Preparation method and application of injectable collagen gel

Abstract

The invention relates to the technical field of cartilage repair, in particular to a preparation method and application of injectable collagen gel. The method comprises the following specific steps: (1) carrying out low-temperature pretreatment and irradiation sterilization on a collagen solution; (2) mixing the physiological pH buffer solution B with moisturizing, lubricating and repairing components and a composite nutrient solution, and adjusting the pH value to 7.5-8.5 to obtain a neutralization solution A; and (3) mixing and neutralizing the collagen solution and the neutralization solution A to complete collagen self-assembly to form the hydrogel. The physicochemical property and the structure of the gel are more suitable for adhesion, growth, proliferation and differentiation of cartilage cells, and are more suitable for tissue engineering cartilage research. The technology does not need to be combined with a micro-fracture surgery, does not need to be bonded by virtue of fibrin glue, can be directly operated in joint fluid, does not need a dry environment, does not damage subchondral bones, protects the joint structure of a patient, increases the proliferation migration rate of cells, greatly reduces the operation cost, and simultaneously reduces the technical threshold of an operation hospital and an operation doctor.

Description

Technical Field

[0001] This invention relates to the field of cartilage repair technology, and in particular to a method for preparing an injectable collagen gel and its application. Background Technology

[0002] Cartilage damage is a common clinical condition characterized by progressive destruction of articular cartilage, accompanied by pathological changes in the subchondral bone, leading to joint pain, stiffness, and limited function. According to the Global Burden of Disease study, osteoarthritis is one of the leading causes of disability.

[0003] Because cartilage tissue lacks blood vessels, nerves, and lymphatic tissue, its self-repair capacity is limited. Traditional treatments such as conservative treatment, arthroscopic debridement, and microfracture surgery often yield unsatisfactory results, failing to achieve complete repair and functional reconstruction of cartilage tissue. Traditional treatments, such as microfracture surgery (which stimulates bone marrow mesenchymal stem cells to migrate to the injury site and form fibrocartilage through drilling), autologous chondrocyte transplantation (ACI), and allogeneic cartilage transplantation, suffer from problems such as insufficient mechanical properties of the repaired tissue, limited donors, and immune rejection. For example, fibrocartilage formed by microfracture surgery has poor wear resistance and is prone to long-term degeneration; ACI requires a second surgery to obtain chondrocytes, and in vitro expansion may lead to loss of cell phenotype. Therefore, the medical field urgently needs technological advancements and innovations in novel cartilage repair materials to provide safer and more effective treatment methods for clinical practice.

[0004] In recent years, hyaluronic acid, a natural molecule found in articular cartilage, has demonstrated significant advantages in the long-term repair of cartilage damage due to its excellent biocompatibility and bioactivity, and its participation in the cartilage repair process through various mechanisms. Cross-linked sodium hyaluronate gel is the most commonly used filler material; however, hyaluronic acid typically degrades rapidly in vivo. Furthermore, due to the high flexibility of sodium hyaluronate molecular chains, even after high cross-linking degrees, the resulting gel still exhibits low hardness and viscoelasticity, leading to limitations in its application as a cartilage filler material, such as easy displacement and poor support. Therefore, developing a safe, long-lasting, and highly adhesive cartilage filler material to overcome the shortcomings of current cross-linked sodium hyaluronate gel products holds enormous market potential.

[0005] Hydrogels, as a hydrophilic three-dimensional network structure, are a good candidate material for cartilage repair and a research hotspot in the treatment of cartilage damage. However, current technologies mainly suffer from problems such as the need for combined use with microfracture surgery, weak mechanical properties of hydrogels, inability to adhere to the affected area, poor cell affinity, and weak bone regeneration capacity.

