Injectable hydrogel for cartilage injury repair and preparation method thereof

Abstract

The invention discloses injectable hydrogel for cartilage injury repair and a preparation method of the injectable hydrogel. The gel is prepared by crosslinking 3 to 4 parts of recombinant III type humanized collagen-sodium hyaluronate gel particles, 0.5 to 1 part of thrombin solution and 4 to 6 parts of fibrinogen solution. The recombinant III type humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel for injection, prepared by the preparation method disclosed by the invention, has the advantages of high elastic modulus, good biocompatibility, good needle passing performance and long in-vivo degradation period. Particularly, the recombinant III type humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel for injection prepared by the invention can effectively promote regeneration of cartilage tissues, and is an innovative cartilage repair gel.

Description

Technical Field

[0001] The invention relates to the field of cartilage repair and treatment, and in particular to an injectable hydrogel for cartilage damage repair and a preparation method thereof. Background Art

[0002] At present, according to data from the World Health Organization, the prevalence of joint and bone diseases worldwide has been on the rise in recent years. In addition, sports injuries, accidents and the growth of the elderly and obese population worldwide are other important factors for the growth of the global cartilage repair / regeneration market. In addition, the World Health Organization's October 2021 Overview of Aging and Health shows that the number of people aged 60 and above in 2020 is 1 billion, and it is expected to increase to 1.4 billion by 2030, accounting for 1 / 6 of the world's total population. This group of people is more likely to suffer from diseases and need cartilage repair surgery. Therefore, the cartilage repair material market has great potential. In addition, the damaged part of the cartilage has no blood supply and no nerves. Once damaged, it is difficult to self-repair; it will cause pain, joint deformity and even disability, which seriously affects the patient's quality of life. Traditional clinical treatment methods such as microfracture technology, autologous osteochondral transplantation technology, allogeneic osteochondral transplantation technology, etc. have problems such as painful treatment process, slow recovery, and limited donor sources. It is difficult to achieve long-term hyaline cartilage repair. Tissue engineering cartilage repair developed on this basis can make up for the above problems, but it also has disadvantages such as secondary surgical injury, risk of cell culture contamination in vitro, long surgical interval, high cost, and limited number of chondrocytes. Therefore, the medical field urgently needs technological progress and innovation of new cartilage repair materials to provide safer and more effective treatment methods for clinical practice.

[0003] Cross-linked sodium hyaluronate gel is the most commonly used filling material, but hyaluronic acid usually degrades quickly in the body. In addition, due to the high flexibility of the molecular chain segments of sodium hyaluronate, even after cross-linking at a high degree of cross-linking, the hardness and viscoelasticity of the gel obtained are still very low, resulting in the related products being prone to displacement and poor support effects when used as cartilage filling materials. Therefore, the development of a safe, long-lasting, and highly adhesive cartilage filling material to make up for the defects of the current cross-linked sodium hyaluronate gel products will have huge market potential.

[0004] Collagen is the main structural protein of human tissues and organs, accounting for about 30-40% of the total protein in the human body. At present, collagen products on the domestic and foreign markets are mainly derived from animal tissues and are widely used in the repair of skin, bone, cartilage, cardiovascular system, oral cavity and luminal tissues. Recombinant humanized type III collagen has good biocompatibility and cell adhesion, can form a reticular fiber structure, and has good repair properties. It is expected to have a wide range of applications in the field of cartilage repair.

[0005] In summary, the problems existing in sodium hyaluronate gel, combined with the characteristics of recombinant type III humanized collagen, the present invention introduces sodium hyaluronate and recombinant type III humanized collagen gel for cross-linking to prepare a composite gel on the basis of chemical cross-linking. The composite gel makes up for the defects of sodium hyaluronate gel, has moderate swelling degree, high elastic modulus, good needle permeability, thermal stability, and can quickly degrade the dislocated gel by injecting sodium hyaluronate enzyme and collagenase, and has many advantages such as wide clinical application range, diversified production process selection, and controllable production cost.

[0006] Fibrinogen and thrombin are key components in the blood coagulation process, which can prevent excessive bleeding and promote wound healing. Fibrinogen and thrombin solution can quickly gel when mixed, because fibrinogen is enzymatically converted into fibrin, and then the fibrin monomers are polymerized and cross-linked to form a stable fibrin network or blood clot. Fibrin clot helps to stop bleeding and provides a temporary matrix for tissue repair. Therefore, the present invention selects to fix the injectable gel to the cartilage damage position by the mode of rapid cross-linking of fibrinogen and thrombin, which not only enhances the adhesiveness of the gel, but also blocks the bone marrow blood. This injectable hydrogel material will show a broad clinical application prospect in cartilage damage repair. Summary of the invention

[0007] In view of the deficiencies and gaps in the prior art, the present invention provides an injectable hydrogel for repairing cartilage damage and a preparation method thereof.

