A type A recombinant humanized collagen bone repair material and its preparation method and use

Abstract

The present invention relates to a type A recombinant humanized collagen bone repair material and a preparation method and use thereof. The preparation method of the bone repair material provided by the present invention comprises a first step of precipitation method and a second step of dialysis method. By combining the two-step method of precipitation method and dialysis method, the composition and formation process of natural bone are simulated, and a bone repair material with a high collagen ratio and activity is successfully prepared, and collagen can be gradiently degraded and released, collagen expression activity is increased, and the osteogenesis effect is improved.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and specifically relates to a type A recombinant humanized collagen bone repair material and a preparation method and application thereof. Background Art

[0002] Bone defect repair materials are one of the most clinically demanded biomedical materials. At present, the incidence of bone defects caused by accidents, diseases and other reasons is increasing. In order to minimize the pain of patients, reduce surgical trauma and complications, it is particularly important to prepare bone repair materials with excellent performance. A hot topic in the research of bone repair materials is to simulate the composition, structural characteristics and mineralization process of natural bone itself, design and regulate the composition and structure of the material, obtain new bionic artificial bone repair materials or perform bionic functional modification on traditional materials. Bone tissue belongs to the collagen-calcium phosphate composite system. Among them, the inorganic component is mainly hydroxyapatite (HA), and the organic component is mainly collagen. Bone formation is a process in which calcium phosphate minerals are deposited in an orderly manner on the collagen fiber matrix, and then the mineralized collagen is arranged in an orderly manner.

[0003] Biomineralization is the phenomenon of minerals depositing on the surface of organisms as functional and structural parts. Mineralized collagen (MC) is a material with collagen and hydroxyapatite as the main components, among which hydroxyapatite is the main inorganic component of human bone tissue. Using the idea of ​​biomimetic, a hydroxyapatite and collagen composite with a hierarchical structure similar to that of natural bone can be prepared.

[0004] Currently, there are many examples of using animal-derived collagen for mineralization to mimic natural bone components and their formation process. However, animal-derived collagen has disadvantages such as viral risks, low preparation purity, immunogenicity leading to immune responses, and infectious diseases. These disadvantages greatly limit the production and application of collagen in the fields of biomaterials. At the same time, traditional animal-derived collagen is mostly prepared by acid, alkali, and enzymatic hydrolysis, and is mostly dissolved by acid during the biomineralization process. Affected by external conditions, the collagen extracted by these methods or the collagen solution obtained reduces the original biological activity of collagen and weakens its ideal function.

[0005] Recombinant humanized collagen is obtained by optimizing and modifying the amino acid composition of recombinantly expressed human collagen using genetic engineering technology. It is 100% identical to the amino acid sequence of the functional region of natural human collagen and does not produce immune rejection or allergic reactions when used in the human body. According to the "Guidelines for the Naming of Recombinant Collagen Biomaterials", "recombinant humanized collagen" can be further divided into two types, type A and type B, depending on whether it contains non-human collagen amino acid sequences: type A refers to materials that do not contain non-human collagen amino acid sequences, including the full-length amino acid sequence encoded by the gene of a specific type of human collagen (without a triple helix structure), or a partial amino acid sequence fragment encoded by the gene of a specific type of human collagen, or a combination of human collagen functional fragments (the amino acid sequence does not contain non-human collagen amino acid sequences such as connecting amino acids and label amino acids); type B refers to materials that add non-human collagen amino acid sequences such as connecting amino acids and label amino acids to the combination of human collagen functional fragments. Chinese invention patents ZL202110968550.2 and ZL201811438582.6 respectively record two types of type A recombinant humanized collagens that were researched and produced by the applicant of the present invention in the early stage, namely type A recombinant type I humanized collagen and type A recombinant type III humanized collagen. The amino acid sequences of the two proteins are sequences that have been screened and optimized for a long time, have very good hydrophilicity and stability, and the amino acid sequence composition is 100% identical to the corresponding part of the natural collagen amino acid sequence. The two proteins have excellent cell adhesion effects and will not cause immune rejection and allergic reactions when used in the human body. However, there is currently no literature disclosing their specific uses in the field of bone repair materials. Summary of the invention

[0006] Problem that the invention aims to solve

[0007] Although the prior art has studied bionic bone repair materials and recombinant humanized collagen, it is still not sufficient. For example, there are still biosafety risks in the use of animal-derived collagen for biomineralization, and the original biological activity of animal-derived collagen is reduced due to the preparation method. There is still a need for bone repair materials with better properties in this field. In this regard, the present invention provides a method for preparing a type A recombinant humanized collagen bone repair material, and a type A recombinant humanized collagen bone repair material prepared by the method and its use. The application of collagen-based bone materials in bone repair is broadened, so that it can be used in bone defect repair in orthopedics and dentistry.

[0008] Solutions for solving problems

[0009] [1]. A method for preparing a bone repair material, wherein the method comprises a first step of precipitation and a second step of dialysis:

[0010] The first step of precipitation method comprises the following steps:

[0011] (i) dissolving type A recombinant humanized collagen in water to obtain solution I;

[0012] (ii) adding a solution containing Ca 2+ The solution was stirred and PO4 3- The solution was stirred to obtain solution II;

[0013] (iii) adjusting the pH value of the solution II to 8.0-10.0 and allowing to stand to obtain a solution III;

[0014] The second step of dialysis comprises the following steps:

[0015] (iv) dialyzing the solution III and then freeze-drying it to obtain material I;

[0016] (v) Grinding the material I to obtain the bone repair material.

[0017] [2] The preparation method according to [1], wherein the content of the type A recombinant humanized collagen in the solution I is 0.5 to 2.5 mg / ml.

[0018] [3] The preparation method according to [1] or [2], wherein in the solution II, the type A recombinant humanized collagen and the Ca 2+ The content ratio is 1g: (0.007-0.056) mol, the Ca 2+ With the PO4 3- The molar ratio is 1.6 to 1.7.

[0019] [4] The preparation method according to any one of [1] to [3], wherein the Ca-containing 2+ The solution comprises an aqueous solution of CaCl2 containing PO4 3- The solution includes aqueous NaH2PO4.

[0020] [5] The preparation method according to any one of [1] to [4], wherein the preparation method is a two-step method, and the dialysis process in the second step dialysis method comprises two days of dialysis with running water and three days of dialysis with deionized water.

[0021] [6]. The preparation method according to any one of [1] to [5], wherein in step (ii), the stirring temperature is 25±5°C and the stirring time is 1.5 to 2.5 h;

[0022] And / or, in the step (iii), the standing time is 5.5 to 6.5 hours.

[0023] [7]. The preparation method according to any one of [1] to [6], wherein in step (iv), the molecular weight cut-off of the dialysis is greater than or equal to 1000 Da.

[0024] [8]. A bone repair material, wherein the bone repair material is prepared by the preparation method according to any one of [1] to [7].

[0025] [9] The bone repair material according to [8], wherein the content of type A recombinant humanized collagen in the bone repair material is greater than or equal to 4 wt % based on dry weight.

[0026]

[10] . Use of the bone repair material according to [8] or [9] in the preparation of materials for filling, repairing and guiding bone regeneration for treating bone defects.

[0027] Effects of the Invention

[0028] Through the implementation of the above technical solution, the present invention achieves the following beneficial effects:

[0029] The present invention provides a method for preparing a bone repair material, which uses type A recombinant humanized collagen as a collagen matrix, adopts a two-step method of precipitation method combined with dialysis method, simulates the composition and formation process of natural bone, and successfully prepares a bone repair material with a high collagen ratio and activity. Type A recombinant humanized collagen has good hydrophilicity and stability, can be dissolved in water, does not lose the function of collagen itself during the neutral mineralization process, and can be purified and collected by dialysis to improve the utilization rate of collagen. The bone repair material is prepared by a two-step method of precipitation method combined with dialysis method, which can make collagen gradient degradation and release, improve collagen expression activity, and improve osteogenesis.

