CN113786516A

CN113786516A - PCL/Col/MC gradient three-layer artificial periosteum and preparation method and application thereof

Info

Publication number: CN113786516A
Application number: CN202111163886.8A
Authority: CN
Inventors: 杜昶; 吴晶晶; 余雪贵; 姚孟宇
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-09-30
Filing date: 2021-09-30
Publication date: 2021-12-14
Anticipated expiration: 2041-09-30
Also published as: CN113786516B

Abstract

The invention discloses a PCL/Col/MC gradient three-layer artificial periosteum and a preparation method and application thereof. Belongs to the field of orthopedic implant materials, and comprises the preparation of electrostatic spinning solution and the preparation of periosteum materials. All adopted raw materials are approved medical implantable materials, the three-layer composite fiber membrane with PCL, Col and MC components changing in a gradient manner layer by layer is prepared by an electrostatic spinning method, 3 layers of continuous spinning are respectively carried out by using PCL, PCL/Col and PCL/Col/MC solutions, and the two solutions are spun at the same time when alternating by 10% of the solution amount. The prepared artificial periosteum has the outer layer component of a pure PCL fiber layer and has a good soft tissue shielding function, the middle layer is PCL/collagen fiber and provides mechanical support for the whole artificial periosteum, and the inner layer is PCL/Col/MC composite fiber and has good bone induction activity and bone guiding capability.

Description

PCL/Col/MC gradient three-layer artificial periosteum and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a PCL/Col/MC gradient three-layer artificial periosteum and a preparation method thereof, which is a periosteum material prepared by an electrostatic spinning method. The method belongs to the field of biological medicine materials, and the prepared material can be widely applied to the repair of periosteum and bone tissues.

Background

Bone defects are common diseases in orthopedics clinic and can be caused by various factors such as trauma, infection, tumor and the like, and large-section bone defects are one of challenges in orthopedics clinic. Currently, bone grafting is the most clinically used treatment, i.e., filling a bone defect with an implanted scaffold to induce bone regeneration. However, these methods have certain limitations without the aid of periosteum, which contributes about 70% to bone formation during the initial healing process of autograft implantation [2-4 ]. The regenerative capacity of periosteum has been used to heal bone of critical dimensions [6-8 ]. Thus, retention of periosteum or implantation of artificial periosteum can improve osteogenesis and osteoconductivity [7 ].

At present, no mature artificial periosteum product is applied in clinic, and the artificial periosteum in the existing research comprises the following components: a degradable polymer porous membrane (Wangshikuan, Wangwei, Liuhao, Ningwen, bioabsorbable artificial periosteum and a preparation method thereof, 2008-02-27.) which is prepared by an electric spray technology and loads bioactive factors such as BMP, VEGF and the like; ② mineralized collagen artificial periosteum (Touchengsen, Dingel, Song Tianxi, Zhang Tong, Chi Fuzhai, a mineralized collagen artificial periosteum and its preparation method 2014-10-15.) prepared by pressing, freeze drying and other processes using nano calcium phosphate and collagen fiber, no research and related products of artificial periosteum with the function of slowly releasing SDF-1 alpha and CGRP are available. Therefore, there is a need to develop an artificial periosteum capable of slowly releasing SDF-1 alpha and CGRP factors to improve the difficulty of clinical treatment of large bone defect repair.

The electrostatic spinning technology can produce polymer fibers with the diameter of nanometer, the prepared fibers have controllable appearance, good flexibility, high specific surface area and porosity and the appearance of the fibers is similar to that of extracellular matrix, so that the fibers are widely concerned in the field of biomedical engineering in J.Xue, J.Xie, W.Liu, Y.Xia, Electrospen Nanofibres: New concepts, Materials, and applications, Accchem Res,50(2017) 1976-.

