Polypeptide and application thereof in dermal tissue repair

Abstract

The invention provides a polypeptide and application thereof in dermis tissue repair. The polypeptide comprises an amino acid sequence as shown in SEQ ID NO. 1. The polypeptide can effectively promote angiogenesis and is used for preparing a material for promoting dermis tissue repair. The polypeptide and hydrogel can be used for preparing 3D printing dermis scaffold bio-ink together, an artificial dermis scaffold is prepared through a 3D printing technology and used for repairing dermis tissue, the problem that the vascularization speed is too low in the wound healing process can be effectively solved, the wound healing speed is increased, and the time needed for wound healing is shortened.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a polypeptide and its application in dermal tissue repair. Background Art

[0002] The skin is the largest organ in the human body, accounting for about 16% of the body weight. The skin is divided into three parts: epidermis, dermis, and subcutaneous tissue. The epidermis is a stratified squamous epithelium in histology, mainly composed of keratinocytes, melanocytes, Langerhans cells and Merkel cells. Among them, keratinocytes are divided into 5 layers according to the differentiation stage and characteristics, from deep to shallow, they are basal layer, spinous layer, granular layer, clear layer and stratum corneum. The dermis is differentiated from the mesoderm and can be divided into papillary layer and reticular layer from shallow to deep. The dermis is an irregular dense connective tissue in histology, composed of fibers (including collagen fibers, reticular fibers, elastic fibers), matrix and cell components, among which the fiber components are the main ones, and there are a small amount of matrix and cell components between the fibers. The subcutaneous tissue is located below the dermis, connected to tissues such as the fascia below, and is composed of loose connective tissue and fat lobules, also known as the subcutaneous fat layer. It contains blood vessels, lymphatic vessels, nerves, small sweat glands and apocrine sweat glands.

[0003] Skin has multiple functions such as protection, absorption, sensation, secretion, excretion, temperature regulation and metabolism. The treatment of large-area skin defects caused by burns, scalds, trauma and ulcers is a major clinical problem. Wound repair materials are often used clinically to promote wound healing. Currently, most wound repair materials only have the effects of promoting healing and antibacterial. The repair of the dermis depends on the regeneration rate of blood vessels. Accelerating vascularization is beneficial to the regeneration of dermal tissue.

[0004] Therefore, there is a need in the art for a repair material to solve the problem of slow vascularization during wound healing and to promote rapid repair of dermal tissue. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a polypeptide and its application in dermal tissue repair, wherein the polypeptide has the effect of promoting angiogenesis, thereby effectively promoting the regeneration and repair of dermal tissue.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] The first aspect of the present invention provides a polypeptide for promoting angiogenesis, wherein the polypeptide comprises an amino acid sequence as shown in SEQ ID NO.1.

[0008] The second aspect of the present invention provides the use of the polypeptide as described above in the preparation of a vascularized dermal repair material.

[0009] In some embodiments, the pro-vascularized dermal repair material is a dermal repair scaffold.

[0010] The third aspect of the present invention provides the use of the polypeptide as described above in the preparation of 3D printed dermal scaffold bio-ink.

[0011] The fourth aspect of the present invention provides a 3D printed dermal scaffold bio-ink, which contains the following components in mass concentrations: 0.1% to 5% of the polypeptide, 1% to 10% of the photocurable hydrogel, and 0.2% to 0.5% of the initiator.

[0012] In some embodiments, the bio-ink comprises the following components in mass concentrations: 0.1% to 3% of the polypeptide, 1% to 5% of the photocurable hydrogel, and 0.2% to 0.5% of the photoinitiator.

[0013] In some embodiments, the photocurable hydrogel is selected from at least one of methacrylated gelatin and methacrylated hyaluronic acid; the initiator is selected from at least one of LAP, photoinitiator 184, and photoinitiator 819.

[0014] A fifth aspect of the present invention provides an artificial dermal scaffold, which is prepared by 3D printing using the bio-ink as described above.

[0015] The sixth aspect of the present invention provides a method for preparing the artificial dermal scaffold as described above, comprising the following steps: (1) pouring the biological ink into a printing barrel, fixing the printing barrel on a 3D printer nozzle, installing a printing needle, setting 3D printing parameters, and performing aseptic printing to obtain an artificial dermal scaffold model; (2) light-curing and cross-linking the artificial dermal scaffold model, freezing it, soaking it to remove the photoinitiator, and vacuum freeze-drying it to obtain the artificial dermal scaffold model.

