A polypeptide and its use in bone defect repair

Abstract

The application provides a kind of polypeptide and its application in bone defect repair, belong to polypeptide technical field.The application is based on the functional domain of TGF-beta 3 factor, designs 10 derived peptides, only selects one derived peptide with bone inductive property, the derived peptide is composed of 21 amino acid residues;The polypeptide can not only induce murine bone marrow mesenchymal stem cells to produce mineralization nodule in vitro, but also can induce new bone formation in rat skull extreme bone defect model (in vivo) and rat intramuscular (heterotopic ossification model), and the osteogenic induction efficiency is equivalent to BMP-2.Summing up, the polypeptide provided by the application has bone inductive property, and has great application prospect in bone defect repair.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and more specifically, to a polypeptide and its application in bone defect repair. Technical Background

[0002] Bone defects are among the most common clinical conditions, caused by a variety of factors including congenital bone dysplasia or malformation, tumors, inflammation, and trauma. Repairing large-volume bone defects remains a major challenge in medicine. Autologous bone grafts possess excellent osteogenic potential and are therefore considered the gold standard for treating bone defects; however, their clinical application is limited by factors such as the need for a second surgical field, prolonged surgery time, and donor site pain. Furthermore, allograft bone, xenograft bone grafts, and most synthetic materials used clinically lack osteoinductive properties, making complete bone tissue repair impossible and thus unsuitable for repairing critical-volume bone defects alone. Therefore, endowing medical materials with high osteoinductive properties through various active agents is crucial for the repair of large-volume bone defects.

[0003] Currently discovered osteoinductive active factors mainly come from the transforming growth factor-β (TGF-β) superfamily. The TGF-β superfamily includes proteins with pleiotropic activities such as BMPs and TGF-βs, playing a crucial role in regulating embryonic development and tissue patterns, postnatal tissue induction and morphogenesis, immunity and fibrosis, and initiating morphogenesis of tissues and organs, including the scaffold and bone matrix. However, the synthesis of protein growth factors mainly relies on genetic engineering techniques based on *E. coli* and mammalian cells. This technology has low yields, high costs, and is difficult to purify, and it also carries potential immunogenicity. Furthermore, protein growth factors have strict requirements for storage conditions and are highly sensitive to sterilization methods, thus affecting their osteoinductive activity. Therefore, the development of novel, low-cost, low-immunogenic, and highly efficient osteoinductive active factors has become a research hotspot in the field of large-volume bone defect repair.

[0004] Current research on bone-promoting peptides primarily focuses on BMP peptides. Studies have confirmed that the alkaline phosphatase (ALP) activity induced by the BMP2 core structural region peptide (sequence 73-92) in mouse stem cells is significantly higher than that induced by the non-core structural region (44-58) or (68-87). Another study showed that the expression of osteocalcin (OCN), a late-stage osteogenic differentiation marker in bone marrow mesenchymal stem cells (BMSCs), induced by the BMP2 core structural region peptide (73-92) is significantly higher than that of peptides from other protein sources. Previously, the inventors significantly improved the bone-promoting efficacy of BMP peptides by modifying the peptide structure, such as adding a linker portion to increase the region where the peptide binds to the receptor. However, although researchers are continuously optimizing BMP peptides, the optimization effect remains relatively limited. On the other hand, TGF-β3, as a novel molecular strategy, replicates the synergistic induction of bone formation by expressing multiple BMP and TGF-β genes individually but synergistically, rapidly and substantially inducing bone formation in primate models. It is currently the transforming growth factor discovered in primate testing. Chinese patents “A Hydrogel Material for Promoting Cartilage Repair and Its Preparation Method and Application” (Publication (Announcement) No.: CN114306732B), “A Method for Inducing Stem Cell-Directed Cartilage Differentiation” (Publication (Announcement) No.: CN110684725B), and “A TGF-β3 Derivative Peptide and Its Use in the Preparation of Drugs for Promoting Cartilage Repair and / or Treating Cartilage Defects” (Publication (Announcement) No.: CN117720637A) all provide the use of TGF-β3 protein or its derivative peptide in cartilage repair / induction; among them, the patent “A Hydrogel Material for Promoting Cartilage Repair and Its Preparation Method and Application” provides the use of TGF-β3 protein or its derivative peptide in cartilage repair / induction; Neither the application of TGF-β3 protein nor the method of inducing chondrogenic differentiation of stem cells utilizes TGF-β3 protein-derived peptides, nor is it a direct application of TGF protein. The former simply prepares exosomes from TGF-β3 protein and then mixes the exosomes with BMSCs to prepare a hydrogel. The latter uses an adenovirus vector to introduce transforming growth factor TGF-β3 into chondrocytes. The transfected chondrocytes provide stem cells with continuous, local, and overexpressed TGF-β3, thereby stimulating stem cells to differentiate into chondrocytes. Therefore, it is still unknown whether TGF-β3 protein or its derivative peptides can be used to induce osteogenic formation and then treat bone defects. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a polypeptide and its application in bone defect repair, belonging to the field of polypeptide technology. Based on the functional domain of TGF-β3, this invention designed 10 derived peptides, and only one was screened out as having osteoinductive properties. This derived peptide consists of 21 amino acid residues. This polypeptide can not only induce the formation of mineralized nodules in murine bone marrow mesenchymal stem cells in vitro, but also induce new bone formation in a rat skull extreme bone defect model (in vivo) and in rat muscle (ectopic osteogenic model), with osteogenic induction efficacy comparable to BMP-2. In summary, the polypeptide provided by this invention has osteoinductive properties and has great application potential in bone defect repair.

