Triple-helix self-assembly recombinant humanized I-type collagen fiber implant as well as preparation method and application of triple-helix self-assembly recombinant humanized I-type collagen fiber implant

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a triple-helix self-assembly recombinant humanized I-type collagen fiber implant as well as a preparation method and application of the triple-helix self-assembly recombinant humanized I-type collagen fiber implant. The invention firstly provides a recombinant humanized type I collagen, wherein the recombinant humanized type I collagen has a unique triple helix structure and an excellent self-assembly property; secondly, by optimizing the self-assembly process of the recombinant humanized I-type collagen, the recombinant humanized I-type collagen implant with orderly arranged collagen fibers is prepared, and the implant has the advantages of excellent injectability, durability, high biological safety, no heating and hemolysis risk, low immunogenicity, good biocompatibility, good biocompatibility and good biocompatibility. Adhesion, migration, proliferation and differentiation of human skin fibroblasts are remarkably promoted, adhesion, migration and proliferation of cartilage cells and chondrogenic differentiation of bone marrow mesenchymal stem cells can be remarkably promoted, and the application to the fields of skin, cartilage, bone and the like can be achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a triple-helix self-assembly recombinant humanized type I collagen fiber implant, a preparation method and applications thereof. Background Art

[0002] Skin aging is the most visible manifestation of human aging, characterized by changes in skin structure, function, and appearance, such as deepening wrinkles, sagging, dilated capillaries, and uneven pigmentation. The root cause of these phenomena is the degradation and loss of collagen in the skin, which leads to decreased skin elasticity, weakened support, and impaired barrier function.

[0003] Collagen, the primary structural protein of the skin, possesses a characteristic triple-helical structure and can self-assemble into collagen fibers with superior morphology and mechanical strength. This plays a vital role in maintaining skin elasticity and supporting its structure, and can be used as an implant for skin rejuvenation. However, the raw materials for currently available collagen implants are primarily animal-derived collagen obtained through extraction methods, which carries risks such as viral transmission and immunogenicity. Furthermore, the recombinant humanized type I collagen reported in the literature lacks self-assembly activity.

[0004] Compared with collagen from animal sources, recombinant collagen produced by genetic engineering can overcome these shortcomings, but its structural domain is smaller and it is difficult to self-assemble into collagen fibers, which limits its application in advanced fields such as tissue engineering and regenerative medicine. Summary of the Invention

[0005] In response to the above technical problems, the present invention obtains a recombinant humanized type I collagen THRCI with a unique triple helix structure and excellent self-assembly properties through special amino acid sequence design; the recombinant humanized type I collagen is prepared through self-assembly to obtain a recombinant humanized type I collagen implant with excellent injectability and durability; the recombinant humanized type I collagen fiber implant has excellent biological activity, not only significantly promoting the adhesion, migration, proliferation, and differentiation of human skin fibroblasts, but also significantly promoting the adhesion, migration, proliferation of chondrocytes and the chondrogenic differentiation of bone marrow mesenchymal stem cells, and has high biosafety, no pyrogenicity and hemolytic risks, and low immunogenicity, and can be applied to the fields of skin, cartilage, bone, etc. Specifically including the following contents:

[0006] In a first aspect, the present invention provides a recombinant humanized type I collagen having a triple helical structure, the sequence of which is shown in SEQ ID NO.1.

[0007] Preferably, the recombinant humanized type I collagen is obtained by treating a precursor collagen with a protease; the amino acid sequence of the precursor collagen is shown in SEQ ID NO.2.

[0008] Preferably, the gene sequence of the precursor collagen is shown as SEQ ID NO.3.

[0009] In a second aspect, the present invention provides use of the recombinant humanized type I collagen described in the first aspect in the preparation of a recombinant humanized type I collagen fiber implant.

[0010] In a third aspect, the present invention provides a recombinant humanized type I collagen fiber implant, wherein the recombinant humanized type I collagen implant is formed by self-assembly of the recombinant humanized type I collagen described in the first aspect.

[0011] Preferably, the preparation method of the recombinant humanized type I collagen implant comprises: dissolving the recombinant humanized type I collagen in a buffer solution of 0-200 mM PB and pH 5.0-7.4 to a concentration of 50-100 mg / mL, standing at 4-25° C. for 24-72 hours, and then centrifuging to precipitate the recombinant humanized type I collagen fiber implant.

[0012] Preferably, the centrifugation parameters are: 1000-10000 rpm for 5-20 min.

[0013] Preferably, the PB is 50 mM.

[0014] Preferably, the pH is 7.4.

[0015] Preferably, the concentration of the recombinant humanized type I collagen is 75 mg / mL.

[0016] Preferably, the standing temperature is 25°C.

[0017] Preferably, the standing time is 24 hours.

[0018] Preferably, the centrifugation parameters are: 10000 rpm for 10 min.

[0019] In a fourth aspect, the present invention provides use of the recombinant humanized type I collagen fiber implant described in the third aspect in the preparation of medical devices for use in the fields of skin, cartilage, and bone.

[0020] In a fifth aspect, the present invention provides a cross-linked recombinant humanized type I collagen fiber implant, which is obtained by cross-linking the recombinant humanized type I collagen fiber implant described in the third aspect with a cross-linking agent.

[0021] Preferably, the cross-linking agent is selected from BDDE or THPC.

[0022] Preferably, the preparation method of the cross-linked recombinant humanized type I collagen fiber implant comprises the following steps:

[0023] (1) preparing the recombinant humanized type I collagen fiber implant according to the third aspect;

[0024] (2) The recombinant humanized type I collagen fiber implant was dispersed in a 0-200 mM PB, pH 5.0-7.4 buffer solution, and 0.001-2.0% BDDE or 0.001-0.05% THPC was added, respectively. The mixture was cross-linked at 4-25°C for 24-72 hours and then centrifuged. The precipitate was washed with a 0-200 mM PB, pH 5.0-7.4 buffer solution to obtain the cross-linked recombinant humanized type I collagen fiber implant.

[0025] Preferably, the centrifugation parameters are: 1000-10000 rpm for 5-20 min.