[0006] Current research on articular cartilage often employs scaffolds such as type I or type II collagen and chitosan for cartilage defect repair. However, the use of collagen and chitosan may lead to cartilage ossification, which is detrimental to cartilage tissue repair. For example, Chinese patent CN101020083A discloses an integrated bone-cartilage composite tissue engineering scaffold with a biomimetic functional interface, which uses type II collagen and chitosan to prepare a cartilage composite tissue. However, this method produces a multi-layered bone-cartilage composite scaffold, which is not suitable for the adhesion and growth of mesenchymal stem cells, as well as their subsequent proliferation and differentiation. Therefore, there is an urgent need to develop a cartilage scaffold suitable for cartilage repair, capable of co-culturing with cells, possessing excellent mechanical properties and porosity, and applicable to tissue-engineered cartilage research, providing a better option for the treatment of cartilage defects.

[0007] Currently, Ubiosis, a South Korean company, produces collagen cartilage repair scaffolds that require fixation with fibrin glue to the affected area. Using fibrin glue in cartilage repair has two drawbacks: first, it can hinder cell migration and proliferation; second, its adhesiveness is not high. Regarding the issue of fibrin glue hindering chondrocyte migration, the article "Fibrin glue does not assist migration and proliferation of chondrocytes in collagenic membranes: an invitro study" investigated this. This study evaluated the effect of fibrin glue on the collagen membrane loaded with chondrocytes. The results showed that cells in the non-fibrin glue group had stronger intramembrane migration ability. Significant differences in cell migration between the non-fibrin glue group and the fibrin glue group were observed in weeks 1 (P < 0.001), 2 (P = 0.004), and 3 (P = 0.03). Regarding the viscosity of fibrin glue, the paper "Biomimetic Natural Biopolymer-Based Wet-Tissue Adhesive for Tough Adhesion, Seamless Sealed, Emergency / Nonpressing Hemostasis, and Promoted Wound Healing" states that the average adhesion strength of medical fibrin glue is approximately 15 kPa. In contrast, other hydrogel materials, such as CoSt hydrogel, exhibit an optimal average adhesion strength of 62 ± 4.8 kPa after 2 hours of contact, significantly higher than fibrin glue, indirectly reflecting the relatively low viscosity of fibrin glue.

[0008] Currently available arthroscopic in-situ gelation products, such as the collagen cartilage repair scaffold produced by Ubiosis Co., Ltd. in South Korea and the ChondroFiller product produced by Amedrix GmbH, Esslingen, Germany, require operation in a relatively dry environment. The procedure necessitates the creation of a dry surgical area through a combination of active drainage and gas isolation, making the process complex and demanding a high level of surgical skill. Furthermore, only medical institutions with tertiary-level orthopedic (or sports medicine) department qualifications for minimally invasive arthroscopic surgery and equipped with complete arthroscopic surgical equipment and a pneumoperitoneum system are permitted to perform this procedure.

[0009] In conclusion, if a product could be developed that does not require combination with microfracture surgery or fibrin glue adhesion and can be directly applied in the synovial fluid, more patients could receive treatment. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides a method for preparing injectable collagen gel, comprising the following steps:

[0011] (1) The collagen solution was pretreated at low temperature and sterilized by irradiation;

[0012] (2) Mix physiological pH buffer B with weakly alkaline inorganic salts, metabolizable carbon sources and compound nutrient solution, and adjust the pH to 7.5-8.5 to obtain neutralized solution A;

[0013] (3) Neutralize the collagen solution by mixing it with neutralization solution A.

[0014] In one embodiment of the present invention, the physiological pH buffer B is selected from at least one of MOPS, HEPES, MOPS, and PIPES.

[0015] In one embodiment of the present invention, the weakly alkaline inorganic salt is selected from at least one of calcium carbonate, sodium bicarbonate, and potassium bicarbonate.

[0016] In one embodiment of the present invention, the metabolizable carbon source is selected from at least one of fructose, glucose, fructose, and galactose.

[0017] In one embodiment of the present invention, the composite nutrient solution is a 10× concentration F-12 series culture medium; it includes any one of HAM'S F-12 culture medium and modified HAM'S F-12 culture medium.

[0018] This invention provides an injectable collagen gel, which is prepared by the aforementioned method for preparing an injectable collagen gel.

