CN114853852B - Polypeptides and their use in promoting bone repair - Google Patents

Abstract

The invention relates to a polypeptide and application thereof in promoting bone repair, belonging to the technical field of biological medicine. The amino acid sequence of the polypeptide provided by the invention is shown as SEQ ID NO. 1. The invention also discloses application of the polypeptide KS29 in bone injury and/or bone repair. Furthermore, the invention also discloses a polypeptide bracket for bone repair. The polypeptide KS29 can recruit periosteum-derived cells to migrate to complete connection of defect areas, promote mineralization of periosteum newly-grown tissues, has the effect of accelerating repair and regeneration of bone defects, and can be used as a functional factor of bone tissue engineering.

Description

Polypeptides and their use in promoting bone repair

Technical Field

The invention belongs to the technical field of biological medicines, and relates to an artificial synthetic polypeptide KS29 capable of promoting bone repair.

Background

Bone defect is a common clinical disease, and data of Chinese osteoporosis white book shows that about 300 thousands of patients with new bone injury are added in China every year, and great burden is brought to public health. Bone defects may be caused by a variety of causes including trauma, infection, tumors, aging, and the like. Although bone tissue has strong self-repair and regeneration capacity, defects of larger size often come with bone nonunion, dysfunction, delayed healing, even non-healing, etc. At this time, a special treatment method is required for intervention to restore the structure and function of the damaged bone tissue.

Autologous bone grafting is considered as a gold standard for repairing critical bone defects, however, the use of autologous bone grafts has certain limitations, which are severely limited by problems of donor site morbidity, donor source deficiency, and increased risk of infection. Allograft bone grafting (from other patients) can partially compensate for the lack of autologous bone, provide some growth factors, and have osteoinductive properties. However, this method also suffers from a series of problems such as limited sources and ethics. At present, tissue engineering bones adopting inorganic nonmetallic or high polymer material scaffolds are widely focused, and personalized and customized scaffold materials such as 3D printing and the like can be well matched with defect areas, and the porous structure of the scaffold materials is utilized to guide cell angiogenesis so as to realize bone regeneration. However, tissue engineering bone is highly dependent on seed cells and cytokines it carries to recruit and induce proliferation and differentiation of repair cells (such as bone marrow mesenchymal stem cells BMSCs) in vivo, thereby forming new bone tissue. Among the drugs currently approved by the FDA for promoting new bone formation, parathyroid hormone (PTH) can cause osteosarcoma formation when ingested at a high dose, and bone morphogenic protein (BMP 2) has a short half-life, which can cause ectopic bone formation, osteolysis and local inflammatory reaction. Thus, an effective and safe factor for promoting bone formation has yet to be developed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an artificial synthetic polypeptide capable of promoting bone repair.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the polypeptide of the present invention contains 29 amino acids and has an amino acid sequence of KCKCHGLSGSCGPGGDKCRCVFHWCCYVS, namely Lys Cys Lys Cys His Gly Leu Ser Gly Ser Cys Gly Pro Gly Gly Asp Lys Cys Arg Cys Val Phe His Trp Cys Cys Tyr Val Ser, and the inventors named KS29, and the following polypeptides are used.

The molecular weight of the polypeptide is 3121.66Da.

As a preferred embodiment of the polypeptide of the present invention, the polypeptide KS29 of the present invention can be synthesized by a conventional synthesis method such as a liquid phase stepwise synthesis method, a solid phase synthesis method, a biological synthesis method, etc., and the polypeptide is synthesized by a solid phase polypeptide synthesis process. Furthermore, in order to ensure biological safety, the purity of the polypeptide of the invention is more than or equal to 95 percent. The product may be purified using HPLC.

The polypeptides of the invention are useful for bone injury and/or bone repair. Further, the composition is suitable for a wide range of bone defect indications, such as a truncated bone defect, a bone-related wound, a tumor bone defect, etc., and can promote repair of the bone defect and/or be used for repairing the bone defect.

