Cartilage collagen membrane, preparation method thereof, culture medium and application thereof

Abstract

The present application relates to a kind of cartilage collagen membrane and its preparation method, culture medium and application, belong to biological material technical field, at least one of the problems such as insufficient mechanical strength of existing cartilage regeneration cell repair material, low activity and migration efficiency of chondrocyte is solved.The preparation method includes: S1, digestion and culture are carried out to cartilage fragment, and obtain chondrocyte suspension;S2, chondrocyte suspension is induced and cultured in stages, including three stages, the first stage culture medium includes: DMEM low sugar base culture medium, GlutaMAX, non-essential amino acid, penicillin, ITS, proline, ascorbic acid and serum;Second stage culture medium is added dexamethasone and transforming growth factor on the basis of first stage culture medium;Third stage culture medium is removed transforming growth factor on the basis of second stage culture medium.The cartilage collagen membrane of the present application has good mechanical support, biological activity and biological function of promoting cell migration.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a cartilage collagen membrane, its preparation method, culture medium, and applications. Background Technology

[0002] Articular cartilage tissue, when injured or degenerative, has extremely limited natural repair capabilities due to its lack of blood vessels, nerves, and cells, often leading to pain, functional impairment, and even irreversible joint degeneration. While traditional microfracture techniques can induce bone marrow-derived mesenchymal stem cells to fill the defect area, the resulting new tissue is often predominantly fibrocartilage, with significantly different mechanical and biological properties compared to native hyaline cartilage. Long-term follow-up shows that joint function is difficult to maintain. The emergence of first- and second-generation autologous chondrocyte implantation (ACI) techniques has filled the gaps in early repair methods.

[0003] Currently, second-generation matrix-induced autologous chondrocyte implantation (MACI) technology utilizes a three-dimensional biodegradable scaffold (such as type I collagen or polylactic-co-glycolic acid copolymer membrane) to uniformly seed in vitro expanded autologous chondrocytes. During the procedure, the scaffold-cell complex is cut into appropriate shapes and directly attached to the cartilage defect surface, then fixed with bio-adhesive or sutures. Numerous clinical studies have shown that MCI is superior to traditional microfracture techniques in terms of histological and functional indicators of cartilage regeneration, and its safety and durability have also been confirmed. However, MACI still has the following limitations: the application of MCI technology requires the assistance of animal collagen or chemically synthesized scaffolds, and the introduction of exogenous scaffolds carries certain risks of disease transmission and biosafety, potentially triggering inflammatory responses or affecting the integration of newly formed cartilage. Furthermore, the mechanical strength of the scaffold and its degradation rate in vivo are not perfectly matched with the rate of new extracellular matrix (ECM) generation, potentially leading to insufficient mechanical support of the graft due to excessively rapid scaffold degradation. Therefore, scaffold-free cartilage repair remains a pressing challenge in the field of cartilage defect repair.

[0004] Cartilage fragment transplantation technology circumvents the limitations of traditional scaffold transplantation, becoming a significant breakthrough in "scaffold-free" cartilage repair. It involves minimally invasive intraoperative harvesting of autologous or allogeneic cartilage, which is then cut into fragments of 0.5–2 mm. 3 Micro-fragments, directly implanted into the defect area, avoid the immune risks and degradation matching problems of traditional scaffold materials. However, cartilage fragment transplantation technology still has many limitations in clinical applications: for example, the shearing force and thermal damage generated by high-speed grinding under arthroscopy reduce the viable cell rate, and a large number of cells in the central area of ​​the fragment die due to hypoxia and mechanical compression. Damaged cells release MMP-13 catabolic enzymes, which accelerate the in vivo degradation of transplanted cartilage-like tissue; chondrocyte migration depends on the degradation and remodeling of the ECM at the edge of the fragment, but the density of the fragment tissue severely limits the activity and migration efficiency of the internal chondrocytes, resulting in insufficient sources of regenerative cells. Summary of the Invention

[0005] Based on the above analysis, the embodiments of the present invention aim to provide a cartilage collagen membrane and its preparation method, culture medium and application, to solve at least one of the problems of existing cartilage regeneration cell repair materials, such as easy early degradation in vivo, insufficient mechanical strength, and low activity and migration efficiency of cartilage cells.

[0006] On one hand, the present invention provides a method for preparing a cartilage collagen membrane, comprising the following steps: S1, digest and culture cartilage fragments to obtain a chondrocyte suspension; S2 involves culturing chondrocyte suspensions and using a phased induction culture method to promote the self-secretion and self-assembly of extracellular matrix derived from chondrocytes to form a cartilage collagen membrane. The phased induction culture includes three stages, each using a different culture medium composition, all based on DMEM low-glucose basal medium. The first-stage culture medium also includes: GlutaMAX, non-essential amino acids, penicillin and streptomycin, ITS, proline, ascorbic acid and fetal bovine serum; The second-stage culture medium is based on the first-stage culture medium, with the addition of dexamethasone and transforming growth factor; The third-stage culture medium is based on the second-stage culture medium, but the transforming growth factor is removed.

