Recombinant collagen and its use in cartilage repair matrix

Abstract

The present invention discloses a recombinant collagen and its use in cartilage repair matrix. The recombinant collagen provided by the present invention comprises a sequence shown in SEQ ID No.1; the amino acid sequence of the recombinant collagen comprises N basic repeating units, and the basic repeating unit contains n1 of the following characteristic amino acid sequences: "G‑Xaa1‑Xaa2‑G‑E‑Xaa3"; the 3' end and the 5' end of the basic repeating unit are connected to form the above-mentioned characteristic amino acid sequence. The recombinant collagen provided by the present invention has a relatively obvious integrin binding activity, has the effects of promoting cell adhesion, proliferation, differentiation, repairing cartilage tissue defects, etc., and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biomaterials, and in particular relates to a recombinant collagen and application thereof in a cartilage repair matrix. Background Art

[0002] Articular cartilage injury has become a common disease in orthopedics. Trauma, arthritis and sports-related injuries can all cause articular cartilage injury or defect. The repair of articular cartilage mainly depends on the proliferation and differentiation of chondrocytes to produce sufficient extracellular matrix to repair cartilage defects. However, healthy adult chondrocytes are usually static and highly differentiated cells. Because of their low degree of vascularization, their nutrition mainly comes from synovial fluid and subchondral bone. If traumatic or pathological damage occurs, the repair and regeneration of articular cartilage is very limited. At present, some treatment methods used in clinical practice include microfracture, osteochondral transplantation, chondrocyte transplantation, etc. These methods can repair cartilage defects to a certain extent and relieve patients' pain, but the long-term effect is not satisfactory. The important reason is that the cartilage reconstructed by these methods is very different from normal hyaline cartilage in organization and structure, which leads to a gap in histological and biomechanical properties with normal human cartilage.

[0003] In recent years, tissue engineering has made considerable progress in obtaining functional articular cartilage. Some bioengineering treatment strategies, including stem cell methods and scaffold technology, have been proposed to provide minimally invasive and more effective ways to repair articular cartilage. As one of the three major elements of tissue engineering, scaffold materials play an important role in the construction of tissue engineering cartilage. Their mechanical properties and chemical composition will affect cell adhesion, proliferation and cell phenotype. Collagen has become a biomaterial with relatively in-depth research in tissue engineering due to its excellent biocompatibility, bioactivity and biodegradability. However, most traditional collagen comes from animals. The amino acid sequence of animal collagen is highly similar to that of human collagen. For example, the similarity between mammals pigs and cattle and humans is 95%, and the similarity between fish and humans is 65%. However, when animal collagen is used in biomaterials or medical devices, these differences may bring obvious risks such as immune rejection, allergic reactions, and viral infection and transmission. At the same time, since different subtypes of natural collagen are unevenly distributed in tissues and organs and coexist in a complex form, the efficiency of extracting collagen from biological tissues depends largely on its natural abundance, and it is also difficult to avoid the presence of a mixture of different subtypes. Therefore, the deficiencies in batch stability, purity, molecular weight and distribution of animal-derived collagen further limit its many applications in the biomedical field.

[0004] In order to solve the above problems, researchers have focused on the research of using genetic engineering technology to prepare recombinant collagen. Recombinant collagen is based on the natural human collagen gene sequence, and codons with specific functional expressions are selected. After appropriate modification and editing, they are transcribed into host cells and the recombinant protein material is obtained by large-scale production using biofermentation technology. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant collagen and its use in a cartilage repair matrix in view of the deficiency of animal-derived collagen. The provided recombinant collagen has relatively obvious integrin binding activity, can promote cell adhesion, proliferation and differentiation, and has the effect of repairing cartilage tissue defects.

[0006] Integrin binding activity is related to the integrin family, which is a widely distributed transmembrane glycoprotein that is the link between the extracellular matrix and intracellular signal transduction. Integrins are composed of α subunits and β subunits connected by non-covalent bonds to form heterodimers. Currently, 18 α subunits and 8 β subunits have been discovered, which can be combined into at least 24 integrin dimers. The integrin family of cell adhesion molecules is the main connecting substance between the extracellular matrix and parenchymal cells such as inflammatory cells and fibroblasts. It is closely related to cell adhesion, spreading, migration, functional expression, and the occurrence, maintenance and development of tissue fibrosis. It can regulate the interaction between cells and cells and between cells and extracellular matrix, such as recognition and adhesion.

