) Cut the solid gel into gel pieces with a side length of 0.5 cm. Then wash three times with hydrochloric acid solution with a pH of 4.01 and three times with physiological saline, each time for 3 hours.

[0043] (3) Dissolve 0.1g sodium hyaluronate (120W) and 6g hydroxyapatite in purified water, with a total mass of 20g, and stir until homogeneous to form a dispersion. Then mix the dispersion with the washed gel block and homogenize with a homogenizer to obtain injectable hydroxyapatite cross-linked recombinant collagen gel.

[0044] Figure 1 The hydroxyapatite crosslinked recombinant collagen gel sample prepared in Example 3 exhibits uniform and continuous injectability.

[0045] Example 4

[0046] (1) Dissolve 6.0g of recombinant collagen powder and 9.0g of hydroxyapatite powder in 14.7g of purified water, and adjust the pH to 5.44 with 1mol / L hydrochloric acid solution. At room temperature and 300rpm, add 0.3g of catalyst EDC, react for 20s, quickly transfer to a mold, and let stand at room temperature for 2h to obtain a solid gel.

[0047] (2) Cut the solid gel into gel blocks with a side length of 0.5 cm, then wash it 3 times with hydrochloric acid solution with a pH of 4.01 and 2 times with physiological saline, each time for 3 hours.

[0048] (3) Dissolve 0.1g sodium hyaluronate (120W) and 6g hydroxyapatite in purified water, with a total mass of 20g, and stir until homogeneous to form a dispersion. Then mix the dispersion with the washed gel block and homogenize with a homogenizer to obtain injectable hydroxyapatite cross-linked recombinant collagen gel.

[0049] Example 5

[0050] (1) Dissolve 4.5g of recombinant collagen powder and 9.0g of hydroxyapatite powder in 14.4g of purified water, and adjust the pH to 5.47 with 1mol / L hydrochloric acid solution. At room temperature and 300rpm, add 0.6g of catalyst EDC, react for 20s, quickly transfer to a mold, and let stand at room temperature for 2h to obtain a solid gel.

[0051] (2) Cut the solid gel into gel pieces with sides of 0.5 cm. Then wash three times with hydrochloric acid solution with pH 4.01 and twice with physiological saline, each time for 3 hours.

[0052] (3) Dissolve 0.1g sodium hyaluronate (120W) and 6g hydroxyapatite in purified water, with a total mass of 20g, and stir until homogeneous to form a dispersion. Then mix the dispersion with the washed gel block and homogenize with a homogenizer to obtain injectable hydroxyapatite cross-linked recombinant collagen gel.

[0053] Example 6

[0054] (1) Dissolve 4.8g of recombinant collagen powder and 6.0g of hydroxyapatite powder in 18.9g of purified water, and adjust the pH to 5.60 with 1mol / L hydrochloric acid solution. At room temperature and 300rpm, add 0.3g of catalyst EDC, react for 20s, quickly transfer to a mold, and let stand at room temperature for 2h to obtain a solid gel.

[0055] (2) Cut the solid gel into gel pieces with a side length of 0.5 cm. Then wash three times with hydrochloric acid solution with a pH of 4.01 and three times with physiological saline, each time for 3 hours.

[0056] (3) Dissolve 0.1g sodium hyaluronate (120W) and 4g hydroxyapatite in purified water, with a total mass of 20g, and stir until homogeneous to form a dispersion. Then mix the dispersion with the washed gel block and homogenize with a homogenizer to obtain injectable hydroxyapatite cross-linked recombinant collagen gel.

[0057] Example 7

[0058] (1) Dissolve 4.8g of recombinant collagen powder and 12.0g of hydroxyapatite powder in 12.9g of purified water, and adjust the pH to 5.44 with 1mol / L hydrochloric acid solution. At room temperature and 300rpm, add 0.3g of catalyst EDC, react for 20s, quickly transfer to a mold, and let stand at room temperature for 2h to obtain a solid gel.

