Non-denatured type Ⅱ collagen ultrafine powder with cartilage tissue targeted growth and structure repair effect and its biological activity preserving preparation method and joint application

Abstract

This invention relates to the field of biomedical materials, providing non-denatured type II collagen ultrafine powder with cartilage tissue-targeted growth and structural repair functions, its bioactivity preservation preparation method, and its application in joints. The ultrafine powder has an average particle size of 0.5-50 μm, a triple helix structure integrity rate of not less than 85%, a thermal denaturation temperature of 38-48℃, and retains chondrocyte binding domains CB10, CB11, and MMP cleavage sites. The preparation method includes raw material pretreatment, time-limited enzymatic hydrolysis, graded purification, and ultra-low temperature ball milling. The product can be used for the repair and treatment of cartilage degenerative diseases such as osteoarthritis and cartilage damage.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to non-denatured type II collagen ultrafine powder with cartilage tissue-targeted growth and structural repair functions, its bioactivity preservation preparation method, and its application in joints. Background Technology

[0002] Type II collagen is a major structural protein of the extracellular matrix of articular cartilage cells, accounting for approximately 50%-60% of the dry weight of cartilage tissue. It plays a central role in maintaining cartilage tissue integrity, regulating chondrocyte function, and controlling cartilage mechanical properties. Type II collagen is characterized by a triple helix structure composed of three α1(II) chains. This unique supramolecular structure formed by the (Gly-XY)n repeating sequence is the molecular basis for its biological functions. Studies have shown that type II collagen possesses specific chondrocyte-binding domains and signal transduction sites, which can regulate chondrocyte adhesion, proliferation, differentiation, and matrix synthesis.

[0003] Osteoarthritis is the most common degenerative joint disease, affecting approximately 300 million people worldwide. The pathological characteristics of osteoarthritis include progressive destruction of articular cartilage, subchondral bone remodeling, and synovial inflammation, ultimately leading to loss of joint function. Cartilage tissue has extremely limited self-repair capabilities and rarely heals itself once damaged. Currently, there are no effective drugs for cartilage regeneration in clinical practice; existing treatments are mainly symptomatic and cannot fundamentally reverse the cartilage degeneration process.

[0004] Existing methods for preparing type II collagen suffer from the following technical defects: First, traditional enzymatic hydrolysis or chemical treatment methods often destroy the triple helix structure of type II collagen, forming denatured collagen or gelatin, losing specific bioactive epitopes, and failing to effectively promote the directed differentiation of chondrocytes and matrix synthesis. Second, existing collagen products have uneven particle size distribution, mostly in the form of ordinary powder or granules, resulting in low bioavailability and difficulty in achieving effective enrichment and sustained action in cartilage tissue. Third, the lack of protection measures for cartilage-specific active structural domains leads to rapid attenuation of product activity during storage and application. Chinese patent CN119925703A discloses a highly stable aquatic animal type II collagen complex and its preparation method, which improves thermal stability through polysaccharide complexation; however, this technical solution does not involve ultrafine powder preparation processes, nor does it solve the technical problems of cartilage tissue targeting and active epitope protection.

[0005] Therefore, there is an urgent need in this field to develop non-denatured type II collagen products that can retain the complete triple helix structure and cartilage-specific active epitopes, and to establish corresponding bioactive preservation preparation methods to meet the clinical needs of cartilage repair therapy. Summary of the Invention

[0006] To address the aforementioned technical deficiencies in existing technologies, this invention provides non-denatured type II collagen ultrafine powder with targeted growth and structural repair effects on cartilage tissue, along with its bioactivity-preserving preparation method and joint applications. The technical solution of this invention utilizes innovative molecular integrity protection technology to ensure that type II collagen retains its complete triple helix structure and cartilage-specific active epitopes. Ultra-low temperature ball milling technology is employed to prepare the ultrafine powder, thereby improving bioavailability and enabling the product to specifically act on cartilage tissue, promoting chondrocyte proliferation and differentiation while maintaining cartilage matrix stability.

