A triple helix self-assembled recombinant humanized type i collagen protein fiber implant, a preparation method and application thereof

Abstract

The application belongs to the technical field of biomedical materials, and particularly relates to a triple helix self-assembled recombinant humanized type I collagen fiber implant, a preparation method and application thereof. The application firstly provides a recombinant humanized type I collagen, which has a unique triple helix structure and excellent self-assembly properties. Secondly, the application optimizes the self-assembly process of the recombinant humanized type I collagen to prepare a recombinant humanized type I collagen implant with ordered arrangement of collagen fibers. The implant has excellent injectability, durability, high biological safety, no pyrogen and hemolysis risk, low immunogenicity, can significantly promote the adhesion, migration, proliferation and differentiation of human skin fibroblasts, can significantly promote the adhesion, migration and proliferation of chondrocytes, and can promote the chondrogenic differentiation of bone marrow mesenchymal stem cells, and can be applied to the fields of skin, cartilage, bone and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a triple-helix self-assembled recombinant humanized type I collagen fiber implant, its preparation method, and its application. Background Technology

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

[0003] Collagen, as the main structural protein of the skin, has a characteristic triple helix structure and can self-assemble into collagen fibers with good morphology and mechanical strength. It plays an important role in maintaining skin elasticity and supporting structure and can be used as an implant for skin rejuvenation. However, the raw materials of currently commercially available collagen implants are mainly animal-derived collagen obtained through extraction methods, which poses risks such as viral transmission and immunogenicity. Moreover, recombinant humanized type I collagen reported in the literature generally does not possess self-assembly activity.

[0004] Compared to animal-derived collagen, recombinant collagen produced through genetic engineering can overcome these drawbacks. However, its structural domains are smaller, making it 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] To address the aforementioned technical problems, this invention, through special amino acid sequence design, obtains a recombinant humanized type I collagen, THRCI, with a unique triple helix structure and excellent self-assembly properties. The recombinant humanized type I collagen, through self-assembly, yields a recombinant humanized type I collagen implant with excellent injectability and durability. This recombinant humanized type I collagen fiber implant exhibits excellent bioactivity, significantly promoting not only the adhesion, migration, proliferation, and differentiation of human skin fibroblasts, but also the adhesion, migration, and proliferation of chondrocytes and the chondrogenic differentiation of bone marrow mesenchymal stem cells. Furthermore, it possesses high biocompatibility, with no risk of pyrogenicity or hemolysis, and low immunogenicity, making it applicable to the fields of skin, cartilage, and bone. Specifically, it includes the following:

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

[0007] Preferably, the recombinant humanized type I collagen is obtained by treating precursor collagen with 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 in SEQ ID NO.3.

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

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

[0011] Preferably, the preparation method of the recombinant humanized type I collagen implant includes: dissolving recombinant humanized type I collagen in a buffer solution of 0-200mM PB, pH 5.0-7.4 to a concentration of 50-100mg / mL, letting it stand at 4-25℃ for 24-72h, 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 50mM.

[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 settling temperature is 25°C.

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

[0018] Preferably, the centrifugation parameters are: 10,000 rpm for 10 min.

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

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

[0021] Preferably, the crosslinking agent is selected from BDDE or THPC.

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

[0023] (1) Prepare the above-mentioned recombinant humanized type I collagen fiber implantation agent in the third aspect;

[0024] (2) Disperse the recombinant humanized type I collagen fiber implant into a buffer solution of 0-200mM PB and pH 5.0-7.4, add 0.001-2.0% BDDE or 0.001-0.05% THPC by volume, crosslink at 4-25℃ for 24-72h, centrifuge, and wash the precipitate with a buffer solution of 0-200mM PB and pH 5.0-7.4 to obtain the crosslinked 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 50mM.

[0027] Preferably, the pH is 7.4.

[0028] Preferably, the crosslinking temperature is 4°C.

[0029] Preferably, the crosslinking time is 24 hours.

