A biosynthetic type i collagen hydrogel for fracture repair and methods of making the same

Abstract

The application discloses a biosynthetic type I collagen hydrogel for fracture repair and a preparation method thereof, and belongs to the technical field of genetic engineering. 10 The application promotes the high-polymerization self-assembly of collagen to form type I collagen fiber collagen fibers with periodic light and dark stripes by fusing and expressing (PPG) 10 at the N terminal and the C terminal of the collagen, and promotes the further covalent crosslinking of the collagen fibers to form the hydrogel by introducing Cys in the (PPG) 10 and the CL-domain through directional design. The collagen fiber structure in the hydrogel prepared by the application is similar to that of natural type I collagen fibers, the hydrogel has good biocompatibility and bone repair function, the preparation process is simple, the type I collagen fiber hydrogel can be produced on a large scale and has low cost, and the type I collagen fiber hydrogel has a wide application prospect in the field of biological materials.

Description

Technical Field

[0001] This invention relates to a biosynthetic type I collagen hydrogel for fracture repair and its preparation method, belonging to the field of genetic engineering technology. Background Technology

[0002] Collagen, composed of three polypeptide chains forming a triple helix structure, is the most abundant protein in mammals, accounting for one-third of the total protein in the human body. It is a major component of the extracellular matrix and plays a crucial role in regulating cell proliferation and differentiation, as well as in the repair and regeneration of tissues and organs. There are 28 types in mammals, with type I being the most common. In higher organism cells, type I collagen undergoes translation, modification, folding, and cleavage to form regularly arranged banded fibrous structures. This banded feature, characterized by alternating dark and light bands, is known to be essential for osteoblast differentiation and matrix mineralization. This structure facilitates cell adhesion and growth, and plays a key role in the repair and regeneration of tissues and organs. Under an electron microscope, type I collagen fibers exhibit alternating light and dark striped patterns. The unique periodic pattern in collagen is formed by the offset and parallel alignment of the collagen triple helix, which is crucial for cell activity, osteoblast differentiation, and matrix mineralization.

[0003] Type I collagen extracted from animals has wide applications as a biomaterial in food, cosmetics, drug delivery, and biomedical engineering. It can be used in cell culture, tissue engineering, and regenerative medicine. It can also serve as a scaffold or matrix material for skin burn dressings, surgical and dental hemostatic sponges, and bone defect fillings, promoting tissue regeneration and repair. Market demand is huge. Currently, most type I collagen on the market comes from animal skin and connective tissues such as Achilles tendons. While it has the advantages of high biocompatibility and easy absorption by the human body, it is susceptible to contamination from animal-derived diseases such as prions. To improve biosafety, how to prepare clean-source type I collagen is an important issue in the field of biomedical materials.

[0004] Currently, there are four main sources of collagen: 1. Animal extraction: inexpensive, but prone to carrying pathogens; 2. Chemical synthesis: high purity and strong controllability, but expensive, limited in length, and unsuitable for mass production; 3. Expression in eukaryotic systems such as transgenic plants and mammalian cells: capable of correctly folding proteins and performing complex post-translational processing, but with high culture costs, long cycles, low expression levels, and difficulty in large-scale production; 4. Microbial expression systems: with significant advantages such as low cost and high expression levels.

[0005] Studies have shown that an increasing number of mammalian and bacterial collagens are efficiently heterologously expressed in hosts such as bacteria and yeast, and correctly fold into collagen triple helix structures, exhibiting advantages such as low cost and high expression levels. However, most recombinant collagens still lack self-assembly driving forces and cannot polymerize into hydrogels with periodic stripes or specific structures similar to natural type I collagen fibers. This limits their inherent advantages in cell viability, osteogenic differentiation, and biomineralization, hindering their potential applications in biomaterials and tissue engineering.

[0006] Collagen hydrogels are biodegradable, water-rich three-dimensional matrices that provide biological signals and act as cell scaffolds, creating the necessary microenvironment for cell growth, differentiation, and migration, making them suitable for bone repair. Therefore, the preparation of collagen hydrogels with visible collagen streaks and good physiological activity has extremely high practical and economic value. Summary of the Invention

[0007] This application describes the expression of PPG through N-terminal and C-terminal fusion of collagen from different sources. 10 It promotes the high-polymer self-assembly of collagen to form collagen fibers, and through (PPG) 10 Introducing Cys into the neutralized CL-domain promotes further covalent cross-linking of collagen fibers to form a hydrogel.

[0008] The first object of the present invention is to provide a collagen single chain having the following structure:

[0009] From the N-terminus to the C-terminus are: the first repeat sequence, the collagen domain, and the second repeat sequence;

[0010] Specifically, 2 to 5 cysteine ​​residues are introduced into the repeating sequences and / or collagen domains of collagen single chains;

[0011] The introduction is the insertion of cysteine, or the replacement of an amino acid with cysteine.

[0012] Optionally, 2 to 5 cysteine ​​residues may be introduced into the repeating sequence and collagen domain of the collagen single chain.

[0013] In one embodiment, one cysteine ​​residue (named CC) is introduced at the front and the end of the second repeat sequence, respectively.

[0014] Alternatively, one cysteine ​​residue (named NC) may be introduced at the end of the first repeat sequence and the second repeat sequence, respectively.

