Implant containing mineralized hydroxyapatite and collagen as well as preparation method and application of implant

Abstract

The invention provides an implant containing mineralized hydroxyapatite and collagen as well as a preparation method and application of the implant, and relates to the technical field of organic high-molecular compound processing and medical materials. According to the preparation method, water-soluble micromolecular saccharides such as D-mannitol and trehalose are added into the collagen solution, so that the stability of the collagen solution is improved; the preparation method comprises the following steps: adding calcium hydrophosphate into a collagen solution, fully stirring and mixing by utilizing weak bonds formed between side chain groups in the collagen and calcium ions in the calcium hydrophosphate, and then adding calcium hydroxide step by step to realize step-by-step regulation and control of the pH value, so that the collagen is fully and uniformly combined with calcium phosphate; according to the collagen implant containing the mineralized hydroxyapatite, the uniform in-situ synthesis of the hydroxyapatite on the collagen is realized, the uniform collagen mineralized hydroxyapatite is obtained, the hydroxyapatite and the collagen are tightly combined, and the obtained collagen implant containing the mineralized hydroxyapatite can be stably injected through a 27G needle and has good degradation performance and safety performance.

Description

Technical Field

[0001] The present invention relates to the technical field of organic polymer compound processing and medical materials, and in particular to an implant containing mineralized hydroxyapatite and collagen, and a preparation method and application thereof. Background Art

[0002] To improve facial aging problems (rough skin, sagging, wrinkles, etc.), facial filler materials are rapidly developing. Currently, the main types of materials used for facial filler are as follows:

[0003] Collagen and hyaluronic acid facial fillers: They are effective immediately, but they degrade relatively quickly, have a short efficacy period, and do not promote collagen production.

[0004] Facial fillers containing PCL, PMMA, and PLLA microspheres primarily utilize PCL, PMMA, and PLLA microspheres to stimulate inflammation and promote collagen production, which can last for a long time. However, the degree of inflammation varies significantly among individuals, leading to a relatively high risk of microsphere migration and vascular occlusion. Furthermore, microspheres are prone to sedimentation and aggregation during storage when blended with other materials, leading to needle clogging during injection.

[0005] Hydroxyapatite-containing facial fillers, primarily Radiesse, are primarily composed of hydroxyapatite microspheres, which have a long history of use internationally. Hydroxyapatite, a component of bone, exhibits excellent biocompatibility, is less likely to induce adverse immune responses, and can promote collagen production. However, hydroxyapatite has a long degradation cycle, especially after high-temperature sintering. It is the most stable phase of calcium phosphate in body fluids, while other calcium phosphates convert to hydroxyapatite. While hydroxyapatite's long degradation cycle or resistance to degradation in the body increases the product's effectiveness, this prolonged degradation cycle significantly increases microsphere aggregation and displacement, leading to potential risks such as nodules and vascular occlusion.

[0006] In addition, existing facial filling materials containing hydroxyapatite have poor injectability and are prone to needle clogging. When the injectability is good, there is a greater possibility of rapid collagen degradation or uncontrolled release, which can easily cause a surge in local calcium and phosphate ion concentrations, triggering inflammation or ectopic mineralization. Summary of the Invention

[0007] In order to reduce the potential risks of cosmetic filler materials in the prior art, such as difficulty in degradation in the body, easy aggregation and displacement of microspheres, and thus leading to nodules, blood vessel blockage, etc.; and to improve the injectability and safety of hydroxyapatite-containing facial filler materials, the present application provides mineralized hydroxyapatite and collagen implants, as well as preparation methods and applications thereof.

