CN114853852A

CN114853852A - Polypeptide and application thereof in promoting bone repair

Info

Publication number: CN114853852A
Application number: CN202210572523.8A
Authority: CN
Inventors: 余钒源; 吴佳益; 王海溦; 李飞飞; 叶玲; 王怡天; 张宇衡
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2022-08-05
Anticipated expiration: 2042-05-25
Also published as: CN114853852B

Abstract

The invention relates to a polypeptide and application thereof in promoting bone repair, and belongs to the technical field of biological medicines. 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 scaffold for bone repair. The polypeptide KS29 can collect migration of periosteum source cells to complete connection of defect regions, promotes mineralization of periosteum neogenetic 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

Polypeptide and application thereof in promoting bone repair

Technical Field

The invention belongs to the technical field of biomedicine, and relates to an artificially synthesized polypeptide KS29 capable of promoting bone repair.

Background

Bone defect is a common clinical disease, and data in the Chinese white cortex of osteoporosis show that about 300 million new bone injury patients are added in China every year, which brings huge burden to public health. Bone defects may be caused by a variety of causes, including trauma, infection, tumor, aging, and the like. Although bone tissue has strong self-repairing and regenerating capabilities, defects of large size are often accompanied by the consequences of bone nonunion, dysfunction, delayed healing, even nonunion. At this time, special therapeutic intervention is required to restore the structure and function of the damaged bone tissue.

Autologous bone grafting is considered as the gold standard for repairing critical bone defects, however, the application of autologous bone grafts has certain limitations, which are severely limited by problems of donor site morbidity, donor source shortage and increased infection risk. Allogeneic bone grafts (taken from other patients) partially compensate for the deficiency of autologous bone, provide some growth factors, and have osteoinductive properties. However, this method also has a series of problems such as limited source and ethical issues. At present, tissue engineering bones adopting inorganic non-metallic or high polymer material scaffolds are widely concerned, and the individually customized scaffold materials such as 3D printing and the like can well match with a defect region, and the loose and porous structure of the scaffold materials is utilized to guide cell angiogenesis so as to realize bone regeneration. However, tissue engineered bones are highly dependent on their seed cells and cytokines carried in order to recruit and induce proliferation and differentiation of repair cells (e.g., BMSCs) in vivo to form new bone tissue. Among the currently FDA-approved drugs with the ability to promote new bone formation, parathyroid hormone (PTH) can cause osteosarcoma formation upon high dose ingestion, and bone morphogenetic protein (BMP2) has a short half-life and can cause ectopic bone formation, osteolysis and local inflammatory responses. Thus, effective and safe factors for promoting bone formation have yet to be developed.

Disclosure of Invention

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

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

the polypeptide of the present invention has 29 amino acids and an amino acid sequence of KCKCHGLSGSCGPGGDKCRCVFHWCCYVS, i.e., 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 the polypeptide as KS29, which is used for the following polypeptides.

The polypeptide of the invention has molecular weight of 3121.66 Da.

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

The polypeptides of the invention are useful for bone injury and/or bone repair. Furthermore, the bone defect repairing liquid is suitable for wide bone defect indications, such as truncated bone defects, bone-related wounds, tumor bone defects and the like, and can promote bone defect repair and/or be used for repairing bone defects.

KS29 was designed from a recognition segment on WNT3A ligand protein that binds to cell membrane Frizzled receptor and LRP5/6 co-receptor, and this protein activates the canonical Wnt signaling pathway, activating intramembrane β -catenin to enter nucleus, thus initiating transcription of downstream functional genes. A large number of researches show that the classical Wnt signal pathway participates in regulation and control of bone reconstruction in physiological and pathological states, on one hand, osteogenic differentiation of osteogenic precursor cells is regulated and controlled, osteoblast proliferation is promoted, the survival rate of osteoblasts and osteocytes is improved, and on the other hand, the function of osteoclasts is inhibited by regulating and controlling the ratio of nuclear factor kappa-B receptor activator ligand (RANKL) and Osteoprotegerin (OPG), so that the effect of bone promotion is achieved. Based on in vitro and in vivo experimental data of KS29, the recombinant protein can play a role similar to WNT3A recombinant protein, and can activate the bone repair function corresponding to the classical Wnt signal pathway.

