Double-layer heterogeneous artificial periosteum with controllable collagen release rate and preparation method of double-layer heterogeneous artificial periosteum

Abstract

The invention discloses a double-layer heterogeneous artificial periosteum with a controllable collagen release rate and a preparation method of the double-layer heterogeneous artificial periosteum, and belongs to the field of biomedical materials. Collagen and nano-hydroxyapatite are wrapped in polycaprolactone through coaxial electrostatic spinning to prepare an HC layer, then polycaprolactone and chitosan are prepared into a high-density CP layer through electrostatic spinning, and finally the HC-CP double-layer heterogeneous artificial periosteum is prepared. The double-layer heterogeneous artificial periosteum with a unique structure is prepared, nanofiber shell structures with different thicknesses are formed by further controlling the content of polycaprolactone in an HC layer, and the release rate of collagen is controllable, so that the in-vitro degradability of the artificial periosteum is controlled. Research results show that the prepared double-layer heterogeneous artificial periosteum has good biological activity and can induce osteogenic differentiation and cell migration; meanwhile, the double-layer heterogeneous artificial periosteum can induce the generation and deposition of new sclerotin in a rat body, and has a relatively slow and controllable collagen release rate.

Description

Technical Field

[0001] The present invention relates to a bilayer heterogeneous artificial periosteum, and particularly to a bilayer heterogeneous artificial periosteum with a controllable collagen release rate and a preparation method thereof, belonging to the field of biomedical materials. Background Art

[0002] At present, the repair of large-area bone defects remains a challenge in clinical practice. The transplantation of autologous bone or allogeneic bone is the main method for repairing bone defects and has the best repair effect for bone defects. However, without a periosteum to guide the repair of the defect site, the transplantation of autologous bone can only repair about 70% of the bone defect. Among various artificial periosteum materials, artificial periosteum derived from natural polymer materials, such as collagen, gelatin, chitosan, silk fibroin, etc., has the advantage of relatively good biocompatibility, but usually has low mechanical strength, fast degradation rate, is difficult to support the regeneration space of large-area bone defects, and has a relatively single function. Therefore, the research and development of artificial synthetic functional periosteum materials is of great significance for clinical practice.

[0003] In recent years, electrospun nanofibers have a high surface-to-volume ratio, uniform structure, adjustable porosity and ductility to adapt to various sizes and shapes. The application of a single natural polymer material in tissue engineering scaffolds has the advantages of good tissue compatibility, low toxicity, easy degradation and the degradation products are easily absorbed by the human body without causing inflammation, etc. However, at the same time, it usually has disadvantages such as poor mechanical properties and the need for additional cross-linking. Nanofiber materials composed of artificial polymers have good mechanical strength, and their structure and degradation rate can be artificially adjusted by changing their composition. Many researchers have combined natural polymer materials with inorganic materials to improve the mechanical strength of natural polymer materials, make up for the brittleness of inorganic materials and improve the biocompatibility of composite materials.

[0004] Type I collagen has excellent biocompatibility and degradability, strong adhesion to most cells, and has a wide range of applications in tissue engineering scaffolds and wound dressing. The composition and microstructure of the type I collagen / nano-hydroxyapatite composite fiber membrane are similar to those of natural bone, and it is regarded as a potential candidate material for bone tissue engineering. The Zhou Y research group prepared a polycaprolactone / collagen fiber composite material using electrospinning technology (Zhou Y, Yao H, Wang J, Wang D, Liu Q, Li Z. Greener synthesis of electrospun collagen / hydroxyapatite composite fibers with an excellent microstructure for bone tissue engineering. Int J Nanomedicine. 2015 Apr 29;10:3203-15. doi: 10.2147 / IJN.S79241.). Physical and chemical experiments and cytotoxicity tests have all proved that the composite material prepared by this method has good mechanical strength, mineralization ability and high biocompatibility, and has high application prospects in the field of bone defect repair. However, the membrane prepared by this method has the defect of fast collagen degradation rate, usually only maintaining for about 20 days. Problems such as irregular degradation, too fast degradation rate, and poor spatial stability are likely to cause collapse at the surgical site and affect the repair of bone defects. The Miele D research group electrospun collagen / polycaprolactone nanofibers in a green solvent (Miele D, Catenacci L, Rossi S, Sandri G, Sorrenti M, Terzi A, Giannini C, Riva F, Ferrari F, Caramella C, Bonferoni MC. Collagen / PCL Nanofibers Electrospun in Green Solvent by DOE Assisted Process. An Insight into Collagen Contribution. Materials (Basel). 2020 Oct 22;13(21):4698. doi: 10.3390 / ma13214698.). After one week in an aqueous environment, the collagen was completely released. Because its degradation rate does not match the formation rate of new bone, it is difficult to achieve good bone defect repair effects.