[0007] Furthermore, the first-stage culture medium includes: 5-10 μl / ml GlutaMAX, 5-10 μl / ml non-essential amino acids, 9-10 μl / ml penicillin and streptomycin, 5-10 μl / ml ITS, 40-50 μg / ml proline, 40-50 μg / ml ascorbic acid, and 10%-15% fetal bovine serum.

[0008] Furthermore, the second-stage culture medium was supplemented with 100-120 nM dexamethasone and 8-10 ng / ml transforming growth factor, based on the first-stage culture medium.

[0009] Furthermore, the second-stage culture medium, based on the first-stage culture medium, has a lower concentration of fetal bovine serum.

[0010] Furthermore, bone morphogenetic protein 7 was added to the second-stage culture medium.

[0011] Furthermore, the concentration of bone morphogenetic protein 7 was 40-50 ng / ml.

[0012] Further, in step S1, the concentration of chondrocytes in the chondrocyte suspension is (1~5)×10⁻⁶. 5 cells / cm 2 .

[0013] The present invention also provides a culture medium for cartilage collagen membrane, comprising: a first-stage culture medium, a second-stage culture medium, and a third-stage culture medium; the first-stage culture medium, the second-stage culture medium, and the third-stage culture medium are used to induce chondrocytes to undergo staged induction culture, thereby enabling the extracellular matrix to self-secrete and self-assemble to form a cartilage collagen membrane; the first-stage culture medium comprises: DMEM low-glucose basal medium, GlutaMAX, non-essential amino acids, penicillin and streptomycin, ITS, proline, ascorbic acid, and fetal bovine serum; the second-stage culture medium is based on the first-stage culture medium, with the addition of dexamethasone and transforming growth factor, and the concentration of fetal bovine serum is reduced; the third-stage culture medium is based on the second-stage culture medium, with the transforming growth factor removed.

[0014] The present invention also provides a cartilage collagen membrane, which is prepared by the above preparation method or cultured in the above culture medium.

[0015] The present invention also provides the application of the above-mentioned cartilage collagen membrane or the cartilage collagen membrane prepared by the above preparation method in articular cartilage injury repair products.

[0016] This invention can achieve at least one of the following beneficial effects: 1. This invention employs a staged induction culture method, optimizing the culture medium system to promote the self-secretion and self-assembly of chondrocyte-derived extracellular matrix (CE-ECM), which can form a cartilage collagen membrane of a certain thickness. This membrane possesses good mechanical strength, biological activity, and specific biological functions that promote chondrocyte migration. Furthermore, the prepared cartilage collagen membrane has a higher degree of maturity, reducing the risk of early degradation in vivo, and also has cartilage regeneration properties. The newly formed cartilage after implantation is a hyaline cartilage-like tissue with abundant type II collagen and glycosaminoglycan secretion.

[0017] 2. This invention, by adding dexamethasone and transforming growth factor (TGF-β3) in the second stage, can strongly induce chondrocyte differentiation and the autocrine secretion of cartilage-specific extracellular matrix such as type II collagen and agglutinin. Cells are gradually encapsulated by their own matrix, forming continuous semi-transparent membranes. This invention, by further adding bone morphogenetic protein 7 (BMP-7), can synergistically with TGF-β3 to further upregulate cartilage-specific matrix genes (such as COL2A1 and ACAN), enhancing extracellular matrix deposition and tissue formation.

[0018] 3. By reducing fetal bovine serum concentration in the second stage, this invention can, to a certain extent, inhibit excessive proliferation of fibroblast-like cells and maintain cell differentiation phenotype.

[0019] 4. This invention maintains dexamethasone, ascorbic acid and proline in the third stage, which further cross-links, thickens and matures the extracellular matrix, ultimately obtaining a complete three-dimensional cartilage collagen membrane with a certain thickness.

[0020] 5. This invention, by designing staged culture media with different components and adjusting the composition and concentration of the culture media at different stages, can induce the culture of cartilage collagen membranes in stages, promote the self-secretion and self-assembly of chondrocyte-derived extracellular matrix (CE-ECM), and obtain a continuous, complete, and three-dimensional cartilage collagen membrane with a certain thickness. The obtained cartilage collagen membrane has good mechanical strength, biological activity, and specific biological functions that promote chondrocyte migration, and is not prone to early degradation in vivo.

[0021] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0022] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0023] Figure 1 This is a morphological diagram of the cartilage collagen membrane according to Embodiment 1 of the present invention.

[0024] Figure 2 This is an optical microscope image of the cartilage collagen membrane according to Embodiment 1 of the present invention.