[0008] Technical solution:

[0009] In the first aspect, a method for preparing mineralized hydroxyapatite:

[0010] A method for preparing mineralized hydroxyapatite comprises the following steps:

[0011] A 0.01-0.15% collagen solution containing 0.1-1 wt% of a small molecule carbohydrate dispersant is prepared, and then a pH adjusting solution formed by slowly adding dilute hydrochloric acid to dissolve calcium hydrogen phosphate is adjusted to a pH of 3-4 to form solution A. The mass ratio of calcium hydrogen phosphate to collagen in solution A is (0.8-1.5):1;

[0012] The calcium hydroxide suspension is slowly added to solution A, the pH value of the solution is adjusted to 9-10, and stirred for 1.5-4 hours; then the solution is allowed to stand for 12-36 hours, the supernatant is removed, and the precipitate is filtered, washed, freeze-dried, ground, and sieved to obtain mineralized hydroxyapatite powder.

[0013] The focus of this patent is on injectable collagen implants containing collagen mineralized hydroxyapatite, which are mainly used for facial medical beauty injections.

[0014] Currently, mineralized hydroxyapatite with a high collagen content (e.g., a 50% collagen to hydroxyapatite ratio) suffers from severe collagen aggregation during freezing. This aggregated mineralized hydroxyapatite is difficult to redisperse upon remelting, and is also difficult to grind and sieve after freeze-drying. The mineralized collagen prepared in this way experiences excessive force during the compounding of the collagen solution, and is prone to needle clogging.

[0015] During our research, we found that in the process of preparing mineralized hydroxyapatite, adding D-mannitol and other substances is beneficial to preventing the aggregation of collagen. After remelting, its dispersibility is significantly improved. The pushing force of the final preparation of the collagen implant containing mineralized hydroxyapatite is significantly reduced, and there is no problem of needle blockage.

[0016] The hydroxyapatite prepared in this invention also exhibits superior degradation performance, with a higher effective ion release concentration than a control group without D-mannitol. This is primarily due to the effective inhibition of collagen aggregation by the addition of D-mannitol, which imparts a larger specific surface area to the mineralized collagen particles. The effective release of calcium and phosphorus ensures the complete degradation of the hydroxyapatite in this implant, overcoming the difficulty in degrading traditional sintered hydroxyapatite and ensuring the safety of this product in clinical applications.

[0017] The present invention utilizes mannitol to effectively inhibit collagen aggregation, which is beneficial to the grinding and screening of mineralized hydroxyapatite. At the same time, the pH value cooperates with mannitol to increase the solubility of mineralized hydroxyapatite and enhance the chemical bonding between mineralized hydroxyapatite and collagen. Under acidic conditions, collagen and calcium phosphate interact weakly with each other, collagen absorbs calcium phosphate to the maximum extent in a neutral environment, and hydroxyapatite is mineralized in an alkaline environment. Collagen is compounded with mineralized hydroxyapatite and an appropriate amount of cross-linking agent is used to enhance the interaction between mineralized hydroxyapatite and collagen. As a result, the obtained collagen implant containing mineralized hydroxyapatite has good injectability, can be injected and drawn with a 27G needle, does not deviate during the in vivo metabolic process, has a long effective filling time, can be completely degraded at the implantation site, and has good material biocompatibility and higher safety.

[0018] Furthermore, the dispersant is a small molecule sugar dispersant with a molecular weight of less than 1000.

[0019] Preferably, the molecular weight of the dispersant is less than 600.

[0020] Furthermore, the dispersant is D-mannitol and / or trehalose.

[0021] Furthermore, the mass ratio of calcium hydrogen phosphate to collagen in the solution A is (1-1.2):1, and the stirring time is 1-3 hours;

[0022] The steps for adjusting pH value using calcium hydroxide suspension are as follows:

[0023] Slowly add the calcium hydroxide suspension to solution A, adjust the pH value of the solution to 6-7, and continue the reaction for 1-2 hours; continue to add the calcium hydroxide suspension until the pH value of the solution reaches 9-10, and continue stirring for 0.5-2 hours.

[0024] Furthermore, the Ca / P ratio of the calcium phosphate salt in the final product in the final solution is (1.3-2):1; and the mass ratio of the calcium hydroxide to the collagen is (0.1-0.35):1.

[0025] In a second aspect, the present application provides a mineralized hydroxyapatite:

[0026] A mineralized hydroxyapatite is obtained by using a preparation method of mineralized hydroxyapatite of the present application.