[0005] In summary, when using electrospinning technology to prepare nanofibrous bone repair materials containing collagen, there are still problems such as a fast degradation rate of collagen and a mismatch between the degradation rate of the composite material and the new bone formation rate. How to simulate the natural structure of the periosteum and prepare a bilayer artificial periosteum with a controllable collagen release rate and osteogenic induction activity is one of the difficult problems in the research and development of artificial bone repair materials with potential application prospects. Summary of the Invention

[0006] In view of the above problems, the first object of the present invention is to provide a bilayer heterogeneous artificial periosteum that mimics the natural structure of the periosteum with a controllable collagen release rate and good osteogenic induction activity, and by controlling the in vivo and in vitro release rates of collagen, better exert the osteogenic induction activity and meet the requirements of bone defect repair.

[0007] Another object of the present invention is to provide a preparation method of the above bilayer heterogeneous artificial periosteum with a controllable collagen release rate.

[0008] The present invention first uses coaxial electrospinning to encapsulate collagen and nano-hydroxyapatite in polycaprolactone to form a nano-hydroxyapatite / type I collagen / polycaprolactone (nano Hydroxylapatite / Type I Collagen / Polycaprolactone, abbreviated as HC) layer. This layer provides a microenvironment for bone defect repair by recruiting cells and promoting osteogenic differentiation of cells, and promotes the repair of bone defects; then uses polycaprolactone to electrospin into a high-density nanofiber membrane to provide good mechanical properties and prevent connective tissue from growing into the bone defect site, and by adding an appropriate amount of chitosan to increase its degradation rate and achieve an antibacterial effect at the same time, forming a chitosan / polycaprolactone (Chitosan / Polycaprolactone, abbreviated as CP) layer. The present invention controls the release rate of collagen by controlling the content of polycaprolactone in the coaxial electrospinning of the HC layer to form a nanofiber shell layer structure with different thicknesses, thereby controlling the in vivo and in vitro degradability and osteogenic induction activity of the composite material, and preparing a bilayer heterogeneous artificial periosteum with a controllable collagen degradation rate.

[0009] The specific technical solution of the present invention is as follows: A bilayer heterogeneous artificial periosteum with a controllable collagen degradation rate, characterized in that it includes an HC layer and a CP layer. The HC layer is a coaxial electrospun nanofiber membrane formed by a polycaprolactone shell layer and a collagen and nano-hydroxyapatite core layer, and the CP layer is a polycaprolactone nanofiber membrane containing chitosan.

[0010] The mass ratio of the nano-hydroxyapatite to the collagen is 1: (1-10).

[0011] The thickness of the polycaprolactone shell layer is 60-350 nm.

[0012] The mass percentage content of chitosan in the CP layer is 10 - 50%.