[0025] Figure 3 An optical microscope photograph of chondrocytes according to Comparative Example 1 of the present invention.

[0026] Figure 4 The images show live cell fluorescence images and cell activity statistics of the cartilage collagen membrane of Example 1 and the chondrocytes of Comparative Example 1, as well as the cell activity statistics of the chondrocytes of Comparative Example 1. (a) is a fluorescence image of the cartilage collagen membrane of Example 1, (b) is a fluorescence image of the chondrocytes of Comparative Example 1, (c) is a cell activity statistics of the cartilage collagen membrane of Example 1, and (d) is a cell activity statistics of the chondrocytes of Comparative Example 1.

[0027] Figure 5The images shown are fluorescence images and fluorescence statistics of type II collagen in the cartilage collagen membrane of Example 1 and the chondrocytes of Comparative Example 1, respectively. (a) is a fluorescence image of the cartilage collagen membrane of Example 1, (b) is a fluorescence image of the chondrocytes of Comparative Example 1, (c) is a fluorescence statistics of type II collagen in the cartilage collagen membrane of Example 1, and (d) is a fluorescence statistics of type II collagen in the chondrocytes of Comparative Example 1.

[0028] Figure 6 These are cell tissue diagrams at different time points in the single-layer cell scratch experiment of the cartilage collagen membrane of Example 1 and the cartilage cells of Comparative Example 1 according to the present invention.

[0029] Figure 7 This is a quantitative statistical graph showing the scratch closure area at different time points in the single-layer cell scratch test of the cartilage collagen membrane of Example 1 and the cartilage cells of Comparative Example 1 according to the present invention.

[0030] Figure 8 This is a fluorescence image of the type II collagen in the cartilage collagen membrane according to Comparative Example 2 of the present invention.

[0031] Figure 9 This is a gross image of cartilage fragments encapsulated by a cartilage collagen membrane according to Embodiment 1 of the present invention.

[0032] Figure 10 The figures show the blood biochemistry and blood routine test results of the experimental and control groups according to the application example of the present invention; wherein, (a) is the blood biochemistry test result and (b) is the blood routine test result.

[0033] Figure 11 Gross and histological images of samples taken 8 weeks post-operation from the experimental and control groups according to an application example of the present invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the present invention clearer, exemplary embodiments of the present invention will be described below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. For clarity and brevity, not all features of actual embodiments are described in the specification.

[0035] An embodiment of the present invention provides a method for preparing a cartilage collagen membrane, comprising the following steps: S1, digest and culture cartilage fragments to obtain a chondrocyte suspension; S2 involves culturing chondrocyte suspensions and using a staged induction culture method to promote the self-secretion and self-assembly of extracellular matrix derived from chondrocytes to form a cartilage collagen membrane.

[0036] In step S2, the phased induction culture includes three stages, each using a different culture medium composition, all based on DMEM low-glucose basal medium.

[0037] The first-stage culture medium includes: DMEM low-glucose basal medium, GlutaMAX, non-essential amino acids, penicillin and streptomycin, ITS, proline, ascorbic acid and fetal bovine serum; The second-stage culture medium is based on the first-stage culture medium, with the addition of dexamethasone and transforming growth factor; The third-stage culture medium is based on the second-stage culture medium, but the transforming growth factor is removed.

[0038] The embodiments of this invention employ a staged induction culture method. By optimizing the culture medium system, the self-secretion and self-assembly of chondrocyte-derived extracellular matrix (CE-ECM) are promoted, resulting in the formation of cartilage collagen membranes of a certain thickness. These membranes exhibit good mechanical strength, biological activity, and specific cartilage regeneration properties, and can promote cell migration. Furthermore, compared to traditional methods, by optimizing the culture medium system, the secretion of cartilage-specific collagen and glycosaminoglycans can be promoted. The prepared cartilage collagen membranes have higher maturity, reducing the risk of early degradation in vivo. Moreover, the newly formed cartilage after implantation is a hyaline cartilage-like tissue with abundant type II collagen and glycosaminoglycan secretion.

[0039] According to some embodiments of the present invention, the first-stage culture medium comprises: DMEM low-glucose basal medium, 5-10 μl / ml GlutaMAX, 5-10 μl / ml non-essential amino acids (NEAA), 9-10 μl / ml penicillin and streptomycin, 5-10 μl / ml ITS, 40-50 μg / ml proline, 40-50 μg / ml ascorbic acid, and 10%-15% fetal bovine serum.