[0027] In a third aspect, the present application provides a method for preparing a collagen implant containing mineralized hydroxyapatite:

[0028] A method for preparing a collagen implant containing mineralized hydroxyapatite comprises the following steps:

[0029] The mineralized hydroxyapatite obtained by the preparation method described in the present application or the mineralized hydroxyapatite-containing material described in the present application is mixed with a cross-linking agent to form a mixture; the mixture is then stirred and mixed with a 5-50 mg / mL neutral collagen solution to prepare a collagen implant containing mineralized hydroxyapatite;

[0030] The amount of the cross-linking agent in the mixture is 0.1-2.5wt% of the amount of the mineralized hydroxyapatite powder;

[0031] The mixture in the collagen implant accounts for 5-30 wt%.

[0032] Furthermore, the collagen in the collagen solution and the neutral collagen solution includes any one or more combinations of type I collagen, type II collagen, and type III collagen; and the cross-linking agent includes genipin and / or an aldehyde cross-linking agent.

[0033] In a fourth aspect, the present application provides a collagen implant containing mineralized hydroxyapatite:

[0034] The collagen implant containing mineralized hydroxyapatite is obtained by the preparation method of the collagen implant described in the present application.

[0035] In a fifth aspect, the present application provides the use of the mineralized hydroxyapatite as described in the present application or the collagen implant as described in the present application in the preparation of a medical injectable filling material.

[0036] The mineralized hydroxyapatite collagen implant described in this application utilizes non-sintered mineralized hydroxyapatite for facial augmentation and improvement of deep wrinkles such as nasolabial folds, ensuring complete degradation of the implant and enhancing product safety. Hydroxyapatite and collagen are essential components of the body and possess excellent biocompatibility, making subcutaneous implantation for facial skin improvement more biosafe and less likely to induce adverse immune responses. Mineralized hydroxyapatite can prolong collagen degradation, promote collagen production in the body, and increase the duration of product efficacy. The use of micro-crosslinking technology for mineralized hydroxyapatite and collagen mitigates the potential risks of implant displacement and vascular occlusion, particularly in dynamic wrinkle areas, associated with subcutaneous implantation.

[0037] Beneficial effects:

[0038] 1. In the preparation method of the present application, water-soluble small molecule sugars such as D-mannitol and trehalose are added to the collagen solution to increase the stability of the collagen solution, inhibit excessive hydrophobic aggregation of collagen during the mineralization process of mineralized hydroxyapatite, increase the uniformity of mineralization sedimentation, and facilitate the later grinding of mineralized hydroxyapatite and the dispersion of mineralized hydroxyapatite powder in the collagen solution; at the same time, small molecule sugars can be used as cryoprotectants, which is beneficial to the freeze-drying molding of mineralized hydroxyapatite and the crushing and grinding of mineralized hydroxyapatite after freeze-drying. The obtained sintered hydroxyapatite has a looser structure and hydrophilic properties. The loose structure enables it to better contact with water and thus has better degradability, forming an injectable implant that can be injected with a 27G needle.

[0039] 2. In the preparation method of the present application, calcium hydrogen phosphate is added to the collagen solution, and the weak bond formed between the side chain groups (mainly carboxyl groups) in the collagen and the calcium ions in the calcium hydrogen phosphate is utilized. After thorough stirring and mixing (1-3 hours), the calcium hydrogen phosphate is evenly distributed, and then calcium hydroxide is added step by step to achieve step-by-step regulation. The pH value of the solution is regulated at 6-7 by calcium hydroxide, and the free H+ in the solution is consumed by neutralization reaction, thereby enhancing the ionization of the collagen carboxylic acid group. The solution is stirred for 1-2 hours to ensure full binding of the collagen and the calcium hydrogen phosphate. Calcium hydroxide is further added to regulate the pH value of the solution to 9-10 to provide an alkaline environment for the synthesis of hydroxyapatite, react with the calcium hydrogen phosphate bound to the collagen, and achieve relatively uniform in situ synthesis of hydroxyapatite on the collagen, thereby obtaining uniform collagen mineralized hydroxyapatite, so that the hydroxyapatite and collagen are tightly bound. The obtained collagen containing mineralized hydroxyapatite can be stably injected through a 27G needle, and has good degradation and safety performance.

