Polypeptide and application thereof in bone defect repair

Abstract

The invention provides a polypeptide and application thereof in bone defect repair, and belongs to the technical field of polypeptides. On the basis of a functional structure domain of a TGF-beta3 factor, 10 derived peptides are designed, only one derived peptide with osteoinductivity is screened out, and the derived peptide is composed of 21 amino acid residues; the polypeptide not only can induce mouse-derived bone marrow mesenchymal stem cells to generate mineralized nodules in vitro, but also can induce new bone formation in a rat skull limit bone defect model (in vivo) and in rat muscles (an ectopic osteogenesis model), and the osteogenesis induction efficiency is equivalent to that of BMP-2. In conclusion, the polypeptide provided by the invention has osteoinductivity and has a huge application prospect in the aspect of bone defect repair.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and in particular to a polypeptide and its application in bone defect repair. Technical Background

[0002] Bone defect is one of the most common clinical diseases, caused by congenital bone dysplasia or developmental malformation as well as tumors, inflammation, trauma and other reasons. The repair of large volume bone defect is one of the major problems that have not yet been overcome in medicine. Autologous bone grafts have good osteogenic potential and are therefore considered the gold standard for the treatment of bone defects, but their clinical application is subject to many limitations, such as the need for a second surgical field, prolonged surgery time, and pain in the bone donor area. In addition, allogeneic bone, xenogeneic bone transplants and most synthetic materials used clinically lack osteoinductivity and cannot achieve complete bone tissue repair, so they cannot be used alone to repair critical volume bone tissue defects. Therefore, giving medical materials efficient osteoinductivity through various active factors is the key to repairing large volume bone defects.

[0003] The active factors with osteoinduction discovered so far 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, which play an important role in regulating embryonic development and tissue patterning, postnatal tissue induction and morphogenesis, immunity and fibrosis, and initiating tissue and organ morphogenesis including skeleton and bone matrix. However, the synthesis of protein growth factors mainly relies on genetic engineering technology based on Escherichia coli and mammalian cells, which has low yield, high cost, high purification difficulty, and potential immunogenicity; moreover, protein growth factors have strict requirements on storage conditions and are very sensitive to sterilization methods, which in turn affects their osteoinductive activity. Therefore, the development of new, low-cost, low-immunogenicity, and high-efficiency osteoinductive active factors has become a research hotspot in the field of large-volume bone defect repair.

[0004] At present, the research on osteogenic peptides is mainly focused on BMP peptides. Studies have confirmed that the alkaline phosphatase activity (ALP) of the mouse stem cell pre-osteoblastic index induced by the BMP2 core structure region peptide (sequence 73-92) is significantly higher than that of the non-core structure region (44-58) or (68-87); another study showed that the expression of osteocalcin (OCN), a late osteogenic differentiation index of bone marrow mesenchymal stem cells (BMSC) induced by the BMP2 core structure region peptide (73-92) is significantly higher than that of peptides from other protein sources; the inventors previously modified the peptide structure, such as adding a linker part to increase the peptide and receptor functional binding area, thereby greatly improving the osteogenic efficacy of BMP peptides. However, although researchers are constantly optimizing BMP peptides, the optimization effect is still relatively limited; on the other hand, TGF-β3, as a new molecular strategy, replicates the synergistic induction of bone formation by expressing multiple BMP and TGF-β genes separately but synergistically in molecular mechanism and morphology, and rapidly and substantially induces bone formation in primate models. It is a transforming growth factor found in primate tests today. The Chinese patents "A hydrogel material that promotes cartilage repair, its preparation method and application" (publication (announcement) number: CN114306732B), "A method for inducing directional cartilage differentiation of stem cells" (publication (announcement) number: CN110684725B) and "A TGF-β3 derivative peptide and its use in the preparation of drugs for promoting cartilage repair and / or treating cartilage defect diseases" (publication (announcement) number: CN117720637A) all provide a use of a TGF-β3 protein or its derivative peptide in cartilage repair / induction; among them, the patent "A hydrogel material that promotes cartilage repair, its preparation method and application" "A method for inducing directional chondrogenic differentiation of stem cells" and "A method for inducing directional chondrogenic differentiation of stem cells" do not use derivative peptides of TGF-β3 protein, nor are they direct applications of TGF protein. The former only uses TGF-β3 protein to prepare exosomes, and then mixes the exosomes with BMSC to prepare hydrogels, while the latter uses adenovirus vectors to introduce transforming growth factor TGF-β3 into chondrocytes. The transfected chondrocytes provide continuous, local and overexpressed TGF-β3 factors to stem cells, thereby stimulating the directional chondrogenic differentiation of stem cells. Therefore, it is still unknown whether TGF-β3 protein or its derivative peptides can be used to induce osteogenesis and then treat bone defects. Summary of the invention