[0015] In one embodiment, 1 to 3 cysteine ​​residues are introduced into the collagen domain;

[0016] Optionally, one cysteine ​​residue is introduced into the collagen domain;

[0017] Optionally, two cysteine ​​residues are introduced into the collagen domain, with the two cysteine ​​residues separated by 40 to 80 amino acids;

[0018] Optionally, three cysteine ​​residues are introduced into the collagen domain, with the three cysteine ​​residues located in amino acids 1-40, 41-80, and 81-120 of the collagen domain, respectively.

[0019] In one embodiment, the collagen single chain has the following structure:

[0020] The sequence from N-terminus to C-terminus is the first repeat sequence (PPG). n Collagen domain (CL-domain) and second repeat sequence (PPG)n.

[0021] In one embodiment, a folding domain is attached to the N-terminus of the first repeat sequence, and the amino acid sequence of the folding domain is shown in SEQ ID NO.1;

[0022] Optionally, the front of the folded field also includes a 6×His tag.

[0023] In one embodiment, a folding domain is attached to the N-terminus of the first repeating sequence, the amino acid sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is shown in SEQ ID NO.16.

[0024] There is a linker peptide between the folded domain and the repeat sequence, the amino acid sequence of which is shown in LVPRGSPG (SEQ ID NO.4);

[0025] Optionally, the first and second repeat sequences (PPG) n n is an integer from 4 to 30; preferably, n is from 5 to 10; preferably, the amino acid sequences of the first and second repeating sequences are as shown in SEQ ID NO.2;

[0026] Optionally, the collagen domain (CL-domain) includes human collagen, bacterial collagen, recombinant human collagen, and recombinant bacterial collagen;

[0027] Optionally, the amino acid sequence of the collagen domain is shown in SEQ ID NO.3.

[0028] In one embodiment, in the structure of a collagen single chain, the repeating sequence located at the N-terminus (front end) of the CL-domain is the first repeating sequence, and the repeating sequence located at the C-terminus (back end) of the CL-domain is the second repeating sequence.

[0029] In one embodiment, the collagen single chain is as shown in any one of SEQ ID NO. 8 to SEQ ID NO. 11; the folded and enzyme-digested collagen single chain is as shown in any one of SEQ ID NO. 18 to SEQ ID NO. 21.

[0030] In one embodiment, an amino acid is replaced with C at the end of the CL-domain, and a cysteine ​​residue is inserted at the end of the second repeat sequence.

[0031] Optionally, the Q (glutamine) at position 119 of the CL-domain (SEQ ID NO.3) is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, with the amino acid sequence of the collagen single chain as shown in SEQ ID NO.8.

[0032] In one implementation, an amino acid is replaced with C at the front end of the CL-domain, and a cysteine ​​residue is inserted at the end of the second repeat sequence.

[0033] Optionally, the first position (N-terminus) P of the CL-domain (SEQ ID NO.3) is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, the amino acid sequence of the collagen single chain being shown in SEQ ID NO.9.

[0034] In one embodiment, the first P in the CL-domain (SEQ ID NO.3) is replaced with C, and based on the insertion of a cysteine ​​residue at the end of the second repeat sequence (i.e., based on the collagen single chain with the amino acid sequence as shown in SEQ ID NO.9), the amino acid (X or Y in GXY) in the CL-domain is replaced with C.

[0035] Optionally, in the first 1 / 3 of the peptide in the CL-domain (amino acids 1 to 40 of the CL-domain), one X or Y is replaced with C;

[0036] Optionally, in the middle 1 / 3 of the peptide in the CL-domain (amino acids 41 to 80 of the CL-domain), one X or Y is replaced with C;

[0037] Optionally, in the last third of the peptide segment of the CL-domain (amino acids 81 to 120 of the CL-domain), one X or Y is replaced with C.

[0038] In one implementation, the first P in the CL-domain is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, and two X or Y residues in the CL-domain are replaced with C.

[0039] Optionally, in the first half of the peptide in the CL-domain (amino acids 1 to 60 of the CL-domain), one X or Y is replaced with C, and in the last half of the peptide in the CL-domain (amino acids 61 to 120 of the CL-domain), one X or Y is replaced with C.

[0040] Optionally, the distance between the two substituted Cs is 40 to 80 amino acids.

[0041] In one implementation, the first P in the CL-domain is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, and three X or Y residues in the CL-domain are replaced with C.

[0042] Optionally, in the first 1 / 3 of the peptide segment (amino acids 1 to 40 of the CL-domain), the middle 1 / 3 of the peptide segment (amino acids 41 to 80 of the CL-domain), and the last 1 / 3 of the peptide segment (amino acids 81 to 120 of the CL-domain), one X or Y is replaced with C.

[0043] Optionally, in the three substituted Cs, the distance between each pair is 20 to 40 amino acids.

[0044] A second object of the present invention is to provide a gene encoding any of the above-mentioned single chains of collagen, a plasmid or cell carrying the gene encoding any of the above-mentioned single chains of collagen;

[0045] Optionally, the plasmids include the pColdIII series or the pET series.

[0046] In one embodiment, the cells are Escherichia coli cells, including but not limited to E. coli BL21 and E. coli BL21(DE3).

[0047] A third object of the present invention is to provide a collagen product obtained by preparing any of the above-mentioned collagen single chains;

[0048] Optionally, the collagen product includes type I collagen, collagen fibers, and collagen hydrogel.

[0049] In one embodiment, the type I collagen is formed by the high-polymer self-assembly of the above-mentioned collagen single chains, and has a striped pattern of alternating light and dark.

[0050] In one embodiment, the collagen fibers are formed by the high-polymer self-assembly of the aforementioned type I collagen.