Mineralized Collagen (MC) prepared through a biomimetic mineralization process simulates the composition and the graded self-assembly structure of natural bone tissues, and numerous studies show that MC has good effects on stimulating osteogenic differentiation, bone reconstruction and spinal fusion of stem cells [22-25 ]. Type i collagen is the main organic component in the bone matrix and plays an important role in bone formation and remodeling. Periosteum is composed primarily of collagen fibers, which confer toughness to the periosteum and favorable microenvironments to the internal cells [26 ]. Thus, the incorporation of MC and Col into the periosteum will improve the biocompatibility and osteointegrative capacity of the PCL fiber membrane.

In addition to the choice of biomaterial, structural design is also critical to the ideal artificial periosteum. This patent was inspired by the layered structure of periosteum and the specific function of each layer [28], and membranes in which PCL, Col, and MC exhibit gradient change in each layer were constructed by electrostatic spinning. The fibroblast or epithelial cell has a faster proliferation speed, a dense and hydrophobic PCL layer is used as an outer layer to prevent surrounding connective tissues from growing into a defect area, PCL/Col is used as an intermediate layer to provide mechanical support for the whole artificial periosteum, and PCL/Col/MC is used as an inner layer to have good bone induction activity and biocompatibility and provide a favorable microenvironment for the recruited cell.

Disclosure of Invention

The invention aims to overcome the defects of the conventional artificial periosteum, and provides a PCL/Col/MC gradient three-layer artificial periosteum and a preparation method thereof, so as to improve the clinical treatment of large-section bone defect repair.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

a preparation method of a PCL/Col/MC gradient three-layer artificial periosteum comprises the following steps:

1) preparing a mineralized collagen material by adopting an in-vitro biomimetic mineralization method;

firstly, dissolving collagen in an acid solution, and continuously stirring;

then, adding CaCl₂And NaH₂PO₄Dropwise adding the solution into an acidic solution of collagen according to the molar ratio of calcium to phosphorus of 1.67 to prepare a mixed solution containing collagen, calcium ions and phosphate ions;

slowly dropwise adding a sodium hydroxide solution into the mixed solution, raising the pH value of the solution to 7.2-7.5, and stirring for 24-48 hours to obtain mineralized collagen flocculent precipitate;

finally, centrifugally washing the mineralized collagen precipitate for 3-5 times by using ultrapure water, freeze-drying for 48-72 hours, and grinding to obtain particles with the particle size of 10-100 mu m;

2) preparation of the electrospinning solution

Adding Polycaprolactone (PCL), polycaprolactone/collagen (PCL/Col) and polycaprolactone/collagen/mineralized collagen (PCL/Col/MC) into Hexafluoroisopropanol (HFIP) as a solvent respectively to prepare three layers of spinning solutions with the concentrations respectively as follows: the outer layer is a 10% w/w PCL solution, the middle layer is a PCL/Col solution, the concentration of the PCL solution is 7.5% w/w PCL, the concentration of the Col solution is 2.5% w/w, the inner layer is a PCL/Col/MC solution, the concentration of the PCL solution is 3.3% -5% w/w, the concentration of the Col solution is 2.5% -4% w/w, the concentration of the MC solution is 2.5% -4% w/w, and the PCL/Col/MC solution is magnetically stirred for 12 hours at room temperature;

3) preparation of periosteal material

Adopting a double-nozzle electrostatic spinning device and a No. 19 electrostatic spinning needle, adjusting the receiving distance to be 10cm, and adding a positive high voltage of 12kV and a negative high voltage of 1 kV; adjusting the liquid flow rate at 0.5mL/h, receiving the rotation speed of a roller at 600r/min, filling the spinning solution into a 5mL injector, and sequentially spinning the PCL, PCL/Col and PCL/Col/MC solutions by two spray heads under the control of an injection pump, so as to better ensure the interlayer binding force, before stopping spinning the former solution, simultaneously spinning the next solution by using another same needle head, and obtaining a fiber membrane with the thickness of 600 mu m.