[0016] In some embodiments, the 3D printing parameters include: a fiber diameter of 100 μm to 300 μm, a fiber spacing of 100 μm to 300 μm, a thickness of 3000 μm to 4000 μm, and a nozzle movement speed of 0.5 mm / s to 15 mm / s.

[0017] After extensive research and screening, the present invention has obtained a polypeptide that can effectively promote angiogenesis. The polypeptide can be used together with hydrogel to prepare 3D printed dermal scaffold bio-ink. Through 3D printing technology, an artificial dermal scaffold is prepared for repairing dermal tissue. The artificial dermal scaffold contains the polypeptide of the present invention, which can effectively solve the problem of slow vascularization speed in the process of wound healing, promote the regeneration and repair of dermal tissue, accelerate the speed of wound healing, and shorten the time required for wound healing. DETAILED DESCRIPTION

[0018] For ease of understanding of the present invention, the present invention will be described more fully below with reference to the embodiments, and preferred embodiments of the present invention are given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should be understood that the experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. The various commonly used reagents used in the embodiments are all commercially available products.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0020] The following is a description with reference to specific embodiments.

[0021] The polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID NO.1.

[0022] SEQ ID NO. 1: ALALALMFRKPIPSTVKGEE.

[0023] The amino acid sequence of the polypeptide used in the following examples is shown in SEQ ID NO.1. The polypeptide can be synthesized by conventional technical means in the art, such as solid phase synthesis (preferably FMOC method), or it can be commissioned to a third-party biological company for synthesis. The synthesized polypeptide is used for subsequent research after being confirmed to be correct.

[0024] Example 1

[0025] This embodiment provides a 3D printed dermal scaffold bio-ink, which contains the following components in mass concentrations: 1.5% of the polypeptide, 2% of methacryloyl hyaluronic acid, and 0.35% of initiator LAP.

[0026] The 3D printed dermal scaffold bio-ink is prepared by the following method: taking the initiator LAP according to the formula, adding it to the PBS buffer, fully dissolving it, filtering and sterilizing it with a 0.22um filter membrane to obtain a LAP solution; weighing methacryloyl hyaluronic acid (HAMA) according to the formula, sterilizing it with acetone; under sterile conditions, adding HAMA to the LAP solution and fully dissolving it; then weighing the polypeptide according to the formula, adding it to water to fully dissolve it, and then adding it to the LAP solution containing HAMA, mixing it evenly, to obtain the product.

[0027] Example 2

[0028] This embodiment provides a 3D printed dermal scaffold bio-ink, which contains the following components in mass concentrations: 3.5% of the polypeptide, 1% of methacryloyl hyaluronic acid, and 0.2% of initiator LAP.

[0029] The preparation method of the 3D printed dermal scaffold bio-ink is the same as that in Example 1.

[0030] Example 3

[0031] This embodiment provides a 3D printed dermal scaffold bio-ink, which contains the following components in mass concentrations: 0.5% of the polypeptide, 8% of methacrylated gelatin, and 0.5% of initiator LAP.

[0032] The preparation method of the 3D printed dermal scaffold bio-ink is the same as that in Example 1.

[0033] Example 4

[0034] The bio-ink prepared in the above-mentioned Examples 1 to 3 is subjected to 3D printing to obtain an artificial dermal scaffold, and the method is as follows: (1) the bio-ink is poured into a printing barrel, the printing barrel is fixed on the nozzle of a 3D printer, a printing needle is installed, and the 3D printing parameters are set: the fiber diameter is 200 μm, the fiber spacing is 100 μm, the thickness is 3500 μm, and the nozzle movement speed is 10 mm / s, and aseptic printing is performed to obtain an artificial dermal scaffold model; (2) the artificial dermal scaffold model is irradiated with blue light (wavelength 405 nm) to be cured and cross-linked, and then placed in a -40°C refrigerator for freezing, and then soaked in deionized water to remove the photoinitiator, and finally vacuum freeze-dried to obtain the artificial dermal scaffold, which is irradiated and sterilized for use.

[0035] The artificial dermis scaffolds obtained by 3D printing using the bio-ink in Examples 1 to 3 are respectively named as artificial dermis scaffolds 1 to 3. The dermis tissue repair effects of the prepared artificial dermis scaffolds 1 to 3 are tested.