[0008] The technical solution of the present invention is as follows: an injectable hydrogel for repairing cartilage damage, which is composed of 3 to 4 parts of recombinant type III humanized collagen-sodium hyaluronate gel particles, 0.5 to 1 part of thrombin solution and 4 to 6 parts of fibrinogen solution by volume.

[0009] Preferably, the injectable hydrogel for repairing cartilage damage is composed of 4 parts by volume of recombinant type III humanized collagen-sodium hyaluronate gel particles, 1 part of thrombin solution and 5 parts of fibrinogen solution.

[0010] Preferably, the mass ratio of recombinant type III humanized collagen to sodium hyaluronate in the recombinant type III humanized collagen-sodium hyaluronate gel particles is 1:1 to 6:1, preferably 1:1.

[0011] Preferably, the concentration of the thrombin solution is 5000u / mL.

[0012] Preferably, the mass concentration of the fibrinogen solution is 20 mg / mL.

[0013] Preferably, the molecular weight of the recombinant humanized type III collagen is between 10 KDa and 300 KDa, preferably between 20 KDa and 100 KDa; the molecular weight of the sodium hyaluronate is between 300 KDa and 1200 KDa, preferably between 600 KDa and 900 KDa.

[0014] The present invention provides a method for preparing the injectable hydrogel for repairing cartilage damage:

[0015] 1) adding the catalyst to purified water and stirring to dissolve, and then adding the recombinant type III humanized collagen to the catalyst solution and stirring to dissolve, then adding sodium hyaluronate and stirring to dissolve, and then adding the cross-linking agent and stirring to evenly carry out the cross-linking reaction;

[0016] 2) After the cross-linking is completed, the primary gel is crushed into small pieces, washed and swollen with an ethanol aqueous solution, sieved and crushed for the first time, and then washed, swollen, and centrifuged to remove the catalyst and free cross-linking agent after the whole particle is formed, and the ethanol is removed by rotary evaporation, and then washed and centrifuged with a phosphate buffer solution or a physiological saline solution, and then sieved and crushed for the second time, and sterilized by wet heat to prepare gel particles;

[0017] 3) The recombinant type III humanized collagen-sodium hyaluronate gel particles are mixed with the thrombin solution, and then mixed with the fibrinogen solution again with the same force and pushed out through a double-row mixing syringe to obtain an injectable hydrogel for cartilage damage repair.

[0018] Preferably, the cross-linking agent in step 1) is one or more of ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, polypropylene glycol diglycidyl ether, diglycidyl ether or divinyl sulfone, preferably 1,4-butanediol diglycidyl ether; the amount of the cross-linking agent added is 1 / 3 to 1 / 10 of the total amount of recombinant type III humanized collagen and sodium hyaluronate, preferably 1 / 4 to 1 / 6.

[0019] Preferably, the catalyst in step 1) is one or more of lithium bromide, calcium chloride, lithium hydroxide, and sodium hydroxide, preferably lithium bromide, with a concentration of 9.3 to 12M.

[0020] Preferably, in step 1), the cross-linking reaction temperature is 18 to 35° C., preferably 22 to 28° C.; the cross-linking reaction time is 36 to 72 hours, preferably 44 to 60 hours.

[0021] Preferably, the ethanol aqueous solution in step 2) is prepared from ethanol and water in a volume ratio of 1:1, and the aperture of the sieve for the first sieving and crushing is 4 mm; the aperture of the sieve for the second sieving and crushing is 200 μm.

[0022] Preferably, in step 3), the volume ratio of thrombin to recombinant humanized type III collagen-sodium hyaluronate gel is 1:5-1:2, preferably 1:4; the volume ratio of the fibrinogen solution to recombinant humanized type III collagen-sodium hyaluronate-thrombin gel is 1:1.

[0023] The present invention provides the use of an injectable hydrogel for cartilage damage repair in the preparation of a cartilage damage filling material; preferably, the cartilage damage filling material is a cartilage tissue regeneration and repair material.

[0024] Beneficial effects of the present invention:

[0025] 1) In the present invention, recombinant humanized type III collagen and sodium hyaluronate raw materials are directly dissolved in a catalyst solution for cross-linking reaction, and the sodium hyaluronate is not modified, so the operation is simpler and more controllable, and the safety hazards of the gel can be avoided; by adding ethanol for washing, the BDDE residue can be completely removed, reducing the toxicity of the gel to the human body.

[0026] 2) The present invention obtains gels with different elastic moduli from weak to strong by adjusting the amount of crosslinking agent input and the concentration of catalyst, and the application prospects of this material are broad. The injectable recombinant type III humanized collagen-sodium hyaluronate gel prepared by the present invention has the advantages of high elastic modulus and good needle permeability.

