CN114525599A - Bionic periosteum and preparation method and application thereof - Google Patents
Bionic periosteum and preparation method and application thereof Download PDFInfo
- Publication number
- CN114525599A CN114525599A CN202210278327.XA CN202210278327A CN114525599A CN 114525599 A CN114525599 A CN 114525599A CN 202210278327 A CN202210278327 A CN 202210278327A CN 114525599 A CN114525599 A CN 114525599A
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- Prior art keywords
- spinning solution
- periosteum
- water
- spinning
- electrospun membrane
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Abstract
The invention provides a bionic periosteum and a preparation method and application thereof, and relates to the technical field of biological materials. The bionic periosteum provided by the invention comprises an outer layer electrospun membrane and an inner layer electrospun membrane which are overlapped together, wherein the inner layer electrospun membrane is loaded with exosome. The outer layer of the electrospun membrane is used for simulating a fibrous layer of a periosteum, and the inner layer of the electrospun membrane is loaded with exosomes and used for simulating a hair growth layer of the periosteum. The bionic periosteum can simulate the fiber structure of a natural periosteum and can simultaneously play the roles of bone conduction and bone induction so as to achieve the bionic effect of both structure and function.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a bionic periosteum and a preparation method and application thereof.
Background
To date, clinical treatment of critical-size bone defects caused by trauma, tumor resection surgery, or congenital malformations has presented significant challenges. Various methods, such as autologous bone grafting, allogeneic bone grafting, active biomaterial tissue engineering and the like, have been applied to the reconstruction of defective bones. However, these approaches have limited repair capabilities in the absence of periosteum due to injury during implantation or carelessness in the design of the biomimetic scaffold. Previous studies on autografts have found that periosteal regeneration is about 70% in the initial stages of bone and cartilage healing. Periosteal detachment is closely associated with a high incidence of bone nonunion, and preservation of periosteum or use of periosteal transplantation can significantly improve the reconstructive effects of bone implants. Autologous periosteum is difficult to obtain and limited in source, allogeneic periosteum has rejection risk, and the tissue engineering periosteum not only can assist primary bone grafting or a bionic bone scaffold, but also can be directly used as a substitute of a natural periosteum. Therefore, the construction of biomimetic periosteum is a hot point of research.
Traditional thinking has been that periosteal structures are divided into two layers: the outer layer (fibrous layer) mainly comprises densely arranged coarse collagen fiber bundles and a small amount of elastic fibers, and simultaneously comprises a small amount of fibroblasts and capillaries inserted therein; the inner layer (hair growth layer) mainly comprises stem cells, osteoprogenitor cells, osteoblasts and other cells, and simultaneously contains abundant capillaries and a small amount of loosely arranged collagen fibers.
In the existing research, a high molecular membrane material is mostly adopted to replace a natural periosteum. The polymer membrane material mainly comprises synthetic polymer membrane materials (PCL, PU, PLA, etc.), natural polymer membrane materials (collagen, gelatin, hyaluronic acid, etc.), and composite materials of the two. The function of the polymer membrane material to replace the natural periosteum is mainly that the membrane guides tissue/bone regeneration, namely the bone conduction function. The existing research shows that the membrane material is covered on the bone defect area, on one hand, the membrane material can prevent non-osteogenic cells (such as fibroblasts) from migrating to the defect area, and is beneficial to the bone cells to enter the defect area for proliferation or differentiation so as to achieve the effect of promoting the repair of bone defect; on the other hand, the membrane material can obstruct the invasion of microorganisms so as to achieve the effect of reducing complications such as infection and the like. However, such membrane materials have substantially only osteoconductive and cell or microbe barrier effects, and do not have osteogenic or angiogenetic activity per se. In addition, the single-layer membrane material cannot simulate the characteristics of the double-layer fiber structure of the natural periosteum.
There have also been studies to combine membrane materials with cells, or to incorporate osteogenic cytokines (e.g., BMP-2, TGF-. beta.1, FGF, etc.) or drugs into the membrane, so that the membrane materials can simultaneously perform osteoinductive functions. However, the cell culture period is long, time and labor are consumed; the cytokine has short half-life period, is easy to degrade in vivo and cannot meet the requirement of a longer bone growth cycle, and the cytokine is easy to diffuse into peripheral soft tissues after being excessively added, so that the problems of ectopic ossification and the like are caused.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
A first objective of the present invention is to provide a biomimetic periosteum to solve at least one of the above problems.