[0005] In view of the above problems, the present invention provides a polypeptide and its application in bone defect repair, which belongs to the field of polypeptide technology. Based on the functional domain of TGF-β3 factor, the present invention designed 10 derivative peptides, and only screened out a derivative peptide with osteoinduction, which is composed of 21 amino acid residues; the polypeptide can not only induce mouse-derived bone marrow mesenchymal stem cells to produce mineralized nodules in vitro, but also induce new bone formation in the rat skull extreme bone defect model (in vivo) and in rat muscle (ectopic osteogenesis model), and the osteoinduction efficacy is equivalent to that of BMP-2. In summary, the polypeptide provided by the present invention has osteoinduction and has great application prospects in bone defect repair.

[0006] In one aspect, the present invention provides a TP4 polypeptide having osteoinductive activity, wherein the amino acid sequence of the polypeptide comprises a GPGGDRQDLGWKWVHEPKGYY sequence (SEQ ID NO: 5). The TP4 polypeptide is derived from a transforming growth factor TGF-β3 protein found in primate tests.

[0007] Although both cartilage and bone are composed of cells and extracellular matrix materials, the types of cells that form cartilage and bone are different: the cells that form cartilage are chondrocytes and chondroblasts, while the cells that form bones can be divided into osteocytes, osteoblasts, and osteoclasts; in addition, the extracellular matrix materials that make up cartilage and bone are also different: the matrix materials outside the chondrocytes are mostly gelatinous substances, fibrous substances, etc., while minerals such as calcium and phosphorus constitute most of the extracellular matrix materials of bones. Therefore, it is understandable that the process of cartilage and bone differentiation is regulated by different factors, and even two similar amino acid residue sequences may perform different functions.

[0008] At the same time, a sequence 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] The present invention is based on the structure of TGF-β3 transforming growth factor itself and the crystallographic data of the key binding domain of its type II receptor. By analyzing the crystallographic data of TGF-β3 and its type II receptor key functional domain, the high affinity binding regions of TGF-β3 and its receptor are determined to be α1, β2, β3, β4, β7, and β8. According to the protein structure orientation, by exploring different permutations and combinations of functional regions, adjusting the length of the amino acid sequences between these regions, and streamlining the core sequence of the functional domain, 10 TGF-β3 derivative peptides (TP0-TP9) are designed, and the derivative peptides are applied to the ectopic osteogenesis model of the erector spinae muscle of the back of rats to screen out the derivative peptides with bone induction. In vitro and in vivo experiments show that only TP4 polypeptide can and can only induce bone differentiation, while TP8 can induce cartilage; further, compared with the amino acid residue sequence of TP0 polypeptide (SEQ ID NO: 1), TP4 polypeptide (SEQ ID NO: 5) lacks the amino acid residues TILYYVGRT, which means that the TILYYVGRT amino acid residue sequence is the "switch" for the direction of bone differentiation, that is, the TP polypeptide with this sequence induces stem cells to differentiate into cartilage, and vice versa. Combining the staining results and sequence differences of TP0 polypeptide (SEQ ID NO: 1) and TP8 polypeptide (SEQ ID NO: 9), it can be seen that the GPGGDRQDLGWK amino acid residue sequence significantly inhibits the effect of cartilage induction. In short, TP4 polypeptide (SEQ ID NO: 5) can induce bone formation, but not cartilage formation; while the function of TP8 polypeptide (SEQ ID NO: 9) is the opposite, it cannot induce cells to produce calcification, and then cannot induce bone formation.

