Use of a polypeptide in promoting cartilage regeneration or repair

Abstract

The present invention discloses the use of Histatin-1 polypeptide in promoting cartilage regeneration or repair. By preparing a combined reagent composed of Histatin-1 polypeptide, a biological scaffold material, etc., mainly by loading linear Hst1 on gelatin methacrylate, it is used for cartilage injury repair or promoting cartilage growth. At the same time, the use of Histatin-1 polypeptide in promoting the expression of one or more of the bone differentiation indicators, including collagen fibers, glycosaminoglycans, type II collagen, and aggrecan, is also disclosed.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, specifically, to the field of cartilage regeneration or repair, and particularly to the use of Histatin-1 polypeptide in promoting cartilage regeneration or repair. Background Art

[0002] Cartilage consists of cartilage tissue and the surrounding perichondrium, and the cartilage tissue is composed of chondrocytes, matrix and fibers. For example, articular cartilage or the cartilage of the temporomandibular joint (TMJ).

[0003] Articular cartilage is a thin layer of highly hydrated, avascular, aneural, viscoelastic connective tissue that covers joint surfaces to provide lubricated joint movement and transmits mechanical forces to subchondral bone. Depending on the cartilage morphology and collagen orientation, articular cartilage exhibits a typical layered structure. The superficial zone: is characterized by slender and flattened chondrocytes with parallel and thin collagen fibers. The middle zone contains round chondrocytes and randomly oriented thick collagen fibers. In contrast, the deep zone has round and vertically arranged chondrocytes, as well as thick collagen fibers orthogonal to the contact surface. The calcified zone has round and hypertrophic chondrocytes and distinct mineralized type I collagen. The calcified zone is connected to bone tissue.

[0004] Osteochondral defects of the temporomandibular joint (TMJ) can be caused by acute condylar injury, overloading or abnormal immune responses. Even some daily activities (such as speaking, eating and yawning) may cause pain and limited mandibular movement, which will trouble patients with condylar osteochondral defects throughout their lives.

[0005] Repairing osteochondral defects in the TMJ is very challenging due to the limited self-repair potential of cartilage. First, the avascular nature of condylar cartilage tissue results in the inability to produce classic healing responses, such as coagulation, inflammation, blood invasion, and aggregation of multifunctional mesenchymal stem cells (MSCs). Second, the migration and proliferation of chondrocytes around the defect area are poor. This results in almost no self-repair ability of TMJ cartilage. Third, when the defect further expands into the subchondral bone tissue, the blood supply from the bone tissue will trigger the classic healing response to some extent and enhance the migration of MSCs. However, the blood supply and MSCs migration from the subchondral bone are limited and are not sufficient to completely repair the osteochondral defect. At the same time, the osteochondral defect reduces the soft surface area of ​​the condyle, resulting in mechanical overload of the remaining TMJ tissue, thereby causing secondary mechanical damage to the TMJ. At present, autologous transplantation is mainly used in clinical practice to repair osteochondral defects, such as autologous chondrocyte implantation and mosaicplasty. These repair pathways provide chondrocytes to the defect area and have certain effectiveness in the repair of osteochondral defects. However, the use of autologous transplantation is greatly limited due to the limited site and size of the autologous graft, postoperative pain in the donor site, and a series of complications such as possible arthritis. Therefore, finding the best method to repair osteochondral defects has always been a hot topic in the field of TMJ repair.

[0006] Cartilage tissue engineering combines biomaterial scaffolds with bioactive agents or stem cells to produce composite materials for tissue reconstruction, which has broad prospects. Scaffolds are an important component of tissue engineering, providing support for cell migration and new tissue generation in the defect area.

[0007] It is expected that the complete repair of osteochondral defects can be achieved by carrying stem cells (MSCs) or bioactive agents on biomaterial scaffolds, but compared with carrying MSCs, tissue engineering technology carrying bioactive agents has a series of advantages such as low cost, wide source, and simple operation. Therefore, it is urgent to find a more ideal bioactive agent that can induce angiogenesis and the migration of MSCs in the body from the bone defect area to the cartilage lesion area, thereby promoting the repair of osteochondral defects.

[0008] Histamine is a group of cationic polypeptides rich in histidine secreted by the human parotid and submandibular glands, with a length of 7 to 38 amino acid residues. Histamine 1, 3, and 5 are the three most important types. In recent years, histamine 1 (Histatin-1) polypeptide has been deeply studied in the biomedical field. It has the effects of promoting cell adhesion, extension, migration, angiogenesis, and anti-inflammatory.