[0006] On one hand, the present invention provides a TP4 polypeptide with bone-inducing activity, wherein the amino acid sequence of the polypeptide comprises the sequence GPGGDRQDLGWKWVHEPKGYY (SEQ ID NO:5). The TP4 polypeptide is derived from the transforming growth factor-β3 protein found in primate testing.

[0007] Although both cartilage and bone are composed of cells and extracellular matrix, the types of cells that form cartilage and bone are different: cartilage is formed by chondrocytes and chondrocytes, while bone is formed by osteocytes, osteoblasts, and osteoclasts. Furthermore, the extracellular matrix components of cartilage and bone are also different: the matrix surrounding chondrocytes is mostly composed of gelatinous and fibrous substances, while minerals such as calcium and phosphorus constitute the majority of the extracellular matrix of bone. Therefore, it is understandable that the differentiation processes of cartilage and bone are regulated by different factors; even two similar amino acid residue sequences may perform different functions.

[0008] At the same time, sequences that can induce bone formation must also induce calcium deposition and mineralization in the extracellular matrix, while inducing cartilage formation will not produce calcification.

[0009] This invention is based on the crystallographic data of the structure of TGF-β3 transforming growth factor itself and the key binding domain of its type II receptor. By analyzing the crystallographic data of TGF-β3 and the key functional domains of its type II receptor, the high-affinity binding regions of TGF-β3 and its receptor were determined to be α1, β2, β3, β4, β7, and β8. Based on the protein structural orientation, by exploring different arrangements and combinations of functional regions, adjusting the length of the amino acid sequences between these regions, and simplifying the core sequences of the functional structural domains, 10 TGF-β3-derived peptides (TP0-TP9) were designed. These derived peptides were then applied to a rat model of ectopic osteogenic erector spinae muscle to screen for derivative peptides with osteoinductive properties. In vitro and in vivo experiments showed that only TP4 peptide could induce bone differentiation, while TP8 could induce cartilage formation. Furthermore, compared to the amino acid sequence of TP0 peptide (SEQ ID NO:1), TP4 peptide (SEQ ID NO:5) lacked the TILYYVGRT amino acid residues, meaning that the TILYYVGRT amino acid residue sequence is a "switch" for bone differentiation direction; that is, the presence of this sequence induces stem cells to differentiate into cartilage, while its absence induces bone. Combining the staining results and sequence differences between TP0 peptide (SEQ ID NO:1) and TP8 peptide (SEQ ID NO:9), the GPGGDRQDLGWK amino acid residue sequence significantly inhibited the cartilage induction effect. In summary, TP4 peptide (SEQ ID NO:5) can induce bone formation but not cartilage formation; while TP8 peptide (SEQ ID NO:9) has the opposite function, it cannot induce calcification in cells, and therefore cannot induce bone formation.

[0010] Furthermore, the concentration of the polypeptide is 10–500 ng / mL. -1 .

[0011] In some preferred embodiments, different concentrations (0, 1, 10, 50, 100 ng / mL) of TP4 peptide were used to induce bone marrow mesenchymal stem cell differentiation in vitro. It was found that only when the concentration of TP4 peptide was 50 ng / mL did it have the best effect in inducing the formation of mineralized nodules in bone marrow mesenchymal stem cells, even comparable to the positive control. The formation of mineralized nodules indicates that the cells are in the process of osteogenic differentiation. In other words, TP4 peptide can induce bone differentiation and then be used for bone defect repair. Preferably, the optimal concentration is 50 ng / mL. -1 .

[0012] On the other hand, the present invention provides a bone defect repair product, the product comprising the aforementioned polypeptide.

[0013] Furthermore, the concentration of the polypeptide in the repair product is 10 ng / mL. -1 ~50mg / mL -1 .