[0026] Preferably, the PB is 50 mM.

[0027] Preferably, the pH is 7.4.

[0028] Preferably, the cross-linking temperature is 4°C.

[0029] Preferably, the cross-linking time is 24 hours.

[0030] Preferably, the centrifugation parameters are: 10000 rpm for 10 min.

[0031] Preferably, the volume fraction of the BDDE is 2.0%, and the volume fraction of the THPC is 0.05%.

[0032] Preferably, the step (2) comprises: dispersing the recombinant humanized type I collagen fiber implant into a 0-200 mM PB, pH 5.0-7.4 buffer solution, adding 2.0% BDDE or 0.05% THPC by volume, cross-linking at 4°C for 24 hours, and centrifuging at 10,000 rpm for 10 minutes; precipitating three times with a 0-200 mM PB, pH 5.0-7.4 buffer solution, and centrifuging at 10,000 rpm for 10 minutes to obtain a precipitate, which is the cross-linked recombinant humanized type I collagen fiber implant.

[0033] In a sixth aspect, the present invention provides use of the cross-linked recombinant humanized type I collagen fiber implant described in the fifth aspect in the preparation of medical devices for use in the fields of skin, cartilage, and bone.

[0034] The beneficial effects of the present invention are as follows: (1) Recombinant humanized type I collagen reported in general literature has no self-assembly activity. Based on this, the present invention first obtains a recombinant humanized type I collagen with a unique triple helical structure and excellent self-assembly properties through a special amino acid sequence design; (2) By optimizing the self-assembly process of recombinant humanized type I collagen, a recombinant humanized type I collagen implant with orderly arranged collagen fibers is prepared; (3) The recombinant humanized type I collagen fiber implant prepared by the method has excellent biological activity, which not only significantly promotes The recombinant humanized type I collagen fiber implant has excellent injectability and durability compared with the type I collagen prepared by extraction; it has high biosafety, no risk of pyrogenesis and hemolysis, and low immunogenicity; (5) the recombinant humanized type I collagen fiber implant described in the present invention can be applied to the fields of skin, cartilage, bone, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 These are the characterization results of triple-helical recombinant humanized type I collagen; A is the SDS-PAGE characterization result, and B is the circular dichroism characterization result.

[0036] Figure 2 Scanning electron microscopy images and fiber diameter statistics of recombinant humanized type I collagen fiber implants assembled in buffer solutions of different concentrations; A and a are 20 mM PB, B and b are 50 mM PB, C and c are 100 mM PB, and D and d are 200 mM PB.

[0037] Figure 3 Scanning electron microscopy images and fiber diameter statistics of recombinant humanized type I collagen fiber implants assembled under different pH conditions; A is pH = 5.0, and B is pH = 7.4.

[0038] Figure 4 Scanning electron microscopy images and fiber diameter statistics of recombinant humanized type I collagen fiber implants assembled at different temperatures; A and a are 4°C, and B and b are 25°C.

[0039] Figure 5 Scanning electron micrographs and fiber diameter statistics of recombinant humanized type I collagen fiber implants assembled at different protein concentrations; A and a are 50 mg / mL, B and b are 75 mg / mL, and C and c are 100 mg / mL.

[0040] Figure 6Characterization of the physicochemical properties of recombinant humanized type I collagen fiber implants; A is the detection of denatured collagen, B is the injectability, C is the thermal stability, and D is the durability.

[0041] Figure 7 The figures are the evaluation results of the biocompatibility and bioactivity of recombinant humanized type I collagen fiber implants; wherein, A represents the cell adhesion (a), cell migration (b), cell proliferation (c), cell migration rate (d), live / dead cell staining (e), immunofluorescence staining (f), and the expression of α-SMA (g), Collagen I (h), and Collagen III (i) genes after HFF-1 cells were co-cultured with recombinant humanized type I collagen fiber implants; B represents the cell adhesion (a), cell migration (b), cell proliferation (c), cell migration rate (d), live / dead cell staining (e), immunofluorescence staining (f), and the expression of cartilage-specific genes Sox 9 (g), Acan (h), and (i) after chondrocytes were co-cultured with recombinant humanized type I collagen fiber implants.

[0042] Figure 8 These are the results of the biosafety evaluation of recombinant humanized type I collagen fiber implants; A is the pyrogen test, B is the hemolysis test, and C is the determination of serum total IgG, IgA, and IgM levels.

[0043] Figure 9 The scanning electron micrographs and fiber diameter statistics of recombinant humanized type I collagen fiber implants cross-linked with different cross-linking agents; A and a are BDDE, and B and b are THPC.

[0044] Figure 10 To characterize the durability of cross-linked implants.

[0045] Figure 11 These are the results of biocompatibility and bioactivity evaluation of cross-linked recombinant humanized type I collagen implants; AF are the cell migration, cell migration rate, live / dead cell staining, cell proliferation, immunofluorescence staining, and cell differentiation results of BDDE cross-linked recombinant humanized type I collagen implants, and GL are the cell migration, cell migration rate, live / dead cell staining, cell proliferation, immunofluorescence staining, and cell differentiation results of THPC cross-linked recombinant humanized type I collagen implants. DETAILED DESCRIPTION

[0046] The present invention is described in detail below through specific embodiments. Any technical solutions that can be conceived by those skilled in the art based on the present invention and in combination with common knowledge in the art belong to the protection scope of the present invention.

[0047] Unless otherwise specified, the methods described in the following examples are conventional methods familiar to those skilled in the art.

[0048] Example 1 Characterization of Recombinant Humanized Type I Collagen Preparation

[0049] 1. Preparation of Recombinant Humanized Type I Collagen

[0050] The amino acid sequence of the recombinant humanized type I collagen is shown in SEQ ID NO.1, and the protein is obtained by treating the precursor collagen with protease; the amino acid sequence of the precursor collagen is shown in SEQ ID NO.2, and the gene sequence is shown in SEQ ID NO.3.