[0051] In one embodiment, the collagen hydrogel is prepared by:

[0052] Collagen prepared by fermentation using the above-mentioned collagen gene, plasmid carrying the gene encoding any of the above-mentioned collagen, or cells, is purified, dialyzed, and freeze-dried.

[0053] The freeze-dried collagen was prepared into a solution with a concentration of 0.1–1 mmol / L and allowed to stand at 4℃–37℃ for at least 2 days to obtain type I collagen fibers.

[0054] After freeze-drying, the collagen is prepared into a solution with a concentration of ≥100g / L and left to stand at 4℃~37℃ for 1 week, or hydrogen peroxide is added and left to stand at room temperature for more than 2 hours to obtain type I collagen hydrogel.

[0055] In one embodiment, the type I collagen hydrogel exhibits excellent fracture repair effects.

[0056] The fourth objective of this invention is to provide a method for improving the mechanical strength of collagen hydrogels while maintaining the light and dark streaks of collagen, by preparing collagen hydrogels using collagen single chains;

[0057] The collagen single chain has the following structure:

[0058] From the N-terminus to the C-terminus are: the first repeat sequence, the collagen domain, and the second repeat sequence;

[0059] Specifically, 2 to 5 cysteine ​​residues are introduced into the repeating sequences and / or collagen domains of collagen single chains;

[0060] The introduction is the insertion of cysteine, or the replacement of an amino acid with cysteine.

[0061] In one embodiment, 2 to 5 cysteine ​​residues are introduced into the repeating sequence and collagen domain of the collagen single chain.

[0062] In one embodiment, the collagen single chain has the following structure:

[0063] The sequence from the N-terminus to the C-terminus consists of a folded domain, a first repeat sequence, a collagen domain, and a second repeat sequence; wherein the amino acid sequence of the folded domain is shown in SEQ ID NO.1; the amino acid sequences of the first and second repeat sequences are shown in SEQ ID NO.2; and the amino acid sequence of the collagen domain is shown in SEQ ID NO.3.

[0064] Two to five cysteine ​​residues are introduced into the repeating sequences and collagen domains of collagen single chains. The introduction is either by inserting cysteine ​​residues or by replacing amino acids with cysteine ​​residues.

[0065] In one embodiment, one cysteine ​​residue is introduced at the beginning and end of the second repeat sequence, respectively;

[0066] Alternatively, one cysteine ​​residue may be introduced at the end of the first repeat sequence and the end of the second repeat sequence.

[0067] Optionally, 1 to 3 cysteine ​​residues are introduced into the collagen domain;

[0068] Optionally, one cysteine ​​residue is introduced into the collagen domain;

[0069] Optionally, two cysteine ​​residues are introduced into the collagen domain, with the two cysteine ​​residues separated by 40 to 80 amino acids;

[0070] Optionally, three cysteine ​​residues are introduced into the collagen domain, with the three cysteine ​​residues located at amino acids 1-40, 41-80, and 81-12 of the collagen domain, respectively.

[0071] A fifth object of the present invention is to provide the application of any of the above-described collagen single chains or the above-described genes, plasmids, cells or the above-described collagen products in the fields of biology, food, chemicals, medicine, biomaterials, tissue engineering or cosmetics.

[0072] This invention also provides a method for controlling the mechanical properties and morphology of hydrogels, wherein the method controls the expression of collagen molecules CL-domain and (PPG) in microbial cells. 10 The number of Cys residues in the amino acid sequence, as well as the CL-domain and (PPG) 10 The number of repetitions.

[0073] Beneficial effects:

[0074] 1. This invention uses N-terminal and C-terminal (PPG) 10 Based on the sequence, a continuous Xaa-Yaa-Gly triplet collagen sequence is inserted in the middle, forming a three-segment chimeric collagen P-CL-P pattern. This is achieved through N-terminal and C-terminal (PPG)... 10 The three helices interact and self-assemble into type I collagen fibers with periodic light and dark stripes. This is achieved through the interaction of (PPG) 10 Introducing Cys into the neutralized CL-domain promotes further covalent cross-linking of collagen fibers to form a hydrogel.

[0075] 2. This invention utilizes cold shock expression of collagen sequences in *E. coli* to prepare clean collagen that can self-assemble into solutions and hydrogels with type I collagen fiber structures. Its structure resembles type I collagen fibers. The process is simple, low-cost, and suitable for large-scale production. This invention provides a method and sequence design pattern for preparing type I collagen fibers. The collagen region of this sequence is replaceable and expandable, providing a platform for the research and application of periodic collagen fibers with light and dark stripes, and has broad prospects in biomaterials applications.

[0076] 3. This invention also regulates the mechanical properties and microstructure of the hydrogel by adjusting the amount of Cys in the collagen region. This is achieved by controlling (PPG) 10 The replacement location and number of Cys in collagen regions can control the cross-linking of type I collagen fibers to form hydrogels; by controlling the replacement number of Cys in CL-domain collagen regions, the mechanical properties and microstructure of the hydrogels formed by the cross-linking of type I collagen fibers can be controlled.

[0077] 4. The collagen prepared by this invention is non-cytotoxic and can promote the adhesion, extension and osteogenic differentiation of osteoblast precursor cells MC3T3-E1, as well as promote fracture repair in SD rats. Attached Figure Description

[0078] Figure 1 Design a schematic diagram for the sequence;

[0079] Figure 2 SDS-PAGE identification of collagen;

[0080] Figure 3 The molecular weight of the designed collagen MALDI-TOF was determined; a to f are the molecular weight determinations of MALDI-TOF.