[0019] As one embodiment of the present invention, the raw materials for preparing the injectable collagen gel include: collagen solution and neutralization solution A;

[0020] The components for preparing neutralization solution A are as follows:

[0021] Buffer system: comprising a weakly alkaline inorganic salt and physiological pH buffer B, wherein the final concentration of the weakly alkaline inorganic salt in the injectable collagen gel is 10~50 g / L, and the final concentration of the physiological pH buffer B in the injectable collagen gel is 0.1~1 M;

[0022] Metabolizable carbon source: a monosaccharide compound, with a final concentration of 20-60 g / L in injectable collagen gel;

[0023] Compound nutrient solution: its volume percentage in injectable collagen gel is 2%~10%;

[0024] Solvent: Injectable grade purified water, add to injectable collagen gel to 100 parts by volume;

[0025] The pH value of the neutralization solution A is adjusted to 7.5~9.0, and after sterile filtration, it is packaged for later use.

[0026] Specifically, the preparation method of the injectable collagen gel is as follows:

[0027] (1) The mouse type I collagen solution was dispensed into 6 mL vials, frozen overnight, and then placed in an irradiation chamber for irradiation sterilization.

[0028] (2) Preparation of physiological pH buffer solution B: The concentration of the physiological pH buffer solution B is 1~5M; specifically selected from one of 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, 4.5M, and 5M.

[0029] (3) Mix the weakly alkaline inorganic salt, metabolizable carbon source, compound nutrient solution, water for injection, and physiological pH buffer solution B evenly, adjust the pH to 8.3, filter with a 0.22μm filter membrane, and fill into 5mL vials of syringes through an aseptic filling machine for later use. Label it as neutralization solution A.

[0030] (4) Fill the collagen solution and neutralization solution A prepared in the above three steps into collagen solution syringe 1 and buffer syringe 2 respectively. Collagen solution syringe 1 and buffer syringe 2 are fixedly assembled using a double-barrel syringe connector 7, a double-barrel syringe mixer 3 and a needle 4. Collagen solution syringe plunger 5 and buffer syringe plunger 6 are placed above collagen solution syringe 1 and buffer syringe 2 respectively and fixedly assembled with synchronous extruder 8. Under the condition of 37°C, using an 18G needle, press synchronous extruder 8 to extrude collagen solution and buffer A simultaneously, which can be pushed out by the syringe to form the required shape.

[0031] As one embodiment of the present invention, the specific steps of step (3) in the preparation method of the injectable collagen gel are as follows:

[0032] The pretreated collagen solution and neutralization solution A were filled into the collagen solution syringe (1) and the buffer syringe (2) respectively. The collagen solution syringe (1) and the buffer syringe (2) were fixedly assembled using a double-barrel syringe connector (7), a double-barrel syringe mixer (3) and a needle (4). The collagen solution syringe plunger (5) and the buffer syringe plunger (6) were placed above the collagen solution syringe (1) and the buffer syringe (2) respectively, and fixedly assembled with the synchronous extruder (8).

[0033] At 37°C, using an 18G needle, press the synchronous extruder 8 to simultaneously extrude collagen solution and neutralization solution A to obtain injectable collagen gel.

[0034] As one embodiment of the present invention, the specific steps of step (1) in the preparation method of the injectable collagen gel are as follows:

[0035] Collagen solution filling: The collagen solution is filled into 6mL vials using filling equipment, sealed with rubber stoppers, and then capped. The vials are then placed in a low-temperature freezer for freezing.

[0036] Collagen irradiation sterilization: The frozen and filled collagen solution is placed into an irradiation incubator, then sealed with dry ice, and then subjected to low-temperature irradiation sterilization.

[0037] Preparation of neutralization solution A: It is prepared by mixing at least one of HEPES, MOPS, and PIPES with at least one of calcium carbonate, sodium bicarbonate, and potassium bicarbonate, 10X F12 medium, and at least one of fructose, glucose, and galactose in a certain proportion.

[0038] Preparation of neutralization solution B: It is prepared by mixing at least one of HEPES, MOPS, and PIPES with sodium chloride and sodium hyaluronate in a certain proportion, and is used in conjunction with amino acid glucose.

