Merkel cartilage cell secreted protein composition as well as preparation method and application thereof

Abstract

The invention provides a secretory protein composition, which is characterized in that the composition comprises a GRN protein and any one or more other Merkel cartilage secretory proteins, the invention also provides a preparation method and application thereof. Compared with the prior art, the Merkel cartilage cell secretory protein composition has the advantages that the Merkel cartilage cell secretory protein composition simulates the microenvironment of nerve development, and a more suitable bioactive material is provided for nerve regeneration; the secreted protein combination can promote directional extension of nerve fibers, and is expected to improve the accuracy and function recovery effect of nerve regeneration; the secreted protein combination can more comprehensively meet the complex requirements of nerve regeneration; the research based on the Merkel cartilage cells provides a theoretical basis and an experimental basis for developing novel nerve regeneration materials.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a Merkel chondrocyte secretory protein combination, a preparation method and an application thereof. Background Art

[0002] Peripheral nerve injury is a common clinical problem that can lead to motor and sensory dysfunction and seriously affect the patient's quality of life. Currently, the repair of peripheral nerve injury still faces great challenges, one of the main reasons being the lack of effective biomaterials to promote nerve regeneration. The ideal nerve repair material should be able to simulate the microenvironment during nerve development and provide the necessary biochemical and physical support for axon regeneration.

[0003] At present, there are the following major deficiencies in the field of peripheral nerve injury repair: (1) Lack of materials based on biological principles: Existing neural conduits and scaffold materials are mostly synthetic materials that lack biological activity and cannot fully simulate the microenvironment of neural development.

[0004] (2) Poor directionality of nerve regeneration: Existing materials are difficult to effectively guide the directional growth of axons, resulting in poor accuracy of nerve regeneration and poor functional recovery.

[0005] (3) Imperfect growth factor delivery system: Although neurotrophic factors promote nerve regeneration, how to achieve their sustained and stable release remains a challenge.

[0006] (4) Lack of multifunctional compositions: A single factor is difficult to meet the complex needs of neural regeneration, and it is necessary to develop multifunctional compositions that can synergistically promote neural regeneration.

[0007] Meckel's cartilage is a transient product of embryonic development. It was initially believed that Meckel's cartilage only played a supporting role in the development of the mandible and had no other functions. However, the applicant's research found that during the embryonic development of mammals such as humans and mice, the mandibular nerves distributed and extended around Meckel's cartilage ( Figure 1 , Figure 2 A), more importantly, the applicant found that mice lacking Merkel's cartilage had abnormal nerve distribution and function ( Figure 2 ), suggesting that Meckel's cartilage may be related to neural development. Summary of the invention

[0008] In view of the above-mentioned technical limitations, the present application proposes a Merkel chondrocyte secretory protein combination and a preparation method and application thereof; which overcomes the deficiencies and defects mentioned in the background technology.

[0009] To achieve the above objectives, this application adopts the following technical solutions: The invention of the present application is to provide a secretory protein composition, which includes GRN protein and any one or more other Merkel cartilage secretory proteins.

[0010] Optionally, the above-mentioned secretory protein composition comprises GRN protein and any one or more of the following proteins: CLU, IGFBP5, IGFBP3, SPARC, APP, SEMA3A, IGFBP4, IGFBP2 and SEMA3F.

[0011] CLU: Clusterin, also known as apolipoprotein J, is a novel adipokine that is involved in regulating a variety of biological processes, such as lipid transport, cell adhesion, and complement regulation; GRN: Granulin, involved in inflammation, wound healing and cell proliferation, regulates protein transport to lysosomes, the activity of lysosomal enzymes, and promotes lysosomal acidification; IGFBP5: regulates IGF-mediated cellular processes, including proliferation, differentiation, and apoptosis, increases the proliferation of osteoblasts, intestinal smooth muscle cells, and neuroblastoma cells, and enhances the adhesion and survival of epithelial cells; IGFBP3: inhibits the positive effects on insulin sensitivity in humans and promotes apoptosis of testicular germ cells; SPARC: interacts with the extracellular matrix and cytokines to regulate cell growth, binds calcium and copper, various types of collagen, albumin, thromboxane, PDGF, and cell membranes; APP: As a cell surface receptor, it performs physiological functions related to neurite outgrowth, neuronal adhesion, and axonogenesis on the surface of neurons. The interaction between APP molecules on adjacent cells promotes synapse formation. SEMA3A: induces the collapse and paralysis of neuronal growth cones and guides the direction of growth cones; IGFBP4: can both inhibit and stimulate the growth-promoting effect of IGF on cells and regulate the interaction between IGF and its cell surface receptors; IGFBP2: When mitochondria are damaged, it promotes the apoptosis of ITGA5 in podocytes and then activates the phosphorylation of FAK-mediated mitochondrial damage; SEMA3F: regulates cell motility and cell adhesion.