[0059] (2) Cut the solid gel into gel pieces with a side length of 0.5 cm. Then wash three times with hydrochloric acid solution with a pH of 4.01 and three times with physiological saline, each time for 3 hours.

[0060] (3) Dissolve 0.1g sodium hyaluronate (120W) and 8g hydroxyapatite in purified water, with a total mass of 20g, and stir until homogeneous to form a dispersion. Then mix the dispersion with the washed gel block and homogenize with a homogenizer to obtain injectable hydroxyapatite cross-linked recombinant collagen gel.

[0061] Example 8

[0062] This embodiment is basically the same as embodiment 3, except that the amount of hydroxyapatite added in step (1) is 12g, that is, the content of hydroxyapatite in the solid gel is 40%.

[0063] Example 9

[0064] This embodiment is basically the same as embodiment 3, except that the amount of hydroxyapatite added in step (3) is 8g, that is, the content of hydroxyapatite in the dispersion is 40%.

[0065] Comparative Example 1

[0066] This comparative example is basically the same as Example 3, except that in step (2), the hydrochloric acid solution washing is replaced with purified water washing.

[0067] Comparative Example 2

[0068] This comparative example is basically the same as Example 3, except that the washing time of hydrochloric acid solution and physiological saline in step (2) is halved, that is, the washing time is 1.5h each time.

[0069] Comparative Example 3

[0070] This comparative example is basically the same as Example 3, except that the amount of EDC added in step (1) is 0.12g, that is, the content of EDC in the formed gel is, and the gel formed after cross-linking is fluid.

[0071] Comparative Example 4

[0072] This comparative example is basically the same as Example 3, except that the amount of sodium hyaluronate added in step (3) is 0.05g, that is, the content of sodium hyaluronate in the dispersion is 0.25%.

[0073] Comparative Example 5

[0074] This comparative example is basically the same as Example 3, except that acid washing is not performed in step (2), and physiological saline is used directly for washing. In step (3), 1 mol / L HCl is added to the dispersion to adjust its pH to 6-7, so as to adjust the pH of the final product.

[0075] Comparative Example 6

[0076] This comparative example is basically the same as Example 3, except that the amount of hydroxyapatite added in step (1) is 6g, that is, the content of hydroxyapatite in the solid gel is 20%.

[0077] Comparative Example 7

[0078] This comparative example is basically the same as Example 3, except that the amount of hydroxyapatite added in step (3) is 4g, that is, the content of hydroxyapatite in the dispersion is 20%.

[0079] Characterization example

[0080] 1. Determination of hydroxyapatite content

[0081] The hydroxyapatite content in the hydroxyapatite-crosslinked recombinant collagen gels prepared in each example and comparative example was determined using an enzymatic hydrolysis method. The results are shown in Table 1. Comparing Examples 1-3, 6-9 and Comparative Examples 6-7, it can be seen that the amount of hydroxyapatite added in both the crosslinking and homogenization steps affects the hydroxyapatite content in the final product.

[0082] 2. pH measurement

[0083] According to the pH determination method in 0631 of the Pharmacopoeia of the People's Republic of China (Volume IV) (2020 Edition), a pH meter was used to determine the pH value of the hydroxyapatite cross-linked recombinant collagen gels prepared in each example and comparative example. The results are shown in Table 1. Comparing Example 3 and Comparative Examples 3-4, it can be seen that the acid washing step and the acid washing time affect the pH of the final product. By comparing Examples 1-3, 6-9 and Comparative Examples 6-7, it can be seen that since hydroxyapatite is alkaline, changes in its content also affect the pH. The acid washing time needs to be no less than 6 hours to neutralize the alkalinity of the product, so that the product reaches the pH conditions suitable for human injection, and it is also more conducive to improving the product's resistance to degradation.