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

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

[0012] In another aspect, the present invention provides a bone defect repair product, which comprises the above-mentioned polypeptide.

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

[0014] It should be understood that the optimization of the TP4 polypeptide concentration in the present invention is based on in vitro cell experiments, and if the polypeptide is applied to a specific product, its use concentration will be higher, generally at the mg level. For example, the optimal concentration of BMP-2 in in vitro cultured cells is 50-200 ng / mL, but its concentration in the product is 2-12 mg / mL. This is because the product will be diluted and dissipated to other places in the body fluid environment, and in this process, the active ingredients of the product will be consumed to varying degrees. Therefore, when preparing the product, the concentration of the active ingredient (TP4 polypeptide) is much higher than that directly acting on the cells. In addition, the inventors have proved through experiments that the concentration of 10 ng mL -1 ~50mg mL -1 The TP4 polypeptides have a certain bone-inducing effect, so the content of the TP4 polypeptide in the product can be adjusted according to the characteristics and needs of the product, and the product also falls within the protection scope of the present invention.

[0015] Furthermore, the bone defect repair product includes but is not limited to any one or more of human-derived bone repair material muscle, animal-derived bone repair material, artificial bone repair material, bone defect repair hydrogel, collagen membrane, 3D printed scaffold, metal implant, and collagen membrane.

[0016] The present invention constructs three bone defect repair scaffolds containing different induction factors (TP4 polypeptide or BMP2 factor or TGF-β3 factor), and respectively places the above three scaffolds in the rat skull limit bone defect model. Micro-CT results show that mineralized tissue is formed in the male SD rat skull limit bone defect model after 4 weeks of bone injury treatment with TP4, BMP-2 and TGF-β3 factors, among which TP4 polypeptide has the best therapeutic effect.

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

[0018] It should be understood that the product refers to all substances containing TP4 polypeptide, 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, pills, etc. These dosage forms have good stability, accurate dosage, and are easy to carry and transport; semi-solid dosage forms include creams, suppositories, gels, etc. These dosage forms are generally used for local tissue treatment and have good patient compliance; liquid dosage forms include solutions, injections, suspensions, etc. These dosage forms take effect quickly and are easy to take; gaseous dosage forms include aerosols, sprays, etc. These dosage forms deliver drugs through special drug delivery devices, have a fast 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 a use of a polypeptide for preparing a bone defect repair product, wherein the amino acid sequence of the polypeptide comprises a GPGGDRQDLGWKWVHEPKGYY sequence (SEQ ID NO: 5), and the polypeptide can only induce bone tissue production but not cartilage.

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

[0023] The present invention stained the skull bone defect of rats treated with different factors, and found that the newly generated bone tissue in the TP4 group included osteoid, woven bone, active osteoblasts and blood vessels, but no new cartilage tissue formation was detected; on the contrary, the TGF-β3 group newly generated cartilage tissue with regular structure, i.e., the interface between hypertrophic cartilage and immature woven bone. In summary, the TP4 polypeptide can be used to induce bone differentiation but not cartilage.

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

[0025] On the other hand, the present invention provides a use of a polypeptide for preparing a product for determining 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 speaking, polypeptides containing the TILYYVGRT amino acid residue sequence (TP1-3, TP5, TP7-9) can induce cartilage differentiation and vice versa to induce bone formation.

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

[0028] In general, the GPGGDRQDLGWK amino acid residue sequence inhibits stem cell differentiation into cartilage.

[0029] The beneficial effects of the present invention include:

[0030] 1. The TGF-β3-derived TP4 polypeptide of the present invention can solve various shortcomings in the use of conventional protein growth factors, such as low yield, high cost and potential immunogenicity; as a promising alternative, the polypeptide can be chemically synthesized to have higher yield, lower cost and immunogenicity;

[0031] 2.TP4 peptide is modified based on the transforming growth factor TGF-β3 found in primate tests, so it has higher bone induction activity;

[0032] 3. The present invention proves that TP4 polypeptide can induce bone formation in vivo and in vitro, and provides a new idea for the subsequent construction of ultra-high-efficiency osteoinductive polypeptide, that is, structural optimization based on TP4 polypeptide;

