Osteoinductive active polypeptide capable of being assembled into nanofiber hydrogel and application thereof

Abstract

The application discloses a bone inductive active polypeptide capable of being assembled into nanofiber hydrogel and application thereof. The bone inductive active polypeptide comprises an assembly domain at a C terminal, a bone inductive active domain at an N terminal and a flexible connection separation domain between the two. The assembly domain is (RADA) 4‑9 The bone inductive active domain is parathyroid hormone (PTH) or parathyroid hormone related peptide (PTHrP). The bone inductive active polypeptide can be spontaneously assembled into a peptide nanofiber, and can be co-assembled into a peptide nanofiber hydrogel by mixing with RADA16 peptide. The hydrogel assembled from the bone inductive active polypeptide and the high-molecular composite nanobiomaterial constructed by the hydrogel can effectively avoid polypeptide burst release, avoid polypeptide degradation by local proteases, replace long-term systemic systemic administration of PTH and PTHrP, improve patient compliance and reduce patient pain. The application provides a new strategy for in-situ bone repair of PTH and PTHrP.

Description

Technical Field

[0001] This invention relates to the field of bone biomaterials and tissue engineering, specifically to a bone-inducing active polypeptide that can be assembled into a nanofiber hydrogel and its applications. Background Technology

[0002] Bone tissue has a certain capacity for self-healing after injury, but delayed union or nonunion occurs in over 10% of cases clinically. Currently, autologous bone grafting is the gold standard for surgical bone repair, but this surgical strategy requires additional surgery and may lead to complications such as bleeding, infection, and chronic pain. Allogeneic bone grafting is another clinical treatment for bone defect repair, but allogeneic bone grafts may induce immune rejection and pathogen dissemination, leading to bone healing failure.

[0003] With the development of materials science and biomedicine, bone tissue engineering is gradually becoming an ideal treatment strategy for bone defects. It mainly consists of three core elements: scaffold materials, seed cells, and active factors. Bone morphogenetic protein-2 (BMP-2) is an active factor currently used clinically to treat bone defects and repair fractures. However, substantial clinical evidence shows that BMP-2 easily leads to a series of clinical side effects, including radiculitis, vertebral osteolysis, hematoma formation, and tumorigenesis. Therefore, it is essential to find a novel active factor to replace BMP2 for fracture and bone defect repair.

[0004] Parathyroid hormone (PTH) is an 84-amino acid polypeptide secreted by the parathyroid glands. When it acts on bone tissue, it regulates serum calcium and phosphorus levels through bone remodeling. Small molecule peptides with similar functions to PTH, developed based on PTH, are called parathyroid hormone-related peptides (PTHrPs). For example, the first 34 amino acids of the N-terminus of PTH constitute its active sequence, and its trade name is teriparatide (PTH(1-34)). It mainly exerts its effects on promoting bone growth and regulating bone remodeling by binding to the classic PTH-I receptor. Clinically, systemic daily subcutaneous injection of teriparatide has been used to treat postmenopausal osteoporosis, increasing bone mass and considered a "breakthrough" advancement in the treatment of severe osteoporosis. Currently, systemic daily subcutaneous injection of PTH(1-34) is gradually being used to promote the repair of in situ fractures and bone defects, and it is expected to become an ideal active factor to replace BMP-2 for fracture and bone defect repair. However, systemic daily subcutaneous injections of PTH (1-34) often lead to increased patient suffering, poor compliance, heavy financial burden, and adverse reactions in other systemic systems. Therefore, in situ application of PTH or PTHrPs for fracture and bone defect repair is an ideal alternative to systemic subcutaneous injections. However, PTH and PTHrPs often exhibit more osteoclast-like than osteogenic effects, resulting in insignificant osteogenic effects; as peptide drugs, PTH and PTHrPs are easily degraded by local proteases, thus losing their biological activity; the physical adsorption loading of PTH and PTHrPs is often associated with explosive release, requiring a large peptide loading to ensure local biological activity, which may lead to side effects.

[0005] Peptide nanofiber hydrogels are gel materials formed by the assembly of small-molecule peptides into nanofibers under certain conditions, followed by further cross-linking. Basic ionic complementary peptides (such as RADA16, FEFKFEFK, etc.) are the most common type capable of assembling into peptide nanofiber hydrogels. In salt particle solutions, basic ionic complementary peptides can spontaneously assemble into a β-sheet-like nanofiber network, exhibiting a gel-like appearance. Their degradation products are amino acids, and they do not produce immune or inflammatory responses, nor are they cytotoxic. Functional peptide fragments (such as PRGDSGYRGDS, KLTWQELYQLKYKGI, etc.) can be linked to the side ends of basic ionic complementary peptides via solid-phase synthesis to construct functional ionic complementary peptides. However, due to the small amino acid sequences of current functional peptide fragments and the lack of specific two-dimensional structures, the assembly behavior of the RADA assembly domain in forming β-sheets is hindered. Therefore, the constructed functional ionic complementary peptides cannot independently self-assemble into long nanofibers in aqueous solutions, and thus cannot further cross-link to form nanofiber hydrogels. When mixed with basic self-assembling peptides, although the two can co-assemble to form longer nanofibers, the side-end sequences, lacking specific two-dimensional structures, still affect the stability of the assembled structure. Furthermore, the currently added functional peptide fragments have limited functionality and cannot meet the multiple functions required for bone tissue repair (including cell recruitment, migration, proliferation, biomineralization, osteogenic differentiation, vascularization, and immune regulation).

