A method for the biomimetic construction of a periosteum-like tissue

Abstract

The present application relates to a kind of methods for the biomimetic construction of periosteum-like tissue, comprising the step of implanting active substance and / or cell-loaded biomaterials into animals or humans to develop periosteum-like tissue.The constructed periosteum-like tissue has a typical fibrous structure, contains abundant periosteum-derived stem cells and functional blood vessels, can repair critical size bone defects, and is expected to be applied in the clinical treatment of severe bone / cartilage defects, or weak regeneration in elderly patients, etc.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of materials, life sciences and medicine, and relates to a novel biomimetic construction method for periosteal tissue. This method constructs periosteal tissue by activating the organism's own regenerative ability through active biomaterials, which can treat bone / cartilage defects or deformities caused by diseases or trauma. Background Technology

[0002] Periosteum, a fibrous tissue covering the surface of major long bones in the human body, is rich in mesenchymal stem cells (MSCs), blood vessels, and nerve endings, and is crucial for bone development and post-traumatic repair. Developing periosteum-like materials to treat autologous bone / cartilage defects has become an effective new strategy. Currently, various periosteum-like materials based on organic or composite materials have been developed, but most suffer from drawbacks such as complex fabrication, insufficient osteogenic differentiation capacity, and lack of or very low bioactivity. Their therapeutic effects on critical bone / cartilage defects or ischemic osteonecrosis are poor, especially in elderly patients. Furthermore, the limited availability of autologous periosteum tissue cannot meet the repair needs of critical defects. Therefore, developing periosteum-like tissues with functions comparable to autologous periosteum tissue is particularly important. Summary of the Invention

[0003] The purpose of this invention is to provide a method for constructing a periosteum-like tissue, which can directly treat or participate in the treatment of bone / cartilage defects caused by various reasons.

[0004] In a first aspect, the present invention provides a biomimetic method for constructing periosteal tissue, the method comprising the step of implanting biomaterial loaded with active substances and / or cells into an animal or human body and developing it to produce periosteal tissue.

[0005] In another preferred embodiment, the animal is a mouse, rat, pig, dog, cow, horse, rabbit, or monkey.

[0006] In another preferred embodiment, the construction method includes the step of implanting biomaterial loaded with active substances and / or cells into an animal or human body and allowing it to develop for 3-15 days (preferably 5-10 days) to produce periosteum-like tissue.

[0007] In another preferred embodiment, the construction method includes the step of implanting biomaterial loaded with active substances and / or cells into an animal or human body and allowing it to develop for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days to produce periosteal tissue.

[0008] In another preferred embodiment, the active substance is one or more combinations selected from the group consisting of: bone morphogenetic protein-2, bone morphogenetic protein-7, osteogenic polypeptide, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), chondroitin sulfate (CS), or other growth factors / peptides or combinations of growth factors / peptides with bone regeneration induction capabilities.

[0009] In another preferred embodiment, the growth factor used is selected from: vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), delta-like ligand-4 (DLL-4), or other growth factors / peptides or combinations of growth factors / peptides with pro-angiogenic capabilities.

[0010] In another preferred embodiment, the active substance is bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), BMP-2 / VEGF, or BMP-2 / CS.

[0011] In another preferred embodiment, the bone morphogenetic protein-2 is recombinant bone morphogenetic protein-2.

[0012] In another preferred embodiment, the bone morphogenetic protein-7 is recombinant bone morphogenetic protein-7.

[0013] In another preferred embodiment, the biomaterial is selected from: collagen, gelatin, chitosan, sulfonated chitosan, chondroitin sulfate, alginate, hyaluronic acid, bacterial cellulose, polylactic acid, polyglycolic acid, polylactide, polylactide, polyhydroxyalkanoates, polycarbonate, polycaprolactone, polyethylene glycol, polyfuric acid, hydroxyapatite, calcium sulfate, tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, calcium metaphosphate, magnesium phosphate, pyrophosphate, calcium silicate, bioglass, demineralized bone matrix, etc., or a copolymerization / blend combination thereof.