[0081] Figure 4 The secondary structure of the designed collagen was determined; a) is the full-wavelength scan spectrum of circular dichroism chromatography; b) is the thermal curve of circular dichroism chromatography.

[0082] Figure 5 The morphologies of self-assembled fibers of mC, CC, and NC under natural, oxidizing, and reducing environments are shown. Among them, a, d, and g are the self-assembled fiber morphologies of mC under natural, oxidizing, and reducing environments, respectively; b, e, and h are the self-assembled fiber morphologies of CC under natural, oxidizing, and reducing environments, respectively; and c, f, and i are the self-assembled fiber morphologies of NC under natural, oxidizing, and reducing environments, respectively.

[0083] Figure 6The assembly particle size and hydrogel mechanical properties of mC, CC, and NC are given; where a represents the particle size of mC, CC, and NC under reducing (DTT) and oxidizing (H2O2) conditions, and b represents the mechanical properties of hydrogels prepared from mC, CC, and NC.

[0084] Figure 7 The internal structures of mC, CC, and NC hydrogels are shown below; where a, d, and g represent the internal structures of mC; b, e, and h represent the internal structures of CC; and c, f, and i represent the internal structures of NC.

[0085] Figure 8 The self-assembled fiber morphology of NC1, NC3 and NC5 under natural, oxidative and reducing environments;

[0086] Figure 9 The assembly particle size and mechanical properties of hydrogels of NC1, NC3 and NC5 are given; where a is the particle size of NC1, NC3 and NC5 under reducing (DTT) conditions and oxidizing (H2O2) conditions; b is the mechanical properties of hydrogels prepared from NC1, NC3 and NC5.

[0087] Figure 10 The internal structures of NC1, NC3, and NC5 hydrogels are shown below; where a and d represent the internal structures of NC1 hydrogel; b and e represent the internal structures of NC3 hydrogel; and c and f represent the internal structures of NC5 hydrogel.

[0088] Figure 11 To validate the biocompatibility and bioactivity of the designed type I collagen; where a represents cell proliferation results; b represents cell adhesion ability; c represents VCL mRNA expression level; d represents cell diffusion area; e represents cytoskeleton staining; f represents RUNX2 and Col1a1 mRNA expression levels; g represents ALP staining area; h represents ALP activity; and i represents ALP staining images of the entire well and the central region of the well.

[0089] Figure 12 To validate the fracture repair function of NC1 collagen hydrogel; where a is micro-CT scan 4 weeks after repair; b is bone tissue parameter analysis; c is H&E and immunohistochemical staining analysis; d is quantitative immunohistochemical staining analysis. Detailed Implementation

[0090] Materials and methods used in this invention:

[0091] Culture medium:

[0092] LB solid medium: 15 g / L agar, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0.

[0093] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0.

[0094] TB liquid culture medium: 12 g / L tryptone, 24 g yeast extract, 4 mL glycerol, 2.31 g KH2PO4, 12.54 g K2HPO4, pH 7.5, bring the volume to 1 L.

[0095] Cultivation methods:

[0096] Seed culture conditions: Single colonies grown from streaks on plates were inoculated into LB liquid medium with a medium volume of 10%, and cultured in 250mL shake flasks at a temperature of 37℃ for 10 hours at a rotation speed of 200rpm.

[0097] Fermentation culture conditions: TB medium was used with a medium volume of 20% and an inoculum size of 1%. The medium was cultured in 500 mL shake flasks at 37°C for 24 h. Then, IPTG at a final concentration of 1 mM was used for induction at 25°C for 10 h. The medium was then transferred to 15°C for 14 h of induction at a rotation speed of 200 rpm.

[0098] Example 1: Collagen Sequence Design and Sample Preparation

[0099] 1. Amino acid sequence design

[0100] The design follows the structure shown in VP-CL-P, where V stands for V-domain (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.16), representing the globular domain that guides the correct folding of the collagen region; P stands for (PPG). 10 , namely PPGPPGPPGPPGPPGPPGPPGPPGPPGPPG(SEQ ID NO.2); CL stands for CL-domain, i.e., collagen region.

[0101] Through (PPG) 10 Introducing cysteine ​​(Cys) into the viscous module and CL-domain promotes further covalent cross-linking of collagen fibers to form a hydrogel (the introduction refers to inserting cysteine ​​or replacing an amino acid with cysteine). A schematic diagram of the collagen sequence combination is shown below. Figure 1 As shown.

[0102] The specific steps are as follows:

[0103] (1) The N and C ends of the sequence are (PPG) 10 The motif, with a CL-domain collagen region in the middle, yields a three-segment chimeric sequence (PPG). 10-CL-domain-(PPG) 10 (abbreviated as P) 10 CLP 10 ), in which CL-domain uses the collagen Scl2 (Genbank ID: AAL50184.1) derived from Streptococcus pyogenes to extract and splice an amino acid sequence (the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.13), and inserts an integrin binding site (i.e., cysteine) in the middle of the collagen sequence to achieve its biological function.

[0104] (2) A globular domain V-domain (amino acid sequence as shown in SEQ ID NO.1) derived from Scl2 was inserted at the N-terminus of the sequence to guide the correct folding of the collagen triple helix. A linker peptide LVPRGSPG (SEQ ID NO.4) was inserted between the globular domain and the fixed sequence unit of the collagen region. 6×His was inserted at the N-terminus of the sequence for purification.

