Hyaluronic acid cartilage repair scaffold, preparation method and application thereof

Abstract

The application provides a hyaluronic acid cartilage repair scaffold and a preparation method and application thereof, relates to the technical field of biomedical materials, and the scaffold comprises a dense surface layer and a loose bottom layer; the dense surface layer is composed of a three-dimensional sponge formed by cross-linking of collagen and hyaluronic acid, and the mass ratio of the collagen and the hyaluronic acid is 10:1-2:1; and the loose bottom layer is composed of collagen. The surface layer of the cartilage repair scaffold forms a stable hydration film in a liquid environment; the long-term stability of the lubricating function in the whole key initial repair stage is ensured, the problem of quick loss of lubricating components caused by simple coating is effectively solved; the cartilage repair scaffold has good mechanical properties and is suitable for current cartilage repair surgery (such as ACI / MACI). The technical problems that the cartilage repair scaffold in the prior art cannot maintain long-term lubricating effect or maintain lubrication while having sufficient mechanical load are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medical materials, in particular to a hyaluronic acid cartilage repair scaffold and a preparation method and application thereof. BACKGROUND

[0002] Articular cartilage injury is one of the orthopedic diseases with very high incidence and prevalence. Articular cartilage injury causes degenerative changes of articular cartilage, such as osteoarthritis, which can lead to disability, pain during joint movement and deformation of the bone joint. Articular cartilage injury is usually irreversible, especially when the defect diameter exceeds 4 mm, self-healing cannot be achieved.

[0003] Tissue engineering scaffold is a promising strategy for cartilage repair, which constructs a microenvironment conducive to cell adhesion, proliferation and differentiation through biomimetic materials, and promotes cartilage regeneration. Among them, collagen membrane scaffold and natural inorganic calcium carbonate scaffold show certain biological activity in cartilage repair, but they have significant defects in surface lubrication performance, which seriously restricts their clinical effect and application prospect. Natural articular cartilage has excellent lubrication properties, with a friction coefficient as low as 0.001~0.03, while the friction coefficient of existing collagen scaffolds is usually as high as 0.1~0.5, with a difference of 1~2 orders of magnitude. This deficiency in lubrication performance leads to a series of problems after implantation: the interface between the scaffold surface and the contralateral cartilage is accelerated by the high friction coefficient, not only affecting the structural integrity of the scaffold itself, but also leading to secondary damage to the surrounding healthy cartilage tissue; the shear stress and wear particles generated by high friction can activate the immune system, trigger synovial inflammation and foreign body reaction, and hinder the repair process; excessive friction also seriously affects the stable integration between the scaffold and the host tissue, increasing the risk of repair failure.

[0004] At present, in order to improve the surface lubricity, the membrane-shaped cartilage repair scaffold mainly adopts three kinds of technical solutions of surface coating modification, material composite modification strategy and structure bionic design, wherein the surface coating modification is the most widely used method, and the lubricating molecules such as hyaluronic acid, heparin and phospholipid are usually fixed on the surface of the scaffold through physical adsorption or chemical cross-linking. Among them, hyaluronic acid is the most commonly used because it is similar to the composition of synovial fluid, and a 5-20 micrometer thick lubricating layer is formed by using a carbodiimide cross-linking agent to combine it with the active groups on the surface of collagen. However, the adhesion between the coating and the substrate is weak, and the coating is easy to fall off under the continuous washing of synovial fluid and mechanical load, so it is difficult to maintain long-term lubrication effect. The material composite modification strategy directly blends synthetic lubricating components such as polyvinyl alcohol and polyethylene glycol with collagen solution to prepare a composite membrane material through electrospinning or blending film. Although this method can improve the overall lubricity of the material, it will change the porous structure of the collagen membrane, resulting in a decrease in pore size distribution and pore connectivity, affecting cell migration and nutrient transport, and most synthetic components have the problem of poor biodegradability. The structure bionic design simulates the structure of the natural cartilage surface layer to enhance the lubrication performance, such as using electrospinning technology to construct an oriented fiber array, or manufacturing microstructures such as micro-dimples on the surface of the membrane to promote synovial fluid retention. This method can improve the hydrodynamic lubrication effect to some extent, but it is still difficult to accurately replicate the anisotropic characteristics of the natural cartilage surface, and the manufacturing process is complex and the cost is high, which limits the clinical application.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] One of the purposes of the present application is to provide a hyaluronic acid cartilage repair scaffold to solve the technical problems that the existing cartilage repair scaffold cannot maintain long-term lubrication effect or has sufficient mechanical load while maintaining lubrication.