[0014] In another preferred embodiment, biomaterials loaded with active substances and / or cells are implanted into the muscle pouch, intermuscular space, intramuscular space, subcutaneous space, or dorsal abdominal muscle of an animal or human.

[0015] In another preferred embodiment, the development time is 3-15 days.

[0016] In another preferred embodiment, the development time is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days.

[0017] In another preferred embodiment, the cells are mesenchymal stem cells.

[0018] In another preferred embodiment, the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or mesenchymal stem cells from other sources; other types of cells with osteogenic differentiation capacity; or cells that help mesenchymal stem cells differentiate into osteogenic forms, such as vascular endothelial cells.

[0019] In another preferred embodiment, the number of cells used for seeding is 1 × 10⁻⁶. 5 -5×10 8 indivual.

[0020] In another preferred embodiment, the mass ratio of the active substance to the biological material ranges from 0.0001 to 1:1.

[0021] In another preferred embodiment, the construction method includes the step of implanting a gelatin sponge loaded with an active substance selected from one or more combinations of rhBMP-7, rhBMP-2, VEGF and CS into an animal and allowing it to develop for 3-10 days (preferably 5-8 days) to produce periosteal tissue.

[0022] In another preferred embodiment, the construction method includes the step of implanting a gelatin sponge loaded with an active substance selected from one or more combinations of rhBMP-7, rhBMP-2, VEGF and CS into an animal and allowing it to develop for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days to produce periosteal tissue.

[0023] In another preferred embodiment, the construction method includes the step of implanting a gelatin sponge loaded with rhBMP-2 and CS into an animal and allowing it to develop for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days to produce periosteal tissue.

[0024] In a second aspect, the present invention provides a periosteum-like tissue, which is prepared by the biomimetic construction method described in the first aspect.

[0025] In another preferred embodiment, the periosteal tissue has a fibrous structure and comprises periosteal-derived stem cells, osteoblastic progenitor cells, and functional blood vessels (CD31). + EMCN + (vascular network).

[0026] In another preferred embodiment, the resulting periosteum-like tissue has a structure and function similar to that of autologous periosteum.

[0027] In another preferred embodiment, the resulting periosteal tissue is a new tissue induced by the activation of autologous regeneration capacity.

[0028] A third aspect of the invention provides the use of the periosteum-like tissue described in the second aspect for preparing biomaterials for treating bone or cartilage-related diseases.

[0029] In another preferred embodiment, the disease is (trauma-induced) bone or cartilage injury, bone or cartilage nonunion, delayed bone or cartilage healing, bone or cartilage tumor, bone or cartilage defect, osteoporosis, or bone or cartilage deformity.

[0030] In another preferred embodiment, the bone or cartilage defect is an ischemic bone / cartilage defect.

[0031] In another preferred embodiment, the bone or cartilage defect is a critical bone or cartilage defect.

[0032] In another preferred embodiment, the bone or cartilage is from a juvenile, middle-aged, or elderly patient.

[0033] The periosteum-like tissue constructed by the method of this invention has a typical fibrous structure, contains abundant periosteum-derived stem cells and functional blood vessels, and can repair critical-sized bone defects. It is expected to be applied to the clinical treatment of patients with severe bone / cartilage defects or elderly patients with weak regenerative capacity.

[0034] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0035] Figure 1 The flowchart shows the process of constructing a periosteum-like tissue in mice.

[0036] Figure 2 This image shows a hematoxylin / eosin (H&E) stained section of the periosteum surrounding the natural femur and skull.

[0037] Figure 3 Image of a section of periosteal tissue stained with hematoxylin / eosin (H&E).

[0038] Figure 4 Image of a section of periosteal tissue stained with hematoxylin / eosin (H&E).

[0039] Figure 5 Images of H&E and CD31 stained sections of periosteal tissue.

[0040] Figure 6 This is a diagram of periosteal tissue.

[0041] Figure 7Image of a section of periosteal tissue stained with hematoxylin / eosin (H&E).

[0042] Figure 8 Image showing LepR and Osterix immunofluorescence staining of periosteal tissue.

[0043] Figure 9 Image showing EMCN and CD31 immunofluorescence staining of periosteal tissue.

[0044] Figure 10 Image showing EMCN and Osterix immunofluorescence staining of periosteal tissue.

