Bioresponsive composites, methods of making and using the same

Abstract

The application discloses a composite material with biological responsiveness and a preparation method and application thereof. The composite material comprises a substrate; the substrate comprises a bioactive metal layer; and a polypeptide comprising a first functional peptide segment, an enzyme cutting substrate peptide segment and a second functional peptide segment sequentially connected, the first functional peptide segment being modified to the bioactive metal layer. The peptide segment capable of being cut by a specific enzyme is embedded in the middle of the segment, so that the enzyme responsiveness of the segment is realized. In this way, the first functional peptide segment away from the bioactive metal layer and the second functional peptide segment close to the bioactive metal layer are sequentially exposed, so that the matching degree of the orthopedic implant material and the specific requirements of the bone tissue repair process in each stage is improved. In addition, the bioactive metal layer on the surface of the orthopedic implant material has a regulation effect on bone repair, and long-acting slow release of the bioactive metal can be further realized through modification of the polypeptide.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to bioresponsive composite materials, their preparation methods, and applications. Background Technology

[0002] The skeleton is a vital structural support system of the human body. Bone defects refer to the disruption of the integrity of the skeletal structure caused by congenital or acquired factors. Bone defects larger than 2 to 2.5 times the bone diameter are unlikely to heal spontaneously. Furthermore, commonly used autologous and allogeneic transplants are severely limited in their treatment scope and clinical outcomes due to donor scarcity and postoperative infections. Therefore, there is an urgent need to explore new alternative materials to provide patients with more options while achieving good bone repair and integration. Ideal orthopedic implant materials should possess sufficient mechanical strength while also promoting bone repair and improving osseointegration.

[0003] Currently, commonly used metal-based orthopedic implants, such as titanium, suffer from excessively high elastic modulus, which can cause stress shielding effects in the later stages of implantation, directly leading to osseointegration failure. Polymer-based orthopedic implants, such as polyetheretherketone (PEEK), are bioinert and easily form fibrous encapsulation at the bone-implant interface, also causing osseointegration failure. Ceramic-based materials, such as hydroxyapatite, while possessing certain bioactivity, have low mechanical strength and are insufficient to support bone tissue on their own. Therefore, current approaches primarily focus on combining two or more materials, or introducing multiple bioactive molecules into metal-based or polymer-based materials through physical / chemical methods to improve their osteoinductive properties. While these methods have achieved some success, more effective approaches are still needed to achieve complete bone repair and excellent osseointegration.

[0004] Recent research in bone biology has revealed that the repair of large bone defects is a complex, multicellular, and sequential process. This bone repair process mainly includes an early hematoma and inflammatory response phase, a mid-stage callus formation phase, and a late-stage bone plate remodeling phase. Immediately after orthopedic implantation, a hematoma and inflammatory response are triggered. The migration, proliferation, and functional phenotypes of inflammatory cells such as platelets, macrophages, and neutrophils involved in this process form the basis for subsequent angiogenesis, osteogenic formation, and bone resorption. During the mid-stage callus formation and late-stage bone plate remodeling phases, vascular endothelial growth factor secreted by early inflammatory cells promotes the migration and aggregation of a large number of endothelial cells at the defect repair site, while bone morphogenetic proteins promote the aggregation, proliferation, and osteogenic differentiation of a large number of bone marrow mesenchymal stem cells at the defect repair site, thereby completing bone repair and achieving osseointegration. However, current transplant materials only focus on some aspects of the bone repair process and cannot programmatically match different stages of bone repair, resulting in incomplete bone repair and excellent osseointegration. Summary of the Invention

[0005] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a bioresponsive composite material, its preparation method, and its application, which can achieve complete bone repair and excellent osseointegration.

[0006] A first aspect of this application provides a composite material comprising:

[0007] Substrate, the substrate comprising a bioactive metal layer;

[0008] The polypeptide comprises a first functional peptide, an enzyme-cleaved substrate peptide, and a second functional peptide connected in sequence, wherein the first functional peptide is modified on the bioactive metal layer.

[0009] The beneficial effects of the embodiments of this application are:

[0010] The composite material provided in this application is surface-modified with bioresponsive multifunctional polypeptide segments. These segments have embedded peptides that can be cleaved by specific enzymes, achieving enzyme-responsive cleavage. In this way, a second functional peptide segment, farther from the bioactive metal layer, and a first functional peptide segment, closer to the bioactive metal layer, are sequentially exposed, thereby improving the matching degree between the orthopedic implant material and the specific needs of each stage of bone tissue repair. Furthermore, the bioactive metal layer on the surface of the orthopedic implant material has a regulatory effect on bone repair, and the polypeptide modification can further achieve long-term sustained release of the bioactive metal. Through the synergistic effect of these two aspects, the temporal process of bone repair is dynamically matched, thereby promoting bone repair and improving the integration effect between the bone implant material and bone tissue.

[0011] It should be noted that the bone repair process, at the cellular level, includes promoting cell migration, adhesion, proliferation, and osteogenic differentiation, regulating the balance between osteogenic and osteoclast formation, and increasing intercellular mineralization levels; according to the sequence of stages, it includes the early hematoma and inflammatory response period, the intermediate callus formation period, and the late bone plate remodeling period; at the individual level, it includes repairing bone defects, promoting trabecular bone growth, and new bone formation. Therefore, bioactive metals refer to metallic elements that play a certain role in promoting bioactivity in the above-mentioned bone repair process, and the corresponding bioactive metal layer refers to a layered structure of a certain thickness formed by raw materials including bioactive metals.

[0012] The thickness of the bioactive metal layer can be a single atomic layer, a multi-atom layer, or a thickness on a larger scale (such as nanometers, micrometers, millimeters, centimeters, etc.). In some embodiments, the thickness of the bioactive metal layer is less than 10 cm, 1 cm, 1 mm, 100 μm, 10 μm, 1 μm, or 100 nm. In some embodiments, the bioactive metal layer may be continuous or discontinuous, uniform or non-uniformly distributed on the substrate, which can be determined according to the corresponding process method. In other embodiments, the bioactive metal layer composed of bioactive metal is the substrate. That is, the substrate containing the bioactive metal layer may be formed on the surface of a substrate, or the bioactive metal layer formed of bioactive metal may serve as the substrate.

[0013] In this context, the enzyme-digested substrate peptide refers to a peptide containing the amino acid sequence of a specific enzyme-digested substrate. When the corresponding enzyme is present in the environment, specific enzymatic cleavage occurs, achieving a biological response. This removes one functional peptide from the composite material, exposing the other functional peptide, thus allowing it to exert its function. Specifically, since the first functional peptide is directly modified onto the bioactive layer, specific enzymatic cleavage of the substrate peptide removes the second functional peptide from the composite material, exposing the first functional peptide.

[0014] The first and second functional peptides refer to peptides with different biological functional activities, corresponding to different effector cells in the repair stage, thus matching the various stages of bone tissue repair and promoting bone repair and integration. It is understandable that the selection of the first and second functional peptides offers high flexibility, allowing for targeted design based on repair objectives and tissue repair needs.

[0015] In some embodiments, the metal element of the bioactive metal layer includes at least one selected from Mg, Ta, Sr, Zn, Cu, Co, Gd, Ga, and Fe. For example, related studies have shown that Mg can promote osteogenic or angiogenesis by directly affecting osteogenic-related cells (such as mesenchymal stem cells, osteoblasts, osteoclasts, etc.) or regulating the osteogenic immune microenvironment; Ta can promote osteogenic differentiation of cells by activating signaling pathways such as Wnt / β-catenin, TGF-β / Smad, MAPK / ERK, and integrins; a locally rich Sr microenvironment also significantly improves osteoblast proliferation, adhesion, and differentiation; Zn can enhance the osteogenic capacity of cells and the formation of surrounding blood vessels through multiple pathways, which is beneficial for new bone formation and bone integration; Fe, Cu, and other elements have also been shown to have the same or similar mechanisms of promoting osteogenic formation.

[0016] In some embodiments, the bioactive metal layer includes at least one of Mg, Zn, and Fe. These multivalent metal elements possess good bone bioactivity and biosafety.

[0017] In some embodiments, a first functional peptide is modified onto a bioactive metal layer via a linker molecule. The first functional peptide has an azide group, and the linker molecule includes a catechol unit and a DBCO group. The peptide and the linker molecule are linked through a click reaction between the azide group and the DBCO group, and the linker molecule is linked to the bioactive metal layer through chelation between the metal and the catechol unit. This linker molecule achieves this by chelating the bioactive metal with the catechol group and by conducting a click chemical reaction between the DBCO group and the azide group at the peptide tail; both processes are simple and efficient.