[0012] Optionally, in the above-mentioned Merkel chondrocyte secretory protein combination, the ratio of each protein in the secretory protein combination is selected as CLU:GRN:IGFBP5:IGFBP3:SPARC:APP:SEMA3A:IGFBP4:IGFBP2:SEMA3F is (0-1.5):(0.3-0.5):(0-0.5):(0-0.5):(0-0.5):(0-0.5):(0-0.3):(0-0.2):(0-0.1):(0-0.1), preferably 1:0.439:0.397:0.384:0.384:0.384:0.2:0.132:0.062:0.038.

[0013] The second invention of the present application is to provide a method for preparing the above-mentioned Merkel chondrocyte secretory protein combination, comprising the following steps: Merkel's primary chondrocyte culture: The Meckel's cartilage isolated from the mandible was rinsed, filtered after digestion, and the cell suspension was collected. After the cell suspension was centrifuged, the supernatant was discarded and the cell pellet was resuspended and inoculated for culture; Merkel Chondrocyte Primary Cell Secretory Protein Collection: Collect the culture medium of Merkel's primary cartilage cells cultured in step S1; Determination of Secretory Proteins of Merkel's Primary Chondrocytes: The culture medium containing secretory proteins of primary Merkel cartilage cells collected in step S2 is digested into peptides by protease, and then the peptides are preliminarily separated by chromatography. The separated peptides are detected by mass spectrometer to obtain peptide information.

[0014] Optionally, in the above preparation method, in step S1, rinsing is performed using PBS buffer containing antibiotics (gibco, 15140122, penicillin-streptomycin mixture 100×), repeatedly rinsing to remove impurities and blood stains, and cutting the cartilage into small pieces; digestion is performed using type II collagenase (Thermo Fisher, P5667270), the digestion temperature is 35°C-40°C, the digestion time is 50min-70min, and continuous shaking is required during the digestion process. The digestion is terminated by using cartilage containing 8%-12% serum Culture medium (Pnosai, complete human cartilage culture medium CM-H107); filtration was performed using a 35μm-45μm filter to remove undigested tissue fragments and collect cell suspension; the centrifugation speed was 1000rpm-1500rpm, the centrifugation time was 4min-6min, and the supernatant was discarded after centrifugation; the cell pellet was resuspended and the cells were inoculated into culture bottles or culture plates. The culture conditions after cell inoculation were 35℃-40℃, 4%-6% CO2, and the cell growth status was observed regularly afterwards.

[0015] The preferred scheme of the above step S1 is: rinsing is performed using PBS buffer containing antibiotics; digestion is performed using type II collagenase, the digestion temperature is 37°C, the digestion time is 60 min, and digestion is terminated using cartilage culture medium containing 10% serum; filtration is performed using a 40 μm filter; the centrifugation speed is 1200 rpm, and the centrifugation time is 5 min; the culture conditions after cell inoculation are 37°C and 5% CO2.

[0016] In step S2, the culture medium of the cultured primary Merkel's chondrocytes is collected after three days of culture, serum and other factors are removed from the culture medium, and the protein is digested into peptides using trypsin, and isotope labeling is performed for quantitative comparison, etc.; In step S3, the peptides are initially separated using chromatography technology; the separated peptides are introduced into a mass spectrometer for detection to obtain information such as the mass and charge of the peptides; the mass spectrometry data are processed and analyzed, including peptide identification, protein assembly, quantitative calculation, differential analysis, etc.