[0030] The present invention provides a bone repair material prepared by the above method. The recombinant humanized collagen bone repair material is prepared by using type A recombinant humanized collagen with excellent water solubility and excellent cell adhesion as a mineralization deposition template. It not only avoids the immunogenicity and infectious disease problems of animal-derived collagen, but also experiments have confirmed that its high collagen activity and proportion give it more excellent osteogenic performance.

[0031] The bone repair material prepared by using type A recombinant humanized collagen provided by the present invention can be widely used in the repair of bone defects caused by various reasons, thus broadening the application of collagen in the field of biomedical materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Thermogravimetric test graphs of the type A recombinant type I humanized collagen bone repair material (denoted as A / P1) prepared based on the one-step acid-soluble precipitation method in Example 1 and the type A recombinant type I humanized collagen bone repair material (denoted as W / P1) prepared based on the one-step water-soluble precipitation method in Example 2.

[0033] Figure 2 Infrared spectra of type A recombinant type I humanized collagen bone repair materials (respectively denoted as P / D1, P / D2, and P / D3) prepared in Examples 3, 4, and 5 based on a two-step method (precipitation method and dialysis method), type A recombinant type I humanized collagen (denoted as Pure Collgen), and hydroxyapatite (denoted as HA) prepared in Example 7.

[0034] Figure 3 Thermogravimetric test graphs of type A recombinant type I humanized collagen bone repair materials (respectively denoted as P / D1, P / D2, and P / D3) prepared in Example 3, Example 4, and Example 5.

[0035] Figure 4 Scanning electron microscope images and EDS energy spectrum analysis graphs of the type A recombinant type I humanized collagen bone repair materials prepared in Example 3, Example 4, and Example 5 (respectively denoted as P / D1, P / D2, and P / D3) and the hydroxyapatite prepared in Example 7 (denoted as HA).

[0036] Figure 5 XRD patterns of the type A recombinant type I humanized collagen bone repair materials prepared in Example 3, Example 4, and Example 5 (respectively denoted as P / D1, P / D2, and P / D3) and the hydroxyapatite prepared in Example 7 (denoted as HA).

[0037] Fig. 6A This is a one-month pure water degradation analysis chart of the hydroxyapatite prepared in Example 7.

[0038] Figure 6B This is a one-month pure water degradation analysis chart of the type A recombinant type I humanized collagen bone repair material prepared in Example 5.

[0039] Figure 7 Infrared spectra of type A recombinant type I humanized collagen bone repair materials prepared in Examples 3, 4 and 5 after degradation for 5 days, 7 days and 28 days (respectively denoted as 5-P / D1, 5-P / D2, 5-P / D3, 7-P / D1, 7-P / D2, 7-P / D3, 28-P / D3), type A recombinant type I humanized collagen (denoted as Pure Collgen) and hydroxyapatite prepared in Example 7 (denoted as HA).

[0040] Fig. 8ASEM images of the type A recombinant type I humanized collagen bone repair materials prepared in Example 3, Example 4 and Example 5 after degradation for 5 and 7 days (respectively denoted as 5-P / D1, 5-P / D2, 5-P / D3, 7-P / D1, 7-P / D2 and 7-P / D3).

[0041] Figure 8B This is a SEM image of the type A recombinant type I humanized collagen bone repair material prepared in Example 5 after 28 days of degradation (denoted as 28-P / D3).

[0042] Fig. 9 The results of CCK-8 detection of cell proliferation in pure culture medium (denoted as Control), extracts of hydroxyapatite of different concentrations (Example 7) (denoted as 50% HA and 100% HA, respectively), and extracts of type A recombinant type I humanized collagen bone repair materials (Example 5) of different ratios (denoted as 50% P / D3 and 100% P / D3, respectively).

[0043] Fig.10 The relative cell activity test results are shown in pure culture medium (denoted as Control), extracts of hydroxyapatite of different concentrations (Example 7) (denoted as 50% HA and 100% HA, respectively), and extracts of type A recombinant type I humanized collagen bone repair materials (Example 5) of different ratios (denoted as 50% P / D3 and 100% P / D3, respectively).

[0044] Fig.11 The results of CCK-8 detection of cell proliferation in pure culture medium (denoted as Control), extracts of different concentrations of hydroxyapatite (Example 7) (denoted as 50% HA and 100% HA, respectively), extracts of different proportions of type A recombinant type I humanized collagen bone repair material (Example 5) (denoted as 50% P / D3 and 100% P / D3, respectively), and extracts of different proportions of type I bovine collagen bone repair material (Example 6) (denoted as 50% P-BC1, 100% P-BC1, 50% P-BC2, 100% P-BC2, 50% P-BC3, 100% P-BC3, 50% P-BC4, 100% P-BC4, respectively).

[0045] Fig.12 The live / dead staining results of cells in extracts of different concentrations of hydroxyapatite (Example 7) (respectively denoted as 50% HA and 100% HA), extracts of different ratios of type A recombinant type I humanized collagen bone repair material (Example 5) (respectively denoted as 50% P / D3 and 100% P / D3) and pure culture medium (respectively denoted as Control).

[0046] Fig.13These are the results of Alizarin red staining of cells in pure culture medium (denoted as Control), type A recombinant type I humanized collagen bone repair material prepared in Example 5 (denoted as P / D3), and hydroxyapatite prepared in Example 7 (denoted as HA).

[0047] Fig.14 The alkaline phosphatase (ALP) test results of pure culture medium (denoted as Control), type A recombinant type I humanized collagen bone repair material prepared in Example 5 (denoted as 50% P / D3 and 100% P / D3, respectively), type I bovine collagen bone repair material prepared in Example 6 (denoted as 50% P-BC1, 100% P-BC1, 50% P-BC2, 100% P-BC2, 50% P-BC3, 100% P-BC3, 50% P-BC4, 100% P-BC4, respectively) and hydroxyapatite prepared in Example 7 (denoted as 50% HA and 100% HA, respectively).

[0048] Fig.15 The results are shown in Figure 2, which are the results of CCK-8 detection of cell proliferation in pure culture medium (denoted as Control), type A recombinant type I humanized collagen extract (denoted as HC), and type I bovine collagen extract (denoted as BC).

[0049] Fig.16 The results of CCK-8 detection of cell proliferation in pure culture medium (denoted as Control), hydroxyapatite (Example 7) extract (denoted as HA), type I bovine collagen bone repair material (Example 6) extract (denoted as P-BC4), type A recombinant type I humanized collagen bone repair material (Example 5) extract (denoted as P / D3), type A recombinant type III humanized collagen bone repair material (Example 8) extract (denoted as P / D-III) and type A recombinant type I / type III humanized collagen bone repair material (Example 9) extract (denoted as P / DI / III). DETAILED DESCRIPTION

[0050] The following describes the embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various structures described below, and various changes can be made within the scope of the invention claim, and the embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0051] Definition of terms

[0052] In the present invention, a numerical range expressed using "a value A to a value B" or "a value A - a value B" means a range including the endpoints A and B.

[0053] In the present invention, a numerical range expressed using "above" or "below" means a numerical range including the number.

[0054] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0055] In the present invention, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances where the event occurs and instances where it does not occur.

[0056] In the present invention, the term "a" or "an" or "the" may mean "one", and may also mean "one or more", "at least one" and "one or more than one".

[0057] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0058] In the present invention, the term "about" may mean that a value includes the error and standard deviation of the device or method used to determine the value. The numerical ranges and parameters used to define the present invention are all approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains errors and standard deviations caused by the aforementioned testing devices or methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in the present invention are modified by "about". Here, "about" generally means that the actual value is within ±5%, ±1% or ±0.5% of a particular value or range.

[0059] In the present invention, the term "recombinant humanized collagen" refers to the full-length or partial amino acid sequence fragment encoded by the gene of a specific type of human collagen prepared by DNA recombination technology, or a combination of functional fragments of human collagen. Furthermore, "type A" means that the recombinant humanized collagen does not contain non-human collagen amino acid sequences.