[0014] It is important to understand that the optimization of TP4 peptide concentration in this invention is based on in vitro cell experiments. However, if the peptide is applied to a specific product, the concentration used will be much higher, generally at the mg level. For example, the optimal concentration of BMP-2 in in vitro cell culture is 50-200 ng / mL, but its concentration in the product is 2-12 mg / mL. This is because the product is diluted and dispersed to other parts of the body in the in vivo fluid environment, and the active ingredient is consumed to varying degrees during this process. Therefore, the concentration of the active ingredient (TP4 peptide) in the product preparation is much higher than that applied directly to cells. Furthermore, the inventors have experimentally demonstrated that a concentration of 10 ng / mL is optimal. -1 ~50mg / mL -1 TP4 peptides all have certain bone-inducing effects. Therefore, the content of TP4 peptides in the product can be adjusted according to the characteristics and needs of the product. The product is also within the protection scope of this invention.

[0015] Furthermore, the bone defect repair product includes, but is not limited to, any one or more of the following: human bone repair materials (muscle), animal bone repair materials, artificial bone repair materials, bone defect repair hydrogels, collagen membranes, 3D printed scaffolds, metallic implants, and collagen membranes.

[0016] This invention constructed three types of bone defect repair scaffolds containing different inducing factors (TP4 peptide, BMP-2 factor, or TGF-β3 factor), and placed these three scaffolds in a rat model of extreme skull bone defects. Micro-CT results showed that mineralized tissue formation was observed in male SD rats with extreme skull bone defects after 4 weeks of treatment with TP4, BMP-2, and TGF-β3 factors, with TP4 peptide showing the best therapeutic effect.

[0017] On the other hand, the present invention provides the use of a polypeptide for preparing a product that induces bone differentiation, wherein the amino acid residue sequence of the polypeptide contains the sequence GPGGDRQDLGWKWVHEPKGYY (SEQ ID NO:5), and the polypeptide can only induce bone tissue production but cannot induce cartilage.

[0018] It should be understood that the product refers to all substances containing TP4 peptides, and the form of the product includes, but is not limited to, solid, semi-solid, liquid or gas.

[0019] Solid dosage forms include powders, granules, tablets, capsules, and pills. These dosage forms have good stability, accurate dosage, and are easy to carry and transport. Semi-solid dosage forms include creams, suppositories, and gels. These dosage forms are generally used for local tissue treatment and have good patient compliance. Liquid dosage forms include solutions, injections, and suspensions. These dosage forms have a faster onset of action and are easy to take. Gaseous dosage forms include aerosols and sprays. These dosage forms deliver drugs through special drug delivery devices, have a rapid onset of action, and high bioavailability.

[0020] Furthermore, the concentration of the polypeptide is 10 ng / mL. -1 ~50mg / mL -1 .

[0021] On the other hand, the present invention provides the use of a polypeptide in the preparation of bone defect repair products, wherein the amino acid sequence of the polypeptide contains the sequence GPGGDRQDLGWKWVHEPKGYY (SEQ ID NO:5), and the polypeptide can only induce bone tissue production but cannot induce cartilage.

[0022] Furthermore, the generated bone tissue comprises one or more of osteoid, woven bone, active osteoblasts, and blood vessels.

[0023] This invention involved staining sections of rat skulls with bone defects after treatment with different factors. The results showed that the newly formed bone tissue in the TP4 group contained osteoid, woven bone, active osteoblasts, and blood vessels, but no new cartilage tissue formation was detected. Conversely, the TGF-β3 group produced well-structured cartilage tissue, including the interface between hypertrophic cartilage and immature woven bone. In conclusion, the TP4 peptide can be used to induce bone differentiation but not cartilage.

[0024] On the other hand, the present invention provides the use of a polypeptide in the preparation of a product that promotes bone mineralization, wherein the amino acid sequence of the polypeptide is GPGGDRQDLGWKWVHEPKGYY, and the polypeptide can only induce bone formation but not cartilage formation.

[0025] On the other hand, the present invention provides the use of a polypeptide for preparing a product that determines the direction of bone differentiation, wherein the amino acid residue sequence of the polypeptide is TILYYVGRT, and the direction of bone differentiation includes differentiation into cartilage or differentiation into bone.

[0026] Generally, polypeptides containing the TILYYVGRT amino acid residue sequence (TP1-3, TP5, TP7-9) can induce cartilage differentiation, and conversely, induce bone formation.

[0027] On the other hand, the present invention provides the use of a polypeptide in a product for regulating the efficiency of stem cell differentiation into cartilage, characterized in that the amino acid residue sequence of the polypeptide is GPGGDRQDLGWK.

[0028] Under normal circumstances, the GPGGDRQDLGWK amino acid residue sequence inhibits stem cell differentiation into cartilage.

[0029] The beneficial effects of this invention include:

[0030] 1. The TGF-β3-derived TP4 peptide of the present invention can overcome various drawbacks of conventional protein growth factor use, such as low yield, high cost and potential immunogenicity; as a promising alternative, the peptide can be chemically synthesized, thereby achieving higher yield, lower cost and immunogenicity.

[0031] 2. The TP4 peptide is based on the transforming growth factor TGF-β3 found in primate tests, and therefore has high bone-inducing activity.