[0036] 30 BALB / C nude mice aged 6-8 weeks were selected, and 10% chloral hydrate solution was intraperitoneally injected for anesthesia. After the nude mice were anesthetized, the back skin was fixed and disinfected in a prone position, and then a full-thickness skin defect was prepared with scissors, and the wound size was 2cm*2cm. The nude mice with full-thickness skin defect were randomly divided into 5 groups, 6 in each group: blank group, control group, experimental group 1, experimental group 2 and experimental group 3. Among them, the blank group covered the wound surface of the model nude mice with medical gauze material; the control group covered the wound surface of the model nude mice with a control dermal scaffold, and the control dermal scaffold contained no polypeptide except that the raw materials and preparation methods of the control dermal scaffold were the same as those in Example 1; the experimental group 1 directly covered the wound surface of the model nude mice with artificial dermal scaffold 1; the experimental group 2 directly covered the wound surface of the model nude mice with artificial dermal scaffold 2; the experimental group 3 directly covered the wound surface of the model nude mice with artificial dermal scaffold 3. The wound tissue repair was observed at different time points, the size of the wound was measured and recorded with a ruler, and the wound contraction rate was calculated, and the average value of 6 mice in each group was taken.

[0037] The calculation formula of wound contraction rate is as follows: [(So-St) / So]×100%, where So is the area of ​​the original wound and St is the area of ​​the wound at different observation time points.

[0038] The results are shown in Table 1:

[0039] Table 1

[0040] Group 3 days after surgery 7 days after surgery 14 days after surgery Blank Group 13.8％ 50.3％ 64.7％ Control group 16％ 63.2％ 88.6％ Experimental Group 1 27.6％ 66.9％ 96.3％ Experimental Group 2 31.3％ 68.3％ 97.8％ Experimental Group 3 25.2％ 64.6％ 91.6％

[0041] The above results show that the artificial dermal scaffold of the present invention can effectively promote the repair of dermal tissue and promote wound healing.

[0042] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0043] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A polypeptide that promotes angiogenesis, characterized in that: The polypeptide comprises the amino acid sequence shown in SEQ ID NO.

1.

2. Use of the polypeptide according to claim 1 in the preparation of vascularization-promoting dermal repair materials.

3. The use according to claim 2, characterized in that The vascularization-promoting dermal repair material is a dermal repair scaffold.

4. Use of the polypeptide as claimed in claim 1 in preparing 3D printed dermal scaffold bio-ink.

5. A 3D printed dermal scaffold bio-ink, characterized in that: The biological ink comprises the following components in mass concentrations: 0.1% to 5% of the polypeptide, 1% to 10% of the photocurable hydrogel, and 0.2% to 0.5% of the initiator.

6. The bio-ink according to claim 5, characterized in that: The biological ink comprises the following components in mass concentrations: 0.1% to 3% of the polypeptide, 1% to 5% of the photocurable hydrogel, and 0.2% to 0.5% of the photoinitiator.

7. The bio-ink according to claim 5 or 6, characterized in that: The photocurable hydrogel is selected from at least one of methacrylated gelatin and methacrylated hyaluronic acid; the initiator is selected from at least one of LAP, photoinitiator 184, and photoinitiator 819.

8. An artificial dermis scaffold, characterized in that: The artificial dermal scaffold is prepared by 3D printing using the biological ink as described in any one of claims 5 to 7.

9. The method for preparing the artificial dermis scaffold according to claim 8, characterized in that: The method comprises the following steps: (1) pouring the biological ink into a printing material cylinder, fixing the printing cylinder on a nozzle of a 3D printer, installing a printing needle, setting 3D printing parameters, and performing aseptic printing to obtain an artificial dermal scaffold model; and (2) light-curing and cross-linking the artificial dermal scaffold model, freezing it, soaking it to remove a photoinitiator, and vacuum freeze-drying it to obtain the artificial dermal scaffold model.

10. The preparation method according to claim 9, characterized in that: The 3D printing parameters include: fiber diameter of 100 μm to 300 μm, fiber spacing of 100 μm to 300 μm, thickness of 3000 μm to 4000 μm, and nozzle movement speed of 0.5 mm / s to 15 mm / s.