[0007] The second purpose of the present application is to provide a preparation method of the hyaluronic acid cartilage repair scaffold.

[0008] The third purpose of the present application is to provide the application of the hyaluronic acid cartilage repair scaffold or the hyaluronic acid cartilage repair scaffold prepared by the above preparation method in cartilage tissue regeneration or cartilage defect repair materials.

[0009] In order to achieve the above purposes of the present application, the following technical solutions are adopted:

[0010] In a first aspect, the present application provides a hyaluronic acid cartilage repair scaffold, comprising a dense surface layer and a loose bottom layer; the dense surface layer is composed of a three-dimensional sponge body formed by cross-linking collagen and hyaluronic acid, and the mass ratio of collagen to hyaluronic acid in the dense surface layer is 10:1-2:1; the loose bottom layer is composed of collagen.

[0011] Further, the porosity of the dense surface layer is >70%, and the porosity of the loose bottom layer is >90%.

[0012] Further, the thickness of the dense surface layer is 0.2-1 mm, and the thickness of the loose bottom layer is 1-3 mm.

[0013] Further, the collagen includes type I collagen.

[0014] The molecular weight of the hyaluronic acid is 0.5-2.5 MDa.

[0015] In a second aspect, the present application provides a preparation method of a hyaluronic acid cartilage repair scaffold, comprising the following steps:

[0016] A. A hyaluronic acid solution is added dropwise to a collagen slurry according to the formula amount, and physical cross-linking is performed to form a collagen-hyaluronic acid cross-linked blend;

[0017] B. The collagen-hyaluronic acid cross-linked blend is subjected to first pre-freezing, freeze-drying, and compression to form a dense surface layer.

[0018] C. Collagen slurry is injected onto the dense surface layer, and a loose bottom layer is formed after second pre-freezing and freeze-drying, thereby obtaining a hyaluronic acid cartilage repair scaffold.

[0019] Further, the concentration of collagen in the collagen slurry is 3-20 mg / mL.

[0020] The concentration of the hyaluronic acid solution is 0.5-1% w / v.

[0021] Further, the addition of the hyaluronic acid solution to the collagen slurry includes adding the hyaluronic acid solution dropwise to the collagen slurry while stirring.

[0022] The stirring speed is 50-200 rpm.

[0023] The stirring time is 5-20 min.

[0024] The stirring temperature is 2-10℃.

[0025] The physical cross-linking includes ultrasonic cross-linking or enzyme cross-linking.

[0026] The ultrasonic cross-linking includes intermittent ultrasonic treatment for 1-5 min.

[0027] The intermittent ultrasonic treatment includes stopping ultrasonic treatment for 2-5 s after ultrasonic treatment for 2-5 s.

[0028] The power of the ultrasonic treatment is 100-500 W.

[0029] Further, the temperature of the first pre-freezing is -80~ -20℃, preferably -80℃, and the time of the first pre-freezing is ≥ 6h.

[0030] The temperature of the second pre-freezing is -15~ -25℃, and the time of the second pre-freezing is ≥ 24h.

[0031] Further, the compression includes compression from 1±0.5mm to 0.2~1mm.

[0032] In a third aspect, the application provides the use of the hyaluronic acid cartilage repair scaffold or the hyaluronic acid cartilage repair scaffold prepared by the preparation method in the above in cartilage tissue regeneration or cartilage defect repair materials.