[0030] Preferably, the centrifugation parameters are: 10,000 rpm for 10 min.

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

[0032] Preferably, step (2) is as follows: the recombinant humanized type I collagen fiber implant is dispersed in a buffer solution of 0-200 mM PB and pH 5.0-7.4, and 2.0% BDDE or 0.05% THPC is added respectively. After crosslinking at 4°C for 24 h, the mixture is centrifuged at 10000 rpm for 10 min. The mixture is then precipitated three times with a buffer solution of 0-200 mM PB and pH 5.0-7.4 and centrifuged at 10000 rpm for 10 min. The precipitate obtained is the crosslinked recombinant humanized type I collagen fiber implant.

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

[0034] The beneficial effects of this invention are: (1) Recombinant humanized type I collagen reported in general literature has no self-assembly activity. Based on this, this invention first obtains a recombinant humanized type I collagen with a unique triple helix structure and excellent self-assembly properties through 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 ordered collagen fiber arrangement 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 adhesion, migration, proliferation, and differentiation of human skin fibroblasts can also significantly promote the adhesion, migration, and proliferation of chondrocytes and the chondrogenic differentiation of bone marrow mesenchymal stem cells, which is comparable to the bioactivity of extracted type I collagen; (4) Compared with type I collagen prepared by extraction, the recombinant humanized type I collagen fiber implant has excellent injectability and durability, and has high biosafety, no risk of pyrogenicity and hemolysis, and low immunogenicity; (5) The recombinant humanized type I collagen fiber implant of the present invention can be applied to the fields of skin, cartilage, and bone. Attached Figure Description

[0035] Figure 1 The results represent the characterization of triple-helix recombinant humanized type I collagen; where A represents the SDS-PAGE characterization result and B represents the circular dichroism characterization result.

[0036] Figure 2 Scanning electron micrographs and statistical results of fiber diameters of recombinant humanized type I collagen fiber implants assembled with buffer solutions of different concentrations; where 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 micrographs and statistical results of fiber diameters of recombinant humanized type I collagen fiber implants assembled under different pH conditions are shown; where A is pH=5.0 and B is pH=7.4.

[0038] Figure 4 Scanning electron microscope images and statistical results of fiber diameters of recombinant humanized type I collagen fiber implants assembled at different temperatures are shown; where A and a represent 4℃, and B and b represent 25℃.

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

[0040] Figure 6The physicochemical properties of the recombinant humanized type I collagen fiber implant are characterized; where A represents denatured collagen detection, B represents injectability, C represents thermal stability, and D represents durability.

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

[0042] Figure 8 The results represent the biosafety evaluation of recombinant humanized type I collagen fiber implants; where 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 Scanning electron micrographs and fiber diameter statistics of recombinant humanized type I collagen fiber implants crosslinked with different crosslinking agents; where A and a represent BDDE, and B and b represent THPC.

[0044] Figure 10 This characterizes the durability of cross-linked implants.

[0045] Figure 11 The results represent the biocompatibility and bioactivity evaluation of the cross-linked recombinant humanized type I collagen implant; where AF represents the cell migration, cell migration rate, live / dead cell staining, cell proliferation, immunofluorescence staining, and cell differentiation results of the BDDE cross-linked recombinant humanized type I collagen implant, and GL represents the cell migration, cell migration rate, live / dead cell staining, cell proliferation, immunofluorescence staining, and cell differentiation results of the THPC cross-linked recombinant humanized type I collagen implant. Detailed Implementation

[0046] The present invention will be 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 are within the protection scope of the present invention.

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

[0048] Example 1: Characterization of the preparation of recombinant humanized type I collagen

[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. This protein is obtained by treating 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 for synthesizing precursor collagen was constructed into the E. coli expression vector pCold, and the successful synthesis of the plasmid was confirmed by DNA sequencing. The plasmid was transformed into E. coli BL21-DE3 strain to obtain a precursor collagen expression strain. The successfully transformed strain was stored in glycerol at -80℃.