[0033] 4. The effect of peptides derived from TGF-β3 on the osteogenic differentiation of bone marrow mesenchymal stem cells was explored, and a method for using the peptides to promote bone repair was proposed, thereby achieving 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 bone-inducing active factor TP4 provided may be used in clinical treatment to provide better treatment options for patients with bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

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

[0039] Figure 3 : Alizarin red staining results of mBMSCs treated with different concentrations of TP4 peptides, BMP2 was used as a positive control with a concentration of 50 ng / mL; T1, T10, T50 and T100 represent mBMSCs treated with 1, 10, 50 and 100 ng / mL TP4 peptides, respectively;

[0040] Figure 4 :Skull scan of SD rats induced by different factors (TP4, BMP2, TGF-β3) for bone regeneration; the gray part is the healed bone area, and the black shadow is the defect area;

[0041] Figure 5 : Staining images of skull sections of SD rats induced with bone regeneration by different factors (TP4, BMP2, TGF-β3); Figure A is the staining image of skull sections of SD rats induced with bone regeneration by TP4; Figure B is the staining image of skull sections of SD rats induced with BMP2; Figure C is the staining image of skull sections of SD rats induced with TGF-β3; The meanings of the symbols in the figures: B (bone); BM (bone marrow); M (muscle); C (cartilage): cartilage; The scale in the figure is 100μm. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0043] Example 1: In vivo screening of TGF-β3-derived peptides capable of inducing bone differentiation

[0044] 1.1 Design of derivative peptides based on TGF-β3 inducing factor

[0045] Based on the structure of TGF-β3 inducing factor itself and the crystallographic data of the key binding domain of its type II receptor, by analyzing the crystallographic data of TGF-β3 and its type II receptor key functional domain, it was determined that the high affinity binding regions of TGF-β3 and its receptor are α1, β2, β3, β4, β7, β8 ( Figure 1 )[1]. According to 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 derivative peptides were designed (Table 1). Among them, the TP0 (SEQ ID NO: 1) polypeptide retained a relatively complete sequence of the functional region, and also added amino acid residues between different functional regions according to experience, so that the functional region was fully exposed and better bound to the receptor; compared with the TP0 polypeptide, the remaining TP polypeptides (TP2 to TP9) simplified the core sequence of the functional region, such as deleting KIEQ, YIDF and A amino acid residues, and were mainly used to explore the functions of different functional region combinations. The polypeptide was produced by Hangzhou Huibo Biotechnology Co., Ltd. using solid phase peptide synthesis technology with a purity of 95%.

[0046] Table 1 Amino acid residue sequence of TGF-β3 derived peptide

[0047]

[0048]

[0049] 1.2 Preparation of rat dorsal erector spinae muscle ectopic osteogenesis model

[0050] The animal experiment was approved by the Animal Experiment Ethics Review Committee of Guangzhou Medical University (acceptance number: GD2019-116, date: 2019.06.10). The scaffold material used in the animal experiment was oral repair membrane, and the scaffold material was made into a circular sheet with a diameter of about 5 mm using a 5 mm punch. The following groups were designed for in vivo research, with four replicates in each group:

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

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

[0053] A 1% sodium pentobarbital saline solution was drawn with a 5 mL syringe, the dosage was calculated as 50 mg / kg body weight, and the injection volume for each rat was about 2.5 mL, and the anesthetic was injected intraperitoneally; the rat entered an anesthetized state within 5-10 minutes, which was manifested by slow corneal reflex and relaxation of muscles throughout the body; the back hair of the anesthetized Sprague-Dawley (SD) rat was shaved with an electric shaver, and then iodine was applied to the surgical area for disinfection twice and then covered with a sterile towel; a shaving was performed at 0.5-2 cm from the left side of the midline of the back. A longitudinal incision of about 1.5 cm in length was made, and it was extended 1.5-2 cm to both sides. A small hole was made in the erector spinae muscle on the left side with ophthalmic scissors, and the muscles on both sides were bluntly separated. When the hole was about 2 mL, the corresponding sample materials (BMP2 group and TP polypeptide group) were implanted. The myometrium, deep fascia and skin were sutured layer by layer, and the implantation site was marked with epidermal sutures and ear studs were punched at the ear roots for marking. After surgery, 80,000 U / d of penicillin solution was injected intramuscularly to prevent infection, and the rats were kept warm and observed for half an hour. After waking up, each SD rat was marked according to the group and fed routinely in cages. Penicillin solution was injected intramuscularly every day within three days after surgery, and the vital signs, activity status and surgical incision of SD rats were observed every day for one week for infection. If there was infection, it was treated in time.