[0060] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] Preparation method of bone repair material

[0062] The present invention provides a method for preparing a bone repair material, the method comprising a first step of precipitation method and a second step of dialysis method:

[0063] The first step of precipitation method comprises the following steps:

[0064] (i) dissolving type A recombinant humanized collagen in water to obtain solution I;

[0065] (ii) adding a solution containing Ca 2+ The solution was stirred and PO4 3- The solution was stirred to obtain solution II;

[0066] (iii) adjusting the pH value of the solution II to 8.0-10.0 and allowing to stand to obtain a solution III;

[0067] The second step dialysis method comprises the following steps:

[0068] (iv) dialyzing the solution III and then freeze-drying it to obtain material I;

[0069] (v) Grinding the material I to obtain the bone repair material.

[0070] The preparation method provided by the present invention utilizes type A recombinant humanized collagen as a collagen matrix, adopts a two-step method of precipitation method combined with dialysis method, simulates the composition and formation process of natural bone, and successfully prepares a bone repair material.

[0071] <Step (i)>

[0072] The mineralized deposition template for preparing the bone repair material of the present invention is type A recombinant humanized collagen. Exemplarily, the type A recombinant humanized collagen includes type A recombinant type I humanized collagen and / or type A recombinant type III humanized collagen.

[0073] In some embodiments, the type A recombinant humanized collagen is type A recombinant type I humanized collagen.

[0074] In some specific embodiments, the amino acid sequence of the type A recombinant type I humanized collagen comprises the sequence shown in any one of the following (a1) to (d1):

[0075] (a1) the amino acid sequence shown in SEQ ID NO.1;

[0076] (b1) an amino acid sequence that is 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO.1, and that retains the cell adhesion effect of the amino acid sequence shown in SEQ ID NO.2;

[0077] (c1) an amino acid sequence in which one or more amino acid residues are added, substituted or deleted in the amino acid sequence as shown in SEQ ID NO.1, and which retains the cell adhesion effect of the amino acid sequence as shown in SEQ ID NO.1;

[0078] (d1) An amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO.1 under stringent conditions, and the amino acid sequence retains the cell adhesion effect of the amino acid sequence shown in SEQ ID NO.1, and the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

[0079] In some preferred embodiments, the amino acid sequence of the type A recombinant humanized collagen comprises the amino acid sequence shown in SEQ ID NO.1.

[0080] In some more preferred embodiments, the amino acid sequence of the type A recombinant humanized collagen is the amino acid sequence shown in SEQ ID NO.1.

[0081] In some embodiments, the type A recombinant humanized collagen is type A recombinant type III humanized collagen.

[0082] In some specific embodiments, the amino acid sequence of the type A recombinant type III humanized collagen comprises the sequence shown in any one of the following (a2) to (d2):

[0083] (a2) the amino acid sequence shown in SEQ ID NO.2;

[0084] (b2) an amino acid sequence that is 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO.2, and that retains the cell adhesion effect of the amino acid sequence shown in SEQ ID NO.2;

[0085] (c2) an amino acid sequence in which one or more amino acid residues are added, substituted or deleted in the amino acid sequence as shown in SEQ ID NO.2, and which retains the cell adhesion effect of the amino acid sequence as shown in SEQ ID NO.2;

[0086] (d2) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO.2 under stringent conditions, and the amino acid sequence retains the cell adhesion effect of the amino acid sequence shown in SEQ ID NO.2, wherein the stringent conditions are medium stringent conditions, medium-high stringent conditions, high stringent conditions or very high stringent conditions.

[0087] In some preferred embodiments, the amino acid sequence of the type A recombinant humanized collagen comprises the amino acid sequence shown in SEQ ID NO.2.

[0088] In some more preferred embodiments, the amino acid sequence of the type A recombinant humanized collagen is the amino acid sequence shown in SEQ ID NO.2.

[0089] In the present invention, illustratively, type A recombinant humanized collagen can be produced by the following steps: (1) construction of genetically engineered Escherichia coli; (2) fermentation culture of genetically engineered Escherichia coli; (3) induction and expression of type A recombinant humanized collagen; (4) purification and optional enzymatic cleavage of type A recombinant humanized collagen.

[0090] The key to initiating and controlling the mineralization process is that the oxygen atoms in the two groups (-COOH and C=O) in type A recombinant humanized collagen can react with Ca 2+ The coordination reaction occurs, and the small molecular weight makes it water-soluble, so that its molecules expose more of the above two groups.

[0091] At the same time, the type A recombinant humanized collagen used in the present invention has good hydrophilicity and stability, so that it can be mineralized by water-soluble method. Comparing the two experimental methods of acid-soluble mineralization and water-soluble mineralization, water-soluble method is preferred for mineralization, so that the activity of collagen itself will not be lost during the mineralization process, and the utilization rate of collagen is improved.

[0092] In some specific embodiments, the step (i) comprises: dissolving type A recombinant humanized collagen in water, stirring at room temperature (25±5° C.) for 0.5 h, to obtain solution I.

[0093] In some embodiments, in the solution I, the content of the type A recombinant humanized collagen is 0.5 to 2.5 mg / ml; preferably 0.6 to 2.4 mg / ml.

[0094] <Step (ii)>

[0095] In some specific embodiments, the step (ii) comprises: slowly adding a solution containing Ca 2+ The solution was stirred at room temperature (25±5℃) at a constant speed and PO4 was slowly added 3- The solution was stirred at a constant speed at room temperature (25±5° C.) to obtain solution II. The stirring time is preferably 1.5 to 2.5 hours, more preferably 2 hours, from the perspective of being beneficial to the mineralization of type A recombinant humanized collagen.

[0096] In some embodiments, in the solution II, the type A recombinant humanized collagen and the Ca 2+ The content ratio of Ca is 1g: (0.007-0.056) mol, preferably 1g: (0.007-0.03) mol, more preferably 1g: (0.01-0.03) mol, and further preferably 1g: 0.01 mol. 2+ With the PO4 3- The molar ratio of calcium to phosphorus is 1.6 to 1.7, preferably 1.67. This ratio makes the calcium to phosphorus ratio in the bone repair material closer to the calcium to phosphorus ratio of natural bone hydroxyapatite.

[0097] In some embodiments, the Ca 2+ The solution comprises an aqueous solution of CaCl2 containing PO4 3- The solution includes aqueous NaH2PO4.

[0098] In some specific embodiments, the concentration of the CaCl2 aqueous solution is 0.05-0.15 mol / L, preferably 0.1 mol / L, and the concentration of the NaH2PO4 aqueous solution is 0.05-0.15 mol / L, preferably 0.1 mol / L. 2 + With PO4 3- It is more favorable to the mineralization ratio of type A recombinant humanized collagen.

[0099] <Step (iii)>

[0100] In some embodiments, the step (iii) comprises: adjusting the pH value of the solution II to 8.0-10.0, and allowing the solution to stand to obtain a solution III.

[0101] In some embodiments, from the perspective of fully precipitating the type A recombinant humanized collagen bone repair material, the pH value of solution II is preferably adjusted to about 8-10, and preferably allowed to stand for 5.5-6.5 hours, more preferably for 6 hours.

[0102] In some specific embodiments, a low concentration of acid / base solution can be used to adjust the pH value of solution II to better maintain the biological activity of type A recombinant humanized collagen; for example, a 0.1 mol / L NaOH aqueous solution can be used to adjust the pH value of solution II.

[0103] <Step (iv)>

[0104] In some embodiments, the step (iv) comprises: placing the solution III into a dialysis bag, dialyzing against running water for two days, dialyzing against deionized water for three days, and then pre-freezing and freeze-drying to obtain material I.

[0105] In order to improve the problem of low collagen collection rate caused by the one-step precipitation method, the present invention introduces a dialysis method, which adopts a two-step method of precipitation followed by dialysis to improve the utilization rate of collagen, achieve gradient degradation and exert active effects to achieve better osteogenesis.

[0106] In some embodiments, from the perspective of fully removing impurities and retaining relatively pure type A recombinant humanized collagen bone repair material, the molecular weight cutoff of the dialysis bag is greater than or equal to 1000 Da, preferably 1000 Da.

[0107] In some embodiments, the pre-freezing temperature is -20°C, the pre-freezing time is 2 hours, and the freeze-drying conditions include: freeze-drying at -96°C for 48 hours.