[0052] Precursor collagen expression: 20 μL of cryopreserved bacterial culture was added to 200 mL of LB broth containing antibiotics and cultured overnight at 37°C using a shaker. Then, the culture was transferred to 1 L of LB broth containing antibiotics at a 2% inoculation rate and further amplified at 37°C using a shaker. OD was calculated. 600 When the value reaches the range of 1.2-2.0, adjust the temperature of the shaker to 25℃, add IPTG to a final concentration of 1mM to induce expression, and incubate overnight at a constant temperature; centrifuge the bacterial culture in a low-temperature centrifuge at 3500rpm, 4℃, for 30min, and collect the bacterial cells.

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

[0054] 2. SDS-PAGE characterization of recombinant humanized type I collagen

[0055] Recombinant humanized type I collagen (THRCI) was characterized by SDS-PAGE.

[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. The protein band is around 40 kDa. Due to the unique amino acid sequence and structure of collagen, its migration rate on SDS-PAGE is lower 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), consistent with the characteristics of collagen.

[0057] 3. Circular dichroism characterization of recombinant humanized type I collagen

[0058] The recombinant humanized type I collagen was prepared into a 0.5 mg / mL solution and scanned at 4 °C using a 1 mm cuvette with full wavelength (190-260 nm) using circular dichroism spectroscopy, 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 Figure B, the recombinant humanized type I collagen exhibits a characteristic absorption peak of collagen around 220 nm, indicating that the recombinant humanized type I collagen described in this application has a triple helix 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] After incubating the recombinant humanized type I collagen solution at 25°C for 24 hours, centrifuge at 4°C and 10,000 rpm for 10 minutes, and collect the precipitate 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, sputtered with gold for 30 seconds, and the morphology of the sample was detected at an operating voltage of 5.0 kV.

[0067] The results are as follows Figure 2As shown, the fiber diameters of recombinant humanized type I collagen assembled in 20mM, 50mM, 100mM, and 200mM PB were (174±43)nm, (232±44)nm, (288±46)nm, and (305±48)nm, respectively. The results indicate that the fiber diameter of the recombinant humanized type I collagen implant gradually increases with the increase of PB concentration. Under the condition of 50mM PB as a buffer solution, ordered collagen fibers were prepared. 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 2.0M NaOH or HCl.

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

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

[0074] The experimental procedure is the same as above.

[0075] The results are as follows Figure 3 As shown, the fiber diameters of recombinant humanized type I collagen assembled at pH 5.0 and 7.4 were (216±49) nm and (233±47) nm, respectively. The results indicate that the pH of the buffer solution has no significant effect on the formation of recombinant humanized type I collagen fibers. Subsequent experiments selected physiological pH (pH=7.4) for the assembly of recombinant humanized type I collagen.

[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 hours and then centrifuged at 4°C, 10,000 rpm for 10 minutes 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 procedure is the same as above.

[0082] The results are as follows Figure 4 As shown, the fiber diameters assembled from 50 mg / mL and 100 mg / mL recombinant humanized type I collagen are (233±46) nm and (230±49) nm, respectively. When the concentration of recombinant humanized type I collagen is 75 mg / mL, the diameter of the collagen fiber is (267±48) nm. The following experiment selected a recombinant humanized type I collagen concentration of 75 mg / mL 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℃ and 25℃ for 24 h, respectively, and then centrifuged at 4℃, 10000 rpm for 10 min 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 procedure is the same as above.

[0089] The results are as follows Figure 5 As shown, the diameters of recombinant humanized type I collagen fibers assembled at 4℃ and 25℃ are (252±47)nm and (263±42)nm, respectively. The experimental results show that the assembly temperature has no significant effect on the size of recombinant humanized type I collagen fibers. The following experiments selected a temperature of 25℃ for assembling recombinant humanized type I collagen.

[0090] Example 3: Physicochemical characterization 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 hours and then centrifuged at 4°C, 10,000 rpm for 10 minutes to obtain the recombinant humanized type I collagen fiber implant.