[0007] The first aspect of this invention provides non-denatured type II collagen ultrafine powder with targeted growth and structural repair effects on cartilage tissue. The non-denatured type II collagen ultrafine powder retains an intact triple helix structure and cartilage-specific active epitopes. The average particle size of the ultrafine powder is 0.5-50 μm, the triple helix structure integrity rate of the type II collagen is not less than 85%, the hydroxyproline content in the ultrafine powder is 8%-14%, the glycosaminoglycan content is 0.1%-3%, the thermal denaturation temperature of the ultrafine powder is 38-48℃, and the type II collagen retains the chondrocyte-binding domains CB10 and CB11 and the matrix metalloproteinase cleavage site (MMP) characteristic sequences.

[0008] In a preferred embodiment of the present invention, the average particle size of the ultrafine powder is 1-20 μm. This particle size range is beneficial for the uniform dispersion of the ultrafine powder within the joint cavity and its effective uptake by cartilage tissue. More preferably, the D90 particle size does not exceed 45 μm, ensuring uniformity of particle size distribution. The triple helix structure integrity rate is not less than 90%, indicating that the natural conformation of type II collagen molecules is well maintained. The hydroxyproline content is 10%-13%, which conforms to the amino acid composition characteristics of natural type II collagen. The thermal denaturation temperature is 40-45℃, higher than the physiological temperature of the human body, ensuring structural stability of the product during in vivo application.

[0009] In another embodiment of the invention, circular dichroism spectroscopy is used to characterize the triple helix structure of the ultrafine powder. The ultrafine powder exhibits a positive peak at 221 nm and a negative peak at 197 nm, which are characteristic spectral signals of the triple helix structure of type II collagen. The molar ellipticity at 221 nm is also considered. for - deg·cm²·dmol -1 This numerical range indicates that the triple helix structure remains intact. The solubility of the ultrafine powder in simulated synovial fluid is 0.5-5 mg / mL, making it suitable for intra-articular injection. Regarding the source of the raw materials, the ultrafine powder can be derived from aquatic animal cartilage such as shark cartilage, ray cartilage, sturgeon cartilage, or squid cartilage; avian cartilage such as chicken breast cartilage; or mammalian cartilage such as bovine cartilage or porcine cartilage.

[0010] Furthermore, the ultrafine powder may also contain cartilage matrix proteoglycans, with a mass ratio of proteoglycans to type II collagen of 0.01-0.5:1. The presence of proteoglycans helps maintain the natural microenvironment of type II collagen and enhances its biological function. The bulk density of the ultrafine powder is 0.15-0.45 g / cm³, and the moisture content of the ultrafine powder does not exceed 8%, ensuring the storage stability of the product.

[0011] A second aspect of this invention provides a method for preparing the non-denatured type II collagen ultrafine powder while preserving its bioactivity. This method employs mild enzymatic hydrolysis conditions, precise process parameter control, and ultra-low temperature ball milling technology to maximize the preservation of the triple helix structure and active epitopes of type II collagen during the preparation process.

[0012] The preparation method includes the following steps:

[0013] Step 1: Raw Material Pretreatment: After cleaning the animal cartilage tissue, place it in a 0.5%-2% sodium chloride solution and soak it at 2-8℃ for 8-24 hours to remove non-collagenous components. Sodium chloride solution treatment effectively removes soluble proteins and lipids from the cartilage tissue while maintaining the structural integrity of collagen. Low temperature conditions help inhibit the activity of endogenous enzymes, preventing collagen degradation during pretreatment.

[0014] Step 2, Enzymatic Extraction: The pretreated cartilage tissue is placed in an acidic solution containing pepsin and subjected to time-limited enzymatic hydrolysis at 15-25℃. The hydrolysis time is 12-72 hours, the enzyme activity is 1000-5000 U / g of raw material, and the pH of the acidic solution is 1.5-3.5. Under acidic conditions, pepsin can selectively cleave the non-triple-helix region (terminal peptide region) of collagen molecules without destroying the triple-helix main structure, thereby achieving the extraction of type II collagen. Strictly controlling the hydrolysis temperature below 25℃ can effectively prevent thermal denaturation of collagen.

[0015] Preferably, the enzymatic hydrolysis temperature in step two is 18-22℃, the hydrolysis time is 24-48h, the pH is 2.0-3.0, and the pepsin activity is 2000-4000 U / g of raw material. This combination of parameters can maximize the preservation of the integrity of the triple helix structure while ensuring extraction efficiency.