[0027] 3) The present invention mixes recombinant type III humanized collagen-sodium hyaluronate gel with thrombin, and then mixes it with fibrinogen again through a double-row mixing syringe to achieve secondary cross-linking, thereby obtaining recombinant type III humanized collagen-sodium hyaluronate gel-thrombin-fibrinogen gel for cartilage damage repair. The injectable hydrogel for cartilage damage repair prepared by the present invention has the advantages of good biocompatibility and a long in vivo degradation cycle. In particular, the recombinant type III humanized collagen in the gel can also stimulate the regeneration of tissue fibroblasts and collagen, and can be used as a cartilage defect repair material to accelerate the repair of cartilage at the defect site. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Comparative Example 1 Recombinant Type III Collagen-Sodium Hyaluronate Double Cross-linked Gel Detection Data

[0029] Figure 2 Example 1 Photograph of recombinant type III humanized collagen-sodium hyaluronate gel sample

[0030] Figure 3 Example 1 Results of viscoelasticity test of recombinant type III humanized collagen-sodium hyaluronate gel

[0031] Figure 4 Example 1 Results of shear viscosity test of recombinant type III humanized collagen-sodium hyaluronate gel

[0032] Figure 5 Example 1 Detection results of recombinant type III humanized collagen-sodium hyaluronate gel diameter

[0033] Figure 6 Example 1 Infrared Spectrum of Recombinant Type III Humanized Collagen-Sodium Hyaluronate Gel

[0034] Figure 7 Example 2 Photo of recombinant type III humanized collagen-sodium hyaluronate gel sample

[0035] Figure 8 Example 3 Photo of recombinant type III humanized collagen-sodium hyaluronate gel sample

[0036] Fig. 9 Example 3 Results of viscoelasticity test of recombinant type III humanized collagen-sodium hyaluronate gel

[0037] Fig.10 Example 4 Photograph of recombinant type III humanized collagen-sodium hyaluronate gel sample

[0038] Fig.11 Example 5 Photo of the finished product of the injectable gel for cartilage damage repair

[0039] Fig.12 Example 1 Results of in vitro enzyme degradation experiment of recombinant type III humanized collagen-sodium hyaluronate gel

[0040] Fig.13 Example 5: Anatomical photos of rats injected subcutaneously with recombinant humanized type III collagen-sodium hyaluronate-thrombin-fibrinogen gel 3 months later

[0041] Fig.14 Example 5 H&E staining of tissue sections 3 months after recombinant humanized type III collagen-sodium hyaluronate-thrombin-fibrinogen gel was injected subcutaneously into rats

[0042] Fig.15 Example 5 Masson staining of tissue sections of rats injected subcutaneously with recombinant humanized type III collagen-sodium hyaluronate-thrombin-fibrinogen gel 3 months later

[0043] Fig.16 Example 5 CD31 immunofluorescence staining photos of tissue sections 3 months after recombinant humanized type III collagen-sodium hyaluronate-thrombin-fibrinogen gel was injected subcutaneously into rats

[0044] Fig.17 Example 5 CD90 immunofluorescence staining photos of tissue sections 3 months after recombinant humanized type III collagen-sodium hyaluronate-thrombin-fibrinogen gel was injected subcutaneously into rats

[0045] Fig.18 Image of the gap created at the distal femoral trochlea of ​​rats

[0046] Fig.19 Example 5 Photos of the sham operation group and the defect control group after injection of recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel

[0047] Fig. 20 Example 5 Micro-CT scans of the recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel injection group, sham operation group, and defect control group

[0048] Fig.21 Example 5 Statistical graph of micro-CT scan data of recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel injection group, sham operation group, and defect control group DETAILED DESCRIPTION

[0049] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0050] The molecular weight of the recombinant humanized type III collagen of the present invention is between 20 KDa and 100 KDa; the molecular weight of sodium hyaluronate is between 600 KDa and 900 KDa.

[0051] Comparative Example 1 (CN116603104A patented prescription process)

[0052] The silk fibroin hyaluronic acid composite gel was prepared according to the method of Example 3 of patent CN116603104A, and the specific steps were as follows:

[0053] a) Prepare 20 L of eluent with the following formula: 0.78% sodium chloride, 0.0085% sodium dihydrogen phosphate (NaH2PO·H2O), 0.135% disodium hydrogen phosphate (Na2HPO·12H2O) for later use.

[0054] b) Weigh 5 g of recombinant type III human collagen raw material (BSS grade), add 50 ml of pure water to dissolve, adjust the pH to 12.0 with sodium hydroxide solution (10% concentration), then add 1.2 ml of 1,4-butanediol diglycidyl ether (BDDE), and mix well using a vortex oscillator.

[0055] c) Weigh 1.5g of sodium hyaluronate raw material with a molecular weight of 1.5 million and mix it with the above solution, and swell until there is no visible white undissolved HA. Turn on the constant temperature water bath and adjust the water bath temperature to 45°C. Place the above gel in the water bath and keep it warm for 10 hours for cross-linking reaction. Take out the cross-linked gel, which is a light yellow solid elastic gel. Cut it into cubes of 0.4x0.4cm in size, add 3L of eluent, stir and swell at low speed for 2h, replace 3L of eluent, continue the second elution for 12h, replace 3L of eluent again, and continue the third elution for 8h until the final weight of the gel is 250g, reach the elution end point, stop elution, and the total time is 22h. The final concentration of recombinant type III human collagen is 20mg / ml (mass percentage 2%). Take the dialyzed gel and use a 40-mesh sieve to continuously homogenize 3 times to obtain a gel with a particle diameter of 200-600um. Take 100g of gel, fill it into a 1.0ml prefilled syringe, and sterilize it by wet heat at 121℃ for 10min to finally obtain recombinant type III collagen-sodium hyaluronate double cross-linked gel.