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

[0095] The denaturation status of the recombinant humanized type I collagen (THRCI) and the self-assembled collagen fiber implant (ATHRCI) prepared in the examples was detected using the denatured collagen-targeting fluorescent peptide probe FAM-GOP-14. Specifically:

[0096] Gelatin, THRCI, and ATHRCI were dissolved in 10 mM PB (pH 7.4) to prepare solutions with a concentration of 5 mg / mL. A portion of the THRCI solution was heated at 90 °C for 10 min to obtain heat-denatured THRCI (DTHRCI). 100 μL of each solution was added to a 96-well plate and incubated overnight at 4 °C. The plates were washed three times with 10 mM PB, and then 20 μM FAM-GOP-14 (Anal. Chem. 2024, 96, 39, 15640–15647) was added to each well, followed by incubation at 4 °C for 4 h. Unbound FAM-GOP-14 was washed three times with 10 mM PB. Fluorescence intensity (E) was measured using a Tecan Infinite F200 / M200 multi-functional microplate reader. x =497nm, E m =525nm).

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

[0098] 3. Injectability

[0099] Take the recombinant humanized type I collagen fiber implant prepared by the above method, push the syringe plunger at a speed of 30 mm / min, and conduct a pushing force test.

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

[0101] 4. Thermal stability characterization of recombinant humanized type I collagen fiber implants

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

[0103] The results are as follows Figure 6 As shown in Figure C, the denaturation temperature of unassembled recombinant humanized type I collagen is 80.5℃, while the denaturation temperature of the assembled recombinant humanized type I collagen fiber implant is 88.5℃; the results indicate 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 the lyophilized recombinant humanized type I collagen fiber implant sample and place it in TES buffer (containing 1 mM CaCl2, pH 7.4) with 5 U / mL collagenase for enzymatic hydrolysis. After hydrolysis, lyophilize the sample and weigh it, then calculate the hydrolysis rate. The formula for calculating the hydrolysis rate (DR%) is as follows:

[0106]

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

[0108] The results are as follows Figure 6 As shown in Figure D, on day 14 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 that of the recombinant humanized type I collagen before assembly. The experimental results show that the recombinant humanized type I collagen fiber implant prepared by this 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 experiment

[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 untreated 24-well plates and incubated at 4°C for 12 h. 100 μL of the cell-rich material was added to each 24-well plate, resulting in a cell density of 1 × 10⁻⁶ cells / well. 5 Human foreskin fibroblasts (HFF-1) or chondrocytes per mL were incubated at 37°C and 5% CO2 for 24 h. BSA was used as a negative control group, and self-assembled type I collagen implant (ACol I) was used as a positive control group. Cell morphology was observed using an inverted microscope. The extracted type I collagen was incubated in 20 mM PB (pH 7.4) at 25°C for 24 h, and the precipitate obtained by centrifugation was 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 this application exhibits similar HFF-1 cell (shown in A a) and chondrocyte (shown in B b) adhesion promotion effects to ACl I, demonstrating excellent cell adhesion performance.

[0113] 2. Cell proliferation experiment

[0114] Add 100 μL to a 96-well plate to achieve a cell density of 1 × 10⁻⁶ cells. 5 HFF-1 or chondrocytes per mL were incubated at 37°C and 5% CO2 for 24 h. After 24 h, the culture medium in the 96-well plates was aspirated, and the extract of the recombinant humanized type I collagen fiber implant prepared in Example 3 was added to the 96-well plates. The blank control group was incubated with DMEM high-glucose medium or DMEM / F12 medium, and the positive control group was incubated with ACl I. The plates were incubated at 37°C and 5% CO2 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 assay.