The second purpose of the invention is to provide a preparation method of the bionic periosteum, which is simple and convenient.
The third purpose of the invention is to provide the application of the bionic periosteum in preparing bone repair products.
In a first aspect, the invention provides a biomimetic periosteum, which comprises an outer layer electrospun membrane and an inner layer electrospun membrane which are overlapped together;
the inner electrospun membrane is loaded with exosomes.
As a further technical scheme, the fibers of the inner layer electrospun membrane are of a coaxial multilayer structure;
the inner layer of the fiber comprises exosomes and a water-soluble high polymer material;
the outer layer of the fiber comprises biodegradable polyester and optionally a water-insoluble polymeric material.
As a further technical scheme, the source of the exosome comprises at least one of human umbilical vein endothelial cells or rat bone marrow mesenchymal stem cells;
preferably, the water-soluble polymer material includes at least one of polyvinyl alcohol, polyethylene oxide, water-soluble chitosan, or sodium hyaluronate.
As a further technical scheme, the outer layer electrospun membrane comprises biodegradable polyester and an optional water-insoluble high polymer material.
As a further technical scheme, the biodegradable polyester comprises at least one of PCL, PU, PLA, PLGA or PLLA;
preferably, the water-insoluble polymer material includes at least one of collagen, gelatin, or chitosan.
In a second aspect, the invention provides a preparation method of a bionic periosteum, which comprises the following steps:
a. carrying out electrostatic spinning on the spinning solution A to prepare an outer-layer electrospun membrane;
b. carrying out coaxial electrostatic spinning on the spinning solution B and the spinning solution C to prepare an inner-layer electrospun membrane; the spinning solution B and the spinning solution C respectively correspond to the outer layer and the inner layer of the fiber of the inner-layer electrospun membrane;
c. superposing at least one outer-layer electrospun membrane and at least one inner-layer electrospun membrane together to prepare a bionic periosteum;
the spinning solution A contains 0.01-0.2 g/mL of biodegradable polyester and optionally 0.001-0.05 g/mL of water-insoluble high polymer material;
the spinning solution B contains 0.01-0.2 g/mL of biodegradable polyester and optionally 0.001-0.08 g/mL of water-insoluble high polymer material;
the spinning solution C contains exosome and 0.01-0.1 g/mL water-soluble high polymer material.
As a further technical scheme, the spinning solution A contains 0.04g/mL of biodegradable polyester and 0.01g/mL of water-insoluble high polymer material;
the spinning solution B contains 0.035g/mL biodegradable polyester and 0.015g/mL water-insoluble high molecular material;
the spinning solution C contains exosome and 0.05g/mL water-soluble high polymer material;
the solvents of the spinning solution A and the spinning solution B respectively and independently comprise at least one of hexafluoroisopropanol, trifluoroethanol, N-dimethylformamide or trichloromethane;
the solvent of the spinning solution C comprises water.
As a further technical scheme, in the step a, the spinning operation parameters at least meet one of the following conditions: the spinning voltage is 15-30 kV, the receiving distance is 7-15 cm, the injection speed is 0.3-1.5 mL/h, and the spinning time is 1-6 h;
preferably, in the step b, the spinning operation parameters at least satisfy one of the following conditions: 20-30 kV, the receiving distance is 7-15 cm, the injection speed of the spinning solution B is 0.5-1.5 mL/h, the injection speed of the spinning solution C is 0.1-0.3 mL/h, and the spinning time is 1-10 h.
As a further technical scheme, the method also comprises the step of sterilizing the outer layer electrospun membrane and the inner layer electrospun membrane;
the method of sterilization includes at least one of ultraviolet irradiation, cobalt 60 irradiation, or ethylene oxide sterilization.
In a third aspect, the invention provides an application of a bionic periosteum in preparing a bone repair product.