[0105] The amino acid sequence was constructed, and its name and specific amino acid sequence are shown in Table 1.

[0106] Table 1 Amino acid sequences

[0107]

[0108]

[0109] Among them, NC1, NC3, and NC5 are based on NC with an additional 3 to 5 Cs.

[0110] 2. Construction of recombinant strains

[0111] The pColdM plasmid was constructed by mutating the pColdIII plasmid using pColdM-S:CTCGAGGGATCCGAATTCA (SEQ ID NO.14) and pColdM-A:GAGCTCCATGGGCACTTTG (SEQ ID NO.15) as primers to introduce the NcoI site.

[0112] The genes corresponding to the amino acid sequences in Table 1 are synthesized, and their nucleotide sequences contain a 5' Nco I restriction site, a 5' flanking GC sequence, and a 3' Bam HI restriction site, respectively.

[0113] The synthesized genes were inserted between NcoI and BamHI in the pColdM plasmid to obtain the corresponding recombinant collagen protein plasmids. The recombinant plasmids were then transformed into E. coli BL21(DE3) competent cells using the CaCl2 method. The cells were plated on LB plates containing antibiotics, cultured, and screened to obtain recombinant strains for preparing heterozygous collagen. Based on the name of collagen, the recombinant strains were named E. coli-mC, E. coli-CC, E. coli-NC, E. coli-NC1, E. coli-NC3, and E. coli-NC5.

[0114] 3. Collagen Sample Preparation

[0115] After induction and fermentation of the recombinant strain, the fermentation broth was centrifuged at 8000 rpm for 5 minutes, and the bacterial cells were collected separately. The bacterial cells were resuspended in 10 mM phosphate buffer, and the cells were homogenized using an autoclave. Cell debris was then removed by centrifugation at 10000 rpm for 20 minutes at 4°C, and impurities were removed by filtration through a 0.45 μm microporous membrane. The sample was injected into a 5 mL His-Trap HP affinity chromatography column installed in a protein purification instrument. Eight column volumes were washed with washing buffer, and proteins were eluted with elution buffer containing an imidazole gradient (140 mM, 400 mM). The peak protein was collected for SDS-PAGE electrophoresis analysis. Then, the globular guide fold domain was removed by trypsin digestion at a final concentration of 0.05 mg / mL at 25°C for 8 hours. Following dialyzing to desalt, the collagen was freeze-dried to obtain lyophilized powder. Based on the sequence, the collagen was named mC, CC, NC, NC1, NC3, and NC5 (the amino acid sequences of the digested and folded collagen are shown in SEQ ID NO. 17–21). Small amounts of the lyophilized powder were dissolved in water and identified by SDS-PAGE and MALDI-TOF.

[0116] Figure 2 The purified protein appeared as a single band on SDS-PAGE. Since collagen is a rod-shaped protein, the molecular weight of the globular protein marker used on SDS-PAGE was larger than expected.

[0117] Figure 3 The results of mass spectrometry show that the molecular weight obtained is consistent with the theoretical molecular weight, proving that the obtained collagen molecular weight is correct.

[0118] Example 2: Determination of collagen secondary structure

[0119] The collagen (mC, CC, NC, NC1, NC3, NC5) designed and prepared in Example 1 was prepared to a concentration of 1 mg / mL and incubated at 4°C for at least 24 hours. Circular dichroism spectroscopy was performed at 4°C using a 1 mm cuvette, scanning the entire wavelength range from 190 nm to 260 nm, with 1 nm intervals between wavelengths and a dwell time of 5 s at each wavelength. Thermochromatography was performed at 220 nm, with temperatures ranging from 4°C to 80°C, equilibrating at each temperature for 8 s, and a temperature increment rate of 1°C / 6 min. A typical triple-helix collagen CD spectrum showed a positive absorption peak at 225 nm.

[0120] The results are as follows Figure 4 As shown, under full-wavelength scanning, the collagen prepared in Example 1 has a characteristic absorption peak near 225 nm; the thermal change experiment results show that as the temperature increases, the characteristic absorption value at 225 nm changes abruptly between 37 and 55 °C, which is a manifestation of the destruction of the secondary structure of collagen, that is, the unwinding of the triple helix.

[0121] Both circular dichroism spectroscopy and thermal distortion tests showed that the collagen (mC, CC, NC, NC1, NC3, NC5) designed in Example 1 could fold correctly to form a triple helix structure of collagen and had high thermal stability.

[0122] Example 3: (PPG) 10 Effects of Cys substitution in different regions on fiber morphology

[0123] The freeze-dried collagen mC, CC, and NC prepared in Example 1 were prepared into a solution with a final concentration of 0.5 mM using 10 mM PB, or 10 mM PB containing 0.1% H2O2 and 100 mM DTT was added. After being placed at 4°C for 3.5 days, a small amount was dropped onto a copper grid, adsorbed for 45 seconds, and then blotted dry with filter paper. The solution was then negatively stained with 0.75% phosphotungstic acid for 20 seconds, blotted dry with filter paper, and observed using a Hitachi H-7650 transmission electron microscope.

[0124] Transmission electron microscopy results as follows Figure 5 As shown, in (PPG) 10 Introducing Cys into the motif affects the morphology of the self-assembled fiber. Under natural conditions, CC and NC form relatively good light and dark striped fibers. Under reducing conditions, mC, CC, and NC form obvious light and dark striped fibers because they are not affected by Cys crosslinking. Under oxidizing conditions, only NC can form good light and dark striped fibers.