[0108] <Step (v)>

[0109] In some embodiments, the step (v) comprises: the material I is ground with agate and passed through a 200-mesh sieve to obtain the bone repair material.

[0110] <Other steps>

[0111] In some embodiments, in order to obtain bone repair materials with multiple collagen matrices, the preparation method further comprises the step of mixing bone repair materials prepared using two different types of recombinant humanized collagen A.

[0112] Bone repair materials

[0113] The present invention provides a bone repair material prepared by the above-mentioned preparation method. By using type A recombinant humanized collagen with excellent water solubility and cell adhesion as a mineralization template to prepare a recombinant humanized collagen bone repair material, not only the immunogenicity and infectious disease problems of animal-derived collagen are avoided, but also experiments have confirmed that it has more excellent osteogenic activity.

[0114] Based on dry weight, the proportion of type A recombinant humanized collagen in the bone repair material provided by the present invention can reach more than 4%, preferably more than 5%, preferably greater than 5%, more preferably more than 10%, and preferably less than 15%. Compared with the prepared bovine collagen bone repair material, the bone repair material provided by the present invention has better cell proliferation promoting properties and ALP activity. At the same time, compared with hydroxyapatite, the bone repair material provided by the present invention has better cell proliferation promoting and osteogenic differentiation ability.

[0115] use

[0116] The bone repair material provided by the present invention can be used to prepare materials for filling and repairing bone defects and guiding bone regeneration.

[0117] In some embodiments, the bone defect comprises a maxillofacial surgery bone defect, a dental surgery bone defect, a skull bone defect, various closed fracture bone defects of the limbs, and / or a spinal bone defect.

[0118] The amino acid sequence and nucleotide sequence involved in the present invention

[0119] Amino acid sequence of type A recombinant type I humanized collagen (SEQ ID NO.1):

[0120] GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS GEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGAS.

[0121] Amino acid sequence of type A recombinant type Ш humanized collagen (SEQ ID NO.2):

[0122] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP。

[0123] Nucleotide sequence of the coding gene of recombinant type I humanized collagen type A (SEQ ID NO.3):

[0124] GGTGAAAAAGGTAGTCCGGGTGCAGATGGTCCGGCCGGTGCACCTGGTACACCGGGTCCTCAGGGCATTGCAGGCCAGCGTGGTGTTGTGGGCCTGCCGGGTCAGCGCGGTGAACGTGGTTTTCCGGGTCTGCCGGGTCCGAGCGGCGAACCTGGTAAACAGGGCCCGAGTGGTGCAAGCGGTGAAAAAGGCAGTCCGGGCGCAGATGGTCCTGCAGGTGCCCCTGGTACACCTGGTCCGCAGGGTATTGCAGGTCAGCGCGGCGTGGTGGGCCTGCCTGGTCAACGTGGCGAACGTGGTTTCCCGGGCCTGCCGGGACCGTCAGGTGAACCTGGCAAACAGGGCCCTAGCGGTGCAAGTGGCGAAAAAGGTAGCCCGGGCGCAGACGGCCCGGCAGGTGCACCTGGCACACCTGGTCCTCAGGGTATTGCGGGCCAGCGCGGCGTTGTTGGTCTGCCTGGCCAGCGTGGCGAACGCGGTTTTCCGGGCCTGCCTGGCCCGTCAGGCGAACCTGGAAAACAGGGCCCAAGTGGTGCAAGTGGTGAAAAAGGAAGTCCGGGCGCCGATGGCCCGGCCGGTGCGCCTGGTACGCCTGGTCCTCAAGGCATTGCCGGTCAGCGCGGAGTTGTTGGCCTGCCGGGCCAGCGTGGAGAACGCGGTTTCCCGGGTCTGCCTGGTCCGAGTGGTGAACCGGGTAAACAGGGTCCGAGCGGTGCATCATAA。

[0125] Nucleotide sequence of the coding gene of recombinant type Ш humanized collagen of type A (SEQ ID NO.4):

[0126]

[0127] Example

[0128] The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out under normal conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used are all commercially available conventional products.

[0129] The amino acid sequence of the type A recombinant type I humanized collagen used in the examples is shown in SEQ ID NO.1, and the amino acid sequence of the type A recombinant type II humanized collagen used in the examples is shown in SEQ ID NO.2.

[0130] The specific preparation method of type A recombinant type I / type III humanized collagen used in the examples refers to Chinese invention patents ZL202110968550.2 and ZL201811438582.6, the contents of which are incorporated herein by reference.

[0131] Example 1

[0132] Preparation method of type A recombinant type I humanized collagen bone repair material A / P1 based on acid dissolution precipitation method:

[0133] Dissolve 0.03g of type A recombinant type I humanized collagen in 50ml acetic acid solution (0.6mg / ml) and stir for 12h. Add 16.93ml CaCl2 solution (0.1mol / L) to the collagen solution and stir for 2h. Prepare phosphoric acid solution according to the calcium-phosphorus ratio of 1.67, and drop 10.14ml phosphoric acid solution (0.1mol / L) into the mixed solution and stir for 2h. Add NaOH (0.1mol / L) to adjust and maintain the pH value of the mixed solution at 8-10, stir for 6h and let it stand for 12h. Pour off the supernatant, wash and centrifuge the precipitate until the pH of the supernatant is 7. Pre-freeze the precipitate at -20℃ and freeze-dry it for 24h. After grinding with agate and passing through a 200-mesh sieve, the type A recombinant type I humanized collagen bone repair material based on the acid precipitation method is obtained, which is recorded as A / P1.

[0134] Example 2

[0135] Preparation method of type A recombinant type I humanized collagen bone repair material W / P1 based on water-soluble precipitation method:

[0136] Dissolve 0.03g of type A recombinant type I humanized collagen in 50ml pure water and stir for 12h. Add 11.97ml CaCl2 solution (0.1mol / L) to the collagen solution and stir for 2h. Prepare phosphoric acid solution according to the calcium-phosphorus ratio of 1.67, and drop 7.17ml phosphoric acid solution (0.1mol / L) into the mixed solution and stir for 2h. Add NaOH (0.1mol / L) to adjust and maintain the pH value of the mixed solution to 8-10, stir for 6h and let it stand for 12h. Pour off the supernatant, wash and centrifuge the precipitate until the pH of the supernatant is 7. Pre-freeze the precipitate at -20℃, freeze-dry for 48h, grind it with agate and pass it through a 200-mesh sieve to obtain type A recombinant type I humanized collagen bone repair material based on the water-soluble precipitation method, recorded as W / P1.

[0137] Example 3

[0138] Preparation method of type A recombinant type I humanized collagen bone repair material P / D1:

[0139] After dissolving 0.03g of type A recombinant type I humanized collagen in 50ml of pure water, a 0.6mg / ml type A recombinant type I humanized collagen solution was prepared, and 8.95ml of CaCl2 solution (0.1mol / L) was slowly added dropwise at room temperature (25±5℃), stirred for 2h, and 5.37ml of NaH2PO4 solution (0.1mol / L) was added dropwise at room temperature (25±5℃), and stirred for 2h. The pH value was adjusted to about 8-10 with 0.1mol / L NaOH, and then allowed to stand for 6h, placed in a dialysis bag (1000Da) for dialysis (dialysis with running water for two days and dialysis with deionized water for three days), placed in a -20℃ refrigerator for pre-freezing for 2h, freeze-dried for 48h, ground with agate and passed through a 200-mesh sieve, and type A recombinant type I humanized collagen bone repair material was obtained, which was recorded as P / D1.