[0007] The first object of the present invention is to provide a recombinant collagen, wherein the amino acid sequence of the recombinant collagen comprises N basic repeating units, wherein the basic repeating units contain n1 of the following characteristic amino acid sequences: "G-Xaa1-Xaa2-GE-Xaa3"; the 3' end and the 5' end of the basic repeating unit are connected to form the above characteristic amino acid sequence;

[0008] Here, N is an integer greater than or equal to 4; and n1 is an integer greater than or equal to 3.

[0009] Furthermore, the N value is an integer greater than or equal to 4 to 300, and further an integer greater than or equal to 4 to 200.

[0010] The N value can make the molecular weight of the recombinant collagen reach 1kDa to 250kDa, and further reach 3kDa to 150kDa.

[0011] Furthermore, the characteristic amino acid sequences are arranged continuously or at intervals in the basic repeating unit.

[0012] Furthermore, the amino acid sequence of the recombinant collagen presents the following characteristics:

[0013] [-GE-Xaa3-Yaa1-(G-Xaa1-Xaa2-GE-Xaa3) n2 -Yaa2-(G-Xaa1-Xaa2-GE-Xaa3) n3 -Yaa3-(G-Xaa1-Xaa2-GE-Xaa3) n3 -Yaa4-(G-Xaa1-Xaa2-GE-Xaa3) n4 -…………-

[0014] Yaa n -(G-Xaa1-Xaa2-GE-Xaa3) n -Yaa n+1 -G-Xaa1-Xaa2-] N

[0015] Wherein, Xaa1 is a nonpolar hydrophobic amino acid; Xaa2 is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); Xaa3 is a basic amino acid;

[0016] Yaa1, Yaa2, Yaa3, Yaa4,………, Yaa n , Yaa n+1 Each occurrence is independently selected from none, one or more different or identical amino acids;

[0017] n2, n3, n4, ..., n are integers greater than or equal to 0, and none of them are 0 at the same time.

[0018] The non-polar hydrophobic amino acid of the present invention includes one of glycine (G), valine (V), phenylalanine (F), alanine (A), leucine (L), isoleucine (I), methionine (M), proline (P), and hydroxyproline (O); and further includes one of phenylalanine (F), alanine (A), leucine (L), isoleucine (I), methionine (M), proline (P), and hydroxyproline (O).

[0019] The basic amino acid includes one of lysine, arginine and histidine, and is further lysine or arginine.

[0020] Furthermore, the recombinant collagen sequence does not contain a protein tag.

[0021] In one embodiment of the present invention, the amino acid sequence of the recombinant collagen as mentioned above is shown as SEQ ID NO.1.

[0022] The second object of the present invention is to provide a method for preparing the aforementioned recombinant collagen, which uses gene recombination technology to obtain recombinant collagen through host cell expression, extraction, and purification; the amino acid sequence of the recombinant collagen is obtained by repeatedly connecting multiple basic repeating units;

[0023] Furthermore, the amino acid sequence of the recombinant collagen includes N basic repeating units, and the basic repeating unit contains n1 of the following characteristic amino acid sequence: "G-Xaa1-Xaa2-GE-Xaa3"; the 3' end and the 5' end of the basic repeating unit are connected to form the above characteristic amino acid sequence;

[0024] Wherein, N is an integer greater than or equal to 4; n1 is an integer greater than or equal to 3;

[0025] Furthermore, the characteristic amino acid sequences are arranged continuously or alternately in the basic repeating unit.

[0026] Therefore, the N value is an integer greater than 4 to 300, and further an integer greater than 4 to 200; the N value can make the molecular weight of the recombinant collagen reach 1kDa to 200kDa, and further reach 3kDa to 150kDa.

[0027] The value of N can be taken from the endpoint values ​​at both ends of the range value defined by the present invention, or from any integer in the range value. For example, the value of N can be 1, 3, 10, 50, 70, 90, 95, 100, 125, 150, etc.

[0028] Further, the recombinant collagen sequence does not contain a protein tag;

[0029] The amino acid sequence of the recombinant collagen presents the following characteristics:

[0030] [-GE-Xaa3-Yaa1-(G-Xaa1-Xaa2-GE-Xaa3)n2-Yaa2-(G-Xaa1-Xaa2-GE-Xaa3) n3 -Yaa3-(G-Xaa1-Xaa2-GE-Xaa3) n3 -Yaa4-(G-Xaa1-Xaa2-GE-Xaa3) n4 -…………-

[0031] Yaa n -(G-Xaa1-Xaa2-GE-Xaa3) n -Yaa n+1 -G-Xaa1-Xaa2-] N

[0032] Wherein, Xaa1 is a nonpolar hydrophobic amino acid; Xaa2 is one of serine (S), alanine (A), proline (P), and hydroxyproline (O); Xaa3 is a basic amino acid;

[0033] Yaa1, Yaa2, Yaa3, Yaa4,………, Yaa n , Yaa n+1 Each occurrence is independently selected from none, one or more different or identical amino acids;

[0034] n2, n3, n4, ..., n are integers greater than or equal to 0, and none of them are 0 at the same time.