As a preferred embodiment of the application of the polypeptide, the concentration of the polypeptide is 25-150 mug/mL, and more preferably 100-150 mug/mL.

Preferably, polypeptide KS29 of the present invention can be used in combination with a tissue-engineering acceptable carrier for the treatment of bone injury and/or bone repair. Furthermore, the polypeptide KS29 can be loaded on a bone repair material in any form and implanted into a bone defect part. For example, polypeptide KS29 can be loaded into bone cement, injected into a bone injury site in combination with a bone repair hydrogel, and used as an implant surface coating, and the like.

KS29 can accelerate the repair of bone defect area and promote bone regeneration by the action mode carried by tissue engineering scaffold. The acceptable carrier in tissue engineering generally means a tissue engineering scaffold, and further can be applicable to all the 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 osteogenic oriented bone marrow mesenchymal stem cells BMSC to form mineralized nodules, so that bone repair is promoted.

Further, by constructing a rat skull defect model, KS29 was implanted into a bone defect region, skull tissue was taken 4 weeks later, Micro-CT was used to analyze bone repair in the defect region, and tissue morphology was observed by tissue section HE, Goldner staining. Through Micro-CT and HE, Goldner staining results, the polypeptide osteogenesis effect is comprehensively analyzed, compared with ineffective peptide, the polypeptide KS29 can remarkably accelerate skull defect repair, and therefore the polypeptide KS29 has the potential of being used as a bone tissue engineering functional factor for treating bone defects.

Preferably, the polypeptide drug KS29 can complete the connection of a skull defect area by recruiting migration of periosteum-derived cells, and KS29 has a good function of promoting periosteum neogenesis tissue mineralization so as to accelerate the repair and regeneration of bone defects and is proved to be an effective functional factor for bone tissue engineering.

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

Preferably, the invention also discloses a polypeptide scaffold comprising polypeptide KS 29. Preferably, the polypeptide scaffold is a biological ceramic, a metal, a carbon-based and degradable polymer composite material and the like.

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

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

Preferably, the concentration of the polypeptide KS29 in the polypeptide scaffold is 25-150 mug/mL, and preferably 100-150 mug/mL.

Methacryloylated gelatin (GelMA) is a photosensitive biomaterial, and its powder can be uniformly mixed with functional factors when dissolved in a liquid 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, and has cell adhesion sites on the structure, so that the proliferation and migration of cells can be promoted.

The KS29 modified GelMA scaffolds could also be prepared to any shape with the aid of a mold or by 3D printing to conform to the morphology of the defect area. By changing the substitution degree and concentration of GelMA, the mechanical property after curing can be flexibly adjusted, so that the gel has certain elasticity, strength and support property to recover the structure and partial functions of the defective bone.

The invention has the beneficial effects that:

1) the polypeptide KS29 can promote osteogenic-oriented bone marrow mesenchymal stem cells BMSC to form mineralized nodules, so that bone repair is promoted.

2) The polypeptide drug KS29 provided by the invention can complete the connection of a skull defect area by recruiting migration of periosteum derived cells; meanwhile, the bone repair material has a good effect of promoting the mineralization of periosteum new tissues, has the effect of accelerating the 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 WNT3A recombinant protein, and can activate the bone repair function corresponding to a classical Wnt signal pathway.

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

Description of the drawings:

FIG. 1 shows silver nitrate (Von kossa) staining of polypeptide osteogenic mineralized nodules in vitro.

FIG. 2 is a heat map and quartered cross-sectional view of the defect area of each group 4 weeks after implantation of the polypeptide into the skull defect of a rat.

FIG. 3 shows the bone density (BMD) of the neogenetic tissue in each defect area 4 weeks after implantation of the polypeptide into the skull defect of the rat.

FIG. 4 shows HE staining of groups 4 weeks after implantation of the polypeptide into the skull defect of rats.

FIG. 5 shows Goldner staining of each set 4 weeks after implantation of the polypeptide into a rat cranial defect.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.

Example 1 in vitro cell assay

1.1 cell culture: selecting primary bone marrow mesenchymal stem cells (BMSC) extracted from 3-week-old C57BL/6 male mice long bone at 37 deg.C and 5% CO ₂ Cultured under conditions, and seeded at 30000/well in 24-well plates. To alpha-MEM medium containing 5% serum and 1% double antibody, 50. mu.g/mL ascorbic acid, 10mM sodium beta-glycerophosphate, and 100nM dexamethasone were added to prepare osteogenic induction medium for osteogenic induction.