[0140] Example 4

[0141] Preparation method of type A recombinant type I humanized collagen bone repair material P / D2:

[0142] After dissolving 0.06g of type A recombinant type I humanized collagen in 50ml of pure water, a 1.2mg / ml type A recombinant type I humanized collagen solution was prepared, and 8.95ml of CaCl2 solution (0.1mol / L) was slowly added dropwise at room temperature (25±5℃), stirred for 2h, and 5.37ml of NaH2PO4 solution (0.1mol / L) was added dropwise at room temperature (25±5℃), and stirred for 2h. The pH value was adjusted to about 8-10 with 0.1mol / L NaOH, and then allowed to stand for 6h, placed in a dialysis bag (1000Da) for dialysis (dialysis with running water for two days and dialysis with deionized water for three days), placed in a -20℃ refrigerator for pre-freezing for 2h, freeze-dried for 48h, ground with agate and passed through a 200-mesh sieve, and type A recombinant type I humanized collagen bone repair material was obtained, which was recorded as P / D2.

[0143] Example 5

[0144] Preparation method of type A recombinant type I humanized collagen bone repair material P / D3:

[0145] After dissolving 0.12g of type A recombinant type I humanized collagen in 50ml of pure water, a 2.4mg / ml type A recombinant type I humanized collagen solution was prepared, and 8.95ml of CaCl2 solution (0.1mol / L) was slowly added dropwise at room temperature (25±5℃), stirred for 2h, and 5.37ml of NaH2PO4 solution (0.1mol / L) was added dropwise at room temperature (25±5℃), and stirred for 2h. The pH value was adjusted to about 8-10 with 0.1mol / L NaOH, and then allowed to stand for 6h, placed in a dialysis bag (1000Da) for dialysis (dialysis with running water for two days and dialysis with deionized water for three days), placed in a -20℃ refrigerator for pre-freezing for 2h, freeze-dried for 48h, ground with agate and passed through a 200-mesh sieve, and type A recombinant type I humanized collagen bone repair material was obtained, which was recorded as P / D3.

[0146] Example 6

[0147] Preparation method of type I bovine collagen bone repair material P-BC:

[0148] An appropriate amount of type I bovine collagen gel (MW = (27-30) × 10 4 Da) was dissolved in 0.5 mol / L acetic acid solution to prepare a 1 mg / ml collagen solution, stirred for 12 h, and then heated to 0.12 mol Ca 2+Slowly add 0.1mol / L CaCl2 solution to the collagen solution corresponding to 1g collagen, stir for 2h at room temperature (25±5℃), and add 0.1mol / L NaH2PO4 solution according to the calcium-phosphorus molar ratio (Ca / P) of 1.67, and stir for 2h at room temperature (25±5℃). Adjust the pH value to about 8-10 with 0.5mol / L NaOH and let it stand for 6h. Centrifuge and wash several times until the pH value of the supernatant is about 7. Take the precipitate and place it in a -20℃ refrigerator for pre-freezing for 2h, freeze-dry for 48h, grind it with agate and pass it through a 200-mesh sieve to obtain bovine collagen bone repair material. Different batches are recorded as P-BC1, P-BC2, P-BC3 and P-BC4.

[0149] Example 7

[0150] Preparation method of hydroxyapatite HA:

[0151] Take 250ml of 0.1mol / L CaCl2 solution and stir it at room temperature (25±5℃) for 2h, then add 0.1mol / L NaH2PO4 solution according to Ca / P of 1.67, and continue stirring for 10h at room temperature (25±5℃). Adjust the pH value to around 8-10 with 0.5mol / L NaOH and let it stand for 12h, then wash it with deionized water for more than three times until the pH value of the supernatant is about 7. Prefreeze the obtained precipitate in a -20℃ refrigerator for 2h, freeze-dry it for 48h, grind it with agate and pass it through a 200-mesh sieve to obtain hydroxyapatite material, recorded as HA.

[0152] Example 8

[0153] Preparation method of type A recombinant type III humanized collagen bone repair material P / D-III:

[0154] After dissolving 0.12g of type A recombinant type III humanized collagen in 50ml of pure water, a 2.4mg / ml type A recombinant type III humanized collagen solution was prepared, and 8.95ml of CaCl2 solution (0.1mol / L) was slowly added dropwise at room temperature (25±5℃), stirred for 2h, and 5.37ml of NaH2PO4 solution (0.1mol / L) was added dropwise at room temperature (25±5℃), and stirred for 2h. The pH value was adjusted to about 8-10 with 0.1mol / L NaOH, and then allowed to stand for 6h, placed in a dialysis bag (1000Da) for dialysis (dialysis with running water for two days and dialysis with deionized water for three days), placed in a -20℃ refrigerator for pre-freezing for 2h, freeze-dried for 48h, ground with agate and passed through a 200-mesh sieve, and type A recombinant type III humanized collagen bone repair material was obtained, which was recorded as P / D-III.

[0155] Example 9

[0156] Preparation method of type A recombinant type I / type III humanized collagen bone repair material P / DI / III:

[0157] Dissolve 0.12g of type A recombinant type I humanized collagen in 50ml of pure water to obtain a 2.4mg / ml type A recombinant type I humanized collagen solution, slowly drop 8.95ml of CaCl2 solution (0.1mol / L), stir at room temperature (25±5℃), drop 5.37ml of NaH2PO4 solution (0.1mol / L), stir at room temperature (25±5℃) for 2h. Adjust the pH value to around 8-10 with 0.1mol / L NaOH, let stand for 6h, put into a dialysis bag (1000Da) for dialysis (dialysis with running water for two days, dialysis with deionized water for three days), put in a -20℃ refrigerator for pre-freezing for 2h, freeze-dry for 48h, and obtain type A recombinant type I humanized collagen bone repair raw material. Secondly, 0.12g of type A recombinant type III humanized collagen was dissolved in 50ml of pure water to obtain a 2.4mg / ml type A recombinant type III humanized collagen solution, 8.95ml of CaCl2 solution (0.1mol / L) was slowly added dropwise at room temperature (25±5℃), stirred for 2h, and 5.37ml of NaH2PO4 solution (0.1mol / L) was added dropwise at room temperature (25±5℃), and stirred for 2h. The pH value was adjusted to about 8-10 with 0.1mol / L NaOH, and then allowed to stand for 6h, placed in a dialysis bag (1000Da) for dialysis (two days of dialysis with running water and three days of dialysis with deionized water), placed in a -20℃ refrigerator for pre-freezing for 2h, and freeze-dried for 48h to obtain type A recombinant type III humanized collagen bone repair raw material. Type A recombinant type I humanized collagen bone repair raw material and type A recombinant type III humanized collagen bone repair raw material were weighed in a mass ratio of 1:1, ground with agate and passed through a 200-mesh sieve to obtain type A recombinant type I / type III humanized collagen bone repair material, recorded as P / DI / III.

[0158] Experimental example

[0159] 1. Thermogravimetric test (TG test) was performed on the type A recombinant type I humanized collagen bone repair material A / P1 prepared by the acid-soluble precipitation method in Example 1 and the type A recombinant type I humanized collagen bone repair material W / P1 prepared by the water-soluble precipitation method in Example 2. The specific results are shown in Tables 1 and Figure 1 During the test, the first step occurred in the range of 30-200°C, and the weight loss in this stage was related to the gradual loss of bound water. The second step occurred in the range of 200-600°C, and the weight loss in this stage was related to the degradation of collagen chains. Most of the weight loss occurred during the second heating process.

[0160] Through Table 1 and Figure 1 It can be found that since the free carboxyl groups in the collagen molecules are the main nucleation sites for the mineralization of collagen fibers, in the collagen solution containing calcium ions, Ca 2+ It can preferentially coordinate with the carboxyl group (-COOH) in collagen, while acetic acid can be Ca 2+ Ions provide more nucleation sites, so the organic content of type A recombinant type I humanized collagen bone repair material prepared by acetic acid is slightly higher in TG test, but compared with the type A recombinant type I humanized collagen bone repair material prepared by water-soluble method, its organic content is close, and the difference between the two is negligible. In order to avoid the loss of the function of collagen itself, type A recombinant type I humanized collagen bone repair material based on water-soluble precipitation method is preferred.