[0040] For example, in the first-stage culture medium, the concentration of GlutaMAX can be 5 μl / ml, 6 μl / ml, 7 μl / ml, 8 μl / ml, 9 μl / ml, or 10 μl / ml; the concentration of non-essential amino acids can be 5 μl / ml, 6 μl / ml, 7 μl / ml, 8 μl / ml, 9 μl / ml, or 10 μl / ml; the concentration of ITS can be 5 μl / ml, 6 μl / ml, 7 μl / ml, 8 μl / ml, 9 μl / ml, or 10 μl / ml; and the concentration of proline can be 40 μg / ml, 41 μg / ml, or 40 μg / ml. The concentrations of fetal bovine serum (FBS) can be 2 μg / ml, 43 μg / ml, 44 μg / ml, 45 μg / ml, 46 μg / ml, 47 μg / ml, 48 μg / ml, 49 μg / ml, and 50 μg / ml; the concentrations of ascorbic acid can be 40 μg / ml, 41 μg / ml, 42 μg / ml, 43 μg / ml, 44 μg / ml, 45 μg / ml, 46 μg / ml, 47 μg / ml, 48 μg / ml, 49 μg / ml, and 50 μg / ml; and the volumetric concentrations of FBS can be 10%, 11%, 12%, 13%, 14%, and 15%.

[0041] In the first stage, high concentrations of serum combined with ascorbic acid, proline, and ITS in the culture medium promote cartilage fragment adhesion, cell migration, and proliferation. The resulting cells are small, round, or polygonal and secrete an initial matrix to connect the cartilage fragments. It should be noted that GlutaMAX is a cell culture additive whose main component is L-alanyl-L-glutamine; ITS is a cell culture supplement whose components are insulin-transferrin-selenium.

[0042] According to some embodiments of the present invention, the first stage of cultivation can last for 3-4 days.

[0043] According to some embodiments of the present invention, the second-stage culture medium is supplemented with 100-120 nM dexamethasone and 8-10 ng / ml transforming growth factor based on the first-stage culture medium. Exemplarily, the concentration of dexamethasone can be 100 nM, 102 nM, 104 nM, 106 nM, 108 nM, 110 nM, 112 nM, 114 nM, 116 nM, 118 nM, or 120 nM; and the concentration of transforming growth factor can be 8 ng / ml, 8.5 ng / ml, 9 ng / ml, 9.5 ng / ml, or 10 ng / ml.

[0044] This invention, by adding dexamethasone and transforming growth factor in the second stage, can strongly induce chondrocyte differentiation and the auto-secretion of cartilage-specific extracellular matrix such as type II collagen and agglutinin. The cells are gradually encapsulated by their own matrix and form a continuous semi-transparent membrane.

[0045] Specifically, the transforming growth factor can be transforming growth factor β3 (TGF-β3). TGF-β3 can induce SOX9 gene expression, promote the synthesis of type II collagen and proteoglycans, while inhibiting terminal hypertrophy, which is beneficial to the formation of hyaline cartilage-specific matrix.

[0046] According to some embodiments of the present invention, the second-stage culture medium, based on the first-stage culture medium, has a reduced concentration of fetal bovine serum. By reducing the serum concentration, excessive proliferation of fibroblast-like cells can be inhibited to a certain extent, maintaining the cell differentiation phenotype.

[0047] Specifically, the volume concentration of fetal bovine serum in the second-stage culture medium can be reduced to 2-3%.

[0048] According to some embodiments of the present invention, bone morphogenetic protein 7 (BMP-7) is also added to the second-stage culture medium. BMP-7 works synergistically with TGF-β3 to further upregulate cartilage-specific matrix genes (such as COL2A1 and ACAN), enhancing extracellular matrix deposition and tissue formation.

[0049] Specifically, the concentration of bone morphogenetic protein 7 (BMP-7) can be 40-50 ng / ml, for example, 40 ng / ml, 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, 45 ng / ml, 46 ng / ml, 47 ng / ml, 48 ng / ml, 49 ng / ml, and 50 ng / ml.

[0050] According to some embodiments of the present invention, the second stage of cultivation can last for 5-6 days.

[0051] According to some embodiments of the present invention, the third-stage culture medium is based on the second-stage culture medium, but the transforming growth factor (TGF-β3) is removed. The present invention, by continuing to maintain dexamethasone, ascorbic acid, and proline in the third stage, allows for further cross-linking, thickening, and maturation of the extracellular matrix. Simultaneously, the removal of TGF-β3 prevents chondrocytes from undergoing terminal hypertrophic differentiation and matrix mineralization under prolonged TGF-β3 stimulation, while promoting the stable maturation of collagen membranes, ultimately obtaining a complete three-dimensional cartilage collagen membrane with a certain thickness.

[0052] According to some embodiments of the present invention, the third stage of cultivation can take 4-5 days.

[0053] In the embodiments of the present invention, the concentration settings of each component in the culture medium at each stage are mainly aimed at achieving a balance between maximizing the promotion of synthesis and inhibiting cartilage hypertrophy. Setting the concentration too low or too high will have a certain impact on the cell phenotype.