[0084] Furthermore, pH values ​​were measured from the upper, middle, and lower layers of the samples in Example 3 and Comparative Example 5. Table 1 shows that the pH values ​​of the upper, middle, and lower layers of the Comparative Example 5 sample differed significantly, indicating that while adjusting the pH of the dispersion during homogenization can control the pH of the final product to some extent, it cannot completely control the pH, posing a risk of inhomogeneity. In contrast, the pH values ​​of the upper, middle, and lower layers of the Example 3 sample were uniform.

[0085] 3. Determination of catalyst residue

[0086] According to the Pharmacopoeia of the People's Republic of China, section 9101, the validation items included linearity, specificity, limit of detection, accuracy (recovery rate), precision, and stability. All validation results met the requirements of the Pharmacopoeia of the People's Republic of China. The results are shown in Table 1. Comparing Examples 1-2 and Examples 4-5, it can be seen that increasing the catalyst dosage leads to an increase in catalyst residue in the product; however, due to the cleaning step, the results are still within the acceptable range. Comparing Example 3 and Comparative Example 2 shows that the cleaning time affects catalyst residue.

[0087] Table 1. Hydroxyapatite content, pH value, and cross-linking agent residue of hydroxyapatite-crosslinked recombinant collagen gel for injection.

[0088]

[0089] 4. Aging test

[0090] YY / T 0681.1-2018 Test Methods for Sterile Medical Device Packaging Part 1: Guidelines for Accelerated Aging Tests states that accelerated aging technology is based on the assumption that the chemical reactions involved in material degradation follow the Arrhenius reaction rate function. Numerous studies of chemical reactions have shown that a temperature increase or decrease of 10°C can double or halve the chemical reaction rate. Therefore, a simplified formula for accelerated aging can be established based on the Arrhenius reaction rate function:

[0091]

[0092] Parameter meanings: AAT: Accelerated aging time; RT: Real-time aging time; Q10: Aging coefficient when the temperature increases or decreases by 10℃; TAA: Accelerated aging temperature; TRT: Temperature under normal storage conditions.

[0093] The above formula reflects the relationship between accelerated aging time and corresponding shelf life in accelerated stability testing. Q10 is generally set to 2. Setting a higher accelerated aging temperature can reduce the accelerated stability test time. However, higher temperatures may alter the properties of medical device raw materials / components and packaging materials or trigger multiple chemical reactions, leading to deviations in test results. Therefore, the accelerated aging temperature should generally not exceed 60°C. Set the aging parameters according to the table below.

[0094] Table 2: Acceleration Stability Parameter Settings

[0095]

[0096] Taking the injectable hydroxyapatite cross-linked recombinant collagen gel prepared in Example 3 as an example, as can be seen from Table 3, the product of the present invention has higher stability than other similar products and has a better filling and support effect after injection.

[0097] Table 3 Summary of accelerated aging data of injectable hydroxyapatite cross-linked recombinant collagen gel at 40℃

[0098]

[0099]

[0100] 5. Sterilization stability test

[0101] The stability of the injectable hydroxyapatite cross-linked recombinant collagen gel samples prepared in Example 3 and Comparative Example 3 was observed after moist heat sterilization. Figure 2 The images show the sterilized samples of injectable hydroxyapatite cross-linked recombinant collagen gel prepared in Example 3 and Comparative Example 3, with Example 3 on the left and Comparative Example 3 on the right. Figure 2 It can be observed that reducing the amount of cross-linking agent decreases the degree of cross-linking of the gel, making it fluid during cross-linking. While it can still be made into a gel, after sterilization, the gel liquefies. In Comparative Example 3, hydroxyapatite precipitates at the bottom, while the sample in Example 3 remains gel-like after sterilization, exhibiting stable properties. Therefore, with an appropriate degree of cross-linking, the gel becomes solid, and the resulting fluid gel, obtained through homogenization, has higher strength and better encapsulation properties. It can stably encapsulate hydroxyapatite microspheres, achieving better support during injection into the human body.