[0016] Step 3: Fractional Purification: The enzymatic hydrolysis products are purified using a combined salting-out and dialysis process. Sodium chloride is used as the salting-out agent at a concentration of 0.8-2.5 M, and the dialysis molecular weight cutoff is 100-300 kDa. Type II collagen has a molecular weight of approximately 300 kDa. By selecting appropriate salting-out concentrations and dialysis molecular weight cutoffs, type II collagen can be effectively separated from impurity proteins and small molecule degradation products. Preferably, the salting-out concentration is 1.0-2.0 M, and the dialysis molecular weight cutoff is 150-250 kDa. Under these conditions, both the recovery rate and purity of type II collagen reach ideal levels.

[0017] Step 4: Ultrafine Grinding: After low-temperature drying, the purified product is ultrafinely ground using ultra-low temperature ball milling technology. The ball milling temperature is -80℃ to -40℃, and the ball milling time is 2-8 hours. Ultra-low temperature ball milling is one of the key innovative technologies of this invention. Under extremely low temperature conditions, collagen molecules are in a glassy state, and the movement of molecular chain segments is frozen, thereby effectively preventing molecular chain breakage and structural denaturation caused by mechanical force during the ball milling process. Preferably, the ball milling temperature is -70℃ to -50℃, which ensures the grinding effect while maximizing the protection of the triple helix structure.

[0018] In addition, step four may include an activity stabilization treatment step. The ultrafine powder is mixed with a 0.01%-1% (w / w) trehalose solution and then freeze-dried. Trehalose, as a bioprotective agent, can replace water molecules and form hydrogen bonds with collagen during the drying process, preventing protein denaturation. Alternatively, inert gas purging packaging can be used to isolate oxygen and prevent oxidation. Alternatively, 0.005%-0.1% (w / w) of an antioxidant can be added, the antioxidant being selected from ascorbic acid, ascorbyl palmitate, or tocopherol, or a combination thereof.

[0019] A third aspect of this invention provides the application of the aforementioned non-denatured type II collagen ultrafine powder in the preparation of cartilage repair materials. The cartilage repair material can be an oral formulation, an intra-articular injection formulation, or a cartilage tissue engineering scaffold. The oral formulation contains 10-500 mg of non-denatured type II collagen ultrafine powder per dose unit, and the intra-articular injection formulation contains 0.5-20 mg / mL of non-denatured type II collagen ultrafine powder.

[0020] The described cartilage repair material can be used for the repair and treatment of cartilage damage caused by osteoarthritis and rheumatoid arthritis, sports-induced cartilage defects, or degenerative cartilage diseases. The non-denatured type II collagen ultrafine powder of this invention can slowly degrade in cartilage tissue, continuously releasing active factors, improving the joint cavity microenvironment, promoting chondrocyte proliferation and differentiation, maintaining cartilage matrix stability, and providing an effective means for specific cartilage repair.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] First, this invention employs an innovative molecular integrity protection technology. Through mild enzymatic hydrolysis conditions and ultra-low temperature ball milling, it ensures that type II collagen retains its complete triple helix structure and cartilage-specific active epitopes. The triple helix structure integrity rate can reach over 85%, which is significantly superior to denatured collagen products in the prior art.

[0023] Secondly, the ultrafine powder prepared by this invention has an average particle size of 0.5-50 μm, which is significantly smaller than that of traditional collagen powder products, greatly improving bioavailability and enabling effective enrichment in cartilage tissue.

[0024] Third, the product of this invention retains specific active epitopes such as chondrocyte binding domains CB10 and CB11, which can specifically act on cartilage tissue and promote chondrocyte proliferation and differentiation. Compared with non-specific collagen products in the prior art, it has a significant targeting advantage.

[0025] Fourth, the thermal denaturation temperature of the product of this invention reaches 38-48℃, which is higher than the physiological temperature of the human body. It has a stable structure when used in the body and can continuously release active factors in cartilage tissue.

[0026] Fifth, the preparation method of this invention is simple, has mild conditions, and is highly controllable, making it suitable for industrial production. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the preparation method for preserving the bioactivity of non-denatured type II collagen ultrafine powder according to the present invention.

[0028] Figure 2 The images shown are circular dichroic chromatograms of the products prepared in Examples 1-3 of this invention.

[0029] Figure 3 The images shown are SDS-PAGE electrophoresis analysis diagrams of the products prepared in Examples 1-3 of this invention.