Further, in the step 2), the concentration of the PCL solution in the inner layer PCL/Col/MC solution is 3.3% w/w, the concentration of the Col solution is 3.3% w/w, and the concentration of the MC solution is 3.3% w/w.

Further, in the step 2), the concentration of the PCL solution in the inner layer PCL/Col/MC solution is 4.0% w/w, the concentration of the Col solution is 4.0% w/w, and the concentration of the MC solution is 4.0% w/w.

Further, in the step 2), the concentration of the PCL solution in the inner layer PCL/Col/MC solution is 5.0% w/w, the concentration of the Col solution is 2.5% w/w, and the concentration of the MC solution is 2.5% w/w.

Further, the MC material is weak-crystallization nano hydroxyapatite, and the mass of the hydroxyapatite accounts for 2.5% -4% of that of the artificial periosteum.

Further, in the step 2), the organic solution is hexafluoroisopropanol HFIP.

Further, the type I glue raw material accounts for 5% -6.5% of the mass of the artificial periosteum.

Further, the Polycaprolactone (PCL) is a degradable natural polymer material or a degradable synthetic polymer material.

The PCL/Col/MC gradient trilayer absorbable artificial periosteum is characterized in that fibers of the PCL/Col/MC gradient trilayer artificial periosteum have a nanofiber structure which is randomly arranged or directionally arranged.

An application of an artificial periosteum material with a PCL/Col/MC gradient three-layer structure in preparing a periosteum tissue repair material.

According to the artificial periosteum material with the PCL/Col/MC gradient three-layer structure, the response function is realized by utilizing the characteristics of fibers of all layers, and the continuity of all layers is good.

The artificial periosteum material with the PCL/Col/MC gradient three-layer structure has good combination of three fiber layers.

The invention provides a PCL/Col/MC gradient three-layer artificial periosteum material, which has the advantages that:

(1) all the adopted raw materials are approved medical materials, and the medical materials have specific functions, good biocompatibility and high safety and are not easy to cause immunological rejection through special process steps;

(2) the MC particles are added into the inner layer of the artificial periosteum of the inventor, so that a favorable microenvironment is provided for recruited cells, and the osteoblast differentiation of stem cells is promoted;

(3) the outer layer of the artificial bone is a compact and hydrophobic PCL layer, and has the function of preventing surrounding connective tissues from growing into a defect area;

(4) the artificial bone intermediate layer is PCL/Col, and provides mechanical support for the whole artificial periosteum;

(5) the invention uses the electrostatic spinning technology, has the advantages of electrostatic spinning fiber membranes, and comprises the following steps: large specific surface area (favorable for cell adhesion), high porosity (favorable for exchange of nutrients and metabolites and cell signal communication), pores smaller than the size of tissue cells (for preventing infection and heterotopic ossification), degradability (no need of secondary operation after implantation),

(5) The invention has simple process and easy conversion.

Drawings

FIG. 1 shows the cross-sectional morphology and element distribution of the artificial periosteum prepared in the present invention;

FIG. 2 is a photograph of an artificial periosteum according to the present invention;

FIG. 3a is a scanning electron microscope image of the outer layer of PCL of the artificial periosteum prepared by the present invention;

FIG. 3b is a scanning electron microscope image of the inner layer of PCL/Col/MC of the artificial periosteum prepared by the present invention;

FIG. 4 is a graph showing the effect of artificial periosteum prepared according to the present invention on cell proliferation;

FIG. 5 is a graph showing the effect of the artificial periosteum according to the present invention on the activity of alkaline phosphatase (ALP), one of the markers of cell osteogenesis;

FIG. 6a is a graph showing the effect of the artificial periosteum prepared according to the present invention on the expression activity of other osteogenic markers in cells;

FIG. 6b is a graph showing the effect of the artificial periosteum of the present invention on the expression activity of other osteogenic markers in cells;

FIG. 7 shows the effect of the artificial periosteum prepared by the present invention on the repair of critical defects of rat skull.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.

Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

Detailed description of the preferred embodiments

Example 1

Preparing a PCL/Col/MC gradient three-layer artificial periosteum;

1. the mineralized collagen material is prepared by an in-vitro biomimetic mineralization method.

Step 1, dissolving collagen in an acid solution, and continuously stirring;

step 2, adding CaCl₂And NaH₂PO₄Dropwise adding the solution into an acidic solution of collagen according to the calcium-phosphorus ratio of 1.67 to prepare a mixed solution containing collagen, calcium ions and phosphate ions;

step 3, slowly dropwise adding a sodium hydroxide solution into the mixed solution, raising the pH value of the solution to 7.4, and stirring for 48 hours to obtain mineralized collagen flocculent precipitate;

step 4, finally, centrifugally washing the mineralized collagen precipitate for 3 times by using ultrapure water, freeze-drying for 48 hours, and grinding the mineralized collagen precipitate into particles with the particle size of 10-100 mu m;

2. preparing an electrostatic spinning solution.

Step 1, respectively adding Polycaprolactone (PCL), polycaprolactone and collagen (PCL/Col), polycaprolactone, collagen and mineralized collagen (PCL/Col/MC) into Hexafluoroisopropanol (HFIP) as a solvent to prepare three layers of spinning solutions required, wherein the concentrations are respectively as follows: an outer layer PCL solution (10% w/w), an intermediate layer PCL/Col solution (7.5% PCL, 2.5% Col, w/w), an inner layer PCL/Col/MC solution (5% PCL, 2.5% Col, 2.5% MC, w/w);

step 2, magnetically stirring for 12 hours at room temperature for later use;

3. preparation of periosteal material

Step 1, adopting a double-nozzle electrostatic spinning device and a No. 19 electrostatic spinning needle, adjusting the receiving distance to be 10cm, and adding a positive high voltage of 12kV and a negative high voltage of 1 kV; regulating the liquid flow rate to be 0.5mL/h, and receiving the rotating speed of the roller to be 600 r/min;

step 2, filling a 5mL injector with spinning solution, and sequentially spinning PCL, PCL/Col and PCL/Col/MC solution by two nozzles under the control of an injection pump;

step 3, in order to better ensure the interlayer bonding force, before the PCL solution is stopped spinning, using another needle head to start to spin the PCL/Col solution at the same time, wherein the coincided spinning time accounts for about 10% of the PCL solution amount, before the PCL/Col solution is stopped spinning, using another needle head to start to spin the PCL/Col/MC solution at the same time, the coincided spinning time accounts for about 10% of the PCL solution amount, and finally obtaining a fiber membrane with the thickness of 600 mu m;

FIG. 3a shows the scanning electron microscope image of the outer layer of PCL/Col/MC gradient three-layer artificial periosteum prepared in example 1, wherein the image shows that the fibers are continuous and uniform in diameter and are distributed at 1200-1600nm, and FIG. 3b shows the scanning electron microscope image of the inner layer of PCL/Col/MC gradient three-layer artificial periosteum prepared in example 1, wherein the image shows that the fibers are continuous and uniform in diameter and are distributed at 100-300nm, the fibers are finer than the pure PCL fibers at the outer layer, and mineralized collagen particles are distributed among the fibers. The inner layer and the outer layer are of a nanofiber structure which is arranged randomly or not in an oriented way.

And secondly, detecting the performance of the PCL/Col/MC gradient three-layer artificial periosteum prepared by the invention.

1. The effect of artificial periosteum on cell proliferation.

Dipping the PCL/Col/MC gradient three-layer artificial periosteum wafer in a solution for cutting the artificial periosteum into 24-hole plate holesSmall discs, hBMSCs were pressed 10⁵The cells are cultured on the surface of a nanofiber membrane sample at a cell density of one/mL by using a special culture medium for human bone marrow mesenchymal stem cells, wherein the special culture medium comprises DMEM, 10% FBS, 1% double antibody (penicillin-streptomycin) and 1% glutamine. Culturing in an incubator at 37 deg.C and 95% humidity with 5% carbon dioxide for 1, 4, and 7 days, and detecting cell proliferation activity by CCK-8 method. P < 0.05 and P < 0.01 are defined as statistically different.