[0033] The hyaluronic acid cartilage repair scaffold provided by the application introduces hyaluronic acid as a key component, constructs a three-dimensional sponge structure of collagen-hyaluronic acid cross-linking, and forms a stable hydration film on the surface layer of the cartilage repair scaffold in a liquid environment. The dynamic friction coefficient is reduced by more than 50% compared with traditional pure collagen scaffolds through tribological test verification, which provides a more optimal mechanical microenvironment for cartilage regeneration. Since the hyaluronic acid is anchored in the cartilage repair scaffold, as the cartilage repair scaffold biodegrades, a stable hydration film can always be formed on the surface layer, and the scaffold can still maintain more than 70% of the hyaluronic acid content and low friction characteristics within a test period of up to 28 days, ensuring the persistence and stability of the lubrication function in the entire critical early repair stage, matching the functional life and tissue regeneration cycle, and effectively solving the problem of rapid loss of lubricating components caused by simple coating. At the same time, the structure of the dense surface layer and the loose bottom layer forms a gradient difference in pore size, and the formation of the vaginal transparent cartilage-like tissue enables the cartilage repair scaffold to have good mechanical properties, wherein the bottom layer can effectively support the adhesion, proliferation and migration of chondrocytes; the excellent lubricating performance of the surface layer exhibits extremely low friction coefficient in the tribological test of simulated joint movement, which can effectively protect the contralateral cartilage and adapt to current cartilage repair surgery (such as ACI / MACI). The technical problems that the cartilage repair scaffold in the prior art cannot maintain long-term lubrication effect or maintain lubrication while having sufficient mechanical load are solved. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0035] Figure 1 The SEM image of the hyaluronic acid cartilage repair scaffold provided for Example 1 of the application. DETAILED DESCRIPTION

[0036] Unless otherwise defined, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meanings of the terms will be clear in view of the description, given the benefit of the present disclosure. For any potentially ambiguous

[0037] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0038] The technical solutions of the present application will be described clearly and completely below in connection with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0039] In one aspect of the present application, a hyaluronic acid cartilage repair scaffold is provided, which comprises a dense surface layer and a loose bottom layer.

[0040] The dense surface layer is composed of a three-dimensional sponge formed by cross-linking collagen and hyaluronic acid, and the mass ratio of collagen to hyaluronic acid in the dense surface layer is 10:1 to 2:1; the loose bottom layer is composed of collagen.

[0041] By introducing hyaluronic acid as a key component, a three-dimensional sponge structure cross-linked with collagen and hyaluronic acid was constructed. This allows the cartilage repair scaffold to form a stable hydration film on its surface in a liquid environment. Tribological testing verified that its dynamic friction coefficient was reduced by more than 50% compared to traditional pure collagen scaffolds, providing a superior mechanical microenvironment for cartilage regeneration. Because the hyaluronic acid is anchored within the cartilage repair scaffold, a stable hydration film can always be formed on its surface as the scaffold biodegrades. The scaffold maintained a hyaluronic acid content of over 70% and low friction characteristics throughout a 28-day testing period, ensuring optimal performance throughout the entire process. The sustained and stable lubrication function in the early stages of cartilage repair achieves a match between functional lifespan and tissue regeneration cycle, effectively solving the problem of rapid loss of lubricating components caused by simple coating. Simultaneously, the dense surface layer and loose sublayer structure create a gradient difference in pore size, leading to the formation of vaginal hyaline cartilage-like tissue, giving the cartilage repair scaffold excellent mechanical properties. The sublayer effectively supports the adhesion, proliferation, and migration of chondrocytes. The excellent lubrication performance of the surface layer exhibits an extremely low coefficient of friction in tribological tests simulating joint movement, effectively protecting the contralateral cartilage and making it suitable for current cartilage repair surgeries (such as ACI / MACI). This solves the technical problem in existing technologies where cartilage repair scaffolds cannot maintain long-term lubrication or simultaneously provide sufficient mechanical load.

[0042] The mass ratio of collagen to hyaluronic acid can be, but is not limited to, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 or 2:1, or any ratio between 10:1 and 2:1, preferably between 5:1 and 4:1.