[0056] Figure 1 Example 3 in the comparative example shows the parameters of the recombinant type III humanized collagen gel prepared in this comparative example after being sterilized at 121°C for 10 minutes. Figure 1 It can be seen that BDDE residues were detected in this preparation method, so it has a small amount of biological toxicity.

[0057] Example 1 (III-COL:HA=1:1, lithium bromide concentration 12M, BDDE is 1 / 5 of the total amount of the two materials)

[0058] A method for preparing a recombinant type III humanized collagen-sodium hyaluronate gel for injection, the preparation method comprising the following steps:

[0059] a) Weigh 50 g of recombinant humanized type III collagen, add it to 600 ml of 12 M lithium bromide solution and stir to dissolve, then add 50 g of sodium hyaluronate and stir to dissolve, then add 20 ml of BDDE (butylene glycol glycidyl ether, content > 95%) and stir evenly, and crosslink at 28° C. for 60 h.

[0060] b) After the gel was crushed into small pieces, it was washed twice with 5L of ethanol-water solution (ethanol: water = 1:1, v / v), each time for 30 minutes; then it was crushed through a 4mm sieve, and the whole granules were washed 6 times with 5L of injection saline solution, each time for 30 minutes. After the last three washings, the free lithium bromide and BDDE were removed by centrifugation; the collected centrifuged gel was passed through a 200μm sieve twice to obtain gel particles.

[0061] c) The gel particles were added with 120% of the gel weight of injection physiological saline to prepare the gel particles, and the solution was obtained after being stirred evenly. The solution was filled with a 1 ml pre-filled syringe and sterilized by wet heat at 121° C. for 8 min.

[0062] Take 1 gel sample and use a 27G needle to test according to method 6.4 in YY / T 0962-2021. The average pushing force is 12.76N, the maximum pushing force is 13.90N, and the minimum pushing force is 11.77N. Take 1.0g of gel and test according to method 6.6 in YY / T 0962-2021 "Cross-linked Sodium Hyaluronate Gel for Plastic Surgery". The swelling ratio is 21.1.

[0063] Take an appropriate amount of gel and measure the osmotic pressure according to method 6.7 of YY / T 0962-2021. The osmotic pressure is 318mOsmol / kg. Take an appropriate amount of gel and measure the pH value according to method 6.8 of YY / T 0962-2021. The pH value is 7.18.

[0064] Take a proper amount of gel, add proteinase K to hydrolyze it, and use a high-performance gas phase instrument to detect the residue of cross-linking agent BDDE. The result shows that BDDE: not detected (limit is <2.0 μg / g).

[0065] Take an appropriate amount of gel, add purified water to dilute, and perform headspace sampling. Use a high-performance gas phase instrument to detect the cross-linking agent ethanol residue. The result shows ethanol: 31.1 μg / g (limit is <200 μg / g).

[0066] Take an appropriate amount of gel and dry it. Determine it according to the inductively coupled plasma mass spectrometry (ICP-MS) method 0412 of the Pharmacopoeia of the People's Republic of China (Volume 4) (2020 edition). The residual lithium ion is 0.118 mg / g (the limit is <2 mg / g, dry product).

[0067] Take an appropriate amount of gel and dry it. Determine it according to the ion chromatography method 0513 of the "Pharmacopoeia of the People's Republic of China (Volume 4)" (2020 edition), and the residual bromide ion is 0.008 mg / g (the limit is <80 mg / g, dry product).

[0068] Figure 2 The picture shows the recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example after being sterilized by moist heat at 121°C for 8 minutes. Figure 2 It can be seen that the recombinant type III humanized collagen-sodium hyaluronate gel is a colorless, translucent gel with uniform properties and no glue separation.

[0069] Figure 3 The finished product of recombinant humanized type III collagen-sodium hyaluronate gel prepared in this embodiment is shown. At 25±0.2°C, a rheometer is used to draw a coordinate diagram of the viscosity modulus G", elastic modulus G' and phase angle δ with frequency f under a shear rate of 0.1Hz to 100Hz. As can be seen from the figure, at a shear rate of 0.1Hz, the elastic modulus G' is 856.52Pa, and the viscosity modulus G": 123.54Pa. The results show that the finished product of recombinant humanized type III collagen-sodium hyaluronate gel in this embodiment has good viscoelasticity.