As a result, as shown in FIG. 4, all the cells on the surface of the material were in a state where the number of cells was increased from day 1 to day 7. No significant difference in cell number was observed between the groups at day 1, and the cell activity on the surface of the PCL/Col/MC material was significantly higher than that of the PCL group and the control group at

days

4 and 7, which indicated that the PCL/Col/MC membrane was favorable for the adhesion and proliferation of hBMSCs cells.

2. Effect of Artificial periosteum on Activity of alkaline phosphatase (ALP), one of cellular osteogenesis markers

ALP assay, hBMSCs cells at 1X10⁶and/mL of the culture medium is inoculated into a 24-well plate, 1mL of the culture medium is inoculated into a 1-well plate, the culture medium is placed in a 5% carbon dioxide incubator overnight for the next day, the culture medium is replaced every other day, cell culture plates are taken out at the 7 th and 14 th days of osteogenesis induction, the culture medium is discarded, PBS is washed for 2 times, and then 0.2% triton X-100 lysate is added into each well for overnight at 4 ℃. The expression level of ALP in hBMSCs cells was determined using alkaline phosphatase kit (P0321M, Byunnan). Firstly extracting total protein in cells, then using a BCA protein concentration kit (P0009, Biyun day) to determine the protein concentration, finally using an alkaline phosphatase kit to detect the ALP content, firstly mixing a buffer solution matrix liquid of the kit into a working solution according to an instruction, then adding 4 mu L of a sample to be detected into a 96-well plate, then adding 200 mu L of the working solution, reacting for 15min at 37 ℃, and finally placing in a microplate reader for detection. ALP activity was determined by the ratio of ALP content to protein content. P < 0.05 and P < 0.01 are defined as statistically different.

As shown in FIG. 5, the ALP activity of the PCL/Col/MC artificial periosteum was higher than that of the cell culture plate control group and the PCL group at all time points, so that it was confirmed that the PCL/Col/MC artificial periosteum was able to support osteogenic differentiation of cells without stimulation of soluble osteogenic growth factors.

3. Influence of artificial periosteum on the expression activity of other osteogenic markers in cells.

The expression of osteogenic differentiation related protein of bone marrow mesenchymal stem cells is detected by western blot method, and the detection objects comprise ALP, OCN, OPN, Col-I and GAPDH at 2 × 10⁵Cell concentration per well cells were seeded in 24-well plates containing the material, cultured for 14 and 21 days using complete medium, first protein was extracted, the medium was discarded, the material cell complex was washed 3 times with pre-cooled PBS, protein lysate containing protease inhibitor PMSF was added, lysed on ice for 30 minutes, repeatedly whipped, the lysed cell suspension was transferred to a 1.5mLEP tube, centrifuged at 4 ℃, 12000rpm for 5min, and the supernatant was taken for protein concentration determination and western blot testing. Protein concentration was determined by BCA method as per the instructions.

Western blot test was performed using the above extracted proteins, first, primary and secondary antibodies were diluted with TBST at a ratio of 1:500 and 1:2500 and placed in a refrigerator at 4 ℃ for future use. And then cleaning and drying the glass plate, ensuring the whole leakage, injecting 10% of separation glue into the glass plate, avoiding the generation of bubbles, adding absolute ethyl alcohol to test whether the separation glue is solidified, removing the absolute ethyl alcohol, adding 5% of concentrated glue into the glass plate, inserting a 10-hole comb into the concentrated glue, taking out the comb after the separation glue is solidified, and sequentially adding a pre-dyed protein marker, a sample to be detected and a loading buffer into the holes. Secondly, the gel is electrophoretically concentrated for 30min at a constant voltage of 80V, and electrophoretically separated for 1.5h at a constant voltage of 100V until the marker protein is separated. After electrophoresis is finished, cutting the PVDF membrane with the same size as the gel, fixing the membrane, the filter paper and the gel in a transfer tank, and rotating the membrane for 2h under the current condition of 300 Ma. At this point, the protein had been transferred to a PVDF membrane, which was blocked with 5% BSA solution for 2 h. Finally, the membrane is incubated with primary and secondary antibodies at 4 ℃ overnight, and after completion, protein expression is detected by chemical development in low-cost ECL chemiluminescence solution on the protein side of the membrane. P < 0.05 and P < 0.01 are defined as statistically different.