[0043] A collagen-hyaluronic acid cross-linked blend is injected into the dense surface layer, which simultaneously penetrates into the interior of the dense surface layer. In articular cartilage, the ratio of collagen to hyaluronic acid is approximately 50:1. In some specific embodiments, the ratio of collagen content in the dense surface layer to collagen content in the loose basal layer is approximately 2:3.

[0044] In some specific embodiments, the porosity of the dense surface layer is >70%, and the porosity of the loose sublayer is >90%.

[0045] In some specific embodiments, the thickness of the dense surface layer is 0.2~1mm; the thickness of the loose underlayer is 1~3mm.

[0046] The thickness of the dense surface layer can be, but is not limited to, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm or 1 mm, or any value between 0.2 and 1 mm.

[0047] The thickness of the loose bottom layer can be, but is not limited to, 1 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, or 3 mm, or any value between 1 mm and 3 mm.

[0048] In some specific embodiments, the collagen comprises collagen type I; and the hyaluronic acid has a molecular weight of 0.5-2.5 MDa.

[0049] According to another aspect of the present application, a preparation method of the hyaluronic acid cartilage repair scaffold is also provided, comprising the following steps:

[0050] A. The hyaluronic acid solution is added dropwise into the collagen slurry according to the formula amount, and physical cross-linking is performed to form a collagen-hyaluronic acid cross-linked blend;

[0051] B. The collagen-hyaluronic acid cross-linked blend is subjected to first pre-freezing, freeze-drying, and compression to form a dense surface layer;

[0052] C. The collagen slurry is injected onto the dense surface layer, and after second pre-freezing and freeze-drying, a loose bottom layer is formed, thereby obtaining the hyaluronic acid cartilage repair scaffold.

[0053] The physical cross-linking is achieved by ultrasonic treatment, which promotes physical entanglement and hydrogen bonding of molecular chains through cavitation effect, integrates collagen and hyaluronic acid, and obtains a stable three-dimensional structure with uniform structure. After freeze-drying, a three-dimensional sponge body is obtained, which completely eliminates the hidden danger of chemical toxicity, and improves the lubrication performance and ensures the two key performance indicators of biological safety. The structure of the dense surface layer and the loose bottom layer makes the cartilage repair scaffold not only structurally bionic, but also functionally bionic, achieving long-term maintenance of lubrication effect while having mechanical properties.

[0054] In order to improve the cross-linking effect, in some specific embodiments, the concentration of collagen in the collagen slurry is 3-20 mg / mL; and the concentration of the hyaluronic acid solution is 0.5-1% w / v.

[0055] The concentration of collagen in the collagen slurry can be, but is not limited to, 3 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, or 20 mg / mL, or any value between 3 mg / mL and 20 mg / mL, and is preferably 5-10 mg / mL.

[0056] In order to uniformly mix the collagen slurry and the hyaluronic acid, in some specific embodiments, the adding dropwise of the hyaluronic acid solution into the collagen slurry comprises adding dropwise the hyaluronic acid solution into the collagen slurry while stirring.

[0057] In some specific embodiments, the stirring speed is 50-200 rpm; in some specific embodiments, the stirring time is 5-20 min; in some specific embodiments, the stirring temperature is 2-10℃.

[0058] In some specific embodiments, the physical cross-linking comprises ultrasonic cross-linking or enzymatic cross-linking; in some specific embodiments, the ultrasonic cross-linking comprises intermittent ultrasonic treatment for 1-5 min; the hyaluronic acid is physically cross-linked with collagen. In some specific embodiments, the intermittent ultrasonic treatment comprises stopping the ultrasonic treatment for 2-5 s after each ultrasonic treatment for 2-5 s; in some specific embodiments, the power of the ultrasonic treatment is 100-500 W.