KS29 is designed from a recognition segment combined with a cell membrane Frizzled receptor and an LRP5/6 co-receptor on a WNT3A ligand protein, and the protein can activate a classical Wnt signal path and beta-catenin in a membrane to enter a nucleus, so that transcription of a downstream functional gene is started. Numerous studies have shown that the canonical Wnt signaling pathway is involved in regulating bone remodeling in physiological and pathological states, on the one hand, regulating osteogenic differentiation of osteoblasts precursors, promoting osteoblast proliferation and increasing survival of osteoblasts and bone cells, and on the other hand, inhibiting osteoclast function by regulating the ratio of nuclear factor kappa-B receptor activator ligand (RANKL) to Osteoprotegerin (OPG), thereby contributing to bone formation. Based on the in vitro and in vivo experimental data of KS29, we speculate that it can exert a function similar to WNT3A recombinant protein, activating bone repair effects corresponding to canonical WNT signaling pathways.

As a preferred embodiment of the use of the polypeptide of the present invention, the polypeptide is used at a concentration of 25 to 150. Mu.g/mL, more preferably 100 to 150. Mu.g/mL.

Preferably, the polypeptide KS29 according to the invention can be used in combination with a tissue-engineering-acceptable carrier for the treatment of bone injuries and/or bone repair. Further, the polypeptide KS29 of the present invention may be supported on the bone repair material in any form for implantation into a bone defect site. For example, the polypeptide KS29 may be supported in bone cement, may be injected into a site of bone injury in combination with a bone repair hydrogel, may be used as a surface coating for implants, and the like.

KS29 can accelerate repair of a bone defect region and promote bone regeneration by means of the action of mounting a tissue engineering scaffold. The tissue engineering acceptable carrier is generally referred to as a tissue engineering scaffold, and further, can be applied to all existing tissue engineering scaffolds, including biodegradable bone tissue engineering scaffold materials and non-biodegradable bone tissue engineering scaffold materials.

In vitro cell experiments show that the polypeptide KS29 can promote bone mesenchymal stem cells BMSC of bone formation orientation to form mineralized nodules, thereby promoting bone repair.

Further, KS29 was implanted into a bone defect region by constructing a rat skull defect model, skull tissue was taken after 4 weeks, bone repair conditions in the defect region were analyzed using Micro-CT, and tissue morphology was observed by tissue section HE, goldner staining. The bone formation effect of the polypeptide is comprehensively analyzed through Micro-CT, HE and Goldner staining results, and compared with ineffective peptide, the polypeptide KS29 can obviously accelerate the repair of skull defects, so that the polypeptide KS29 has the potential of treating bone defects as a bone tissue engineering functional factor.

As a preferable scheme, the polypeptide medicine KS29 can complete the connection of the skull defect area by recruiting periosteum-derived cells, and the KS29 has good function of promoting the mineralization of periosteum newly-generated tissues, so that the repair and regeneration of bone defects are accelerated, and the polypeptide medicine KS29 is verified to be an effective functional factor of bone tissue engineering.

Further, the invention also discloses a bone repair composition, which contains a therapeutically effective dose of polypeptide KS29 and a carrier acceptable in tissue engineering.

Also disclosed is a polypeptide scaffold comprising the polypeptide KS29 as a preferred embodiment. Preferably, the polypeptide scaffold is a ceramic, metal, carbon-based and degradable polymer composite material, etc.

Further, the degradable polymer composite gelatin scaffold is preferably: sodium alginate, chitosan, hyaluronic acid and methacrylic anhydride gelatin (GelMA) scaffold.

Preferably, in the polypeptide scaffold of the present invention, KS29 is homogeneously dispersed in a methacrylic acid anhydrified gelatin (GelMA) scaffold.

Preferably, the concentration of the polypeptide KS29 in the polypeptide scaffold is 25 to 150. Mu.g/mL, preferably 100 to 150. Mu.g/mL.