[0051] The gene sequence of the synthesized precursor collagen was constructed into the Escherichia coli expression vector pCold, and the successful synthesis of the plasmid was confirmed by DNA sequencing. The plasmid was transformed into the Escherichia coli BL21-DE3 strain to obtain the precursor collagen expression strain, and the successfully transformed strain was stored in glycerol at -80°C.

[0052] Expression of procollagen: 20 μL of the low-temperature stored bacterial solution was added to 200 mL of LB liquid medium containing antibiotics. After overnight culture at 37°C constant temperature shaker, the inoculum was transferred to 1 L of LB liquid medium containing antibiotics at a 2% inoculum volume and continued to be cultured at 37°C constant temperature shaker. 600 When the value reaches the range of 1.2-2.0, the shaker temperature is adjusted to 25°C, IPTG is added at a final concentration of 1 mM to induce expression, and culture is carried out at a constant temperature overnight; the bacterial solution is centrifuged in a low-temperature centrifuge at 3500 rpm, 4°C, for 30 min, and the bacteria are collected.

[0053] Purification of recombinant humanized type I collagen: Bacteria were dissolved in a buffer solution (20 mM sodium phosphate buffer, 20 mM imidazole, 0.5 M sodium chloride, pH 7.4) at a ratio of 1:10. The cells were disrupted using a high-pressure homogenizer. The resulting suspension was centrifuged again, and the supernatant, the crude protein solution, was collected and further purified using a nickel affinity chromatography column to obtain the procollagen. Thrombin was added to a final concentration of 8 U / mL to treat the procollagen, purifying and removing the enzymatic cleavage products to obtain recombinant humanized type I collagen.

[0054] 2. SDS-PAGE Characterization of Recombinant Humanized Type I Collagen

[0055] SDS-PAGE was used to characterize the recombinant humanized type I collagen (THRCI).

[0056] The results are as follows Figure 1As shown in Figure A, the recombinant humanized type I collagen is a single band, indicating the successful preparation of high-purity recombinant humanized type I collagen. This protein band is located around 40 kDa. Due to the unique amino acid sequence and structure of collagen, its migration rate on SDS-PAGE is slower than that of globular proteins of the same molecular weight. Therefore, the apparent molecular weight of this recombinant humanized type I collagen is greater than the theoretical molecular weight (24.7 kDa), which is consistent with the characteristics of collagen.

[0057] 3. Circular Dichroism Characterization of Recombinant Humanized Type I Collagen

[0058] Recombinant humanized type I collagen was prepared into a 0.5 mg / mL solution and subjected to circular dichroism spectroscopy scanning at full wavelength (190-260 nm) using a 1 mm cuvette at 4°C with a wavelength interval of 1 nm and a dwell time of 5 s at each wavelength.

[0059] The results are as follows Figure 1 As shown in B, the recombinant humanized type I collagen shows a characteristic absorption peak of collagen at around 220 nm, indicating that the recombinant humanized type I collagen described in the present application has a triple helical structure.

[0060] Example 2 Preparation of recombinant humanized type I collagen fiber implant

[0061] 1. Preparation of recombinant humanized type I collagen fiber implants using buffer solutions of different concentrations

[0062] (1) Preparation method

[0063] The recombinant humanized type I collagen prepared in Example 1 was dissolved in 20, 50, 100, and 200 mM PB buffer solutions (pH = 7.4) to prepare 50 mg / mL recombinant humanized type I collagen solutions;

[0064] The recombinant humanized type I collagen solution was incubated at 25° C. for 24 h, and then centrifuged at 4° C. and 10,000 rpm for 10 min. The precipitate was collected to obtain the recombinant humanized type I collagen fiber implant.

[0065] (2) Scanning electron microscopy characterization of recombinant humanized type I collagen fiber implants

[0066] The recombinant humanized type I collagen fiber implant sample prepared in (1) above was fixed on the sample stage of a scanning electron microscope, sprayed with gold for 30 seconds, and the sample morphology was detected at an operating voltage of 5.0 kV.

[0067] The results are as follows Figure 2As shown in the figure: the diameters of the fibers assembled by recombinant humanized type I collagen at 20mM, 50mM, 100mM and 200mM PB were (174±43)nm, (232±44)nm, (288±46)nm and (305±48)nm, respectively; the results showed that with the increase of PB concentration, the fiber diameter of the recombinant humanized type I collagen implant gradually increased, and orderly arranged collagen fibers were prepared under the condition of 50mM PB as the buffer solution. Therefore, 50mM PB was selected as the buffer solution for assembling recombinant humanized type I collagen fiber implants in the following experiments.

[0068] 2. Preparation of recombinant humanized type I collagen fiber implants under different pH conditions

[0069] (1) Preparation method

[0070] The recombinant humanized type I collagen prepared in Example 1 was dissolved in 50 mM PB to prepare a 50 mg / mL recombinant humanized type I collagen solution;

[0071] The pH of the recombinant humanized type I collagen solution was adjusted to 5.0 and 7.4, respectively, using 2.0 M NaOH or HCl;

[0072] The recombinant humanized type I collagen solution was incubated at 25° C. for 24 h, and then centrifuged at 4° C. and 10,000 rpm for 10 min to obtain a recombinant humanized type I collagen fiber implant.

[0073] (2) Scanning electron microscopy characterization of recombinant humanized type I collagen fiber implants

[0074] The experimental steps are the same as above.

[0075] The results are as follows Figure 3 As shown in the figure: the diameters of the fibers assembled by recombinant humanized type I collagen at pH 5.0 and 7.4 were (216±49) nm and (233±47) nm, respectively; the results showed that the pH of the buffer solution had no significant effect on the formation of recombinant humanized type I collagen fibers. Subsequent experiments chose to assemble recombinant humanized type I collagen at physiological pH (pH = 7.4).

[0076] 3. Preparation of recombinant humanized type I collagen fiber implants at different protein concentrations

[0077] (1) Preparation method

[0078] The recombinant humanized type I collagen prepared in Example 1 was dissolved in 50 mM PB (pH = 7.4) to prepare recombinant humanized type I collagen solutions with concentrations of 50, 75, and 100 mg / mL, respectively;

[0079] The recombinant humanized type I collagen solutions of different concentrations were incubated at 25° C. for 24 h, and then centrifuged at 4° C. and 10,000 rpm for 10 min to obtain recombinant humanized type I collagen fiber implants.