[0039] Gel preparation: Collagen solution is mixed with neutralization solution A or neutralization solution B in a certain proportion and injected using a double-barrel syringe.

[0040] As one embodiment of the present invention, the injectable collagen gel is applied in the field of cartilage repair.

[0041] By adopting the above technical solution, the present invention has the following beneficial effects:

[0042] The physicochemical properties and structure of the gel are more suitable for the adhesion, growth, proliferation, and differentiation of chondrocytes. Compared with existing clinical techniques or collagen cartilage repair scaffolds currently used in clinical practice, which require the use of fibrin glue, this technology is more suitable for tissue-engineered cartilage research. This invention does not require integration with microfracture surgery or fibrin glue adhesion; it can be operated directly in synovial fluid without requiring a dry environment. It does not damage the subchondral bone, protects the patient's joint structure, increases cell proliferation and migration rates, significantly reduces surgical costs, and lowers the technical threshold for hospitals and surgeons, allowing more patients to receive treatment and providing a better option for the treatment of cartilage defects. Attached Figure Description

[0043] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0044] Figure 1 This is a schematic diagram of the dual-tube syringe system for preparing the hydrogel according to the present invention;

[0045] Figure 2 Example image of injection;

[0046] Figure 3 This is a test image for detecting the gelation process using an ELISA reader;

[0047] Figure 4 This is a graph showing the shrinkage of collagen gel.

[0048] Figure 5 Image of collagen identification test after different doses of irradiation

[0049] Figure 6 This is a graph from the oscillation frequency scan (elastic modulus) test of Example 1;

[0050] Figure 7 This is a graph showing the oscillation frequency scan (elastic modulus) test results from Example 2.

[0051] Figure 8 This is a viscosity curve detection graph;

[0052] Figure 9 The diagram shows a simulated injection test in a 0.9% sodium chloride solution.

[0053] Figure 10 This is a diagram of a simulated injection test in synovial fluid.

[0054] Figure 11 The image shows a fatigue test result in a 0.9% sodium chloride solution.

[0055] Figure 12 Image of collagen gel stained with hematoxylin and eosin after culture on adherent cells;

[0056] Figure 13 This image shows an observation of collagen gel repairing the surface of cartilage tissue in a large animal experiment.

[0057] Figure 14 Immunohistochemistry of collagen for repairing cartilage tissue with collagen gel in large animal experiments (×200).

[0058] Figure 1 The attached reference numerals are: 1. Collagen solution syringe; 2. Buffer syringe; 3. Dual-barrel syringe mixer; 4. Needle; 5. Collagen solution syringe plunger; 6. Buffer syringe plunger; 7. Dual-barrel syringe connector; 8. Synchronous extruder. Detailed Implementation

[0059] The present invention will be further explained below with reference to specific embodiments.

[0060] Example 1

[0061] (1) The mouse type I collagen solution was aliquoted into 6 mL vials, frozen overnight, and then placed in an irradiation chamber for sterilization. The sterilized collagen solution was then subjected to protein electrophoresis for identification. The results are as follows: Figure 5 As shown, the irradiated collagen still maintains its complete protein structure.

[0062] The specific steps are as follows: The rat tail is placed in a medical-grade bacterial bag and inactivated by gamma irradiation (10-25 KGy). After inactivation, the tail is peeled, cut, and soaked in alcohol and PBS in a biosafety cabinet. Collagen fibers are then extracted and soaked in 10% wt hydrogen peroxide solution for 60 min. The collagen fibers are then dissolved in 0.3% wt acetic acid, centrifuged, and impurities are removed to obtain a collagen solution. The obtained collagen solution is then filled into 6 mL vials and frozen overnight, followed by irradiation sterilization (15-30 KGy) in an irradiation chamber.

[0063] (2) Preparation of 1.2M MOPS solution: Dissolve 12.556g MOPS in water for injection, cool the clear solution to room temperature, adjust the pH to 7.8 with 45% sodium hydroxide solution, and add water for injection to 50mL.