[0032] 3. This invention demonstrates that TP4 peptide can induce bone formation both in vivo and in vitro, providing a new approach for the subsequent construction of ultra-efficient osteogenic peptides, namely, structural optimization based on TP4 peptide;

[0033] 4. The effect of a peptide derived from TGF-β3 on osteogenic differentiation of bone marrow mesenchymal stem cells was investigated, and a method for promoting bone repair using this peptide was proposed to achieve the repair of bone defects.

[0034] 5. The present invention provides a group of bioactive agents containing TP4 polypeptides, which can not only functionalize the biomaterials of bone tissue engineering, but also induce bone regeneration in vitro and in vivo.

[0035] 6. The provided bone-inducing active factor TP4 may be used in clinical treatment, providing better treatment options for patients with bone defects. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 Schematic diagram of some functional regions of TGF-β3 inducible factor;

[0038] Figure 2: In vivo staining images of erector spinae muscle sections from rats treated with different factors (BMP2 and TP peptides); the meanings of the symbols in the figures are: B (bone); BM (bone marrow); M (muscle); C (cartilage); ACS (absorbable collagen sponge); F (fibrous tissue); the scale bar in the figures is 500 μm;

[0039] Figure 3 Alizarin Red staining results of mBMSCs treated with different concentrations of TP4 peptide, with BMP2 as a positive control at a concentration of 50 ng / mL; T1, T10, T50, and T100 represent mBMSC cells treated with 1, 10, 50, and 100 ng / mL of TP4 peptide, respectively.

[0040] Figure 4 Skull scans of SD rats with bone regeneration induced by different factors (TP4, BMP2, TGF-β3); gray areas represent healed bone regions, and black shading represents defect sites.

[0041] Figure 5 Stained skull sections of SD rats induced by different factors (TP4, BMP2, TGF-β3) in bone regeneration; Figure A shows the stained skull section of SD rats induced by TP4 in bone regeneration; Figure B shows the stained skull section of SD rats induced by BMP2 in bone regeneration; Figure C shows the stained skull section of SD rats induced by TGF-β3 in bone regeneration; The meanings of the labels in the figures are: B (bone); BM (bone marrow); M (muscle); C (cartilage); The scale bar in the figures is 100 μm. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0043] Example 1: In vivo screening of TGF-β3-derived peptides that can induce bone differentiation

[0044] 1.1 Design of derived peptides based on TGF-β3 inducible factor

[0045] Based on the crystallographic data of the structure of TGF-β3 itself and the key binding domain of its type II receptor, this study analyzed the crystallographic data of key functional domains of TGF-β3 and its type II receptor, determining that the high-affinity binding regions of TGF-β3 and its receptor are α1, β2, β3, β4, β7, and β8. Figure 1 [1]. Based on the protein structure orientation, different arrangements and combinations of functional regions were explored, the types and numbers of amino acids between these regions were adjusted, the sequences of functional regions were simplified, and 10 TGF-β3-derived peptides were designed (Table 1). Among them, the TP0 (SEQ ID NO:1) peptide retained the relatively complete sequence of the functional region, and also added amino acid residues between different functional regions according to experience, so that the functional regions were fully exposed and better bound to the receptor. Compared with the TP0 peptide, the core sequences of the functional regions of the other TP peptides (TP2-TP9) were simplified, such as the deletion of KIEQ, YIDF and A amino acid residues, and were mainly used to explore the functions of different combinations of functional regions. The peptides were produced by Hangzhou Huibo Biotechnology Co., Ltd. using solid-phase peptide synthesis technology, and the purity was 95%.

[0046] Table 1. Amino acid residue sequences of TGF-β3 derived peptides

[0047]

[0048]

[0049] 1.2 Preparation of a rat model of ectopic osteogenic erector spinae muscle

[0050] The animal experiments were approved by the Animal Experiment Ethics Review Committee of Guangzhou Medical University (Acceptance No.: GD2019-116, Date: June 10, 2019). The scaffold material used in the animal experiments was an oral repair membrane, which was fabricated into circular sheets approximately 5 mm in diameter using a 5 mm punch. The following groups were designed for in vivo studies, with four replicates per group:

[0051] (A) Repair membrane + 2μg BMP2 factor (BMP2 group)

[0052] (B~L) Repair membrane + 2μg TP peptide (TP group); dissolve TP peptide in enzyme-free water to prepare a concentration of 100ng / μL, and add 20μL TP4 peptide solution to each scaffold.