[0059] In order to form the dense surface layer, in some specific embodiments, the temperature of the first pre-freezing is -80--20℃, preferably -80℃, and the time of the first pre-freezing is ≥6 h; in order to form the loose bottom layer, in some specific embodiments, the temperature of the second pre-freezing is -15--25℃, and the time of the second pre-freezing is ≥24 h; the slow pre-freezing process allows the ice crystals to grow slowly, thereby forming large-sized ice crystals in the collagen solution in the bottom layer, which, after subsequent sublimation, leave a loose and interconnected large-pore structure, thereby forming the loose bottom layer. Through the sequential freezing process and the physical compounding, the integrated formation of the functional gradient can be achieved without using any adhesive.

[0060] In some specific embodiments, the freeze-drying conditions are not limited, and a freeze-drying machine can be used to achieve freeze-drying.

[0061] In some specific embodiments, the compression comprises compression from 1±0.5 mm to 0.2-1 mm.

[0062] According to another aspect of the present application, there is further provided the use of the hyaluronic acid cartilage repair scaffold as described above or prepared by the preparation method as described above in cartilage tissue regeneration or cartilage defect repair materials.

[0063] The present application is further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to the existing methods or directly purchased from the market.

[0064] Example 1

[0065] A hyaluronic acid cartilage repair scaffold, which is composed of a three-dimensional sponge body cross-linked from type I collagen and hyaluronic acid with a molecular weight of 0.5 MDa, and the mass ratio of collagen to hyaluronic acid is 2:1; the scaffold comprises a dense surface layer and a loose bottom layer, the thickness of the dense surface layer is 0.5 mm; and the thickness of the loose bottom layer is 1.5 mm.

[0066] The preparation is carried out specifically according to the following steps:

[0067] 1. Preparation of collagen slurry

[0068] Type I collagen extracted from bovine hide is dissolved in a 0.5 mol / L dilute acetic acid solution; slowly stirred at 4°C for 36 hours to avoid denaturation of collagen molecules and form a homogeneous collagen suspension;

[0069] The collagen concentration is adjusted to 8 mg / mL. A 0.1M NaOH solution is slowly added to the solution to neutralize the pH of the solution to 7.0-7.4, and stirred until uniform.

[0070] 2. Cross-linking of collagen-hyaluronic acid

[0071] Take the type I collagen slurry prepared in step 1, prepare a 1% (w / v) hyaluronic acid solution (HA solution), slowly add the HA solution to the type I collagen slurry under ice bath and low-speed magnetic stirring (stirring speed is 150 rpm, stirring time is 15 min) to obtain a Col-HA blended solution. The Col-HA blended solution is placed in an ice water bath environment, and an ultrasonic cell disruptor is used to treat the solution with a power of 300W for 5 minutes in an intermittent mode (working for 2 seconds, intermittent for 2 seconds). The cavitation effect and mechanical shear force generated by the ultrasonic wave can break the high molecular chain, generate free radicals, and promote the entanglement of collagen fibers and HA molecular chains. A more stable and uniform three-dimensional network is formed through physical entanglement, hydrogen bonding enhancement, and possible slight cross-linking induced by free radicals.

[0072] 3. Preparation of dense surface layer

[0073] Pour the treated cross-linked blended solution into a freeze-drying mold with a thickness of 1 mm, place it in liquid nitrogen for rapid freezing for more than 6 hours, and quickly transfer the frozen sample to a freeze dryer for processing until the sample is completely dried. The obtained collagen membrane is compressed to 0.5 mm for standby.

[0074] 4. Compound of loose bottom layer

[0075] The compressed dense surface layer is placed in a freeze-drying mold, and the type I collagen slurry prepared in step 1 is injected to a thickness of 2 mm. The mold is placed in a -20°C refrigerator for slow pre-freezing for 24 hours, and then placed in a freeze dryer for processing until the sample is completely dried. After demolding, it is stored at 2-8°C.

[0076] Example 2

[0077] The difference from Example 1 is that the scaffold consists of a three-dimensional sponge formed by cross-linking of type I collagen and hyaluronic acid with a molecular weight of 0.8 MDa, the mass ratio of collagen and hyaluronic acid being 20:3.