1.2 cell model: osteogenic induction medium was allowed to act on BMSCs for 5 days to allow osteogenic orientation. Then, the test group was cultured for 14 days in osteogenesis induction medium supplemented with 150. mu.g/mL KS29, and the Control group was cultured in osteogenesis induction medium supplemented with 150. mu.g/mL null peptide (Control peptide). Wherein KS29 adopts a solid phase polypeptide synthesis process to synthesize the polypeptide, and HPLC is used for purifying the product, and the purity of the synthesized polypeptide is 95.65%; the amino acid sequence of the null peptide is shown as SEQ ID NO. 2, namely CKPLRLSKEEHPLK, the null 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 null peptide refers to the amino acid sequence unless otherwise stated).

1.3 result verification: after the induction is finished, the cells are fixed by using 4% paraformaldehyde, dyed in a silver nitrate dye solution (Biyuntian) for 20min in the dark, and developed by ultraviolet irradiation. The whole-hole image was taken with a body microscope (olympus) and the local magnification image was taken with an optical microscope (olympus). Five fields were taken per well and the Area of mineralized nodules (Area) and total gray value (IntDen) were statistically analyzed using imageJ (Vol 6.0) with p < 0.05.

As shown in the figure 1, silver nitrate (Von kossa) staining is adopted, A is a silver nitrate staining whole-hole shooting and local magnification schematic diagram, B is silver staining mineralized nodule area analysis, and C is silver staining mineralized nodule total gray value analysis.

The results show that: compared with the ineffective peptide, the number and maturity of mineralized nodules of the KS29 group are obviously increased, and the area of the mineralized nodules and the total gray value are obviously increased.

Namely, KS29 acts on bone marrow mesenchymal stem cells (BMSC) oriented in an osteogenesis way, and a silver nitrate staining experiment proves that the in vitro mineralization promoting effect is good.

In conclusion, this example demonstrates that polypeptide KS29 promotes osteogenic directed BMSC formation of mineralized nodules, thereby promoting bone repair.

Example 2 preparation of GelMA hydrogel scaffolds as polypeptide vectors

2.1GelMA preparation: dissolving 2g of gelatin in 10mL of PBS at 60 ℃, adding 125 mu L of Methacrylic Anhydride (MA), stirring for 2 hours, adding 40mL of PBS to terminate the reaction, pouring the reaction solution into a 12-14kDa dialysis bag, dialyzing with deionized water, and freeze-drying by a freeze-dryer to obtain powder, namely GelMA.

2.2 polypeptide modification: dissolving 2g GelMA freeze-dried powder in 10mL PBS at 60 ℃, adding polypeptide according to the concentration of 0.1mg/mL, fully and uniformly mixing KS29 and GelMA solution, then adding 2.5% photoinitiator LAP, and fully and uniformly mixing again. Sucking 20 μ L of the mixture with a pipette, injecting into a 5mm round hole of a polytetrafluoroethylene custom mold, irradiating with an ultraviolet lamp for 1min, solidifying, separating the material from the mold, and placing on ice for use. The Control group used GelMA hydrogel scaffolds loaded with null peptides (Control peptides).

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

3.1 animal models: 12-week-old SD male rats, each about 320 + -20 g, were used, 3 per group. Using 2% pentobarbital (injected according to the proportion of 300g/ml of the weight of the rat) to carry out intraperitoneal injection anesthesia, taking the prone position, shaving the head with a razor, preparing skin in an iodophor sterilization area, and paving a disposable sterile hole towel in the sterilization area. The nasal bone is followed by skin incision of 1.5-2.0cm in the longitudinal direction along the median line of the top of the head, the scalpel handle gently separates subcutaneous tissues, the periosteum is cut regularly along the sagittal suture of the skull, and the periosteum is separated bluntly, so that the parietal bone, the occipital bone and part of the frontal bone are fully exposed. Circular full-thickness bone defects with the diameter of 5mm are respectively prepared on two sides of the midline of the skull by using trephines, and sterilized materials are implanted. The experimental group was implanted with GelMA scaffold carrying polypeptide KS29, and the control group was implanted with GelMA scaffold carrying ineffective peptide. Reposition skin, suture, and sterilize again.