[0054] According to some embodiments of the present invention, in step S2, the entire culture process is carried out in a cell culture incubator at 37°C and 5% CO2, using ordinary culture dishes or well plates that have undergone tissue culture treatment, and adopting a static culture mode to avoid shear force from disrupting early cell aggregation and extracellular matrix deposition.

[0055] According to some embodiments of the present invention, in step S2, the culture medium is completely replaced every 2-3 days, and the culture medium is replaced with the culture medium of that stage when the medium is replaced at each stage.

[0056] According to some embodiments of the present invention, the thickness and shape of the cartilage collagen membrane obtained in step S2 are adjustable. When it is used to repair cartilage damage, the operation of wrapping cartilage fragments with the cartilage collagen membrane during surgery is simple, without the need for repeated suspension injection or concern about cell loss, which can reduce operation time and improve transplantation efficiency.

[0057] The embodiments of this invention differ from traditional culture methods by employing a staged culture approach. This approach combines TGF-β3, BMP-7, dexamethasone, ascorbic acid, and proline. In the early stages of culture, it promotes cartilage fragment adhesion, cell migration, and proliferation. In the later stages, it promotes the synthesis of type II collagen and proteoglycans, gradually forming continuous cartilage collagen membranes. Specifically, BMP-7, in synergy with TGF-β3, can further upregulate cartilage-specific matrix genes (such as COL2A1 and ACAN), enhancing extracellular matrix deposition and tissue formation. Simultaneously, by employing a dynamic serum concentration culture method, the serum content is reduced in the later stages, which can, to some extent, reduce excessive proliferation of fibroblasts and maintain the differentiation phenotype.

[0058] According to some embodiments of the present invention, in step S1, the cartilage fragments are digested and cultured to obtain a chondrocyte suspension, specifically including the following steps: S11, cut the cartilage tissue into small pieces, add collagenase to the cartilage fragments, and digest the cartilage fragments for 4-6 hours; S12. After the cartilage fragments and tissue blocks have basically dissolved and the suspension is turbid, filter to remove undigested residue and centrifuge to obtain cell precipitate. S13, the obtained cell pellet was resuspended in DMEM low-glucose medium containing 10% fetal bovine serum to terminate residual collagenase activity, and then centrifuged and washed to obtain cell suspension. S14: Perform live cell counting on the cell suspension obtained in S13 and adjust the cell density to the expected cell density.

[0059] The embodiments of the present invention can obtain a chondrocyte suspension with the required cell density by digesting cartilage fragments and resuspending the cells, which facilitates subsequent cell culture.

[0060] According to some embodiments of the present invention, in step S11, the cartilage tissue can be human-derived cartilage tissue, such as normal cartilage in the non-weight-bearing area of ​​the human knee joint. In embodiments of the present invention, autologous cartilage tissue fragments are used to induce film formation in vitro, prompting chondrocytes to produce large amounts of hyaline cartilage-specific type II collagen and glycosaminoglycans (GAG) in vitro, thereby culturing a cartilage collagen membrane for application in autologous cartilage loss repair. For example, it can be applied to autologous transplantation repair through a one-stage harvesting and two-stage implantation method. The cartilage collagen membrane of the present invention is used to encapsulate fresh autologous cartilage fragments and implant them into the cartilage defect site for filling. The inner layer of cartilage fragments is obtained from the cartilage tissue surrounding the damaged area and the non-weight-bearing area, providing good mechanical support for the collagen membrane material and simultaneously providing a source of living cells. The outer layer is encapsulated by a three-dimensional cartilage collagen membrane of a certain thickness, which helps stabilize the cartilage fragment implant and increases the bioactivity of chondrocytes. Human-derived cartilage tissue avoids the risk of disease transmission associated with animal-derived tissues, and poses no risk of immune rejection or biosafety issues. The cartilage collagen membrane exhibits excellent biocompatibility. Furthermore, utilizing fresh cartilage tissue fragments can provide additional natural extracellular matrix and cell sources, thus promoting repair in two ways and enhancing the mechanical strength of the cartilage collagen membrane and its integration with the host interface.

[0061] According to some embodiments of the present invention, in step S11, the collagenase used for digestion can be 0.2% type II collagenase. Specifically, the cartilage tissue can be minced into pieces of approximately 0.5-1 mm. 3 To obtain uniform fragments, 0.2% type II collagenase was dissolved in low-glucose DMEM basal medium, and penicillin and streptomycin were added to prevent contamination. The shredded cartilage tissue fragments were then transferred to the above medium for digestion.

[0062] According to some embodiments of the present invention, in step S11, during digestion, the culture medium can be gently agitated multiple times to promote cell dispersion. Specifically, it can be gently agitated once every 1 hour.

[0063] In step S12, undigested residue can be removed by filtration through a 70-100μm cell sieve. The filtrate is centrifuged at 300×g for 5-10 minutes, and the supernatant is discarded to obtain chondrocyte precipitate.