[0045] Figure 11 Image showing EMCN and Aggrecan immunofluorescence staining of periosteal tissue.

[0046] Figure 12 This is a diagram illustrating the experimental process of treating autologous skull defects in mice with periosteum-like tissue.

[0047] Figure 13 μCT scans at 3 and 6 weeks after autologous skull defects in mice treated with periosteum-like tissue.

[0048] Figure 14 The bone volume / total volume (BV / TV) of the periosteum-like tissue repair site is shown.

[0049] Figure 15 The bone mineralization density (BMD) of the periosteal tissue repair site is shown.

[0050] Figure 16 This is an image of an H&E-stained section after transplantation of periosteal tissue.

[0051] Figure 17 This is an image of the appearance of a periosteal tissue.

[0052] Figure 18 This is an H&E stained section of periosteal tissue.

[0053] Figure 19 Immunofluorescence staining images of EMCN and CD31 on periosteal tissue.

[0054] Figure 20 This is an image of EMCN and Osterix immunofluorescence staining of periosteal tissue.

[0055] Figure 21 This is an image of EMCN and Aggrecan immunofluorescence staining of periosteal tissue.

[0056] Figure 22 This is a diagram illustrating the experimental process of treating autologous skull defects in aged mice with periosteum-like tissue.

[0057] Figure 23μCT scans at 3 and 6 weeks after autologous skull defects in aged mice treated with periosteum-like tissue.

[0058] Figure 24 The bone volume / total volume (BV / TV) of the periosteum-like tissue repair site is shown.

[0059] Figure 25 The bone mineralization density (BMD) of the periosteal tissue repair site is shown.

[0060] Figure 26 This is an image of an H&E-stained section after transplantation of periosteal tissue. Detailed Implementation

[0061] The inventors of this application, through extensive and in-depth research, have developed a biomimetic method for constructing periosteum-like tissue. This method involves implanting biomaterials loaded with active substances and / or cells into animals or humans, where they develop to produce periosteum-like tissue, which is constructed by activating the body's autologous regenerative capacity. The periosteum-like tissue constructed in this application exhibits a typical fibrous structure and contains abundant periosteum-derived stem cells and functional blood vessels. Research results indicate that the constructed periosteum-like tissue can repair critical-sized bone defects and holds promise for clinical applications in the treatment of severe bone / cartilage defects or elderly patients with weak regenerative capacity. Based on this, this invention has been completed.

[0062] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions (such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0064] Example 1

[0065] Preparation of implant materials

[0066] 10 μg of recombinant human bone morphogenetic protein-7 (rhBMP-7) synthesized by a eukaryotic or prokaryotic expression system was added to a gelatin sponge (5 mm diameter × 5 mm thickness, 10 mg weight), and then lyophilized to form an active material containing BMP-7.

[0067] 30 μg of recombinant human bone morphogenetic protein-2 (rhBMP-2) synthesized by a eukaryotic or prokaryotic expression system was added to a gelatin sponge (5 mm diameter × 5 mm thickness, 10 mg weight), and then lyophilized to form an active material containing BMP-2.

[0068] 30 μg of rhBMP-2 synthesized by eukaryotic or prokaryotic expression system and 100 ng of vascular endothelial growth factor (VEGF) were added to a gelatin sponge (5 mm diameter × 5 mm thickness, 10 mg weight), and then lyophilized to form an active material containing BMP-2 / VEGF.

[0069] 30 μg of rhBMP-2 synthesized by eukaryotic or prokaryotic expression system and 30 μg of chondroitin sulfate (CS) were added to a gelatin sponge (5 mm diameter × 5 mm thickness, 10 mg weight), and then lyophilized to form an active material containing BMP-2 / CS.

[0070] Example 2

[0071] Construction of periosteum-like tissue in mice

[0072] Periosteum-like tissue was formed in mice using the active material containing BMP-7, BMP-2, BMP-2 / VEGF, or BMP-2 / CS as described in Example 1. Figure 1 As shown, the above-mentioned material was implanted subcutaneously into the back of 8-week-old male C57BL / 6 mice. The active material formed in the mice developed into periosteum-like tissue after one week of development. After one week of feeding, the constructed periosteum-like tissue was harvested. A portion was used for macroscopic photography and tissue sectioning to characterize the periosteum-like tissue, while the other portion was used for autologous cranial defect transplantation.