[0080] (2) Scanning electron microscopy characterization of recombinant humanized type I collagen fiber implants

[0081] The experimental steps are the same as above.

[0082] The results are as follows Figure 4 As shown in the figure: the diameters of the fibers assembled with 50 mg / mL and 100 mg / mL recombinant humanized type I collagen were (233±46) nm and (230±49) nm, respectively. When the concentration of recombinant humanized type I collagen was 75 mg / mL, the diameter of the collagen fibers was (267±48) nm. In the following experiments, the recombinant humanized type I collagen concentration of 75 mg / mL was selected for assembly.

[0083] 4. Preparation of recombinant humanized type I collagen fiber implants at different temperatures

[0084] (1) Preparation method

[0085] The recombinant humanized type I collagen prepared in Example 1 was dissolved in 50 mM PB (pH = 7.4) to prepare a 75 mg / mL recombinant humanized type I collagen solution;

[0086] The recombinant humanized type I collagen solution was incubated at 4°C and 25°C for 24 hours, respectively, and then centrifuged at 4°C, 10,000 rpm for 10 minutes to obtain recombinant humanized type I collagen fibers.

[0087] (2) Scanning electron microscopy characterization of recombinant humanized type I collagen fiber implants

[0088] The experimental steps are the same as above.

[0089] The results are as follows Figure 5 As shown in the figure: the diameters of recombinant humanized type I collagen fibers assembled at 4°C and 25°C were (252±47) nm and (263±42) nm, respectively. The experimental results showed that the assembly temperature had no significant effect on the size of recombinant humanized type I collagen fibers. The following experiments selected the temperature of 25°C for the assembly of recombinant humanized type I collagen.

[0090] Example 3 Characterization of the physicochemical properties of recombinant humanized type I collagen fiber implants

[0091] 1. Preparation of recombinant humanized type I collagen fiber implant (ATHRCI)

[0092] The recombinant humanized type I collagen prepared in Example 1 was dissolved in 50 mM PB (pH = 7.4) to prepare a recombinant humanized type I collagen solution with a concentration of 75 mg / mL;

[0093] The recombinant humanized type I collagen solution was incubated at 25° C. for 24 h, and then centrifuged at 4° C. and 10,000 rpm for 10 min to obtain a recombinant humanized type I collagen fiber implant.

[0094] 2. Detection of denatured collagen after assembly of recombinant humanized type I collagen

[0095] Denatured collagen targeting fluorescent peptide probe FAM-GOP-14 was used to detect whether the recombinant humanized type I collagen (THRCI) and the self-assembled collagen fiber implant (ATHRCI) prepared in the example were denatured. Specifically:

[0096] Gelatin (Gel), THRCI and ATHRCI were dissolved in 10mM PB (pH 7.4) to prepare a solution with a concentration of 5 mg / mL. Take part of the THRCI solution and heat it at 90°C for 10 minutes to obtain heat-denatured THRCI (DTHRCI). Take 100 μL of each in a 96-well plate, incubate overnight at 4°C, wash 3 times with 10mM PB, and add 20 μM FAM-GOP-14 (Anal. Chem. 2024, 96, 39, 15640–15647) to each well and incubate at 4°C for 4 hours. Unbound FAM-GOP-10 was washed 3 times with 10mM PB. The fluorescence intensity (E x =497nm, E m =525nm).

[0097] The results are as follows Figure 6 As shown in A, the fluorescence intensities of Gel and DTHRCI are 28306 and 26416, respectively, while the fluorescence intensities of THRCI and ATHRCI are 6359 and 6355, which are significantly lower than those of Gel and DTHRCI. The results show that there is no risk of denaturation in the process of preparing the implant by self-assembly of recombinant humanized type I collagen in this embodiment.

[0098] 3. Injectability

[0099] The recombinant humanized type I collagen fiber implant prepared by the above method was taken and the syringe push rod was pushed at a speed of 30 mm / min to perform a pushing force test.

[0100] The results are as follows Figure 6As shown in B, the pushing force of the recombinant humanized type I collagen fiber implant prepared in this embodiment is 1.1N, indicating that the recombinant humanized type I collagen fiber implant described in this application has excellent injectability and meets the requirements of surgical hand suitability.

[0101] 4. Characterization of Thermal Stability of Recombinant Humanized Type I Collagen Fiber Implants

[0102] The prepared recombinant humanized type I collagen fiber implant was freeze-dried, and 5-10 mg of the freeze-dried sample was weighed and placed in an aluminum crucible. The temperature was raised at a rate of 10°C / min, and its thermal stability at 25-150°C was determined using a differential scanning calorimeter (DSC).

[0103] The results are as follows Figure 6 As shown in C, the denaturation temperature of the unassembled recombinant humanized type I collagen is 80.5°C, and the denaturation temperature of the assembled recombinant humanized type I collagen fiber implant is 88.5°C; the results show that the thermal stability of the assembled recombinant humanized type I collagen fiber implant is improved.

[0104] 5. Enzymatic Hydrolysis Experiment of Recombinant Humanized Type I Collagen Fiber Implants

[0105] Accurately weigh m0 (mg) of lyophilized recombinant humanized type I collagen fiber implant sample and place it in TES buffer (containing 1 mM CaCl2, pH 7.4) containing 5 U / mL collagenase for enzymatic hydrolysis. After hydrolysis, freeze-dry the sample and weigh it. Calculate the hydrolysis rate (DR%) using the following formula:

[0106]

[0107] Where m0 is the initial weight of the sample before enzymatic hydrolysis, m t is the weight of the sample after enzymatic hydrolysis.