[0064] In step S14, trypan blue staining can be used to count live cells in the cell suspension obtained in S13.

[0065] According to some embodiments of the present invention, in step S14, the cell density is adjusted to (1-5)×10⁻⁶. 5 cells / cm 2 That is, the concentration of chondrocytes in the final chondrocyte suspension obtained in step S1 is (1-5)×10⁻⁶. 5 cells / cm2 This facilitates subsequent high-density plate culture and promotes the self-assembly of extracellular matrix derived from chondrocytes.

[0066] Embodiments of the present invention also provide a culture medium for chondrocyte collagen membrane, which can be used in the above-described method for preparing chondrocyte collagen membrane. Specifically, the culture medium includes: a first-stage culture medium, a second-stage culture medium, and a third-stage culture medium; the first-stage culture medium, the second-stage culture medium, and the third-stage culture medium are used to induce chondrocytes to be cultured in stages, so that the extracellular matrix is ​​self-secreted and self-assembled to form a chondrocyte collagen membrane.

[0067] The first stage culture medium includes: DMEM low-glucose basal medium, GlutaMAX, non-essential amino acids, penicillin and streptomycin, ITS, proline, ascorbic acid and serum; The second-stage culture medium, based on the first-stage culture medium, is supplemented with dexamethasone and transforming growth factor, and the serum concentration is reduced. The third-stage culture medium is based on the second-stage culture medium, but the transforming growth factor is removed.

[0068] The embodiments of the present invention can induce and culture cartilage collagen membranes in stages using culture media with different components, thereby obtaining continuous, complete, and three-dimensional cartilage collagen membranes with a certain thickness.

[0069] Furthermore, the culture medium used in this embodiment of the invention has the same composition and concentration as the culture medium used in the above preparation method, and will not be repeated here.

[0070] Embodiments of the present invention also provide a cartilage collagen membrane, which can be prepared by the above-described preparation method or cultured using the above-described culture medium. The cartilage collagen membrane of the present invention exhibits good mechanical support, biocompatibility, and biological functions that promote cell migration.

[0071] Embodiments of the present invention also provide the application of the above-described cartilage collagen membrane or the cartilage collagen membrane prepared by the above-described preparation method in articular cartilage injury repair products.

[0072] The technical solution of the present invention will be further illustrated below with specific embodiments.

[0073] Example 1 The preparation method of the cartilage collagen membrane in this embodiment is as follows: S1, take normal cartilage from the non-weight-bearing area of ​​the rabbit's knee joint, and cut it into pieces of approximately 0.5mm. 3To homogenize the fragments, transfer the shredded cartilage fragments to sterile centrifuge tubes, add 0.2% type II collagenase (dissolved in low-glucose DMEM basal medium, with added penicillin and streptomycin to prevent contamination), and incubate at 37°C in a shaker for 4-6 hours, gently agitating the tubes every hour to promote cell dispersion. Once the tissue fragments are mostly dissolved and the suspension is turbid, filter through a 100μm cell sieve to remove undigested residue. Centrifuge the filtrate at 300×g for 5 minutes and discard the supernatant. Resuspend the resulting cell pellet in low-glucose DMEM medium containing 10% fetal bovine serum to terminate residual collagenase activity, and wash twice more by centrifugation. Finally, stain with trypan blue to count viable cells and adjust the cell density to the desired high density of 5×10⁶ cells / mL. 5 cells / cm 2 .

[0074] S2, the chondrocyte suspension obtained in S1 was cultured in stages: (1) First stage (days 0-4): DMEM low glucose basal medium (Sigma-Aldrich, USA) was used as the base, with the addition of GlutaMAX (10 μl / ml), NEAA (10 μl / ml), penicillin-streptomycin (P / S, 10 μl / ml), ITS (10 μl / ml), proline (40 μg / ml) and ascorbic acid (50 μg / ml), and supplemented with 10% fetal bovine serum (FBS). (2) Second stage (days 5-10): The volume concentration of fetal bovine serum in the culture medium of the first stage was reduced to 2%, and dexamethasone (100 nM), transforming growth factor β3 (TGF-β3, 10 ng / ml) and BMP-7 (50 ng / ml) were added to enhance the synthesis of cartilage-specific matrix; (3) Third stage (days 11-14): Remove TGF-β3 from the culture medium in the second stage and continue to maintain dexamethasone, ascorbic acid and proline.

[0075] After 14 days of cultivation, if Figure 1 As shown, chondrocytes formed a membrane-like structure, obtaining a continuous, complete, and three-dimensional cartilage collagen membrane with a certain thickness.