[0115] like Figure 7As shown, for HFF-1 cells (shown in Ac), the relative cell proliferation rates of the recombinant humanized type I collagen fiber implant group were 106.9%, 220.5%, and 271.0% at 1 day, 3 days, and 5 days, respectively, while the relative cell proliferation rates of the ACl 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 ACl I group significantly promoted the proliferation of HFF-1 cells. For chondrocytes (shown in Bc), the relative cell proliferation rates of the recombinant humanized type I collagen fiber implant group were 118.0%, 188.0%, and 275.3% at 1 day, 3 days, and 5 days, respectively, while the relative cell proliferation rates of the ACl I group were 107.0%, 188.1%, and 232.7%, respectively. These 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 experiment

[0117] The cell migration-promoting effect of the recombinant humanized type I collagen fiber implant prepared in Example 3 was determined by the cell scratch assay. Three horizontal lines were drawn on the back of each well of a 6-well plate using a marker pen, with a line spacing of 0.5-1 cm. 2 mL of cells at a density of 5 × 10⁶ cells were seeded into each well of the 6-well plate. 5 HFF-1 cells or chondrocytes were cultured at a density of 1 / mL to ensure a confluence of 95%-100% after 24 hours. After 24 hours, a 10 μL pipette tip was used to gently push downwards along the wells to create longitudinal scratches, aligned with a ruler. Recombinant humanized type I collagen fiber implant, ACl I (positive control) extract, and either DMEM high-glucose medium or DMEM / F12 medium (negative control) were then added to the 6-well plates. The changes in scratch area at 0 hours and 24 hours were recorded under an inverted fluorescence microscope.

[0118] like Figure 7 As shown in Figures A(b,d) and B(b,d), compared to the blank control group, the cell scratch marks in the recombinant humanized type I collagen fiber implant group and the ACl I group were significantly reduced. The migration rates of HFF-1 cells and chondrocytes promoted in the blank control group were 28.9% and 29.1%, respectively; in the recombinant humanized type I collagen fiber implant group, the migration rates were 86.9% and 85.4%, respectively; and in the ACl I group, the migration rates were 83.6% and 86.7%, respectively (as shown in Figures A(b,d) and B(b,d)). These experimental results demonstrate that the recombinant humanized type I collagen fiber implant described in this application has excellent cell migration-promoting effects.

[0119] 4. Live / dead cell staining

[0120] Add 1 mL of the solution to a laser confocal microplate, with a cell density of 1 × 10⁻⁶ cells. 5 HFF-1 cells or chondrocytes per mL were incubated at 37°C and 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 ACl I, and the blank control group was only added to DMEM high-glucose medium or DMEM / F12 medium. The cells were incubated at 37°C and 5% CO2 for another 5 days. The cells were stained with a live / dead cell staining kit for 1 hour, and the stained images were obtained using a laser confocal microscope.

[0121] Live / dead cell staining results 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 this 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 co-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 min, followed by permeabilization with 0.1% Triton X-100 for 5 min. Next, a blocking experiment was performed by incubation in PBS buffer (10 mM, pH 7.4) containing 1% BSA for 30 min at room temperature. The actin cytoskeleton was stained with 100 nM phalloidin-tetramethylrhodamine for 1 h. Cell nuclei were stained by incubation at 37°C for 20 min with 5 μg / mL Hoechst 33258. Finally, fluorescence images were captured 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, such as... Figure 7 As shown: Compared with the blank control group, the cells in the recombinant humanized type I collagen fiber implant group described in this application exhibited a fine actin cytoskeleton structure, an expanded diffusion area, and a significantly increased cell density (as shown in f in A and f in B).

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

[0126] The ability of recombinant humanized type I collagen fiber implants to promote chondrogenic differentiation of HFF-1 cells and bone marrow mesenchymal stem cells was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR). Cell density was 1×10⁶ cells / year. 5 HFF-1 cells or bone marrow mesenchymal stem cells were cultured at a density of 10 cells / mL in 6-well plates and incubated for 7 days with recombinant humanized type I collagen fiber implant. Total RNA was extracted from the cells in 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, and gene expression associated with fibroblast differentiation was assessed using a real-time quantitative PCR system. -ΔΔCT The relative expression levels of the target genes were calculated using a method with GAPDH as an internal reference gene. Primer sequences for genes related to fibroblast differentiation and bone marrow mesenchymal stem cell chondrogenic differentiation are shown in Table 1.