[0035] The third object of the present invention is to provide the use of the aforementioned recombinant collagen in the preparation of products for regulating cell adhesion, proliferation and differentiation.

[0036] The fourth object of the present invention is to provide the use of the aforementioned recombinant collagen in preparing a cartilage repair matrix product.

[0037] When the recombinant collagen of the present invention is used in the aforementioned purposes, it can be used in combination with polymers and / or their derivatives, wherein the polymers include natural polymers and synthetic polymers; further, the natural polymers include but are not limited to hyaluronic acid, chitosan, alginate, cellulose, etc.; further, the synthetic polymers include but are not limited to PLA, PGA, PLCL, PVA.

[0038] When the recombinant collagen of the present invention is used in the aforementioned purposes, the concentration used is 0.01 to 100 mg / mL, for example, it can be 0.01 mg / mL, 0.05 mg / mL, 2 mg / mL, 4.5 mg / mL, 7 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 50 mg / mL, 80 mg / mL, 100 mg / mL, etc. The concentration used is related to the application site and scenario.

[0039] The fifth object of the present invention is to provide a cartilage repair matrix, comprising a polymer and / or its derivatives, and the aforementioned recombinant collagen.

[0040] Furthermore, the polymer includes natural polymers and synthetic polymers; further, the natural polymer is at least selected from any one of hyaluronic acid, chitosan, alginate, and cellulose; further, the synthetic polymer is at least selected from any one of PLA, PGA, PLCL, and PVA.

[0041] The sixth object of the present invention is to provide a method for preparing the aforementioned cartilage repair matrix, comprising the following steps:

[0042] (1) dissolving the hyaluronic acid derivative in a buffer solution and adding EDC / sNHS solution for reaction;

[0043] (2) adding the recombinant collagen to the reaction system in step (1) to react, dialyzing and drying the solution obtained after the reaction to obtain a composite matrix;

[0044] (3) After the composite matrix is ​​dissolved, a photoinitiator solution is added to obtain a cartilage repair matrix;

[0045] Humanized collagen has a small molecular weight and poor mechanical properties. It is difficult to form a gel after modification and cannot meet the needs of cartilage tissue engineering. In the embodiment of the present invention, hyaluronic acid is firstly appropriately chemically modified with methacrylic anhydride to introduce unsaturated carbon-carbon double bonds, and then the carboxyl groups in the hyaluronic acid are activated by adding EDC / sNHS, and then recombinant humanized collagen is added to make the amino groups on the protein and the activated carboxyl groups on the hyaluronic acid undergo amidation reaction to couple the two. The composite hydrogel finally prepared by photocrosslinking not only has good mechanical properties and biocompatibility, but also the photocrosslinking method makes the operation easier, which can meet the needs of cartilage tissue engineering, and also meet the filling of irregular defects in clinical practice.

[0046] Furthermore, the polymer derivative in step (1) is hyaluronic acid modified with methacrylic anhydride;

[0047] Further, the molecular weight of the modified hyaluronic acid is 100 to 1500 kDa, preferably 300 to 600 kDa, more preferably 300 kDa;

[0048] Furthermore, the degree of grafting of the hyaluronic acid modification in step (1) is 20 to 60%, preferably 30 to 50%;

[0049] Furthermore, in step (1), after EDC and sNHS are added, the concentration of sNHS is 0.5-5 mM, and the concentration of EDC is 0.5-5 mM; further, the concentration of sNHS is 1-3 mM, and the concentration of EDC is 2-4 mM.

[0050] In the present invention, in step (1), the pH value of the mixed solution after adding the EDC / sNHS solution is 4 to 5, preferably 4.75;

[0051] The pH value of the mixed solution after the reaction is 7-8, and the reaction time is 2-5 hours; preferably, the pH value is 7.4, and the reaction time is 3 hours.

[0052] In the present invention, in step (2), the mass ratio of recombinant collagen to polymer derivative is 0.5-30:2-10; further 0.5-20:6; further 1-11:6;

[0053] Furthermore, the reaction time in step (2) is 5 to 15 hours, preferably 8 to 12 hours;

[0054] Furthermore, in step (2), the solution after the reaction is placed in a dialysis bag with a molecular weight cutoff of 8000 to 14000, dialyzed with deionized water for 2 to 4 days, and then freeze-dried to obtain a composite matrix.

[0055] The photoinitiator used in the present invention is at least one of LAP and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), and the final concentration is 0.01-0.05% by mass volume, preferably 0.05%.