[0078] Example 3

[0079] The difference from Example 1 is that the scaffold consists of a three-dimensional sponge formed by cross-linking of type I collagen and hyaluronic acid with a molecular weight of 1 MDa, the mass ratio of collagen and hyaluronic acid being 5:1.

[0080] Example 4

[0081] The difference from Example 1 is that the scaffold consists of a three-dimensional sponge formed by cross-linking of type I collagen and hyaluronic acid with a molecular weight of 1.5 MDa, the mass ratio of collagen and hyaluronic acid being 10:3.

[0082] Example 5

[0083] The difference from Example 1 is that the scaffold consists of a three-dimensional sponge formed by cross-linking of type I collagen and hyaluronic acid with a molecular weight of 1.8 MDa, the mass ratio of collagen and hyaluronic acid being 5:2.

[0084] Example 6

[0085] The difference from Example 1 is that the scaffold consists of a three-dimensional sponge formed by cross-linking of type I collagen and hyaluronic acid with a molecular weight of 2 MDa, the mass ratio of collagen and hyaluronic acid being 5:2.

[0086] Example 7

[0087] The difference from Example 1 is that the scaffold consists of a three-dimensional sponge formed by cross-linking of type I collagen and hyaluronic acid with a molecular weight of 2.5 MDa, the mass ratio of collagen and hyaluronic acid being 2:1.

[0088] Comparative Example 1

[0089] The difference from Example 1 is that the scaffold consists of type I collagen, the scaffold including a dense surface layer and a loose bottom layer, the thickness of the dense surface layer being 0.5 mm; the thickness of the loose bottom layer being 1.5 mm.

[0090] The preparation is carried out according to the following steps:

[0091] 1. Preparation of collagen slurry

[0092] Type I collagen extracted from cowhide is dissolved in a 0.5 mol / L dilute acetic acid solution; slowly stirred at 4°C for 36 hours to avoid denaturation of collagen molecules and form a homogeneous collagen suspension;

[0093] The collagen concentration was adjusted to 8 mg / mL. A 0.1M NaOH solution was slowly added to the solution, and the pH of the solution was neutralized to 7.0-7.4, and stirred until uniform.

[0094] 2. Preparation of dense surface layer

[0095] The type I collagen slurry prepared in step 1 was poured into a freeze-drying mold with a thickness of 1 mm, and was rapidly frozen in liquid nitrogen for more than 6 h. The frozen sample was quickly transferred to a freeze dryer for processing until the sample was completely dried. The obtained collagen membrane was compressed to 0.5 mm for standby.

[0096] 4. Compound of loose bottom layer

[0097] The compressed dense surface layer was placed in a freeze-drying mold, and type I collagen slurry was injected to a thickness of 1-3 mm. The mold was placed in a -20°C refrigerator for slow pre-freezing for 24 h, and then placed in a freeze dryer for processing until the sample was completely dried, and was demolded and taken out. It was stored at 2-8°C.

[0098] Comparative Example 2

[0099] The difference from Example 4 is that the mass ratio of collagen and hyaluronic acid is 12:1.

[0100] Comparative Example 3

[0101] The difference from Example 4 is that the mass ratio of collagen and hyaluronic acid is 2:1.5.

[0102] Comparative Example 4

[0103] The difference from Example 4 is that the scaffold only includes a dense surface layer, and the thickness of the dense surface layer is 0.5 mm.

[0104] Test 1: Hygroscopicity determination

[0105] The dried samples of Examples 1-7 and Comparative Examples 1-4 were taken, and were determined according to YY / T 1511-2017 6.3 Liquid Absorption Method in Collagen Sponge, and the results are shown in Table 1.