[0009] CN107108751A discloses that histatin can be used to treat dental demineralization and help achieve remineralization of the tooth enamel surface; Patent Application CN108785657A submitted by the inventor of the present application in 2018 discloses the application of Histatin-1 polypeptide in the preparation of a composite material for promoting the repair of large-area skin defects; however, there is currently no relevant report on the application of Histatin-1 polypeptide in promoting the repair of osteochondral defects or promoting cartilage growth. Summary of the Invention

[0010] Aiming at the problems existing in the prior art, the present invention provides a Histatin-1 polypeptide and its related composition that can be used to prepare a preparation for repairing cartilage damage or promoting cartilage growth, and discloses that the composition has the effects of promoting the expression of type II collagen and / or aggrecan.

[0011] On the one hand, the present invention provides the use of Histatin-1 polypeptide in the preparation of a reagent for promoting cartilage regeneration or repair.

[0012] In the prior art, it is considered that the Histatin-1 (Hst1) polypeptide has the ability to promote the adhesion and migration of epithelial cells, fibroblasts and osteoblasts. At the same time, it can promote cell metabolic activity and maintain cell viability under various adverse environments. However, these functions are essentially different from promoting cartilage repair and cartilage growth.

[0013] The cartilage described in the present invention includes all cartilage tissues, such as articular cartilage or cartilage of the temporomandibular joint (TMJ), etc.

[0014] The repair of cartilage injury or defect requires guiding stem cell homing, stem cell chondrogenic differentiation, chondrocyte proliferation, and the process of hypertrophy, etc. Mesenchymal stem cells have multiple potential differentiation directions, such as osteogenic, chondrogenic, myogenic, and adipogenic directions. These directions are respectively regulated by Runx2, Sox9, MyoD, and PPARγ as key transcription factors, which induce the expression of corresponding downstream pathways. However, the same stem cell can only differentiate in one direction. The high expression of one transcription factor will inhibit the expression of other transcription factors, thereby inhibiting other differentiation directions. Among these factors, the key transcription factor for osteogenic differentiation is Runx2. This protein is a member of the RUNX transcription factor family and has a Runt DNA-binding domain. This is crucial for osteoblast differentiation and skeletal morphogenesis. It acts as a scaffold for nucleic acids and regulatory factors involved in bone gene expression. This protein can bind to DNA either as a monomer or as a subunit of a heterodimeric complex. Transcriptional variants of genes encoding different protein isoforms are generated by using alternative promoters and alternative splicing. The cellular dynamics of Runx2 protein is also important for proper osteoblast differentiation. Runx2 protein is detected in osteoblasts, with upregulated expression in immature osteoblasts and downregulated expression in mature osteoblasts. It is the first transcription factor required to determine osteoblast commitment, followed by Sp7 and Wnt signaling. Runx2 is responsible for inducing the differentiation of pluripotent mesenchymal cells into immature osteoblasts and activating the expression of several key downstream proteins that maintain osteoblast differentiation and bone matrix genes. The knockout of DNA-binding activity leads to the inhibition of osteoblast differentiation. Therefore, Runx2 is usually referred to as the master regulator of bone. BMP-2 can directly upregulate Runx2 through the p-Smad signaling pathway, thereby inducing osteogenic differentiation.

[0015] Although the Histatin-1 polypeptide has the ability to promote the adhesion and migration of epithelial cells, fibroblasts, and osteoblasts. At the same time, it can promote cell metabolic activity and maintain cell viability under various adverse environments. However, the research of the present invention finds that this polypeptide can promote the repair or growth of cartilage damage, and its mechanism of action may be one or a combination of the following: First, the Histatin-1 polypeptide promotes angiogenesis, which is important for bone defects. Cartilage requires blood vessels to deliver nutrients and stem cells, but there cannot be blood vessels in cartilage. Therefore, the mechanism of Hst1 promoting blood vessels is more to assist cartilage formation rather than directly inducing cartilage regeneration. Second, initial cartilage islands mainly appear around the cartilage wall. Therefore, the Histatin-1 polypeptide may promote chondrocytes to express a large number of cartilage differentiation-promoting factors (such as factors like Runx2, Sox9, MyoD, and PPARγ, etc.), thereby inducing stem cells guided by the Histatin-1 polypeptide to enter cartilage differentiation. Third, the present invention also finds that the Histatin-1 polypeptide significantly promotes the expression of cartilage-related proteins, such as type II collagen and / or aggrecan; thus indicating that the Histatin-1 polypeptide can significantly promote the high expression of osteogenic differentiation indicators.

[0016] Furthermore, the Histatin-1 polypeptide can promote the expression of osteogenic differentiation indicators, and the osteogenic differentiation indicators include one or more of collagen fibers, glycosaminoglycans, type II collagen, and aggrecan.