[0017] The third invention of the present application is to provide the use of the above-mentioned Merkel chondrocyte secretory protein combination in the preparation of a drug for preventing / treating peripheral nerve damage.

[0018] The fourth inventive point of the present application is to provide the use of the above-mentioned Merkel chondrocyte secretory protein combination in the preparation of a drug for promoting the directional growth and repair of peripheral nerves.

[0019] The fifth invention point of the present application is to provide the use of the above-mentioned Merkel chondrocyte secretory protein combination in the preparation of a biomaterial for promoting the directional growth of peripheral nerves, wherein the biomaterial is selected from one or more of a nerve conduit and a scaffold material.

[0020] The sixth invention point of the present application is to provide the use of the above-mentioned Merkel chondrocyte secretory protein combination in the preparation of Merkel cartilage organoids.

[0021] The technology disclosed in this application mainly includes the following contents: 1) Composition of the secretory protein combination of Merkel chondrocytes: The specific components and proportions of the protein combination that can promote neural development identified by secretome testing (as shown in Tables 1 and Figure 3 as shown).

[0022] 2) Preparation methods of protein combinations: including culture conditions of Merkel chondrocytes, collection of secretory proteins, etc.

[0023] 3) The mechanism of protein combination promoting directional growth of nerve fibers: clarify how the combination acts on nerve cells to promote their directional growth. That is, the secretory protein combination can promote the growth of neuronal processes, and regulate the cytoskeleton remodeling of nerve fibers through molecules such as CDC42 and RAC1 to make neuronal processes grow in the direction of Merkel chondrocytes ( Figure 4 , Figure 5 ).

[0024] Based on the above research, this application proposes a nerve regeneration technology based on a combination of secretory proteins from Merkel chondrocytes, providing a new treatment strategy for the repair of peripheral nerve injuries.

[0025] Compared with the prior art, the Merkel chondrocyte secretory protein combination and the preparation method and application thereof provided in the present application have the following advantages: 1) Provides a nerve regeneration material based on biological principles: The Merkel chondrocyte secretory protein combination is a natural protein combination, not a synthetic material, which simulates the microenvironment of nerve development and provides a more suitable bioactive material for nerve regeneration; 2) Improved the directionality of nerve regeneration: This secretory protein combination can promote the directional extension of nerve fibers, which is expected to improve the accuracy of nerve regeneration and the effect of functional recovery ( Figure 4 , Figure 5 ); 3) Providing a multifunctional combination that synergistically promotes nerve regeneration: This technology uses a combination of proteins rather than a single factor, which can more comprehensively meet the complex needs of nerve regeneration (Table 1, Figure 3 ); 4) Provides new ideas for the development of neural regenerative materials: Research based on Merkel chondrocytes provides a theoretical basis and experimental basis for the development of new neural regenerative materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the extension of nerves around Meckel's cartilage during mandibular development.

[0027] Figure 2 The abnormal nerve distribution and function of mice lacking Meckel's cartilage; Figure 2 A: WT mice and mice lacking Merkel's cartilage (Sox9 flox / flox 3D reconstruction of mandible, nerves, blood vessels, and Merkel's cartilage of Wnt1-Cre mice. Figure 2 B is the immunofluorescence staining of three neurotransmitters (VAChT, Substance P, CGRP) in WT mice and mice lacking Merkel's cartilage. Figure 2 C is a statistical graph of the fluorescence intensity of three neurotransmitters in WT mice and mice lacking Merkel's cartilage.

[0028] Figure 3 The secretory proteins of Merkel's primary chondrocytes are enriched in pathways that promote neural development; among them, Figure 3 A is a schematic diagram of the collection and mass spectrometry of Merkel cartilage secretory proteins. Figure 3 B shows the pathway by which Meckel's cartilage promotes neural development.

[0029] Figure 4 Kerr's cartilage primary cells secrete proteins to promote the growth of neuronal processes; Figure 4 A is a schematic diagram of co-culture of Merkel's cartilage medium and neurons. Figure 4 B is the MAP2 staining image of neurons in each group. Figure 4 C is a statistical graph of the length of neuronal processes in each group; Figure 4 The effect of applying GRN alone was similar to that of adding MC culture medium (the average values ​​of neurite lengths on the vertical axis were 53, 80, and 87, respectively), so it was believed that the main GRN played a key role.