[0006] Therefore, the development of a safe, biocompatible, biodegradable, multifunctional osteogenic induction active material that can protect the biological activity of PTH and PTHrPs and effectively prevent explosive release will have broad clinical application prospects for surgical bone repair. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a osteoinductively active polypeptide that can be assembled into a nanofiber hydrogel and its applications. The osteoinductively active polypeptide that can be assembled into a nanofiber hydrogel includes a C-terminal assembly domain, an N-terminal osteoinductively active domain, and a flexible linker domain between the two. The assembly domain is (RADA). 4-9 The bone-inducing active domain is PTH or PTHrPs, including PTH, teriparatide PTH(1-34), PTHrP1, PTHrP2, abalotide, parathyroid hormone-related protein 1-37, and other PTH-derived polypeptides.

[0008] When the RADA unit of the assembly domain is a 16-peptide (i.e., repeated 4 times) or a 36-peptide (i.e., repeated 9 times), unexpectedly, the resulting assembly domain can be linked to the osteoinductive active domain PTHrPs without affecting its ability to self-assemble into nanofibers, and at the same time endowing it with osteoinductive ability.

[0009] When PTHrPs are linked to RADA16, which is repeated 4 times in the assembly domain, through a flexible linker domain, they can co-assemble with the unmodified RADA16 peptide to form a nanofiber hydrogel. The resulting co-assembled peptide nanofiber hydrogel retains osteogenic induction activity.

[0010] When PTHrPs are linked to RADA36 (repeated 9 times) via a flexible linker domain, they can self-assemble into nanofiber hydrogels without co-assembling with unmodified RADA16 peptides. The resulting self-assembled peptide nanofiber hydrogels retain osteogenic induction activity. They can also be co-assembled with RADA16 peptides, and the resulting co-assembled peptide nanofiber hydrogels similarly retain osteogenic induction activity.

[0011] Preferably, the bone-inducing active domain PTHrPs is one of PTH(1-34), PTHrP1, PTHrP2, abalopeptide, and parathyroid hormone-related protein 1-37. The sequence of PTH(1-34) is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF, and the sequence of PTHrP1 is S... [PO4] VSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFDDD, the sequence of PTHrP2 is S [PO4] VSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFEEE, the sequence of the abalopeptide is AVSEHQLLHGKGKSIQDLRRRELLEKLLXKLHTA (X is 2-methylalanine), and the sequence of the parathyroid hormone-related protein 1-37 is AVSEHQLLHGKGKSIQDLRRRFFLHHLIAEIHTAEIR.

[0012] Preferably, the flexible connection partition is GG.

[0013] This invention also provides a method for inducing the co-assembly of the above-mentioned osteoinductive active polypeptide, which can be assembled into a nanofiber hydrogel, and the RADA16 peptide to form a peptide nanofiber hydrogel, comprising the following steps:

[0014] S1: Dissolve the osteoinductive active peptide and RADA16 peptide, which can be assembled into nanofiber hydrogels, in sterile ultrapure water respectively, and disperse them by low-temperature ultrasonication to obtain peptide storage solutions.

[0015] S2: The osteogenic inductive active peptide storage solution that can be assembled into a nanofiber hydrogel is mixed with the RADA16 peptide storage solution, vortexed, and then dispersed under low-temperature ultrasonication to obtain a mixed peptide storage solution. In the mixed peptide storage solution, the osteogenic active peptide that can be assembled into a nanofiber hydrogel accounts for 25%-100% of the mass ratio of the mixed peptides.

[0016] S3: Mix the above-mentioned low-temperature ultrasonically dispersed mixed polypeptide storage solution with a salt ion solution, and let it stand at low temperature to promote the co-assembly of bone-inducing active polypeptides that can be assembled into nanofibers with RADA16 peptide to form peptide nanofiber hydrogel.

[0017] Preferably, the concentration of the polypeptide storage solution is 1-2% w / v.

[0018] Preferably, the low-temperature ultrasonic dispersion time is 25-30 minutes, and the ultrasonic temperature is 0-4°C.

[0019] Preferably, in the mixed polypeptide storage solution of osteogenic induction active polypeptide storage solution that can be assembled into nanofiber hydrogel and RADA16 peptide storage solution, the mass ratio of osteogenic induction active polypeptide storage solution that can be assembled into nanofiber hydrogel is 25%-60%.

[0020] Preferably, the salt ion solution is PBS.