[0053] Draw a 1% sodium pentobarbital saline solution into a 5mL syringe, with a dosage calculated at 50mg / kg body weight. Each rat receives approximately 2.5mL of the anesthetic via intraperitoneal injection. The rats will enter anesthesia within 5-10 minutes, exhibiting sluggish corneal reflexes and generalized muscle relaxation. Shave the back hair of the anesthetized Sprague-Dawley (SD) rats using an electric shaver. Disinfect the surgical area twice with povidone-iodine solution and cover with sterile drapes. Perform an incision 0.5-2cm from the left side of the midline of the back. A longitudinal incision of approximately 1.5 cm was made, extending 1.5-2 cm laterally. A small hole was made in the left erector spinae muscle using ophthalmic scissors, and the muscles on both sides were bluntly dissected. When the incision was approximately 2 mL, the corresponding sample materials (BMP2 group and TP polypeptide group) were implanted. The muscle fascia, deep fascia, and skin were sutured layer by layer. The implantation site was marked with epidermal sutures, and an earring was used for marking at the ear root. Postoperatively, 80,000 U / day of penicillin solution was injected intramuscularly to prevent infection. The rats were kept warm and observed for half an hour. After recovery, each SD rat was marked according to its group and fed routinely in separate cages. For three days postoperatively, penicillin solution was injected intramuscularly daily, and the vital signs, activity level, and surgical incision site were observed daily for one week. Any signs of infection were treated promptly.

[0054] Postoperatively, the rats were given an intramuscular injection of 80,000 U / d penicillin solution to fight infection. They were kept warm and observed for half an hour. After waking up, they were put back into their cages. For one week after surgery, the vital signs and activity status of the SD rats and the surgical incision were observed daily for any signs of infection. If any signs of infection were found, they were treated promptly. Four weeks after surgery, the SD rats were observed, and samples were taken for relevant tests.

[0055] 1.3. Histological observation

[0056] Four weeks after implantation of the sample material, rats were anesthetized with 2% sodium pentobarbital and euthanized by cervical dislocation, with the skull intact removed. Tissue from the implantation site was harvested. The tissue was fixed in 4% paraformaldehyde for 36 hours, followed by decalcification in 10% EDTA (pH 7.4) for 30 days. After dehydration using an automated vacuum tissue dehydrator in the pathology department, the specimens were paraffin-embedded. SD rat sections were 4 μm thick, dewaxed, and then stained simultaneously with hematoxylin (H3136, Sigma-Aldrich, USA), eosin (HE) (TMS-009, Sigma-Aldrich, USA), and Masson's trichrome stain (HT15-1KT, Sigma-Aldrich, USA). New bone formation and healing of bone defects were observed under an optical microscope. The staining method, known as Masson's trichrome staining, is a classic technique in connective tissue staining, designed to effectively visualize fibrous structures (collagen fibers and muscle fibers) within the tissue. This method uses different staining agents to distinguish tissue components, making muscle fibers appear red after staining, while collagen fibers appear green or blue. Compared to conventional HE staining, Masson staining provides a more detailed visualization of tissue structures, especially when observing lesions, and can more accurately capture changes in collagen fibers. This classic staining technique has wide applications in pathological research and histological analysis, providing important information for studying tissue fibrosis and other related fields.

[0057] from Figure 2 The stained sections showed that only the BMP-2 and TP4 groups had newly formed bone tissue containing osteoid, woven bone, active osteoblasts, and blood vessels, but no new cartilage tissue was detected. In contrast, the other TP polypeptide groups produced well-structured cartilage tissue, including the interface between hypertrophic cartilage and immature woven bone. The staining results of TP8 polypeptide were the most representative, showing vacuolated cartilage lacunae in the sections, with the cartilage capsule matrix around the chondrocytes rich in purple glycosaminoglycans and other pink interstitial matrix. The sections of the TGF-β3 group showed a transformation process from cartilage tissue to bone tissue.

[0058] Because TP4 and TP8 exhibit more pronounced induction effects, and because the amino acid residue sequence in the functional region of the TP0 peptide is the most complete (retaining KIEQ, YIDF, and A amino acid residues), the TP0 peptide can be used as a control and compared with TP4 and TP8 respectively. The function of TP0 is to induce chondrogenesis. When the 5' end of the TP0 peptide lacks the amino acid residues TILYYVGRT (i.e., the TP4 peptide, the specific sequence of which is shown in SEQ ID NO:5 in the sequence listing), it can only induce bone differentiation. This indicates that the TILYYVGRT amino acid residue sequence acts as a "switch" between chondrogenesis and bone differentiation. When the TP0 peptide lacks the middle GPGGDRQDLGWK amino acid residues (i.e., the TP8 peptide), its effect in inducing chondrogenesis is greatly increased. Therefore, this segment of amino acid residues inhibits the process of stem cells differentiating into chondrogenesis.

[0059] In summary, only TP4 peptide can be used to induce bone differentiation, and its effect is comparable to that of BMP-2 factor.

[0060] Example 2: In vitro experiments verify that peptide TP4 has osteoinductive properties.