[0076] Comparative Example 1 This comparative example uses conventionally cultured chondrocytes, and the specific preparation process is as follows: Normal cartilage from the non-weight-bearing area of ​​a rabbit's knee joint was taken and cut into pieces approximately 0.5 mm in size. 3To homogenize the fragments, transfer the shredded cartilage fragments to sterile centrifuge tubes, add 0.2% type II collagenase (dissolved in low-glucose DMEM basal medium, with added penicillin and streptomycin to prevent contamination), and incubate at 37°C in a shaker for 4-6 hours, gently agitating the tubes every hour to promote cell dispersion. Once the tissue fragments are mostly dissolved and the suspension is turbid, filter through a 100μm cell sieve to remove undigested residue. Centrifuge the filtrate at 300×g for 5 minutes and discard the supernatant. Resuspend the resulting cell pellet in low-glucose DMEM medium containing 10% fetal bovine serum to terminate residual collagenase activity, and wash twice more by centrifugation. Finally, stain with trypan blue to count viable cells and adjust the cell density to the desired high density of 5×10⁶ cells / mL. 5 cells / cm 2 The entire culture process was carried out using DMEM low-glucose medium containing 10% fetal bovine serum.

[0077] The cartilage collagen membrane of Example 1 and the chondrocytes of Comparative Example 1 were observed under an optical microscope, such as... Figure 2 and Figure 3 As shown, the cartilage collagen membrane is a continuous membrane structure, while ordinary cultured chondrocytes do not form a membrane structure.

[0078] The cartilage collagen membrane of Example 1 and the chondrocytes of Comparative Example 1 were subjected to LIVE / DEAD cell viability assays. Figure 4 As shown, Figure 4 Images (a) and (b) show fluorescence images of conventionally cultured chondrocytes and cartilage collagen membranes, respectively. Images (c) and (d) show statistical graphs of cell viability of conventionally cultured chondrocytes and cartilage collagen membranes, respectively. It is evident that cartilage collagen membranes exhibit superior cell viability compared to conventionally cultured chondrocytes.

[0079] The cartilage collagen membrane of Example 1 and the chondrocytes of Comparative Example 1 were subjected to type II collagen (COL-2) immunofluorescence detection. Figure 5 As shown, Figure 5 Images (a) and (b) show fluorescence staining images of conventionally cultured chondrocytes and cartilage collagen membranes, respectively. Images (c) and (d) show statistical graphs of COL-2 fluorescence staining in conventionally cultured chondrocytes and cartilage collagen membranes, respectively. It is evident that the cartilage collagen membrane exhibits significantly higher COL-2 expression compared to conventionally cultured chondrocytes, demonstrating that the collagen membrane possesses characteristics of natural hyaline cartilage tissue.

[0080] A cell scratch experiment was performed on the cartilage collagen membrane of Example 1 to observe the migration of chondrocytes at the scratch site at different time points and to quantitatively count the scratch closure area at different time points. Figure 6 As shown, compared to Comparative Example 1, the scratches on the cartilage collagen membrane in Example 1 completely closed after 48 hours, indicating that the cartilage collagen membrane can promote chondrocyte migration. Figure 7 As shown, at different time points, the cartilage collagen membrane had a larger scratch closure area compared to Comparative Example 1, indicating that it has a good biological function of promoting chondrocyte migration.

[0081] Comparative Example 2 The difference between this comparative example and Example 1 is that this comparative example uses a single-stage culture method, employing the first-stage culture medium, and is cultured for 14 days. That is, no transforming growth factor and bone morphogenetic protein 7 were subsequently added to the culture medium in this comparative example, and the fetal bovine serum concentration remained consistent throughout.

[0082] The cartilage collagen membrane obtained in this comparative example was subjected to type II collagen (COL-2) immunofluorescence detection. Figure 8 As shown, the COL-2 fluorescence staining of the cartilage collagen membrane was low, indicating that the extracellular matrix secretion was low.

[0083] Application examples The cartilage fragments were wrapped with a cartilage collagen membrane from Example 1 and implanted into the rabbits in the experimental group. Figure 9 A gross image of cartilage fragments encapsulated by a cartilage collagen membrane is shown. Meanwhile, Comparative Example 1 served as the control group, where rabbits underwent conventional microfracture surgery.

[0084] Eight weeks after the cartilage collagen membrane was transplanted into rabbits, blood samples were taken from both the experimental and control groups for testing. Figure 10 As shown, there were no statistically significant differences in blood biochemistry and routine blood tests between the experimental and control groups, indicating that the cartilage collagen membrane has excellent biocompatibility.