[0070] Figure 4 The finished product of recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example is shown. The rheometer is used at a shear rate of 0.001s at (25±0.2)°C. -1 ~1000s -1 Perform a creep scan and plot shear stress, viscosity η and shear rate respectively Coordinate diagram of . Figure 4 It can be seen that at a shear rate of 0.001s -1 ~1000s -1 The shear viscosity is ≥250mPa·s.

[0071] Figure 5 The particle size spectrum of the finished product of recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example is shown. Figure 5 It can be seen that the recombinant type III humanized collagen-sodium hyaluronate gel particles prepared in this example have a uniform particle size distribution, with d(0.5) being 695 μm and d(0.9) being 1327 μm.

[0072] Figure 6 The infrared spectrum of the finished product of recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example is shown. Figure 6 It can be seen that the Fourier transform infrared spectroscopy (FT-IR) of the gel prepared in this example measured a band at 3500 cm -1 -3200cm -1 , 2890cm -1 -2850cm -1 , 1660cm -1 -1620cm -1 , 1567cm -1 -1527cm -1 , 1430cm -1 -1390cm-1 , 1260cm -1 -1220cm -1 There are characteristic peaks.

[0073] Example 2 (III-COL:HA=1:1, lithium bromide concentration 12M, BDDE is 1 / 4 of the total amount of the two materials)

[0074] A method for preparing a recombinant type III humanized collagen-sodium hyaluronate gel for injection, the preparation method comprising the following steps:

[0075] a) Weigh 50 g of recombinant humanized type III collagen, add it to 600 ml of 12 M lithium bromide solution and stir to dissolve, then add 50 g of sodium hyaluronate and stir to dissolve, then add 25 ml of BDDE (butylene glycol glycidyl ether, content > 95%) and stir evenly, and crosslink at 28° C. for 60 h.

[0076] b) After the gel was crushed into small pieces, it was washed twice with 5L of ethanol-water solution (ethanol: water = 1:1, v / v), each time for 30 minutes; then it was crushed through a 4mm sieve, and the whole granules were washed 6 times with 5L of injection saline solution, each time for 30 minutes. After the last three washings, the free lithium bromide and BDDE were removed by centrifugation; the collected centrifuged gel was passed through a 200μm sieve twice to obtain gel particles.

[0077] The gel particles were added with 120% of the gel weight of injection physiological saline to prepare, and the solution was obtained after being stirred evenly. The solution was filled with a 1 ml prefilled syringe and sterilized by wet heat at 121° C. for 8 min. Figure 7 The picture shows the recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example after being sterilized by moist heat at 121°C for 8 minutes. Figure 7 It can be seen that the recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example is a white gel with slight gel separation. The results show that as the amount of BDDE added increases, the cross-linking degree increases and the water-locking capacity decreases.

[0078] Example 3 (III-COL:HA=1:1, lithium bromide concentration 9.3M, BDDE is 1 / 5 of the total amount of the two materials)

[0079] A method for preparing a recombinant type III humanized collagen-sodium hyaluronate gel for injection, the preparation method comprising the following steps:

[0080] a) Weigh 50 g of recombinant humanized type III collagen, add it to 600 ml of 9.3 M lithium bromide solution and stir to dissolve, then add 50 g of sodium hyaluronate and stir to dissolve, then add 20 ml of BDDE and stir evenly, and cross-link at 28°C for 60 h.

[0081] b) After the gel was crushed into small pieces, it was washed twice with 5L of ethanol-water solution (ethanol: water = 1:1, v / v), each time for 30 minutes; then it was crushed through a 4mm sieve, and the whole granules were washed 6 times with 5L of injection saline solution, each time for 30 minutes. After the last three washings, the free lithium bromide and BDDE were removed by centrifugation; the collected centrifuged gel was passed through a 200μm sieve twice to obtain gel particles.

[0082] c) The gel particles were added with 120% of the gel weight of injection physiological saline to prepare the gel particles, and the solution was obtained after being stirred evenly. The solution was filled with a 1 ml pre-filled syringe and sterilized by wet heat at 121° C. for 8 min. Figure 8 The picture shows the recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example after being sterilized by moist heat at 121°C for 8 minutes. Figure 8 It can be seen that the recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example is a colorless, translucent gel with uniform properties and no gel separation.

[0083] Fig. 9 The finished product of recombinant type III humanized collagen-sodium hyaluronate gel prepared in this embodiment is shown. At 25±0.2°C, a rheometer is used to draw a coordinate graph of the viscosity modulus G", the elastic modulus G' and the frequency f at a shear rate ranging from 0.1Hz to 100Hz. Fig. 9 It can be seen that when the shear rate is 0.1 Hz, the elastic modulus G' is 426.86 Pa and the viscous modulus G" is 98.072 Pa. The results show that the cross-linking degree and viscoelasticity of the recombinant type III humanized collagen-sodium hyaluronate gel in the lithium bromide 9.3 M solution are reduced.