Methacryloylated gelatin (GelMA) is a photosensitive biological material that can be homogeneously mixed with functional factors when its powder is dissolved in liquids such as water. GelMA has excellent operability and can be rapidly crosslinked to form a three-dimensional structure under the action of a photoinitiator. GelMA has good biocompatibility, has cell adhesion sites on the structure, and can promote proliferation and migration of cells.

The KS29 modified GelMA stent can be prepared into any shape with the aid of a die or by 3D printing so as to be attached to the shape of the defect area. The mechanical property of the cured GelMA can be flexibly adjusted by changing the substitution degree and concentration of the GelMA, so that the GelMA has certain elasticity, strength and support property so as to recover the structure and partial functions of the defective bone.

The invention has the beneficial effects that:

1) The polypeptide KS29 can promote bone mesenchymal stem cells BMSC of bone formation orientation to form mineralized nodules, thereby promoting bone repair.

2) The polypeptide medicine KS29 provided by the invention can complete the connection of the skull defect area by recruiting the migration of periosteum-derived cells; meanwhile, the bone mineral has good effect of promoting mineralization of periosteum newly-grown tissues, has the effect of accelerating repair and regeneration of bone defects, and can be used as a functional factor of bone tissue engineering.

3) The polypeptide KS29 can play a role similar to that of WNT3A recombinant protein, and can activate the bone repair effect corresponding to a classical Wnt signal channel.

4) The polypeptide KS29 provided by the invention belongs to a small molecule polypeptide, has simple preparation process, low cost and high yield, and has higher conversion value and clinical application prospect.

Description of the drawings:

FIG. 1 is a silver nitrate (Von kossa) staining of an in vitro osteogenic mineralized nodule of a polypeptide.

FIG. 2 is a heat map and a four-quarter sectional view of each group of defect areas after 4 weeks of polypeptide implantation into rat skull defects.

FIG. 3 shows the bone density (BMD) of new tissue in each group of defect areas 4 weeks after the implantation of the polypeptides into the rat skull defects.

FIG. 4 shows HE staining of each group 4 weeks after implantation of the polypeptides into the rat skull defects.

FIG. 5 shows Goldner staining of each group 4 weeks after implantation of the polypeptides into rat skull defects.

Detailed Description

The following describes the invention in further detail with reference to examples, which are not intended to limit the invention thereto.

Example 1 in vitro cell experiments

1.1 cell culture: selecting bone marrow mesenchymal stem cells (BMSC) extracted from 3-week-old C57BL/6 male mice, and extracting at 37deg.C and 5% CO ₂ Cultures were incubated at 30000/well and seeded into 24-well plates. To an alpha-MEM medium containing 5% serum and 1% diabody, 50. Mu.g/mL ascorbic acid, 10mM sodium beta-glycerophosphate and 100nM dexamethasone were added to prepare an osteogenesis medium for osteogenesis.

1.2 cell model: osteogenic induction medium was applied to BMSCs for 5 days to bring about osteogenic orientation. The experimental group was then incubated with 150. Mu.g/mL KS29 in osteogenic induction medium, and the Control group was incubated with 150. Mu.g/mL Control peptide in osteogenic induction medium for a further 14 days. Wherein KS29 adopts a solid-phase polypeptide synthesis process to synthesize polypeptide, HPLC is used for purifying the product, and the purity of the synthesized polypeptide is 95.65%; the amino acid sequence of the inactive peptide is shown as SEQ ID NO. 2, CKPLRLSKEEHPLK. The inactive peptide is synthesized into a polypeptide by a solid phase polypeptide synthesis process, and the product is purified by HPLC, wherein the purity of the synthesized polypeptide is 96.25% (in the following examples, the inactive peptide is the amino acid sequence unless otherwise specified).