[0030] Figure 5 It is a protein secreted by Merkel's chondrocytes that attracts the directional growth of nerve fibers by affecting the remodeling of the neuronal cytoskeleton; Figure 5 A is a schematic diagram of the co-culture of Merkel cartilage organoids and neurons. Figure 5 B is the identification of Merkel's cartilage organoids, which express the chondrocyte marker Col2a1. Figure 5 C is an immunofluorescence staining image of Merkel's cartilage organoids co-cultured with neurons, showing that the neuronal processes are attracted by Merkel's cartilage. Figure 5 D is the immunofluorescence staining image of NF200 (nerve) and RAC1 (one of the indicators of cytoskeleton remodeling) in the mandible. Figure 5 E is a plot with MC as the center (the horizontal axis distance is 0) and the average value of RAC1 expression in the nerves of three circles with the same interval as the vertical axis. Figure 5 F is the immunofluorescence staining image of NF200 (nerve) and CDC42 (one of the indicators of cytoskeleton remodeling) in the mandible. Figure 5 G is a graph with MC as the center (the horizontal axis distance is 0) and the average value of CDC42 expression in the nerves of three circles with the same interval as the vertical axis. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application and is not used to limit the scope of the present application.

[0032] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application, and the terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0033] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment. Example

[0034] 1. Combination ratio of Merkel chondrocyte secretory factors: The specific ratio is shown in Table 1.

[0035] Table 1 Secretory factor (gene name) Ratio range Optimal ratio CLU 0-1.5 1 GRN 0.3-0.5 0.439 IGFBP5 0-0.5 0.397 IGFBP3 0-0.5 0.384 SPARC 0-0.5 0.384 APP 0-0.5 0.384 SEMA3A 0-0.3 0.2 IGFBP4 0-0.2 0.132 IGFBP2 0-0.1 0.062 SEMA3F 0-0.1 0.038 2. Merkel cartilage primary cells and organoid culture: (1) Culture of primary Merkel cartilage cells and collection of secretory proteins ( Figure 3 A): 1) Culture of primary Merkel cartilage cells: First, under sterile conditions, Meckel's cartilage is separated from the mandible. The cartilage tissue is repeatedly rinsed with PBS buffer containing antibiotics to remove impurities and blood. The cartilage is cut into small pieces, placed in type II collagenase, and digested at 35-40°C (preferably 37°C) for 50-70min (preferably 60min) with continuous shaking. Subsequently, cartilage culture medium containing 8-12% (preferably 10%) serum is added to terminate the digestion. Filter through a 35-45μm (preferably 40μm) filter to remove undigested tissue fragments and collect the cell suspension. Centrifuge the cell suspension at 1000-1500rpm (preferably 1200rpm) for 4-6 minutes (preferably 5 minutes), and discard the supernatant. Resuspend the cell pellet, inoculate the cells into a culture flask or culture plate, and culture at 35-40°C (preferably 37°C) and 4-6% (preferably 5%) CO2. Observe the cell growth status regularly.

[0036] 2) Collection of secretory proteins from Merkel cartilage primary cells: The culture medium of the cultured primary Merkel chondrocytes was collected after three days of culture; 3) Determination of secretory proteins of Merkel's primary chondrocytes: Trypsin is used to digest the proteins in the collected culture medium into peptides; isotope labeling is performed for quantitative comparison; the peptides are initially separated using chromatography; the separated peptides are introduced into a mass spectrometer for detection to obtain information such as the mass and charge of the peptides; the mass spectrometry data is processed and analyzed, including peptide identification, protein assembly, quantitative calculation, differential analysis, etc.