[0017] Research has proven that the Histatin-1 polypeptide can increase the percentage of the area of one or more of the newly formed subchondral bone, collagen fibers of cartilage, and glycosaminoglycans (GAG) in the cartilage defect area accounting for the total defect area.

[0018] Research has proven that the Histatin-1 polypeptide can promote cartilage regeneration or repair by promoting the expression of type II collagen and / or aggrecan.

[0019] Furthermore, the Histatin-1 polypeptide is derived from a polypeptide isolated from saliva or a synthetic polypeptide.

[0020] In some ways, the Histatin-1 polypeptide can be derived from a polypeptide isolated from saliva; of course, the Histatin-1 polypeptide can also be synthesized artificially.

[0021] Furthermore, the synthetic polypeptide is linear Hst1; the linear Hst1 has the amino acid sequence shown in SEQ ID NO.1 in the sequence listing.

[0022] The synthetic polypeptide can be a synthetic linear Hst1 with the amino acid sequence: DSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN (SEQ ID NO.1).

[0023] Furthermore, the Histatin-1 polypeptide needs to be combined with a biological scaffold material to form a composition for use. The biological scaffold material includes gelatin, collagen, hyaluronic acid, chitosan, sodium alginate, heparin, polyvinyl alcohol, dextran, carboxymethyl cellulose, glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan, chitosan quaternary ammonium salt, hydrophilic or water-soluble animal and plant proteins, collagen, serum proteins, two-armed or multi-armed polyethylene glycol, polyethyleneimine, dendrimer, synthetic polypeptide, polylysine or (meth)acrylate or (meth)acrylamide, and one or more of the modifiers of the above components.

[0024] Biological scaffold reagents are generally reagents that carry active ingredients. This reagent can be safe with biological tissues, or maintain the release and slow release of the active cost, so that the active ingredient can exert its greater or better physiological functions. In some ways, the biological scaffold reagent can be some safe biological materials with good biocompatibility, such as natural polysaccharides (such as: hyaluronic acid, heparin, alginic acid, dextran, carboxymethyl cellulose, glycol chitosan, propylene glycol chitosan, chitosan lactate, carboxymethyl chitosan, chitosan quaternary ammonium salt, etc.) and their modifiers or degradation products; it can also be protein or polypeptide, such as various hydrophilic or water-soluble animal and plant proteins, collagen, serum proteins, gelatin and their modifiers, modified products and degraded polypeptides, etc.; it can also be hydrophilic or water-soluble synthetic polymers, such as two-armed or multi-armed polyethylene glycol, polyethyleneimine, dendrimer, synthetic polypeptide, polylysine or (meth)acrylate or (meth)acrylamide, or other polymer molecules, etc.

[0025] Furthermore, the biological scaffold material is gelatin methacrylate.

[0026] Gelatin methacrylate (Gel-MA) has a chemical composition similar to that of collagen and has good biocompatibility. Different from collagen, Gel-MA has no risk of pathogen transmission. Moreover, Gel-MA contains a large number of adhesion ligands, such as the arginine-glycine-aspartic acid sequence, which promotes cell adhesion and migration.

[0027] Gel-MA has good fluidity before curing, enabling it to flexibly match the defect areas of complex cavities; after photo-curing, Gel-MA changes from a liquid form to a hydrogel form and reaches a hardness of 50-60 KPa, which is beneficial to the formation of cartilage and solid tissues.

[0028] By using Gel-MA as a biological scaffold and carrying Histatin-1 polypeptide as a bioactive agent, it can effectively promote the repair of osteochondral defects.

[0029] Furthermore, in the composition, the mass ratio of Histatin-1 polypeptide to gelatin methacrylate is 2.5 - 50:211, or 50 - 1000 μg of Histatin-1 polypeptide is loaded on 21.1 μl of gelatin methacrylate.

[0030] Furthermore, in the composition, the mass ratio of Histatin-1 polypeptide to gelatin methacrylate is 25:211, or 500 μg of Histatin-1 polypeptide is loaded on 21.1 μl of gelatin methacrylate.

[0031] Furthermore, the composition further comprises stem cells and / or a photoinitiator.

[0032] In the composition, other auxiliary reagents are also included, such as stem cells, photoinitiators, etc.

[0033] The photoinitiator can modify the biological scaffold reagent to make the formed gel structure stable, and can assist in the treatment of cartilage damage and tissue repair. For example, it is modified with an o-nitrobenzyl-based photo-responsive group and irradiated with light, or hyaluronic acid modified with N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy)butyramide is mixed with chitosan, sodium alginate, polyvinyl alcohol, etc.