[0018] In some embodiments, the linker molecule comprises 2 to 10 catechol units, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 catechol units. The linker molecule chelates with the bioactive metal through the phenolic hydroxyl groups of two or more catechol groups, for example, forming 4-coordinate or 6-coordinate chelates depending on the bioactive metal ion.

[0019] In some embodiments, the linker molecule includes a main chain and branches, wherein at least one end group of the main chain is a DBCO group and the branches include catechol units.

[0020] In some embodiments, the linker molecules include The repeating units have two or more repeating units, for example, 2 to 10 repeating units, such as 2, 3, 4, 5, 6, 7, 8, 9, and 10 repeating units.

[0021] In some embodiments, the connecting molecule includes one end The repeating unit is connected to the DBCO group at the other end.

[0022] In some embodiments, the structural formula of the linking molecule is:

[0023] Where n is a positive integer greater than or equal to 2, and x is between 1 and 5.

[0024] In some embodiments, the structural formula of the linking molecule is as follows:

[0025]

[0026] In some embodiments, the first functional peptide, the enzyme-digested substrate peptide, and the second functional peptide in the polypeptide each independently contain 3 to 20 amino acids, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Optionally, the first functional peptide, the enzyme-digested substrate peptide, and the second functional peptide in the polypeptide each independently contain, for example, 4 to 10 amino acids.

[0027] Choosing shorter peptides for both the first and second functional peptides is more advantageous because it allows the functional peptides closer to the bioactive metal layer to maintain lower reactivity due to steric hindrance and other factors, while preserving the activity of the functional peptides farther from the bioactive metal layer. This allows the second functional peptide to primarily exert its function. Furthermore, by gradually releasing the second functional peptide, which is farther from the bioactive metal layer, into the composite material through enzymatic cleavage, the first functional peptide is exposed and can then exert its function. This further enhances the matching degree between the first and second functional peptides and the bone tissue repair process, promoting bone repair and improving the integration effect between the bone implant material and bone tissue.

[0028] Based on the above reaction principle, the enzyme corresponding to the substrate peptide can be any enzyme expressed in the microenvironment of the implantation site during bone repair, such as any of the following: oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, and translocase.

[0029] In some embodiments, the enzyme-cleaved substrate peptide includes the substrate peptide of a hydrolase.

[0030] In some embodiments, the hydrolytic enzyme includes at least one of proteases and phosphatases. Examples include matrix metalloproteinases, cathepsins, and alkaline phosphatases involved in bone repair. The corresponding enzyme-digesting substrate peptides include at least one of matrix metalloproteinase substrate peptides, cathepsin substrate peptides, and alkaline phosphatase substrate peptides.

[0031] Matrix metalloproteinases (MMPs) are proteases found in the bone matrix, named for their requirement of metal ions such as Ca and Zn as cofactors. Based on substrate and fragment homology, MMPs can be classified into at least five types, including collagenases, gelatinases, membrane-type metalloproteinases, matrix hydrolases, and others. MMPs promote cell proliferation, migration, and differentiation, and play a role in angiogenesis, apoptosis, and tissue repair. Therefore, during the corresponding stages of bone repair, the expression levels of one or more of these MMPs increase, allowing for the enzymatic cleavage of substrate peptides and the temporal exposure of functional peptides to match the sequence of bone repair processes, resulting in better bone repair and integration.

[0032] While most members of the cathepsin (CTS) family are activated at the low pH of lysosomes and remain within organelles, there are exceptions such as cathepsin K. These cathepsins are mainly expressed in osteoclasts and can exert their effects extracellularly through osteoclast secretion during bone resorption. They utilize their ability to break down elastin, collagen, and gelatin to break down bone and cartilage, participating in bone remodeling and bone resorption.

[0033] As the main cells forming bone, osteoblasts secrete extracellular matrix proteins, including alkaline phosphatase (ALP). Furthermore, the expression level of alkaline phosphatase increases significantly during the osteogenic differentiation of mesenchymal stem cells.

[0034] Therefore, the substrate peptides corresponding to these enzymes can serve as the cleavage substrate peptides required in polypeptides, thereby enabling the enzymes present in the microenvironment to cleave the substrate peptides and complete the temporal exposure.

[0035] In some embodiments, the matrix metalloproteinase substrate peptide is at least one of matrix metalloproteinases-1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0036] In some embodiments, the enzyme-cleaved substrate peptide is a matrix metalloproteinase-2 substrate peptide whose amino acid sequence includes GPLG, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus, such as enzyme-cleaved substrate peptides including GPLGIAGQ, CGGPLGVRGK (refer to ACS Appl. Mater. Interfaces 2016, 8, 1447-1457; Theranostics 2012; 2(2): 190-197).

[0037] In some embodiments, the amino acid sequence of the enzyme-cleaved substrate peptide is GPLG, GPLGIAGQ, or CGGPLGVRGK.

[0038] In some embodiments, the enzyme-cleaved substrate peptide is a matrix metalloproteinase-9 substrate peptide whose amino acid sequence includes any one of GERGPPGPQGARGFZGTPGL (Z for hydroxyproline), PLGLWADR, GPLGLLGC, GPLGMWSRTKC, AVRWLLTA (see Journal of Controlled Release 239(2016)39-48; Biomacromolecules.201819(3):860-871; Journal of Controlled Release 267(2017)181-190; Mol Pharm.2013 10(8):3164-3174; Anal Chem.2013 85(24):11893-11901), and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus.

[0039] In some embodiments, the amino acid sequence of the enzyme-digested substrate peptide is any one of GERGPPGPQGARGFZGTPGL (Z being hydroxyproline), PLGLWADR, GPLGLLGC, GPLGMWSRKC, and AVRWLLTA.

[0040] In some embodiments, the first functional peptide and the second functional peptide are each selected from at least two different peptides selected from inflammation-regulating functional peptides, angiogenesis-promoting functional peptides, osteogenic functional peptides, antimicrobial peptides, and antioxidant peptides.

[0041] In some embodiments, the second functional peptide segment includes an inflammation-regulating functional peptide segment, and the first functional peptide segment includes at least one of a pro-angiogenic functional peptide segment and a bone-regenerating functional peptide segment. By incorporating the inflammation-regulating functional peptide segment and the pro-angiogenic (bone)-regenerating functional peptide segment, and through enzymatic cleavage of the embedded enzyme substrate peptide segment, temporal regulation of inflammation and promotion of angiogenesis and / or bone regeneration are achieved. This dynamically matches the sequence of the hematoma and inflammation phase, callus formation phase, and callus remodeling phase of bone repair, thereby promoting bone repair and improving bone integration.

[0042] In some embodiments, the inflammatory regulatory peptides include anti-inflammatory peptides.

[0043] In some embodiments, the inflammatory regulatory peptides include peptides having anti-transglutaminase 2 (TGase 2) activity and / or anti-phospholipase A2 (PLA2) activity.

[0044] In some embodiments, the amino acid sequence of the inflammatory regulatory peptide includes KVLDGQDP, and may also include 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, at the N-terminus and / or C-terminus, while maintaining its original inflammatory regulatory activity.

[0045] In some embodiments, the amino acid sequence of the inflammatory regulatory peptide is KVLDGQDP.

[0046] In some embodiments, the angiogenesis-promoting functional peptides include osteopontin (OPN)-derived peptides.

[0047] In some embodiments, the angiogenesis-promoting functional peptide includes the N-terminal peptide of osteopontin (OPN).

[0048] In some embodiments, the amino acid sequence of the pro-angiogenic functional peptide includes SVVYGLR, and may also include 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids, at the N-terminus and / or C-terminus, while maintaining its original pro-angiogenic activity.

[0049] In some embodiments, the amino acid sequence of the angiogenesis-promoting functional peptide is SVVYGLR.

[0050] In some embodiments, the osteogenic functional peptides include osteogenic growth peptides (OGP)-derived peptides.

[0051] In some embodiments, the osteogenic functional peptide includes the C-terminal peptide of osteogenic growth peptide (OGP).

[0052] In some embodiments, the amino acid sequence of the osteogenic functional peptide includes YGFGG, and may also include 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, at the N-terminus and / or C-terminus, while maintaining its original osteogenic activity.

[0053] In some embodiments, the amino acid sequence of the osteogenic functional peptide includes YGFGG.

[0054] In some embodiments, the amino acid sequence of the polypeptide includes SVVYGLR-GPLG-KVLDGQDP, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus.

[0055] In some embodiments, the amino acid sequence of the polypeptide is SVVYGLR-GPLG-KVLDGQDP.