[0061] 2.1 Resuscitation and Culture of Mouse Bone Marrow Mesenchymal Cells

[0062] Preheat the water bath to 37°C. Prepare high-glucose DMEM (Dulbecco's modified Eagle) complete medium (Gibco, Thermo Fisher Scientific) containing 10% FBS and 1% penicillin and streptomycin (both purchased from Sigma-Aldrich). Scientific, USA) placed the cells in a water bath to 37°C, and transferred 9 mL of preheated complete culture medium to a 15 mL centrifuge tube. Mouse bone marrow mesenchymal stem cells (mBMSCs) (purchased from Cyagen Biosciences, China) were removed from liquid nitrogen and immediately placed in a -80°C freezer to allow the liquid nitrogen to evaporate. After 2-3 minutes at -80°C, the frozen cells were removed, and the cryovial was quickly placed in 37°C warm water, rapidly agitated to thaw the contents, and carefully observed. Once completely thawed, the cryovial was removed (Note: avoid submerging the cap in water to reduce the risk of contamination; the cell thawing process should be completed quickly, as prolonged thawing will result in poor cell viability after thawing). The outer wall of the cryovial opening was then wiped with 75% alcohol for disinfection. The cryovial was opened in a clean bench, and the cell suspension was transferred using a pipette to a 9 mL centrifuge tube. In a 15mL centrifuge tube, carefully avoid generating air bubbles. To minimize cell loss, add 1mL of complete culture medium to the cryovial, gently pipette, and collect the cells into the centrifuge tube. Centrifuge at 250×g (1,134rpm) for 5 minutes. After centrifugation, remove the supernatant and gently mix the cell pellet with 1-2mL of preheated 37°C complete culture medium. Seed the cells into a T25 culture flask (Corning, USA) or a culture dish with a similar bottom area, and add sufficient complete culture medium. Gently shake the cell culture dish to distribute the cells evenly and place it in an incubator at 37°C, 5% CO2, and saturated humidity for culture and recovery. Replace the complete culture medium with fresh medium every other day until the cells reach 80%-90% confluence. Two days after recovery, replace the recovered cells with fresh, preheated 37°C complete culture medium until the cells reach 80%-90% confluence, and then perform digestion and passage.

[0063] 2.2 Cell passage

[0064] Preheat the complete culture medium, 1×PBS (Gibco, Thermo Fisher Scientific, USA), and 0.25% Trysin-0.04% EDTA (Gibco, Thermo Fisher Scientific, USA) to 37°C. Aspirate the culture medium and wash the cells 2-3 times with 1×PBS buffer (approximately 3 mL of PBS for T25 flasks and approximately 6 mL for T75 flasks), being careful not to damage adherent cells. Aspirate the 1×PBS buffer and add 0.25% Trysin-0.04% EDTA solution (approximately 1 mL of EDTA solution for T25 flasks and approximately 2-3 mL for T75 flasks). Gently rotate the container to coat the cell surface with Trysin-EDTA. After digestion, observe under a microscope that approximately 70%-80% of the cells have become rounded. Gently tap the walls of the culture dish to loosen the cells. Cell detachment: Once obvious cell detachment is observed, immediately add preheated complete culture medium (approximately 3 mL for T25 flasks and approximately 6 mL for T75 flasks) to stop digestion. Use a pipette to aspirate the liquid and repeatedly pipette down the bottom of the culture dish to completely detach the cells. Avoid vigorous pipetting to prevent air bubbles. Transfer the cell suspension to a new 15 mL centrifuge tube, wash the bottom with 1×PBS buffer, collect the wash solution, and centrifuge at 250×g for 5 min. Carefully discard the supernatant, add 2 mL of complete culture medium to resuspend the cells (avoiding vigorous pipetting to prevent air bubbles). Then, perform trypan blue staining to count the number of viable cells at a ratio of 2.5–4.0 × 10⁶ cells / mL. 4 live cells / cm 2 Seed cells at the appropriate density; add an appropriate amount of complete culture medium, gently shake the cell culture dish to distribute the cells evenly, and place the cells in an incubator at 37°C, 5% CO2, and saturated humidity for culture.

[0065] 2.3 Preparation of Osteogenesis Inducing Solution (OM)

[0066] Preparation of sodium β-glycerophosphate stock solution: Dissolve 10g of sodium β-glycerophosphate (thermofisher, USA) in 46.3mL of PBS, filter through a 0.22μm sterile filter (Corning, USA) to prepare 1M sodium β-glycerophosphate stock solution, aliquot into sterile 1.5mL centrifuge tubes, and store at -20℃.

[0067] Preparation of ascorbic acid stock solution: Dissolve 100 mg ascorbic acid (Thermofisher, USA) in 20 mL PBS, filter with a 0.22 μm sterile filter to prepare 5 mg / mL ascorbic acid stock solution, aliquot into sterile 1.5 mL centrifuge tubes. It is recommended to prepare and use immediately, and store at -20℃ for a short period of time.

[0068] Preparation of osteogenic induction solution: Add 1 μL of 1 mM dexamethasone (A13449, Thermofisher, USA), 1 mL of 1 M β-glycerophosphate stock solution, and 1 mL of 5 mg / mL ascorbic acid stock solution to 100 mL of DMEM (Gibco, UK) culture medium containing 10% fetal bovine serum (Gibco, UK) and 1% penicillin and antibiotics.