[0056] In the present invention, the concentration of the composite matrix after adding the photoinitiator in step (3) is 2 to 30 mg / ml, further 5 to 20 mg / ml, and further 7 to 17 mg / ml.

[0057] The cartilage repair matrix of the present invention is a product containing the recombinant collagen, which can be used as a component of medical devices including but not limited to implant materials, tissue engineering scaffold materials, soft tissue filling materials, cells or other active substance carrier materials.

[0058] When used clinically, the cartilage repair matrix of the present invention can be prepared into a cartilage repair hydrogel after adding cells, or can be directly applied to the cartilage defect site without adding cells.

[0059] The seventh object of the present invention is to provide a method for preparing a cartilage repair hydrogel, wherein the cartilage repair matrix prepared by the aforementioned method is compounded with chondrocytes or bone marrow mesenchymal stem cells, injected into the cartilage damage site, and irradiated with light to form a cartilage repair hydrogel.

[0060] The wavelength of the light is selected adaptively according to the initiator used.

[0061] The cartilage repair hydrogel of the present invention further comprises a physiologically acceptable carrier material.

[0062] The carrier materials here include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).

[0063] The composition can also be used in combination with other functional materials as needed, such as ingredients with antibacterial and antimicrobial effects, anti-aging ingredients, anticoagulant ingredients, antioxidant ingredients, growth factors, etc.

[0064] The composition can be prepared according to conventional techniques of pharmaceutics or cosmetics, including mixing the recombinant collagen of the present invention as an active ingredient with a carrier, and preparing the desired dosage form according to conventional techniques. The composition of the present invention can be formulated into a variety of dosage forms as needed, including but not limited to oral administration preparations, mucosal administration preparations, injection preparations, inhalation preparations, and external preparations.

[0065] The products described in the present invention include but are not limited to medicines, foods, health products or cosmetics, and daily necessities.

[0066] Beneficial effects:

[0067] (1) The recombinant collagen provided by the present invention has relatively obvious integrin binding activity, has the effect of promoting cell adhesion, proliferation, differentiation, and repairing cartilage tissue defects, and has good application prospects.

[0068] (2) The present invention covalently cross-links hyaluronic acid and humanized collagen to form a stable composite hydrogel matrix with good mechanical properties and can well maintain the morphology of the composite; it has good biocompatibility, and the photocross-linking method makes the operation easier, which can meet the needs of cartilage tissue engineering and also meet the clinical filling of irregular defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 It is the result of fibroblast cell adhesion and growth;

[0070] Figure 2 is the OD value of fibroblast cell adhesion;

[0071] Figure 3 It is the result of chondrocyte adhesion and growth;

[0072] Figure 4 is the OD value of chondrocyte adhesion;

[0073] Figure 5 The results of the adhesion test of sequence A and sequence B at different concentrations to human gingival fibroblasts;

[0074] Figure 6 The morphological images of chondrocyte / hydrogel complex after 1, 7, 14, and 21 days of in vitro culture;

[0075] Figure 7 is the OD value of chondrocyte adhesion after 1, 7, and 14 days of in vitro culture of chondrocyte / hydrogel complex;

[0076] Figure 8 The CCK-8 images of chondrocytes after 1, 7, and 14 days of in vitro culture of chondrocyte / hydrogel complexes;

[0077] Fig. 9 The staining results of chondrocyte / hydrogel complex;

[0078] Fig.10 The results of immunohistochemical staining of type II collagen in chondrocyte / hydrogel composites;

[0079] Fig.11 The results of quantitative detection of GAG in chondrocyte / hydrogel complex. DETAILED DESCRIPTION

[0080] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0081] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0082] Example 1

[0083] A nucleotide sequence encoding the recombinant collagen is constructed, a vector containing the nucleotide sequence is constructed and screened, a host cell (which may be a prokaryotic cell or a eukaryotic cell) containing the vector is constructed and screened, the host cell is cultured under appropriate conditions to express the protein, and the expressed recombinant collagen is collected and purified.

[0084] The gene recombination and transcription involved in the present invention refers to synthesizing the nucleotide sequence encoding the recombinant collagen, cloning the nucleotide sequence into the expression vector by conventional techniques, and then transforming the expression vector into the host cell to construct and screen the engineered strain. The transformation method includes but is not limited to electroporation, CaCl2 transformation, etc.; the expression vector can be a common vector such as pET26, pET32, pGEX-6p, pPIC9, pPIC9K, etc., and can be a plasmid, phage, virus or other vector; the host cell can be a microbial cell such as Enterobacteriaceae cells (such as Escherichia coli BL21, DH5α, etc.), yeast (such as Pichia pastoris, Saccharomyces cerevisiae, etc.) or eukaryotic cells such as CHO cells.