[0084] Example 4 (III-COL:HA=1:1, lithium bromide concentration 12M, BDDE is 1 / 5 of the total amount of the two materials, reducing the amount of re-prepared physiological saline)

[0085] A method for preparing a recombinant type III humanized collagen-sodium hyaluronate gel for injection, the preparation method comprising the following steps:

[0086] a) Weigh 50 g of recombinant humanized type III collagen, add it to 600 ml of 12 M lithium bromide solution and stir to dissolve, then add 50 g of sodium hyaluronate and stir to dissolve, then add 20 ml of BDDE and stir evenly, and cross-link at 28° C. for 60 h.

[0087] b) After the gel was crushed into small pieces, it was washed twice with 5L of ethanol-water solution (ethanol: water = 1:1, v / v), each time for 30 minutes; then it was crushed through a 4mm sieve, and the whole granules were washed 6 times with 5L of injection saline solution, each time for 30 minutes. After the last three washings, the free lithium bromide and BDDE were removed by centrifugation; the collected centrifuged gel was passed through a 200μm sieve twice to obtain gel particles.

[0088] c) The gel particles were added with 60% of the gel weight of injection physiological saline to prepare the gel particles, and the solution was obtained after being stirred evenly. The solution was filled with a 1 ml pre-filled syringe and sterilized by wet heat at 121° C. for 8 min.

[0089] Fig.10 The picture shows the recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example after being sterilized by moist heat at 121°C for 8 minutes. Fig.10 It can be seen that the recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example has a large granular feel and uneven properties. The sample is difficult to pass through the needle, and blockage occurs when using a 27G needle. According to the 6.4 method in YY / T0962-2021, the maximum pushing force is >60N. Therefore, the gel prepared in this example is difficult to use for clinical injection filling.

[0090] Example 5 (Recombinant humanized type III collagen-sodium hyaluronate-thrombin-fibrinogen gel)

[0091] A method for preparing a recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel for injection, the preparation method comprising the following steps:

[0092] a) The gel particle solution prepared in Example 1 of the present invention and the thrombin solution (with a concentration of 5000 u / mL) were mixed at a volume ratio of 4:1, and after mixing evenly, the mixture was filled into a 2 mL syringe A.

[0093] b) Dissolve 200 mg of fibrinogen in 10 mL of enzyme-free water and fill into 2 mL syringe B after complete dissolution.

[0094] c) Install syringe A and syringe B on the double-row injection stand.

[0095] d) After being pushed out through a double-row syringe, a uniformly mixed recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel can be obtained.

[0096] Fig.11 The finished injectable hydrogel product for cartilage damage repair prepared in this example is shown.

[0097] Test Example 1 In vitro anti-degradation test

[0098] The recombinant type III humanized collagen-sodium hyaluronate gel prepared in Example 1 was degraded in vitro using sodium hyaluronidase and collagenase. The weight of the remaining gel at different time points was observed to determine the in vitro anti-enzymatic ability of the recombinant type III humanized collagen-sodium hyaluronate gel under the action of the combination of hyaluronidase and collagenase.

[0099] The specific implementation plan is as follows:

[0100] a) Mixed enzyme solution preparation

[0101] Hyaluronidase solution: Accurately weigh 20 mg of hyaluronidase (1500 units / mg) into a 20 ml volumetric flask, dilute to the mark with 0.9% sodium chloride solution, and shake well.

[0102] Mixed enzyme solution: Take a vial of collagenase for injection (600 units / bottle) and dissolve it with 15 ml of the prepared hyaluronidase solution to obtain a mixed enzyme solution.

[0103] b) Sample preparation

[0104] Take 10 gel samples, mix them evenly after taking them out, weigh 1g respectively into 8 50ml centrifuge tubes, mark 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, except 0h (add 49ml pure water), add 2ml combined enzyme solution to the other tubes, keep in a constant temperature gas bath at 37℃, take them out on time, add 47ml pure water to mix, centrifuge (8000r / min, 15min), filter the filter cake by suction, put it in a 75℃ oven, dry it together and weigh it.

[0105] c) Calculation

[0106] The sample weight at time 0 is recorded as 100%, and the remaining amount of gel (%) = sample weight at different time points / sample weight at time 0 × 100%. Fig.12 As shown; it can be concluded that recombinant type III humanized collagen-sodium hyaluronate gel can be almost completely degraded within 24 hours using a mixed solution of hyaluronidase-collagenase (hyaluronidase activity is 1500u / ml, collagenase activity is 40u / ml).

[0107] The results show that the recombinant type III humanized collagen-sodium hyaluronate gel prepared in this example can be completely degraded in an environment close to that inside the human skin, has high biosafety, and provides guidance for clinical applications.

[0108] Test Example 2 Short-term biological test

[0109] The recombinant type III humanized collagen-sodium hyaluronate gel prepared in Example 1 was subjected to the extraction method (MTT method) specified in GB / T16886.5-2017 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test", and the extraction medium was cell culture medium. The results showed that the gel had no cytotoxicity under the conditions of the MTT cytotoxicity test.