[0073] Figure 2 H&E sections of the periosteum surrounding the natural femur and skull show the microstructure of the lateral periosteum of the femur and skull in situ.

[0074] Figure 3 The hematoxylin / eosin (H&E) stained sections shown indicate that the periosteal tissue formed in mice containing BMP-7 active material has a microstructure similar to that of the natural periosteal tissue.

[0075] Figure 4 The H&E sections shown indicate that the periosteum-like tissue formed in mice containing BMP-2 active material has a microstructure similar to that of the natural periosteum. The constructed periosteum-like tissue contains chondrocytes, suggesting its potential to repair cartilage defects.

[0076] Figure 5The H&E and CD31 stained sections shown indicate that the periosteum-like tissue formed in mice containing BMP-2 / VEGF active material has a microstructure similar to that of the natural periosteum, and the constructed periosteum-like tissue contains abundant blood vessels.

[0077] Figure 6 The study showed that periosteal tissue was formed in mice one week after implantation of BMP-2 / CS active material. Figure 7 The H&E stained sections shown indicate that the periosteum-like tissue formed in young mice containing BMP-2 / CS active material has a microstructure similar to that of the natural periosteum. The constructed periosteum-like tissue contains chondrocytes, suggesting its potential to repair cartilage defects. Figure 8 The LepR and Osterix immunofluorescence staining results show that the periosteal tissue formed in mice containing BMP-2 / CS active material has abundant periosteal-derived stem cells (LepR). + PDCs and osteogenic precursor cells (Osterix + It can be used as an effective graft for the treatment of ischemic bone / cartilage defects. Figure 9 The EMCN and CD31 immunofluorescence staining results show that the periosteum-like tissue formed in mice containing BMP-2 / CS active material is rich in CD31. + EMCN + Vascular networks, similar to the highly vascularized natural periosteum, can serve as effective grafts for the treatment of ischemic bone / cartilage defects. Figure 10 The EMCN and Osterix immunofluorescence staining results show that the periosteal tissue formed in young mice containing BMP-2 / CS active material has abundant osteogenic progenitor cells, similar to the natural periosteum, and can serve as an effective graft for the treatment of bone / cartilage defects. Figure 11 The EMCN and Aggrecan immunofluorescence staining results show that the periosteum-like tissue formed in mice containing BMP-2 / CS active material has abundant chondrogenic progenitor cells and a very strong chondrogenic differentiation capacity. Similar to the natural periosteum, it can be used as an effective graft for the treatment of bone / cartilage defects.

[0078] Example 3

[0079] In vivo construction of periosteum-like tissue for the treatment of autologous skull defects in mice

[0080] The purpose of this embodiment is to evaluate the therapeutic effect of in vivo periosteum-like tissue in mice on a 5 mm diameter defect in the autologous skull.

[0081] Grouping:

[0082] SPF-grade male C57BL / 6 mice, aged 8 weeks, were randomly assigned to groups. The experimental groups are as follows:

[0083] quantity 6 6

[0084] Preparation of periosteum-like tissue: The BMP-2 / CS-containing scaffold described in Example 1 was implanted subcutaneously, and periosteum-like tissue was generated after 1 week of development. The periosteum-like tissue was removed, and a 5mm diameter disc-shaped periosteum-like tissue was obtained by trimming it using a 5mm inner diameter punch.

[0085] Autologous periosteum tissue transplantation: After anesthetizing mice, the skin on the mouse's head was incised with a scalpel to expose the skull. A 5mm skull defect was created using a 5mm outer diameter circular saw, and the autologous periosteum tissue prepared in the previous step was transplanted to the skull defect. After suturing the skin, the mice were placed on a temperature-controlled platform until they woke up. Samples were taken and tested at predetermined time points. In the control group, only a 5mm skull defect was created in the mice, and then the wound was sutured.

