CN114751960A

CN114751960A - Polypeptide and application thereof in bone repair

Info

Publication number: CN114751960A
Application number: CN202210548440.5A
Authority: CN
Inventors: 李飞飞; 余钒源; 吴佳益; 叶玲; 王海溦; 王怡天; 张宇衡
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2022-07-15
Anticipated expiration: 2042-05-20
Also published as: CN114751960B

Abstract

The invention relates to a polypeptide and application thereof in bone repair, belonging to the technical field of biological medicines. The amino acid sequence of the polypeptide provided by the invention is shown in SEQ ID NO. 1. The invention also discloses the application of the polypeptide GC16 in bone injury and/or bone repair. Furthermore, the invention also discloses a polypeptide scaffold for bone repair. The polypeptide GC16 can attract and recruit cells to enter 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 bone repair

Technical Field

The invention belongs to the technical field of biological medicines, and relates to an artificially synthesized polypeptide GC16 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 grafting 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 personalized scaffold materials prepared by 3D printing and the like can well match with a defect region, and a loose and porous structure of the scaffold material 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, thereby forming new bone tissue. Among the drugs currently approved by the FDA to promote new bone formation, parathyroid hormone (PTH) can cause osteosarcoma formation when ingested at high doses, 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 realize 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 invention contains 16 amino acids and has the amino acid sequence GPGGDKCKCHGLSGSC, namely Gly Pro Gly Gly Asp Lys Cys Lys Cys His Gly Leu Ser Gly Ser Cys, and the inventor names the polypeptide as GC16, which is used for the following polypeptides.

The polypeptide of the invention has molecular weight of 1505.69 Da.

The polypeptide GC16 of the invention can adopt conventional synthetic methods, such as liquid phase segmented synthesis, solid phase synthesis, biological synthesis and the like, and as a preferred embodiment of the polypeptide of the invention, the polypeptide is synthesized by adopting 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 in bone injury and/or bone repair. Furthermore, the bone defect repairing liquid is suitable for wide bone defect indications, such as amputation 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.

GC16 was designed from a recognition segment of WNT3A ligand protein, which binds to cell membrane Frizzled receptor and LRP5/6 co-receptor, and the protein can activate canonical Wnt signaling pathway, activate intramembrane beta-catenin nuclear entry, and thus initiate transcription of downstream functional genes. A large number of studies indicate that the canonical Wnt signaling pathway is involved in regulating the various stages of osteoblast lineage cell survival, proliferation, and differentiation, and its main functions include: regulate osteogenic differentiation of osteogenic precursor cells, promote osteoblast proliferation and improve the survival rate of osteoblasts and osteocytes. Therefore, based on the in vivo experimental data of GC16, we speculate that it can play a role similar to WNT3A protein, activating bone repair corresponding to canonical WNT signaling pathway.

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

Preferably, the polypeptide GC16 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 GC16 can be loaded on a bone repair material in any form and implanted into a bone defect part. For example, the polypeptide GC16 can be supported in bone cement, injected into the bone injury site in combination with bone repair hydrogel, or used as an implant surface coating, etc.

The GC16 can accelerate the repair of a bone defect area and promote bone regeneration in an action mode of carrying a 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, a rat skull defect model is constructed, GC16 is implanted into a bone defect area, skull tissues are taken after 4 weeks and 8 weeks, Micro-CT scanning is used for scanning the tissues, three-dimensional views of the tissues are reconstructed, the relative bone density of new bones in the defect area is analyzed, and the tissue morphology is observed through tissue sections, HE and Masson staining. The osteogenesis effect of the polypeptide is comprehensively analyzed through Micro-CT, HE and Masson dyeing results, and compared with ineffective peptide, the polypeptide GC16 can obviously accelerate skull defect repair, so that the polypeptide GC16 has the potential of being used as a bone tissue engineering functional factor for treating bone defects.

Preferably, the polypeptide drug GC16 can attract and recruit cells into the scaffold material, promote the formation of inflammatory organization and fibrous tissue at 4 weeks, complete defect connection, and promote the repair of a defect area by fibrous tissue with higher mineralization degree at 8 weeks, thereby accelerating the repair and regeneration of bone defects, and is proved to be an effective functional factor of bone tissue engineering.

Further, the invention discloses a bone repair composition comprising a therapeutically effective amount of polypeptide GC16 and a tissue engineering acceptable carrier.

Preferably, the invention also discloses a polypeptide scaffold which comprises the polypeptide GC 16. 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, GC16 is uniformly dispersed in a methacrylic anhydrified gelatin (GelMA) scaffold in the polypeptide scaffold of the present invention.