1.3 verification of results: after the induction was completed, the cells were fixed with 4% paraformaldehyde, stained with silver nitrate dye solution (Biyun) for 20min in the dark, and developed by ultraviolet irradiation. Whole-well photographing was performed using a bulk microscope (olympus), and partial magnification photographing was performed using an optical microscope (olympus). Five fields were taken per well and statistically analyzed for Area (Area) and total gray scale value (IntDen) of mineralized nodules using imageJ (Vol 6.0), p < 0.05.

In fig. 1, silver nitrate (Von kossa) is used for dyeing, a is a schematic diagram of whole hole shooting and partial enlargement of silver nitrate dyeing, B is silver-dyed mineralized nodule area analysis, and C is silver-dyed mineralized nodule total gray value analysis.

The results show that: the number and maturity of the KS29 group mineralized nodules were significantly increased compared to the ineffective peptide, with a significant increase in both the node area and the total gray scale value.

Namely, KS29 acts on bone marrow mesenchymal stem cells (BMSCs) oriented by bone formation, and silver nitrate staining experiments prove that the bone marrow mesenchymal stem cells have good mineralization promotion effect in vitro.

Taken together, this example demonstrates that polypeptide KS29 promotes the formation of mineralized nodules in osteogenic committed BMSCs, thereby promoting bone repair.

EXAMPLE 2 preparation of polypeptide Carrier GelMA hydrogel scaffold

2.1GelMA preparation: after 2g of gelatin is dissolved in 10mL of PBS at 60 ℃, 125 mu L of Methacrylic Anhydride (MA) is added and stirred for 2 hours, 40mL of PBS is added to stop the reaction, the reaction solution is poured into a 12-14kDa dialysis bag, deionized water is used for dialysis, and the obtained powder is GelMA after freeze-drying by a freeze dryer.

2.2 polypeptide modification: 2g of GelMA lyophilized powder is dissolved in 10mL of PBS at 60 ℃, polypeptide is added according to the concentration of 0.1mg/mL, KS29 and GelMA solution are fully and uniformly mixed, then 2.5% of photoinitiator LAP is added, and fully and uniformly mixed again. And sucking 20 mu L of the mixture by a pipetting gun, injecting the mixture into a round hole with the diameter of 5mm on a polytetrafluoroethylene customized mold, irradiating for 1min by an ultraviolet lamp, separating the material from the mold after the material is solidified, and placing the material on ice for later use. The Control group used a GelMA hydrogel scaffold carrying a Control peptide.

EXAMPLE 3 bone repair Effect of polypeptide after implantation into rat skull defects

3.1 animal model: SD male rats of 12 weeks of age were used, about 320.+ -.20 g each, 3 per group. Abdominal injection anesthesia was performed using 2% pentobarbital (injected at a ratio of 300g/ml rat body weight), the prone position was taken, the razor head was prepared, the iodophor sterilized area was sterilized, and a disposable sterile drape was laid over the sterilized area. The skin incision is 1.5 cm to 2.0cm along the midline of the top of the head from the nasal bone, the subcutaneous tissue is gently separated by the surgical knife handle, the periosteum is neatly cut along the sagittal suture of the skull, the periosteum is blunt separated, and the parietal bone, the occipital bone and part of the frontal bone are fully exposed. The circular full-layer bone defect with the diameter of 5mm is prepared on two sides of the midline of the parietal bone by using trephine, and the sterilized material is implanted. The experimental group was implanted with GelMA scaffold material loaded with polypeptide KS29, and the control group was implanted with GelMA scaffold material loaded with ineffective peptide. The skin is reset, sutured, and disinfected again.

3.2 tissue material selection: after 4 weeks, rats were sacrificed and cranium parietal bones were harvested and 4% paraformaldehyde-soaked tissues were fixed overnight at 4 ℃, soaked in PBS and stored.

3.3 verification of results: the free specimens of the skull were scanned using Micro-CT, scanning conditions: 70kVp,200 μA, with an accuracy of 10 μm. Heat map preparation was performed using Micro-CT self-contained analysis software, quarter point cross-sectional map preparation was performed using chemicals, and new tissue bone density (BMD) in the defect area was analyzed using dataview and Ctan software.