[0056] In some embodiments, the polypeptide is N3-SVVYGLR-GPLG-KVLDGQDP.

[0057] In some embodiments, the amino acid sequence of the polypeptide includes YGFGG-GPLG-KVLDGQDP, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus.

[0058] In some embodiments, the amino acid sequence of the polypeptide includes YGFGG-GPLG-KVLDGQDP.

[0059] In some embodiments, the polypeptide is N3-YGFGG-GPLG-KVLDGQDP.

[0060] In some embodiments, the polypeptides modified on the bioactive metal layer include multiple peptides, each having the same or different first functional peptides, the same or different enzyme cleavage substrate peptides, and the same or different second functional peptides. For example, different enzyme cleavage substrate peptides can be selected to selectively expose different functional peptides based on the strength of enzyme activity at different time stages during bone repair. Alternatively, different functional peptides can be exposed simultaneously through a combination of different first functional peptides or different second functional peptides from multiple polypeptides, thereby achieving multi-faceted bone repair.

[0061] In some embodiments, the substrate includes at least one of polymers, ceramics, metals, and non-metallic nanomaterials. It is understood that when the substrate is a metal, particularly containing the aforementioned bioactive metal, it may not be necessary to form an additional bioactive metal layer.

[0062] In some of these embodiments, the polymers include natural polymers and synthetic polymers.

[0063] In some embodiments, the natural polymer includes at least one of collagen, gelatin, alginate, silk fibroin, elastin, agarose, chitin, chitosan, etc.

[0064] In some embodiments, the synthetic polymer includes at least one of polycaprolactone, polylactic acid, polyethylene glycol, poly(N-isopropylacrylamide), polytetrafluoroethylene, expanded polytetrafluoroethylene, polyamide, nylon, polysulfone, polyvinyl alcohol, polycarbonate, polyurethane, polyether, polyetherketone, polyetheretherketone, polyethylene terephthalate, polymethyl methacrylate, and polyolefin.

[0065] In some embodiments, the ceramics include phosphate bioceramics, such as calcium phosphate (e.g., hydroxyapatite, β-tricalcium phosphate), magnesium phosphate, etc.; and silicate ceramics, such as magnesium silicates, calcium silicates, etc., at least one of these.

[0066] In some embodiments, the metal includes at least one of stainless steel, cobalt and its alloys, titanium and its alloys, nickel and its alloys, magnesium and its alloys, etc.

[0067] In some embodiments, the non-metallic nanomaterials include at least one of carbon-based nanomaterials (such as carbon nanotubes, graphene, graphene oxide), black phosphorus, boron nitride, and carbon nitride.

[0068] In some embodiments, the shape of the substrate can be at least one regular or irregular shape, such as a block, sheet, or plate.

[0069] In some of these implementations, the substrate may be a 3D printing material.

[0070] A second aspect of this application provides a method for preparing a composite material, comprising the following steps:

[0071] A substrate comprising a bioactive metal layer is provided, as well as a polypeptide and a linker molecule, the polypeptide comprising a first functional peptide, an enzyme-cleaved substrate peptide and a second functional peptide sequentially linked, the first functional peptide containing an azide group, and the linker molecule comprising a catechol unit and a DBCO group.

[0072] Linking molecules are attached to the bioactive metal layer through chelation of bioactive metal with catechol units;

[0073] The linker molecule is linked to the first functional peptide via a click reaction between the DBCO group and the azide group.

[0074] In some embodiments, a bioactive metal layer is formed on the surface of a substrate by plasma immersion ion implantation. In other embodiments, the substrate includes a bioactive metal, such as a bioactive metal material formed from a raw material containing a bioactive metal, and thus can serve as the bioactive metal layer without the need to form an additional bioactive metal layer by plasma immersion ion implantation.

[0075] In some embodiments, the plasma immersion ion implantation method uses a negative bias voltage of 10–30 kV, an implantation pulse width of 20–200 μs, an implantation pulse frequency of 50–1000 Hz, a radio frequency power of 100–1000 W, and an implantation time of 30–180 minutes.

[0076] In some embodiments, the chelation of the bioactive metal with the catechol unit involves mixing and incubating a substrate containing the bioactive metal layer with a solution of the linking molecules for 12 to 24 hours.

[0077] In some embodiments, the chelation of bioactive metal with catechol units includes mixing and incubating a substrate with a bioactive metal layer on its surface with a solution of linking molecules for 12 to 24 hours.

[0078] In some embodiments, the substrate and the solution of the linking molecules are mixed and incubated at a temperature of 0 to 10°C, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C.

[0079] In some embodiments, the solvent in the solution of the linking molecules is water or a buffer solution, such as a phosphate buffer, like PBS buffer.

[0080] It is understandable that the amount of linker molecules introduced onto the substrate through chelation can be reasonably adjusted by controlling the solution concentration of the linker molecules and the incubation time.

[0081] In some embodiments, the click reaction of the DBCO group with the azide group involves mixing and incubating a substrate with a surface-chelated linker molecule with a solution (dispersion) of the peptide for 12 to 24 hours.

[0082] In some embodiments, the substrate and peptide solution are mixed and incubated at a temperature of 0–10°C, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C.

[0083] In some embodiments, the solvent in the polypeptide solution is water or a buffer solution, such as a phosphate buffer, like PBS buffer.

[0084] Understandably, the amount of peptide introduced onto the substrate via click reaction can be reasonably adjusted by controlling the concentration of the peptide solution and the mixing and incubation time.

[0085] In some embodiments, the metal element of the bioactive metal layer includes at least one selected from Mg, Ta, Sr, Zn, Zr, Cu, Co, Gd, Ga, and Fe.

[0086] In some embodiments, the linker molecule includes 2 to 10 catechol units, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 catechol units.

[0087] In some embodiments, the linker molecule includes a main chain and branches, wherein at least one end group of the main chain is a DBCO group and the branches include catechol units.

[0088] In some embodiments, the linker molecules include The repeating units have two or more repeating units, for example, 2 to 10 repeating units, such as 2, 3, 4, 5, 6, 7, 8, 9, and 10 repeating units.

[0089] In some embodiments, the connecting molecule includes one end The repeating unit is connected to the DBCO group at the other end.

[0090] In some embodiments, the structural formula of the linking molecule is:

[0091] Where n is a positive integer greater than or equal to 2, and x is between 1 and 5.

[0092] In some embodiments, the structural formula of the linking molecule is as follows:

[0093]

[0094] In some embodiments, the first functional peptide, the enzyme-digested substrate peptide, and the second functional peptide in the polypeptide each independently contain 3 to 20 amino acids, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids.

[0095] In some embodiments, the enzyme corresponding to the substrate peptide can be any enzyme expressed in the microenvironment of the implantation site during bone repair, such as any of the following: oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, and translocase.

[0096] In some embodiments, the enzyme-cleaved substrate peptide includes the substrate peptide of a hydrolase.

[0097] In some embodiments, the hydrolytic enzyme includes at least one of proteases and phosphatases. Examples include matrix metalloproteinases, cathepsins, and alkaline phosphatases involved in bone repair. The corresponding enzyme-digesting substrate peptides include at least one of matrix metalloproteinase substrate peptides, cathepsin substrate peptides, and alkaline phosphatase substrate peptides.

[0098] In some embodiments, the matrix metalloproteinase family substrate peptide is at least one of matrix metalloproteinases-1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0099] In some embodiments, the enzyme-cleaved substrate peptide is a matrix metalloproteinase-2 substrate peptide whose amino acid sequence includes GPLG, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus, such as enzyme-cleaved substrate peptides including GPLGIAGQ and CGGPLGVRGK.

[0100] In some embodiments, the amino acid sequence of the enzyme-cleaved substrate peptide is GPLG, GPLGIAGQ, or CGGPLGVRGK.

[0101] In some embodiments, the enzyme-cleaved substrate peptide is a matrix metalloproteinase-9 substrate peptide, whose amino acid sequence includes any one of GERGPPGPQGARGFZGTPGL (Z is hydroxyproline), PLGLWADR, GPLGLLGC, GPLGMWSRKC, and AVRWLLTA, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus.

[0102] In some embodiments, the amino acid sequence of the enzyme-digested substrate peptide is any one of GERGPPGPQGARGFZGTPGL (Z being hydroxyproline), PLGLWADR, GPLGLLGC, GPLGMWSRKC, and AVRWLLTA.