[0013] A preparation method of the bilayer heterogeneous artificial periosteum with controllable collagen release rate, which is characterized by comprising the following steps: (1) Preparation of the CP layer nanofiber membrane: Dissolve chitosan in acetic acid solution to prepare a chitosan solution; dissolve polycaprolactone (PCL) in dichloromethane containing N, N - dimethylformamide to prepare a PCL solution; mix the above two solutions in a volume ratio of 1:1, load them into a syringe, and perform electrospinning, and receive the CP layer nanofiber membrane with aluminum foil; (2) Preparation of the HC layer nanofiber membrane: Add nano - hydroxyapatite to acetic acid solution, ultrasonically treat to completely disperse the nanoparticles, then add type I collagen and stir to obtain a core layer solution of collagen and nano - hydroxyapatite; dissolve polycaprolactone (PCL) in glacial acetic acid to obtain a shell layer solution of polycaprolactone (PCL); load the prepared core layer solution and shell layer solution into the nozzles of coaxial electrospinning respectively, perform coaxial electrospinning, and receive and prepare the HC layer nanofiber membrane on the CP layer prepared in step (1), and finally prepare the HC - CP bilayer heterogeneous artificial periosteum.

[0014] The mass - volume concentration of the polycaprolactone (PCL) shell layer solution is 6% - 12%.

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: (1) Use coaxial electrospinning to wrap collagen and nano - hydroxyapatite in polycaprolactone, so that the collagen release rate becomes slower; control the collagen release rate by controlling the content of polycaprolactone, match the bone repair process, and better meet the requirements of bone defect repair.

[0016] (2) Simulate the layered structure and specific functions of each layer of the natural periosteum: the HC layer close to the injury site uses the release of collagen and nano - hydroxyapatite to provide a micro - environment for bone defect repair, and promotes the repair of bone defects by recruiting cells and promoting cell differentiation; the CP layer far from the injury site provides good mechanical properties through a high - density nanofiber membrane and prevents connective tissue from growing into the bone defect site.

[0017] (3) The bilayer heterogeneous artificial periosteum prepared by the present invention has good cell compatibility and good osteogenic induction activity, which is beneficial to the migration, proliferation and osteogenic differentiation of pre-osteoblasts. The composite artificial periosteum was implanted into the skull defect site of rats. Observation by micro-CT and Masson staining showed that the bilayer heterogeneous artificial periosteum could induce the generation and deposition of new bone mass and promote the repair of bone defects. Description of the Drawings

[0018] Figure 1 are the scanning electron microscope images of the CP layer and HC layer nanofiber membranes prepared by the present invention.

[0019] Among them, A. Scanning electron microscope image of the CP layer; B. Scanning electron microscope image of the HC layer.

[0020] Figure 2 is the transmission electron microscope image of the HC layer nanofibers prepared by the present invention.

[0021] Among them, A. HC-0.6; B. HC-0.8; C. HC-1.0; D. HC-1.2; E. Statistical chart of the PCL shell layer thickness.

[0022] Figure 3 is the collagen release rate graph of the HC layer nanofiber membrane prepared by the present invention.

[0023] Figure 4 is the mechanical property test graph of different nanofiber membranes prepared by the present invention.

[0024] Among them, A. Ultimate tensile strength of different nanofiber membranes; B. Fracture strain of different nanofiber membranes; C. Young's modulus of different nanofiber membranes.

[0025] Figure 5 is the osteogenic induction activity and cell migration activity graph of the CP layer nanofiber membrane and the HC-CP bilayer heterogeneous artificial periosteum prepared by the present invention.

[0026] Among them, A. Microscopic imaging results of the scratch experiment; B. Quantitative analysis of the scratch closure rate by ImageJ software; C. ALP staining results; D. Quantitative analysis results of ALP activity.

[0027] Figure 6 are the micro-CT images, quantitative statistical charts and Masson staining charts of the CP layer nanofiber membrane and the HC-CP bilayer heterogeneous artificial periosteum implanted into the rat skull at 4, 8, and 12 weeks.

[0028] Among them, A. Micro-CT detection results; B. Image J calculation of the new bone coverage rate at the skull defect site in Figure A; C. Masson staining results of tissue sections. Detailed implementation manners

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and through specific embodiments.

[0030] Example 1:

[0031] (1) Dissolve 0.3 g of chitosan in 10 ml of 60% acetic acid aqueous solution to prepare a 3% (w / v) chitosan solution ①, and stir with a magnetic stirrer for 24 h.