[0161] Table 1 Thermogravimetric test results

[0162] A / P1 Example 1 W / P1 Example 2 Bound water 4.34％ 4.19％ Organic (Collagen) 3.11％ 3.06％ Inorganic (HA, phosphate) 92.54％ 92.75％ Organic:Inorganic 3.36％ 3.30％

[0163] 2. The type A recombinant type I humanized collagen bone repair materials prepared in Examples 3, 4 and 5, the type A recombinant type I humanized collagen and the hydroxyapatite prepared in Example 7 were characterized by Fourier transform infrared spectroscopy (FTIR) (resolution of 4 cm -1 The sample scanning time is 24 scans, and the scanning range is 500~4000cm -1 ), the result is as follows Figure 2 shown.

[0164] The spectrum of type A recombinant humanized type I collagen is at 1645 cm -1 、1541cm -1 The peaks appearing at 1645 cm are the characteristic absorption peaks of amide I and amide II. -1 The beam moved to the low beam and red-shifted. The amide II and amide III bands that appeared in the spectrum of type A recombinant type I humanized collagen weakened or even disappeared, indicating that the vibration of the amide bond in the type A recombinant type I humanized collagen bone repair material was suppressed due to the deposition of HA. This means that there is a certain chemical reaction between the organic phase and the inorganic phase in the material, and the site of action occurs on the amide bond.

[0165] 3. Thermogravimetric tests were performed on the type A recombinant type I humanized collagen bone repair materials prepared in Examples 3, 4 and 5. The specific results are shown in Tables 2 and Figure 3During the test, the first step occurred in the range of 30-200°C, and the weight loss in this stage was related to the gradual loss of bound water. The second step occurred in the range of 200-600°C, and the weight loss in this stage was related to the degradation of collagen chains. Most of the weight loss occurred during the second heating process.

[0166] Through Table 2 and Figure 3 It can be found that as the concentration of type A recombinant type I humanized collagen in the solution used for mineralization increases, its proportion in the overall bone repair material also increases.

[0167] Table 2 Thermogravimetric test results

[0168]

[0169] 4. The elements in the type A recombinant type I humanized collagen bone repair materials prepared in Examples 3, 4 and 5 and the hydroxyapatite prepared in Example 7 were analyzed by energy dispersive X-ray spectrometry (EDS) (EDS acquisition profile resolution 256, pixel dwell time 200 μm, acceleration voltage adjusted to 10 kV, and limited scanning elements were Ca and P elements). The results are as follows: Figure 4 shown.

[0170] The calcium-phosphorus ratio of hydroxyapatite in the type A recombinant type I humanized collagen bone repair material was measured to be around 1.67, which is close to the calcium-phosphorus ratio of natural bone hydroxyapatite. At the same time, scanning electron microscopy (SEM) testing showed that the hydroxyapatite deposited on the surface of the material had a good morphology and was evenly distributed.

[0171] 5. The type A recombinant type I humanized collagen bone repair materials prepared in Examples 3, 4 and 5 and the hydroxyapatite prepared in Example 7 were subjected to phase analysis, structural analysis and phase analysis of their crystals by X-ray diffraction (XRD) (scanning speed of 5° / min, scanning angle of 20° to 80°). The results are as follows: Figure 5 shown.

[0172] Due to the existence of collagen assembly, the crystallization of the inorganic phase HA of the type A recombinant type I humanized collagen bone repair material prepared in Example 3, Example 4 and Example 5 is restricted, so the particle size of the HA crystal is very small, which also causes the broadening effect of the peak position on the X-ray diffraction pattern. In addition, the XRD diffraction pattern of the type A recombinant type I humanized collagen bone repair material is very similar to that of natural bone, indicating that its crystal composition and size are similar to those of natural bone. (002), (211), (004) are characteristic crystal planes of hydroxyapatite. It can be found that the inorganic component of the type A recombinant type I humanized collagen bone repair material formed after the liquid in the dialysis bag is freeze-dried is hydroxyapatite.

[0173] 6. The degradation of the type A recombinant type I humanized collagen bone repair material prepared in Example 5 and the hydroxyapatite prepared in Example 7 in pure water for one month was tested: incubated in a constant temperature shaker at 37°C, the shaking rate of the shaker was 90 rpm, the water was changed every two days, and the samples were taken out and dried in an oven every week to record the weight. The results are as follows Fig. 6A and Figure 6B shown.

[0174] By calculating the percentage of weight loss to original weight, it was found that the degradation rates of the type A recombinant type I humanized collagen bone repair material prepared in Example 5 and the hydroxyapatite prepared in Example 7 in pure water within one month were 16.16% and 4.95%, respectively, as shown in the following formula:

[0175]

[0176] Where: M i is the initial weight of the sample, M w,t is the dry weight of the sample at each time.

[0177] 7. The type A recombinant type I humanized collagen bone repair materials prepared in Examples 3, 4 and 5 (as the experimental group) were subjected to the above steps for degradation for 5 days, 7 days and 28 days, and then FTIR characterization (resolution of 4 cm -1 The sample scanning time is 24 scans, and the scanning range is 500~4000cm -1 ), the control group was undegraded type A recombinant type I humanized collagen and hydroxyapatite prepared in Example 7. The results are as follows Figure 7 shown.

[0178] It can be found that when the experimental group was degraded for 5 and 7 days, the peak of amide I existed, the peaks of amide II and amide III were weak, and the peak at 1030 cm -1 、600cm -1 、560cm -1 The peak at is still obvious, indicating that the type A recombinant type I humanized collagen in the type A recombinant type I humanized collagen bone repair material has not been completely degraded, and the hydroxyapatite component is still present. After 28 days of degradation, the peak of amide I in the experimental group still exists, indicating that the type A recombinant type I humanized collagen in the type A recombinant type I humanized collagen bone repair material is retained due to the deposition of calcium phosphate, which effectively slows down the degradation process of the type A recombinant type I humanized collagen component.

[0179] 8. After the above steps of degradation of the type A recombinant type I humanized collagen bone repair materials prepared in Examples 3, 4 and 5 for 5 days, 7 days and 28 days, the surface micromorphology thereof was tested by scanning electron microscopy. The results are as follows: Fig. 8A and Figure 8B shown.

[0180] and Figure 4 By comparison, it was found that the surface was mainly characterized by the lamellar morphology of hydroxyapatite, and type A recombinant type I humanized collagen fibers were still observed after 28 days of degradation.

[0181] 9. With reference to GB / T16886.5: Sample preparation and reference samples for biological evaluation of medical devices, 40 mg of type A recombinant type I humanized collagen bone repair material powder prepared in Example 5 (prepared by two-step method, concentration of 2.4 mg / ml), 40 mg of type I bovine collagen bone repair material powder prepared in Example 6, and 40 mg of hydroxyapatite powder prepared in Example 7 were weighed, and after UV sterilization, they were prepared into 5 mg / ml suspension using DMEM culture medium containing 15% bovine serum by volume, and placed in a 37°C cell culture incubator for 24 h. After centrifugation at 1000 r / min for 5 min, the supernatant was aspirated and filtered through a 0.22 supernatant filter to obtain 100% type A recombinant type I humanized collagen bone repair material extract, 100% type I bovine collagen bone repair material extract, and 100% hydroxyapatite extract, respectively. Take 3 ml of the above extracts in the corresponding centrifuge tubes, add 3 ml of cell culture medium DMEM to obtain 50% type A recombinant type I humanized collagen bone repair material extract, 50% type I bovine collagen bone repair material extract and 50% hydroxyapatite extract. MC3T3-E1 cells (seeding density 2.5×10 4 Cells / mL) were inoculated in a 96-well plate containing DMEM and incubated in a cell culture incubator at 37°C. One day later, 100% and 50% type A recombinant type I humanized collagen bone repair material extract, 100% and 50% type I bovine collagen bone repair material extract, 100% and 50% hydroxyapatite extract, and pure DMEM medium were used to replace the type A recombinant type I humanized collagen bone repair material group, type I bovine collagen bone repair material group and hydroxyapatite group, cell-containing pure culture medium group (as a control group), and the cell-free pure culture medium group (as a blank group) continued to be cultured with the original culture medium for 1, 3, and 5 days to evaluate cytotoxicity. The proliferation of MC3T3-E1 cells in different type A recombinant type I extracts was evaluated using the CCK-8 test.