As shown in FIGS. 6a and 6b, the artificial periosteum of PCL/Col/MC showed a significant osteogenic differentiation promoting effect on hBMSCs cells.

4. Influence of the repair of critical defect of skull of rat with artificial periosteum.

The animal experiments were performed in compliance with the guidelines of the animal care and use committee (IACUC). Adult male SD rats 7 weeks old weighing 250g to 300g were selected for the experiment. Preparing the artificial periosteum material into a circular sheet with the diameter of 5mm and the thickness of 1mm, and performing irradiation sterilization.

The manufacturing steps of the critical defect of the rat skull are as follows: rats were first anesthetized by intraperitoneal injection with a dose of 0.3ml per 100g of animal body weight using a physiological saline solution containing 10% chloral hydrate. After the rat is anesthetized, the skin surgical site is prepared, fixed on an operating table, the surgical site is sterilized with iodophor, an incision of about 2.5cm is made along the midline of the skull, and the scalp and subcutaneous tissues are pulled down to one side with a blunt instrument. A5 mm diameter ring drill (Bernal Dental, Britain) was used to make one defect on each of the left and right sides of the rat skull, and the process was continued with saline solution to reduce the temperature while taking care not to injure the meninges below the skull. After the defect is manufactured, putting each group of bracket materials, and suturing the wound layer by layer. Each post-operative injection of 20 million units of penicillin was performed daily for 3 days.

After 4, 8 and 12 weeks of operation, rats were sacrificed by injection of excess chloral hydrate anesthetic, the cranium was removed, gently rinsed with normal saline, fixed with 4% paraformaldehyde for 24 hours, and then the defect was scanned with a Micro computer tomography scanner (Micro-CT) at a working current of 313 μ a, a voltage of 80kV, and an image pixel of 17.4 μm. The collected data were used to reconstruct 3D tomographic images and to analyze bone volume/tissue volume (BV/TV) and Bone Mineral Density (BMD) based on Micro-CT images.

After the Micro-CT scan was completed, bone tissue was decalcified using 10% EDTA decalcifying solution for 6 weeks, the decalcifying solution was changed three times per week, and the decalcified tissue was dehydrated, paraffin embedded, tissue sectioned, and H & E stained and Masson stained in the same manner as 2.2.6.2 and 2.2.6.3. And finally, observing and recording under a slice scanner. P < 0.05 and P < 0.01 are defined as statistically different.

The results are shown in figure 7, after 12 weeks, the PCL/Col/MC artificial periosteum has no significant difference among the groups, and the new bone mass and the bone density are both significantly higher than those of the blank group and the PCL group, thereby proving that the PCL/Col/MC artificial periosteum has good in-vivo in-situ bone regeneration effect.

Example 2

Preparing the PCL/Col/MC gradient three-layer artificial periosteum.

1. Preparation of mineralized collagen material by in vitro biomimetic mineralization method

Step 1, dissolving collagen in an acid solution, and continuously stirring;

2. preparation of the electrospinning solution

Step 1, respectively adding Polycaprolactone (PCL), polycaprolactone/collagen (PCL/Col) and polycaprolactone/collagen/mineralized collagen (PCL/Col/MC) into Hexafluoroisopropanol (HFIP) as a solvent to prepare three layers of spinning solutions required by the three layers, wherein the concentrations are respectively as follows: an outer layer PCL solution (10% w/w), an intermediate layer PCL/Col solution (7.5% PCL, 2.5% Col, w/w), an inner layer PCL/Col/MC solution (3.3% w/w of each component);

step 2, magnetically stirring for 12 hours at room temperature for later use;

3. preparation of periosteal material

the fibers of the PCL/Col/MC gradient three-layer artificial periosteum have a nanofiber structure which is arranged randomly or directionally.