[0032] (2) Dissolve 2.0 g of polycaprolactone (PCL) in 10 ml of dichloromethane containing N, N-dimethylformamide (30%, v / v) to prepare a 20% (w / v) PCL solution ②, and stir with a magnetic stirrer for 24 h.

[0033] (3) Add 0.1 g of nano-hydroxyapatite particles to 10 ml of acetic acid aqueous solution (50% v / v), ultrasonically treat for 30 minutes until the nanoparticles are completely dispersed, and then add 0.4 g of collagen to prepare a 4% (w / v) collagen solution ③, and stir with a magnetic stirrer at 4 °C for 24 h.

[0034] (4) Dissolve 0.6 g, 0.8 g, 1.0 g, and 1.2 g of polycaprolactone (PCL) in 10 ml of glacial acetic acid respectively to prepare a 6% PCL solution ④, an 8% PCL solution ⑤, a 10% PCL solution ⑥, and a 12% PCL solution ⑦, and stir with a magnetic stirrer for 24 h.

[0035] (5) Mix the above two solutions ① and ② in a ratio of 1:1 to obtain solution ⑧.

[0036] (6) Load solution ⑧ into a syringe with a 22-gauge stainless steel needle, with a voltage of 16 kV, a flow rate of 1.5 ml / h, and a distance between the needle tip and the collector of 24 cm, electrospin for 4 h, collect with aluminum foil to obtain a CP layer nanofiber membrane, and place it in a fume hood to dry overnight for standby.

[0037] (7) The above-prepared Solutions ③ and ④ were respectively loaded into the coaxial electrospinning nozzles. The voltage was 18 kV, the core flow rate (Solution ③) was 0.4 ml / h, the shell flow rate (Solution ④) was 1.6 ml / h, the distance between the needle tip and the collector was 24 cm, electrospinning was carried out for 3 h, and a HC-0.6 nanofiber membrane was obtained by receiving with aluminum foil, and it was placed in a fume hood to dry overnight for standby.

[0038] (7) The above-prepared Solutions ③ and ⑤ were respectively loaded into the coaxial electrospinning nozzles. The voltage was 18 kV, the core flow rate (Solution ③) was 0.4 ml / h, the shell flow rate (Solution ⑤) was 1.6 ml / h, the distance between the needle tip and the collector was 24 cm, electrospinning was carried out for 3 h, and a HC-0.8 nanofiber membrane was obtained by receiving with aluminum foil, and it was placed in a fume hood to dry overnight for standby.

[0039] (8) The above-prepared Solutions ③ and ⑥ were respectively loaded into the coaxial electrospinning nozzles. The voltage was 18 kV, the core flow rate (Solution ③) was 0.4 ml / h, the shell flow rate (Solution ⑥) was 1.6 ml / h, the distance between the needle tip and the collector was 24 cm, electrospinning was carried out for 3 h, and a HC-1.0 nanofiber membrane was obtained by receiving with aluminum foil, and it was placed in a fume hood to dry overnight for standby.

[0040] (9) The above-prepared Solutions ③ and ⑦ were respectively loaded into the coaxial electrospinning nozzles. The voltage was 18 kV, the core flow rate (Solution ③) was 0.4 ml / h, the shell flow rate (Solution ⑦) was 1.6 ml / h, the distance between the needle tip and the collector was 24 cm, electrospinning was carried out for 3 h, and a HC-1.2 nanofiber membrane was obtained by receiving with aluminum foil, and it was placed in a fume hood to dry overnight for standby.

[0041] (10) After electrospinning 3 ml of Solution ⑧, 2 ml of the coaxial electrospun Solutions ③ and ⑦ were used on the upper layer to prepare a HC-CP double-layer nanofiber membrane, which was Example 1.