[0127] Table 1 Primer sequences of genes associated with fibroblast differentiation and chondrogenic differentiation.

[0128]

[0129] α-SMA is an important marker for fibroblast differentiation into myofibroblasts. For example... Figure 7 As shown, after 7 days of co-culturing HFF-1 cells with recombinant humanized type I collagen fiber implant, the α-SMA expression levels in the recombinant humanized type I collagen fiber implant group and the ACl I group increased by 1.26-fold and 1.57-fold, respectively, with no significant difference between the two groups (shown as g in A). As major 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 better than those in the blank control group (shown as hi in A). Similarly, for bone marrow mesenchymal stem cells, the recombinant humanized type I collagen fiber implant significantly promoted the expression of key transcription factors for chondrogenic differentiation, such as Sox9, proteoglycan Acan, and type II collagen Col2α1 (shown as gi in B). These results indicate that the recombinant humanized type I collagen fiber implant has excellent ability to promote HFF-1 cell differentiation and bone marrow mesenchymal stem cell chondrogenic differentiation.

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

[0131] 1. Pyrogen Experiment

[0132] Animal experiments were conducted in accordance with the ethical standards of the Ethics Committee of the School of Chemistry and Chemical Engineering, Lanzhou University. New Zealand White rabbits (2.0-3.0 kg, 3-4 months old) were purchased from the Animal Center of Lanzhou University. Before temperature measurement, rabbits were fasted for 2 hours. Afterward, rectal temperature was measured every 30 minutes for a total of 8 measurements. The normal temperature range was 38.0-39.6℃, with a maximum permissible difference of 0.4℃ between the highest and lowest temperatures. Three rabbits meeting the temperature standard were selected from each group and numbered. Before the experiment began, body temperature was monitored every 30 minutes, ensuring the temperature difference between consecutive measurements did not exceed 0.2℃. The average of the two measured temperatures was taken as the rabbit's normal body temperature. Recombinant humanized type I collagen fiber implant extract (recombinant humanized type I collagen fiber implant extracted in 0.9% NaCl solution for 72 hours) and 0.9% NaCl (control group) were injected via the marginal ear vein, at a dose of 2 mL. Rectal temperature was measured every 30 minutes after injection, for a total of 6 measurements. The highest body temperature measured six times is subtracted from the normal body temperature to obtain the body temperature rise value. If the body temperature rise of each rabbit in the group does not exceed 0.6℃, and the sum of the body temperature rises of the three rabbits in the group does not exceed 1.3℃, then the recombinant humanized type I collagen fiber implant is considered to be 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℃, 0.1℃, and 0℃, respectively, while the body temperatures of the three rabbits in the recombinant humanized type I collagen fiber implant group increased by 0.3℃, 0℃, and 0.2℃, respectively. The total temperature increase of each group of rabbits was less than 1.3℃, indicating that the recombinant humanized type I collagen fiber implant is non-pyrogenic.

[0134] 2. Hemolysis test

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

[0136]

[0137] OD a OD b and OD cThe absorbance values ​​are those of the test sample, 0.9% NaCl, and H2O, respectively. H2O is used as a positive control with a hemolysis rate of 100%, and 0.9% NaCl is used as a negative control with a hemolysis rate of 0%.

[0138] The results are as follows Figure 8 As shown in Figure B, the hemolysis rate of the recombinant humanized type I collagen fiber implant was only 0.53% as measured by the hemolysis test, 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 a risk of hemolysis.