Compared with the prior art, the invention has the following beneficial effects:
the bionic periosteum provided by the invention comprises an outer layer electrospun membrane and an inner layer electrospun membrane which are overlapped together, wherein the inner layer electrospun membrane is loaded with exosome. The outer layer of the electrospun membrane is used for simulating a fibrous layer of a periosteum, and the inner layer of the electrospun membrane is loaded with exosomes and used for simulating a hair growth layer of the periosteum. The bionic periosteum can simulate the fiber structure of a natural periosteum and can simultaneously play the roles of bone conduction and bone induction so as to achieve the bionic effect of both structure and function.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic view of an electrospun membrane;
FIG. 2 is a graph of bone volume fraction for each group at different time points;
fig. 3 is a graph of bone density values for various groups of defect regions at different time points.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In a first aspect, the invention provides a biomimetic periosteum, which comprises an outer layer electrospun membrane and an inner layer electrospun membrane which are overlapped together;
the inner electrospun membrane is loaded with exosomes.
In the invention, the outer layer electrospinning membrane and the inner layer electrospinning membrane are prepared by electrospinning technology. The schematic diagram of the electrospun membrane is shown in FIG. 1, wherein in FIG. 1, the outer layer electrospun membrane is shown; ② inner layer electrospinning membrane; thirdly, an inner layer electrospinning membrane fiber structure (containing small particles as exosomes); the first and the second layers may be superposed one or more layers.
The research of the inventor finds that the exosome promotes osteogenesis in vivo and in vitro. Meanwhile, the exosome can be used as a natural nano-scale carrier to deliver drugs or growth factors, so that the effect of promoting the repair of the defective bone is achieved. Therefore, the exosome-loaded double-layer or multi-layer fibrous membrane structure is designed and prepared, and exosomes with the function of promoting bone regeneration are added on the basis of simulating a natural periosteum fibrous structure, so that the aim of promoting bone reconstruction at a defect part is fulfilled.
The outer layer of the bionic periosteum provided by the invention is used for simulating a fibrous layer of the periosteum, and the inner layer of the bionic periosteum is loaded with exosomes and is used for simulating a hair growth layer of the periosteum. The bionic periosteum can simulate the fiber structure of a natural periosteum and can simultaneously play the roles of bone conduction and bone induction so as to achieve the bionic effect of both structure and function.
In some preferred embodiments, the fibers of the inner electrospun membrane are of a coaxial multilayer structure;
the inner layer of the fiber comprises exosomes and a water-soluble high polymer material;
the outer layer of the fiber comprises biodegradable polyester and optionally a water-insoluble polymeric material.
According to the invention, the inner layer membrane is prepared by combining degradable polyester and water-soluble polymer, and the exosome is loaded inside the fiber of the inner layer electrospun membrane, so that the structure is beneficial to slow release and function exertion of the exosome in the inner layer electrospun membrane, and the bionic periosteum has the effect of promoting bone regeneration while exerting the bone conduction effect.
In the present invention, "optional" means that the compound may or may not be contained.
In some preferred embodiments, the source of the exosomes includes, but is not limited to, human umbilical vein endothelial cells, rat bone marrow mesenchymal stem cells, such as human adipose stem cells, human induced pluripotent stem cells, mesenchymal stem cells, gingival stem cells, and the like.
Preferably, the water-soluble polymer material includes, but is not limited to, polyvinyl alcohol, polyethylene oxide, water-soluble chitosan or sodium hyaluronate, or other water-soluble polymer materials well known in the art.
In some preferred embodiments, the outer electrospun membrane comprises a biodegradable polyester and optionally a water insoluble polymeric material.
According to the invention, the degradable polyester is combined with the water-insoluble high polymer material to prepare the outer layer membrane, so that invasion of non-osteocyte can be prevented, and osteocyte is guided to migrate to the bone defect position.
In some preferred embodiments, the biodegradable polyester includes, but is not limited to, at least one of PCL, PU, PLA, PLGA, or PLLA;
preferably, the water-insoluble polymer material includes at least one of collagen, gelatin, or chitosan.