[0030] Figure 4 Differential scanning calorimetry (DSC) charts of the products prepared in Examples 1-3 of this invention.

[0031] Figure 5 This is a particle size distribution analysis diagram of the product prepared in Example 1 of the present invention.

[0032] Figure 6 This is a bar chart showing the results of the in vitro chondrocyte proliferation experiment in Example 4 of the present invention.

[0033] Figure 7 This is a bar chart showing the histological score of the articular cartilage repair effect in animal experiments of Example 5 of the present invention. Detailed Implementation

[0034] Please refer to the attached document.Figures 1-7 The technical solution of the present invention will be further described in detail below with reference to specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0035] Example 1: Preparation of non-denatured type II collagen ultrafine powder derived from shark cartilage

[0036] Step 1: Raw Material Pretreatment: Take 1000g of fresh shark cartilage, clean it to remove surface blood and fascia, and cut it into small pieces with sides of about 2cm. Place the cartilage pieces in a 1% sodium chloride solution (5 times the weight of the cartilage) and soak them at 4℃ for 16 hours. After soaking, discard the solution, rinse the cartilage pieces three times with deionized water, and drain for later use.

[0037] Step 2, Enzymatic Extraction: Prepare the enzymatic hydrolysate by adding pepsin to 0.5M acetic acid solution, achieving an enzyme activity of 3000 U / g of raw material, and adjusting the pH to 2.5. Place the pretreated cartilage blocks in the enzymatic hydrolysate, with a cartilage to hydrolysate mass ratio of 1:10. Perform enzymatic hydrolysis at 20℃ with magnetic stirring for 36 hours. After hydrolysis, adjust the pH to 7.0 with 5M sodium hydroxide solution to terminate the enzyme reaction. Centrifuge at 12000 rpm for 30 minutes and collect the supernatant.

[0038] Step 3: Fractional Purification: Slowly add sodium chloride solid to the supernatant to achieve a final concentration of 1.5M, and incubate overnight at 4°C. Centrifuge at 12000 rpm for 30 min to collect the precipitate, and dissolve the precipitate in 0.5M acetic acid solution. Transfer the solution to a dialysis bag (molecular weight cutoff 200 kDa) and dialyze in 0.02M phosphate buffer (pH 7.4) for 48 h, changing the dialysate every 8 h. Collect the solution in the dialysis bag after dialysis.

[0039] Step 4: Ultrafine Grinding: Pre-freeze the dialyzed solution at -80℃ for 24 hours, then dry it in a freeze dryer for 48 hours to obtain dried type II collagen. Place the dried product in an ultra-low temperature ball mill, pre-cool the milling jar to -60℃, add an appropriate amount of zirconia grinding beads (3mm diameter), with a ball-to-material ratio of 10:1, and ball mill at -60℃ for 4 hours. After ball milling, pass the product through a 200-mesh sieve to obtain ultrafine powder of non-denatured type II collagen derived from shark cartilage.

[0040] Step 5, Activity Stabilization Treatment: Mix the ultrafine powder with 0.5% trehalose solution at a mass ratio of 1:5, pre-freeze at -80℃, freeze-dry for 24 hours, and store in vacuum packaging.

[0041] Product testing: Particle size distribution was determined using a laser particle size analyzer. The results showed an average particle size of 8.5 μm, D10 of 2.1 μm, D50 of 7.8 μm, and D90 of 21.3 μm. Collagen purity was determined using a hydroxyproline assay, with a hydroxyproline content of 11.8%. The triple helix structure was determined using circular dichroism spectroscopy, with a molar ellipticity of [value missing] at 221 nm. deg·cm²·dmol -1 The triple helix structure integrity rate was 92%. The thermal denaturation temperature was determined to be 42.5℃ using differential scanning calorimetry.

[0042] Example 2: Preparation of non-denatured type II collagen ultrafine powder derived from sturgeon cartilage

[0043] Step 1: Raw material pretreatment: Take 800g of fresh sturgeon head cartilage, clean it, and cut it into small pieces. Place the cartilage pieces in a 1.5% sodium chloride solution and soak them at 6℃ for 20 hours.

[0044] Step 2, Enzymatic Extraction: Pepsin was added to a 0.5M acetic acid solution with an enzyme activity of 2500 U / g of raw material, and the pH was adjusted to 2.8. The pretreated cartilage was placed in the enzymatic hydrolysate at a mass ratio of 1:8 and enzymatically hydrolyzed at 18°C ​​for 48 hours.