[0034] The preparation method of the composition reagent for promoting cartilage regeneration or repair according to the present invention is as follows: Dissolve 200 mg of freeze-dried Gel-MA macromolecular polymer in 1 ml of PBS, filter it with a bacterial filter at 80 °C to obtain a Gel-MA hydrogel prepolymer solution; Take 50 - 1000 μg of Histatin-1 polypeptide, dissolve it in 21.1 μl of Gel-MA hydrogel prepolymer solution, and simultaneously add appropriate auxiliary reagents such as a photoinitiator.

[0035] On the other hand, the present invention provides the use of Histatin-1 polypeptide in the preparation of a reagent for promoting the expression of one or more of collagen fibers, glycosaminoglycans, type II collagen, and aggrecan.

[0036] The present invention has been proven by a large number of studies that Histatin-1 polypeptide can promote the growth of the area of collagen fibers, glycosaminoglycans, the expression level of type II collagen, and aggrecan.

[0037] Furthermore, Histatin-1 polypeptide needs to be combined with a biological scaffold material to form a composition for use.

[0038] Furthermore, the biological scaffold material is gelatin methacrylate.

[0039] Furthermore, in the composition, the mass ratio of Histatin-1 polypeptide to gelatin methacrylate is 2.5-50:211, or 50-1000 μg of Histatin-1 polypeptide is loaded on 21.1 μl of gelatin methacrylate.

[0040] Furthermore, in the composition, the mass ratio of Histatin-1 polypeptide to gelatin methacrylate is 25:211, or 500 μg of Histatin-1 polypeptide is loaded on 21.1 μl of gelatin methacrylate.

[0041] Furthermore, the composition further comprises stem cells and / or a photoinitiator.

[0042] The beneficial effects of the present invention are as follows:

[0043] 1. It is disclosed that Histatin-1 polypeptide can be used for the preparation of reagents for cartilage injury repair or promoting cartilage growth; and a combined reagent for cartilage injury repair or promoting cartilage growth and its preparation method are provided.

[0044] 2. It is disclosed that Histatin-1 polypeptide can be used for the preparation of reagents for promoting the expression of one or more of collagen fibers, glycosaminoglycans, type II collagen, and aggrecan, and a combined reagent for promoting the expression of one or more of collagen fibers, glycosaminoglycans, type II collagen, and aggrecan and its preparation method are provided. Description of the Drawings

[0045] Figure 1 Schematic diagram for the preparation of a rabbit temporomandibular joint condyle osteochondral defect (3 mm * 3 mm) model in Example 2

[0046] Figure 2 Comparison diagram of the photos and macroscopic evaluation results of the rabbit condyle osteochondral defect in Example 2

[0047] Figure 3 Optical microscope photo of the H&E-stained tissue section of the rabbit condyle at the 4th week after surgery in Example 3

[0048] Figure 4 Comparison diagram of the quantitative analysis of the area of the newly formed cartilage and subchondral bone and the MODS score results for the repair of osteochondral defects at 1, 2, and 4 weeks after surgery in Example 3

[0049] Figure 5Comparison diagram of special staining results of osteochondral defects repaired in the Gel-MA group and Hst1 / Gel-MA group in Example 3

[0050] Figure 6 Comparison diagram of results of quantitative analysis of glycosaminoglycan (GAG) and collagen fibers in the defect area, newly formed cartilage, and newly formed subchondral bone at 2 weeks and 4 weeks after surgery in Example 3

[0051] Figure 7 Comparison diagram of optical microscope photos of immunohistochemical staining tissue sections of type II collagen and aggrecan and quantitative analysis of expression in the Gel-MA group and Hst1 / Gel-MA group in Example 3

[0052] Figure 8 Comparison diagram of optical microscope photos of H&E staining tissue sections of rabbit condyles at different Hst1 concentrations in Example 4 Specific implementation mode

[0053] The present invention will be further described in detail below in conjunction with embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0054] Example 1 Preparation of Gel-MA hydrogel prepolymer solution and Hst1

[0055] The Gel-MA lyophilized powder used in this example was purchased from Wenzhou Institute (Wenzhou Institute, Wenzhou, China).

[0056] Dissolve 200 mg of freeze-dried Gel-MA macromolecular polymer in 1 ml of PBS, which contains 0.5% (w / v) 2-hydroxy-1-(4-(hydroxyethoxy)phenyl)-2-methyl-1-propanone (photoinitiator 2959, CIBA Chemical Co., Ltd., Basel, Switzerland, concentration 50 mg / ml, add 0.42 μl), filter with a bacterial filter at 80 °C to obtain a prepolymer solution. Keep the prepolymer solution in a constant temperature water bath at 40 °C. Prepare a fresh hydrogel prepolymer solution before surgery and store it in a sterile bottle.