Preferably, the concentration of polypeptide GC16 in the polypeptide scaffold is 25-150. mu.g/mL, preferably 100-150. mu.g/mL, and more preferably 100. mu.g/mL.

Methacryloylated gelatin (GelMA) is a photosensitive biomaterial, and when its powder is dissolved in a liquid such as water, it can be uniformly mixed with functional factors. 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 GC16 modified GelMA scaffolds can 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 cured bone has certain elasticity, strength and support property, and the structure and partial functions of the defective bone are recovered.

The invention has the beneficial effects that:

1) the polypeptide drug GC16 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 GC16 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 GC16 provided by the invention belongs to small molecular polypeptide, and has the advantages of simple preparation process, low cost, high yield, 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 implantation of the polypeptide into rat skull defect for 4 weeks and 8 weeks.

FIG. 2 is a comparison of relative bone density (relative BMD) of neogenetic tissue in the defect area of each group 4 weeks and 8 weeks after implantation of the polypeptide into the skull of the rat.

FIG. 3 shows HE staining of groups 4 and 8 weeks after implantation of the polypeptide into the skull defect of rats.

FIG. 4 shows Masson staining of each group 4 weeks and 8 weeks after implantation of the polypeptide into the skull defect of rat.

Detailed Description

The following examples are given to further illustrate the invention and are not to be construed as limiting the invention to the examples described.

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 polypeptide according to the concentration of 0.1mg/mL, fully and uniformly mixing GC16 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, GC16 adopts solid phase polypeptide synthesis technology to synthesize polypeptide, HPLC is used for purifying the product, and the purity of the synthesized polypeptide is 96.25%; 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 the Polypeptides implanted into skull defects of rat on bone repair

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 loaded with polypeptide GC16, and the control group was implanted with GelMA scaffold loaded with null peptide. Skin reduction, suture, and re-disinfection.

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. And (4) carrying out three-dimensional reconstruction and relative bone density analysis by using Micro-CT self-contained analysis software.

As shown in fig. 1, the three-dimensional reconstruction diagram of the skull defect shows that: after 4 weeks of material implantation, the ineffective peptide group defect area hardly had new bone formation, and the polypeptide GC16 significantly promoted bone formation at the edge of the defect area, so that the bone defect area was significantly reduced. At 8 weeks, GC16 promoted the new repair bone even further into the center of the defect, while the control new bone was only present in the marginal area of the defect.

As shown in fig. 2 relative bone density analysis results: relative bone density of the neonatal bone was significantly increased in the GC16 group compared to the null peptide control group at 4 weeks after material implantation, while the relative bone density was further increased in the GC16 group at 8 weeks compared to 4 weeks and still had a significant statistical difference from the control group. P < 0.05, p < 0.01.

In conclusion, GC16 can accelerate bone repair at a bone defect site, increase bone density of new bone, and promote bone repair.

Example 3 morphological Observation of neogenetic tissue in defect area after implantation of polypeptide into rat skull defect

3.1 sample preparation: rat skull tissue was soaked in 12% EDTA at pH 7.0 for 6 weeks to decalcify the bones soft enough.

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

3.3 result verification: the tissue section is baked for 2 hours at the temperature of 65 ℃, xylene and gradient ethanol are dewaxed and hydrated, and the histological morphology of the new tissue in the defect area is observed by HE and Masson staining.

As shown by the HE staining results in fig. 3: after 4 weeks of material implantation, the polypeptide GC-16 attracted well recruited cells into the scaffold compared to the control null peptide group, no significant scaffold retention was seen in the defect area, and the defect was repaired by organized inflammatory and fibrous tissue. After 8 weeks of material implantation, a large amount of blank gel remained in the center of the defective area of the ineffective peptide control group, the fibrous tissues are only connected with the defective area around the material, a small amount of mature bone is formed in the connected tissues, and the defective area of the GC16 group is repaired by the fibrous tissues with higher mineralization degree.

As shown in fig. 4Masson staining results: polypeptide GC16 can recruit cells into the material, the connection of the bone defect area is completed by purple red organized tissue and partial blue fiber tissue at 4 weeks, the connection of the new tissue in the defect area is completed by fiber tissue with higher blue-staining mineralization degree at 8 weeks, while the control ineffective peptide group and the center of the defect area at 4 weeks and 8 weeks both retain a large amount of scaffold material without cells growing in, and the connection is completed by only the fiber tissue at the periphery. The results of the histological staining together suggest that GC16 has the effects of recruiting cell growth, and accelerating bone defect repair and new bone formation.

SEQUENCE LISTING

<110> Sichuan university

<120> polypeptide and application thereof in promoting bone repair

<130> GC16

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

<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, 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 mu g/mL.

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