[0045] Step 3, fractional purification: purification was carried out by a combination of sodium chloride salting-out (final concentration 1.2M) and dialysis (molecular weight cutoff 180kDa).

[0046] Step 4: Ultrafine grinding: After freeze drying, ball mill at -55℃ for 5 hours.

[0047] Step 5, Activity Stabilization Treatment: Add 0.05% by mass of ascorbic acid as an antioxidant, and package with nitrogen purging.

[0048] Product testing: Average particle size is 12.3 μm, D90 is 35.6 μm. Hydroxyproline content is 10.5%. Molar ellipticity at 221 nm is... deg·cm²·dmol -1 The triple helix structure has an integrity rate of 88%. The heat denaturation temperature is 40.8℃.

[0049] Example 3: Preparation of non-denatured type II collagen ultrafine powder derived from chicken breast cartilage

[0050] Step 1: Raw material pretreatment: Take 500g of fresh chicken breast cartilage, wash and cut it into small pieces. Place the cartilage pieces in a 0.8% sodium chloride solution and soak them at 2℃ for 12 hours.

[0051] Step 2, Enzymatic Extraction: Pepsin was added to a 0.5M acetic acid solution with an enzyme activity of 3500 U / g of raw material, and the pH was adjusted to 2.2. The pretreated cartilage was placed in the enzymatic hydrolysate at a mass ratio of 1:12 and enzymatically hydrolyzed at 22℃ for 28 hours.

[0052] Step 3, fractional purification: purification was carried out by a combination of sodium chloride salting-out (final concentration 1.8M) and dialysis (molecular weight cutoff 220kDa).

[0053] Step 4: Ultrafine grinding: After freeze drying, ball mill at -65℃ for 6 hours.

[0054] Step 5, Activity Stabilization Treatment: Mix with 0.2% trehalose solution and freeze dry.

[0055] Product testing: Average particle size is 5.6 μm, D90 is 18.2 μm. Hydroxyproline content is 12.6%. Molar ellipticity at 221 nm is... deg·cm²·dmol -1 The triple helix structure has a 95% integrity rate. The heat denaturation temperature is 44.2℃.

[0056] Comparative Example 1: Preparation of Type II Collagen by Traditional Enzymatic Hydrolysis

[0057] The same raw materials as in Example 1 were used, but the enzymatic hydrolysis temperature was increased to 35°C, the hydrolysis time was extended to 72 hours, and the pH was 2.0. After enzymatic hydrolysis, a conventional freeze-drying and pulverizing process (room temperature ball milling) was used to prepare the powder product. Product testing results showed that the average particle size was 156 μm, the hydroxyproline content was 9.8%, and the molar ellipticity at 221 nm was only [value missing]. deg·cm²·dmol -1 The triple helix structure integrity rate was only 28%, and the thermal denaturation temperature was 32.5℃. This result indicates that the high enzymatic hydrolysis temperature and room temperature ball milling process led to severe damage to the triple helix structure of type II collagen.

[0058] Comparative Example 2: Preparation by room temperature ball milling process

[0059] The same enzymatic extraction and purification process as in Example 1 was used, but the ultrafine grinding step was changed to ball milling at room temperature (25°C) for 6 hours. Product testing results showed: average particle size of 10.2 μm, hydroxyproline content of 11.5%, and molar ellipticity at 221 nm of [value missing]. deg·cm²·dmol -1 The triple helix structure integrity rate was 65%, and the heat distortion temperature was 37.8℃. Compared with Example 1, room temperature ball milling led to a decrease of approximately 27 percentage points in the triple helix structure integrity rate, indicating that ultra-low temperature ball milling plays an important role in protecting the triple helix structure.

[0060] Comparative Example 3: Conventional Grinding Process

[0061] The same enzymatic extraction and purification process as in Example 1 was used, but after drying, the product was pulverized using a conventional mechanical pulverizer and passed through an 80-mesh sieve. Product testing results showed an average particle size of 125 μm, a D90 of 280 μm, a hydroxyproline content of 11.2%, a triple helix structure integrity rate of 78%, and a heat denaturation temperature of 40.5℃. Compared to Example 1, the particle size of the product from the conventional pulverization process was significantly larger, which is detrimental to improving bioavailability.