[0085] The fermentation culture involved in the present invention refers to inducing fermentation culture in a sterile fermentation tank by combining the screened and protein-high expression-identified engineered strain with a suitable fermentation culture medium under suitable temperature, pressure and pH conditions.

[0086] The separation and purification of proteins involved in the present invention refers to obtaining bacterial liquid after lysing, homogenizing, separating and other treatments of bacteria, and further obtaining recombinant collagen by conventional protein separation and purification techniques. Separation and purification techniques include but are not limited to filtration, centrifugation, salting out, dialysis, liquid chromatography, ion exchange chromatography, affinity chromatography, etc.

[0087] In the embodiment of the present invention, gene recombination and transcription are performed by taking Escherichia coli as a host cell as an example, specifically:

[0088] 1. Preparation of recombinant collagen by E. coli fermentation

[0089] Gene recombination and transcription: The DNA fragments were codon optimized and spliced ​​and recombined using the PCR method. The expression vector was constructed using pET-32a and then transferred into the Escherichia coli expression strain BL21. After culture and screening, genetically engineered Escherichia coli bacteria with high protein expression were obtained.

[0090] Fermentation culture: Select a single colony of genetically engineered Escherichia coli from the LB plate, place it in a 100 mL Erlenmeyer flask containing 10 mL LB medium, and culture it at 37°C and 220 rpm for 12-16 h; inoculate the bacterial solution into a fermenter containing LB medium at a ratio of 1:100 for amplification culture, culture it at 37°C and 220 rpm for 3 h until OD600 is about 0.6, add 0.5 mM IPTG, induce culture at 16°C for 20 h, and then collect the bacteria by centrifugation.

[0091] Protein separation and purification: After reselecting the bacteria with Tris buffer, stir at high speed to completely dissolve the bacteria, collect the supernatant by centrifugation and cool to 4°C; the supernatant is filtered using 1μm, 0.45μm and 0.22μm filter membranes in turn and further purified by affinity chromatography to obtain recombinant collagen.

[0092] 2. Cell culture method

[0093] 1) Sample preparation: Recombinant collagen was prepared into a certain concentration solution (such as 0.5 mg / ml) with PBS, and the animal source collagen solution was diluted to the same concentration (such as 0.5 mg / ml) with PBS, and PBS was used as the blank control group;

[0094] 2) Plating: Add the experimental solution to each well of a 96-well plate, add 100 μL to each well, 5 replicate wells per group, and incubate at 4°C overnight;

[0095] 3) Blocking: After the plating is completed, remove the liquid in the well plate and wash it twice with 200 μL PBS solution, add 100 μL of heat-inactivated 1% BSA-PBS solution, and incubate in an incubator at 37°C and 5% CO2 for 1 hour;

[0096] 4) Cell seeding: After the incubation, remove the liquid in the well plate and wash twice with 200 μL PBS solution. Add 100 μL of 5×10 4 -1×10 6 Cell suspension of 100 cells / ml was incubated in an incubator at 37°C and 5% CO2 for 1 h;

[0097] 5) Detection: After the incubation, remove the liquid in the well plate and wash twice with 200 μL PBS solution, add 150 μL CCK-8, incubate in an incubator at 37°C, 5% CO2 for 1 hour, then draw 100 μL of detection solution and add it to a new 96-well plate to detect the absorbance value (OD) at 450 nm.

[0098] Test Example 1:

[0099] The method of Example 1 was used to prepare the following three recombinant collagens containing different amino acid sequences.

[0100] The recombinant collagen obtained by directly linking the repeating units with the above-mentioned multiple characteristic amino acid sequence combinations 16 times was used to improve a series of experiments such as cell adhesion, migration, and functional expression; (sequence A was linked 16 times, sequence A: GERGAP GFR GPAGPN GIP GEK GPAGER GAP)

[0101] Another recombinant collagen protein obtained by directly repeating the repeating unit 4 times with different characteristic amino acid sequences has different effects on cell adhesion and proliferation. (Repeat the connecting sequence B 4 times, sequence B: GEK GSP GAD GPA GAPGTP GPQ GIA GQR GVV GLP GQR GER GFP GLP GPS GEP GKQ GPS GAS);

[0102] After the recombinant collagen with the above repeating unit linked 16 times was added with a tag, the adhesion and proliferation effects on cells were significantly reduced; (sequence C was linked 16 times, sequence C: HHHHHH GER GAP GFR GPAGPN GIP GEK GPAGERGAP)

[0103] The cell culture method in Example 1 was used to perform fibroblast adhesion test, chondrocyte adhesion test, and human gingival fibroblast adhesion promotion test on the above three collagens.