[0182] The CCK-8 test results are expressed by the formula: Relative cell viability = [(A s -A b ) / (A c -A b)](Ativ was used to calculate the relative viability of cells after culturing cells with 50% and 100% extracts of the type A recombinant type I humanized collagen bone repair material in Example 5 and 50% and 100% extracts of hydroxyapatite in Example 7, respectively, so as to compare the cytotoxicity of the type A recombinant type I humanized collagen bone repair material in Example 5 and the hydroxyapatite in Example 7. s : Absorbance of experimental wells (containing cells, culture medium, CCK-8 solution); A c : Absorbance of control well (containing cells, culture medium, CCK-8 solution); A b : Absorbance of blank well (containing culture medium, CCK-8 solution, but no cells).

[0183] CCK-8 test results Figures 9-11 shown. Fig. 9 and Fig.10 The results showed that the differences between the experimental group and the control group and the blank group at each time point (1, 3, and 5 days) were statistically significant, and the absorbance value of the experimental group increased significantly with the increase of incubation time. There were significant differences between the experimental group and the control group and the blank group, which may be due to the fact that the type A recombinant type I humanized collagen contained in it promoted cell proliferation. With the extension of culture time, all cells had obvious proliferation ability, indicating that the material has good cell compatibility and can maintain the normal growth and proliferation of MC3T3-E1 cells. Fig.11 In the experiment, the CCK-8 test results showed that the absorbance of 100% type A recombinant type I humanized collagen bone repair material group was higher than that of the control group, 100% hydroxyapatite group and 100% type I bovine collagen bone repair material group, indicating that the type A recombinant type I humanized collagen bone repair material prepared by the two-step method has better cell proliferation activity than the type I bovine collagen bone repair material prepared by the one-step method.

[0184] 10. According to GB / T16886.5: Sample preparation and reference samples for biological evaluation of medical devices, 40 mg of type A recombinant type I humanized collagen bone repair material prepared in Example 5 (prepared by two-step method, concentration is 2.4 mg / ml) and 40 mg of hydroxyapatite powder prepared in Example 7 were weighed, and after UV sterilization, they were prepared into 5 mg / ml suspension using DMEM culture medium containing 15% bovine serum by volume, placed in a 37°C cell culture incubator for 24 hours, centrifuged at 1000 r / min for 5 minutes, and the supernatant was aspirated and filtered through a 0.22 μm filter membrane to obtain 100% type A recombinant type I humanized collagen bone repair material extract and 100% hydroxyapatite extract. 3 ml of the above extracts were taken in corresponding centrifuge tubes, and 3 ml of cell culture medium DMEM was added to obtain 50% type A recombinant type I humanized collagen bone repair material extract and 50% hydroxyapatite extract. MC3T3-E1 cells (seeding density 2×10 4 Cells / mL) were seeded in a 24-well plate and incubated in a cell culture incubator at 37°C using cell culture medium DMEM. After one day, the original culture medium was replaced with 100% and 50% type A recombinant type I humanized collagen bone repair material extract, 100% and 50% hydroxyapatite extract and pure culture medium, respectively, and culture was continued for 1, 3, and 5 days. The adherent cells were washed 1-3 times with PBS. 250 μL of Calcein AM / PI detection working solution (Beyotime Biotechnology, C2015M) was added to each well and incubated at 37°C in the dark for 30 minutes. After incubation, the staining was observed under a fluorescence microscope to obtain live / dead staining images. The results are shown in the figure. Fig.12 .

[0185] Observing the cell morphology under a fluorescence microscope, MC3T3-E1 cells proliferated and survived well, and the density of live cells increased with the increase of culture time. After 5 days of culture, the cell density in the extract of type A recombinant type I humanized collagen bone repair material of different concentrations in Example 5 was significantly higher than the cell density in the hydroxyapatite extract of different concentrations and pure culture medium in Example 7. Very small amounts of dead cells were detected using low-brightness red fluorescence, indicating that the prepared type A recombinant type I humanized collagen bone repair material had no obvious cytotoxic effect on MC3T3-E1 cells. And the type A recombinant type I humanized collagen bone repair material has good cell compatibility, which is consistent with the results of the above-mentioned CCK-8 test.

[0186] 11. According to GB / T16886.5: Sample preparation and reference samples for biological evaluation of medical devices, 40 mg of type A recombinant type I humanized collagen bone repair material powder prepared in Example 5 (prepared by two-step method, concentration of 2.4 mg / ml) and 40 mg of hydroxyapatite powder prepared in Example 7 were weighed and sterilized with ultraviolet light and then incubated in osteogenic differentiation medium (HyCyte TM , EOMX-D101) was prepared into a 5 mg / ml suspension, placed in a 37°C cell culture incubator for 24 hours, centrifuged at 1000 r / min for 5 minutes, and the supernatant was aspirated and filtered through a 0.22 μm filter membrane to obtain 100% type A recombinant type I humanized collagen bone repair material osteogenic differentiation extract and 100% hydroxyapatite osteogenic differentiation extract. Take 3 ml of the above extracts in the corresponding centrifuge tubes, and add 3 ml of pure osteogenic differentiation medium to obtain 50% type A recombinant type I humanized collagen bone repair material osteogenic differentiation extract and 50% hydroxyapatite osteogenic differentiation extract. MC3T3-E1 cells (seeding density of 2×10 4 Cells / mL) were seeded in a 24-well plate and incubated in a 37°C cell culture incubator using cell culture medium DMEM. After the cells were fused to 90%, the corresponding original culture medium was replaced with 100% and 50% type A recombinant type I humanized collagen bone repair material osteogenic differentiation extract, 100% and 50% hydroxyapatite osteogenic differentiation extract and pure osteogenic differentiation medium to continue culturing. After 7 days, the adherent cells were washed and fixed with PBS, and then incubated with Alizarin Red stain (Solarbio, G1452) at 37°C for 30 minutes, and the staining was observed under a fluorescence microscope. The results are shown in the figure. Fig.13 .

[0187] Alizarin red was used to stain the calcium nodules deposited on the surface of the differentiated cells. Compared with the pure culture medium and the hydroxyapatite group of Example 7, the type A recombinant type I humanized collagen bone repair material group of Example 5 had a deep color and a large amount, indicating that it had good bone formation ability. It is speculated that this is because the type A recombinant type I humanized collagen bone repair material promotes cell proliferation, and more cells form more calcium nodules under the action of hydroxyapatite.

[0188] 12. According to GB / T16886.5: Sample preparation and reference samples for biological evaluation of medical devices, 40 mg of type A recombinant type I humanized collagen bone repair material powder prepared in Example 5 (prepared by two-step method, concentration of 2.4 mg / ml), 40 mg of type I bovine collagen bone repair material powder prepared in Example 6, and 40 mg of hydroxyapatite powder prepared in Example 7 were weighed and sterilized with ultraviolet light and then incubated in osteogenic differentiation medium (HyCyte TM, EOMX-D101) was prepared into a 5 mg / ml suspension, placed in a 37°C cell culture incubator for 24 hours, centrifuged at 1000 r / min for 5 minutes, and the supernatant was aspirated and filtered through a 0.22 μm filter membrane to obtain 100% type A recombinant type I humanized collagen bone repair material osteogenic differentiation extract, 100% type I bovine collagen bone repair material osteogenic differentiation extract, and 100% hydroxyapatite osteogenic differentiation extract. MC3T3-E1 cells (seeding density of 2×10 4 Cells / mL) were seeded in a 24-well plate and incubated in a 37°C cell culture incubator using cell culture medium DMEM. After the cells were fused to 90%, the corresponding original culture medium was replaced with 100% type A recombinant type I humanized collagen bone repair material osteogenic differentiation extract, 100% type I bovine collagen bone repair material osteogenic differentiation extract, 100% hydroxyapatite osteogenic differentiation extract and pure osteogenic differentiation medium to continue culturing. After 7 days, cell lysis solution (Beyotime, P0013) was added to each well and placed on ice. The lysate was aspirated for 4 minutes, and after centrifugation, 50 μL of the supernatant was taken and incubated with 50 μL of the lysis reaction substrate (Beyotime, P0321S) at 37°C for 30 minutes. Finally, the stop solution (Beyotime, P0321S) was added, and the absorbance was measured at 415 nm in a 96-well plate. The results are shown in the table. Fig.14 :

[0189] The results show that the type A recombinant type I humanized collagen bone repair material of Example 5 has a significantly improved ability to promote osteogenic differentiation compared with the type I bovine collagen bone repair material of Example 6 and the hydroxyapatite of Example 7.