In a second aspect, the invention provides a preparation method of a bionic periosteum, which comprises the following steps:
a. carrying out electrostatic spinning on the spinning solution A to prepare an outer-layer electrospun membrane;
b. carrying out coaxial electrostatic spinning on the spinning solution B and the spinning solution C to prepare an inner-layer electrospun membrane; the spinning solution B and the spinning solution C respectively correspond to the outer layer and the inner layer of the fiber of the inner-layer electrospun membrane;
c. superposing at least one outer-layer electrospun membrane and at least one inner-layer electrospun membrane together to prepare a bionic periosteum;
the spinning solution A contains biodegradable polyester and optional water-insoluble high molecular material, wherein the concentration of the biodegradable polyester can be, but is not limited to, 0.01g/mL, 0.02g/mL, 0.04g/mL, 0.06g/mL, 0.08g/mL, 0.1g/mL or 0.2g/mL, and is preferably 0.04 g/mL; the concentration of the water-insoluble polymer material may be, for example, but not limited to, 0.001g/mL, 0.005/mL, 0.01g/mL, 0.02g/mL, 0.03g/mL, 0.04g/mL, or 0.05g/mL, and is preferably 0.01 g/mL.
The spinning solution B contains biodegradable polyester and optionally a water-insoluble high molecular material, wherein the concentration of the biodegradable polyester can be, but is not limited to, 0.01g/mL, 0.02g/mL, 0.04g/mL, 0.06g/mL, 0.08g/mL, 0.1g/mL or 0.2g/mL, and is preferably 0.035 g/mL; the concentration of the water-insoluble polymer material may be, for example, but not limited to, 0.001g/mL, 0.005/mL, 0.01g/mL, 0.02g/mL, 0.03g/mL, 0.04g/mL, 0.05g/mL, 0.06g/mL, 0.07g/mL, or 0.08g/mL, preferably 0.015 g/mL.
The spinning solution C contains exosomes and a water-soluble high polymer material, wherein the amount of the exosomes is selected according to the requirement; the concentration of the water-soluble polymer material is 0.01g/mL, 0.02g/mL, 0.04g/mL, 0.06g/mL, 0.08g/mL, or 0.1g/mL, preferably 0.05 g/mL.
The solvent of the spinning solution a and the spinning solution B independently include but are not limited to hexafluoroisopropanol, trifluoroethanol, N-dimethylformamide or trichloromethane, or other organic solvents known to those skilled in the art.
The solvent of the spinning solution C comprises water.
The preparation method of the bionic periosteum provided by the invention is simple and convenient, has low cost, can prepare the bionic periosteum which has different fiber apertures and loads exosomes with different amounts, and is used for double bionic of periosteum structure and function.
In some preferred embodiments, in the step a, the spinning operation parameters at least satisfy one of the following conditions: the spinning voltage is 15-30 kV, the receiving distance is 7-15 cm, the injection speed is 0.3-1.5 mL/h, and the spinning time is 1-6 h;
preferably, in the step b, the spinning operation parameters at least satisfy one of the following conditions: 20-30 kV, the receiving distance is 7-15 cm, the injection speed of the spinning solution B is 0.5-1.5 mL/h, the injection speed of the spinning solution C is 0.1-0.3 mL/h, and the spinning time is 1-10 h.
The repairing effect of the bionic periosteum on the periosteum is improved by further optimizing and adjusting the spinning processes of the outer layer electrospun membrane and the inner layer electrospun membrane.
In some preferred embodiments, the method further comprises the step of sterilizing the outer electrospun membrane and the inner electrospun membrane, so that the safety of the bionic periosteum is improved by sterilization.
Methods of sterilization include, but are not limited to, ultraviolet irradiation, cobalt 60 irradiation, or ethylene oxide sterilization, or other sterilization methods known to those skilled in the art.
In a third aspect, the invention provides an application of a bionic periosteum in preparing a bone repair product.