[0054] After the operation, 80,000U / d of penicillin solution was injected intramuscularly to prevent infection. The rats were kept warm and observed for half an hour. After waking up, they were returned to the cage. The vital signs and activity status of the SD rats and the infection of the surgical incision were observed every day for one week after the operation. If there was any infection, it was treated in time. The SD rats after the operation were observed and samples were collected 4 weeks after the operation for related tests.

[0055] 1.3. Histological observation

[0056] Four weeks after the implantation of the sample material, the rats were anesthetized with 2% sodium pentobarbital and killed by cervical dislocation. The skull of the rats was completely removed; the tissue at the implantation site was cut. The specimens were fixed with 4% paraformaldehyde for 36 hours and then decalcified in 10% EDTA (pH 7.4) for 30 days. After dehydration using a fully automatic vacuum tissue dehydrator in the pathology department, the specimens were embedded in paraffin. The thickness of the slices of SD rats was 4 μm. After dewaxing, the slices were stained with hematoxylin (H3136, Sigma-Aldrich, USA), eosin (HE) (TMS-009, Sigma-Aldrich, USA) and Masson (HT15-1KT, Sigma-Aldrich, USA) at the same time, and the newly formed bone and the healing of the bone defect were observed under an optical microscope. The staining method is called Masson trichrome staining, which is a classic technique in connective tissue staining, aiming to effectively display the fiber structure (collagen fibers and muscle fibers) in the tissue. This method uses different dyes to distinguish tissue components, making muscle fibers appear red after staining, while collagen fibers appear green or blue. Compared with general HE staining, Masson staining provides a more detailed display of tissue structure, especially when observing lesions, it can more accurately capture the changes in collagen fibers. This classic staining technique is widely used in pathological research and histological analysis, and provides important information for studying tissue fibrosis and other aspects.

[0057] from Figure 2 From the section staining images, it can be seen that only the newly generated bone tissues in the BMP-2 group and the TP4 group contained osteoid, woven bone, active osteoblasts and blood vessels, but no new cartilage tissue formation was detected; on the contrary, the other TP polypeptide groups newly generated cartilage tissue with regular structure, that is, the interface between hypertrophic cartilage and immature woven bone, among which the staining result of TP8 polypeptide was the most representative, that is, vacuolar cartilage lacunas were visible in the sections, and the cartilage capsule matrix around the chondrocytes was rich in purple glycosaminoglycans and the rest of the pink interval matrix; while the sections of the TGF-β3 group showed the process of transformation from cartilage tissue to bone tissue.

[0058] Because the induction effect of TP4 and TP8 is more prominent, and because the amino acid residue sequence in the functional region of the TP0 polypeptide is the most complete, that is, the KIEQ, YIDF and A amino acid residues are retained, the TP0 polypeptide can be used as a control and compared with TP4 and TP8 respectively. The function of TP0 is to induce cartilage differentiation, and when the 5' end of the TP0 polypeptide lacks the TILYYVGRT amino acid residues (i.e., the TP4 polypeptide, the specific sequence is shown in the sequence table SEQ ID NO: 5), it can only induce bone differentiation. It can be seen that the TILYYVGRT amino acid residue sequence is the "switch" for cartilage and bone differentiation; when the TP0 polypeptide lacks the middle GPGGDRQDLGWK amino acid residue (i.e., the TP8 polypeptide), its effect of inducing cartilage differentiation is greatly increased, so this segment of amino acid residues inhibits the process of stem cells differentiating into cartilage.