[0064] (3) Mix 3.6g sodium bicarbonate, 7g glucose, 50mL HAM'S F-12 10× solution, 90mL water for injection, and 10mL 1.2M MOPS until homogeneous. Adjust the pH to 7.8, filter with a 0.22μm filter membrane, and fill into 5mL vials using an aseptic filling machine for later use. Label it as neutralization solution A.

[0065] (4) Fill the collagen solution and neutralization solution A prepared in the above three steps into collagen solution syringe 1 and buffer syringe 2 respectively. Collagen solution syringe 1 and buffer syringe 2 are fixedly assembled using a double-barrel syringe connector 7, a double-barrel syringe mixer 3 and a needle 4. Collagen solution syringe plunger 5 and buffer syringe plunger 6 are placed above collagen solution syringe 1 and buffer syringe 2 respectively, and fixedly assembled with synchronous extruder 8 (e.g. Figure 1 (As shown); at 37°C, using an 18G needle, press the synchronous extruder 8 to simultaneously extrude collagen solution and buffer A, as shown. Figure 2 As shown, it can be ejected by a syringe to form the desired shape.

[0066] Example 2

[0067] (1) The mouse type I collagen solution was dispensed into 6 mL vials, frozen overnight, and then placed in an irradiation chamber for irradiation sterilization.

[0068] (2) Preparation of 1.2M MOPS solution: Dissolve 12.556g MOPS in water for injection. The solution is clear, indicating that it is completely dissolved. Cool the clear solution to room temperature and adjust the pH to 7.8 with 45% sodium hydroxide solution. Add water for injection to a final volume of 50mL.

[0069] (3) Mix 13.5g sodium chloride, 15ml of 1mg / mL sodium hyaluronate, 37.5mL of 1.2M MOPS and 73.75mL of water for injection until homogeneous. Filter the mixture through a 0.22um filter membrane and fill it into a 2mL vial syringe for later use. Label it as neutralization solution B.

[0070] (4) Mix equal volumes of 5.5% amino acid injection and 10% glucose injection with neutralization solution B before use.

[0071] (5) The collagen solution and neutralization solution B (mixed with amino acids and glucose injection) from step (1) can be used in accordance with the method of Example 1.

[0072] Example 3

[0073] (1) The mouse type I collagen solution was aliquoted into 6 mL vials, frozen overnight, and then placed in an irradiation chamber for sterilization. The sterilized collagen solution was then subjected to protein electrophoresis for identification. The results are as follows: Figure 5 As shown, the irradiated collagen still maintains its complete protein structure.

[0074] (2) Preparation of 2 M MOPS solution: Dissolve 20.9 g MOPS in water for injection, cool the clear solution to room temperature, adjust the pH to 7.8 with 45% sodium hydroxide solution, and add water for injection to 50 mL.

[0075] (3) Mix 3.6g sodium bicarbonate, 7g glucose, 50mL HAM'S F-12 10× solution, 90mL water for injection, and 10mL 2M MOPS until homogeneous. Adjust the pH to 7.8, filter using a 0.22μm filter membrane, and fill into 5mL vials using an aseptic filling machine for later use. Label it as neutralization solution A.

[0076] (4) Fill the collagen solution and neutralization solution A prepared in the above three steps into collagen solution syringe 1 and buffer syringe 2 respectively. Collagen solution syringe 1 and buffer syringe 2 are fixedly assembled using a double-barrel syringe connector 7, a double-barrel syringe mixer 3 and a needle 4. Collagen solution syringe plunger 5 and buffer syringe plunger 6 are placed above collagen solution syringe 1 and buffer syringe 2 respectively, and fixedly assembled with synchronous extruder 8 (e.g. Figure 1 (As shown); at 37°C, using an 18G needle, press the synchronous extruder 8 to simultaneously extrude collagen solution and buffer A, as shown. Figure 2 As shown, it can be ejected by a syringe to form the desired shape.

[0077] Application Example 1

[0078] By using an ELISA reader to detect the absorbance changes of collagen gels from Examples 1, 2, and 3, the time required for the formation of a stable gel can be determined. The results are as follows: Figure 3 As shown, a stable collagen gel can be formed within 5 to 8 minutes.