[0057] The freeze-dried linear Hst1 polypeptide was from the University of Amsterdam (Amsterdam, Netherlands, amino acid sequence as shown in SEQ ID NO.1) and stored at -20 °C. Dissolve 500 μg of freeze-dried Hst1 polypeptide in 21.1 μl (volume of the defect area) of Gel-MA prepolymer solution to prepare an implant reagent.

[0058] Example 2 Macroscopic evaluation of Hst1 on promoting cartilage repair

[0059] Fifty-four adult male New Zealand white rabbits (Zhejiang Chinese Medical University, IACUC - 20180625 - 04) were randomly divided into three groups, with 18 rabbits in each group. The right condyles of the 54 rabbits were surgically treated, while the left condyles were kept intact.

[0060] There were three groups in total. Among them, the first group was the control group where the defect area received no treatment; the second group was the Gel - MA group where Gel - MA hydrogel was implanted in the defect area; the third group was the Hst1 / Gel - MA group where Hst1 - functionalized Gel - Ma was implanted in the defect area (provided by Example 1).

[0061] After general anesthesia of the rabbits, a pre - auricular skin incision was made at the right temporomandibular joint to expose the condylar head, and an osteochondral defect with a diameter of 3 mm and a depth of 3 mm was created on the condyle using a drill bit with a diameter of 3 mm (as shown in Figure 1 A - C).

[0062] The first group was the control group. After the preparation of the defect area, no further treatment was carried out.

[0063] In the second group, Gel - MA hydrogel was injected into the defect area.

[0064] In the third group, Hst1 - functionalized Gel - MA hydrogel was injected into the defect site (as shown in Figure 1 D).

[0065] The Gel - MA and its prepolymer solution in the second and third groups were photo - polymerized using ultraviolet light (365 nm, 90 s) (as shown in Figure 1 E). The wound was sutured layer by layer with nylon sutures. After all steps were completed, the animals were sent back to the animal breeding center. In the first three days after surgery, the animals were intraperitoneally injected with penicillin (1:100000) every 24 hours.

[0066] At the 1st, 2nd, and 4th weeks after surgery, 6 animals were randomly selected from each group and euthanized. The right temporomandibular joint and its surrounding tissues were removed, and the gross specimens were macroscopically scored and statistically analyzed.

[0067] Among them, the macroscopic scoring evaluation of the gross specimens used the International Cartilage Repair Society (ICRS) macroscopic scoring system. The gross appearance of the defect site was photographed, and three independent scorers performed blind - review scoring. This scoring system evaluated the cartilage repair situation in the defect area from four aspects: the degree of repair, the integration of the border area, the macroscopic appearance, and the overall repair evaluation. The evaluation criteria are shown in Table 1.

[0068] Table 1. Macroscopic scoring criteria for gross specimens

[0069]

[0070] Statistical analysis was performed using SPSS 18.0 statistical analysis software (SPSS Inc, USA) for one-way ANOVA and Tukey's HSD post hoc test. All data were expressed as mean ± standard deviation (SD). P<0.05 indicated statistically significant differences, and P<0.01 and 0.001 indicated highly statistically significant differences.

[0071] All rabbits recovered well after surgery and were able to ingest sufficient food daily to maintain their original body weight. No complications occurred 4 weeks after surgery. All rabbits survived until they were sacrificed.

[0072] Tissues were harvested at 1, 2, and 4 weeks after surgery, respectively. Photographs and macroscopic evaluation results of the condylar osteochondral defects in rabbits are shown as Figure 2 follows, where the red circular area represents the original defect area, and the blue irregular area represents the enlarged absorption area; Group 1 control group is shown in Figure 2 A, D, G in; Group 2 Gel-MA group is shown in Figure 2 B, E, H in; Group 3 Hst1 / Gel-MA group is shown in Figure 2 C, F, I in; The ICRS macroscopic scoring results are shown in Figure 2 J-L in, representing the scoring results of the samples at 1, 2, and 4 weeks, respectively. (n = 6, **P<0.01; ***P<0.001).