[0108] The results are as follows Figure 6 As shown in D, on the 14th day of enzymatic hydrolysis, the degradation rate of the recombinant humanized type I collagen fiber implant was only 58.0±5.3%, which was significantly lower than the degradation rate of the recombinant humanized type I collagen before assembly; the experimental results show that the recombinant humanized type I collagen fiber implant prepared by the present invention has excellent resistance to enzymatic hydrolysis.

[0109] Example 4 Evaluation of the biocompatibility and bioactivity of recombinant humanized type I collagen fiber implants

[0110] 1. Cell Adhesion Assay

[0111] The recombinant humanized type I collagen fiber implant (ATHRCI, 5 mg) prepared in Example 3 was extracted for 72 h and then added to a 24-well plate without tissue treatment and incubated at 4°C for 12 h. 100 μL of the solution was added to the 24-well plate at a cell density of 1×10 5 Human foreskin fibroblasts (HFF-1) or chondrocytes were incubated at 37°C, 5% CO2 for 24 hours. BSA was used as a negative control group, and a self-assembled extracted type I collagen implant (ACol I) was used as a positive control group. Cell morphology was observed under an inverted microscope. The extracted type I collagen was incubated in 20mM PB (pH 7.4) at 25°C for 24 hours, and the resulting precipitate was centrifuged as ACol I.

[0112] The results are as follows Figure 7 As shown, compared with BSA, the recombinant humanized type I collagen fiber implant described in the present application exhibited an adhesion-promoting effect on HFF-1 cells (shown as a in A) and chondrocytes (shown as b in B) comparable to that of ACoI I, and had excellent cell adhesion performance.

[0113] 2. Cell Proliferation Assay

[0114] Add 100 μL to a 96-well plate at a cell density of 1 × 10 5 HFF-1 or chondrocytes were incubated at 37°C, 5% CO₂ for 24 hours. After 24 hours, the culture medium in the 96-well plate was aspirated and the extract of the recombinant humanized type I collagen fiber implant prepared in Example 3 was added to the 96-well plate. A blank control group was treated with only DMEM high-glucose medium or DMEM / F12 medium, and the positive control group was treated with ACol I. Incubation was continued in a 37°C, 5% CO₂ incubator for 1, 3, and 5 days. The cell proliferation-promoting effect of the recombinant humanized type I collagen fiber implant was detected using CCK-8.

[0115] like Figure 7As shown, for HFF-1 cells (shown in c in A), the relative cell proliferation rates of the recombinant humanized type I collagen fiber implant group were 106.9%, 220.5%, and 271.0% on day 1, 3, and 5, respectively, and the relative cell proliferation rates of the ACol I group were 107.2%, 219.0%, and 278.3%, respectively. Compared with the blank control group (100.0%, 163.6%, and 237.5%), both the recombinant humanized type I collagen fiber implant group and the ACol I group significantly promoted the proliferation of HFF-1 cells. For chondrocytes (shown in c in B), the relative cell proliferation rates of the recombinant humanized type I collagen fiber implant group were 118.0%, 188.0%, and 275.3% on day 1, 3, and 5, respectively, and the relative cell proliferation rates of the ACol I group were 107.0%, 188.1%, and 232.7%, respectively, which were significantly better than those of the blank control group (100.0%, 140.3%, and 164.0%). The recombinant humanized type I collagen fiber implant significantly promoted the proliferation of chondrocytes.

[0116] 3. Cell Migration Assay

[0117] The cell migration promoting effect of the recombinant humanized type I collagen fiber implant prepared in Example 3 was determined by cell scratch assay. Three horizontal lines were drawn on the back of each well of a 6-well plate with a spacing of 0.5-1 cm. 2 mL of cells were seeded into the 6-well plate at a density of 5×10 5 HFF-1 cells or chondrocytes were cultured at a concentration of 100 cells / mL to ensure 95%-100% confluence after 24 hours of cell culture. After 24 hours, a 10 μL pipette tip was aligned with a ruler and gently pushed downward to create a longitudinal scratch. Recombinant humanized type I collagen fiber implants, an extract of ACol I (positive control), and DMEM high-glucose medium or DMEM / F12 medium (negative control) were then added to the 6-well plate. Changes in scratch area at 0 and 24 hours were recorded under an inverted fluorescence microscope.

[0118] like Figure 7 As shown in A(b, d) and B(b, d), compared with the blank control group, the cell scratches in the recombinant humanized type I collagen fiber implant group and the ACol I group were significantly reduced; the migration rates of HFF-1 cells and chondrocytes in the blank control group were 28.9% and 29.1%, respectively, the migration rates of HFF-1 cells and chondrocytes in the recombinant humanized type I collagen fiber implant group were 86.9% and 85.4%, respectively, and the migration rates of HFF-1 cells and chondrocytes in the ACol I group were 83.6% and 86.7%, respectively (shown in b and d in A and b and d in B). The experimental results show that the recombinant humanized type I collagen fiber implant described in the present application has an excellent effect in promoting cell migration.

[0119] 4. Live / dead cell staining

[0120] Add 1 mL of the medium to the laser confocal microplate, with a cell density of 1×10 5 HFF-1 cells or chondrocytes were incubated at 37°C, 5% CO₂ for 24 hours. After 24 hours, the culture medium in the laser confocal microscopy dish was aspirated and the extract of the recombinant humanized type I collagen fiber implant prepared in Example 3 was added to the laser confocal microscopy dish. The positive control group was ACoI I, and the blank control group was treated with only DMEM high-glucose medium or DMEM / F12 medium. The cells were incubated in a 37°C, 5% CO₂ incubator for another 5 days. The cells were stained using a live / dead cell staining kit for 1 hour, and the stained images were acquired using a laser confocal microscope.

[0121] Live / dead cell staining results are as follows Figure 7 As shown, after HFF-1 cells and chondrocytes were co-cultured with the extract of recombinant humanized type I collagen fiber implant for 5 days, it was further confirmed that the cell proliferation promoting effect of the recombinant humanized type I collagen fiber implant group described in the present application was significantly better than that of the blank control group (as shown in e in A and e in B).