[0190] 13. Refer to GB / T16886.5: Sample preparation and reference samples for biological evaluation of medical devices. Weigh 40 mg of type A recombinant type I humanized collagen and 40 mg of type I bovine collagen. After UV sterilization, use DMEM medium containing 15% bovine serum to make a 5 mg / ml suspension. Place in a 37°C cell culture incubator for 24 hours. Centrifuge at 1000 r / min for 5 minutes, aspirate the supernatant, and filter through a 0.22 μm filter membrane to obtain 100% type A recombinant type I humanized collagen extract and 100% type I bovine collagen extract. MC3T3-E1 cells (seeding density of 2.5×10 4Cells / mL) were inoculated in a 96-well plate containing DMEM and incubated in a cell culture incubator at 37°C. One day later, 100% type A recombinant type I humanized collagen extract, 100% type I bovine collagen extract and pure DMEM medium were used to replace the type A recombinant type I humanized collagen group, type I bovine collagen group and pure DMEM group (as a control group), respectively. The cell-free pure culture medium group (as a blank group) continued to be cultured with the original culture medium for 1, 3, and 5 days to evaluate cytotoxicity. The proliferation of MC3T3-E1 cells in type A recombinant type I humanized collagen extract and type I bovine collagen extract was evaluated using the CCK-8 test.

[0191] CCK-8 test results Fig.15 As shown in the figure, the differences between the experimental group and the control group at each time point (1, 3, and 5 days) were statistically significant, and the absorbance value of the experimental group increased significantly with the increase of incubation time. On the 5th day of culture, the number of cells in the type A recombinant type I humanized collagen extract group was significantly higher than that in the type I bovine collagen extract group. The results showed that water-soluble type A recombinant type I humanized collagen had better cell proliferation activity than acid-soluble type I bovine collagen.

[0192] 14. Referring to the biological evaluation of medical devices GB / T16886.5: Sample preparation and reference samples, weigh 40 mg of type A recombinant type I humanized collagen bone repair material powder prepared in Example 5 (prepared by precipitation dialysis method, concentration is 2.4 mg / ml), 40 mg of type A recombinant type III humanized collagen bone repair material powder prepared in Example 8 (prepared by precipitation dialysis method, concentration is 2.4 mg / ml), 40 mg of type A recombinant type I / type III humanized collagen bone repair material powder prepared in Example 9, 40 mg of type I bovine collagen bone repair material powder prepared in Example 6, and 40 mg of hydroxyapatite powder prepared in Example 7. 40 mg of the final product was sterilized by ultraviolet and then prepared into a 5 mg / ml suspension in DMEM medium containing 15% bovine serum by volume. The suspension was placed in a cell culture incubator at 37°C for 24 hours. After centrifugation at 1000 r / min for 5 minutes, the supernatant was aspirated and filtered through a 0.22 μm filter membrane to obtain 100% type A recombinant type I humanized collagen bone repair material extract, 100% type A recombinant type III humanized collagen bone repair material extract, 100% type A recombinant type I / type III humanized collagen bone repair material extract, 100% type I bovine collagen bone repair material extract and 100% hydroxyapatite extract. MC3T3-E1 cells (seeding density of 2.5×10 4Cells / mL) were seeded in a 96-well plate containing DMEM and incubated in a cell culture incubator at 37°C. One day later, 100% A-type recombinant type I humanized collagen bone repair material extract, 100% A-type recombinant type III humanized collagen bone repair material extract, 100% A-type recombinant type I / type III humanized collagen bone repair material extract, 100% A-type recombinant type I / type III humanized collagen bone repair material extract, 100% I-type bovine collagen bone repair material extract, 100% hydroxyapatite extract and pure DMEM medium were used to replace the original culture medium of the A-type recombinant type I humanized collagen bone repair material group, the A-type recombinant type III humanized collagen bone repair material group, the A-type recombinant type I / type III humanized collagen bone repair material group, the I-type bovine collagen bone repair material group, the hydroxyapatite group and the pure DMEM group (as the control group) for 1, 3, and 5 days to evaluate cytotoxicity. The CCK-8 assay was used to evaluate the proliferation of MC3T3-E1 cells in the extracts of type A recombinant type I humanized collagen bone repair material, type A recombinant type III humanized collagen bone repair material, and type A recombinant type I / type III humanized collagen bone repair material.

[0193] CCK-8 test results Fig.16 As shown in the figure, the differences between the experimental group and the control group at each time point (1, 3, and 5 days) were statistically significant, and the absorbance value of the experimental group increased significantly with the increase of incubation time. On the 5th day of culture, the number of cells in the A type recombinant type I humanized collagen bone repair material extract group was significantly higher than that in the type I bovine collagen bone repair material extract group and the hydroxyapatite extract group (p<0.001). The results showed that the A type recombinant type I humanized collagen bone repair material prepared by the two-step method had better cell proliferation activity than the type I bovine collagen bone repair material and hydroxyapatite prepared by the one-step method.

Claims

1. A method for preparing a bone repair material, characterized in that: The method comprises a first step of precipitation and a second step of dialysis: The first step of precipitation method comprises the following steps: (i) dissolving type A recombinant humanized collagen in water to obtain solution I; (ii) adding a solution containing Ca 2+ The solution was stirred and PO4 3- The solution was stirred to obtain solution II; (iii) adjusting the pH value of the solution II to 8.0-10.0, and allowing to stand for 5.5-6.5 hours to obtain a solution III; The second step of dialysis comprises the following steps: (iv) dialyzing the solution III and then freeze-drying it to obtain material I; (v) grinding the material I to obtain the bone repair material; Furthermore, in the solution II, the type A recombinant humanized collagen and the Ca 2+ The content ratio is 1g: (0.007~0.056)mol; The type A recombinant humanized collagen is type A recombinant type I humanized collagen or type A recombinant type III humanized collagen, the amino acid sequence of the type A recombinant type I humanized collagen is the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence of the type A recombinant type III humanized collagen is the amino acid sequence shown in SEQ ID NO.2; The Ca 2+ The solution comprises an aqueous solution of CaCl2 containing PO4 3- The solution includes a NaH2PO4 aqueous solution, the concentration of the CaCl2 aqueous solution is 0.05 to 0.15 mol / L, and the concentration of the NaH2PO4 aqueous solution is 0.05 to 0.15 mol / L.

2. The preparation method according to claim 1, characterized in that: In the solution I, the content of the type A recombinant humanized collagen is 0.5-2.5 mg / ml.

3. The preparation method according to claim 1 or 2, characterized in that: In the solution II, the Ca 2+ With the PO4 3- The molar ratio is 1.6 to 1.

7.

4. The preparation method according to claim 1 or 2, characterized in that: The preparation method is a two-step method, wherein the dialysis process in the second step dialysis method comprises two days of flowing water dialysis and three days of deionized water dialysis.

5. The preparation method according to claim 1 or 2, characterized in that: In the step (ii), the stirring temperature is 25±5° C., and the stirring time is 1.5 to 2.5 hours.

6. The preparation method according to claim 1 or 2, characterized in that: In the step (iv), the molecular weight cut-off of the dialysis is greater than or equal to 1000 Da.

7. A bone repair material, characterized in that: The bone repair material is prepared by the preparation method according to any one of claims 1 to 6.

8. The bone repair material according to claim 7, characterized in that: Based on dry weight, the content of type A recombinant humanized collagen in the bone repair material is greater than or equal to 4wt%.

9. Use of the bone repair material according to claim 7 or 8 in preparing materials for filling, repairing and guiding bone regeneration for treating bone defect diseases.

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