The bionic periosteum provided by the invention can not only simulate the fiber structure of a natural periosteum, but also play roles of bone conduction and bone induction at the same time to achieve the bionic effect of both structure and function, so that the bionic periosteum can be used for preparing bone repair products.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
Example 1
(I) scheme for preparing outer layer electrospun membrane
The preparation process comprises the following steps: (1) pipetting 20mL of hexafluoroisopropanol into two 50mL conical flasks (i.e., 10mL per flask) labeled as solvent 1 and solvent 2; (2) weighing 1.25g of PLGA, and adding the PLGA into the solvent 1; weighing 1g of collagen, and adding the collagen into a solvent 2; placing the 2 solution systems on a magnetic stirrer, and stirring for 6 hours to completely dissolve the solution systems; (3) respectively adding 8mL of PLGA solution and 2mL of collagen solution into a new 50mL conical flask, and stirring for 2 hours on a magnetic stirrer to mix into homogeneous spinning solution; (4) 5mL of the prepared spinning solution is absorbed by using a 5mL injector, the injector, a communicating pipe and a nozzle are arranged on the right position of the instrument, the spinning parameter is adjusted to have the voltage of 20kV, the receiving distance is 15cm, the injection speed is 1.0mL/h, and the continuous spinning is stopped after 6 h. (5) And cutting the electrospun membrane according to actual requirements, and sterilizing by utilizing ultraviolet irradiation for later use.
(II) exosome extraction scheme
Different endothelial cells from human umbilical vein were inoculated into corresponding culture medium supplemented with 10% fetal bovine serum and 1% antibiotics for culture. After the cells are fused to 70-80%, the culture medium is replaced by a 5% exosome-free serum culture medium, and the culture is continued for 48 hours. After the predetermined time point was reached, cell supernatants were collected for differential ultracentrifugation to extract exosomes.
The extraction step comprises: (1)300g, centrifuged at 4 ℃ for 15min to remove dead cells; (2) centrifuging at 3000g and 4 deg.C for 15min to remove cell debris; (3)10000g, centrifugating at 4 ℃ for 70min, and then filtering by a 0.22 mu m filter to remove other large extracellular vesicles; (4)120000g, centrifuging for 120min at 4 ℃, removing supernatant and obtaining the precipitate as the exosome. Taking a proper amount of PBS or normal saline to resuspend the exosome, and storing the exosome in a refrigerator at the temperature of-80 ℃ for later use. Exosome concentrations were detected using BCA protein concentration kit prior to use.
(III) preparation scheme of core-shell structure inner-layer electrospun membrane loaded with exosomes
The preparation process comprises the following steps: (1) pipetting 20mL of hexafluoroisopropanol into two 50mL conical flasks (i.e., 10mL per flask) labeled as solvent 1 and solvent 2; (2) weighing 1g of PLGA, adding the PLGA into the solvent 1, weighing 1g of gelatin, adding the gelatin into the solvent 2, and stirring for 4 hours on a magnetic stirrer to completely dissolve the gelatin; (3) adding 7mL of PLGA solution and 3mL of gelatin solution into a new 50mL conical flask, and stirring for 2 hours on a magnetic stirrer to obtain homogeneous shell spinning solution; (4) weighing 0.3g of PVA, adding the PVA into 5mL of deionized water, and dissolving in a water bath at 90 ℃ for about 2 hours; (5) adding 200 mu L of exosome solution and 1mL of PVA solution into a 50mL conical flask, and stirring for 2 hours on a magnetic stirrer to mix into homogeneous nuclear layer spinning solution; (6) respectively sucking 5mL of shell spinning solution and 2mL of core spinning solution by using a 5mL injector, installing the injector, a communicating pipe and a nozzle at the correct positions of an instrument, adjusting the spinning parameter to be 24kV, adjusting the receiving distance to be 15cm, adjusting the shell layer injection speed to be 0.8mL/h and the core layer injection speed to be 0.1mL/h, and stopping continuous spinning after 5 h. (7) And cutting the electrospun membrane according to actual requirements, and sterilizing by utilizing ultraviolet irradiation for later use.
(IV) preparation scheme of bionic periosteum
And (3) overlapping the outer layer of electrospun membrane and the inner layer of electrospun membrane to prepare the bionic periosteum.