[0125] Example 4: (PPG) 10 The Influence of Cys Replacement in Different Regions on Assembly, Hydrogel Mechanical Properties and Internal Structure

[0126] The collagen mC, CC, and NC obtained in Example 1 were tested for their properties, as detailed below:

[0127] 1. Collagen particle size detection

[0128] The lyophilized collagen mC, CC, and NC prepared in Example 1 were prepared into a solution with a final concentration of 0.5 mg / mL. The buffer solution was 10 mM PB containing 0.1% H2O2 or 100 mM DTT. After being placed at 4°C for 3.5 days, the hydrated particle size of the assembled particles was determined by dynamic light scattering. Figure 6 As shown in Figure a, mC, CC, and NC all aggregate into large particles with a hydration radius (Rh) of approximately 1000 nm. The particle size under oxidizing conditions is larger than that under reducing conditions, with the NC aggregates having the largest particle size at 1828 nm.

[0129] 2. Collagen hydrogel performance testing

[0130] (1) Appearance and modulus of hydrogel

[0131] The lyophilized collagen mC, CC and NC prepared in Example 1 were prepared into a solution with a final concentration of 100 g / L, placed at 4°C for 3.5 days, and then 0.1% H2O2 was added for cross-linking at room temperature for 2 hours.

[0132] The rheological properties of the hydrogel are as follows: Figure 6 As shown in b, at a concentration of 10% w / v, mC, CC, and NC all exhibit hydrogels, with storage modulus G' > loss modulus G''. The modulus of NC is larger than that of mC and CC. The appearance of the hydrogels is as follows. Figure 7 As shown in a, b, and c, mC, CC, and NC formed a translucent hydrogel. The sample did not flow in the inverted transparent centrifuge tube, forming a collagen hydrogel.

[0133] (2) Scanning electron microscopy of hydrogels

[0134] A small amount of hydrogel prepared in step (1) was frozen in liquid nitrogen and then freeze-dried in a freeze dryer. After sputtering gold onto the cross-section, the internal structure of the collagen hydrogel was observed using a scanning electron microscope. The results are as follows: Figure 7 As shown in d, e, f, g, h, and i, the hydrogel samples of mC, CC, and NC all exhibit a loose and porous collagen sponge-like structure; however, the internal structure of mC is an isotropic network-like microporous structure, while the internal structures of CC and NC are anisotropic layered stacked structures.

[0135] Example 5: Effect of different amounts of Cys substitution in the CL-domain region on fiber morphology

[0136] The freeze-dried collagen NC1, NC3, and NC5 prepared in Example 1 were prepared into a solution with a final concentration of 0.5 mM using 10 mM PB, or 10 mM PB containing 0.1% H2O2 and 100 mM DTT was added. After being placed at 4°C for 3.5 days, a small amount was dropped onto a copper grid, adsorbed for 45 seconds, and then blotted dry with filter paper. The solution was then negatively stained with 0.75% phosphotungstic acid for 20 seconds, blotted dry with filter paper, and observed using a Hitachi H-7650 transmission electron microscope.

[0137] Transmission electron microscopy results as follows Figure 8 As shown, replacing different amounts of Cys in the CL-domain region affects the morphology of the self-assembled fibers; the more Cys present, the more severe the fiber damage. Under natural conditions, NC1 and NC3 can form light and dark striped fibers; under reducing conditions, NC1 and NC3 form obvious light and dark striped fibers, while NC5 still cannot form light and dark striped fibers; under oxidizing conditions, only NC1 can form relatively good light and dark striped fibers.

[0138] Example 6: Effects of different amounts of adhesive used in CL-domain region replacement on mechanical properties and internal structure

[0139] The collagen NC1, NC3, and NC5 obtained in Example 1 were tested for their properties, as detailed below:

[0140] 1. Collagen particle size detection

[0141] The lyophilized collagen NC1, NC3 and NC5 prepared in Example 1 were prepared into a solution with a final concentration of 0.5 mg / mL. The buffer solution was 10 mM PB containing 0.1% H2O2 or 100 mM DTT. After being placed at 4°C for 3.5 days, the hydrated particle size of the assembled particles was determined by dynamic light scattering.

[0142] The results are as follows Figure 9 As shown in a, NC1, NC3 and NC5 all aggregate into large particles. NC1 has the largest hydrated particle size (Rh) at 1427 nm, while NC5 has the smallest hydrated particle size at 375 nm. The particle size under oxidizing conditions is larger than that under reducing conditions, and the aggregate formed by NC1 has the largest particle size at 3580 nm.

[0143] 2. Collagen hydrogel performance testing

[0144] (1) Appearance and modulus of hydrogel

[0145] The freeze-dried collagen NC1, NC3 and NC5 prepared in Example 1 were prepared into a solution with a final concentration of 100 g / L, placed at 4°C for 3.5 days, and then 0.1% H2O2 was added for cross-linking at room temperature for 2 hours.

[0146] The rheological properties of the hydrogel are as follows: Figure 9 As shown in b, at a concentration of 10%, NC1, NC3, and NC5 all exhibit hydrogels, with storage modulus G' > loss modulus G". The more Cys atoms replaced in the CL-domain region, the weaker the mechanical properties of the hydrogel. With increasing Cys content, the modulus of the hydrogel decreases, and the angular frequency reaching the sol point decreases. The appearance of the hydrogel is as follows... Figure 10 As shown in a, b, and c, NC1, NC3, and NC5 formed a translucent hydrogel. The sample did not flow in the inverted transparent centrifuge tube, forming a collagen hydrogel. After gelation at 4°C for more than 2 weeks, NC1 could be picked up with pointed tweezers and had strong mechanical properties, while NC3 and NC5 could not be picked up with pointed tweezers.