[0021] Preferably, the assembly temperature for standing at low temperature is 0-4℃, and the assembly time is 6-12 hours.

[0022] This invention also provides a polymeric composite peptide nanofiber biomaterial with osteogenic induction activity, obtained by combining the osteogenic induction activity peptide nanofiber hydrogel assembled above with a polymeric hydrogel. The polymeric hydrogel includes type I collagen hydrogel, alginate hydrogel, chitosan hydrogel, hyaluronic acid hydrogel, etc.

[0023] Preferably, the polymer hydrogel in the polymeric composite peptide nanofiber biomaterial is a type I collagen hydrogel.

[0024] This invention also provides a method for preparing the above-mentioned polymeric composite peptide nanofiber biomaterial, comprising the following steps:

[0025] S1: Prepare a mixture of osteogenic inducible active peptide storage solution and RADA16 peptide storage solution that can be assembled into nanofiber hydrogels. The osteogenic inducible active peptide that can be assembled into nanofiber hydrogels accounts for 25%-100% of the mass of the mixed peptides.

[0026] S2: A polymer hydrogel is obtained by adjusting the pH, adding ions, or chemical cross-linking.

[0027] S3: The mixture of the osteogenic induction active peptide storage solution and the RADA16 peptide storage solution that can be assembled into nanofiber hydrogel is thoroughly mixed with the polymer hydrogel, and then dispersed by low-temperature ultrasonication so that the osteogenic induction active peptide and RADA16 peptide that can be assembled into nanofiber hydrogel are uniformly dispersed inside the polymer hydrogel.

[0028] S4: The polymer hydrogel containing the osteoinductive active peptide and RADA16 peptide is placed at a low temperature to promote the assembly of the osteoinductive active peptide and RADA16 peptide to form a nanofiber hydrogel, thereby obtaining a polymer composite peptide nanofiber hydrogel.

[0029] S5: After pre-cooling the polymeric composite hydrogel, freeze-dry it to obtain the polymeric composite peptide nanofiber biomaterial with osteogenic induction activity.

[0030] Preferably, the concentration of the osteogenic active peptide storage solution that can be assembled into a nanofiber hydrogel and the RADA16 peptide storage solution is 1-2% w / v. The mixing ratio of the osteogenic active peptide storage solution that can be assembled into a nanofiber hydrogel and the RADA16 peptide storage solution is 25%-60%.

[0031] Preferably, the low-temperature ultrasonic dispersion time is 25-30 minutes, and the ultrasonic dispersion temperature is 0-4℃.

[0032] Preferably, the polymeric hydrogel containing bone-inducing active peptides and RADA16 peptide is placed at 0-4°C for 6-12 hours to form a polymeric composite peptide nanofiber hydrogel.

[0033] This invention also provides the application of the above-mentioned osteoinductive active polypeptides, peptide nanofiber hydrogels, or polymeric composite peptide nanofiber biomaterials that can be assembled into nanofiber hydrogels in the preparation of bone repair materials for treating bone defects and repairing fractures.

[0034] A bone repair material for treating bone defects and repairing fractures, comprising any one of the above-mentioned osteoinductive active peptides, peptide nanofiber hydrogels, or polymeric composite peptide nanofiber biomaterials that can be assembled into nanofiber hydrogels.

[0035] The present invention has the following advantages:

[0036] (1) When the active fragments PTH(1-34) and PTHrP are covalently bound to the side end of the assembly domain, they do not affect the formation of nanofibers in the assembly domain. The formed nanofibers are thicker and have abundant osteogenic active epitopes anchored at the side end, which can promote osteogenic differentiation.

[0037] (2) The assembled nanofiber hydrogel network can effectively protect the biological activity of PTH(1-34) and PTHrPs, prevent them from being degraded by proteases in tissues, thereby improving the in situ application half-life of PTH(1-34) and PTHrP.

[0038] (3) The assembled nanofiber hydrogel network can effectively avoid the rapid release of PTH(1-34) and PTHrPs. The bone-inducing active peptides are covalently anchored to the nanofiber network, and the release rate is low, which can effectively reduce the dose of peptides used in situ and reduce the occurrence of adverse reactions.

[0039] (4) The assembled nanofiber hydrogel is injectable and can be filled into non-weight-bearing bone defects through minimally invasive injection, reducing the pain of open surgery and avoiding complications such as infection and bleeding.

[0040] (5) The assembled nanofiber hydrogel can be used to modify other scaffold materials to fill the fracture ends or bone defects, and can recruit repair cells in situ and promote osteogenic differentiation, thus avoiding the risks and side effects of systemic PTH administration.

[0041] (6) The degradation products are amino acids, which do not produce immune or inflammatory reactions;

[0042] (7) The preparation process is simple and easy to scale up production;

[0043] (8) Functional expansion: The osteoinductive active peptides that can be assembled into nanofiber hydrogels can not only promote osteogenic repair, but also relatively inhibit osteoclasts, promote the proliferation and mineralization of repair cells, promote vascularization, and resist osteoporosis. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the structure of a bone-inducing active polypeptide that can be assembled into a nanofiber hydrogel.