[0139] 3. Immunogenicity

[0140] BALB / c mice (20-25g, 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. On days 1, 14, and 35, the experimental groups were subcutaneously injected with 100 μL of an emulsion of recombinant humanized type I collagen fiber implantation mixed with an incomplete Freund's adjuvant. The adjuvant-free group was subcutaneously injected on days 1, 14, and 35 with a sample of 100 μL of a mixture of recombinant humanized type I collagen fiber implantation and PBS. Blood was collected from the saphenous vein before the first injection and subsequently 7 days after each injection. All blood samples were allowed to stand at room temperature for 1-2 hours, then centrifuged at 3000 rpm for 15 minutes before serum collection and storage at -20°C. Antibody levels in the serum were detected using enzyme-linked immunosorbent assay (ELISA).

[0141] The immunogenicity of recombinant humanized type I collagen fiber implants was assessed by measuring serum levels of total IgG, IgA, and IgM. The results are as follows: Figure 8 As shown in Figure C, after three immunizations with or without the addition of adjuvants, the levels of IgG, IgA, and IgM in the mouse serum did not show statistically significant differences compared with those before injection, indicating that the recombinant humanized type I collagen fiber implant described in this application is non-immunogenic.

[0142] Example 6: Crosslinking Recombinant Humanized Type I Collagen Fiber Implants with Different Crosslinking Agents

[0143] 1. Crosslinking recombinant humanized type I collagen fibers with different crosslinking 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 hours and then centrifuged at 4°C, 10,000 rpm for 10 minutes to obtain the 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 were added respectively. After crosslinking at 4°C for 24 h, the product was centrifuged and washed three times with 50 mM PB. The crosslinked recombinant humanized type I collagen fiber implant was obtained by centrifugation.

[0147] 2. Scanning electron microscopy characterization of cross-linked recombinant humanized type I collagen fiber implants

[0148] The experimental procedure was the same as in Example 2. Scanning electron microscopy characterization of recombinant humanized type I collagen fiber implants crosslinked with different crosslinking agents is as follows: Figure 9 As shown, after crosslinking recombinant humanized type I collagen fibers with a crosslinking agent, the recombinant humanized type I collagen implant still maintains a dense fiber morphology. After crosslinking 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 crosslinking is beneficial to increasing the diameter of collagen fibers.

[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 in Example 3. The results are as follows: Figure 10 As shown: On day 14, the degradation rate of the BDDE-crosslinked recombinant humanized type I collagen fiber implant was 30.0±4.0%, the degradation rate of the THPC-crosslinked recombinant humanized type I collagen fiber implant was 33.3±4.2%, and the degradation rate of the uncrosslinked recombinant humanized type I collagen fiber implant was 48.7±4.2%. The experimental results indicate that the enzymatic resistance of the recombinant humanized type I collagen fiber implant was significantly improved after crosslinking with BDDE or THPC.

[0151] Example 7: Evaluation of the 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. The results are as follows: Figure 11As shown in Figures AB, after co-culturing HFF-1 cells with BDDE-crosslinked recombinant humanized type I collagen fiber implant (B-ATHRCI) for 24 h, a large number of HFF-1 cells migrated to the scratch area, with a cell migration rate of 77.2±11.4%, while the cell migration in the control group was not significant, with a cell migration rate of only 18.0±5.5% (shown in AB). Similarly, after co-culturing cells with THPC-crosslinked recombinant humanized type I collagen fiber implant (T-ATHRCI) for 24 h, the cell migration rate was 83.5±9.9%, while the cell migration rate in the control group was only 36.2±2.5% (shown in GH). The experimental results indicate that recombinant humanized type I collagen fiber implants crosslinked 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 in Example 4. Live / dead cell staining and CCK-8 assays were used to assess the cell proliferation-promoting ability of the BDDE and THPC cross-linked recombinant humanized type I collagen fiber implant. Live / dead cell staining results are shown below. Figure 11 As shown, with increasing incubation time of HFF-1 cells with the BDDE / THPC cross-linked recombinant humanized type I collagen fiber implant, the number of surviving cells gradually increased, and the cell density was significantly higher than that of the control group (shown in C and I). CCK-8 assay results further demonstrated that, with increasing incubation time, the cell viability of the BDDE and THPC cross-linked recombinant humanized type I collagen fiber implant group gradually increased (shown in D and J). These results indicate 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 crosslinked 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 groups showed a fine actin cytoskeleton structure, an increased diffusion area, and a significant increase in cell density. The results indicate that the cross-linked recombinant humanized type I collagen fiber implant significantly promotes cell adhesion and spread.