[0040] 3. The present invention utilizes a cross-linking agent to achieve weak cross-linking between the micropowder and the collagen implant, which facilitates the fixation of the micropowder and prevents aggregation of the micropowder during storage and displacement of the micropowder after implantation, which can lead to problems such as nodules and blood vessel blockage. The present invention utilizes a cross-linking agent to react with primary amines to achieve cross-linking between collagens, forming a collagen network. The collagen network fixes the micropowder, achieving stable and uniform weak cross-linking between the micropowder and the collagen, thereby extending the duration of action of the collagen implant in the body, improving safety, and enhancing the cosmetic effect of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the XRD pattern of mineralized hydroxyapatite obtained by the preparation method in Example 1;

[0042] Figure 2 is the FT-IR spectrum of mineralized hydroxyapatite obtained by the preparation method in Example 1;

[0043] Figure 3 is a SEM morphology image of the mineralized hydroxyapatite obtained by the preparation method in Example 1;

[0044] Figure 4 This is a picture of the appearance of the collagen implant obtained by the preparation method of Example 1;

[0045] Figure 5 This is the mapping distribution diagram of Ca, P, C, O, and N elements in the collagen implant prepared by the method of Example 1;

[0046] Figure 6 This is a SEM morphology of the freeze-dried collagen implant obtained by the preparation method of Example 1;

[0047] Figure 7 This is a diagram of the collagen implant obtained by the preparation method of Example 1 passing through a 27G needle;

[0048] Figure 8 The ion release curve of Ca ions in the collagen implant obtained by the preparation method of Example 1, Example 3 and Comparative Example 1 within 7 days is shown;

[0049] Figure 9 7-day ion release curves of P ions from the collagen implants prepared using the methods of Example 1, Example 3, and Comparative Example 1. DETAILED DESCRIPTION

[0050] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0051] Example 1, a method for preparing a collagen implant containing mineralized hydroxyapatite, comprising the following steps:

[0052] Preparation of mineralized hydroxyapatite:

[0053] 1) Prepare a 0.05 wt% collagen solution containing 0.5 wt% D-mannitol, adjust the pH to 3.5 with acetic acid, and stir thoroughly.

[0054] 2) Dissolve dicalcium phosphate in 0.06 mol / L dilute hydrochloric acid (1 g of dicalcium phosphate per 50 mL of dilute hydrochloric acid) and slowly add the solution to the collagen solution, adjusting the pH to 3.5. Stir magnetically for 2 hours to allow the carboxylic acid groups in the collagen to form weak bonds with the calcium ions. The amount of dicalcium phosphate added depends on the amount of collagen added in Step 1. The mass ratio of dicalcium phosphate to collagen is 1.028:1, forming Solution A.

[0055] 3) Prepare a calcium hydroxide suspension (dissolve 1g of calcium hydroxide in 50mL of water) and slowly add the calcium hydroxide suspension to solution A;

[0056] 4) Calcium hydroxide suspension was used to adjust the solution pH to 6.5. At this stage, the carboxylic acid groups were converted to carboxylate radicals, which enhanced the interaction with calcium ions and the adsorption performance of collagen. The reaction lasted for 1.5 h.

[0057] 5) Continue adding calcium hydroxide suspension until the mass ratio of calcium hydroxide to collagen is 0.294:1, ensuring that the Ca / P ratio of the calcium phosphate salt in the final product is 1.67:1. Finally, use 5% sodium hydroxide solution to adjust the pH value of the solution to 9.5 to provide a suitable environment for hydroxyapatite mineralization. Continue stirring for 1 hour;

[0058] The amount of calcium hydroxide added depends on the amount of collagen and calcium hydrogen phosphate added in step 1. The mass ratio of calcium hydroxide to collagen is 0.294:1, ensuring that the Ca / P ratio of the calcium phosphate salt in the final product is 1.67:1.