[0045] Figure 2 High-performance liquid chromatography (HPLC) chromatograms of RADA16-GG-PTHrP1(A) and RADA36-GG-PTH(1-34)(B).

[0046] Figure 3 Mass spectrometry (MS) detection chromatograms of RADA16-GG-PTHrP1(A) and RADA36-GG-PTH(1-34)(B).

[0047] Figure 4Macroscopic images of RADA16-GG-PTHrP1 / RADA16 nanofiber hydrogel (A) and RADA36-GG-PTH(1-34) / RADA16 nanofiber hydrogel (B).

[0048] Figure 5 Nanofibers formed by the self-assembly of RADA16 and RADA16-GG-PTHrP1, respectively.

[0049] Figure 6 Transmission electron microscopy (TEM) images of RADA16-GG-PTHrP1 / RADA16 nanofiber hydrogel (A) and RADA16-GG-PTHrP1 / RADA16 nanofiber hydrogel (B).

[0050] Figure 7 Macroscopic image (A) and scanning electron microscope image (B) of the polymer collagen complex peptide nanofiber biomaterial.

[0051] Figure 8 Live / dead staining images of mesenchymal stem cells after treatment with peptide nanofibers.

[0052] Figure 9 This image shows the proliferation effect of mesenchymal stem cells after peptide nanofiber intervention.

[0053] Figure 10 This study aimed to promote osteogenic differentiation of mesenchymal stem cells induced by RADA16-GG-PTHrP1 / RADA16 nanofibers.

[0054] Figure 11 This study aimed to promote osteogenic differentiation of mesenchymal cells induced by RADA36-GG-PTH(1-34) / RADA16 nanofibers.

[0055] Figure 12 Human umbilical vein endothelial cells were induced to form angiogenesis using RADA16-GG-PTHrP1 / RADA16 nanofibers.

[0056] Figure 13 X-ray images of ectopic osteoosis induced in a bilateral hind limb muscle pouch model of SD rats using RADA16-GG-PTHrP1 / RADA16 nanofiber biomaterials composed of high molecular weight collagen.

[0057] Figure 14 MicroCT images of ectopic osteoosis induced by high molecular weight collagen composite RADA16-GG-PTHrP1 / RADA16 nanofiber biomaterial in a bilateral hind limb muscle pouch model of SD rats.

[0058] Figure 15X-ray image of a high-molecular-weight collagen composite RADA16-GG-PTHrP1 / RADA16 nanofiber biomaterial used to repair critical-sized skull defects in SD rats. Detailed Implementation

[0059] The technical solution of the present invention will be further described below with reference to specific embodiments. However, the provided embodiments are merely illustrative of the method of the present invention and do not limit the remaining contents disclosed in the present invention in any way. The present invention can be implemented in many different forms and is not limited to the embodiments described herein.

[0060] During the experiment, the inventors unexpectedly discovered that introducing a longer amino acid sequence of PTHrP1 to the RADA16 peptide via the flexible linker domain GG did not affect the spontaneous assembly of the RADA assembly domain into a β-sheet-like nanofiber structure. Although the nanofibers assembled by RADA16-GG-PTHrP1 could not be further cross-linked to form a hydrogel, when mixed and co-assembled with RADA16, the inventors were surprised to find that the two could form a peptide nanofiber hydrogel. Furthermore, the inventors tried to increase the number of RADA units in the assembly domain and unexpectedly found that when the RADA units were repeated up to 9 (i.e., RADA36 peptide), the constructed RADA36-GG-PTH(1-34) could spontaneously assemble into nanofibers and could be further cross-linked to form a peptide nanofiber hydrogel. RADA36-GG-PTH(1-34) could also assemble into a peptide nanofiber hydrogel when mixed with RADA16. The structure of the osteoinductively active peptides [RADA16-GG-PTHrP1 peptide and RADA36-GG-PTH(1-34) peptide] that can be assembled into nanofiber hydrogels is as follows: Figure 1 As shown.

[0061]

Example 1

[0062] The peptide molecules required in the experiment were synthesized using an automated peptide synthesizer and purified by high-performance liquid chromatography (HPLC). Their purity and sequence were then determined using mass spectrometry (MS) and an amino acid and peptide analyzer. The HPLC and MS chromatograms of RADA16-GG-PTHrP1 and RADA36-GG-PTH(1-34) are shown below. Figure 2 , Figure 3 As shown.

[0063] The amino acid sequence of RADA16-GG-PTHrP1 is as follows:

[0064] AcN-RADARADARADARADAGGS[PO4] VSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF DDD-NH2, by Figure 2 (A) The purity of RADA16-GG-PTHrP1 obtained is greater than 95%, derived from... Figure 3 (A) The molecular weight of RADA16-GG-PTHrP1 obtained is 6352.20 Da.