As shown in the heat map and the quartered cross-sectional view of the skull defect area in FIG. 2, yellow, blue and purple lines in the heat map respectively mark the cross-sectional positions, images in yellow, blue and purple boxes are cross-sectional views at corresponding positions, and yellow arrows indicate that KS29 osteogenic peptides in the cross-section have obvious osteogenic effects. In the cross-sectional view, the KS29 group yellow arrows indicate significant bone formation in the cross-section. The results show that: after 4 weeks of implantation of the material, there was almost no new tissue formation in the defective areas of the null peptide groups, while polypeptide KS29 significantly promoted bone formation in the defective areas.

The Bone Mineral Density (BMD) analysis results as shown in fig. 3 show: after 4 weeks of material implantation, bone density was significantly increased in the KS29 group of neotissue compared to the ineffective peptide control group. * p < 0.05.

The above results show that: KS29 promotes bone repair, and has the effect of accelerating repair and regeneration of bone defects.

EXAMPLE 4 morphological observation of New tissue in defect area after polypeptide implantation into rat skull defect

4.1 sample preparation: after 4 weeks of the effect of the polypeptide KS29 and the ineffective peptide (Control peptide) in the rat skull defect, the skull tissue was taken, 4% paraformaldehyde-soaked tissue was fixed overnight at 4 ℃, and then soaked in EDTA at a ph=7.0 concentration of 12% for 6 weeks to decalcify the bone sufficiently soft.

4.2 tissue sections: tissue dehydration, paraffin embedding, and slicing into tissue sections with a thickness of 6 μm.

4.3 verification of results: the tissue sections were baked at 65℃for 2 hours, deparaffinized with xylene and gradient ethanol, and HE and Goldner stained for the histological morphology of the new tissue in the defect area.

The HE staining results show as in fig. 4: after 4 weeks of material implantation, the center of the defects of the control group and the experimental group is made of cell-free ingrowth material, fiber tissues around the defects are formed, and the connection of the defect areas is completed, but compared with the control group, the KS29 group of new fiber tissues have new tissue formation which is obviously similar to mature bone tissues.

The Goldner staining results show in fig. 5: the neonatal tissue surrounding the control ineffective peptide group defect was fibrous tissue with a relatively low degree of light green mineralization, while mature bone formation with a higher degree of dark green mineralization was seen in the neonatal connective tissue surrounding the polypeptide KS29 group defect.

Combining HE and Goldner staining results, the polypeptide KS29 contributes to bone action in vivo, probably by recruiting periosteum-derived cells to complete the attachment of the defect area, while KS29 has good periosteal neogenesis tissue mineralization promoting effect.

SEQUENCE LISTING

<110> university of Sichuan

<120> polypeptides and their use in promoting bone repair

<130> KS29

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 29

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Lys Cys Lys Cys His Gly Leu Ser Gly Ser Cys Gly Pro Gly Gly Asp

1 5 10 15

Lys Cys Arg Cys Val Phe His Trp Cys Cys Tyr Val Ser

20 25

<210> 2

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Cys Lys Pro Leu Arg Leu Ser Lys Glu Glu His Pro Leu Lys

1 5 10

Claims (5)

1. A polypeptide, characterized in that: the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

2. A bone repair composition characterized by: the bone repair composition comprising a therapeutically effective amount of the polypeptide of claim 1 and a tissue engineering acceptable carrier.

3. A polypeptide scaffold, characterized in that: comprising the polypeptide of claim 1.

4. A polypeptide scaffold according to claim 3, wherein: the scaffold is a methacrylic anhydride gelatin scaffold; the concentration of the polypeptide is 25-150 mug/mL.

5. Use of the polypeptide of claim 1 for the preparation of a composition for the treatment of bone injury and/or bone repair.

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