[0106] Table 1

[0107]

[0108] The results show that the moisture absorption of each example does not change significantly compared with Comparative Example 1, indicating that the three-dimensional sponge formed by cross-linking collagen and hyaluronic acid in Examples 1-7 does not affect the moisture absorption of the scaffold. Compared with Example 4, the moisture absorption thickness of Comparative Example 2 increases, and the moisture absorption thickness of Comparative Example 3 decreases, indicating that the collagen and hyaluronic acid in the proportion range of Examples 1-7 can maintain the moisture absorption of the scaffold. Comparative Example 4 only significantly reduces the moisture absorption, indicating that the dense surface layer and the loose bottom layer cooperate to maintain the moisture absorption.

[0109] Test 2 Lubricity Determination

[0110] The samples of Examples 1-7 and Comparative Examples 1-4 after drying were taken, and the ball-on-disc friction and wear test machine was used to determine the lubricity of the product according to the principle of ball-on-disc friction test described in ASTM F732 standard.

[0111] 1. The scaffold sample (experimental group) after sufficient hydration was fixed, and the zirconium monoxide counter-rotating ball formed a friction pair in a simulated joint synovial fluid environment. The instrument automatically recorded the dynamic friction force and calculated the dynamic friction coefficient. The results are shown in Table 2.

[0112] Table 2

[0113]

[0114] The data shows that the friction coefficient of Examples 1-7 is significantly reduced compared with Comparative Example 1. Compared with Example 4, the friction coefficient of Comparative Example 2 is significantly increased, indicating that too low content of hyaluronic acid cannot reduce the friction coefficient. In Comparative Example 3, too high content of hyaluronic acid cannot further significantly reduce the friction coefficient, indicating that within the limited content range of hyaluronic acid in the present application, the friction coefficient of the scaffold can be significantly reduced. Although the friction coefficient of Comparative Example 4 is reduced, it loses the moisture absorption performance.

[0115] 2. Hyaluronic Acid Content Determination

[0116] The scaffold samples were incubated in phosphate buffer solution (containing 0.02% NaN3) at pH 7.4 at 37°C in a constant temperature shaker for 28 days, and the buffer solution was replaced regularly. At the predetermined time points (4, 7, 14, 21, 28 days), samples were taken for quantitative analysis of the remaining hyaluronic acid content by enzyme-linked immunosorbent assay. By calculating the percentage of hyaluronic acid content at each time point relative to the initial content, the maintenance rate can be evaluated, and the results are shown in Table 3. The data show that the scaffolds of Examples 3-7 can still maintain more than 70% of the hyaluronic acid content during the test period of up to 28 days, and Examples 1-2 can maintain more than 50%. Compared with Example 4, the hyaluronic acid content of Comparative Examples 2 and 3 is significantly lower than that of Example 4, indicating that reducing the hyaluronic acid content directly affects the release efficiency of hyaluronic acid, and excessive addition of hyaluronic acid does not significantly increase the residual amount of hyaluronic acid, but shows a decreasing trend; Comparative Example 4 is comparable to Example 4 when the test reaches 28 days, indicating that the loose bottom layer does not affect the release of hyaluronic acid in the dense surface layer.

[0117] Table 3

[0118]

[0119] Test 3 Tensile breaking force determination

[0120] The dried samples of Examples 1-7 and Comparative Examples 1-4 were cut into strip-shaped test samples with a width of 9 mm and a length of 30 mm. The two ends of the test sample were fixed on the clamps of the testing machine, and stretched at a stable speed of 10 mm / min until the test sample broke. The load force at the time of breaking was recorded, and the test results were averaged. The results are shown in Table 4.

[0121] Table 4

[0122]

[0123] The data show that compared with Comparative Example 1, the mechanical properties of Examples 1-7 are improved. Compared with Example 4, the mechanical properties of Comparative Example 2 are significantly reduced due to the excessively low content of hyaluronic acid, and the mechanical properties of Comparative Example 3 are also reduced due to the excessively high content of hyaluronic acid. Controlling the content of hyaluronic acid on the surface plays a key role in improving the mechanical properties. Comparative Example 4 is a single-layer structure, and its mechanical properties are significantly reduced.