[0110] The recombinant type III humanized collagen-sodium hyaluronate gel prepared in Example 1 was used to evaluate the potential of the test sample to cause skin sensitization in guinea pigs under the test conditions in accordance with the requirements of the skin sensitization test (maximum dose method) recommended in GB / T16886.10-2017 "Biological Evaluation of Medical Devices Part 10: Irritation and Skin Sensitization Test". The results showed that the test sample did not cause skin sensitization, and the incidence of positive stimulation results was 0%.

[0111] Take the recombinant type III humanized collagen-sodium hyaluronate gel prepared in Example 1, and perform a biocompatibility test on the sample extract according to the requirements of the intradermal reaction recommended in GB / T16886.10-2017 "Biological Evaluation of Medical Devices Part 10: Irritation and Skin Sensitization Test". The test samples were extracted with polar extracts (0.9% sodium chloride injection) and non-polar extracts (cottonseed oil), and the obtained extracts were administered to New Zealand albino rabbits by intradermal injection to evaluate the potential for irritation reactions produced by the materials under the test conditions. The results showed that the final score of the intradermal reaction of the polar extract of the test sample was 0.00; the final score of the intradermal reaction of the non-polar extract was 0.00, and the final result showed that the intradermal reaction was good.

[0112] The recombinant humanized type III collagen-sodium hyaluronate gel prepared in Example 1 was tested according to the method recommended in GB / T16886.11-2021 "Biological Evaluation of Medical Devices Part 11: Systemic Toxicity Test". The sample was directly used as the test solution and injected subcutaneously into mice. The results showed that the test sample had no acute systemic toxicity reaction to mice.

[0113] The recombinant type III humanized collagen-sodium hyaluronate gel prepared in Example 1 was tested according to the method recommended in GB / T16886.11-2021 "Biological Evaluation of Medical Devices Part 11: Systemic Toxicity Test". The test sample was extracted with 0.9% sodium chloride injection, and the supernatant was injected into the ear vein of New Zealand rabbits. The results showed that the test solution pyrogen test of the test sample gel met the requirements and there was no material pyrogenic effect.

[0114] Test Example 3 Gel Subcutaneous Implantation Test

[0115] The injectable gel prepared in Example 5 was taken and the two parts of the materials were mixed by a double-row drug mixing injection stand to obtain a recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel, which was then injected subcutaneously into rats for a subcutaneous implantation test.

[0116] Fig.13 This is a photo of the tissue extracted from the recombinant humanized type III collagen-sodium hyaluronate-thrombin-fibrinogen gel implanted into the subcutaneous tissue of rats 3 months later. Fig.13 It can be seen that 3 months after implantation in the tissue, the implant did not undergo significant degradation. At the same time, an obvious vascular network was formed inside the implant, indicating that the implant has excellent biocompatibility.

[0117] Fig.14 The figure shows an H&E stained photograph of the extracted tissue section 3 months after the recombinant humanized type III collagen-sodium hyaluronate-thrombin-fibrinogen gel was implanted subcutaneously in rats. From the photograph, it can be seen that only a small number of inflammatory cells exist around the implant, indicating that the implant has good biocompatibility and will not cause serious local tissue inflammation.

[0118] Fig.15 The Masson staining photographs of the extracted tissue sections 3 months after the recombinant humanized type III collagen-sodium hyaluronate-thrombin-fibrinogen gel was implanted subcutaneously in rats show that a large amount of collagen fiber regeneration can be seen in the tissue surrounding the implant.

[0119] Fig.16 The CD31 immunofluorescence staining photograph of the extracted tissue section 3 months after the recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel was implanted subcutaneously in rats is shown. From the photograph, it can be seen that there is obvious CD31 vascular endothelial cell membrane protein distributed around and inside the implant, indicating that the filler has good biocompatibility and can induce autologous vascular tissue to grow into the filler.

[0120] Fig.17 The CD90 immunofluorescence staining photographs of the extracted tissue sections 3 months after the recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel was implanted subcutaneously in rats are shown. From the photographs, it can be seen that there is obvious CD90 protein distributed around and inside the implant, indicating that the filler has good biocompatibility and can induce autologous fibroblasts to grow and proliferate in the filler.

[0121] Test Example 4 Gel cartilage repair test

[0122] 8-week-old SD rats were purchased and raised for 1 week to adapt to the environment. The rats were anesthetized by injection of 3% pentobarbital, with the injection volume based on the rat body weight, 0.2 mL / 100 g. Fig.18 As shown, after the rat was deeply anesthetized, the skin and muscles at the knee joint were opened with a scalpel, and a defect with a diameter of 2 mm and a depth of 1.5 mm was established at the distal femoral pulley. Low-temperature physiological saline was dripped while drilling to prevent femoral necrosis.

[0123] like Fig.19 As shown, in Example 5, two parts of the material are blended by a double-row injection stand to obtain a recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel, and then injected into the defect. A sham operation group without a defect and a control group without a material after the defect are set up. After the filling is completed, the muscle tissue and skin are sutured separately using a suture needle. 200 μL of antibiotics (800 U / mL) are injected intramuscularly to prevent postoperative infection.