[0086] Figure 12 This diagram illustrates the experimental process of using BMP-2 / CS-containing bioactive material to form periosteum-like tissue in mice for the treatment of autologous skull defects. As shown in the figure, the constructed periosteum-like tissue, after trimming, effectively covers the defect site, achieving rapid repair.

[0087] Figure 13 The images show μCT scans at 3 weeks and 6 weeks after the formation of a periosteum-like tissue containing BMP-2 / CS active material in mice, used to treat autologous skull defects. The images demonstrate that the constructed periosteum-like tissue can rapidly repair bone defects.

[0088] Figure 14 and 15 The results showed that the bone volume / total volume (BV / TV) and bone mineralization density (BMD) of the periosteum-like tissue formed in mice containing BMP-2 / CS active material were significantly higher than those of the blank control group, indicating that the constructed periosteum-like tissue has a better repair effect.

[0089] Figure 16 The H&E stained sections shown indicate that the periosteal tissue formed in mice containing BMP-2 / CS active material can survive at the defect site after transplantation, differentiate into osteoblasts, and effectively integrate with the defect margin, achieving a good repair effect.

[0090] The results of Examples 2 and 3 show that the periosteal tissue constructed from the BMP-2 / CS active material prepared in Example 1 has a structure and function similar to the natural periosteal tissue, and can effectively repair autologous skull defects. It is expected to be applied to the repair of various bone / cartilage defects.

[0091] Example 4

[0092] Periosteum-like tissue develops in aged mice

[0093] Using the BMP-2 / CS active material described in Example 1, subcutaneous implantation was performed on the back of 52-week-old male C57BL / 6 mice to construct periosteum-like tissue. After one week of feeding, the constructed periosteum-like tissue was harvested. A portion was used for macroscopic photography, tissue sectioning, and flow cytometry analysis, while the other portion was used for autologous cranial defect transplantation therapy.

[0094] Figure 17 Macroscopic photographs show that the periosteal tissue formed in aged mice containing BMP-2 / CS active material in Example 4 is white and transparent.

[0095] Figure 18 The H&E stained sections shown indicate that the periosteal tissue formed in aged mice containing BMP-2 / CS active material has a microstructure similar to that of the natural periosteal tissue.

[0096] Figure 19 The EMCN and CD31 immunofluorescence staining results show that the periosteum-like tissue formed in aged mice containing BMP-2 / CS active material has abundant CD31. + EMCN + Vascular networks, similar to the natural periosteum, can serve as effective grafts for the treatment of bone / cartilage defects.

[0097] Figure 20 The EMCN and Osterix immunofluorescence staining results show that the periosteal tissue formed in aged mice containing BMP-2 / CS active material has abundant osteogenic progenitor cells, similar to the natural periosteum, and can serve as an effective graft for the treatment of bone / cartilage defects.

[0098] Figure 21 The EMCN and Aggrecan immunofluorescence staining results show that the periosteum-like tissue formed in aged mice containing BMP-2 / CS active material has abundant chondrogenic progenitor cells and a very strong chondrogenic differentiation capacity. Similar to the natural periosteum, it can be used as an effective graft for the treatment of bone / cartilage defects.

[0099] Example 5

[0100] In vivo construction of periosteum-like tissue for the treatment of autologous cranial defects in aged mice.

[0101] The purpose of this embodiment is to evaluate the therapeutic effect of periosteum-like tissue constructed in elderly mice on a 5mm diameter defect in the autologous skull.

[0102] Grouping:

[0103] SPF-grade male C57BL / 6 mice, 52 weeks old, were randomly assigned to groups. The experimental groups are as follows:

[0104] quantity 6 6

[0105] Preparation of periosteum-like tissue: The scaffold containing BMP-2 / CS described in Example 1 was subcutaneously implanted in the back of 52-week-old male C57BL / 6 mice, and periosteum-like tissue was generated after 1 week of development. The periosteum-like tissue was removed and trimmed into 5mm diameter disc-shaped pieces using a 5mm inner diameter punch.