[0147] (2) Scanning electron microscopy of hydrogels

[0148] A small amount of hydrogel was frozen in liquid nitrogen, then lyophilized in a freeze dryer. Gold was sputtered onto the cross-section, and the internal structure of the collagen hydrogel was observed using a scanning electron microscope. Figure 10 As shown in d, e, and f, the hydrogel samples NC1, NC3, and NC5 all exhibit a loose and porous collagen sponge-like structure. The internal structure of NC5 is an isotropic network-like microporous structure, while the internal structures of NC1 and NC3 are anisotropic layered stacked structures.

[0149] Example 7: Validation of the biocompatibility and bioactivity of the designed type I collagen

[0150] The lyophilized collagen mC, CC, NC, NC1, NC3 and NC5 prepared in Example 1 were prepared into a collagen solution with a final concentration of 40 ng / μL. Bovine serum albumin (BSA) and rat tail type I collagen at the same concentration were used as negative and positive controls, respectively.

[0151] 100 μL of collagen solution was added to each well of a 96-well plate, with three replicates per group. After adsorption at 4°C for 24 h, the solution was aspirated, dried overnight, and then sterilized by UV irradiation for 30 min. Finally, the plates were blocked with 5% BSA for 2 h. MC-3T3-E1 cells were resuspended in DMEM containing 10% FBS at a density of 2000 cells per well, and 100 μL was seeded onto cell culture plates. Cell viability was measured using a CCK8 assay kit at days 0, 1, 3, and 5.

[0152] The results are as follows Figure 11 As shown in Figure a, the designed collagen had a significant effect on cell proliferation and no cytotoxicity. Collagen was adsorbed onto a 96-well plate in the same manner, and 10,000 cells were seeded. After culturing for 6 hours, the cells were washed three times with 10 mM PBS, and cell viability was measured using a CCK8 assay kit. Figure 11 b、 Figure 11 As shown in c, compared to samples with poor fiber morphology, NC1 exhibited a significant promoting effect on cell adhesion, superior to natural Type I collagen, and also significantly increased the expression level of VCL mRNA related to the cytoskeletal protein Vinculin. Figure 11 As shown in de, when the cell diffusion area was assessed using the same method and the cytoskeleton was stained with phalloidin, cells adhering to the NC1 matrix exhibited a larger diffusion area and more pronounced cytoskeleton extension, comparable to Type I. These results indicate that the synthesized collagen NC1 possesses excellent biocompatibility.

[0153] Furthermore, we verified the osteogenic differentiation capacity of MC3T3-E1 osteoblast precursor cells on the surface of designed collagen fibers. One week after inducing osteogenic differentiation in MC3T3-E1 cells, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining. Figure 11 As shown in fi, the ALP staining area and ALP activity of NC and NC1 are comparable to or even greater than those of type I collagen, significantly exceeding those of mC, CC, NC3, NC5 or BSA sample groups.

[0154] Meanwhile, type I collagen fibers NC and NC1 induced the expression of osteoblast-specific transcription factor RUNX2 and the type I collagen α1 chain Col1a1 of the bone matrix protein gene, with levels exceeding those of other synthetic collagens and even higher than those of natural type I collagen. The review demonstrates that NC and NC1 collagen can promote osteogenic differentiation of MC3T3-E1 osteoblast precursor cells.

[0155] Example 8: Verification of the fracture repair function of NC1 collagen hydrogel

[0156] The lyophilized collagen NC1 prepared in Example 1 was prepared into a solution with a final concentration of 100 g / L and placed at 4°C for one week to form NC1 collagen hydrogel, thus verifying its fracture repair function.

[0157] Six-week-old male SD rats underwent a 1.5–2 cm incision on the lateral side of the femur. The mucosa and muscle were bluntly dissected to expose the midshaft of the femur. Intramedullary fixation was performed using 1.2 mm Kirschner wires, and the fracture ends were secured with 3-0 absorbable sutures. Postoperatively, the wound was thoroughly rinsed with 0.9% sodium chloride solution, and the muscle and skin were sutured. Penicillin was administered intramuscularly at 100,000 units per rat for 3 consecutive days postoperatively to prevent infection.

[0158] 200 μL of each of the following treatments were injected into the fracture area: blank control group (physiological saline), NC1 collagen hydrogel, and positive control group (rat tail type I collagen hydrogel). The injections were administered every other day for two weeks. Four weeks later, the rats were sacrificed, and micro-CT scans were performed to observe fracture repair. The remaining samples were decalcified and subjected to tissue sectioning, H&E staining, and immunohistochemical staining (VEGF, RUNX2, and ALP) to observe angiogenesis and the expression levels of bone synthesis-related proteins.

[0159] The results are as follows Figure 12 As shown, micro-CT results revealed that, compared to the untreated drug-treated model, NC1 collagen hydrogel exhibited a significant fracture repair effect, closely resembling that of natural type I hydrogel from rat tails. Bone-related parameter analysis also indicated that the fracture areas treated with NC1 collagen hydrogel showed significantly increased bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone count, and connective tissue density.