[0059] Optionally, the calcium hydroxide suspension is prepared by dissolving 0.5-3 g of calcium hydroxide in 50 mL of water.

[0060] 6) Let the above solution stand for 25 hours;

[0061] 7) Remove the supernatant and perform gradient centrifugation using a centrifuge (4000 rpm, 2 times, 10 minutes each time, 5000 rpm, 2 times, 10 minutes each time). During each centrifugation, add purified water 4 times the weight of the centrifuged material for washing to remove water-soluble additives in the mineralized hydroxyapatite;

[0062] 8) The obtained sample was quickly frozen in dry ice and then freeze-dried in a freeze dryer for 24 hours. The dried sample was ground and sieved through a 300-mesh sieve to obtain mineralized hydroxyapatite powder.

[0063] Preparation of collagen implants:

[0064] 1) fully mixing the mineralized hydroxyapatite powder and 1.5 wt% genipin powder in an amount equal to the mineralized hydroxyapatite powder to form a mixture;

[0065] 2) The above mixture was mixed with a 35 mg / mL neutral collagen solution at an amount accounting for 20 wt% of the collagen implant, and the powder and collagen solution were thoroughly mixed using a homogenizer. Genipin was used to achieve weak cross-linking between the collagen in the mineralized hydroxyapatite powder and the neutral collagen solution, thereby achieving dispersion of the mineralized hydroxyapatite powder in the collagen solution and avoiding precipitation problems. Finally, a gel-like, 27G needle-injectable, and drawable collagen implant was prepared.

[0066] Example 2, a method for preparing a collagen implant containing mineralized hydroxyapatite, comprising the following steps:

[0067] Preparation of mineralized hydroxyapatite:

[0068] 1) Prepare a 0.05 wt% collagen solution containing 0.5 wt% trehalose, adjust the pH to 3.2-3.6 with acetic acid solution, and stir well.

[0069] 2) Dissolve dicalcium phosphate in 0.06 mol / L dilute hydrochloric acid (1 g of dicalcium phosphate per 50 mL of dilute hydrochloric acid) and slowly add the solution to the collagen solution, adjusting the pH to 3. Magnetic stirring was performed for 3 hours to allow the carboxylic acid groups in the collagen to form weak bonds with the calcium ions. The amount of dicalcium phosphate added depends on the amount of collagen added in step 1. The mass ratio of dicalcium phosphate to collagen is 1.028.

[0070] 3) Prepare a calcium hydroxide suspension (dissolve 1g of calcium hydroxide in 50mL of water) and slowly add the calcium hydroxide suspension to solution A;

[0071] 4) Using calcium hydroxide suspension to adjust the solution pH to 6, at this stage the carboxylic acid groups are converted to carboxylate radicals, the interaction with calcium ions is enhanced, and the adsorption performance of collagen is enhanced. The reaction lasts for 2 hours.

[0072] 5) Continue adding calcium hydroxide until the mass ratio of calcium hydroxide to collagen reaches 0.294:1, ensuring that the Ca / P ratio of the calcium phosphate salt in the final product is 1.67:1. Finally, use a 5% sodium hydroxide solution suspension to adjust the pH value of the solution to 10 to provide a suitable environment for hydroxyapatite mineralization. Continue stirring for 1 hour;

[0073] 6) Let the above solution stand for 36 hours;

[0074] 7) Remove the supernatant and perform gradient centrifugation using a centrifuge (4000 rpm, 2 times, 10 minutes each time, 5000 rpm, 2 times, 10 minutes each time). During each centrifugation, add purified water 4 times the weight of the centrifuged material for washing to remove water-soluble additives in the mineralized hydroxyapatite;

[0075] 8) The obtained sample was quickly frozen in dry ice and then freeze-dried in a freeze dryer for 24 hours. The dried sample was ground and sieved through a 300-mesh sieve to obtain mineralized hydroxyapatite powder.