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

[0060] Example 2: In vitro experiments confirm that the polypeptide TP4 has osteoinductivity

[0061] 2.1 Recovery and culture of mouse bone marrow mesenchymal cells

[0062] Preheat a water bath to 37°C, place high-glucose DMEM (Dulbecco's modified Eagle) complete medium (Gibco, ThermoFisher Scientific, USA) containing 10% FBS and 1% double antibodies (penicillin and streptomycin, both purchased from Sigma-Aldrich) in a water bath and warm to 37°C, and take 9 mL of the preheated complete medium into a 15 mL centrifuge tube; take out the frozen mouse bone marrow mesenchymal stem cells (mBMSC) (purchased from Cyagen Biosciences, China) from liquid nitrogen and immediately put them into a -80°C refrigerator to allow the liquid nitrogen entering the cryopreservation tube to evaporate; after being placed at -80°C for 2 to 3 minutes, take out the frozen cells, quickly put the cryopreservation tube into 37°C warm water, shake quickly to melt the contents in the tube as soon as possible, and observe carefully, and take out the cryopreservation tube after the contents are completely melted (Note: Try to avoid water covering the tube cap to reduce the risk of contamination; the cell recovery process should be completed quickly, and the thawing process time is too long, which will result in poor cell activity after recovery. ), and then wipe the outer wall of the cryopreservation tube with 75% alcohol; open the cryopreservation tube in the clean bench, and use a pipette to transfer the cell cryopreservation suspension to a 15mL centrifuge tube containing 9mL complete culture medium, and pay attention to avoid bubbles as much as possible; in order to reduce cell loss, add 1mL of complete culture medium to the cryopreservation tube, blow it slightly, collect it in a centrifuge tube, and centrifuge it at 250×g (1,134rpm) for 5min; after centrifugation, remove the supernatant, and gently mix the cell pellet with 1-2mL of complete culture medium preheated to 37℃; inoculate the cells into a T25 culture flask (Corning, USA) or a culture vessel with equivalent bottom area, and add sufficient complete culture medium; gently shake the cell culture vessel to evenly distribute the cells, and place it at 37℃, 5% CO 2 , culture and resuscitate in a saturated humidity incubator, wherein fresh complete medium is replaced every other day until the cells reach 80% to 90% confluence; 2 days after resuscitation, the revived cells are replaced with fresh complete medium preheated to 37°C until the cells reach 80% to 90% confluence, and then digested and passaged.

[0063] 2.2 Cell passaging

[0064] Complete culture medium, 1×PBS (Gibco, Thermo Fisher Scientific, USA), and 0.25% Trypsin-0.04% EDTA (Gibco, Thermo Fisher Scientific, USA) were preheated to 37°C; the culture medium was aspirated, and the cells were washed 2 to 3 times with 1×PBS buffer (about 3 mL of PBS was added to the T25 culture flask and about 6 mL was added to the T75 culture flask), taking care not to damage the attached cells; the 1×PBS buffer was aspirated, and 0.25% Trypsin-0.04% EDTA solution was added (about 1 mL of the EDTA solution was added to the T25 culture flask and about 2 to 3 mL of EDTA solution was added to the T75 culture flask), and the cells were gently rotated to cover the Trypsin-EDTA surface and digested. When about 70% to 80% of the cells were observed to be rounded under a microscope, the wall of the culture vessel was gently tapped by hand to make the cells round. Debonding; when obvious cells are seen falling off, immediately add preheated complete culture medium (about 3 mL for T25 culture flask and about 6 mL for T75 culture flask) to terminate digestion; use a pipette to absorb the liquid, repeatedly blow the bottom wall of the culture vessel to completely detach the cells from the bottom wall of the vessel. Do not blow too violently and try to avoid bubbles; transfer the cell suspension to a new 15 mL centrifuge tube, wash the bottom wall with 1× PBS buffer, collect the washing liquid into the centrifuge tube, and centrifuge at 250×g for 5 minutes; carefully discard the supernatant, add 2 mL of complete culture medium to resuspend the cells, do not blow too violently and try to avoid bubbles, and then perform trypan blue staining on the cells to count the number of live cells, according to 2.5-4.0×10 4 viable cells / cm 2 Inoculate cells at a density of 100 μl; add an appropriate amount of complete culture medium, gently shake the cell culture vessel to evenly distribute the cells, and place the cells in a 37°C, 5% CO 2 , cultured in an incubator with saturated humidity.