[0065] The amino acid sequence of RADA36-GG-PTH(1-34) is as follows:

[0066] AcN-RADARADARADARADARADARADARADARADARADAGGSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH2, by Figure 2 (B) The purity of RADA36-GG-PTH(1-34) obtained is greater than 95%, derived from... Figure 3 (B) The molecular weight of RADA36-GG-PTH(1-34) obtained is 7994.40 Da.

[0067]

Example 2

[0068] 5 mg of RADA16-GG-PTHrP1 and 5 mg of RADA16 powder were dissolved in 500 μL of sterile ultrapure water to obtain RADA16-GG-PTHrP1 and RADA16 peptide storage solutions with a mass-to-volume ratio of 1%. The solutions were then ultrasonically dispersed at 4 °C for 30 minutes. The total volume was 100 μL. The solutions were vortexed and mixed at RADA16-GG-PTHrP1 to RADA16 volume ratios of 0:100, 10:90, 25:75, 50:50, and 100:0, followed by further ultrasonic dispersion at low temperature for 30 minutes to obtain peptide mixed storage solutions with RADA16-GG-PTHrP1 to RADA16 mass ratios of 0:100, 10:90, 25:75, 50:50, and 100:0. The obtained 100 μL peptide mixed storage solution was mixed with an equal volume of 100 μL 2×PBS solution and incubated at 4 °C for 6 hours. RADA16-GG-PTHrP1 and RADA16 assembled into nanofibers in salt solution. Mixtures with RADA16-GG-PTHrP1 to RADA16 mass ratios of 0:100, 10:90, 25:75, and 50:50 could further cross-link to form nanofiber hydrogels. In a 100:0 solution, i.e., only RADA16-GG-PTHrP1 exists, RADA16-GG-PTHrP1 can self-assemble into nanofibers but cannot further cross-link to form a hydrogel. A macroscopic image of the hydrogel is shown below. Figure 4 As shown in Figure A, atomic force microscopy images of peptide nanofibers self-assembled from RADA16 (i.e., 0:100) and RADA16-GG-PTHrP1 (i.e., 100:0) are shown below. Figure 5 As shown, the peptide nanofibers self-assembled from RADA16-GG-PTHrP1 (i.e., 100:0) are significantly wider than those self-assembled from RADA16 (i.e., 0:100), indicating that PTHrP1 is exposed at the side ends of the peptide nanofibers. Transmission electron microscopy (TEM) images of the peptide nanofiber hydrogels formed by RADA16-GG-PTHrP1 to RADA16 mass ratios of 0:100, 10:90, 25:75, and 50:50 are shown below. Figure 6 As shown in Figure A, nanofibers can entangle with each other to form a peptide nanofiber hydrogel network.

[0069]

Example 3

[0070] 5 mg of RADA36-GG-PTH(1-34) and 5 mg of RADA16 powder were dissolved in 500 μL of sterile ultrapure water to obtain RADA36-GG-PTH(1-34) and RADA16 peptide storage solutions with a mass-to-volume ratio of 1%. The solutions were then ultrasonically dispersed at 4 °C for 30 minutes. The total volume was 50 μL. The solutions were vortexed and mixed at volume ratios of 0:100, 30:70, 60:40, and 100:0 for RADA36-GG-PTH(1-34) and RADA16, followed by further ultrasonic dispersion at low temperature for 30 minutes to obtain peptide mixed storage solutions with mass ratios of 0:100, 30:70, 60:40, and 100:0 for RADA36-GG-PTH(1-34) and RADA16. The obtained 50 μL peptide mixed storage solution was mixed with an equal volume of 50 μL 2×PBS solution and incubated at 4 °C for 6 hours. RADA36-GG-PTH(1-34) and RADA16 were assembled into nanofibers in a salt solution. Mixtures of RADA36-GG-PTH(1-34) and RADA16 at mass ratios of 0:100, 30:70, 60:40, and 100:0 could be further cross-linked to form nanofiber hydrogels. Macroscopic images of the hydrogels are shown below. Figure 4 As shown in B. The corresponding transmission electron microscopy (TEM) image of the hydrogel nanofibers is shown below. Figure 6 As shown in B, nanofibers can entangle with each other to form a peptide nanofiber hydrogel network. Meanwhile, the width of the peptide nanofibers formed by the self-assembly of RADA36-GG-PTH(1-34) (i.e., 100:0) is significantly wider than that of the peptide nanofibers formed by the self-assembly of RADA16 (i.e., 0:100). This phenomenon indicates that PTH(1-34) is exposed at the side ends of the peptide nanofibers.

[0071] [Example 4] Construction of polymeric composite peptide nanofiber biomaterials using peptide nanofibers and polymeric collagen hydrogel

[0072] Different ratios of RADA16-GG-PTHrP1 / RADA16 nanofiber hydrogels obtained in Example 2 were used. Five grams of type I collagen sponge were weighed and dissolved in 1 liter of 0.1% acetic acid solution. The solution was titrated with 0.1M sodium hydroxide solution at 4°C to adjust the pH to 6.5-7. The solution was then placed at 37°C to promote collagen fiber self-assembly. After 30 minutes, the solution was centrifuged at 10,000 rpm for 40 minutes at 4°C, and the lower precipitate was obtained as the collagen hydrogel.