[0085] Histological examination was performed on samples from the experimental and control groups 8 weeks after transplantation. Figure 11 Gross and histological images of knee cartilage harvested 8 weeks after surgery in rabbits of the experimental and control groups are shown (scale bar is 500 μm). Figure 11As shown, in the control group (rabbits undergoing microfracture surgery), the cartilage tissue repair results after 8 weeks show that the cartilage damage was not completely covered by the newly grown cartilage tissue, and the newly generated cartilage differed significantly from the surrounding normal tissue. Histological results showed that the regenerated tissue was not hyaline cartilage-like, which is inconsistent with natural articular cartilage. In the experimental group (rabbits with cartilage collagen membrane wrapping cartilage fragments to fill the cartilage defect), the cartilage tissue repair results after 8 weeks showed that the original cartilage damage was no longer obvious, the newly grown cartilage tissue completely covered the defect, and there was no significant difference from the surrounding normal cartilage tissue, indicating good tissue integration. The cartilage collagen membrane did not undergo early degradation in vivo. Histological staining results showed that it was rich in and continuous with glycosaminoglycans and COL-2, indicating hyaline cartilage-like regenerated tissue, consistent with the structure of natural cartilage tissue. Furthermore, it can be seen that after the cartilage fragments wrapped in the cartilage collagen membrane were implanted into the rabbits, the cartilage collagen membrane could stabilize the cartilage fragments, allowing for stable implantation at the cartilage defect site without additional scaffolding, indicating that the cartilage collagen membrane has good mechanical support.

[0086] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a cartilage collagen membrane, characterized in that, Includes the following steps: S1, digest and culture cartilage fragments to obtain a chondrocyte suspension; S2, the chondrocyte suspension is cultured, and a staged induction culture method is used to promote the self-secretion and self-assembly of extracellular matrix derived from chondrocytes to form a cartilage collagen membrane; the staged induction culture includes three stages, each using a different culture medium composition, and all the culture medium is based on DMEM low-glucose basal medium. The first-stage culture medium also includes: GlutaMAX, non-essential amino acids, penicillin and streptomycin, ITS, proline, ascorbic acid and fetal bovine serum; The second-stage culture medium is based on the first-stage culture medium, with the addition of dexamethasone and transforming growth factor; The third-stage culture medium is based on the second-stage culture medium, but the transforming growth factor is removed.

2. The preparation method according to claim 1, characterized in that, The first-stage culture medium includes: 5-10 μl / ml GlutaMAX, 5-10 μl / ml non-essential amino acids, 9-10 μl / ml penicillin and streptomycin, 5-10 μl / ml ITS, 40-50 μg / ml proline, 40-50 μg / ml ascorbic acid, and 10%-15% fetal bovine serum.

3. The preparation method according to claim 1, characterized in that, The second-stage culture medium is based on the first-stage culture medium, with the addition of 100-120 nM dexamethasone and 8-10 ng / ml transforming growth factor; And / or, the second stage culture medium is based on the first stage culture medium, but with a reduced concentration of fetal bovine serum.

4. The preparation method according to claim 1, characterized in that, Bone morphogenetic protein 7 was also added to the culture medium in the second stage.

5. The preparation method according to claim 4, characterized in that, The concentration of bone morphogenetic protein 7 is 40-50 ng / ml.

6. The preparation method according to claim 1, characterized in that, In step S1, the concentration of chondrocytes in the chondrocyte suspension is (1~5)×10⁻⁶. 5 cells / cm 2 .

7. A culture medium for cartilage collagen membranes, characterized in that, include: The first-stage culture medium, the second-stage culture medium, and the third-stage culture medium are used to induce chondrocytes to undergo staged culture, enabling the extracellular matrix to be secreted and self-assembled to form a cartilage collagen membrane. The first stage culture medium includes: DMEM low-glucose basal medium, GlutaMAX, non-essential amino acids, penicillin and streptomycin, ITS, proline, ascorbic acid and fetal bovine serum; The second-stage culture medium is based on the first-stage culture medium, with the addition of dexamethasone and transforming growth factor, and the concentration of the fetal bovine serum is reduced; The third-stage culture medium is based on the second-stage culture medium, except that the transforming growth factor is removed.

8. The culture medium according to claim 7, characterized in that, The first stage culture medium includes: 5-10 μl / ml GlutaMAX, 5-10 μl / ml non-essential amino acids, 9-10 μl / ml penicillin and streptomycin, 5-10 μl / ml ITS, 40-50 μg / ml proline, 40-50 μg / ml ascorbic acid, and 10%-15% fetal bovine serum; And / or, the second stage culture medium is based on the first stage culture medium, with the addition of 100-120 nM dexamethasone and 8-10 ng / ml transforming growth factor; And / or, the second stage culture medium is based on the first stage culture medium, but with a reduced concentration of fetal bovine serum; And / or, the second-stage culture medium also contains bone morphogenetic protein 7; the concentration of bone morphogenetic protein 7 is 40-50 ng / ml.

9. A cartilage collagen membrane, characterized in that, It is prepared by the preparation method according to any one of claims 1-6, or cultured in the culture medium according to claim 7 or 8.

10. The application of a cartilage collagen membrane according to claim 9 or a cartilage collagen membrane prepared by any one of claims 1-6 in a product for repairing articular cartilage damage.

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