Example 2
(I) scheme for preparing outer layer electrospun membrane
The difference from example 1 is that:
the spinning solution is a trifluoroethanol solution containing 0.01g/mL of PCL and 0.05g/mL of chitosan;
the spinning process comprises the following steps: 5mL of the prepared spinning solution is absorbed by using a 5mL injector, the injector, a communicating pipe and a nozzle are arranged on the right position of an instrument, the spinning parameter is adjusted to be 15kV, the receiving distance is 15cm, the injection speed is 0.3mL/h, and the continuous spinning is stopped after 2 h. And cutting the electrospun membrane according to actual requirements, and sterilizing by utilizing ultraviolet irradiation for later use.
(II) exosome extraction scheme
The experimental procedure was the same as in example 1, and the exosomes were derived from rat bone marrow mesenchymal stem cells.
(III) preparation scheme of core-shell structure inner-layer electrospun membrane loaded with exosomes
The difference from example 1 is that:
the shell spinning solution is a trifluoroethanol solution containing 0.01g/mL PLA and 0.08g/mL collagen.
The core layer spinning solution contained exosome and 0.01g/mL polyethylene oxide.
The spinning process comprises the following steps: respectively sucking 5mL of shell spinning solution and 2mL of core spinning solution by using a 5mL injector, installing the injector, a communicating pipe and a nozzle at the correct positions of an instrument, adjusting the spinning parameter to be 20kV, adjusting the receiving distance to be 15cm, adjusting the shell layer injection speed to be 0.5mL/h and the core layer injection speed to be 0.3mL/h, and stopping continuous spinning after 1 h. And cutting the electrospun membrane according to actual requirements, and sterilizing by utilizing ultraviolet irradiation for later use.
(IV) preparation scheme of bionic periosteum
And superposing the two outer-layer electrospun membranes and the two inner-layer electrospun membranes together to prepare the bionic periosteum.
Example 3
(I) scheme for preparing outer layer electrospun membrane
The difference from example 1 is that:
the spinning solution is an N, N-dimethylformamide solution containing 0.2g/mL PLLA;
the spinning process comprises the following steps: 5mL of the prepared spinning solution is absorbed by using a 5mL injector, the injector, a communicating pipe and a nozzle are arranged on the right position of the instrument, the spinning parameter is adjusted to have the voltage of 30kV, the receiving distance is 7cm, the injection speed is 1.5mL/h, and the continuous spinning is stopped after 4 h. And cutting the electrospun membrane according to actual requirements, and irradiating and sterilizing the electrospun membrane by using cobalt 60 for later use.
(II) exosome extraction scheme
The experimental procedure was the same as in example 1, with exosomes derived from human adipose stem cells.
(III) preparation scheme of core-shell structure inner-layer electrospun membrane loaded with exosomes
The difference from example 1 is that:
the shell spinning solution was an N, N-dimethylformamide solution containing 0.2g/mL of PU.
The core layer spinning solution contains exosome and 0.1g/mL sodium hyaluronate.
The spinning process comprises the following steps: respectively sucking 5mL of shell spinning solution and 2mL of core spinning solution by using a 5mL injector, installing the injector, a communicating pipe and a nozzle at the correct positions of an instrument, adjusting the spinning parameter to be 30kV, adjusting the receiving distance to be 7cm, adjusting the shell layer injection speed to be 1.5mL/h and the core layer injection speed to be 0.1mL/h, and stopping continuous spinning after 10 h. And cutting the electrospun membrane according to actual requirements, and irradiating and sterilizing the electrospun membrane by using cobalt 60 for later use.
(IV) preparation scheme of bionic periosteum
And superposing the two outer-layer electrospun membranes and the one inner-layer electrospun membrane together to prepare the bionic periosteum.
Test example 1
To study the osteo-repair promoting effect of biomimetic periosteum, the following groups were performed: blank surgery group (CON), outer membrane group (EXT, outer electrospun membrane prepared according to the method provided in example 1, same thickness as BIO group), exosome-loaded inner membrane group (INN, inner electrospun membrane prepared according to the method provided in example 1, same thickness as BIO group), and biomimetic periosteum group (BIO, biomimetic periosteum provided in example 1). A rat skull defect model is adopted, materials are respectively obtained at 4 weeks and 8 weeks after the operation, and the bone regeneration conditions among all the groups are compared. The results are shown in FIGS. 2 and 3.