[0079] The swelling and shrinkage properties of the collagen gels formed in Examples 1, 2, and 3 were tested using PBS, and the results are as follows: Figure 4 As shown in the figure. The results show that the prepared gel has excellent shrinkage properties.

[0080] Figure 6-7 The following diagrams show the collagen gel products prepared in Examples 1 and 2, respectively. Coordinate graphs were plotted using a rheometer at 25±0.2℃ and shear rates ranging from 0.1Hz to 100Hz. Figure 6 It can be seen that the elastic modulus G' is always greater than the viscous modulus G. The results show that the collagen gel product in this embodiment has good viscoelasticity and strong resistance to external forces and deformation.

[0081] Figure 8 The viscosity curves of Example 2, plotted using a rheometer at 25±0.2℃, are shown. The results indicate that the initial viscosity of the sample is 1.69E+05 mPa·s, which is relatively high and facilitates sample adsorption to the affected area. As the sample gradually gels, the viscosity decreases.

[0082] Application Example 2

[0083] (1) Under the condition of soaking in 0.9% sodium chloride, the temperature was heated to 37°C. A circular hole with a diameter of 12 mm and a depth of 5 mm was drilled in the joint cartilage of the pig using a trephine drill to simulate joint defects. The gel of Example 2 was injected into the joint of the pig bone. After the gel was trimmed, the soaking experiment was carried out.

[0084] (2) Under simulated joint fluid conditions, the mixture was heated to 37°C, and a 12 mm diameter and 5 mm deep hole was drilled in the pig joint cartilage using a trephine to simulate joint defects. The gel from Example 2 was injected into the pig joint, and the gel was then modified for an immersion experiment.

[0085] (3) Under the condition of soaking in 0.9% sodium chloride, heat to 37°C, drill a round hole with a diameter of 12 mm and a depth of 5 mm in the joint cartilage of pigs using a trephine to simulate joint defects, and inject the gel of Example 2 into the joint of the pig's foot. After the gel is modified, a fatigue test is conducted.

[0086] like Figures 9-11As shown, collagen gel was directly injected into the joint repair pore under simulated synovial fluid immersion conditions, and all samples gelled. After 72 hours of immersion and 5000 folding cycles simulating fatigue tests, the gel showed no signs of detachment. This indicates that this method can be used for injection in a liquid environment, and the resulting gel adheres firmly to the defect site without detaching. Figure 11 In the diagram, A represents the initial state of gel formation when the sample is injected into the joint repair pore; B represents the gel state after the joint has been repeatedly folded 2000 times in a liquid environment; C represents the gel state after the joint has been repeatedly folded 4000 times in a liquid environment; and D represents the gel state after the joint has been repeatedly folded 5000 times in a liquid environment.

[0087] Application Example 3

[0088] (1) Collagen gel was prepared in a 12-well plate using Example 2.

[0089] (2) Experimental group: Apply fibrin adhesive evenly to the surface of CFI, immediately invert it into the well filled with adherent cells, and gently press it to make it fully contact the bottom of the well.

[0090] (3) Control group: No fibrin adhesive was applied to the upper surface. It was also placed upside down in the hole filled with adherent cells and gently pressed to make it fully contact the bottom surface.

[0091] (4) Add 1.5 ml of autologous culture medium to each well and continue culturing. Change the culture medium every 3 to 4 days.

[0092] (5) The tissue was fixed on day 13 and day 20 of co-culture, then embedded, sectioned, and stained with hematoxylin and eosin (HE). Images of the HE-stained sections were taken.

[0093] (6) Digest the adherent cells into a cell suspension, count the cells, and divide them into three equal portions. Add one portion to a CFI plate, add the second portion to a CFI plate with a layer of fibrin adhesive evenly coated on the surface, and add the third portion to the wells of a 12-well plate. Add 1.5 ml of autologous culture medium to each well and continue culturing, changing the culture medium every 3 to 4 days. Count the gel cells on day 10 of culture.