[0076] Preparation of collagen implants:

[0077] 1) fully mixing mineralized hydroxyapatite powder and 2.5 wt% genipin powder to form a mixture;

[0078] 2) The above mixture was mixed with a 50 mg / mL neutral collagen solution at a 30 wt% concentration, and the powder and collagen solution were thoroughly mixed using a homogenizer. Genipin was used to achieve weak cross-linking between the collagen in the mineralized hydroxyapatite powder and the neutral collagen solution, thereby achieving dispersion of the mineralized hydroxyapatite powder in the collagen solution and avoiding precipitation problems. Finally, a gel-like, 27G needle-injectable, and drawable collagen implant was prepared.

[0079] Example 3, a method for preparing a collagen implant containing mineralized hydroxyapatite, differs from Example 1 in that, in the preparation of the mineralized hydroxyapatite, maltodextrin with a molecular weight of 1500 is substituted for D-mannitol in an equal amount.

[0080] Comparative Example 1, a method for preparing a collagen implant containing mineralized hydroxyapatite, differs from Example 1 in that D-mannitol is not used in the preparation of the mineralized hydroxyapatite.

[0081] Performance testing:

[0082] 1. Mineralized hydroxyapatite powder:

[0083] The XRD pattern of mineralized hydroxyapatite powder is as follows: Figure 1 ,Will Figure 1 After comparing the XRD pattern with the standard pattern of hydroxyapatite, it was determined that the XRD diffraction peak of the material prepared this time belonged to hydroxyapatite; and it was non-sintered hydroxyapatite with poor crystallinity.

[0084] FT-IR spectrum such as Figure 2 ,from Figure 2 The FT-IR spectrum shows that the vibration absorption peaks at 605.4 and 564.5 are the characteristic double peaks of hydroxyapatite, the vibration absorption peak at 1033.8 is caused by phosphate, and the vibration peaks at 1656.8 and 1422.5 are caused by the amide bond and carboxylate in collagen.

[0085] SEM morphology Figure 3 ,like Figure 3 In the upper picture, we can see large collagen-mineralized hydroxyapatite, and almost no hydroxyapatite nanoparticles can be seen, which indicates the close binding between hydroxyapatite and collagen. Figure 3 The picture below shows hydroxyapatite distributed on the surface of collagen, indicating that hydroxyapatite can be deposited on collagen more evenly.

[0086] from Figure 3The resulting collagen-mineralized hydroxyapatite has an irregular morphology, with distinct strips and flakes. A 20,000x magnification SEM image reveals the aggregation of numerous nanoscale circular particles, primarily caused by the formation of hydroxyapatite.

[0087] 2. Collagen implants:

[0088] 1) Appearance and structure

[0089] The state diagram of collagen implants Figure 4 It can be seen that the mineralized hydroxyapatite-containing collagen implant obtained by compounding the collagen solution with the mineralized hydroxyapatite is a milky white, semi-solid substance with no granular feel.

[0090] The element's Mapping distribution diagram is as follows Figure 5 , where (a) electron image (SE); (b) EDS layered image; (c) C element distribution map; (d) N element distribution map; (e) P element distribution map; (f) O element distribution map; (g) Ca element distribution map.

[0091] Figure 5 The distribution diagrams of the separated C, N, P, O, and Ca elements in Figure (b) are as follows: Figure 5 As shown in Figures (c), (d), (e), (f), and (g), it further shows that the uniform distribution of mineralized hydroxyapatite in the collagen solution can also ensure the uniformity and easy degradation of the final material.

[0092] The SEM morphology of the collagen implant after freeze drying is shown in the figure below. Figure 6 After 24 hours of freeze-drying, the implant becomes porous, which facilitates inward cell migration and growth in the body, thereby increasing collagen secretion and achieving long-term wrinkle filling. The mineralized hydroxyapatite is evenly distributed within the collagen, without noticeable granularity.

[0093] 2) Injection effect

[0094] Injection status diagram Figure 7 The results showed that the mineralized hydroxyapatite-containing collagen implant obtained by compounding the collagen solution with mineralized hydroxyapatite can be smoothly pushed out from the 27G needle of the syringe, and the implant has a good filamentation effect.