[0065] 2.3 Preparation of osteogenic induction medium (OM)

[0066] Preparation of β-sodium glycerophosphate stock solution: 10 g of β-sodium glycerophosphate (thermofisher, USA) was dissolved in 46.3 mL of PBS, filtered through a 0.22 μm sterile filter (Corning, USA) to prepare 1 M β-sodium glycerophosphate stock solution, dispensed into sterile 1.5 mL centrifuge tubes, and stored at -20°C.

[0067] Preparation of ascorbic acid stock solution: 100 mg ascorbic acid (thermofisher, USA) was dissolved in 20 mL PBS, filtered through a 0.22 μm sterile filter to prepare 5 mg / mL ascorbic acid stock solution, and dispensed into sterile 1.5 mL centrifuge tubes. It is recommended to prepare and use immediately and store at -20°C for a short term.

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

[0069] 2.4TP4 treatment of mBMSCs during osteogenic induction

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

[0071] 2.5 Alizarin red staining and quantitative analysis

[0072] Alizarin red is an anionic dye that can easily complex with a variety of metal ions to form red complexes. Alizarin red staining is an authoritative and classic technical method that aims to analyze the orange-red calcium deposition phenomenon in fixed cell samples by using the chelation principle to produce a complex between calcium ions and alizarin red. This method is mainly suitable for the detection of calcium deposition and calcified nodules in primary or cultured animal cells. The result of alizarin red staining is an important indicator for detecting the process of cell bone differentiation, that is, the more red substances there are, the higher the degree of cell bone differentiation. In this example, two experimental groups were set up: different concentrations (0, 1, 10, 50, 100 ng / mL) of TP4 polypeptide (SEQ ID NO: 5) and BMP-2 factor; on the 10th day, the mineralized nodule formation in the 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, and observed and recorded using a stereo microscope (Leica, Germany) ( Figure 3 ).

[0073] Depend on Figure 3 It can be seen that when the concentration of TP4 polypeptide is 1, 10 or 100 ng / mL, its bone induction effect is poor, that is, fewer mineralized nodules are produced, but when the concentration of TP4 is 50 ng / mL, its bone induction effect is significant and comparable to that of the positive control BMP-2. In summary, when treating bone defects, the TP4 polypeptide with a concentration of 50 ng / mL has the best therapeutic effect.

[0074] At the same time, the inventors also used a concentration of 100 ng mL -1 ~50mg mL -1 The TP4 peptides used to treat mBMSCs all had a certain effect of inducing mineralized nodules.

[0075] Example 3: In vivo experiments confirm that the polypeptide TP4 has osteoinductive properties

[0076] 3.1 Preparation of rat skull extreme bone defect model

[0077] All animal experiments were approved by the Animal Experiment Ethics Review Committee of Guangzhou Medical University (acceptance number: GD2019-648, date: 2019.02.22). The oral prosthetic membrane was used as the scaffold material, and the scaffold was made into a circular sheet with a diameter of about 5 mm using a 5 mm puncher; the following groups were designed for in vivo study, with four replicates in each group:

[0078] (I) Repair membrane only (control group)