[0069] 2.4 TP4 treatment of mBMSCs in osteopathic induction

[0070] mBMSCs were seeded in 48-well culture plates (2.5 × 10⁻⁶). 4 Osteogenic differentiation was induced in vitro by adding osteogenic induction solution to cells / wells every other day. At the same time, different concentrations (0, 1, 10, 50, 100 ng / mL) of TP4 polypeptide (SEQ ID NO:5) or 50 ng / mL of BMP-2 factor were added to the osteogenic induction solution for subsequent analysis of osteogenic differentiation potential. The BMP-2 factor was used as a positive control.

[0071] 2.5 Alizarin Red Staining and Quantitative Analysis

[0072] Alizarin red is an anionic dye that readily complexes with various metal ions to form red complexes. Alizarin red staining is an authoritative and classic technique designed to analyze orange-red calcium deposition in fixed cell samples by chelating calcium ions with alizarin red to form a complex. This method is primarily applicable to the detection of calcium deposition and calcified nodules in primary or cultured animal cells. Alizarin red staining results are an important indicator for detecting osteogenic differentiation in cells; the more red material present, the higher the degree of ossification. This embodiment set up two experimental groups: different concentrations (0, 1, 10, 50, 100 ng / mL) of TP4 peptide (SEQ ID NO: 5) and BMP-2 factor; on day 10, the formation of mineralized nodules in mBMSC culture was determined by alizarin red staining. The culture was washed three times in phosphate-buffered saline (PBS, pH=7.4), fixed in 95% ethanol (or 4% paraformaldehyde) for 30 min, and then stained with 0.1% alizarin red (pH=4.2, Sigma-Aldrich) at room temperature for 5-10 min. The images were observed and recorded using a stereomicroscope (Leica, Germany). Figure 3 ).

[0073] Depend on Figure 3It was found that when the concentration of TP4 peptide was 1, 10, or 100 ng / mL, its bone-inducing effect was poor, resulting in fewer mineralized nodules. However, when the concentration of TP4 was 50 ng / mL, its bone-inducing effect was significant and comparable to that of the positive control BMP-2. In conclusion, a concentration of 50 ng / mL of TP4 peptide showed the best therapeutic effect in treating bone defects.

[0074] Meanwhile, the inventors also used a concentration of 100 ng / mL. -1 ~50mg / mL -1 The TP4 peptide, when used to treat mBMSCs, has a certain effect on inducing mineralized nodules.

[0075] Example 3: In vivo experiments verify that peptide TP4 has osteoinductive properties.

[0076] 3.1 Establishment of a rat model of extreme skull bone defects

[0077] All animal experiments were approved by the Animal Experiment Ethics Review Committee of Guangzhou Medical University (Acceptance No.: GD2019-648, Date: 2019.02.22). The oral repair membrane was used as a scaffold material, and the scaffold was fabricated into a circular sheet with a diameter of approximately 5 mm using a 5 mm punch. The following groups were designed for in vivo studies, with four replicates per group:

[0078] (I) Membrane repair only (control group)

[0079] (II) Repair membrane + 2 μg TP4 peptide (TP4 group); dissolve TP4 peptide in enzyme-free water to prepare a concentration of 100 ng / μL, and add 20 μL of TP4 solution to each scaffold.

[0080] (III) Repair membrane + 2μg BMP2 factor (BMP2 group)

[0081] (IV) Repair membrane + 2μg TGF-β3 factor (TGF-β3 group)

[0082] A 1% sodium pentobarbital saline solution was drawn into a 5mL syringe, with a dosage calculated at 50mg / kg body weight. Approximately 2.5mL was injected per rat via intraperitoneal injection. Within 5-10 minutes, the rats entered anesthesia, characterized by sluggish corneal reflexes and generalized muscle relaxation. The hair on the top of the head of the anesthetized Sprague-Dawley (SD) rats was shaved off with an electric shaver. The surgical area was then disinfected twice with povidone-iodine, and 1% lidocaine hydrochloride was injected into the skin for local anesthesia. After disinfection and anesthesia, a 1.5-2cm incision was made along the midline of the skull above the operculum. The epidermis, subcutaneous fascia, and periosteum were incised until the lambdoid suture was visible. Using a curette, the skull was carefully dissected close to the bone surface to fully expose the area requiring modeling. The exposed area extended no further than the line connecting the eyes anteriorly, 3-5mm posterior to the lambdoid suture, and approximately 8mm lateral to the midline of the skull on both sides. Skull defects were drilled on both sides of the midline of the rat skull using a 5mm diameter skull drill. A high-speed drill was used initially to create a circular groove in the skull, after which the speed was reduced. Care was taken to be gentle and slow to avoid damaging the meninges, which could affect the modeling effect. Furthermore, the soft tissue should be fully separated and the surgical field exposed before drilling; otherwise, the soft tissue will affect the normal operation of the drill and damage to the soft tissue at the top of the skull, leading to modeling failure. After the skull defect model was created, the corresponding sample materials (blank group, TP4 group, BMP2 group, TGF-β3 group) were implanted into the appropriate groups, and the periosteum and skin were sutured. Postoperatively, penicillin was injected intramuscularly for infection control, and the rats were kept warm and observed for half an hour. After recovery, they were returned to their cages. For one week postoperatively, the vital signs and activity levels of the SD rats were observed daily, and the surgical incision was checked for infection. If infection was observed, it was treated promptly. Four weeks postoperatively, the SD rats were observed, and relevant tests were performed.