[0103] In some embodiments, the first functional peptide and the second functional peptide are each selected from at least two different peptides selected from inflammation-regulating functional peptides, angiogenesis-promoting functional peptides, osteogenic functional peptides, antimicrobial peptides, and antioxidant peptides.

[0104] In some embodiments, the second functional peptide segment includes an inflammation-regulating functional peptide segment, and the first functional peptide segment includes at least one of an angiogenesis-promoting functional peptide segment and an osteogenic functional peptide segment.

[0105] In some embodiments, the inflammatory regulatory peptides include anti-inflammatory peptides.

[0106] In some embodiments, the inflammatory regulatory peptides include peptides having anti-transglutaminase 2 (TGase 2) activity and / or anti-phospholipase A2 (PLA2) activity.

[0107] In some embodiments, the amino acid sequence of the inflammatory regulatory peptide includes KVLDGQDP, and may also include 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, at the N-terminus and / or C-terminus, while maintaining its original inflammatory regulatory activity.

[0108] In some embodiments, the amino acid sequence of the inflammatory regulatory peptide is KVLDGQDP.

[0109] In some embodiments, the angiogenesis-promoting functional peptides include osteopontin (OPN)-derived peptides.

[0110] In some embodiments, the angiogenesis-promoting functional peptide includes the N-terminal peptide of osteopontin (OPN).

[0111] In some embodiments, the amino acid sequence of the pro-angiogenic functional peptide includes SVVYGLR, and may also include 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids, at the N-terminus and / or C-terminus, while maintaining its original pro-angiogenic activity.

[0112] In some embodiments, the amino acid sequence of the angiogenesis-promoting functional peptide is SVVYGLR.

[0113] In some embodiments, the osteogenic functional peptides include osteogenic growth peptides (OGP)-derived peptides.

[0114] In some embodiments, the osteogenic functional peptide includes the C-terminal peptide of osteogenic growth peptide (OGP).

[0115] In some embodiments, the amino acid sequence of the osteogenic functional peptide includes YGFGG, and may also include 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, at the N-terminus and / or C-terminus, while maintaining its original osteogenic activity.

[0116] In some embodiments, the amino acid sequence of the osteogenic functional peptide includes YGFGG.

[0117] In some embodiments, the amino acid sequence of the polypeptide includes SVVYGLR-GPLG-KVLDGQDP, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus.

[0118] In some embodiments, the amino acid sequence of the polypeptide is SVVYGLR-GPLG-KVLDGQDP.

[0119] In some embodiments, the polypeptide is N3-SVVYGLR-GPLG-KVLDGQDP.

[0120] In some embodiments, the amino acid sequence of the polypeptide includes YGFGG-GPLG-KVLDGQDP, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus.

[0121] In some embodiments, the amino acid sequence of the polypeptide includes YGFGG-GPLG-KVLDGQDP.

[0122] In some embodiments, the polypeptide is N3-YGFGG-GPLG-KVLDGQDP.

[0123] In some embodiments, the polypeptides modified on the bioactive metal layer include multiple peptides, each having the same or different first functional peptides, the same or different enzyme cleavage substrate peptides, and the same or different second functional peptides.

[0124] In some embodiments, the substrate includes at least one of polymers, ceramics, metals, and non-metallic nanomaterials. It is understood that when the substrate is a metal, particularly containing the aforementioned bioactive metals, it is not necessary to form an additional bioactive metal layer. The polymers include natural polymers and synthetic polymers. Natural polymers include at least one of collagen, gelatin, alginate, silk fibroin, elastin, agarose, chitin, and chitosan. Synthetic polymers include at least one of polycaprolactone, polylactic acid, polyethylene glycol, poly(N-isopropylacrylamide), polytetrafluoroethylene, expanded polytetrafluoroethylene, polyamide, nylon, polysulfone, polyvinyl alcohol, polycarbonate, polyurethane, polyether, polyetherketone, polyetheretherketone, polyethylene terephthalate, polymethyl methacrylate, and polyolefins. Ceramics include phosphate bioceramics, such as calcium phosphate (e.g., hydroxyapatite, β-tricalcium phosphate), magnesium phosphate, etc.; and silicate ceramics, such as magnesium silicates, calcium silicates, etc., at least one of these. Metals include at least one of stainless steel, cobalt and its alloys, titanium and its alloys, nickel and its alloys, and magnesium and its alloys. Non-metallic nanomaterials include at least one of carbon-based nanomaterials (such as carbon nanotubes, graphene, and graphene oxide), black phosphorus, boron nitride, and carbon nitride.

[0125] In some embodiments, the substrate may be a regular or irregular shape, such as a block, sheet, or plate. In some embodiments, the substrate may be a 3D printing material.

[0126] A third aspect of this application provides a method for forming a bone repair layer on a substrate, comprising the following steps:

[0127] A substrate comprising a bioactive metal layer is provided, as well as a polypeptide and a linker molecule, the polypeptide comprising a first functional peptide, an enzyme-cleaved substrate peptide and a second functional peptide sequentially linked, the first functional peptide containing an azide group, and the linker molecule comprising a catechol unit and a DBCO group.

[0128] Linking molecules are attached to the bioactive metal layer through chelation of bioactive metal with catechol units;

[0129] The linker molecule is linked to the first functional peptide via a click reaction between the DBCO group and the azide group.

[0130] In some embodiments, a bioactive metal layer is formed on the surface of a substrate by plasma immersion ion implantation. In other embodiments, the substrate includes a bioactive metal, such as a bioactive metal material formed from a raw material containing a bioactive metal, and thus can serve as the bioactive metal layer without the need to form an additional bioactive metal layer by plasma immersion ion implantation.

[0131] In some embodiments, the plasma immersion ion implantation method uses a negative bias voltage of 10–30 kV, an implantation pulse width of 20–200 μs, an implantation pulse frequency of 50–1000 Hz, a radio frequency power of 100–1000 W, and an implantation time of 30–180 minutes.

[0132] In some embodiments, the chelation of the bioactive metal with the catechol unit involves mixing and incubating a substrate containing the bioactive metal layer with a solution of the linking molecules for 12 to 24 hours.

[0133] In some embodiments, the chelation of bioactive metal with catechol units includes mixing and incubating a substrate with a bioactive metal layer on its surface with a solution of linking molecules for 12 to 24 hours.

[0134] In some embodiments, the substrate and the solution of the linking molecules are mixed and incubated at a temperature of 0 to 10°C, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C.

[0135] In some embodiments, the solvent in the solution of the linking molecules is water or a buffer solution, such as a phosphate buffer, like PBS buffer.

[0136] It is understandable that the amount of linker molecules introduced onto the substrate through chelation can be reasonably adjusted by controlling the solution concentration of the linker molecules and the incubation time.

[0137] In some embodiments, the click reaction of the DBCO group with the azide group involves mixing and incubating a substrate with a surface-chelated linker molecule with a solution (dispersion) of the peptide for 12 to 24 hours.

[0138] In some embodiments, the substrate and peptide solution are mixed and incubated at a temperature of 0–10°C, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C.

[0139] In some embodiments, the solvent in the polypeptide solution is water or a buffer solution, such as a phosphate buffer, like PBS buffer.

[0140] Understandably, the amount of peptide introduced onto the substrate via click reaction can be reasonably adjusted by controlling the concentration of the peptide solution and the mixing and incubation time.

[0141] In some embodiments, the metal element of the bioactive metal layer includes at least one selected from Mg, Ta, Sr, Zn, Zr, Cu, Co, Gd, Ga, and Fe.

[0142] In some embodiments, the linker molecule includes 2 to 10 catechol units, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 catechol units.

[0143] In some embodiments, the linker molecule includes a main chain and branches, wherein at least one end group of the main chain is a DBCO group and the branches include catechol units.

[0144] In some embodiments, the linker molecules include The repeating units have two or more repeating units, for example, 2 to 10 repeating units, such as 2, 3, 4, 5, 6, 7, 8, 9, and 10 repeating units.

[0145] In some embodiments, the connecting molecule includes one end The repeating unit is connected to the DBCO group at the other end.

[0146] In some embodiments, the structural formula of the linking molecule is:

[0147] Where n is a positive integer greater than or equal to 2, and x is between 1 and 5.

[0148] In some embodiments, the structural formula of the linking molecule is as follows:

[0149]

[0150] In some embodiments, the first functional peptide, the enzyme-digested substrate peptide, and the second functional peptide in the polypeptide each independently contain 3 to 20 amino acids, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 amino acids.

[0151] In some embodiments, the enzyme corresponding to the substrate peptide can be any enzyme expressed in the microenvironment of the implantation site during bone repair, such as any of the following: oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, and translocase.