[0124] Test 4 Cytotoxicity determination

[0125] The samples after drying of Examples 1-7 and Comparative Example 1 were detected for cytotoxicity of the scaffold material by MTT method. First, an extract solution of the scaffold material was prepared, that is, 2.0 g of the sterile double-layered osteochondral repair scaffold material prepared in the above examples and subjected to electron beam radiation sterilization was selected, 20 mL of DMEM complete culture solution was added, and the mixture was extracted in a 37°C constant temperature box for 72±2 h to prepare a scaffold extract solution of 100 mg / mL. The hBMSC cells were cultured at 37°C and under a CO2 concentration of 5.0%. The MTT test method in GB / T16886.5 was used for the test, the absorbance was measured at 450 nm by using an enzyme-labeled instrument, and the relative proliferation rate of the hBMSC cells was calculated, and the results are shown in Table 5.

[0126] Table 5

[0127]

[0128] The results show that the cell growth rate of Examples 1-7 has no significant change compared with Comparative Example 1, that is, the introduction of medium and high molecular weight hyaluronic acid can interact with cell surface receptors (such as CD44) to maintain the stable state of the cells and does not produce cytotoxicity, indicating that the material has excellent biological safety.

[0129] Test 5

[0130] The sample after drying of Example 1 was observed under a scanning electron microscope, and the internal pore size of the sample was compared, as shown in Table 6. Figure 1

[0131] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.​

Claims

1. A hyaluronic acid cartilage repair scaffold, characterized in that, The scaffold comprises a dense outer layer and a porous inner layer; The dense surface layer is composed of a three-dimensional sponge formed by cross-linking of collagen and hyaluronic acid, and the mass ratio of collagen to hyaluronic acid in the dense surface layer is 10:1 to 2:

1. The loose underlying layer is composed of collagen; The molecular weight of the hyaluronic acid is 1~2.5 MDa.

2. The hyaluronic acid cartilage repair scaffold according to claim 1, characterized in that, The porosity of the dense surface layer is >70%, and the porosity of the loose sublayer is >90%.

3. The hyaluronic acid cartilage repair scaffold according to claim 1, characterized in that, The thickness of the dense surface layer is 0.2~1mm; the thickness of the loose bottom layer is 1~3mm.

4. The hyaluronic acid cartilage repair scaffold according to any one of claims 1 to 3, characterized in that, The collagen includes type I collagen.

5. The method for preparing the hyaluronic acid cartilage repair scaffold according to any one of claims 1 to 4, characterized in that, Includes the following steps: A. Add the hyaluronic acid solution dropwise to the collagen slurry according to the formula, and then perform ultrasonic cross-linking to form a collagen-hyaluronic acid cross-linked mixture; the ultrasonic cross-linking includes intermittent ultrasonic treatment for 1-5 minutes; the intermittent ultrasonic treatment includes stopping the ultrasonic treatment for 2-5 seconds after each ultrasonic treatment; the power of the ultrasonic treatment is 100-500W; B. The collagen-hyaluronic acid cross-linked blend solution is subjected to a first pre-freezing, freeze-drying and compression to form a dense surface layer; the temperature of the first pre-freezing is -80~-20℃, and the first pre-freezing time is ≥6h; C. Inject collagen slurry into the dense surface layer, and after a second pre-freezing and freeze-drying, a loose bottom layer is formed, thus obtaining the hyaluronic acid cartilage repair scaffold; the temperature of the second pre-freezing is -15~-25℃, and the time of the second pre-freezing is ≥24h.

6. The preparation method according to claim 5, characterized in that, The collagen concentration in the collagen slurry is 3~20 mg / mL; the concentration of the hyaluronic acid solution is 0.5~1% w / v.

7. The preparation method according to claim 5, characterized in that, The step of adding hyaluronic acid solution dropwise to collagen slurry includes adding hyaluronic acid solution dropwise to collagen slurry while stirring; The stirring speed is 50-200 rpm; The stirring time is 5~20 minutes; The stirring temperature is 2~10℃.

8. The preparation method according to claim 5, characterized in that, The compression includes compressing from 1±0.5mm to 0.2~1mm.

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