[0094] HE staining results, see Figure 12 The average number of cells in three fields of view of HE-stained images was calculated, as shown in Table 1. The results show that the number of cells migrating in the control group was significantly greater than that in the experimental group, and the relative distance of migration was also greater in the control group.

[0095] Table 1. Cell count at different time points after collagen gel culture on adherent cells (HE images).

[0096] Group 13d (pieces) 20 days (pieces) C12-0006D-J 19 30 C12-0006D-NC 28 55.3

[0097] Cell counts were performed on day 10 of adherent cell culture on collagen gel. The cell counts before and after inoculation are shown in Table 2. Table 2 shows that the experimental group had fewer cells than the control group, with the latter having 2.4 times more cells. Furthermore, the cell proliferation rate results indicate that cells growing and proliferating on the collagen gel surface were faster than those growing and proliferating on the collagen gel surface coated with fibrin adhesive.

[0098] Table 2. Count of chondrocytes before and after culture on collagen gel.

[0099] project experimental group control group Handling method CFI is coated with bio-protein adhesive. CFI does not apply biological protein glue Cell quantity per well at inoculation <![CDATA[5.2×10 4 ]]> <![CDATA[5.2×10 4 ]]> Cell viability at inoculation (%) 98.1% 98.1% Cell count per well on day 10 <![CDATA[3.24×10 5 ]]> <![CDATA[7.83×10 5 ]]> Cell viability (%) on day 10 98.82% 98.31% Cell doubling rate 0.2640 0.3913

[0100] Application Example 4

[0101] Thirty healthy Bama miniature pigs were randomly divided into two groups. The animals were anesthetized and then placed supine on the operating table with their limbs fixed. After routine skin disinfection with iodine, a 6cm diameter skin incision was made on the middle lateral side of the left hind leg knee joint to expose the joint. The patella was pushed aside, and joint incision was performed through the connection of the knee joint ligaments to expose the lateral femoral condyle. A cylindrical defect with a diameter of 6.0mm was created on the lateral femoral condyle using a corneal trephine periosteal elevator.

[0102] Then, either the collagen gel implantation group (experimental group) or the microfracture surgery group (control group) of Example 2 was implanted. After the operation, the animals were housed separately, fed standard animal feed, provided with clean drinking water, and kept at a room temperature of 15-26℃. The rooms were disinfected with ultraviolet light daily. Postoperative antibiotic treatment was given, with one injection of gentamicin intramuscularly twice a day for 3 consecutive days to prevent postoperative infection. At 6 and 12 months postoperatively, samples were taken from the implantation site and the microfracture surgery site to measure the corresponding indicators.

[0103] like Figure 13 As shown, at 6 months, the cartilage defects in the collagen gel implantation group showed some degree of repair. In some animals, the surface smoothness of the defect area was visible. In most animals, the repaired cartilage gradually improved from the center of the defect outwards, with clear boundaries between the repaired and normal cartilage edges, without gaps. The repaired cartilage was slightly pale in color compared to normal cartilage. In the microfracture group, the repaired cartilage surface was uneven, and the repaired edges were not clearly defined compared to normal cartilage edges. At 12 months, the cartilage repair surface in the collagen gel implantation group was smooth, with a color similar to normal cartilage. The repaired edges were largely integrated with normal cartilage, with no obvious boundary, and the degree of repair was significantly improved compared to 6 months. In the microfracture group, the defect area increased compared to 6 months, but the repair effect was not significantly different.

[0104] like Figure 14As shown, cartilage tissue samples were collected at 6 and 12 months post-surgery from the microfracture group and the collagen gel implantation group. Immunohistochemical staining was used to detect the expression of type II collagen in each tissue. Results showed that at 6 and 12 months, chondrocytes and matrix in the collagen gel implantation group were stained brownish-yellow, indicating a positive result, but the staining was stronger at 12 months than at 6 months. The microfracture group was negative at 6 months and showed a weak positive result at 12 months. This further suggests that collagen gel implantation is beneficial for the repair of damaged cartilage tissue, and the cartilage generated at the repair site is hyaline cartilage, which is similar to the main matrix components of normal cartilage.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an injectable collagen gel, characterized in that, The steps are as follows: (1) The collagen solution was pretreated at low temperature and sterilized by irradiation; (2) Mix physiological pH buffer B with weakly alkaline inorganic salts, metabolizable carbon sources and compound nutrient solution, and adjust the pH to 7.5-8.5 to obtain neutralized solution A; (3) Neutralize the collagen solution by mixing it with neutralization solution A.