[0152] In some embodiments, the enzyme-cleaved substrate peptide includes the substrate peptide of a hydrolase.

[0153] In some embodiments, the hydrolytic enzyme includes at least one of proteases and phosphatases. Examples include matrix metalloproteinases, cathepsins, and alkaline phosphatases involved in bone repair. The corresponding enzyme-digesting substrate peptides include at least one of matrix metalloproteinase substrate peptides, cathepsin substrate peptides, and alkaline phosphatase substrate peptides.

[0154] In some embodiments, the matrix metalloproteinase family substrate peptide is at least one of matrix metalloproteinases-1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0155] In some embodiments, the enzyme-cleaved substrate peptide is a matrix metalloproteinase-2 substrate peptide whose amino acid sequence includes GPLG, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus, such as enzyme-cleaved substrate peptides including GPLGIAGQ and CGGPLGVRGK.

[0156] In some embodiments, the amino acid sequence of the enzyme-cleaved substrate peptide is GPLG, GPLGIAGQ, or CGGPLGVRGK.

[0157] In some embodiments, the enzyme-cleaved substrate peptide is a matrix metalloproteinase-9 substrate peptide, whose amino acid sequence includes any one of GERGPPGPQGARGFZGTPGL (Z is hydroxyproline), PLGLWADR, GPLGLLGC, GPLGMWSRKC, and AVRWLLTA, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus.

[0158] In some embodiments, the amino acid sequence of the enzyme-digested substrate peptide is any one of GERGPPGPQGARGFZGTPGL (Z being hydroxyproline), PLGLWADR, GPLGLLGC, GPLGMWSRKC, and AVRWLLTA.

[0159] In some embodiments, the first functional peptide and the second functional peptide are each selected from at least two different peptides selected from inflammation-regulating functional peptides, angiogenesis-promoting functional peptides, osteogenic functional peptides, antimicrobial peptides, and antioxidant peptides.

[0160] In some embodiments, the second functional peptide segment includes an inflammation-regulating functional peptide segment, and the first functional peptide segment includes at least one of an angiogenesis-promoting functional peptide segment and an osteogenic functional peptide segment.

[0161] In some embodiments, the inflammatory regulatory peptides include anti-inflammatory peptides.

[0162] In some embodiments, the inflammatory regulatory peptides include peptides having anti-transglutaminase 2 (TGase 2) activity and / or anti-phospholipase A2 (PLA2) activity.

[0163] In some embodiments, the amino acid sequence of the inflammatory regulatory peptide includes KVLDGQDP, and may also include 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, at the N-terminus and / or C-terminus, while maintaining its original inflammatory regulatory activity.

[0164] In some embodiments, the amino acid sequence of the inflammatory regulatory peptide is KVLDGQDP.

[0165] In some embodiments, the angiogenesis-promoting functional peptides include osteopontin (OPN)-derived peptides.

[0166] In some embodiments, the angiogenesis-promoting functional peptide includes the N-terminal peptide of osteopontin (OPN).

[0167] In some embodiments, the amino acid sequence of the pro-angiogenic functional peptide includes SVVYGLR, and may also include 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids, at the N-terminus and / or C-terminus, while maintaining its original pro-angiogenic activity.

[0168] In some embodiments, the amino acid sequence of the angiogenesis-promoting functional peptide is SVVYGLR.

[0169] In some embodiments, the osteogenic functional peptides include osteogenic growth peptides (OGP)-derived peptides.

[0170] In some embodiments, the osteogenic functional peptide includes the C-terminal peptide of osteogenic growth peptide (OGP).

[0171] In some embodiments, the amino acid sequence of the osteogenic functional peptide includes YGFGG, and may also include 1 to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, at the N-terminus and / or C-terminus, while maintaining its original osteogenic activity.

[0172] In some embodiments, the amino acid sequence of the osteogenic functional peptide includes YGFGG.

[0173] In some embodiments, the amino acid sequence of the polypeptide includes SVVYGLR-GPLG-KVLDGQDP, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus.

[0174] In some embodiments, the amino acid sequence of the polypeptide is SVVYGLR-GPLG-KVLDGQDP.

[0175] In some embodiments, the polypeptide is N3-SVVYGLR-GPLG-KVLDGQDP.

[0176] In some embodiments, the amino acid sequence of the polypeptide includes YGFGG-GPLG-KVLDGQDP, and may also include 1 to 10 amino acids at the N-terminus and / or C-terminus.

[0177] In some embodiments, the amino acid sequence of the polypeptide includes YGFGG-GPLG-KVLDGQDP.

[0178] In some embodiments, the polypeptide is N3-YGFGG-GPLG-KVLDGQDP.

[0179] In some embodiments, the polypeptides modified on the bioactive metal layer include multiple peptides, each having the same or different first functional peptides, the same or different enzyme cleavage substrate peptides, and the same or different second functional peptides.

[0180] In some embodiments, the substrate includes at least one of polymers, ceramics, metals, and non-metallic nanomaterials. It is understood that when the substrate is a metal, particularly containing the aforementioned bioactive metals, it is not necessary to form an additional bioactive metal layer. The polymers include natural polymers and synthetic polymers. Natural polymers include at least one of collagen, gelatin, alginate, silk fibroin, elastin, agarose, chitin, and chitosan. Synthetic polymers include at least one of polycaprolactone, polylactic acid, polyethylene glycol, poly(N-isopropylacrylamide), polytetrafluoroethylene, expanded polytetrafluoroethylene, polyamide, nylon, polysulfone, polyvinyl alcohol, polycarbonate, polyurethane, polyether, polyetherketone, polyetheretherketone, polyethylene terephthalate, polymethyl methacrylate, and polyolefins. Ceramics include phosphate bioceramics, such as calcium phosphate (e.g., hydroxyapatite, β-tricalcium phosphate), magnesium phosphate, etc.; and silicate ceramics, such as magnesium silicates, calcium silicates, etc., at least one of these. Metals include at least one of stainless steel, cobalt and its alloys, titanium and its alloys, nickel and its alloys, and magnesium and its alloys. Non-metallic nanomaterials include at least one of carbon-based nanomaterials (such as carbon nanotubes, graphene, and graphene oxide), black phosphorus, boron nitride, and carbon nitride.

[0181] In some embodiments, the substrate may be a regular or irregular shape, such as a block, sheet, or plate. In some embodiments, the substrate may be a 3D printing material.

[0182] A fourth aspect of this application provides a medical product comprising the aforementioned composite material, or a composite material prepared by the aforementioned method, or a substrate having a bone repair layer formed by the aforementioned method.

[0183] In some of these implementations, the medical supplies include implantable medical devices.

[0184] The fifth aspect of this application provides the use of the aforementioned composite material, or the composite material prepared by the aforementioned preparation method, or the substrate with a bone repair layer formed by the aforementioned method, in the preparation of medical products.

[0185] In this application, the modified polypeptide is a bifunctional polypeptide chain with bioresponsiveness; the temporal exposure of the functional polypeptide regulates effector cells corresponding to the repair stage, thereby matching each stage of bone tissue repair and promoting bone repair and integration; the bifunctional polypeptide offers high flexibility in selection and can be specifically designed according to the repair purpose and tissue repair needs; the metal plasma immersion ion implantation technology is simple, efficient, and highly reproducible, with no restrictions on the composition and shape of the molding material, and a wide range of multivalent metals can be selected; the synthesized linker molecule undergoes chelation between the catechol group and the bioactive metal, and its DBCO terminal group undergoes a click chemical reaction with the azide group at the polypeptide tail, both processes being simple and efficient; the technologies involved in this application have significant advantages of simplicity, efficiency, and high reproducibility, ensuring subsequent batch and industrial production.

[0186] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0187] Figure 1a This is the synthetic route for connecting molecules in Example 1.

[0188] Figure 1b This is the 1H NMR spectrum of the linker molecule obtained in Example 1.

[0189] Figure 2a This is a scanning electron microscope image of the untreated PEEK (abbreviated as P) surface in Example 2.

[0190] Figure 2b This is a scanning electron microscope image of the PEEK(P+Zn) surface after treatment with metal plasma immersion ion implantation technology in Example 2.

[0191] Figure 2c This is a scanning electron microscope image of the surface of P+Zn (abbreviated as P+Zn+D) after grafting and connecting molecules in Example 3.

[0192] Figure 2d This is a scanning electron microscope image of the surface of P+Zn+D (abbreviated as P+Zn+D+DPI) after the target polypeptide chain was modified in Example 4.

[0193] Figure 3 It is the full X-ray photoelectron spectrum of each group of samples measured in Example 5.