[0095] The pushing force test values ​​during the injection process are shown in Tables 1 to 3. The implants of the examples can be pushed out smoothly from a 27G needle. However, needle blockage occurs during the injection process of both Example 3 and Comparative Example 1, and the effective maximum force is relatively large.

[0096] Table 1. Collagen implant 27G needle pushing force data for Example 1

[0097] Table 2. Collagen implant 27G needle pushing force data for Example 3

[0098]

[0099] Table 3. Collagen implant 27G needle pushing force data of Comparative Example 1

[0100]

[0101] Regarding the issue of pushing force: Currently, mineralized hydroxyapatite with a high collagen content (e.g., a 50% collagen to hydroxyapatite ratio) experiences severe collagen aggregation during freezing. Aggregated mineralized hydroxyapatite is difficult to redisperse upon remelting, and grinding and sieving after freeze-drying is also difficult. The mineralized collagen prepared by this process exhibits excessive pushing force during the compounding of the collagen solution and is prone to needle clogging. Our research has found that the addition of D-mannitol and other agents during the preparation of mineralized hydroxyapatite can help prevent collagen aggregation, significantly improve its dispersibility after remelting, and significantly reduce pushing force during the final preparation of the mineralized hydroxyapatite-containing collagen implant, without needle clogging. As shown in Table 1, Example 1, in which D-mannitol was added as a dispersant, produced an implant with a pushing force of approximately 5N using a 27G needle, without needle clogging. As shown in Table 2, the pushing force of Example 3 is generally higher than 10N, and the needle blocking problem is more serious; as shown in Table 3, the pushing force of Comparative Example 1 is generally higher than 10N, and the needle blocking problem is more serious.

[0102] 3) Sustained-release effect

[0103] Ion release test method:

[0104] 1 g of mineralized hydroxyapatite collagen implant was weighed and placed in a 15 mL centrifuge tube. 5 mL of purified water was added (n = 3). The centrifuge tube was placed in a thermostatic shaker at 80 rpm and 37°C. After 1 hour of shaking, the tube was removed and allowed to cool for 10 minutes. 2 mL of the supernatant from each tube was transferred to a new 15 mL centrifuge tube. 2 mL of purified water was then added to each tube and the tube was placed in a thermostatic shaker at 80 rpm and 37°C. The tubes containing the supernatant were then centrifuged in a refrigerated centrifuge at 8000 rpm and 10°C for 10 minutes. 1 mL of the supernatant was transferred to an EP tube for ICP-OES analysis. The tubes were removed after 3, 6, 24, 96, and 168 hours of shaking, and the above procedure was repeated. The ion release at different time points was calculated as follows:

[0105]

[0106] Where: Q——ion release amount at a certain time point

[0107] C1——ion concentration at a certain time point

[0108] C0——ion concentration at the previous time point

[0109] V——volume of solution (5mL)

[0110] The cumulative ion release is: the ion release at a certain time point + the ion release at all previous time points.

[0111] For each example or comparative example, three samples were tested in parallel, and the average value of the ion release concentration was taken. The test values ​​are shown in Tables 4 and 5.

[0112] The Ca ion release concentration curves of Example 1, Example 3 and Comparative Example 1 are as follows: Figure 8 .

[0113] The P ion release concentration curves of Example 1, Example 3 and Comparative Example 1 are as follows: Figure 9 .