[0042] Morphology characterization of the nanofiber membrane: The morphologies of the HC layer and CP layer nanofibers were analyzed by scanning electron microscope (SEM). The specific method was: cut small pieces of 1 cm×1 cm of the HC and CP nanofiber membranes, first use an ion sputtering instrument to spray the surface with a current of 5 - 10 mA for 60 s, and then observe the surface morphology of the nanofibers. The results showed that: as Figure 1 shown in A and B, the SEM images showed the dense fiber structures of different nanofiber membranes, and the nanofiber membranes exhibited continuous and smooth nanofibers; the diameters of the CP layer nanofibers were between 50 nm - 250 nm, and the diameters of the HC layer nanofibers were between 100 nm - 500 nm. The fiber membranes had small pores between fibers of about 2 - 5 µm.

[0043] Internal structure characterization of HC layer nanofibers: The TEM images of HC layer nanofibers are shown in Figure 2. The diameter of HC-0.6 fibers is about 237.4 nm. The surface of HC-0.8 fibers is smooth, and the diameter of the fibers is about 243.4 nm, among which the diameter of the PCL shell layer is about 136.7 nm. The diameter of HC-1.0 fibers is about 295.7 nm, among which the diameter of the PCL shell layer is about 174.5 nm. The diameter of HC-1.2 fibers is about 509.1 nm, among which the diameter of the PCL shell layer is about 342.8 nm. The statistical result of the PCL shell layer thickness is 60 - 350 nm. The above results show that: by controlling the content of polycaprolactone in the coaxial electrospinning of the HC layer, nanofiber shell layer structures with different thicknesses can be formed.

[0044] Testing of the collagen release rate in the HC layer nanofiber membrane: Take a 10mm×10mm HC layer nanofiber membrane and soak it in 1 ml of simulated body fluid (SBF). After 1, 2, 4, and 8 days respectively, measure the concentration of the released collagen and draw a curve of the collagen release rate over time.

[0045] From Figure 3 It can be seen that the in vitro release of collagen in different HC layer nanofiber membranes all shows a typical biphasic release behavior: a burst release stage and a sustained release stage. In the burst release stage, the collagen is released rapidly, and the cumulative release amount reaches 40% - 60%. In the sustained release stage, the release rate of collagen tends to be flat. Specifically, the PCL shell layers of the HC-0.6 and HC-0.8 groups of nanofibers are thinner, and the collagen release rate is faster. The cumulative release amount exceeds 95% at 12 days. The cumulative release amount of collagen in the HC-1.0 group is reduced to about 88% at 12 days, and the increase in the PCL concentration reduces the collagen release rate. In the HC-1.2 group, as the PCL concentration increases, the structure of the nanofibers becomes more compact, delaying the release of collagen. At the same time, the hydrophobic property of PCL further inhibits water molecules from entering the fiber core, further delaying the release of collagen. At 12 days, the collagen release amount in the HC-1.2 group is only 75%. The above results show that: different nanofiber shell layer structures can achieve the purpose of controlling the collagen release rate, thereby controlling the in vivo and in vitro degradability and osteogenic induction activity of the composite material. In particular, the HC-1.2 group of nanofibers can better control the release of collagen, which is beneficial to the repair of bone defects.

[0046] Mechanical property testing of the nanofiber membrane: The nanofiber membranes (HC-0.6, HC-0.8, HC-1.0, HC-1.2, CP, and HC-CP) were cut into strip specimens (40×10 mm) for uniaxial tensile tests. The ends of the membranes were mounted in the fixtures of a universal testing machine (Instron 5567, Norwood, MA) equipped with a 200 N load cell. The uniaxial tensile tests of the membranes were carried out at a tensile rate of 10 mm / min, and the ultimate tensile strength, fracture strain, and Young's modulus were mainly analyzed.

[0047] As Figure 4 According to the mechanical property test results, in the HC group, as the PCL content increased from 0.6 to 1.2, the stress value increased from 1.10 MPa to 2.20 MPa, indicating that PCL had a significant strengthening effect. The fracture strain analysis showed that the strain of the HC-1.2 group reached the highest, indicating that when the PCL content was moderate (HC-1.2), the material had better toughness and balanced mechanical properties. In terms of Young's modulus, the HC-1.0 group showed the highest value (1.73 MPa), while the HC-1.2 group was lower (1.04 MPa), indicating that an appropriate amount of PCL could improve rigidity, but an excessive amount might lead to an increase in the brittleness of the material. The CP group showed the highest stress and Young's modulus, which might be due to the synergistic effect of the rigid molecular chains of chitosan and the hydrophobicity of PCL, making the fiber structure more dense and the crystallinity increase, thus improving the overall mechanical properties. The HC-CP group achieved optimization in mechanical properties. The stress and Young's modulus of the HC-CP group were between those of the CP group and the HC-1.2 group, and were significantly higher than those of the single HC group.