3.2 tissue drawing: after 4 weeks, the rats were sacrificed and the parietal bones were harvested, fixed overnight at 4 ℃ by 4% paraformaldehyde-soaked tissue, and stored in PBS.

3.3 verification of results: scanning a skull free specimen by using Micro-CT, wherein the scanning conditions are as follows: 70kVp, 200 μ A, accuracy 10 μm. Thermography was performed using Micro-CT self-contained analysis software, cross-sectional views of four equal points of the defect area were made using mics, and bone density (BMD) of neogenetic tissue in the defect area was analyzed using Dataviewer and Ctan software.

Referring to fig. 2, the heat map and the quartered section of the skull defect area are shown, wherein yellow, blue and purple lines mark the section positions respectively, the yellow, blue and purple boxes show the sections of the corresponding positions, and yellow arrows indicate that the KS29 osteogenic peptide in the sections has obvious osteogenic effect. In the sectional view, there was significant bone formation in the section indicated by the yellow arrows of KS29 group. The results show that: after 4 weeks of implantation of the material, the null pepset defect region had little new tissue formation, while polypeptide KS29 significantly promoted bone formation in the defect region.

As shown in fig. 3 bone density (BMD) analysis results: the bone density of the neogenetic tissue of KS29 group was significantly increased compared to the null peptide control group 4 weeks after material implantation. P < 0.05.

The above results show that: KS29 can promote bone repair, and has effects of accelerating bone defect repair and regeneration.

Example 4 morphological Observation of neogenetic tissue in defect area after implantation of polypeptide into rat skull defect

4.1 sample preparation: polypeptide KS29 and null peptide (Control peptide) were implanted into rat skull defects for 4 weeks, and the skull tissue was taken, 4% paraformaldehyde soaked in tissue at 4 ℃ overnight for fixation, and then soaked in EDTA with pH 7.0 concentration of 12% for 6 weeks to decalcify the bone to be soft enough.

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

4.3 verification of the results: the tissue section is baked for 2 hours at the temperature of 65 ℃, xylene and gradient ethanol are dewaxed and hydrated, and HE and Goldner are used for staining and observing the histological morphology of the new tissues in the defect area.

As shown by the HE staining results in fig. 4: after 4 weeks of implantation of the material, no cell-in material was present in the center of the defects in the control and experimental groups, and fibrous tissue was formed around the defects to complete the connection of the defect regions, but compared to the control group, the formation of new fibrous tissue apparently similar to that of mature bone tissue was observed in the KS29 group.

As shown by Goldner staining results in fig. 5: the neogenetic tissue around the defect of the control ineffective peptide group is fibrous tissue with relatively low light green mineralization degree, and mature bone formation with higher dark green mineralization degree is seen in the neogenetic connecting tissue around the defect of the polypeptide KS29 group.

Combining HE and Goldner staining results, polypeptide KS29 in vivo osteogenesis promoting effect is possible to complete the connection of defect regions by recruiting migration of periosteum-derived cells, and KS29 has good effect of promoting mineralization of periosteum neogenetic tissues.

SEQUENCE LISTING

<110> Sichuan university

<120> polypeptide and use thereof for 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

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

2. The polypeptide of claim 1, wherein: the polypeptides are useful for bone injury, and/or for bone repair.

3. Use of a polypeptide according to claim 1 or 2 for activating the canonical Wnt signaling pathway.

4. Use according to claim 3, characterized in that: the concentration of the polypeptide is 25-150 mug/mL.

5. Use according to claim 4, characterized in that: the concentration of the polypeptide is 100-150 mug/mL.

6. The polypeptide according to claim 1 or 2, characterized in that: which are used in combination with a tissue-engineering acceptable carrier to treat bone injury and/or to effect bone repair.

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

8. A polypeptide scaffold, comprising: comprising the polypeptide of claim 1.

9. The polypeptide scaffold of claim 8, wherein: the scaffold is a methacrylic acid anhydrization gelatin scaffold; the concentration of the polypeptide is 25-150 mu g/mL.

10. Use of a polypeptide according to claim 1 for the preparation of a bone injury and/or bone repair composition.