[0106] Autologous periosteum tissue transplantation: After anesthetizing mice, the skin on the mouse's head was incised with a scalpel to expose the skull. A 5mm skull defect was created using a 5mm outer diameter circular saw, and the autologous periosteum tissue prepared in the previous step was transplanted to the skull defect. After suturing the skin, the mice were placed on a temperature-controlled platform until they woke up. Samples were taken and tested at predetermined time points. In the control group, only a 5mm skull defect was created in the mice, and then the wound was sutured.

[0107] Figure 22 This image shows the experimental process of using BMP-2 / CS-containing bioactive material to form periosteum-like tissue in aged mice for the treatment of autologous skull defects in these mice. As shown in the image, the constructed periosteum-like tissue, after trimming, effectively covers the defect site, achieving rapid repair.

[0108] Figure 23 The images show μCT scans at 3 and 6 weeks after the formation of a periosteum-like tissue containing BMP-2 / CS active material in aged mice, used to treat autologous skull defects in aged mice. The images demonstrate that the constructed periosteum-like tissue can rapidly repair bone defects.

[0109] Figure 24 and 25 The results showed that the BV / TV (bone volume / total volume) and BMD (bone mineralization density) of the periosteum-like tissue repaired in aged mice containing BMP-2 / CS active material were significantly higher than those in the blank control group, indicating that the constructed periosteum-like tissue has a better repair effect.

[0110] Figure 26 The H&E stained sections shown indicate that the periosteal tissue formed in aged mice containing BMP-2 / CS active material can survive at the defect site after transplantation, differentiate into osteoblasts, and effectively integrate with the defect margin, achieving a good repair effect.

[0111] The results of Examples 4 and 5 demonstrate that the periosteum-like tissue constructed in aged mice using the BMP-2 / CS active material prepared in Example 1 possesses a structure and function similar to autologous periosteum, and can effectively repair bone defects in elderly patients where repair of critical bone defects is difficult. This method holds promise for application in the repair of bone / cartilage defects in various elderly patients with poor autologous bone / cartilage conditions.

[0112] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A biomimetic construction method for periosteum-like tissue, characterized in that, The construction method includes the step of implanting biomaterials loaded with active substances into animals and allowing them to develop for 6, 7, or 8 days to produce periosteum-like tissue. The active substance is a combination of bone morphogenetic protein-2 and chondroitin sulfate. The biomaterial is a gelatin sponge; The mass ratio of the active substance to the biological material ranges from 0.0001 to 1:1; The animals mentioned are mice, rats, pigs, dogs, cattle, horses, rabbits, or monkeys; The periosteal tissue has a fibrous structure and includes chondrocytes, periosteal-derived stem cells, osteoblastic progenitor cells, and CD31. + EMCN + Vascular network.

2. The biomimetic construction method as described in claim 1, characterized in that, The animal in question is a mouse or a rat.

3. The biomimetic construction method as described in claim 1, characterized in that, The bone morphogenetic protein-2 mentioned is recombinant bone morphogenetic protein-2.

4. The biomimetic construction method as described in claim 1, characterized in that, Biomaterials loaded with active substances are implanted into the muscle pouch, intermuscular space, intramuscular space, subcutaneous space, or dorsal abdominal muscle of an animal.

5. A type of periosteum-like tissue, characterized in that, The periosteal tissue is prepared by the biomimetic construction method according to any one of claims 1-4, and the periosteal tissue has a fibrous structure, comprising chondrocytes, periosteal-derived stem cells, osteoblastic progenitor cells, and CD31. + EMCN + Vascular network.

6. The use of the periosteal tissue as described in claim 5, characterized in that, Used to prepare biomaterials for the treatment of bone or cartilage-related diseases.

7. The use as described in claim 6, characterized in that, The disease is bone or cartilage injury, bone or cartilage nonunion, delayed bone or cartilage healing, bone or cartilage tumor, bone or cartilage defect, osteoporosis, or bone or cartilage deformity.

8. The use as described in claim 7, characterized in that, The bone or cartilage defect is an ischemic bone / cartilage defect.

9. The use as described in claim 7, characterized in that, The bone or cartilage defect is a critical bone or cartilage defect.

10. The use as described in any one of claims 6-9, characterized in that, The bone or cartilage referred to is the bone or cartilage of a child, middle-aged, or elderly patient.

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