[0073] As Figure 2 can be seen, in the Hst1 / Gel-MA group of Group 3, the boundary between the defect area and the surrounding normal cartilage tissue was obvious at 1 week after surgery, and only 25% of the defect boundaries were integrated ( Figure 2 C); at 2 weeks, the boundary between the defect area tissue and the surrounding cartilage became blurred, 50% of the defect boundaries were integrated, and the newly formed tissue repaired 50% of the defect depth ( Figure 2 F); at 4 weeks, the defect had no obvious boundary and was completely integrated with the surrounding cartilage. The inner surface of the defect area was smooth and intact, and the color and texture of the newly formed tissue were similar to those of the surrounding normal cartilage, repairing 75% of the defect depth ( Figure 2 I). In contrast, the defects in Group 2 were not fully filled with light red tissue, and there was still a depression on the defect surface at 4 weeks ( Figure 2 H). In Group 1, diffuse degenerative changes were observed at 4 weeks ( Figure 2 G). From 1 to 4 weeks, the ICRS macroscopic scores of Group 3 were significantly higher than those of Group 2 and Group 1. At the same time point, the differences between groups were statistically significant (p<0.05) (J-L in Figure 2). It can be seen that Hst1 has a very significant effect on promoting cartilage injury repair.

[0074] Example 3: Histological evaluation of Hst1 in promoting cartilage repair and its effects on promoting the expression of collagen fibers, glycosaminoglycan (GAG), type II collagen, and aggrecan

[0075] In this example, three groups of rabbits provided in Example 2 were used. At 1, 2, and 4 weeks after surgery, 6 animals were randomly selected from each group and euthanized. The right temporomandibular joint and its surrounding tissues were removed, and histological analysis of the gross specimens was performed, followed by statistical analysis.

[0076] The histological examination procedure was as follows: In a 4°C environment, the specimens for histological analysis were fixed in 10% neutral formalin (Sigma-Aldrich, USA) for 24 hours and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) buffer solution (Sigma-Aldrich, USA) for 28 days. The samples were embedded in paraffin (Sigma-Aldrich, USA), cut into 4-μm thick sagittal sections, and stained with hematoxylin and eosin, Masson's trichrome, and alcian blue (Sigma-Aldrich, USA). Immunohistochemical detection of the content of type II collagen and aggrecan was also performed on the sections. All sections were observed and photographed using an optical microscope (CX51; Olympus) and a digital charge-coupled device camera (TrueChrome Metrics; Tucsen Photonics). These sections were blindly scored by 3 experienced pathologists using a modified O'Driscoll scoring system (MODS).

[0077] The procedure for histological analysis was as follows: In all sample sections, the original defect area was a 3×3 mm square. The top of the square was flush with the cartilage surface on both sides. The percentage of the area of newly formed subchondral bone, cartilage, collagen fibers, and glycosaminoglycan (GAG) in the defect area was detected using image analysis software (image pro plus 6.0). The integrated optical density (IOD) of type II collagen and aggrecan was measured by image pro plus 6.0 to represent their respective contents.

[0078] Tissues were retrieved at 4 weeks after surgery for histological processing and sectioning. Optical microscope photographs of H&E-stained tissue sections of rabbit condyles are shown as Figure 3 follows. Among them, according to the implants in the defect area, it was divided into the first group, the control group ( Figure 3 A, A1 - A2 in Figure 3 ), the second group, the Gel-MA group ( Figure 3The dashed square area in the middle is the original defect area (3mm * 3mm). Scale bar of A - C = 500μm. A1: The defect surface is covered by fibrous tissue; A2: Osteoclasts can be seen between the subchondral bones in the defect area; B1: Fibrous tissue crawls into the defect area among the undegraded scaffold materials; B2: Subchondral bone neogenesis in the defect area; C1: The chondrocytes in the defect area are arranged in layers; C2: Subchondral bone neogenesis in the defect area, with a large number of osteoblasts and osteoclasts infiltrating. Solid arrow: Osteoclast; Hollow arrow: Osteoblast; Five - pointed star: Immature chondrocytes; Triangle: New blood vessels. Scale bar of A1 - C2: 50μm.

[0079] The quantitative analysis of the area of newly formed cartilage and subchondral bone and the MODS scoring results for the repair of osteochondral defects at 1, 2, and 4 weeks after surgery are as Figure 4 shown. Among them, Figure 4 A in it is the quantitative analysis of the area of newly formed cartilage, B is the quantitative analysis of the area of subchondral bone, and C, D, and E are the MODS scoring results for the repair of osteochondral defects at 1, 2, and 4 weeks after surgery respectively. (n = 6, *P < 0.05, ***P < 0.01, ***P < 0.001).