[0062] Example 4: In vitro chondrocyte proliferation experiment

[0063] The non-denatured type II collagen ultrafine powder prepared in Examples 1-3 and the products of Comparative Examples 1-3 were respectively prepared into 1 mg / mL solutions and used as culture medium additives for in vitro chondrocyte culture experiments.

[0064] Experimental methods: Chondrocytes of the knee joint of SD rats were isolated, and... Cells were seeded at a density of 1 cell / well in 96-well plates and cultured for 24 hours. The culture medium was then replaced with different samples. A blank control group (DMEM medium only) and each sample treatment group were set up, with 6 replicates per group. After 72 hours of culture, cell proliferation activity was detected by the CCK-8 assay, and the absorbance was measured at 450 nm using a microplate reader.

[0065] Formula for calculating cell proliferation rate:

[0066] ,

[0067] Experimental results showed that the cell proliferation rate of the blank control group was set at 100%. The cell proliferation rate of the treatment group in Example 1 was 168.5%±12.3%, that of the treatment group in Example 2 was 156.2%±10.8%, and that of the treatment group in Example 3 was 175.3%±14.2%. The proliferation rate of the treatment group in Comparative Example 1 was 112.5%±8.6%, that of the treatment group in Comparative Example 2 was 135.8%±9.5%, and that of the treatment group in Comparative Example 3 was 142.3%±11.2%. Statistical analysis showed that the differences between Examples 1-3 and Comparative Examples 1-3 were statistically significant (P<0.01).

[0068] The above results demonstrate that the non-denatured type II collagen ultrafine powder prepared by this invention can significantly promote chondrocyte proliferation, and the effect is significantly better than that of products prepared by traditional processes. The product with a high triple helix structure integrity rate (Example 3, 95%) showed the most significant cell proliferation-promoting effect, confirming the importance of preserving the complete triple helix structure for exerting biological functions.

[0069] Example 5: Animal Experiment Verification

[0070] A knee joint cartilage defect model was established in SD rats to verify the cartilage repair effect of the product of this invention.

[0071] Model establishment: Forty-eight healthy male SD rats, weighing 200-220g, were randomly divided into 6 groups of 8 rats each. Surgery resulted in full-thickness cartilage defects in both knee joints, with a defect diameter of 2mm and a depth of 1mm.

[0072] Experimental grouping and administration: The blank control group received 50 μL of 0.9% sodium chloride solution intra-articularly weekly. Example 1 group received 50 μL of the Example 1 product solution (10 mg / mL). Example 3 group received 50 μL of the Example 3 product solution (10 mg / mL). Comparative Example 1 group received 50 μL of the Comparative Example 1 product solution (10 mg / mL). The positive control group received 50 μL of sodium hyaluronate (10 mg / mL). The combination therapy group received 50 μL of a mixed solution of the Example 1 product and sodium hyaluronate (5 mg / mL each). Injections were given weekly for 8 weeks.

[0073] Evaluation of efficacy: Animals were euthanized after treatment, and knee joints were harvested for histological examination. The Wakitani scoring system was used to score cartilage repair, with scoring indicators including cell morphology (0-2 points), matrix staining (0-3 points), surface smoothness (0-3 points), cartilage thickness (0-2 points), and integration with host cartilage (0-2 points), for a total score of 12 points. The lower the score, the better the repair effect.

[0074] The experimental results showed that the scores were as follows: blank control group 9.8±1.2, Example 1 group 4.5±0.8, Example 3 group 3.2±0.6, Comparative Example 1 group 7.5±1.0, positive control group 5.8±0.9, and combined treatment group 2.8±0.5. Statistical analysis was performed using one-way ANOVA. The results showed that the differences between Example 1 group, Example 3 group, and combined treatment group and the blank control group were statistically significant (P<0.001). The differences between Example 1 group and Example 3 group and Comparative Example 1 group were statistically significant (P<0.01). The combined treatment group showed the best repair effect.

[0075] Histological observations showed that in the blank control group, the defect area was filled with fibrous tissue, and no hyaline cartilage regeneration was observed. In Example 1 and Example 3 groups, hyaline cartilage-like tissue regeneration was observed in the defect area, with chondrocytes arranged regularly, matrix staining close to normal cartilage, and good integration with the surrounding host cartilage. In Comparative Example 1, only a small amount of fibrocartilage formation was observed. The combined treatment group showed the most complete cartilage regeneration, with a morphology close to that of normal cartilage tissue.