[0122] 5. Immunofluorescence Staining

[0123] HFF-1 cells were cultured with the recombinant humanized type I collagen fiber implant prepared in Example 3 for 5 days. After 5 days, the cells were fixed with 4% paraformaldehyde for 10 minutes and then permeabilized with 0.1% Triton X-100 for 5 minutes. Next, the cells were incubated in PBS buffer (10mM, pH 7.4) containing 1% BSA for 30 minutes at room temperature for a blocking experiment. The actin cytoskeleton was stained for 1 hour using 100nM phalloidin-tetramethylrhodamine. 5μg / mL of Hoechst 33258 was added and incubated at 37°C for 20 minutes to stain the cell nuclei. Finally, fluorescence images were taken using a laser confocal microscope.

[0124] Immunofluorescence staining was used to detect the cell adhesion and spreading characteristics of recombinant humanized type I collagen fiber implants. Figure 7 As shown: Compared with the blank control group, the cells in the recombinant humanized type I collagen fiber implant group described in the present application showed a fine actin cytoskeleton structure, the diffusion area was expanded, and the cell density was significantly increased (as shown in f in A and f in B).

[0125] 6. Real-time quantitative polymerase chain reaction (RT-qPCR) gene expression analysis

[0126] Real-time quantitative polymerase chain reaction (RT-qPCR) was used to analyze the ability of recombinant humanized type I collagen fiber implants to promote the chondrogenic differentiation of HFF-1 cells and bone marrow mesenchymal stem cells. 5 HFF-1 cells or bone marrow mesenchymal stem cells were cultured in 6-well plates at a concentration of 100 cells / mL and incubated with recombinant humanized type I collagen fiber implants for 7 days. Total RNA was extracted from each well using an RNA extraction kit. The concentration and purity of the RNA samples were assessed using a NanoDrop spectrophotometer. cDNA was synthesized using a PrimeScript RT kit. Gene expression levels were analyzed using TB Green Premix Ex TaqII. The expression of genes related to fibroblast differentiation was evaluated using a real-time quantitative PCR system and 2 -ΔΔCT The relative expression levels of target genes were calculated using the PCR method, with GAPDH as the internal reference gene. The primer sequences for genes related to fibroblast differentiation and chondrogenic differentiation of bone marrow mesenchymal stem cells are shown in Table 1.

[0127] Table 1. Primer sequences of genes related to fibroblast differentiation and chondrogenic differentiation.

[0128]

[0129] α-SMA is an important marker for the differentiation of fibroblasts into myofibroblasts. Figure 7 As shown, after 7 days of co-culture of HFF-1 cells with recombinant humanized type I collagen fiber implants, the expression levels of α-SMA in the recombinant humanized type I collagen fiber implant and ACol I groups increased by 1.26 times and 1.57 times, respectively, with no significant difference between the two groups (shown in g in A). As the main components of the extracellular matrix, the expression levels of Collagen I and Collagen III in the recombinant humanized type I collagen fiber implant group were significantly higher than those in the blank control group (shown in hi in A). Similarly, for bone marrow mesenchymal stem cells, recombinant humanized type I collagen fiber implants can significantly promote the expression of Sox9, a key transcription factor for chondrogenic differentiation, the proteoglycan Acan, and type II collagen Col2α1 (shown in gi in B). These results indicate that recombinant humanized type I collagen fiber implants have excellent ability to promote HFF-1 cell differentiation and promote chondrogenic differentiation of bone marrow mesenchymal stem cells.

[0130] Example 5 Biosafety Evaluation of Recombinant Humanized Type I Collagen Fiber Implants

[0131] 1. Pyrogen test

[0132] Animal experiments were conducted in accordance with the ethical standards of the School of Chemistry and Chemical Engineering Ethics Committee of Lanzhou University. New Zealand white rabbits (2.0-3.0 kg, 3-4 months old) were purchased from the Animal Center of Lanzhou University. Rabbits were fasted for 2 hours before temperature measurement. Rectal temperatures were measured every 30 minutes thereafter for a total of eight measurements. The normal temperature range is 38.0-39.6°C, with a maximum allowable difference of 0.4°C between the highest and lowest temperatures. Three rabbits meeting the temperature standard were selected from each group and numbered. Before the experiment, body temperature was monitored every 30 minutes, with the temperature difference between consecutive measurements controlled to no more than 0.2°C. The average of these two temperatures was used as the normal body temperature of the rabbits. A 2-mL dose of recombinant humanized type I collagen fiber implant extract (recombinant humanized type I collagen fiber implant extracted in 0.9% NaCl solution for 72 hours) or 0.9% NaCl (control group) was injected into the marginal ear vein. Rectal temperatures were measured every 30 minutes after injection for a total of six measurements. The temperature rise value is calculated by subtracting the normal temperature from the highest temperature measured in the six times. If the temperature rise of each rabbit in the group does not exceed 0.6°C, and the sum of the temperature rises of the three rabbits in a group does not exceed 1.3°C, the recombinant humanized type I collagen fiber implant is considered non-pyrogenic.

[0133] The results are as follows Figure 8 As shown in Figure A, the body temperatures of the three rabbits injected with 0.9% NaCl increased by 0°C, 0.1°C, and 0°C, respectively. The body temperatures of the three rabbits in the recombinant humanized type I collagen fiber implant group increased by 0.3°C, 0°C, and 0.2°C, respectively. The total temperature increase of the rabbits in each group was less than 1.3°C, indicating that the recombinant humanized type I collagen fiber implant was not pyrogenic.

[0134] 2. Hemolysis Experiment

[0135] Take 1mL of fresh rabbit blood, add 5mL of 0.1mg / mL sodium heparin solution, and centrifuge at 2500rpm for 10min to obtain red blood cells. Wash the red blood cells 3-5 times with 2mL of 0.9% NaCl until the supernatant is no longer red. Dilute the suspended red blood cells with 0.9% NaCl, take the diluted red blood cell suspension, add 0.9% NaCl, H2O, and recombinant humanized type I collagen fiber implant extract, mix well, and incubate at 37°C for 1h. Then incubate at room temperature for 3h, collect the supernatant by centrifugation and measure its absorbance at 545nm. Use the following formula to calculate the hemolysis rate (HR%):

[0136]

[0137] OD a ,OD b and OD cThey are the absorbance of the sample to be tested, 0.9% NaCl and H2O respectively, wherein H2O is used as the positive control and the hemolysis rate is calculated as 100%, and 0.9% NaCl is used as the negative control and the hemolysis rate is calculated as 0%.