[0080] From Figure 4 A and B, it can be seen that at 1, 2, and 4 weeks, the percentage of the area of newly formed subchondral bone and cartilage in the defect area of the third group was significantly higher than that of the second group and the first group (P < 0.05) ( Figure 4 A - B in it). Moreover, at 1, 2, and 4 weeks, the MODS score of the third group was significantly higher than that of the first and second groups. At the same time point, there were statistical differences in the scores among groups ( Figure 4 C - E in it). It can be seen that the Hst1 / Gel - MA group can significantly promote the repair of subchondral bone and cartilage.

[0081] At 1 week, new tissue was observed in the pores of the undegraded scaffold in the third group. At 2 weeks, newly formed cartilage and bone tissues gradually grew, penetrated, and replaced the scaffold. Newly formed cartilage islands could be seen in the undegraded scaffold. At 4 weeks, the newly formed cartilage proliferated and migrated to the center of the defect. In addition, from Figure 3 C, C1, and C2, it can be seen that the newly formed chondrocytes showed a typical cartilage structure and were arranged in columns ( Figure 3 C1 in it); there was a fibrous layer above the layer of newly formed chondrocytes, which was similar to the fibrous layer in normal mandibular condyle cartilage ( Figure 3 C1 in it); at the bottom of the defect, the reconstruction of subchondral bone was observed, with a large number of osteoblasts and osteoclasts infiltrating ( Figure 3 C2 in it); the formation of new blood vessels could be detected at the junction between subchondral bone and the functionalized hydrogel ( Figure 3 C2 in it). At the same time, from Figure 3It can be seen that compared with Group 3, in Group 2 at 4 weeks, only a small amount of cell infiltration could be detected in the undegraded scaffolds. A large amount of fibrous tissue and some undegraded scaffold materials filled the defect area ( Figure 3 B-B1 in). The defect area in Group 1 remained hollow, and only a layer of fibrous tissue covered the bottom of the defect area ( Figure 3 A-A1 in). Therefore, compared with the control group, Hst1 has a very significant effect on promoting the repair of cartilage injury.

[0082] The special staining and quantitative analysis of the osteochondral defects repaired in Group 2 (Gel-MA group) and Group 3 (Hst1 / Gel-MA group) are as Figure 5 shown, in which Masson trichrome staining was performed at 2 weeks ( Figure 5 A, B) and 4 weeks ( Figure 5 E, F) after surgery. Alcian blue staining was performed at 2 weeks ( Figure 5 C, D) and 4 weeks ( Figure 5 G, H) after surgery. Scale bar for A-H = 1 mm; scale bar for A1-H1 = 100 μm.

[0083] At 2 weeks and 4 weeks after surgery, glycosaminoglycan (GAG) and collagen fibers in the defect area, newly formed cartilage, and newly formed subchondral bone were quantitatively analyzed, and the results are as Figure 6 shown, in which, Figure 6 A and B are the percentage of GAG area in the defect area at 2 weeks and 4 weeks, C and D are the percentage of collagen fiber area in the newly formed cartilage at 2 weeks and 4 weeks, and E and F are the percentage of collagen fiber area in the subchondral bone at 2 weeks and 4 weeks. (n = 6, **P < 0.01, ***P < 0.001, NS = not significant).

[0084] As can be seen from Figure 5 and Figure 6 , in Group 3, at 2 and 4 weeks, it was confirmed by Alcian blue staining and Masson trichrome staining ( Figure 5 A-H1 in) that the percentage of the collagen fiber area in the newly formed cartilage and subchondral bone in the total area of the defect area, and the percentage of the glycosaminoglycan (GAG) area in the defect area in the total area of the defect area were significantly higher than those in Group 2 and Group 1 (P < 0.05) ( Figure 6 A-F). Therefore, compared with the control group, Hst1 can significantly promote the expression of collagen fibers and glycosaminoglycan (GAG) in the newly formed subchondral bone and cartilage.

[0085] At 2 weeks and 4 weeks after surgery, the tissues were taken out, sectioned and processed by immunohistochemistry. The photomicrographs of immunohistochemical staining of type II collagen and aggrecan in the tissue sections of the Gel-MA group and the Hst1 / Gel-MA group are as Figure 7As shown, among them, for Group 2 Gel-MA group, see Figure 7 A and B, for Group 3 Hst1 / Gel-MA group, see Figure 7 C and D, for the results of type II collagen, see Figure 7 A and C, for the results of aggrecan, see Figure 7 B and D; Scale bar for A-D: 100 μm. (n = 6, ***P < 0.001); At the 4th week after surgery, the expression of type II collagen ( Figure 7 E) and aggrecan ( Figure 7 F) was quantitatively analyzed.