[0104] The experimental results are as follows Figures 1 to 3 .

[0105] Figure 1The results showed that the recombinant collagen experimental group obtained by repeatedly connecting sequence A with more characteristic amino acid sequence combinations had very obvious fibroblast adhesion and good cell morphology; the recombinant collagen experimental group obtained by repeatedly connecting sequence B after reducing the characteristic amino acid sequence had similar fibroblast adhesion and cell morphology as the animal collagen group; and the fibroblast adhesion in the recombinant collagen group obtained by repeatedly connecting sequence C after adding a label was significantly reduced, and the fibroblast adhesion in the blank control group was very small.

[0106] Figure 2 The results showed that the recombinant collagen experimental group obtained by repeatedly connecting sequence A with more characteristic amino acid sequence combinations was most able to promote the adhesion of fibroblasts, followed by animal collagen and recombinant collagen obtained by repeatedly connecting sequence B. The adhesion amount of fibroblasts in these three groups was significantly higher than that of the recombinant collagen obtained by repeatedly connecting sequence C and the blank control; the adhesion amount of fibroblasts in the sequence C recombinant collagen group was slightly higher than that of the blank control.

[0107] Figure 3 The results showed that the adhesion of chondrocytes was most obvious in the animal collagen group, almost covering the entire field of view; followed by the sequence A recombinant collagen group, where the chondrocytes adhered obviously and were relatively concentrated, with good cell morphology; the adhered chondrocytes in the sequence B recombinant collagen group were relatively scattered, with only a small number concentrated, and good cell morphology; the adhered chondrocytes in the sequence C recombinant collagen group were dispersed and the number was small; in the blank control group, only a small number of chondrocytes adhered and were dispersed.

[0108] Figure 4 The results showed that animal collagen was most beneficial for promoting the adhesion of chondrocytes, which was significantly higher than other groups; the sequence A recombinant collagen group with more characteristic amino acid sequence combinations was significantly higher than the sequence B recombinant collagen group, sequence C recombinant collagen group and blank control group; the sequence B recombinant collagen group was significantly higher than the sequence C recombinant collagen group and blank control group; the sequence C recombinant collagen group was slightly higher than the blank control group, but there was no statistical difference.

[0109] Figure 5 The results of the human gingival fibroblast adhesion promotion experiment showed that the sequence A recombinant collagen group with more characteristic amino acid sequence combinations was more conducive to the adhesion of human gingival fibroblasts than sequence B at different concentrations; as the concentration of recombinant collagen increased, the adhesion of human gingival fibroblasts also gradually increased.

[0110] Example 2 Application of recombinant collagen in promoting cartilage repair

[0111] The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeatedly connected 16 times and used to promote cartilage repair. The specific steps are:

[0112] (1) dissolving methacrylated hyaluronic acid (HA-MA) with a molecular weight of 300 KDa and a modification degree of 37% in a MES buffer solution to obtain a 1 mM methacrylated hyaluronic acid solution;

[0113] (2) solid sNHS and solid EDC are sequentially added to the mixed solution to make the concentration of sNHS 2 mM and the concentration of EDC 3 mM, and the pH of the mixed solution is maintained at 4.75 with 1.0 M NaOH solution or 1.0 M HCl solution;

[0114] (3) Stirring the reaction at room temperature for 2 h;

[0115] (4) Adjust the pH of the reaction system to 7.4 with 1.0 M NaOH solution;

[0116] (5) adding recombinant humanized collagen to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution is 1:6, and stirring the reaction for 8 hours;

[0117] (6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain freeze-dried sponge HA-rhColⅢ;

[0118] (7) Dissolve the freeze-dried sponge in 0.01 M PBS and add LAP photoinitiator solution (final concentration: 0.05% by mass volume) to make the final concentration of HA-rhColⅢ solution 7 mg / ml;

[0119] (8) The precursor solution in step (7) was placed in a silicone mold with a size of 6 mm × 2.5 mm, and illuminated under ultraviolet light for 1 min to obtain a hyaluronic acid hydrogel containing recombinant humanized collagen (HA-rhCol III).