[0037] (2) Merkel cartilage organoid culture ( Figure 5 A): First, Merkel cartilage was isolated according to the method of primary Merkel cartilage cell culture, cut into pieces, digested with type II collagenase, and centrifuged at 3-5°C, 1000-1500rpm, and collected cells for 4-6 minutes. Next, the cell suspension was mixed with matrix gel (BD Matrigel, 354234) at a ratio of (0.5-1): (0.5-1) (preferably 1:1), and seeded in a 24-well plate, 40-60μl suspension per well, placed in an incubator and allowed to stand for 25-35 minutes, and after it solidified, Merkel cartilage culture medium (Pnosel, complete human chondrocyte culture medium CM-H107) was added to the 24-well plate. Example

[0038] 1. Culture of primary Merkel cartilage cells: (1) First, under sterile conditions, separate Meckel's cartilage from the mandible. Rinse the cartilage tissue repeatedly with PBS buffer containing antibiotics to remove impurities and blood.

[0039] (2) Cut the cartilage into small pieces, place them in type II collagenase, and digest them at 37°C for 1 hour with continuous shaking.

[0040] (3) Then, cartilage culture medium containing 10% serum was added to terminate the digestion.

[0041] (4) Filter through a 40 μm filter to remove undigested tissue fragments and collect the cell suspension.

[0042] (5) Centrifuge the cell suspension at 1200 rpm for 5 minutes and discard the supernatant.

[0043] (6) Resuspend the cell pellet, inoculate the cells into culture flasks or culture plates, and culture them at 37°C and 5% CO2. Regularly observe the cell growth status; the concentration of GRN in the culture medium is determined by ELISA to be 10.23 nM / L (the rest can be calculated by ratio).

[0044] 2. 3D reconstruction of HE-stained sections ( Figure 1 , Figure 2 A): Considering that HE-stained sections can only show the positional relationship between the Meckel's cartilage and nerves and other structures in the plane, the applicant further clarified the overall spatial position through three-dimensional reconstruction. The specific method is as follows: The obtained human mandible was embedded in paraffin, then serially sliced ​​and HE-stained. The scanned HE-stained image was imported into mimics Research20, the direction of the slice was adjusted and the area of ​​interest (mandible, Meckel's cartilage, nerve, etc.) was circled. Then all serial slices were selected for three-dimensional reconstruction, and finally the color and smoothness of the reconstructed image were adjusted.

[0045] 3. Merkel cartilage organoid culture ( Figure 5 A): (1) First, Merkel's cartilage was isolated according to the method of primary Merkel's cartilage cell culture, minced, digested with type II collagenase, and centrifuged at 4°C, 1200 rpm, for 5 minutes to collect the cells.

[0046] (2) Next, mix the cell suspension with matrix gel in a 1:1 ratio and seed the plate in a 24-well plate with 50 μl of the suspension in each well. Place the plate in an incubator and let it stand for 30 minutes. After it solidifies, add Merkel's cartilage culture medium to the 24-well plate.

[0047] Figure 5 In this study, Merkel's cartilage organoids were used to co-culture with neurons to verify whether Merkel's cartilage attracts the processes of neurons (directional effect).

[0048] 4. Primary neuronal cell culture: (1) The cerebral cortex of mice aged 0-3 days after birth was obtained, and the cortical tissue was separated under a stereomicroscope to remove the pia mater, blood vessels, and choroid.

[0049] (2) Mince the brain tissue and digest it with 0.25% trypsin at 37°C with shaking for 10 min.

[0050] (3) Terminate the digestion with neuronal culture medium containing 10% serum and pipette the cell suspension until no tissue clumps are visible to the naked eye.

[0051] (4) Centrifuge at 100 g and filter the supernatant through a 40 μm cell sieve.

[0052] (5) The filtrate was centrifuged at 400 g for 12 minutes, the supernatant was discarded, the precipitate was resuspended and inoculated with cells. The serum-free neural cell culture method was used for mixed neuronal cell culture. According to the growth curve characteristics of astrocytes, 5 μmol / L final concentration of cytarabine was added to the culture medium between 18 and 24 hours after cell planting to inhibit the proliferation of astrocytes. After that, the culture medium was replaced by half every 3 days, and the experiment was carried out on the 7th day.

[0053] 5. Co-culture of Merkel cartilage organoids and primary neuronal cells ( Figure 5 C): Merkel cartilage organoids (spherical organoids with three-dimensional structures formed by culturing Merkel chondrocytes in matrix gel) were subcultured and inoculated into primary neurons that had grown for 4 days. The co-cultured Merkel cartilage organoids and neurons were fixed and stained with immunofluorescence and photographed with a confocal microscope. The regulation of Merkel cartilage organoids on the guidance of neuronal processes has been clarified.