As can be seen from FIG. 2 (BV/TV is bone volume fraction in the figure) and FIG. 3, compared with the blank surgery group, the implantation of fibrous membrane can promote bone regeneration; compared with a single-layer fibrous membrane, the bionic periosteum has a more remarkable effect of promoting bone defect repair.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A bionic periosteum is characterized by comprising an outer layer electrospun membrane and an inner layer electrospun membrane which are overlapped together;
the inner electrospun membrane is loaded with exosomes.
2. The biomimetic periosteum according to claim 1, wherein the fibers of the inner electrospun membrane are of a coaxial multilayer structure;
the inner layer of the fiber comprises exosomes and a water-soluble high polymer material;
the outer layer of the fiber comprises biodegradable polyester and optionally a water-insoluble polymeric material.
3. The biomimetic periostin according to claim 2, wherein a source of the exosomes comprises at least one of human umbilical vein endothelial cells or rat bone marrow mesenchymal stem cells;
preferably, the water-soluble polymer material includes at least one of polyvinyl alcohol, polyethylene oxide, water-soluble chitosan, or sodium hyaluronate.
4. The biomimetic periosteum according to claim 1, wherein the outer electrospun membrane comprises biodegradable polyester and optionally a water-insoluble polymeric material.
5. The biomimetic periost of claim 2 or 4, wherein the biodegradable polyester comprises at least one of PCL, PU, PLA, PLGA or PLLA;
preferably, the water-insoluble polymer material includes at least one of collagen, gelatin, or chitosan.
6. The method for preparing a biomimetic periosteum according to any one of claims 1-5, comprising the steps of:
a. carrying out electrostatic spinning on the spinning solution A to prepare an outer-layer electrospun membrane;
b. carrying out coaxial electrostatic spinning on the spinning solution B and the spinning solution C to prepare an inner layer electrospun membrane; the spinning solution B and the spinning solution C respectively correspond to the outer layer and the inner layer of the fiber of the inner-layer electrospun membrane;
c. superposing at least one outer-layer electrospun membrane and at least one inner-layer electrospun membrane together to prepare a bionic periosteum;
the spinning solution A contains 0.01-0.2 g/mL of biodegradable polyester and optionally 0.001-0.05 g/mL of water-insoluble high polymer material;
the spinning solution B contains 0.01-0.2 g/mL of biodegradable polyester and optionally 0.001-0.08 g/mL of water-insoluble high polymer material;
the spinning solution C contains exosome and 0.01-0.1 g/mL water-soluble high polymer material.
7. The preparation method according to claim 6, wherein the spinning solution A is a solution containing 0.04g/mL of biodegradable polyester and 0.01g/mL of water-insoluble polymer material;
the spinning solution B contains 0.035g/mL biodegradable polyester and 0.015g/mL water-insoluble high molecular material;
the spinning solution C contains exosome and 0.05g/mL water-soluble high polymer material;
the solvents of the spinning solution A and the spinning solution B respectively and independently comprise at least one of hexafluoroisopropanol, trifluoroethanol, N-dimethylformamide or trichloromethane;
the solvent of the spinning solution C includes water.
8. The preparation method according to claim 6, wherein in the step a, the spinning operation parameters at least satisfy one of the following conditions: the spinning voltage is 15-30 kV, the receiving distance is 7-15 cm, the injection speed is 0.3-1.5 mL/h, and the spinning time is 1-6 h;
preferably, in the step b, the spinning operation parameters at least satisfy one of the following conditions: 20-30 kV, the receiving distance is 7-15 cm, the injection speed of the spinning solution B is 0.5-1.5 mL/h, the injection speed of the spinning solution C is 0.1-0.3 mL/h, and the spinning time is 1-10 h.
9. The method of manufacturing according to claim 6, further comprising the step of sterilizing the outer electrospun membrane and the inner electrospun membrane;
the method of sterilization includes at least one of ultraviolet irradiation, cobalt 60 irradiation, or ethylene oxide sterilization.
10. Use of the biomimetic periosteum according to any one of claims 1 to 5 or the biomimetic periosteum prepared by the preparation method according to any one of claims 6 to 9 for preparing a bone repair product.
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