Example 3

Preparing PCL/Col/MC gradient three-layer artificial periosteum

Step 1, dissolving collagen in an acid solution, and continuously stirring;

2. preparation of the electrospinning solution

Step 1, respectively adding Polycaprolactone (PCL), polycaprolactone/collagen (PCL/Col) and polycaprolactone/collagen/mineralized collagen (PCL/Col/MC) into Hexafluoroisopropanol (HFIP) as a solvent to prepare three layers of spinning solutions required by the three layers, wherein the concentrations are respectively as follows: an outer layer PCL solution (10% w/w), an intermediate layer PCL/Col solution (7.5% PCL, 2.5% Col, w/w), an inner layer PCL/Col/MC solution (4% w/w of each component);

step 2, magnetically stirring for 12 hours at room temperature for later use;

3. preparing periosteum material.

Claims

1. A preparation method of PCL/Col/MC gradient three-layer artificial periosteum is characterized by comprising the following steps: the method comprises the following steps:

firstly, dissolving collagen in an acid solution, and continuously stirring;

2) preparation of the electrospinning solution

3) preparation of periosteal material

2. The method for preparing PCL/Col/MC gradient trilayer artificial periosteum according to claim 1, wherein the method comprises the following steps: in the step 2), the concentration of the PCL solution in the inner layer PCL/Col/MC solution is 3.3% w/w, the concentration of the Col solution is 3.3% w/w, and the concentration of the MC solution is 3.3% w/w.

3. The method for preparing PCL/Col/MC gradient trilayer artificial periosteum according to claim 1, wherein the method comprises the following steps: in the step 2), the concentration of the PCL solution in the inner layer PCL/Col/MC solution is 4.0% w/w, the concentration of the Col solution is 4.0% w/w, and the concentration of the MC solution is 4.0% w/w.

4. The method for preparing PCL/Col/MC gradient trilayer artificial periosteum according to claim 1, wherein the method comprises the following steps: in the step 2), the concentration of the PCL solution in the inner layer PCL/Col/MC solution is 5.0% w/w, the concentration of the Col solution is 2.5% w/w, and the concentration of the MC solution is 2.5% w/w.

5. The method for preparing PCL/Col/MC gradient trilayer artificial periosteum according to claim 1, wherein the method comprises the following steps: the MC material is weak crystalline nano hydroxyapatite which accounts for 2.5-4% of the mass of the artificial periosteum.

6. The method for preparing PCL/Col/MC gradient trilayer artificial periosteum according to claim 1, wherein the method comprises the following steps: in the step 2), the organic solution is hexafluoroisopropanol HFIP.

7. The method for preparing PCL/Col/MC gradient trilayer artificial periosteum according to claim 1, wherein the method comprises the following steps: the Polycaprolactone (PCL) is a degradable natural polymer material or a degradable synthetic polymer material.

8. The method for preparing PCL/Col/MC gradient trilayer artificial periosteum according to claim 1, wherein the method comprises the following steps: the first-type glue raw material accounts for 5-6.5% of the mass of the artificial periosteum.

9. The PCL/Col/MC gradient trilayer absorbable artificial periosteum prepared by the preparation method of any one of claims 1-8, wherein fibers of the PCL/Col/MC gradient trilayer artificial periosteum have a nanofiber structure in random arrangement or directional arrangement.

10. Use of the artificial periosteal material having a PCL/Col/MC gradient trilayer structure according to claim 9 for the preparation of a periosteal tissue repair material.