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

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

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

[0082] A 1% sodium pentobarbital saline solution was drawn with a 5mL syringe, the dosage was calculated as 50mg / kg body weight, and the injection volume for each rat was about 2.5mL, and the anesthetic was injected intraperitoneally; the rat entered an anesthetized state within 5-10min, which was manifested by sluggish corneal reflex and relaxation of muscles throughout the body; the hair on the top of the head of the anesthetized Sprague-Dawley (SD) rat was shaved with an electric shaver, and then the skin of the surgical area was disinfected with iodine 2 times, and 1% lidocaine hydrochloride was injected into the skin of the surgical area for local anesthesia; after disinfection and anesthesia, a 1.5-2 cm incision was made on the midline of the skull above the rat's calvaria, and the epidermis, subcutaneous fascia, and periosteum were cut open until the lambdoid suture was seen, and a curette was used to press against the skull bone surface and carefully peel off to fully expose the skull area that needs to be modeled; the exposed area did not exceed the line connecting the two eyes in front, and was 3 to 5mm behind the lambdoid suture, and the left and right sides were about 8mm each extending from the middle suture of the skull. ; A skull drill with a diameter of 5 mm was used to drill skull defects on both sides of the middle suture of the rat skull. When drilling the skull, a high-speed drill should be used. After drilling a circular groove on the skull, the speed should be reduced. Note that the action should be gentle and slow so as not to injure the meninges, so as not to affect the modeling effect. In addition, the soft tissue should be fully separated to expose the surgical field before drilling the bone, otherwise the soft tissue will affect the normal operation of the bone drill and the defective skull soft tissue will cause modeling failure; After the skull defect model was completed, the corresponding sample materials (blank group, TP4 group, BMP2 group, TGF-β3 group) were implanted in the corresponding groups, and the periosteum and skin were sutured; penicillin solution was injected intramuscularly for anti-infection after surgery, and the rats were kept warm and observed for half an hour, and were put back into the breeding cage after waking up; The vital signs and activity status of the SD rats and the infection of the surgical incision were observed every day for one week after surgery, and if there was infection, it was treated in time; The SD rats after surgery were observed and sampled 4 weeks after surgery and related 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 technology. The biggest difference between it and ordinary clinical CT is that its resolution is extremely high, reaching the micron (μm) level. Four weeks after the implantation of the sample material, the rats were anesthetized with 2% sodium pentobarbital, killed by cervical dislocation, and the rat skulls were completely removed; the specimens were scanned by SkyScan1172 (Bruker-Micro-CT in Kontich, Belgium). Each sample scan was digitally redirected using SkyScan DataViewer software. The voxel resolution of the scanned volume was 10μm, the volume of interest was 500 layers, and the images were used to reconstruct the tomogram using 3D Creator software ( Figure 4 ).

[0085] After scanning, the samples were subjected to histological analysis. The specimens were immersed in 4% paraformaldehyde for 1 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. The sections were sliced ​​from the center 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's trichrome staining (Sigma-Aldrich, USA), and the newly formed bone and the healing of the bone defect were observed under an optical microscope.

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

[0087] from Figure 5 The section staining diagram shows that the BMP-2 group ( Figure 5 B) and TP4 group ( Figure 5 A) The newly generated bone tissues included osteoid, woven bone, active osteoblasts and blood vessels, but no new cartilage tissue formation was detected; on the contrary, TGF-β3 formed a well-structured cartilage tissue, i.e., an interface between hypertrophic cartilage and immature woven bone. In summary, TP4 polypeptide can be used to induce bone differentiation and then to treat bone defects. Its effect is comparable to that of BMP-2 factor, but different from that of TGF-β3 factor.

[0088] At the same time, the inventors also used a concentration of 10 ng mL -1 ~50mg mL -1 The TP4 peptide has a certain therapeutic effect on the rat skull extreme bone defect model.

[0089] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in 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 comprises the sequence GPGGDRQDLGWKWVHEPKGYY.

2. The polypeptide according to claim 1, characterized in that The concentration of the polypeptide is 10-500 ng mL -1 .

3. A bone defect repair product, characterized in that: The product comprises the polypeptide according to any one of claims 1 to 2.

4. The product according to claim 3, characterized in that The concentration of the peptide was 10 ng mL -1 ~50mg mL -1 .

5. The product according to claim 3, characterized in that The products include but are not limited to any one or more of human-derived bone repair materials, animal-derived bone repair materials, artificial bone repair materials, bone defect repair hydrogels, collagen membranes, 3D printed scaffolds, and metal implants.

6. Use of a polypeptide for preparing a product for inducing bone differentiation, characterized in that: The amino acid residue sequence of the polypeptide comprises the sequence GPGGDRQDLGWKWVHEPKGYY.

7. The use according to claim 6, characterized in that The concentration of the peptide was 10 ng mL -1 ~50mg mL -1 .

8. The use according to claim 6, characterized in that The form of the product includes but is not limited to solid, semi-solid, liquid or gas.

9. Use of a polypeptide for preparing an agent for promoting bone mineralization nodules, characterized in that: The amino acid residue sequence of the polypeptide comprises the sequence GPGGDRQDLGWKWVHEPKGYY.

10. The use according to claim 9, characterized in that The concentration of the peptide was 10 ng mL -1 ~50mg mL -1 .

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