CN114957404A

CN114957404A - Polypeptide and application thereof in promoting bone repair

Info

Publication number: CN114957404A
Application number: CN202210532025.0A
Authority: CN
Inventors: 吴佳益; 余钒源; 李飞飞; 叶玲; 王怡天; 王海溦; 张宇衡
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-05-17
Filing date: 2022-05-17
Publication date: 2022-08-30
Anticipated expiration: 2042-05-17
Also published as: CN114957404B

Abstract

The invention relates to a polypeptide and application thereof in promoting bone repair, belonging 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 KS32 in bone injury and/or bone repair. Furthermore, the invention also discloses a polypeptide scaffold for bone repair. The polypeptide KS32 can attract and collect cells into a bone repair material, has the effect of accelerating the 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 KS32 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 regeneration-reconstructing capabilities, defects with larger sizes are often accompanied by the consequences of bone nonunion, dysfunction, delayed healing, even nonhealing, and the like. 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 invention provides a polypeptide, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

The polypeptide of the present invention has 32 amino acids and an amino acid sequence of KCKCHGLSGSCEVKTCWWSKCRCVFHWCCYVS, i.e., Lys Cys Lys Cys His Gly Leu Ser Gly Ser Cys Glu Val Lys Thr Cys Trp Trp Ser Lys Cys Arg Cys Val Phe His Trp Cys Cys Tyr Val Ser, and the inventors named the polypeptide as KS32, which is used for the following polypeptides.

The polypeptide of the invention has molecular weight of 3758.64 Da.

The polypeptide KS32 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, to ensure biological safety, the purity of the polypeptide of the invention is more than or equal to 95%, and the product can be purified by 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.

KS32 was designed from the recognition segment of WNT3A ligand protein that binds to the cell membrane Frizzled receptor and LRP5/6 co-receptor, and this protein could activate the canonical Wnt signaling pathway, activate intramembrane beta-catenin to enter nucleus, and thus initiate transcription of downstream functional genes. Numerous studies have shown that the canonical Wnt signaling pathway is involved in regulating the various stages of osteoblastic lineage cell survival, proliferation, differentiation, with its major functions including: regulating osteogenic differentiation of osteogenic precursor cells, promoting osteoblast proliferation and increasing the survival rate of osteoblasts and osteocytes. Therefore, based on the in vivo experimental data of KS32, we speculate that it can play a role similar to WNT3A recombinant protein, and activate the bone repair function corresponding to the canonical Wnt signaling pathway.

According to the preferable embodiment of the application of the polypeptide, the concentration of the application of the polypeptide is 25-150 mug/mL, preferably 100-150 mug/mL, and further preferably 100 mug/mL.

Preferably, polypeptide KS32 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 KS32 can be loaded on a bone repair material in any form and implanted into a bone defect part. For example, polypeptide KS32 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.

KS32 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.

Further, by constructing a rat skull defect model, implanting KS32 into a bone defect region, taking skull tissue after 4 weeks and 8 weeks, analyzing the bone repair condition of the defect region by using Micro-CT, observing the section morphology of the new tissues in the defect region by using a Scanning Electron Microscope (SEM), analyzing the element distribution of the section of the new tissues by using energy spectrum (EDS) and analyzing the weight percentage (wt%) of each element, and further observing the histological morphology by using tissue sections, HE and Goldner staining. The polypeptide osteogenesis effect is comprehensively analyzed by combining Micro-CT, SEM, EDS, HE and Goldner staining results, compared with ineffective peptides, the polypeptide KS32 can remarkably accelerate skull defect repair, and therefore the polypeptide KS32 has the potential of being used as a bone tissue engineering functional factor for treating bone defects.

As a preferred scheme, the polypeptide medicine KS32 can attract and recruit cells to enter a scaffold material, promote the formation of fibrous tissues with high mineralization degree, and complete defect connection, so that the repair and regeneration of bone defects are accelerated, and the polypeptide medicine 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 KS32 and a tissue engineering acceptable carrier.

Preferably, the invention also discloses a polypeptide scaffold comprising polypeptide KS 32. 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, KS32 is uniformly dispersed in a methacrylic anhydrified gelatin (GelMA) scaffold.