[0076] The above animal experiment results confirm that the non-denatured type II collagen ultrafine powder prepared by the present invention can effectively promote the repair and regeneration of articular cartilage defects, and the repair effect is significantly better than that of traditional process products. The combined application with hyaluronic acid can further enhance the repair effect.

[0077] Example 6: Preparation of oral formulation

[0078] The non-denatured type II collagen ultrafine powder prepared in Example 1 was mixed with an appropriate amount of excipients to prepare an oral capsule formulation.

[0079] Formula composition: 200mg of non-denatured type II collagen ultrafine powder, 80mg of microcrystalline cellulose, 50mg of pregelatinized starch, 4mg of magnesium stearate, totaling 334mg / capsule.

[0080] Preparation method: Non-denatured type II collagen ultrafine powder, microcrystalline cellulose, and pregelatinized starch are mixed evenly through an 80-mesh sieve, magnesium stearate is added and mixed further, and then filled into No. 0 hard capsules to obtain an oral capsule preparation.

[0081] Quality testing: Content uniformity, disintegration time, and other indicators all meet the requirements of the Chinese Pharmacopoeia. Stability testing showed that after 12 months of storage at 25℃ / 60%RH, the content of non-denatured type II collagen remained above 95% of the labeled amount, and the triple helix structure integrity rate showed no significant decrease.

[0082] Example 7: Preparation of Intra-articular Injection Formulation

[0083] The non-denatured type II collagen ultrafine powder prepared in Example 3 was mixed with an appropriate amount of excipients to prepare a solution for intra-articular injection.

[0084] Formula composition: 10 mg / mL of non-denatured type II collagen ultrafine powder, 5 mg / mL of sodium hyaluronate, 8.5 mg / mL of sodium chloride, appropriate amount of phosphate buffer to adjust pH to 7.2-7.4, and water for injection to make up to 1 mL.

[0085] Preparation method: Under sterile conditions, non-denatured type II collagen ultrafine powder is added to sodium hyaluronate solution and gently stirred to disperse it evenly. Sodium chloride and phosphate buffer are added to adjust the osmotic pressure and pH value. The mixture is then filtered through a 0.22μm filter membrane for sterilization and dispensed into 2mL pre-filled syringes to obtain the intra-articular injection preparation.

[0086] Quality testing: pH 7.35, osmotic pressure 295 mOsm / kg, sterility test passed, bacterial endotoxin <0.25 EU / mL. Particle size distribution analysis showed that the non-denatured type II collagen ultrafine powder was uniformly dispersed in the solution, with an average particle size of 6.2 μm and a D90 of 19.5 μm.

[0087] Comprehensive Mechanism Analysis: The excellent cartilage repair effect of the non-denatured type II collagen ultrafine powder of this invention can be explained by the following aspects. First, the ultra-low temperature ball milling technology keeps the collagen molecules in a glassy state, effectively avoiding mechanical damage to the triple helix structure, ensuring molecular integrity, and allowing the product to retain complete chondrocyte binding domains and signal transduction sites. Second, the ultrafine powder form significantly increases the specific surface area, enhancing the interaction efficiency with chondrocytes and facilitating the effective enrichment of active ingredients in cartilage tissue. Third, the preserved triple helix structure can be recognized by integrin receptors on the surface of chondrocytes, activating intracellular signaling pathways and promoting cartilage-specific gene expression and matrix protein synthesis. Finally, the non-denatured type II collagen slowly degrades in the cartilage tissue microenvironment, releasing active peptides and continuously exerting cartilage protection and repair effects. The synergistic effect of these multiple mechanisms gives the product of this invention significant advantages in cartilage repair applications.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A non-denatured type II collagen ultrafine powder with targeted growth and structural repair effects on cartilage tissue, characterized in that, The non-denatured type II collagen ultrafine powder retains the complete triple helix structure and cartilage-specific active epitopes. The average particle size of the ultrafine powder is 0.5-50 μm, the triple helix structure integrity of the type II collagen is not less than 85%, the hydroxyproline content in the ultrafine powder is 8%-14%, the glycosaminoglycan content is 0.1%-3%, the thermal denaturation temperature of the ultrafine powder is 38-48℃, and the type II collagen retains the chondrocyte binding domains CB10 and CB11 and the matrix metalloproteinase cleavage site (MMP) characteristic sequences.