[0083] 3.2 Analysis of Bone Regeneration and Bone Defect Healing

[0084] Micro-CT (micro computed tomography), also known as micro-CT, is a non-destructive 3D imaging technique. Its biggest difference from conventional clinical CT lies in its extremely high resolution, reaching the micrometer (μm) level. Four weeks after implantation of the sample material, rats were anesthetized with 2% sodium pentobarbital and euthanized by cervical dislocation, allowing for the complete removal of the rat skull. Specimens were scanned using a SkyScan1172 (Bruker-Micro-CT from Kontich, Belgium). Digital retargeting was performed using SkyScan DataViewer software for each sample scan, with a voxel resolution of 10 μm and 500 slices for the volume of interest. The images were reconstructed using 3D Creator software. Figure 4 ).

[0085] After scanning, histological analysis of the samples was performed. The specimens were soaked in 4% paraformaldehyde for one week, and then decalcified in 10% EDTA (pH=7.4) for 20 days. After dehydration in the pathology department, the specimens were embedded in paraffin. Sections were prepared starting from the central area of ​​the defect, with a thickness of 4 μm. After dewaxing, the sections were stained with hematoxylin (Sigma-Aldrich, USA), eosin (HE) staining (TMS-009, Sigma-Aldrich, USA), and Masson trichrome staining (Sigma-Aldrich, USA). The newly formed bone and the healing of the bone defect were observed under an optical microscope.

[0086] from Figure 4 The micro-CT scan images show that mineralized tissue was formed in the male SD rat skull limit bone defect model after 4 weeks of treatment with TP4, BMP-2, and TGF-β3 factors. Among them, TP4 peptide showed the best therapeutic effect. It is worth noting that although TP4 peptide is derived from TGF-β3 protein, its therapeutic effect is more similar to that of BMP2 factor. This may be because TP4 peptide can bind to the BMP2 protein receptor, thereby stimulating the downstream cascade reaction of the BMP2 protein receptor.

[0087] from Figure 5 The stained sections show that the BMP-2 group ( Figure 5 B) and TP4 group ( Figure 5 In group A), newly formed bone tissue included osteoid, woven bone, active osteoblasts, and blood vessels, but no new cartilage tissue formation was detected. In contrast, the TGF-β3 group produced well-structured cartilage tissue, including the interface between hypertrophic cartilage and immature woven bone. In summary, TP4 peptides can be used to induce bone differentiation and subsequently treat bone defects, with effects comparable to BMP-2 but differing from TGF-β3.

[0088] Meanwhile, the inventors also used a concentration of 10 ng / mL. -1 ~50mg / mL -1 The TP4 peptide showed a certain therapeutic effect when used to treat a rat model of extreme skull bone defects.

[0089] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

[0090] [1]Hart PJ,Deep S,Taylor AB,Shu Z,Hinck CS,Hinck AP.Crystal structureof the human TbetaR2 ectodomain--TGF-beta3 complex.Nat Struct Biol.2002 Mar;9(3):203-8.doi:10.1038 / nsb766.PMID:11850637.

Claims

1. A polypeptide having osteoinductive activity, characterized in that, The amino acid sequence of the polypeptide is GPGGDRQDLGWKWVHEPKGYY.

2. A bone defect repair product, characterized in that, The product comprises the polypeptide as described in claim 1.

3. The product of claim 2, wherein, The concentration of the polypeptide is 100 ng / mL. −1 ~ 50 mg mL −1 .

4. The product as described in claim 2, characterized in that, The products include any one or more of the following: human-derived bone repair materials, animal-derived bone repair materials, artificial bone repair materials, bone defect repair hydrogels, collagen membranes, 3D printed scaffolds, and metallic implants.

5. The use of a polypeptide in the preparation of a drug for treating bone defects, characterized in that, The amino acid residue sequence of the polypeptide is GPGGDRQDLGWKWVHEPKGYY.

6. Use according to claim 5, characterized in that, concentration of the polypeptide is 100 ng mL −1 50 mg mL −1 .

7. Use according to claim 5, characterized in that, The product can be in any of the following forms: solid, semi-solid, liquid, or gas.

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