[0138] The results are as follows Figure 8 As shown in B, the hemolysis rate of the recombinant humanized type I collagen fiber implant measured by the hemolysis experiment is only 0.53%, which is far lower than the national standard (5%), indicating that the recombinant humanized type I collagen fiber implant described in this application does not have the risk of hemolysis.

[0139] 3. Immunogenicity

[0140] BALB / c mice (20-25 g, 8-9 weeks old) were purchased from the Animal Center of Lanzhou University. Ten mice were randomly assigned to each group, half male and half female. The experimental group was subcutaneously injected with 100 μL of recombinant humanized type I collagen fiber implant and an emulsion of incomplete Freund's adjuvant on days 1, 14, and 35, respectively. The adjuvant-free group was subcutaneously injected with 100 μL of recombinant humanized type I collagen fiber implant and PBS on days 1, 14, and 35. Blood was collected from the saphenous vein of the mice before the first injection and then 7 days after each injection. After all blood samples were allowed to stand at room temperature for 1-2 hours, they were centrifuged at 3000 rpm for 15 minutes, and the serum was collected and stored at -20°C. The antibody level in the serum was detected by enzyme-linked immunosorbent assay.

[0141] The immunogenicity of recombinant humanized type I collagen fiber implants was evaluated by measuring the levels of total IgG, IgA, and IgM in serum. Figure 8 As shown in Figure C, with or without the addition of an adjuvant, after three immunizations, there was no statistical difference in IgG, IgA, and IgM in the mouse serum compared with before injection after injection of the recombinant humanized type I collagen fiber implant, indicating that the recombinant humanized type I collagen fiber implant described in the present application is non-immunogenic.

[0142] Example 6 Cross-linking of recombinant humanized type I collagen fiber implants with different cross-linking agents

[0143] 1. Cross-linking of recombinant humanized type I collagen fibers with different cross-linking agents

[0144] Recombinant humanized type I collagen was dissolved in 50 mM PB (pH 7.4) to prepare a recombinant humanized type I collagen solution with a concentration of 75 mg / mL;

[0145] The recombinant humanized type I collagen solution was incubated at 25° C. for 24 h, and then centrifuged at 4° C. and 10,000 rpm for 10 min to obtain a recombinant humanized type I collagen fiber implant.

[0146] The recombinant humanized type I collagen fiber implant prepared above was redispersed in 50 mM PB (pH 7.4), and 2.0% BDDE or 0.05% THPC was added, respectively. After cross-linking at 4°C for 24 h, the mixture was centrifuged and washed three times with 50 mM PB. Cross-linked recombinant humanized type I collagen fiber implants were obtained by centrifugation.

[0147] 2. Scanning Electron Microscopy Characterization of Cross-linked Recombinant Humanized Type I Collagen Fiber Implants

[0148] The experimental steps were the same as those in Example 2. Scanning electron microscopy characterization of recombinant humanized type I collagen fiber implants cross-linked with different cross-linking agents was as follows: Figure 9 As shown in the figure, after cross-linking recombinant humanized type I collagen fibers with a crosslinking agent, the recombinant humanized type I collagen implant maintained a dense fiber morphology. After cross-linking with BDDE and THPC, the fiber diameter of the recombinant humanized type I collagen implant increased to (303±50) nm and (293±55) nm, respectively, indicating that cross-linking is beneficial for increasing collagen fiber diameter.

[0149] 3. Degradation rate of cross-linked recombinant humanized type I collagen fiber implants

[0150] The durability of the cross-linked recombinant humanized type I collagen fiber implant was evaluated according to the method of Example 3. Figure 10 As shown in the results, on the 14th day, the degradation rate of BDDE-crosslinked recombinant humanized type I collagen fiber implant was 30.0±4.0%, the degradation rate of THPC-crosslinked recombinant humanized type I collagen fiber implant was 33.3±4.2%, and the degradation rate of uncrosslinked recombinant humanized type I collagen fiber implant was 48.7±4.2%. The experimental results showed that after cross-linking with BDDE or THPC, the resistance of recombinant humanized type I collagen fiber implant to enzymatic degradation was significantly improved.

[0151] Example 7 Evaluation of Biocompatibility and Bioactivity of Cross-linked Recombinant Humanized Type I Collagen Fiber Implants

[0152] 1. Cell Migration

[0153] The cell migration promoting effect of the BDDE and THPC cross-linked recombinant humanized type I collagen fiber implant prepared in Example 6 was evaluated according to the method of Example 4. Figure 11As shown: After 24 hours of co-culture with HFF-1 cells using BDDE-crosslinked recombinant humanized type I collagen fiber implant (B-ATHRCI), a large number of HFF-1 cells migrated to the scratched area, with a cell migration rate of 77.2±11.4%. In contrast, the cells in the blank group had no significant migration, with a cell migration rate of only 18.0±5.5% (shown in Figures AB). Similarly, after 24 hours of co-culture with cells using THPC-crosslinked recombinant humanized type I collagen fiber implant (T-ATHRCI), the cell migration rate was 83.5±9.9%, while the cell migration rate in the blank group was only 36.2±2.5% (shown in Figures GH). These experimental results demonstrate that recombinant humanized type I collagen fiber implants cross-linked with BDDE or THPC significantly promote HFF-1 cell migration.