[0124] At the third month, the rats were killed, and the knee joint tissues were taken and fixed with tissue fixative. Micro-CT scanning was performed on the knee joint tissue to detect the bone condition of the new cartilage. Fig. 20 As shown in the figure, at the third month, the control group still showed obvious defects, while the defect area in the recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel-treated group was significantly reduced, similar to the sham operation group. Therefore, the recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel-treated group can accelerate the cartilage repair at the defect site.

[0125] like Fig.21 The Micro-CT statistical results shown in the figure show that compared with the control group, the trabecular thickness of the recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel-treated group was significantly increased, close to that of the sham operation group. The trabecular separation and bone density of the recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel-treated group were also slightly improved compared with the control group. This result also proves that recombinant type III humanized collagen-sodium hyaluronate-thrombin-fibrinogen gel is beneficial to cartilage repair.

Claims

1. An injectable hydrogel for repairing cartilage damage, which is composed of 3 to 4 parts by volume of recombinant type III humanized collagen-sodium hyaluronate gel particles, 0.5 to 1 part of thrombin solution and 4 to 6 parts of fibrinogen solution.

2. The injectable hydrogel according to claim 1, characterized in that The injectable hydrogel for repairing cartilage damage is composed of 4 parts of recombinant type III humanized collagen-sodium hyaluronate gel particles, 1 part of thrombin solution and 5 parts of fibrinogen solution by volume; preferably, the mass ratio of recombinant type III humanized collagen to sodium hyaluronate in the recombinant type III humanized collagen-sodium hyaluronate gel particles is 1:1 to 6:1, preferably 1:1; preferably, the concentration of the thrombin solution is 5000u / mL; the mass concentration of the fibrinogen solution is 20mg / mL; preferably, the molecular weight of the recombinant type III humanized collagen is between 10KDa and 300KDa, preferably between 20KDa and 100KDa; the molecular weight of the sodium hyaluronate is between 300KDa and 1200KDa, preferably between 600KDa and 900KDa.

3. The method for preparing the injectable hydrogel for cartilage damage repair according to claim 1: 1) adding the catalyst to purified water and stirring to dissolve, and then adding the recombinant type III humanized collagen to the catalyst solution and stirring to dissolve, then adding sodium hyaluronate and stirring to dissolve, and then adding the cross-linking agent and stirring to evenly carry out the cross-linking reaction; 2) After the cross-linking is completed, the primary gel is crushed into small pieces, washed and swollen with an ethanol aqueous solution, sieved and crushed for the first time, and then washed, swollen, and centrifuged to remove the catalyst and free cross-linking agent after the whole particle is formed, and the ethanol is removed by rotary evaporation, and then washed and centrifuged with a phosphate buffer solution or a physiological saline solution, and then sieved and crushed for the second time, and sterilized by wet heat to prepare gel particles; 3) The recombinant type III humanized collagen-sodium hyaluronate gel particles are mixed with the thrombin solution, and then mixed with the fibrinogen solution again with the same force and pushed out through a double-row mixing syringe to obtain an injectable hydrogel for cartilage damage repair.

4. The preparation method according to claim 3, characterized in that: In step 1), the cross-linking agent is one or more of ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, polypropylene glycol diglycidyl ether, diglycidyl ether or divinyl sulfone, preferably 1,4-butanediol diglycidyl ether; the amount of the cross-linking agent added is 1 / 3 to 1 / 10 of the total amount of recombinant type III humanized collagen and sodium hyaluronate, preferably 1 / 4 to 1 / 6.

5. The preparation method according to claim 3, characterized in that: The catalyst in step 1) is one or more of lithium bromide, calcium chloride, lithium hydroxide, and sodium hydroxide, preferably lithium bromide, with a concentration of 9.3 to 12M.

6. The preparation method according to claim 3, characterized in that: In step 1), the cross-linking reaction temperature is 18 to 35° C., preferably 22 to 28° C.; the cross-linking reaction time is 36 to 72 hours, preferably 44 to 60 hours.

7. The preparation method according to claim 3, characterized in that: In step 2), the ethanol aqueous solution is prepared from ethanol and water in a volume ratio of 1:1, and the aperture of the sieve for the first sieving and crushing is 4 mm; the aperture of the sieve for the second sieving and crushing is 200 μm.

8. The preparation method according to claim 3, characterized in that: In step 3), the volume ratio of thrombin to recombinant type III humanized collagen-sodium hyaluronate gel is 1:5-1:2, preferably 1:4; the volume ratio of the fibrinogen solution to recombinant type III humanized collagen-sodium hyaluronate-thrombin gel is 1:

1.

9. Use of the injectable hydrogel for cartilage damage repair according to claim 1 in the preparation of cartilage damage filling materials; preferably, the cartilage damage filling material is a cartilage tissue regeneration and repair material.

Images