[0102] 6. Pushing force test

[0103] According to Appendix A of "YY / T 0962-2021 Cross-linked Sodium Hyaluronate Gel for Plastic Surgery" for the determination of extrusion force, the injectable hydroxyapatite cross-linked recombinant collagen gel samples prepared in Example 3 and Comparative Example 4 were filled into pre-filled syringes, 27G needles were installed, air was removed from the tip, and the samples were tested using a MED-01 pharmaceutical packaging performance tester. Figure 3 The extrusion force curves are shown for the injectable hydroxyapatite cross-linked recombinant collagen gel samples prepared in Example 3 and Comparative Example 4, where a is Comparative Example 4 and b is Example 3. Figure 3 It can be seen that reducing the amount of sodium hyaluronate used in the dispersion significantly improves the pushing force. These results indicate that, in addition to its lubricating effect and improved injection feel, sodium hyaluronate, due to its water-absorbing properties, expands appropriately while providing gel support during immediate injection, resulting in a better injection effect.

[0104] 7. Cell experiments

[0105] Take 0.1g of the injectable hydroxyapatite cross-linked recombinant collagen gel prepared in Example 3, add 1.2ml of MEM complete medium containing 10% FBS, and extract in a 37℃ incubator for 24h. Centrifuge at 1000rpm for 5min, and collect the supernatant as 100% extract. Then, dilute the extract to 1%, 10%, 25%, 50%, 75%, and 100% as test samples. Digest L-929 cells grown to the logarithmic growth phase with 0.25% trypsin (containing EDTA). After digestion, centrifuge the cell suspension (1000rpm, 5min), discard the supernatant, resuspend the cells in MEM medium, and count to obtain 1×10⁶ cells. 4 Cell suspension was seeded at 100 μl per well in 96-well plates and cultured in a cell culture incubator (37℃, 5% CO2, >90% humidity). Cell morphology was observed under a microscope. After 24 hours of incubation, when cells had grown into a monolayer, the original culture medium in the 96-well plates was discarded. 100 μl of sample, blank control, and positive control samples were added to the corresponding wells of the 96-well plates. The 96-well plates were then cultured in a cell culture incubator (37℃, 5% CO2, >90% humidity) for 24 hours, with 5 replicates per group. After 24 hours of culture, the 96-well plates were removed, and cell morphology was observed under a microscope. The liquid was then removed, and 50 μl of MTT (final concentration 1 mg / ml) was added to each well. The plates were then incubated in a CO2 incubator. After 2 hours, the supernatant was removed, and 100 μl of DMSO / isopropanol was added to each well to dissolve the crystals. The absorbance at 570 nm was measured using a microplate reader, and the cytotoxicity was calculated.

[0106] According to the cytotoxicity criteria in GB / T 16886.5 "Biological Evaluation of Medical Devices - Part 5: In Vitro Cytotoxicity Tests", a decrease in cell viability greater than 30% is considered cytotoxic. As shown in Table 4, injectable hydroxyapatite cross-linked recombinant collagen gel did not exhibit cytotoxicity against L-929 cells in 100%-1% extract.

[0107] Table 4. Results of L-929 Cytotoxicity Tests

[0108]

[0109] 8. Animal experiments

[0110] SPF-grade C57 female mice were used in animal experiments. All technical indicators met the technical requirements for barrier environments in GB 14925-2010 "Laboratory Animal Environment and Facilities". Animals had free access to food and water, and their feed met the nutritional requirements of GB 14924.3-2010 "Laboratory Animal Compound Feed".

[0111] In the experiment, four subcutaneous implantation sites were evenly selected on the back of each mouse, and approximately 60 μl of this product was injected into each site, with a total gel injection volume of approximately 0.25 ml per mouse. Control group mice received an equal volume of physiological saline injected into the same locations on their backs. Mice were euthanized by cervical dislocation 14, 28, 42, 63, and 91 days after injection, and skin tissue was collected and fixed in 4% paraformaldehyde solution for subsequent tissue sectioning.