[0194] Figure 4 The results show the static water contact angles of each group of samples measured in Example 6. The inset above the bar chart is a photograph of water droplets during the test of the corresponding group of samples.

[0195] Figure 5 These are the zinc ion release curves of each group of samples measured in Example 7.

[0196] Figure 6a This refers to the concentration of M1 marker secreted factor IL-6 in the supernatant of macrophages cultured directly on each group of samples for 1 day and 3 days in Example 8.

[0197] Figure 6b This refers to the concentration of M2 marker secretory factor IL-10 in the supernatant of macrophages cultured directly on each group of samples for 1 day and 3 days in Example 8.

[0198] Figure 7 This shows the proliferation of human umbilical vein endothelial cells in each group of samples after direct culture for 1 to 5 days in Example 9.

[0199] Figure 8 This is the tube formation result of human umbilical vein endothelial cells directly cultured on each group of samples for 5 days in Example 9.

[0200] Figure 9a The results are from the cell scratch experiment of human umbilical vein endothelial cells on each group of samples within 24 hours in Example 9.

[0201] Figure 9b The results of the Vanslon migration experiment of human umbilical vein endothelial cells on each group of samples after 24 hours in Example 9 are shown. Dark purple indicates the migrating endothelial cells.

[0202] Figure 10a This shows the alkaline phosphatase staining of human bone marrow mesenchymal stem cells after 7 days of direct culture on different groups of samples in Example 10.

[0203] Figure 10b This refers to the alkaline phosphatase activity in the cell lysate of human bone marrow mesenchymal stem cells after 7 days of direct culture on different groups of samples in Example 10.

[0204] Figure 11a This describes the mineralization of the extracellular matrix of human bone marrow mesenchymal stem cells after direct culture on different groups of samples for 14 and 21 days, as shown in Example 10.

[0205] Figure 11b The results are the quantitative results of Alizarin Red S staining of human bone marrow mesenchymal stem cells after 14 and 21 days of direct culture on different groups of samples in Example 10. Detailed Implementation

[0206] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0207] The embodiments of this application are described in detail below. The described embodiments are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0208] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number, and "approximately" means within the range of ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. of the stated number. The use of "first" and "second" is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0209] In the description of this application, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0210] Example 1: Synthesis of Linking Molecules

[0211] refer to Figure 1a The synthetic route used levodopa (L-Dopa), 10-azido-2,4,6,8-tetraoxadecan-1-amine, and 5,6-dihydro-11,12-dihydrodibenzo[A,E]cyclooctene-5-ol as starting materials to synthesize the linker molecule. The main chain of this linker molecule contains four catechol groups and a diphenylcyclooctynyl (DBCO) group at one end.

[0212] refer to Figure 1b In order to follow Figure 1a The 1H NMR spectrum of the stable product before deprotection obtained from the final synthetic route shows that the target linker molecule was successfully prepared.

[0213] Example 2: Formation of a bioactive metal layer on a substrate

[0214] Cut PEEK into round slices (diameter = 1.5cm, thickness = 2mm), polish with sandpaper (800, 1200 and 2000 grit), and then ultrasonically treat with acetone, ethanol and pure water in sequence.

[0215] A Zn-containing metal layer was prepared on the surface of a PEEK wafer using plasma immersion ion implantation (PIII). The plasma treatment process involved a load bias of 15 kV, a pulse width of 70 μs, and a treatment time of 30 minutes, resulting in PEEK with a Zn-containing metal layer on its surface (denoted as P+Zn). (Results referenced...) Figure 2a and Figure 2b The images show scanning electron microscope (SEM) images of an untreated PEEK disc (denoted as P) and a P+Zn disc, respectively. The results indicate that the Zn metal layer is uniformly distributed on the PEEK.

[0216] Example 3: Grafting and connecting molecules on a substrate

[0217] Two mg of the linker molecule synthesized in Example 1 was dissolved in 100 μL of methanol and shaken to obtain a linker molecule stock solution with a concentration of 20 mg / mL. This stock solution was further diluted with deionized water to obtain a linker molecule working solution with a concentration of 0.1 mg / mL. The P+Zn obtained in Example 2 was immersed in the linker molecule working solution and reacted at 4°C for 24 hours. The material was then removed and washed three times with deionized water to complete the grafting process. Grafting of the linker molecule was completed through the chelation effect of Zn and the catechol groups on the linker molecule, resulting in P+Zn modified with the linker molecule (denoted as P+Zn+D). The results are as follows: Figure 2c The image is a scanning electron microscope image of P+Zn+D, based on... Figure 2c Combination Figure 2b This indicates that the grafting of connecting molecules did not have a significant impact on the surface morphology of the substrate.

[0218] Example 4: Linking peptides on a substrate

[0219] A bifunctional peptide was designed, consisting of a head of -KVLDGQDP (second functional peptide, SEQ ID No. 3) with inflammatory regulatory activity, a middle portion of -GPLG- (enzyme-cleaved substrate peptide, SEQ ID No. 2) that can be specifically cleaved by matrix metalloproteinase-2, and a tail of N3-SVVYGLR- (first functional peptide, SEQ ID No. 1) which has pro-angiogenic function and is modified with azide. The resulting responsive bifunctional peptide N3-SVVYGLR-GPLG-KVLDGQDP was synthesized by a biotechnology company.

[0220] Take 10 mg of bifunctional peptide powder and add 100 mL of deionized water, gently mixing until the peptide is completely dissolved to obtain a peptide working solution with a concentration of 0.1 mg / mL. Immerse the P+Zn+D from Example 3 in the peptide working solution and react at 4°C for 24 hours. Remove the material and wash it three times with deionized water to complete the reaction process, obtaining P+Zn+D modified with the peptide (labeled as P+Zn+D+DPI). Reference Figure 2d The image is a scanning electron microscope image of P+Zn+D+DPI, and... Figure 2a , Figure 2b , Figure 2c The comparison shows that the modification of the target peptide chain did not significantly affect the substrate surface morphology. Therefore, this embodiment utilizes the click chemistry reaction between the DBCO group on P+Zn+D and the azide group on the bifunctional peptide to achieve the grafting of the bifunctional peptide.

[0221] Example 5: X-ray photoelectron spectroscopy test

[0222] X-ray photoelectron spectroscopy (XPS) wide-field scanning was performed on the surfaces of the samples (P, P+Zn, P+Zn+D, and P+Zn+D+DPI) obtained in Examples 2-4 to obtain... Figure 3 The XPS full spectrum shown indicates that the intensity of the characteristic peaks represents the content of the element on the surface. The results show that zinc (Zn) appears on the material surface after metal plasma immersion ion implantation treatment, indicating that zinc was successfully modified onto the material surface, consistent with the scanning electron microscope observations in Example 2. Furthermore, nitrogen (N) appears on the surface of the P+Zn+D+DPI sample, indicating successful grafting.

[0223] Example 6: Wettability Test

[0224] The wettability of the material surface was tested using a static water contact angle meter. 2 μL of ultrapure water was slowly and vertically suspended onto the sample surface using a syringe. The meter's built-in imaging system was used to capture images of the droplet and analyze the contact angle. Figure 4This shows the static water contact angle results for each group of treated samples in Examples 2-4. The horizontal axis represents the sample name, the vertical axis represents the contact angle in degrees, and the inset image is a photograph of the water droplet during the test of the corresponding group of samples. Figure 4 It can be seen that the water contact angle of pure PEEK (P group) is 88.0±3.6°; after modification with a Zn metal layer (P+Zn group), the water contact angle becomes 88.9±3.2°; after grafting and linking molecules (P+Zn+D group), the water contact angle decreases to 72.7±1.5°; after modification with a bifunctional peptide (P+Zn+D+DPI group), the contact angle is 71.8±3.0°, which is similar to the value of P+Zn+D group.

[0225] Example 7: Zn ion release experiment

[0226] The release of zinc ions from the samples prepared in Examples 2-4 was detected when incubated under simulated in vivo conditions. The samples prepared in Examples 2-4 were immersed in phosphate-buffered saline (PBS) and stored at 37°C. The supernatant was collected at specific time points, and the zinc concentration was determined using inductively coupled plasma atomic emission spectrometry (ICP). The results are as follows: Figure 5 As shown, the P+ZN group had the fastest zinc ion release rate. Modification of the linker molecule (P+Zn+D group) reduced the zinc ion release rate, and subsequent modification with a bifunctional peptide (P+Zn+D+DPI group) further slowed down the zinc ion release rate.