[0073] Take a 24-well plate, add 400 μL of collagen hydrogel to the bottom layer, 200 μL of RADA16-GG-PTHrP1 / RADA16 nanofiber hydrogel to the middle layer, and 400 μL of collagen hydrogel to the top layer. First, physically mix using a 1 mL syringe. Then, seal the gap between the plate cap and bottom of the 24-well plate with multiple layers of sealing film, and further wrap the 24-well plate with multiple layers of plastic wrap. Place the wrapped plate in an ultrasonic cleaner for sonication, intermittently adding ice packs to cool it during sonication, ensuring the ultrasonic temperature is maintained at 4°C. During sonication, the plate should be submerged below the liquid level inside the ultrasonic cleaner. After 30 minutes of low-temperature sonication, remove the plate and let it stand at 4°C to induce the assembly of bone-inducing active peptides that can assemble into nanofiber hydrogels with RADA16. After standing for 6 hours, the well plate was transferred to a -20°C freezer for pre-cooling overnight. Finally, it was freeze-dried at -55°C for 24 hours to obtain collagen polymer composite peptide nanofiber biomaterial. The macroscopic image and surface SEM morphology of the biomaterial are shown below. Figure 7 As shown, the biomaterial exhibits a porous network structure with uniform distribution and irregular shape. The pores are not closed, and a fibrous structure formed by the interaction of peptide nanofibers and collagen fibers can be observed on the surface of the porous structure.

[0074] [Example 5] Cytotoxicity experiment of peptide nanofibers

[0075] Different ratios of RADA16-GG-PTHrP1 / RADA16 nanofiber hydrogels obtained in Example 2 were diluted to 1000 ng / mL using cell culture medium.

[0076] Third-generation rat bone marrow-derived mesenchymal stem cells (BMSCs) with a density of 2 × 10⁻⁶. 4 / mL was added to 24-well plates for incubation for 12 hours to ensure BMSCs adhered to the bottom wall of the plate. Then, 500 μL of cell culture medium containing 1000 ng / mL peptide nanofibers was added to each well. After two days of culture, live / dead cell staining was performed, and normal cell growth was observed in all groups, with cell viability remaining above 95% in all groups. Figure 8 As shown.

[0077] [Example 6] Experiment on the promotion of mesenchymal stem cell proliferation by RADA16-GG-PTHrP1 / RADA16 peptide nanofibers

[0078] Third-generation rat bone marrow-derived mesenchymal stem cells (BMSCs) with a density of 10-1 3 / mL, added to 96-well plates for incubation for 12 hours to ensure BMSCs adhere to the bottom wall of the culture plate. 1% w / v (10 mg / mL) of RADA16-GG-PTHrP1 prepared in Example 2 with RADA16 at mass ratios of 0:100, 10:90, 25:75, and 50:50 were used for further analysis.

[0079] RADA16-GG-PTHrP1 / RADA16 peptide nanofiber hydrogels were diluted to 1000 ng / mL using cell culture medium. Four groups of culture media containing 1000 ng / mL peptide nanofibers were used to induce adherent BMSCs, with the medium changed every two days. CCK8 assays were performed on days 1, 3, and 5. The absorbance at 450 nm reflected cell proliferation. The RADA16-GG-PTHrP1 / RADA16 volume ratios of 10:90, 25:75, and 50:50 showed significantly better cell proliferation on days 3 and 5 than the 0:100 group. Figure 9 As shown.

[0080] [Example 7] RADA16-GG-PTHrP1 / RADA16 peptide nanofibers induce osteogenic differentiation of mesenchymal stem cells

[0081] Third-generation rat bone marrow-derived mesenchymal stem cells (BMSCs) with a density of 4 × 10⁻⁶. 4 / mL, added to 24-well plates for incubation for 12 hours to ensure BMSCs adhere to the bottom wall of the culture plate. 1% w / v (10 mg / mL) of RADA16-GG-PTHrP1 prepared in Example 2 with RADA16 at mass ratios of 0:100, 10:90, 25:75, and 50:50 were used for further analysis.

[0082] RADA16-GG-PTHrP1 / RADA16 peptide nanofiber hydrogels were prepared. Cell culture medium was diluted to 1000 ng / mL. Four groups of BMSCs were induced to adhere to the culture medium using medium containing 1000 ng / mL peptide nanofibers, with the medium changed every 2 days for 14 days. After fixation with paraformaldehyde, BMSCs were stained with ALP using the BCIP / NBT alkaline phosphatase staining kit. ALP staining revealed grayish-brown to dark black granular or sheet-like precipitates in the cytoplasm, indicating ALP-positive cells. The groups with RADA16-GG-PTHrP1 to RADA16 mass ratios of 10:90, 25:75, and 50:50 showed significantly more ALP-positive cells than the 0:100 group. Figure 10 As shown, nanofibers incorporating PTHrP1 possess osteogenic induction differentiation capabilities, with the 50:50 group exhibiting the best osteogenic induction ability.