2. The method for preparing an injectable collagen gel according to claim 1, characterized in that, The physiological pH buffer B is selected from at least one of MOPS, HEPES, and PIPES.

3. The method for preparing an injectable collagen gel according to claim 1, characterized in that, The weakly basic inorganic salt is selected from at least one of calcium carbonate, sodium bicarbonate, and potassium bicarbonate.

4. The method for preparing an injectable collagen gel according to claim 1, characterized in that, The metabolizable carbon source is selected from at least one of fructose, glucose, and galactose.

5. The method for preparing an injectable collagen gel according to claim 1, characterized in that, The compound nutrient solution is a 10× concentration F-12 series culture medium; it includes any one of HAM'S F-12 medium and modified HAM'S F-12 medium.

6. An injectable collagen gel, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 5.

7. The injectable collagen gel according to claim 6, characterized in that, Its preparation materials include: collagen solution and neutralization solution A; The components for preparing neutralization solution A are as follows: Buffer system: comprising a weakly alkaline inorganic salt and physiological pH buffer B, wherein the final concentration of the weakly alkaline inorganic salt in the injectable collagen gel is 10~50 g / L, and the final concentration of the physiological pH buffer B in the injectable collagen gel is 0.1~1 M; Metabolizable carbon source: a monosaccharide compound, with a final concentration of 20-60 g / L in injectable collagen gel; Compound nutrient solution: its volume percentage in injectable collagen gel is 10%~30%; Solvent: Injectable grade purified water, add to injectable collagen gel to 100 parts by volume; The pH value of the neutralization solution A is adjusted to 7.5~9.0, and after sterile filtration, it is packaged for later use.

8. A method for preparing an injectable collagen gel according to any one of claims 1 to 5, characterized in that, The specific steps of step (3) are as follows: The pretreated collagen solution and neutralization solution A were filled into the collagen solution syringe (1) and the buffer syringe (2) respectively. The collagen solution syringe (1) and the buffer syringe (2) were fixedly assembled using a double-barrel syringe connector (7), a double-barrel syringe mixer (3) and a needle (4). The collagen solution syringe plunger (5) and the buffer syringe plunger (6) were placed above the collagen solution syringe (1) and the buffer syringe (2) respectively, and fixedly assembled with the synchronous extruder (8). At 37°C, using an 18G needle, press the synchronous extruder 8 to simultaneously extrude collagen solution and neutralization solution A to obtain injectable collagen gel.

9. A method for preparing an injectable collagen gel according to any one of claims 1 to 5, characterized in that, The specific steps of step (1) are as follows: Collagen solution filling: The collagen solution is filled into 6mL vials using filling equipment, sealed with rubber stoppers, and then capped. The vials are then placed in a low-temperature freezer for freezing. Collagen irradiation sterilization: The frozen and filled collagen solution is placed into an irradiation incubator, then sealed with dry ice, and then subjected to low-temperature irradiation sterilization. Preparation of neutralization solution A: It is prepared by mixing at least one of HEPES, MOPS, and PIPES with at least one of calcium carbonate, sodium bicarbonate, and potassium bicarbonate, 10X F12 medium, and at least one of fructose, glucose, and galactose in a certain proportion. Preparation of neutralization solution B: It is prepared by mixing at least one of HEPES, MOPS, and PIPES with sodium chloride and sodium hyaluronate in a certain proportion, and is used in conjunction with amino acid glucose. Gel preparation: Collagen solution is mixed with neutralization solution A or neutralization solution B in a certain proportion and injected using a double-tube syringe.

10. An injectable collagen gel according to claim 6 or 7, characterized in that, It is used in the field of cartilage repair.

Images