[0048] Example 2: Study on the osteogenic activity and cell migration activity of nanofiber membranes Using mouse embryonic osteoblast precursor cells (MC3T3-E1) as the research object, the osteogenic activity of the nanofiber membranes was studied by alkaline phosphatase (ALP) staining and ALP activity measurement, and the cell migration promoting activity of the nanofiber membranes was studied by scratch experiments.

[0049] (1) The irradiated and sterilized HC-CP double-layer nanofiber membrane and CP nanofiber membrane were fixed in a 12-well plate, and then the MC3T3-E1 cell suspension was inoculated onto the surfaces of the HC-CP and CP membranes. The HC layer of the HC-CP double-layer nanofiber membrane was on the top, facing the cell side. ALP staining and ALP activity measurement were carried out on days 7 and 14 respectively.

[0050] (2)Fix the irradiated sterilized HCCP and CP membranes on a 96-well plate, and then inoculate the MC3T3-E1 cell suspension onto the surfaces of the HCCP and CP membranes. The HC layer of the HCCP double-layer nanofiber membrane is on the top, facing the cell side. Scratch vertically with a sterile pipette tip, wash off the detached cells with PBS, and add culture medium to continue culturing. Observe and take pictures under an inverted microscope at 0, 12, and 24 h respectively. Quantitatively analyze the scratch closure rate using ImageJ software.

[0051] The results are as Figure 5 shown. Through alkaline phosphatase (ALP) staining and ALP activity determination, it was found that the nanofiber membrane (HC-CP) group showed a higher ALP expression level than the control group (CP). The staining results showed that the ALP staining in the HC-CP group was more obvious after 7 days and 14 days of culture, indicating that it has a promoting effect on the osteogenic differentiation of MC3T3-E1 cells. The quantitative analysis results of ALP activity further verified this result. The ALP activity in the HCCP group was significantly higher than that in the CP group at both time points. The effect of the nanofiber membrane on the migration ability of MC3T3-E1 cells was evaluated by a scratch assay. In the microscopic imaging observations at 0 h, 12 h, and 24 h, the scratch closure rate of the HC-CP group was faster than that of the CP group. Especially at 24 h, the cells in the HC-CP group had significantly filled the scratched area, while there was still a large unclosed area in the CP group. ImageJ quantitative analysis showed that the scratch closure rate of the HC-CP group was significantly higher than that of the CP group, indicating that this nanofiber membrane can effectively promote the migration ability of osteoprogenitor cells. The experimental results showed that the HCCP nanofiber membrane has significant advantages in promoting the osteogenic differentiation and migration of MC3T3-E1 cells. The HC-CP group not only increased the ALP expression and activity but also accelerated the cell migration ability, which may be attributed to its unique double-layer structure and material properties.

[0052] Example 3: Study on the bone defect repair function of the nanofiber membrane Select male SD rats weighing 240 - 260 g. In the middle and upper region of the rat skull, prepare a bone defect with an inner diameter of approximately 5 mm using a dental drill. Divide the experimental animals into four groups: a blank group, a CP group, an HC-CP group, and an HAO group. The blank group is the negative control group without implanting any material. The HAO group is the positive control group implanted with the Hyaff® bone repair material from Zhenghai BioTech Co., Ltd. The CP group and the HC-CP group are implanted with the irradiated sterilized CP and HC-CP nanofiber membranes respectively. After the operation, suture the muscles layer by layer, and use micro-CT and histological section Masson staining to study the bone defect repair situation at 1, 2, and 3 months after the operation. The results are as Figure 6 shown.