[0083] Using the same method, BMSCs were induced and cultured for 21 days, fixed with paraformaldehyde, and then stained with alizarin red. Alizarin red is the sodium salt of alizarin sulfonate, which can chelate with calcium salts in calcium carbonate or calcium phosphate to form orange-red complexes. The groups with RADA16-GG-PTHrP1 to RADA16 mass ratios of 10:90, 25:75, and 50:50 formed significantly more orange-red complexes than the 0:100 group. Figure 10 As shown, nanofibers incorporating PTHrP1 possess osteogenic induction differentiation capabilities, with the 50:50 group exhibiting the best osteogenic induction ability.

[0084] Similarly, BMSCs were induced and cultured for 28 days, fixed with paraformaldehyde, and then stained with Von Kossa stain. Von Kossa stain is a silver nitrate solution. Silver nitrate reacts with insoluble calcium salts via metathesis to form reducible silver salts. Under strong light, ultraviolet light, or a strong reducing agent, the silver salts are reduced to black elemental silver. After staining, the calcium salt deposition areas appear black. The groups with RADA16-GG-PTHrP1 to RADA16 mass ratios of 10:90, 25:75, and 50:50 formed significantly more black calcium salt deposition areas than the 0:100 group. Figure 10 As shown, nanofibers incorporating PTHrP1 possess osteogenic induction differentiation capabilities, with the 50:50 group exhibiting the best osteogenic induction ability.

[0085]

Example 8

[0086] Third-generation rat bone marrow-derived mesenchymal stem cells (BMSCs) with a density of 4 × 10⁻⁶. 4 / mL was added to 24-well plates for incubation for 12 hours to ensure BMSCs adhered to the bottom wall of the culture plate. 1% w / v (10 mg / mL) RADA36-GG-PTH(1-34) / RADA16 peptide nanofiber hydrogels prepared in Example 3 with RADA36-GG-PTH(1-34) and RADA16 mass ratios of 0:100, 30:70, 60:40, and 100:0 were diluted to 1000 ng / mL using cell culture medium. The adhered BMSCs were induced using four groups of culture media containing 1000 ng / mL peptide nanofibers, with the medium changed every 2 days for 14 days. After BMSCs were fixed with paraformaldehyde, ALP staining was performed using the BCIP / NBT alkaline phosphatase staining kit. ALP staining revealed grayish-brown to dark black granular or sheet-like precipitates in the cytoplasm, indicating ALP-positive cells. Among them, the groups with RADA36-GG-PTH(1-34) to RADA16 mass ratios of 30:70, 60:40, and 100:0 had significantly more ALP-positive cells than the group with a mass ratio of 0:100. Figure 11 As shown, nanofibers incorporating PTH(1-34) possess osteogenic induction differentiation ability, with the 60:40 group exhibiting the best osteogenic induction ability.

[0087] Using the same method, BMSCs were induced and cultured for 21 days, fixed with paraformaldehyde, and then stained with alizarin red. Alizarin red is the sodium salt of alizarin sulfonate, which can chelate with calcium salts in calcium carbonate or calcium phosphate to form orange-red complexes. The groups with RADA36-GG-PTH(1-34) to RADA16 mass ratios of 30:70, 60:40, and 100:0 formed significantly more orange-red complexes than the 0:100 group. Figure 11 As shown, nanofibers incorporating PTH(1-34) possess osteogenic induction differentiation ability, with the 60:40 group exhibiting the best osteogenic induction ability.

[0088] [Example 9] RADA16-GG-PTHrP1 / RADA16 peptide nanofibers induce human umbilical vein endothelial cells to form blood vessels.

[0089] Fifth-generation human umbilical vein endothelial cells (HUVECs) with a density of 20 × 10⁻⁶. 4 / mL, added to 6-well plates for incubation for 12 hours to ensure HUVECs adhere to the bottom wall of the plate. 1% w / v (10 mg / mL) of RADA16-GG-PTHrP1 prepared in Example 2 with RADA16 at mass ratios of 0:100, 10:90, 25:75, and 50:50 were used for further analysis.

[0090] RADA16-GG-PTHrP1 / RADA16 peptide nanofiber hydrogels were diluted to 1000 ng / mL using cell culture medium. Four groups of HUVECs were induced to adhere to the culture medium using medium containing 1000 ng / mL peptide nanofibers. The medium was changed every 2 days, and trypsin digestion was performed after 5 days of culture. Then, the cells were cultured at a density of 3 × 10⁻⁶. 4 / mL was added to a 24-well plate coated with matrix gel, and tube formation was observed after 8 hours. Tube formation was observed in the groups with RADA16-GG-PTHrP1 to RADA16 mass ratios of 10:90, 25:75, and 50:50, significantly better than the 0:100 group. Figure 12 As shown, nanofibers incorporating PTHrP1 have the ability to induce angiogenesis, with the 50:50 group exhibiting the best angiogenesis-inducing ability.