[0054] 6. Co-culture of Merkel chondrocyte culture medium and primary neuronal cells ( Figure 4 ): In order to verify whether Merkel's cartilage can promote the growth of neuronal processes, the culture medium of Merkel's cartilage was added to the co-culture method of neurons. The specific method is as follows: The culture medium of primary cultured Merkel chondrocytes was added to primary neurons grown for 4 days, and the neurons were cultured for another three days. In the GRN group, 10nM / L of GRN protein (Progranulin / PGRN protein, Human, purchased from MCE, HY-P74618) was added to the cultured neurons. The concentration was selected as 10nM / L because the concentration of GRN in Merkel chondrocyte culture medium was about 10nM / L as determined by ELISA kit (purchased from AllianceBiotech, LBK-H04284). After three days, the cells in each group were fixed and immunofluorescently stained for MAP2 to count the length of neuronal processes.

[0055] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A secretory protein combination, characterized in that The combination includes GRN protein and the following proteins: CLU, IGFBP5, IGFBP3, SPARC, APP, SEMA3A, IGFBP4, IGFBP2 and SEMA3F; the concentration ratio of each protein in the secretory protein combination is selected as CLU:GRN:IGFBP5:IGFBP3:SPARC:APP:SEMA3A:IGFBP4:IGFBP2:SEMA3F (0-1.5):(0.3-0.5):(0-0.5):(0-0.5):(0-0.5):(0-0.5):(0-0.3):(0-0.2):(0-0.1):(0-0.1).

2. The secretory protein combination according to claim 1, characterized in that: The concentration ratio of each protein in the secretory protein combination is selected as CLU:GRN:IGFBP5:IGFBP3:SPARC:APP:SEMA3A:IGFBP4:IGFBP2:SEMA3F, which is 1:0.439:0.397:0.384:0.384:0.384:0.2:0.132:0.062:0.

038.

3. The secretory protein combination according to claim 1 or 2, characterized in that: The secretory protein combination is derived from Merkel chondrocytes.

4. The method for preparing the secretory protein combination according to any one of claims 1 to 3, characterized in that: The following steps are involved: Merkel's primary chondrocyte culture: The Meckel's cartilage isolated from the mandible was rinsed, filtered after digestion, and the cell suspension was collected. After the cell suspension was centrifuged, the supernatant was discarded and the cell pellet was resuspended and inoculated for culture; Merkel Chondrocyte Primary Cell Secretory Protein Collection: Collect the culture medium of Merkel's primary cartilage cells cultured in step S1; Determination of Secretory Proteins of Merkel's Primary Chondrocytes: The culture medium containing secretory proteins of primary Merkel cartilage cells collected in step S2 is digested into peptides by protease, and then the peptides are preliminarily separated by chromatography. The separated peptides are detected by mass spectrometer to obtain peptide information.

5. The method for preparing a secretory protein combination according to claim 4, characterized in that: In step S1, the washing is performed using a PBS buffer containing antibiotics; Digestion was performed using 0.2% type II collagenase at a temperature of 35°C-40°C for 50 min-70 min, and digestion was terminated using cartilage culture medium containing 10% serum; filtration was performed using a 35μm-45μm filter; the centrifugal speed was 1000rpm-1500rpm, and the centrifugal time was 4min-6min; the culture conditions after cell inoculation were 35°C-40°C, 4%-6% CO2.

6. Use of the secretory protein combination according to any one of claims 1 to 3 in the preparation of a medicament for preventing / treating peripheral nerve damage.

7. Use of the secretory protein combination according to any one of claims 1 to 3 in the preparation of a drug for promoting directional growth and repair of peripheral nerves.

8. Use of the secretory protein combination according to any one of claims 1 to 3 in the preparation of a biomaterial for promoting directional growth of peripheral nerves, characterized in that: The biological material is selected as a nerve conduit material or a stent material.

9. Use of the secretory protein combination according to any one of claims 1 to 3 in preparing Merkel's cartilage organoids.

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