Preferably, in the polypeptide scaffold, the concentration of the polypeptide KS32 is 25-150 μ g/mL, preferably 100-150 μ g/mL, and more preferably 100 μ g/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 KS32 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 medicine KS32 provided by the invention can attract and recruit cells to enter bone repair materials (such as a scaffold and the like), has the effect of accelerating the repair and regeneration of bone defects, and can be used as a functional factor of bone tissue engineering.

2) The polypeptide KS32 can play a role similar to WNT3A recombinant protein, and can activate the bone repair function corresponding to a classical Wnt signal pathway.

3) The polypeptide KS32 provided by the invention belongs to a micromolecule polypeptide, and the preparation process is simple, the cost is low, the yield is high, and the polypeptide KS32 has higher transformation value and clinical application prospect.

Description of the drawings:

FIG. 1 is a three-dimensional reconstruction diagram of Micro-CT scanning of each group after the polypeptide is implanted into the skull defect of a rat for 4 weeks and 8 weeks.

FIG. 2 is a heat map and quartet sectional view of the defect area of each group 4 weeks and 8 weeks after implantation of the polypeptide into the skull defect of rat.

FIG. 3 shows bone volume fraction (BV/TV) and bone density (BMD) of neogenetic tissue in each defect area 4 weeks and 8 weeks after implantation of the polypeptide into the skull defect of rat.

FIG. 4 shows the results of Scanning Electron Microscopy (SEM) and energy spectroscopy (EDS) of each group of defect regions 4 weeks after implantation of the polypeptide into the skull defect of rat.

FIG. 5 shows the results of HE and Goldner staining in each group 8 weeks after implantation of the polypeptide into a skull defect in a rat.

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 preparation of GelMA hydrogel scaffolds as polypeptide vectors

1.1GelMA 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.

1.2 polypeptide modification: dissolving 2g of GelMA freeze-dried powder in 10mL of PBS at 60 ℃, adding the polypeptide according to the concentration of 0.1mg/mL, fully and uniformly mixing KS32 and GelMA solution, then adding 2.5% of 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, separating the material from the hole plate after the material is solidified, and placing on ice for use. The Control group used GelMA hydrogel scaffolds loaded with null peptide (Control peptide). Wherein KS32 adopts solid phase polypeptide synthesis technology to synthesize polypeptide, HPLC is used for purifying products, and the purity of the synthesized polypeptide is 98.96%; 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).

Example 2 Effect of bone repair after implantation of polypeptide into skull defect of rat

2.1 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-layer bone defects with the diameter of 5mm are respectively prepared on two sides of the middle line of the parietal bone by trephines, and sterilized materials are implanted. The experimental group was implanted with GelMA scaffold carrying polypeptide KS32, and the control group was implanted with GelMA scaffold carrying ineffective peptide. Reposition skin, suture, and sterilize again.

2.2 tissue selection: the rats were sacrificed 4 weeks/8 weeks later, the parietal bones were harvested, the tissues were soaked in 4% paraformaldehyde overnight at 4 ℃ for fixation, and the tissues were soaked in PBS for storage.

2.3 result verification: scanning a skull free sample by using Micro-CT, wherein the scanning conditions are as follows: 70kVp, 200 μ A, precision 10 μm. Three-dimensional reconstruction and defect area heat map making are carried out by using Micro-CT self-contained analysis software, a defect area quartering point section map making is carried out by using Mimics software, and the neogenetic tissue bone volume fraction (BV/TV) and bone density (BMD) of the defect area are analyzed by using Dataviewer and Ctan software.

As shown in fig. 1, the three-dimensional reconstruction map of the skull defect shows that: after 4 weeks of implantation of the material, the null pepset defect region had little new tissue formation, whereas polypeptide KS32 significantly promoted new tissue formation in the defect region. At 8 weeks, the control group formed a small amount of neogenetic tissue in the defect margin area, while polypeptide KS32 formed a large amount of neogenetic tissue in the circular defect and covered almost the entire defect area.