[0091] [Example 10] Induction of ectopic osteogenic formation in rat hind limb muscle pouches using collagen polymer composite RADA16-GG-PTHrP1 / RADA16 peptide nanofiber biomaterial

[0092] Thirty-two healthy male SD rats at 6 weeks of age were randomly divided into four groups of eight each. A bilateral muscle pouch model was constructed, and each group was implanted with a polymeric composite peptide nanofiber biomaterial prepared in Example 4, consisting of peptide nanofiber hydrogels and collagen hydrogels assembled with RADA16-GG-PTHrP1 / RADA16 in mass ratios of 0:100, 10:90, 25:75, and 50:50. Rats were anesthetized intraperitoneally with 1% pentobarbital. After satisfactory anesthesia, routine disinfection and draping were performed, and 2cm incisions were made in both hind limbs. The aforementioned material was implanted bilaterally, and the wounds were sutured layer by layer. Five weeks post-surgery, all rats were euthanized, and skull fragments were harvested for radiographic examination. Figure 13 , Figure 14 As shown, the groups loaded with RADA16-GG-PTHrP1 (i.e., 10:90, 25:75, and 50:50) all significantly promoted the repair of skull defects in rats.

[0093] [Example 11] Collagen polymer composite RADA16-GG-PTHrP1 / RADA16 peptide nanofiber biomaterial promotes repair of critical cranial defects in rats.

[0094] Twenty-four healthy male SD rats at 12 weeks of age were randomly divided into four groups of six each. A critical-sized cranial defect model was constructed. Group A was implanted with saline solution, while groups B, C, and D were implanted with a polymeric composite peptide nanofiber biomaterial prepared in Example 4, which consisted of peptide nanofiber hydrogels and collagen hydrogels assembled from RADA16-GG-PTHrP1 at mass ratios of 0:100, 25:75, and 50:50, respectively. Rats were anesthetized intraperitoneally with 1% pentobarbital. After satisfactory anesthesia, routine disinfection and draping were performed. A longitudinal incision was made in the midline of the skull, and two circular bone defect areas with a diameter of 5 mm were created on either side of the lower outer quadrant where the midline suture and the lambdoid suture intersect. The aforementioned saline solution or the aforementioned biomaterial was implanted in each group, and the incision was sutured. Rats in all groups were euthanized 8 weeks post-surgery, and their skulls were harvested for radiographic examination. Figure 15 As shown, the groups loaded with RADA16-GG-PTHrP1 (i.e., 25:75 and 50:50) significantly promoted the repair of skull defects in rats.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bone-inducing active polypeptide that can be assembled into a nanofiber hydrogel, characterized in that, It consists of an assembly domain at the C-terminus, a osteoinductive activity domain at the N-terminus, and a flexible connection spacer between the two. The assembly structure domain is (RADA)4; The bone-inducing active domain is PTHrP1. The sequence of PTHrP1 is S [PO4] VSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFDDD; The flexible connection partition is GG.

2. A peptide nanofiber hydrogel, characterized in that, The bone-inducing active polypeptide that can be assembled into a nanofiber hydrogel as described in claim 1 is assembled with the RADA16 peptide.

3. The method for preparing the peptide nanofiber hydrogel according to claim 2, characterized in that, The process includes the following steps: dispersing the osteoinductive active polypeptide and RADA16 peptide, which can be assembled into a nanofiber hydrogel as described in claim 1, in a salt ion solution and allowing them to stand at low temperature to allow them to assemble into a peptide nanofiber hydrogel.

4. The method for preparing peptide nanofiber hydrogel according to claim 3, characterized in that, The bone-inducing active polypeptide that can be assembled into a nanofiber hydrogel has a mass percentage of 25%-100% in the mixed polypeptide storage solution formed with RADA16 peptide; the salt ion solution is PBS; and the low-temperature standing condition is standing at 0-4℃ for 6-12 hours.

5. A polymeric composite peptide nanofiber biomaterial with osteogenic induction activity, characterized in that, The product is obtained by thoroughly mixing the peptide nanofiber hydrogel described in claim 2 with the polymer hydrogel, allowing it to stand at low temperature, and then freeze-drying it.

6. The application of the osteoinductive active polypeptide that can be assembled into a nanofiber hydrogel according to claim 1, the peptide nanofiber hydrogel according to claim 2, or the polymeric composite peptide nanofiber biomaterial according to claim 5 in the preparation of bone repair materials for treating bone defects and repairing fractures.

7. A bone repair material for treating bone defects and repairing fractures, characterized in that, It comprises any one of the following: the osteoinductive active polypeptide that can be assembled into a nanofiber hydrogel as described in claim 1, the peptide nanofiber hydrogel as described in claim 2, or the polymeric composite peptide nanofiber biomaterial as described in claim 5.

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