[0227] Example 8: Anti-inflammatory Repair Experiment

[0228] Mouse macrophage-like cell line RAW264.7 was seeded onto the surface of the samples in Examples 2-4 and cultured in a 37°C, 5% CO2 cell culture incubator. The culture supernatant and total cell protein were collected after 1 and 3 days of culture. The expression of the M1 inflammatory phenotype marker interleukin-6 (IL-6) and the M2 repair phenotype marker interleukin-10 (IL-10) in the supernatant was measured using an enzyme-linked immunosorbent assay (ELISA) kit. The results are shown in Figure 6. Figure 6a The IL-6 content in Example 4 showed that, compared with P+Zn+D in Example 3, the grafting of the bifunctional polypeptide in Example 4 reduced the expression of M1 inflammatory phenotype markers, that is, it inhibited the M1 inflammatory phenotype to a certain extent, and the IL-6 content decreased with increasing culture time. Figure 6b The samples from Examples 2 to 4 were compared. The expression trends of IL-10 and IL-6 were opposite, indicating that the grafting of the peptide in Example 4 promoted the M2 repair phenotype of macrophages.

[0229] Example 9: Adhesion, proliferation, migration and tube formation experiments of HUVECs

[0230] Human umbilical vein endothelial cell line (HUVEC) was seeded and cultured on the sample surfaces described in Examples 2-4. The proliferation of HUVECs on different samples was measured using a cell counting kit (CCK-8) for 1-5 days, and the results are as follows: Figure 7 As shown, from Figure 7 It can be seen that HUVECs can adhere to and proliferate on all samples in Examples 2 to 4. Among them, the cells adhered the most and proliferated the fastest on the P+Zn+D+DPI group sample in Example 4.

[0231] The tube-forming ability of HUVECs on different groups of samples in Examples 2-4 was evaluated using a matrix adhesive, and the results are as follows: Figure 8 As shown, from Figure 8 It can be seen that among the four groups of samples, the P+Zn+D+DPI group in Example 4 showed the highest angiogenesis capacity of HUVECs.

[0232] The migration ability of HUVECs on different groups of samples in Examples 2–4 was evaluated using cell scratch assays and Vanslon migration assays. The results are as follows: Figure 9a and 9b As shown in the figure, among the four groups of samples, the HUVEC migration rate was the fastest in the P+Zn+D+DPI group of Example 4.

[0233] In summary, based on the above experiments, the P+Zn+D+DPI group sample modified with pro-angiogenic peptides in Example 4 was most conducive to the adhesion, proliferation, migration, and tube formation of HUVECs.

[0234] Example 10: In vitro osteogenic experiment of BMSCs

[0235] Human bone marrow mesenchymal stem cells (BMSCs) were seeded and cultured on the surfaces of the samples treated in Examples 2–4. The proliferation of BMSCs on different samples was measured using a cell counting kit (CCK-8) for 1–5 days, and the results are shown in Figure 10. As can be seen from Figure 10, similar to HUVECs in Example 9, BMSCs could adhere to and proliferate on all samples in Examples 2–4. The cells showed the highest adhesion and fastest proliferation in the P+Zn+D+DPI group sample in Example 4.

[0236] BMSCs were directly cultured on different groups of samples for 7 days, then fixed with 4% paraformaldehyde and their ALP expression was semi-quantitatively studied using an ALP qualitative kit. The results are as follows: Figure 10a Total protein was collected from BMSCs cells, and ALP expression was quantitatively studied using an alkaline phosphatase (ALP) quantification kit. The results are as follows: Figure 10b .from Figure 10a and Figure 10bIt can be seen that the ALP expression of BMSCs in the samples of Examples 2 to 4 increases sequentially.

[0237] After culturing BMSCs directly on different groups of samples for 14 and 21 days, the BMSCs were fixed with 4% paraformaldehyde, and the mineralized nodules were stained with Alizarin Red S (ARS). The results are as follows: Figure 11a As shown; simultaneously, ARS was quantified using Siberian chloride solution, and the results are as follows. Figure 11b As shown. From Figure 11a and Figure 11b It can be seen that the size and maturity of mineralized nodules of BMSCs in the samples of Examples 2 to 4 increase sequentially.

[0238] In summary, the introduction of the zinc-containing metal layer promoted osteogenic formation of BMSCs in vitro, and the promoting effect was further enhanced with the introduction of the bifunctional peptide.

[0239] The experimental results from Examples 7-10 show that the introduction of the bifunctional peptide effectively improved the long-term sustained release of bioactive metal ions, enhanced anti-inflammatory and repair effects, and also improved angiogenesis and osteogenic effects. Furthermore, compared to directly modifying the two functional peptides, the sequential exposure of the functional peptides achieved by the design of the enzyme-linked substrate peptide structure in the bifunctional peptide significantly improved bone repair and bone integration. The specific experimental procedures were performed as described in the examples above.

[0240] Example 11

[0241] This embodiment provides a composite material that differs from the P+Zn+D+DPI of Example 4 in that the linked bifunctional polypeptide is N3-YGFGG-GPLG-KVLDGQDP, and the Zn layer formed by PIII is replaced by a Co layer.

[0242] Experiments were conducted according to Examples 4-10 above. The results showed that, compared with the P group, P+Co group, and P+Co+D group, the bifunctional peptide in the P+Co+D+DPI group in this example could also further slow down the zinc ion release rate; and further inhibited the M1 inflammatory phenotype and promoted the M2 repair phenotype; HUVECs had the highest adhesion and the fastest proliferation, the highest angiogenesis capacity, and the fastest migration rate; and further promoted the osteogenic formation of BMSCs in vitro.

[0243] Example 12

[0244] This embodiment provides a composite material that differs from the P+Zn+D+DPI in Example 4 in that the linked bifunctional polypeptides include two types: N3-SVVYGLR-GPLG-KVLDGQDP and N3-YGFGG-GPLG-KVLDGQDP. The relevant test results are similar to those of Examples 4-10 and Example 11.

[0245] Example 13

[0246] This embodiment provides a composite material that differs from the P+Zn+D+DPI of Example 4 in that the substrate is replaced by a 50mm×50mm×2mm pure titanium square sheet instead of a polyetheretherketone disc, and the step of forming a Zn layer from PIII is omitted.

[0247] Example 14

[0248] This embodiment provides a composite material that differs from the P+ZN+D+DPI of Embodiment 4 in that the metal layer is replaced by Mg instead of Zn.

[0249] Example 15

[0250] This embodiment provides a composite material that differs from the P+Zn+D+DPI in Example 4 in that the linked bifunctional polypeptide is N3-SVVYGLR-CGGPLGVRGK-KVLDGQDP.

[0251] Example 16

[0252] This embodiment provides a composite material that differs from the P+Zn+D+DPI in Example 4 in that the linked bifunctional polypeptide is N3-SVVYGLR-PLGLWADR-KVLDGQDP.

[0253] The test results of Examples 13-16 are similar to those of Examples 7-10, and will not be repeated here.

[0254] Example 17

[0255] This embodiment provides a composite material that differs from the P+Zn+D+DPI in Example 4 in that the linked bifunctional polypeptide is N3-SVVYGLR-GPLG-GGC (antioxidant and angiogenesis promoter).

[0256] The composite material provided in this embodiment can also play a good role in promoting angiogenesis and osteogenic formation. In addition, it has a certain antioxidant capacity in the early stage of implantation during the sequential repair process, which effectively solves the problem of poor bone repair effect caused by oxidative stress.

[0257] Example 18

[0258] This embodiment provides a composite material that differs from the P+Zn+D+DPI in Example 4 in that the linked bifunctional polypeptide is N3-SVVYGLR-GPLG-KKVVFWVKFK (antibacterial and angiogenesis-promoting).

[0259] The composite material provided in this embodiment can also play a good role in promoting angiogenesis and osteogenic formation. In addition, it has a certain antibacterial ability in the early stage of implantation during the sequential repair process, and has an outstanding repair effect on bone defects caused by infection.

[0260] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present application. Furthermore, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.

Claims

1. A composite material, characterized in that, include: Substrate, the substrate comprising a bioactive metal layer; The polypeptide comprises a first functional peptide, an enzyme-digested substrate peptide, and a second functional peptide sequentially linked together. The first functional peptide is modified onto the bioactive metal layer. The first functional peptide is modified onto the bioactive metal layer via a linker molecule. The first functional peptide contains an azide group. The linker molecule comprises a catechol unit and a DBCO group. The first functional peptide and the linker molecule are linked via a click reaction between the azide group and the DBCO group. The linker molecule is linked to the bioactive metal layer via chelation between the metal and the catechol unit. The first functional peptide and the second functional peptide are each selected from at least two different peptides selected from inflammation-regulating functional peptides, angiogenesis-promoting functional peptides, bone-promoting functional peptides, antimicrobial peptides, and antioxidant peptides.