[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 control group, both BDDE and THPC cross-linked 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 cross-linked recombinant humanized type I collagen fiber implants have excellent HFF-1 cell differentiation-promoting capabilities.

[0160] The above experimental results demonstrate that this invention provides a recombinant humanized type I collagen protein with a unique triple helix structure and excellent self-assembly properties. Furthermore, by optimizing the self-assembly process of the recombinant humanized type I collagen protein, this invention prepares a recombinant humanized type I collagen protein implant with ordered collagen fiber arrangement. This implant exhibits excellent injectability, durability, and high biocompatibility, with no risk of heat generation or hemolysis, and low immunogenicity. It significantly promotes not only the adhesion, migration, proliferation, and differentiation of human skin fibroblasts, but also the adhesion, migration, and proliferation of chondrocytes and the chondrogenic differentiation of bone marrow mesenchymal stem cells, making it applicable to the fields of skin, cartilage, and bone.

Claims

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

1.

2. A recombinant humanized type I collagen fiber implant, characterized in that, The recombinant humanized type I collagen implant is formed by the self-assembly of the recombinant humanized type I collagen as described in claim 1.

3. The recombinant humanized type I collagen implant as described in claim 2, characterized in that, The preparation method of the implant includes: dissolving recombinant humanized type I collagen in 0-200 mM PB buffer, pH 5.0-7.4 to a concentration of 50-100 mg / mL, letting it stand at 4-25 ℃ for 24-72 h, centrifuging at 1000-10000 rpm for 5-20 min, and precipitating it as recombinant humanized type I collagen fiber implant.

4. The recombinant humanized type I collagen fiber implant as described in claim 3, characterized in that, The preparation method is as follows: recombinant humanized type I collagen is dissolved in 50 mM PB, pH 7.4 buffer to a concentration of 75 mg / mL, incubated at 25 ℃ for 24 h, and then centrifuged at 10000 rpm for 10 min to obtain the precipitate as recombinant humanized type I collagen fiber implant.

5. 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 2-4 with a cross-linking agent, wherein the cross-linking agent includes BDDE and THPC.

6. The cross-linked recombinant humanized type I collagen fiber implant as described in claim 5, characterized in that, The preparation method of the cross-linked recombinant humanized type I collagen fiber implant includes the following steps: (1) Prepare the recombinant humanized type I collagen fiber implant as described in any one of claims 2-4; (2) Disperse the recombinant humanized type I collagen fiber implant into a buffer solution of 0-200 mM PB and pH 5.0-7.4, add 0.001-2.0% BDDE or 0.001-0.05% THPC by volume, crosslink at 4-25 ℃ for 24-72 h, centrifuge at 1000-10000 rpm for 5-20 min, and wash the precipitate with a buffer solution of 0-200 mM PB and pH 5.0-7.4 to obtain the crosslinked recombinant humanized type I collagen fiber implant.

7. The cross-linked recombinant humanized type I collagen fiber implant as described in claim 6, characterized in that, Step (2) is as follows: the recombinant humanized type I collagen fiber implant is dispersed in a buffer solution of 50 mM PB and pH 7.4, and 2.0% BDDE or 0.05% THPC is added respectively. After crosslinking at 4℃ for 24 h, the mixture is centrifuged at 10000 rpm for 10 min. The precipitate is washed three times with a buffer solution of 50 mM PB and pH 7.4 and centrifuged at 10000 rpm for 10 min. The precipitate obtained is the crosslinked recombinant humanized type I collagen fiber implant.

8. The use of the recombinant humanized type I collagen fiber implant as described in claim 2 or the cross-linked recombinant humanized type I collagen fiber implant as described in claim 5 in the preparation of medical devices for use in the fields of skin, cartilage, and bone.

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