2. The non-denatured type II collagen ultrafine powder according to claim 1, characterized in that, The average particle size of the ultrafine powder is 1-20 μm, the D90 particle size is no more than 45 μm, the integrity rate of the triple helix structure is not less than 90%, the hydroxyproline content is 10%-13%, and the thermal denaturation temperature is 40-45℃.

3. The non-denatured type II collagen ultrafine powder according to claim 1 or 2, characterized in that, The circular dichroism spectroscopy of the ultrafine powder shows a positive peak at 221 nm and a negative peak at 197 nm, with a molar ellipticity at 221 nm. for - deg·cm 2 ·dmol -1 The solubility of the ultrafine powder in simulated joint fluid is 0.5-5 mg / mL, and the ultrafine powder is derived from aquatic animal cartilage, avian cartilage, or mammalian cartilage.

4. The non-denatured type II collagen ultrafine powder according to any one of claims 1-3, characterized in that, The ultrafine powder also contains cartilage matrix proteoglycan, the mass ratio of the proteoglycan to type II collagen is 0.01-0.5:1, the bulk density of the ultrafine powder is 0.15-0.45 g / cm³, and the moisture content of the ultrafine powder does not exceed 8%.

5. The method for preparing non-denatured type II collagen ultrafine powder according to any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Raw material pretreatment: After cleaning, the animal cartilage tissue is placed in a 0.5%-2% sodium chloride solution and soaked at 2-8℃ for 8-24 hours to remove non-collagen components. Step 2: Enzymatic extraction: The pretreated cartilage tissue is placed in an acidic solution containing pepsin and subjected to time-limited enzymatic hydrolysis at 15-25℃ for 12-72 hours, with an enzyme activity of 1000-5000 U / g of raw material. The pH of the acidic solution is 1.5-3.

5. Step 3: Fractional purification: The enzymatic hydrolysis product is purified using a combined salting-out and dialysis process. The salting-out agent is sodium chloride, with a salting-out concentration of 0.8-2.5M, and the dialysis molecular weight cutoff is 100-300 kDa. Step 4: Ultrafine grinding: After the purified product is dried at low temperature, it is ultrafine ground using ultra-low temperature ball milling technology. The ball milling temperature is -80℃ to -40℃ and the ball milling time is 2-8h to obtain non-denatured type II collagen ultrafine powder.

6. The method for preparing a bioactive product according to claim 5, characterized in that, In step two, the enzymatic hydrolysis temperature is 18-22℃, the enzymatic hydrolysis time is 24-48h, the pH is 2.0-3.0, and the pepsin activity is 2000-4000U / g raw material. In step three, the salting-out concentration is 1.0-2.0M, the dialysis molecular weight cutoff is 150-250kDa, and in step four, the ball milling temperature is -70℃ to -50℃.

7. The method for preparing a bioactive product according to claim 5 or 6, characterized in that, Step four is followed by an activity stabilization step: the ultrafine powder is mixed with a trehalose solution of 0.01%-1% by mass and then freeze-dried, or packaged with inert gas replacement, or an antioxidant of 0.005%-0.1% by mass is added, wherein the antioxidant is selected from one or more of ascorbic acid, ascorbyl palmitate or tocopherol.

8. The use of the non-denatured type II collagen ultrafine powder according to any one of claims 1-4 or the non-denatured type II collagen ultrafine powder prepared by the method according to any one of claims 5-7 in the preparation of cartilage repair materials.

9. The application according to claim 8, characterized in that, The cartilage repair material is an oral preparation, an intra-articular injection preparation, or a cartilage tissue engineering scaffold. The oral preparation contains 10-500 mg of non-denatured type II collagen ultrafine powder per dose unit, and the intra-articular injection preparation contains 0.5-20 mg / mL of non-denatured type II collagen ultrafine powder.

10. The application according to claim 8 or 9, characterized in that, The cartilage repair material is used for the repair and treatment of cartilage damage caused by osteoarthritis and rheumatoid arthritis, sports-induced cartilage defects, or degenerative cartilage diseases.

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