[0114] Table 4. Original data of calcium and phosphorus ion cumulative release curve of collagen implant in Example 1

[0115]

[0116] Table 5. Original data table of calcium and phosphorus ion cumulative release curves of collagen implants in comparative example 1

[0117]

[0118] Regarding the ion release of this product: Hydroxyapatite is the main component of bone tissue. Due to its good biocompatibility, hydroxyapatite materials have received widespread attention in the field of medical beauty (facial wrinkle filling) in recent years. However, hydroxyapatite is the most stable phase in the body fluid environment, and its degradation in the body is long or difficult to completely degrade, which will also increase the application risk of the material to a certain extent. This embodiment simulates the natural bone tissue structure and uses collagen mineralization deposition to form hydroxyapatite, wherein the hydroxyapatite phase is a weakly crystalline phase, and the presence of collagen can accelerate the degradation of hydroxyapatite. Example 1 is added with D-mannitol, which effectively inhibits the aggregation of collagen and has a higher specific surface area during the formation of collagen mineralized hydroxyapatite, thereby ensuring that the calcium and phosphate ion release efficiency is higher than that of Example 3 and Comparative Example 1, ensuring that the hydroxyapatite in Example 1 can be completely degraded after implantation into the human body, thereby improving application safety.

[0119] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for preparing mineralized hydroxyapatite, characterized in that: The method comprises the following preparation steps: A 0.01-0.15% collagen solution containing 0.1-1 wt% carbohydrate dispersant was prepared, and then a pH adjusting solution formed by slowly adding dilute hydrochloric acid to dissolve calcium hydrogen phosphate was adjusted to a pH of 3-4 to form solution A. The mass ratio of calcium hydrogen phosphate to collagen in solution A was (0.8-1.5):1; The calcium hydroxide suspension is slowly added to solution A, the pH value of the solution is adjusted to 9-10, and stirred for 1.5-4 hours; then the solution is allowed to stand for 12-36 hours, the supernatant is removed, and the precipitate is filtered, washed, freeze-dried, ground, and sieved to obtain mineralized hydroxyapatite powder.

2. The method for preparing mineralized hydroxyapatite according to claim 1, wherein: The dispersant is a small molecule sugar dispersant with a molecular weight of less than 1000.

3. The method for preparing mineralized hydroxyapatite according to claim 1 or 2, characterized in that: The dispersant is D-mannitol and / or trehalose.

4. The method for preparing mineralized hydroxyapatite according to claim 3, wherein: The mass ratio of calcium hydrogen phosphate to collagen in the solution A is (1-1.2):1, and the stirring time is 1-3 hours; The steps for adjusting the pH value of solution A using calcium hydroxide suspension are: Slowly add the calcium hydroxide suspension to solution A, adjust the pH value of the solution to 6-7, and continue the reaction for 1-2 hours; continue to add the calcium hydroxide suspension until the pH value of the solution reaches 9-10, and continue stirring for 0.5-2 hours.

5. The method for preparing mineralized hydroxyapatite according to claim 4, characterized in that: The Ca / P ratio of the calcium phosphate salt in the final product in the final solution is (1.3-2):1; the mass ratio of the calcium hydroxide to the collagen is (0.1-0.35):

1.

6. A mineralized hydroxyapatite, characterized in that: The hydroxyapatite is prepared by the method for preparing the mineralized hydroxyapatite according to any one of claims 1 to 5.

7. A method for preparing a collagen implant containing mineralized hydroxyapatite, characterized in that: The method comprises the following preparation steps: The mineralized hydroxyapatite according to claim 6 is mixed with a cross-linking agent to form a mixture; and the mixture is then stirred and mixed with a 5-50 mg / mL neutral collagen solution to prepare a collagen implant containing mineralized hydroxyapatite; The amount of the cross-linking agent in the mixture is 0.1-2.5wt% of the amount of the mineralized hydroxyapatite powder; The mixture in the collagen implant accounts for 5-30 wt%.

8. The method for preparing the collagen implant containing mineralized hydroxyapatite according to claim 7, characterized in that: The collagen in the collagen solution and the neutral collagen solution includes any one or more combinations of type I collagen, type II collagen, and type III collagen; and the cross-linking agent includes genipin and / or an aldehyde cross-linking agent.

9. A collagen implant containing mineralized hydroxyapatite, characterized in that: The method is obtained by the preparation method according to claim 7 or 8.

10. Use of the mineralized hydroxyapatite according to claim 6 or the collagen implant according to claim 9 in the preparation of medical injectable filling materials.

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