[0053] Micro-CT scan results (Figure 6A) showed that there were significant differences in new bone formation among different groups at 4, 8, and 12 weeks after surgery. In the blank group, there was almost no obvious bone generation in the bone defect area throughout the experimental period, while both the HC-CP group and the HAO group showed better new bone formation trends. Among them, the degree of bone defect filling in the HC-CP group was higher than that in the CP group and was close to that in the HAO group, indicating that the HC-CP nanofiber membrane had strong bioactivity in promoting bone repair. The new bone coverage rate at the skull defect site in the Micro-CT images was calculated using ImageJ software (Figure 6B). The data analysis results showed that the bone defect filling rate in the HC-CP group was significantly higher than that in the CP group at 4, 8, and 12 weeks (P < 0.05). The repair effect of the HC-CP group was close to that of the HAO group, further confirming its potential application value in bone defect repair. The Masson staining results (Figure 6C) further revealed the bone tissue repair conditions of different experimental groups. At 12 weeks, both the HC-CP group and the HAO group showed more deposition of new bone tissue, with tightly arranged collagen fibers and a relatively blurred boundary with the native bone tissue, indicating relatively complete bone reconstruction. In the CP group, less new bone was formed, and there was almost no obvious bone repair in the blank group. The HC-CP group showed better osteogenic ability, indicating that its nanofiber membrane material could promote the deposition and remodeling of the bone matrix. The experimental results showed that the HC-CP nanofiber membrane could significantly promote the repair of skull defects. Compared with the CP group, the HC-CP group showed faster new bone formation ability and higher new bone coverage rate, and its repair effect was close to that of the commercial HAO bone repair material. Generally speaking, the HC-CP nanofiber membrane has good application potential in bone defect repair, providing a new feasible strategy for the field of bone tissue engineering.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. A double-layer heterogeneous artificial periosteum with controllable collagen degradation rate, characterized in that: The invention comprises an HC layer and a CP layer, wherein the HC layer is a coaxial electrostatic spinning nanofiber membrane formed by a polycaprolactone shell layer and a collagen and nano-hydroxyapatite core layer, and the CP layer is a polycaprolactone nanofiber membrane containing chitosan.

2. The double-layer heterogeneous artificial periosteum according to claim 1, characterized in that: The thickness of the polycaprolactone shell layer is 60-350 nm.

3. The double-layer heterogeneous artificial periosteum according to claim 1, characterized in that: The mass ratio of the nano-hydroxyapatite to collagen is 1: (1-10).

4. The double-layer heterogeneous artificial periosteum according to claim 1, characterized in that: The mass percentage of chitosan in the CP layer is 10-50%.

5. A method for preparing the double-layer heterogeneous artificial periosteum with controllable collagen degradation rate as claimed in claim 1, characterized in that: The following steps are involved: (1) Preparation of CP layer nanofiber membrane: The chitosan solution is prepared by dissolving chitosan in acetic acid solution; the polycaprolactone solution is prepared by dissolving polycaprolactone in dichloromethane containing N, N-dimethylformamide; the two solutions are mixed in a volume ratio of 1:1, loaded into a syringe, and electrospun, and the CP layer nanofiber membrane is obtained by receiving with aluminum foil; (2) Preparation of HC layer nanofiber membrane: Nano-hydroxyapatite is added to an acetic acid solution, and ultrasonic treatment is performed to completely disperse the nanoparticles. Type I collagen is then added and stirred to obtain a collagen and nano-hydroxyapatite core layer solution. Polycaprolactone is dissolved in glacial acetic acid to obtain a polycaprolactone shell layer solution. The prepared core layer solution and shell layer solution are respectively loaded into a coaxial electrospinning nozzle for coaxial electrospinning, and an HC layer nanofiber membrane is prepared on the CP layer prepared in step (1), and finally an HC-CP double-layer heterogeneous artificial periosteum is prepared.

6. The method for preparing the double-layer heterogeneous artificial periosteum according to claim 5, characterized in that: The mass volume concentration of the polycaprolactone shell solution is 6%-12%.

Images