2. The composite material according to claim 1, characterized in that, The metal element of the bioactive metal layer includes at least one of Mg, Ta, Sr, Zn, Zr, Cu, Co, Gd, Ga, and Fe.

3. The composite material according to claim 1, characterized in that, The linker molecule comprises 2 to 10 catechol units.

4. The composite material according to claim 1, characterized in that, The linker molecule comprises a main chain and branches, wherein at least one end group of the main chain is a DBCO group, and the branches comprise catechol units.

5. The composite material according to claim 1, characterized in that, The connecting molecules include Repeating units.

6. The composite material according to claim 1, characterized in that, The enzyme-digested substrate peptides include at least one of matrix metalloproteinase substrate peptides, cathepsin substrate peptides, and alkaline phosphatase substrate peptides.

7. The composite material according to claim 6, characterized in that, The matrix metalloproteinase substrate peptide is at least one of matrix metalloproteinases-1, 2, 3, 7-20.

8. The composite material according to claim 6, characterized in that, The amino acid sequence of the enzyme-digested substrate peptide includes GPLG.

9. The composite material according to claim 1, characterized in that, The second functional peptide segment includes an inflammation-regulating functional peptide segment, and the first functional peptide segment includes at least one of angiogenesis-promoting functional peptide segment and osteogenic functional peptide segment.

10. The composite material according to claim 1, characterized in that, The amino acid sequence of the inflammatory regulatory peptide includes KVLDGQDP.

11. The composite material according to claim 1, characterized in that, The amino acid sequence of the angiogenesis-promoting peptide includes SVVYGLR.

12. The composite material according to claim 1, characterized in that, The amino acid sequence of the osteogenic peptide includes YGFGG.

13. The composite material according to claim 1, characterized in that, The substrate includes at least one of polymers, ceramics, metals, and non-metallic nanomaterials.

14. A method for preparing composite materials, characterized in that, Includes the following steps: A substrate comprising a bioactive metal layer is provided, along with a polypeptide and a linker molecule. The polypeptide comprises a first functional peptide, an enzyme-cleaved substrate peptide, and a second functional peptide sequentially linked together. The first functional peptide contains an azide group, and the linker molecule comprises a catechol unit and a DBCO group. The first functional peptide and the second functional peptide are each selected from at least two different peptides chosen from inflammation-regulating functional peptides, angiogenesis-promoting functional peptides, osteogenic functional peptides, antimicrobial peptides, and antioxidant peptides. The linker molecule is attached to the bioactive metal layer by chelation between the bioactive metal and the catechol unit; The linker molecule is linked to the first functional peptide via a click reaction between the DBCO group and the azide group.

15. The preparation method according to claim 14, characterized in that, The bioactive metal layer is formed on the surface of the substrate by plasma immersion ion implantation.

16. The preparation method according to claim 15, characterized in that, In the plasma immersion ion implantation method, the negative bias voltage is 10~30 kV, the implantation pulse width is 20~200 μs, the implantation pulse frequency is 50~1000 Hz, the radio frequency power is 100~1000 W, and the implantation time is 30~180 minutes.

17. The preparation method according to claim 14, characterized in that, The metal element of the bioactive metal layer includes at least one of Mg, Ta, Sr, Zn, Zr, Cu, Co, Gd, Ga, and Fe.

18. The preparation method according to claim 14, characterized in that, The linker molecule comprises 2 to 10 catechol units.

19. The preparation method according to claim 14, characterized in that, The linker molecule comprises a main chain and branches, wherein at least one end group of the main chain is a DBCO group, and the branches comprise catechol units.

20. The preparation method according to claim 14, characterized in that, The connecting molecules include Repeating units.

21. The preparation method according to claim 14, characterized in that, The enzyme-digested substrate peptides include at least one of matrix metalloproteinase substrate peptides, cathepsin substrate peptides, and alkaline phosphatase substrate peptides.

22. The preparation method according to claim 21, characterized in that, The matrix metalloproteinase substrate peptide is at least one of matrix metalloproteinases-1, 2, 3, 7-20.

23. The preparation method according to claim 21, characterized in that, The amino acid sequence of the enzyme-digested substrate peptide includes GPLG.

24. The preparation method according to claim 14, characterized in that, The second functional peptide segment includes an inflammation-regulating functional peptide segment, and the first functional peptide segment includes at least one of angiogenesis-promoting functional peptide segment and osteogenic functional peptide segment.

25. The preparation method according to claim 14, characterized in that, The amino acid sequence of the inflammatory regulatory peptide includes KVLDGQDP.

26. The preparation method according to claim 14, characterized in that, The amino acid sequence of the angiogenesis-promoting peptide includes SVVYGLR.

27. The preparation method according to claim 14, characterized in that, The amino acid sequence of the osteogenic peptide includes YGFGG.

28. The preparation method according to claim 14, characterized in that, The substrate includes at least one of polymers, ceramics, metals, and non-metallic nanomaterials.

29. A method for forming a bone repair layer on a substrate, characterized in that, Includes the following steps: A substrate comprising a bioactive metal layer is provided, along with a polypeptide and a linker molecule. The polypeptide comprises a first functional peptide, an enzyme-cleaved substrate peptide, and a second functional peptide sequentially linked together. The first functional peptide contains an azide group, and the linker molecule comprises a catechol unit and a DBCO group. The first functional peptide and the second functional peptide are each selected from at least two different peptides chosen from inflammation-regulating functional peptides, angiogenesis-promoting functional peptides, osteogenic functional peptides, antimicrobial peptides, and antioxidant peptides. The linker molecule is attached to the bioactive metal layer by chelation between the bioactive metal and the catechol unit; The linker molecule is linked to the first functional peptide via a click reaction between the DBCO group and the azide group.

30. The method according to claim 29, characterized in that, The bioactive metal layer is formed on the surface of a substrate by plasma immersion ion implantation.

31. The method according to claim 30, characterized in that, In the plasma immersion ion implantation method, the negative bias voltage is 10~30 kV, the implantation pulse width is 20~200 μs, the implantation pulse frequency is 50~1000 Hz, the radio frequency power is 100~1000W, and the implantation time is 30~180 minutes.

32. The method according to claim 29, characterized in that, The metal element of the bioactive metal layer includes at least one of Mg, Ta, Sr, Zn, Zr, Cu, Co, Gd, Ga, and Fe.

33. The method according to claim 29, characterized in that, The linker molecule comprises 2 to 10 catechol units.

34. The method according to claim 29, characterized in that, The linker molecule comprises a main chain and branches, wherein at least one end group of the main chain is a DBCO group, and the branches comprise catechol units.

35. The method according to claim 29, characterized in that, The connecting molecules include Repeating units.

36. The method according to claim 29, characterized in that, The enzyme-digested substrate peptides include at least one of matrix metalloproteinase substrate peptides, cathepsin substrate peptides, and alkaline phosphatase substrate peptides.

37. The method according to claim 36, characterized in that, The matrix metalloproteinase substrate peptide is at least one of matrix metalloproteinases-1, 2, 3, 7-20.

38. The method according to claim 36, characterized in that, The amino acid sequence of the enzyme-digested substrate peptide includes GPLG.

39. The method according to claim 29, characterized in that, The second functional peptide segment includes an inflammation-regulating functional peptide segment, and the first functional peptide segment includes at least one of angiogenesis-promoting functional peptide segment and osteogenic functional peptide segment.

40. The method according to claim 29, characterized in that, The amino acid sequence of the inflammatory regulatory peptide includes KVLDGQDP.

41. The method according to claim 29, characterized in that, The amino acid sequence of the angiogenesis-promoting peptide includes SVVYGLR.

42. The method according to claim 29, characterized in that, The amino acid sequence of the osteogenic peptide includes YGFGG.

43. The method according to claim 29, characterized in that, The substrate includes at least one of polymers, ceramics, metals, and non-metallic nanomaterials.

44. Medical supplies, characterized in that, Includes the composite material according to any one of claims 1 to 13, or the composite material prepared by the preparation method according to any one of claims 14 to 28, or the substrate having a bone repair layer formed by the method according to any one of claims 29 to 43.

45. The composite material according to any one of claims 1 to 13, or the composite material prepared by the preparation method according to any one of claims 14 to 28, or the substrate with a bone repair layer formed by the method according to any one of claims 29 to 43, in the preparation of medical products.

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