[0120] Example 3 Application of recombinant collagen in promoting cartilage repair

[0121] The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeatedly connected 16 times and used to promote cartilage repair. The specific steps are:

[0122] (1) dissolving methacrylated hyaluronic acid HA-MA with a molecular weight of 100 KDa and a modification degree of 50% in a MES buffer solution to obtain a 2 mM methacrylated hyaluronic acid solution;

[0123] (2) solid sNHS and solid EDC are sequentially added to the mixed solution to make the concentration of sNHS 3 mM and the concentration of EDC 5 mM, and the pH of the mixed solution is maintained at 4.75 with 0.5 M NaOH solution or 0.5 M HCl solution;

[0124] (3) Stirring the reaction at room temperature for 2 h;

[0125] (4) Adjust the pH of the reaction system to 7.4 with 1.0 M NaOH solution;

[0126] (5) adding recombinant humanized collagen to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution is 11:6, and stirring the reaction for 12 hours;

[0127] (6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain the product HA-rhColⅢ;

[0128] (7) Dissolve the freeze-dried sponge in 0.01 M PBS and add LAP photoinitiator solution (final concentration: 0.01% by mass volume) to make the final concentration of HA-rhColⅢ solution 17 mg / ml;

[0129] (8) The precursor solution in step (7) was placed in a silicone mold with a size of 6 mm × 2.5 mm, and irradiated under ultraviolet light for 1 min to obtain HA-rhColⅢ hydrogel.

[0130] Example 4 Application of recombinant collagen in promoting cartilage repair

[0131] The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid sequence core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP (sequence A), which is repeatedly connected 16 times and used to promote cartilage repair. The specific steps are:

[0132] (1) dissolving methacrylated hyaluronic acid HA-MA with a molecular weight of 600 KDa and a modification degree of 30% in MES buffer solution to obtain a 1 mM methacrylated hyaluronic acid solution;

[0133] (2) sNHS and EDC were added to the mixed solution in sequence, so that the concentration of sNHS was 1 mM and the concentration of EDC was 3 mM, and the pH of the mixed solution was maintained at 4.75 with 1.0 M NaOH solution or 1.0 M HCl solution;

[0134] (3) Stirring the reaction at room temperature for 3 h;

[0135] (4) Adjust the pH of the reaction system to 7.4 with 1.0 M NaOH solution;

[0136] (5) adding recombinant humanized collagen to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution is 0.5:6, and stirring the reaction for 8 hours;

[0137] (6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain the product HA-rhColⅢ;

[0138] (7) Dissolve the freeze-dried sponge in 0.01 M PBS and add LAP photoinitiator solution (final concentration: 0.05% by mass volume) to make the final concentration of HA-rhColⅢ solution 7 mg / ml;

[0139] (8) The precursor solution in step (7) was placed in a silicone mold with a size of 6 mm × 2.5 mm, and irradiated under ultraviolet light for 2 min to obtain HA-rhColⅢ hydrogel.

[0140] Example 5 Application of recombinant collagen in promoting cartilage repair

[0141] The method of Example 1 is used to prepare recombinant collagen that meets the amino acid sequence characteristics of the present invention, wherein the amino acid core sequence of the recombinant humanized collagen is preferably GERGAPGFRGPAGPNGIPGEKGPAGERGAP, which is repeatedly connected 16 times and used to promote cartilage repair. The specific steps are:

[0142] (1) dissolving methacrylated hyaluronic acid HA-MA with a molecular weight of 1000 KDa and a modification degree of 40% in a MES buffer solution to obtain a 0.6 mM methacrylated hyaluronic acid solution;

[0143] (2) sNHS and EDC were added to the mixed solution in sequence to make the concentration of sNHS 5 mM and the concentration of EDC 5 mM, and the pH of the mixed solution was adjusted to 5 with 0.2 M NaOH solution or 0.2 M HCl solution.

[0144] Maintained at 4.75;

[0145] (3) Stirring the reaction at room temperature for 3 h;

[0146] (4) adjusting the pH of the reaction system to 7.4 with 0.5 M NaOH solution;

[0147] (5) adding recombinant humanized collagen to the reaction system so that the mass ratio of recombinant humanized collagen to methacrylated hyaluronic acid in the mixed solution is 11:6, and stirring the reaction for 12 hours;

[0148] (6) The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 8000-14000, dialyzed with deionized water for three days, and then freeze-dried using a freeze dryer to obtain the product HA-rhColⅢ;

[0149] (7) The freeze-dried sponge in step (6) was dissolved in 0.01 M PBS, and I2959 photoinitiator solution (final concentration: 0.03% by mass volume) was added to make the final concentration of HA-rhColⅢ solution 17 mg / ml;

[0150] (8) The precursor solution in step (7) was placed in a silicone mold with a size of 6 mm × 2.5 mm, and irradiated under ultraviolet light for 1 min to obtain HA-rhColⅢ hydrogel.