[0086] It can be seen from Figure 7 that in Group 3, with the passage of time after surgery, the expression of type II collagen and aggrecan gradually increased. At the 4th week after surgery, the expression of type II collagen and aggrecan in the newly formed cartilage tissue was close to that of normal cartilage tissue ( Figure 7 C-D). While in Group 2, at the 1st week and the 2nd week, almost no type II collagen and aggrecan were detected in the repair tissue. At the 4th week, the staining of type II collagen and aggrecan could be seen ( Figure 7 A-B). In Group 1, no positive results of immunostaining for type II collagen and aggrecan were observed at all time points. At the 4th week, compared with Group 2 and Group 1, the expression of type II collagen and aggrecan in Group 3 was more significant ( Figure 7 E-F). Therefore, it can be clearly seen from Figure 7 that Hst1 can significantly promote the expression of type II collagen and aggrecan.

[0087] Example 4 Influence of the proportional relationship between Gel-MA hydrogel and Hst1 on promoting cartilage repair

[0088] To determine the optimal concentration of Hst1 for promoting osteochondral repair, in this example, Gel-M and 3 different doses of Hst1 (A, A1) 50, (B, B1) 500, (C, C1) 1000 (μg) / sample were implanted into the critical-sized defects of rabbit condyles. The tissues were taken out 2 weeks after surgery for histological processing and sectioning to explore the cartilage repair effects of 3 different concentrations of Hst1.

[0089] Three different Hst1 concentrations were set as follows: Group A: 50 μg Hst1 + 21.1 μl Gel-MA prepolymer solution; Group B: 500 μg Hst1 + 21.1 μl Gel-MA prepolymer solution; Group C: 1000 μg Hst1 + 21.1 μl Gel-MA prepolymer solution (the condylar defect model volume was 21.1 μl / sample).

[0090] The optical microscope comparison photos of the rabbit condylar H&E stained tissue sections are as Figure 8 shown, where the dotted square area is the original defect area (3 mm * 3 mm), scale bar A-C = 500 μm; scale bar A1-C1 = 50 μm. Figure 8 The results showed that at the 2nd week, several new cartilage islands could be clearly observed in Group B ( Figure 8 B-B1); in Group C ( Figure 8 C-C1), a large amount of new subchondral bone was formed in the defect area, but almost no new cartilage tissue was detected; while in Group A ( Figure 8 A-A1), no new tissue was formed in the defect area, and only a small amount of cell infiltration was detected in the undegraded scaffold at the bottom. Therefore, 500 μg Hst1 / sample (500 μg Hst1 + 21.1 μl Gel-MA) was selected as the optimal concentration.

[0091] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

[0092] Sequence Listing

[0093] SEQ ID NO.1

[0094] Linear Hst1:

[0095] DSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN Sequence Listing <110> Hangzhou Huibo Technology Co., Ltd. <120> Use of Polypeptide in Promoting Cartilage Regeneration or Repair <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 38 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 1 Asp Ser His Glu Lys Arg His His Gly Tyr Arg Arg Lys Phe His Glu 1 5 10 15 Lys His His Ser His Arg Glu Phe Pro Phe Tyr Gly Asp Tyr Gly Ser 20 25 30 Asn Tyr Leu Tyr Asp Asn 35

Claims

1. Use of Histatin-1 polypeptide in the preparation of a reagent for promoting cartilage regeneration or repair, characterized in that, the reagent further comprises gelatin methacrylate, and in the reagent, the mass ratio of Histatin-1 polypeptide to gelatin methacrylate is 25:211, or 500 μg of Histatin-1 polypeptide is loaded on 21.1 μl of gelatin methacrylate; the amino acid sequence of the Histatin-1 polypeptide is as shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that, the Histatin-1 polypeptide can promote the expression of bone differentiation indexes, and the bone differentiation indexes include one or more of collagen fibers, glycosaminoglycans, type II collagen, and aggrecan.

3. The use according to claim 1 or 2, characterized in that, the Histatin-1 polypeptide is derived from a polypeptide isolated from saliva or is a synthetic polypeptide.

4. The use according to claim 1, characterized in that, the reagent further comprises stem cells and / or a photoinitiator reagent.

5. Use of Histatin-1 polypeptide in the preparation of a reagent for promoting the expression of one or more of collagen fibers, glycosaminoglycans, type II collagen, and aggrecan, characterized in that, the reagent further comprises gelatin methacrylate, and in the reagent, the mass ratio of Histatin-1 polypeptide to gelatin methacrylate is 25:211, or 500 μg of Histatin-1 polypeptide is loaded on 21.1 μl of gelatin methacrylate; the amino acid sequence of the Histatin-1 polypeptide is as shown in SEQ ID NO.1.

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