[0112] The injectable hydroxyapatite cross-linked recombinant collagen gel sample prepared in Example 3 (experimental group) and physiological saline (control group) were injected into mice. At 14, 28, 42, 63, and 91 days post-injection, skin tissue was embedded and sectioned for Masson staining and Sirius red staining to analyze collagen production and collagen fiber density. The results are as follows: Figure 4 As shown.

[0113] Figure 4 Tissue sections of the injectable hydroxyapatite cross-linked recombinant collagen gel prepared in Example 3 were used in animal experiments. The control group was injected with physiological saline, and group B was injected with the sample from Example 3. Figure 4 Sirius red staining results showed that the collagen fiber structure in the control group was stable, with no obvious new formation or remodeling. On days 14 and 28, collagen deposition in group B was significantly better than in the control group. By day 63, collagen deposition in group B was even richer, with distinct layers, no obvious fatty degeneration, and good tissue remodeling. By day 91, the tissue structure was close to normal skin, with dense and uniform collagen deposition, indicating relatively complete tissue repair. These results demonstrate that the injectable hydroxyapatite cross-linked recombinant collagen gel of this invention significantly promotes collagen synthesis and tissue structure recovery.

Claims

1. A method for preparing injectable hydroxyapatite cross-linked recombinant collagen gel, characterized in that, Includes the following steps: (1) Hydroxyapatite and recombinant collagen were stirred and mixed in water, and the pH of the mixed solution was adjusted to 4-6 with an acidic solution. Then, catalyst EDC was added, and after reacting for 20-60 seconds, the mixture was transferred to a mold and allowed to stand at room temperature to obtain a solid gel. In the solid gel, the mass concentration of recombinant collagen was 15%-20%, the mass concentration of hydroxyapatite was 20%-40%, and the mass concentration of catalyst was 1%-2%. The recombinant collagen was produced by fermentation of Pichia pastoris with accession number CGMCC No. 5021. (2) Cut the solid gel into pieces, then wash it first with an acidic solution with pH=3~5, and then wash it with physiological saline. (3) Hydroxyapatite and sodium hyaluronate are mixed and dissolved in water to form a dispersion. Then, the washed gel block and the dispersion are mixed evenly and finally homogenized to obtain injectable hydroxyapatite cross-linked recombinant collagen gel. In the dispersion, the mass concentration of hydroxyapatite is 20%~40% and the mass concentration of sodium hyaluronate is 0.3%~0.5%.

2. The preparation method according to claim 1, characterized in that, In step (1) or (3), hydroxyapatite is in the form of microspheres with a particle size of 10~50μm.

3. The preparation method according to claim 1, characterized in that, In step (1), the stirring speed is 200~500 rpm / min, the stirring time is 10~30 min, and the standing time is 1~3 h.

4. The preparation method according to claim 1, characterized in that, In step (1), the reaction time is 20s and the settling time is 2h.

5. The preparation method according to claim 1, characterized in that, In step (2), the volume of the gel block is 0.

5. 3 ~1.0 3 The volume is cubic centimeters; the number of times to clean with hydrochloric acid solution is more than 3, and the number of times to clean with physiological saline is more than 2, with each cleaning time lasting 2 to 4 hours.

6. The preparation method according to claim 1, characterized in that, In step (2), the weight ratio of hydrochloric acid solution or physiological saline to gel block is 10:1 to 20:

1.

7. The preparation method according to claim 1, characterized in that, In step (3), the molecular weight of sodium hyaluronate is 80~150W.

8. The preparation method according to claim 1, characterized in that, In step (3), the homogenizer has a frequency of 30~40Hz and the molecular weight of sodium hyaluronate is 120W.

9. The hydroxyapatite crosslinked recombinant collagen gel for injection prepared by any one of the preparation methods according to claims 1 to 8.

10. The use of the injectable hydroxyapatite crosslinked recombinant collagen gel according to claim 9 as an injectable filler material.

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