[0154] 2. Live / dead cell staining and cell proliferation

[0155] The cell proliferation promoting effect of the BDDE and THPC cross-linked recombinant humanized type I collagen fiber implant prepared in Example 6 was evaluated according to the method of Example 4. Live / dead cell staining and CCK-8 assay were used to evaluate the cell proliferation promoting ability of the BDDE and THPC cross-linked recombinant humanized type I collagen fiber implant. Figure 11 As shown, as the incubation time of HFF-1 cells with the BDDE / THPC cross-linked recombinant humanized type I collagen fiber implant increased, the number of surviving cells gradually increased, and the cell density was significantly higher than that of the blank group (C and I). CCK-8 assay results further demonstrated that cell viability gradually increased over time in the BDDE and THPC cross-linked recombinant humanized type I collagen fiber implant group (D and J). These results demonstrate that the BDDE and THPC cross-linked recombinant humanized type I collagen fiber implant has excellent biocompatibility and significantly promotes cell proliferation.

[0156] 3. Immunofluorescence Staining

[0157] The cell adhesion and spreading characteristics of the BDDE and THPC cross-linked recombinant humanized type I collagen fiber implant prepared in Example 6 were evaluated according to the method of Example 4 ( Figure 11 (As shown in E and K): Compared with the blank group, the cells in the BDDE and THPC cross-linked recombinant humanized type I collagen fiber implant group showed a fine actin cytoskeleton structure, the diffusion area was expanded, and the cell density was significantly increased. The results showed that the cross-linked recombinant humanized type I collagen fiber implant significantly promoted cell adhesion and spreading.

[0158] 4. Cell Differentiation

[0159] The ability of the BDDE and THPC cross-linked recombinant humanized type I collagen fiber implant prepared in Example 6 to promote fibroblast differentiation was evaluated according to the method of Example 4. Figure 11 As shown in Figures F and L, compared with the blank group, both BDDE- and THPC-crosslinked recombinant humanized type I collagen fiber implants significantly promoted the expression of genes related to fibroblast differentiation marker α-SMA and extracellular matrix synthesis (Collagen I and Collagen III). These results indicate that BDDE- and THPC-crosslinked recombinant humanized type I collagen fiber implants have excellent ability to promote HFF-1 cell differentiation.

[0160] The above experimental results show that the present invention provides a recombinant humanized type I collagen, which has a unique triple helix structure and excellent self-assembly properties; secondly, the present invention optimizes the self-assembly process of recombinant humanized type I collagen to prepare a recombinant humanized type I collagen implant with orderly arranged collagen fibers. The implant has excellent injectability, durability, high biosafety, no pyrogenicity and hemolytic risks, and low immunogenicity. It not only significantly promotes the adhesion, migration, proliferation, and differentiation of human skin fibroblasts, but also significantly promotes the adhesion, migration, proliferation of chondrocytes and the chondrogenic differentiation of bone marrow mesenchymal stem cells, and can be applied to the fields of skin, cartilage, bone, etc.

Claims

1. A recombinant humanized type I collagen with a triple helical structure, characterized in that: The sequence of the recombinant humanized type I collagen is shown in SEQ ID NO.

1.

2. The recombinant humanized type I collagen according to claim 1, wherein The recombinant humanized type I collagen is obtained by treating a precursor collagen with a protease; the amino acid sequence of the precursor collagen is shown in SEQ ID NO.

2.

3. The recombinant humanized type I collagen according to claim 2, wherein The gene sequence of the precursor collagen is shown in SEQ ID NO.

3.

4. A recombinant humanized type I collagen fiber implant, characterized in that: The recombinant humanized type I collagen implant is formed by self-assembly of the recombinant humanized type I collagen according to claim 1.

5. The recombinant humanized type I collagen implant according to claim 4, wherein The preparation method of the implant comprises: dissolving recombinant humanized type I collagen in a buffer solution with 0-200 mM PB and pH 5.0-7.4 to a concentration of 50-100 mg / mL, standing at 4-25° C. for 24-72 hours, and centrifuging at 1000-10000 rpm for 5-20 minutes to precipitate a recombinant humanized type I collagen fiber implant.

6. The recombinant humanized type I collagen fiber implant according to claim 5, characterized in that: The preparation method comprises the following steps: dissolving recombinant humanized type I collagen in a 50 mM P-B, pH 7.4 buffer solution to a concentration of 75 mg / mL, standing at 25° C. for 24 hours, and centrifuging at 10,000 rpm for 10 minutes to precipitate a recombinant humanized type I collagen fiber implant.

7. A cross-linked recombinant humanized type I collagen fiber implant, characterized in that: The cross-linked recombinant humanized type I collagen fiber implant is obtained by cross-linking the recombinant humanized type I collagen fiber implant according to any one of claims 4 to 6 with a cross-linking agent, wherein the cross-linking agent includes BDDE and THPC.

8. The cross-linked recombinant humanized type I collagen fiber implant according to claim 7, wherein: The preparation method of the cross-linked recombinant humanized type I collagen fiber implant comprises the following steps: (1) preparing the recombinant humanized type I collagen fiber implant according to any one of claims 4 to 6; (2) The recombinant humanized type I collagen fiber implant was dispersed in a 0-200 mM PB, pH 5.0-7.4 buffer solution, and 0.001-2.0% BDDE or 0.001-0.05% THPC was added, respectively. After cross-linking at 4-25°C for 24-72 hours, the mixture was centrifuged at 1000-10000 rpm for 5-20 minutes. The precipitate was washed with a 0-200 mM PB, pH 5.0-7.4 buffer solution to obtain the cross-linked recombinant humanized type I collagen fiber implant.

9. The cross-linked recombinant humanized type I collagen fiber implant according to claim 8, characterized in that: The step (2) comprises: dispersing the recombinant humanized type I collagen fiber implant into a 50 mM PB, pH 7.4 buffer solution, adding 2.0% BDDE or 0.05% THPC by volume, cross-linking at 4° C. for 24 hours, and centrifuging at 10,000 rpm for 10 minutes; washing the precipitate three times with a 50 mM PB, pH 7.4 buffer solution, and centrifuging at 10,000 rpm for 10 minutes to obtain the cross-linked recombinant humanized type I collagen fiber implant.

10. Use of the recombinant humanized type I collagen fiber implant according to claim 4 or the cross-linked recombinant humanized type I collagen fiber implant according to claim 7 in the preparation of medical devices for use in the fields of skin, cartilage, and bone.

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