[0160] H&E staining results showed no obvious inflammatory cell infiltration, and there was no significant difference between the groups. Immunohistochemical staining results are as follows: Figure 12 c. The vascular endothelial growth factor (VEGF) in the fracture area of ​​SD rats treated with NC1 collagen hydrogel was significantly increased, indicating that angiogenesis was active in the local microenvironment of the fracture and that NC1 collagen hydrogel has the ability to promote angiogenesis.

[0161] Compared with the control group, the protein expression level and alkaline phosphatase (ALP) activity of Runx2, a core transcription factor for osteogenic differentiation, were significantly increased in the NC1 collagen hydrogel group (p<0.0005), and were higher than those in rat tail collagen type I hydrogel.

[0162] In summary, the results indicate that NC1 collagen hydrogel can significantly promote the osteogenic phenotype transformation of mesenchymal stem cells by upregulating the Runx2-mediated osteogenic differentiation pathway, and has a significant promoting effect on fracture repair.

[0163] The amino acid sequence used in this invention is as follows:

[0164] The amino acid sequence of the V-domain (SEQ ID NO.1):

[0165] ADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD

[0166] The amino acid sequence of the CL-domain (SEQ ID NO.3):

[0167] PRGEQGPQGLPGKDGEAGAQGPAGPRGPQGPQGLPGPQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGAQG

[0168] Nucleotide sequence of CL-domain (SEQ ID NO. 13)

[0169] CCTCGTGGTGAACAGGGTCCGCAGGGCCTGCCGGGTAAAGATGGCGAAGCCGGTGCCCAGGGCCCGGCAGGTCCTCGTGGTCCTCAGGGTCCGCAAGGTCTGCCGGGTCCGCAGGGTCCTGCCGGTCCTATGGGTCCGGCCGGTTTTCCGGGTGAACGTGGTGAAAAAGGTGAACCGGGTACCCAGGGCGCCAAAGGTGACCGTGGTGAAACCGGTCCGGTTGGCCCGCGTGGTGAACGCGGTGAAGCAGGCCCGGCCGGTAAAGATGGTGAACGTGGCCCGGTTGGTCCGGCAGGTCCGAGAGGTCCGCAGGGACCTCAGGGTCTGCCGGGACCTCAGGGCCCTGCAGGTGCTCAGGGT

[0170] Nucleotide sequence of V-domain (SEQ ID NO. 16)

[0171] GCCGATGAACAGGAAGAAAAAGCCAAAGTGCGTACCGAACTGATTCAGGAACTGGCACAGGGCCTGGGCGGTATTGAAAAGAAAAATTTTCCGACCCTGGGTGACGAAGATCTGGATCATACCTATATGACCAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCCGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGCATTCAGGATCATGCACTGGAT

[0172] mC after folding and enzymatic digestion (SEQ ID NO. 17)

[0173] After folding and enzyme digestion, CC (SEQ ID NO. 18)

[0174] PPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPRGEQGPQGLPGKDGEAGAQGPAGPRGPQGPQGLPGPQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGACGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPC

[0175] After folding and enzyme digestion, NC (SEQ ID NO. 19)

[0176] PPGPPGPPGPPGPPGPPGPPGPPGPPGPPGCRGEQGPQGLPGKDGEAGAQGPAGPRGPQGPQGLPGPQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGAQGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPC

[0177] After folding and enzyme digestion, NC1 (SEQ ID NO. 20)

[0178] PPGPPGPPGPPGPPGPPGPPGPPGPPGPPGCRGEQGPQGLPGKDGEAGAQGPAGPRGPQGPQGLPGPQGPAGPMGPAGFPGERGEKGECGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGAQGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPC

[0179] After folding and enzyme digestion, NC3 (SEQ ID NO. 21)

[0180] PPGPPGPPGPPGPPGPPGPPGPPGPPGPPGCRGEQGPQGLPGKDGEAGAQGPAGPRGPCGPQGLPGPQGPAGPMGPAGFPGERGEKGECGTQGAKGDRGETGPVGPRGERGEAGPCGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGAQGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPC

[0181] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A collagen single chain, characterized in that, The amino acid sequence of the collagen single chain is shown in any one of SEQ ID NO.8 to SEQ ID NO.

11.

2. The collagen single chain according to claim 1, characterized in that, The amino acid sequence of the collagen single chain after enzymatic cleavage and folding is shown in any one of SEQ ID NO.18~SEQ ID NO.

21.

3. A gene encoding a single strand of collagen as described in any one of claims 1 to 2.

4. A plasmid carrying the single-stranded collagen gene as described in any one of claims 1 to 2.

5. The plasmid according to claim 4, characterized in that, Plasmids include the pColdIII series or the pET series.

6. A cell carrying a single-stranded collagen gene as described in any one of claims 1 to 2.

7. The collagen product prepared from collagen single chains according to any one of claims 1 to 2, characterized in that, The collagen products include type I collagen, collagen fibers, and collagen hydrogels.

8. A method for improving the mechanical strength of collagen hydrogels while maintaining the light and dark streaks of collagen in vitro, characterized in that, Collagen hydrogels were prepared using single-chain collagen. The amino acid sequence of the collagen single chain is shown in any one of SEQ ID NO.8 to SEQ ID NO.

11.

9. The use of the collagen single chain of any one of claims 1 to 2, the gene of claim 3, the plasmid of any one of claims 4 to 5, the cell of claim 6, or the collagen product of claim 7 in the preparation of products for fracture repair.

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