[0151] Test Example 2:

[0152] The results of the cell experiment of the recombinant humanized collagen modified hyaluronic acid hydrogel prepared in Example 2 are as follows:

[0153] Combination Figure 6 and Figure 7As a result, chondrocytes in HA-MA hydrogels were cultured for 21 days, and due to the lack of adhesion sites, chondrocytes aggregated and grew in situ. However, chondrocytes in HA-rhColⅢ hydrogels were partially spread out at 7 days, and the cell spreading growth phenomenon was more obvious and evenly distributed at 14 and 21 days. This indicates that rhColⅢ is conducive to cell adhesion, migration and proliferation, and HA-rhColⅢ hydrogels can promote chondrocyte proliferation better than HA-MA hydrogels.

[0154] Figure 8 The results showed that the nuclei of cells in the HA-rhColⅢ hydrogel group did not deform or shrink, and at 7 and 14 days of culture, the actin of cells in the two groups of hydrogels was spindle-shaped, indicating that the cells were well spread inside the gel, while the cells in the HA-MA group proliferated in situ in the form of cell clusters at each time point. This result shows that rhColⅢ provides a suitable site for cell adhesion, allowing chondrocytes to adhere faster and providing conditions for cell proliferation.

[0155] Histology and type II collagen immunohistochemical staining ( Figure 9-10 ) The results showed that there were more obvious polysaccharide and protein staining areas in HA-rhColⅢ gel, indicating that the addition of rhCol III can promote the functional expression of chondrocytes. GAG quantitative detection results ( Fig.11 ) further confirmed that the addition of rhColⅢ was more conducive to promoting the secretion of extracellular matrix of cartilage cells.

[0156] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Use of a recombinant collagen protein having an amino acid sequence as shown in SEQ ID NO. 1 in preparing a cartilage repair matrix product.

2. A method for preparing a cartilage repair matrix, characterized in that: The steps include: (1) Dissolving the polymer derivative in a buffer solution and adding EDC / sNHS solution for reaction; The polymer derivative described in step (1) is hyaluronic acid modified with methacrylic anhydride; (2) adding the recombinant collagen protein shown in SEQ ID NO.1 to the reaction system in step (1) for reaction, dialyzing and drying the solution obtained after the reaction to obtain a composite matrix; (3) After the composite matrix is ​​dissolved, a photoinitiator solution is added to obtain a cartilage repair matrix.

3. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: The molecular weight of the modified hyaluronic acid is 100-1500 kDa; the degree of grafting of the modified hyaluronic acid in step (1) is 20%-60%.

4. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: In step (1), after EDC and sNHS are added, the concentration of sNHS is 0.5-5 mM, and the concentration of EDC is 0.5-5 mM.

5. The method for preparing the cartilage repair matrix according to claim 4, characterized in that: The concentration of sNHS is 1-3 mM, and the concentration of EDC is 2-4 mM.

6. The method for preparing the cartilage repair matrix according to claim 3, characterized in that: The molecular weight of the modified hyaluronic acid is 300-600 kDa; the modified grafting degree of the hyaluronic acid is 30%-50%.

7. The method for preparing the cartilage repair matrix according to claim 6, characterized in that: The molecular weight of the modified hyaluronic acid is 300 kDa.

8. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: In step (1), the pH value of the mixed solution after adding the EDC / sNHS solution is 4 to 5; The pH value of the mixed solution after the reaction is 7-8, and the reaction time is 2-5 hours.

9. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: In step (1), the pH value of the mixed solution after adding the EDC / sNHS solution is 4.75; The pH value of the mixed solution after the reaction was 7.4, and the reaction time was 3 h.

10. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: In step (2), the mass ratio of recombinant collagen to polymer derivative is 0.5-30:2-10.

11. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: In step (2), the mass ratio of recombinant collagen to polymer derivative is 0.5 to 20:

6.

12. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: In step (2), the mass ratio of recombinant collagen to polymer derivative is 1 to 11:

6.

13. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: In step (2), the reaction time is 5 to 15 hours.

14. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: The reaction time in step (2) is 8 to 12 hours.

15. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: In step (2), the solution after the reaction is placed in a dialysis bag with a molecular weight cutoff of 8000 to 14000, dialyzed with deionized water for 2 to 4 days, and then freeze-dried to obtain a composite matrix.

16. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: The concentration of the composite matrix after adding the photoinitiator in step (3) is 2 to 30 mg / ml.

17. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: The concentration of the composite matrix after adding the photoinitiator in step (3) is 5 to 20 mg / ml.

18. The method for preparing the cartilage repair matrix according to claim 2, characterized in that: The concentration of the composite matrix after adding the photoinitiator in step (3) is 7 to 17 mg / ml.

19. A cartilage repair matrix prepared by the preparation method according to any one of claims 2 to 18.

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