As shown in fig. 2, the heat map and quartered cross-sectional view of the skull defect area show: the KS32 group showed more significant neogenetic tissue formation than the control group in both 4 weeks and 8 weeks in the heat map, in which yellow, blue and purple lines respectively mark the cross-sectional positions, the images in the yellow, blue and purple boxes are cross-sectional views of the corresponding positions, blue arrows indicate cross-sections where KS32 osteogenic peptide had significant osteogenic effect, and it can be seen that KS32 exhibited osteogenic effect on the cross-sections at each level.

As shown in FIG. 3, the results of bone volume fraction (BV/TV) and bone density (BMD) analysis show: at 4 weeks after material implantation, although on average the KS32 group had a higher BV/TV value, this value was not statistically significantly different compared to the null peptide control group, whereas at 8 weeks the KS32 group had a significantly higher BV/TV value than the control group. KS32 significantly increased neogenetic tissue bone density (BMD) in the defect area for both 4 and 8 weeks, and further increased bone density at 8 weeks compared to 4 weeks. P < 0.01.

The experimental results show that the polypeptide KS32 can promote bone repair and has the effect of accelerating repair and regeneration of bone defects.

Example 3 morphology and elemental analysis of neogenetic tissue in the defect area 4 weeks after implantation of the polypeptide into the skull of rats

3.1 sample preparation: the rats were sacrificed 4 weeks after the polypeptide implantation into the skull defects, the skull bone tissue was taken, the tissue was soaked in 4% paraformaldehyde overnight at 4 ℃ for fixation, and stored in PBS. Epoxy resin A liquid B liquid is prepared from the following components in a volume ratio of 2.5: 1, uniformly mixing, embedding the tissue sample in epoxy resin, solidifying overnight, placing in liquid nitrogen for quick freezing, brittle-breaking the epoxy resin wrapping the tissue along the center of the defect together with the tissue, and carrying out sample detection at normal temperature.

3.2 result verification: the profile was observed by Scanning Electron Microscopy (SEM) for morphology at 400 x and 8000 x magnification, respectively, and profile elemental analysis was performed using energy spectroscopy (EDS) with analytical elements including C, N, O, Ca, P, calculated as weight percent (wt%) of each element.

As shown by the SEM and EDS results of fig. 4: the section (defect center) of the control group was smooth and flat, and the morphology of the gel scaffold was presumed to be that no new tissue was formed. The uneven cross section (center of defect) of the experimental group suggests that KS32 group had further new tissue (such as bone tissue) formation in the defect area. The distribution of each element is analyzed by EDS to find that the yellow marked Ca element and the purple marked P element of the KS32 group have higher color content, the weight percentage (wt%) of each element is calculated, the Ca element content of a control group is only 0.1%, the Ca element content of an experimental group is 15.3%, the P element content of the control group is only 0.5%, and the P element content of the experimental group is 8.7%, the polypeptide KS32 is prompted to remarkably promote the deposition of the Ca and P elements, and the Ca (red arrow) and P (green arrow) element peaks of the KS32 group in an energy spectrum also show higher peaks.

This example suggests that KS32 polypeptide can promote the deposition of Ca and P elements in vivo, thereby promoting the mineralization of bone defect repair tissue.

Example 4 morphological Observation of neonatal tissue in the defect area 8 weeks after implantation of the polypeptide into the skull of a rat

4.1 sample preparation: the rats are sacrificed after the polypeptide is implanted into the skull defects of the rats for 8 weeks, skull bone tissues are taken, the tissues are soaked in 4 percent paraformaldehyde and fixed overnight at 4 ℃, the tissues are soaked in EDTA with the pH value of 7.0 and the concentration of 12 percent, and the tissues are soaked for 6 weeks for decalcification until the bones are 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.

HE and Goldner staining results as shown in figure 5 show: after 8 weeks of material implantation, polypeptide KS32 attracted cells well into the scaffold to accelerate bone repair compared with the null peptide group, and the defect repair was completed by fibrous tissue with higher mineralization without significant scaffold retention in the defect area.

SEQUENCE LISTING

<110> Sichuan university

<120> a polypeptide and use thereof in promoting bone repair

<130> KS32

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 32

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Lys Cys Lys Cys His Gly Leu Ser Gly Ser Cys Glu Val Lys Thr Cys

1 5 10 15

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

20 25 30

<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 mu g/mL.

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

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