阅读说明：本技术 骨组织再生引导膜及其制备方法 (Bone tissue regeneration guide membrane and preparation method thereof ) 是由 任亚东 施猛 夏春森 王陈 刘亚龙 于 2021-07-13 设计创作，主要内容包括：本申请提供一种骨组织再生引导膜及其制备方法。上述的骨组织再生引导膜包括引导生长定向纤维膜层和选择透过无序纤维膜层,引导生长定向纤维膜层与选择透过无序纤维膜层连接。上述的骨组织再生引导膜具有屏蔽成纤维细胞、不间断输送营养元素和交换代谢产物,以及定向引导骨细胞生长迁移的三重作用,提高了骨组织再生引导膜对骨缺损的修复愈合效果。(The application provides a bone tissue regeneration guiding membrane and a preparation method thereof. The bone tissue regeneration guiding membrane comprises a growth guiding directional fiber membrane layer and a selective permeation disordered fiber membrane layer, wherein the growth guiding directional fiber membrane layer is connected with the selective permeation disordered fiber membrane layer. The bone tissue regeneration guide membrane has triple functions of shielding fibroblasts, continuously conveying nutrient elements and exchanging metabolites and directionally guiding growth and migration of bone cells, and improves the repairing and healing effects of the bone tissue regeneration guide membrane on bone defects.)

1. The bone tissue regeneration guiding membrane is characterized by comprising a growth guiding directional fiber membrane layer and a selective permeation disordered fiber membrane layer, wherein the growth guiding directional fiber membrane layer is connected with the selective permeation disordered fiber membrane layer.

2. The bone tissue regeneration guide membrane according to claim 1, wherein the permselective disordered fiber membrane layer is miscible with the microetched growth directing fiber membrane layer interface such that the growth directing fiber membrane layer and the permselective disordered fiber membrane layer are bonded together.

3. The bone tissue regeneration guide membrane according to claim 1, wherein the growth directing oriented fiber membrane layer is miscible with the microetched selectively permeable disordered fiber membrane layer at its interface to bind the growth directing oriented fiber membrane layer and the selectively permeable disordered fiber membrane layer together.

4. The bone tissue regeneration guiding membrane according to claim 1, wherein the growth guiding oriented fiber membrane layer comprises the following components:

high molecular substances and bioactive substances.

5. The bone tissue regeneration guiding membrane as claimed in claim 1, wherein the growth guiding oriented fiber membrane layer comprises the following components in parts by mass:

20-99 parts of high molecular substance;

1-80 parts of bioactive substances.

6. The bone tissue regeneration guiding membrane as claimed in claim 1, wherein the growth guiding oriented fiber membrane layer comprises the following components in parts by mass:

80-90 parts of high molecular substance;

10-20 parts of bioactive substances.

7. The guided membrane for bone tissue regeneration according to any one of claims 4 to 6, wherein the polymer in the guided growth oriented fiber membrane layer is at least one of collagen, bone matrix gelatin, silk fibroin, chitosan, polylactic acid, polycaprolactone, polyamide, poly-L-lactic acid, polyglycolic acid, and polylactic acid-polyglycolic acid copolymer.

8. The guide membrane for bone tissue regeneration according to claim 7, wherein the polylactic acid has an average molecular weight of 100000 to 1200000.

9. The guiding membrane for bone tissue regeneration according to any one of claims 4 to 6, wherein the bioactive substance in the guiding growth oriented fiber membrane layer is at least one of a bio-organic active substance, a bioactive inorganic ceramic and a bioactive inorganic glass.

10. The bone tissue regeneration guide membrane of claim 9, wherein the bioactive inorganic ceramic is at least one of a silicate bioactive ceramic, a borate bioactive ceramic, and a phosphate bioactive ceramic.

11. The bone tissue regeneration guiding membrane according to claim 10, wherein the silicate bioactive ceramic contains dicalcium silicate and/or tricalcium silicate.

12. The bone tissue regeneration guiding membrane according to claim 10, wherein the phosphate bioactive ceramic contains hydroxyapatite and/or β -tricalcium phosphate.

13. The guiding film for bone tissue regeneration according to claim 9, wherein the bioactive inorganic glass is at least one of a silicate bioactive glass, a borate bioactive glass, and a phosphate bioactive glass.

14. The bone tissue regeneration guide membrane according to claim 9, wherein the bioactive inorganic glass contains silica, calcium oxide, phosphorus pentoxide, and sodium oxide as main components, and further contains at least one of potassium oxide, magnesium oxide, aluminum oxide, and calcium fluoride.

15. The bone tissue regeneration guide membrane according to any one of claims 4 to 6, wherein the bioactive substance in the growth guide oriented fiber membrane layer is in a granular form, and the particle size distribution thereof is 0.3 to 30 μm.

16. The bone tissue regeneration guide membrane according to claim 1, wherein the growth guide oriented fiber membrane layer is formed by oriented stacking arrangement of fiber filaments.

17. The bone tissue regeneration guiding membrane according to claim 1, wherein the permselective disordered fiber membrane layer is formed by disordered interweaving or random stacking arrangement of fiber filaments.

18. The bone tissue regeneration guide membrane according to claim 16 or 17, wherein the fiber filaments have a diameter of 100nm to 150 μm.

19. The guiding membrane for bone tissue regeneration according to claim 1, wherein the selectively permeable disordered fibrous membrane layer comprises the following components:

a high molecular substance.

20. The guided membrane for bone tissue regeneration according to claim 19, wherein the high molecular substance selectively permeating through the disordered fiber membrane layer is at least one of collagen, bone matrix gelatin, silk fibroin, chitosan, polylactic acid, polycaprolactone, polyamide, poly-L-lactic acid, polyglycolic acid, and polylactic-polyglycolic acid copolymer.

21. The membrane according to claim 20, wherein the polylactic acid has an average molecular weight of 100000 to 1200000.

22. A method for producing a bone tissue regeneration guide membrane according to any one of claims 1 to 21, comprising the steps of:

obtaining a guided growth oriented fiber film layer and a selective permeation disordered fiber film layer;

and connecting the guided growth oriented fiber membrane layer and the selective permeation disordered fiber membrane layer to obtain the bone tissue regeneration guiding membrane.

23. The method for preparing a guiding membrane for bone tissue regeneration according to claim 22, wherein the step of obtaining the guiding growth oriented fiber membrane layer comprises the following steps:

adding a high molecular substance and a bioactive substance into an organic solvent to obtain a first spinning solution;

and carrying out directional electrostatic spinning on the first spinning solution to obtain the oriented fiber film layer for guided growth.

24. The method of preparing a bone tissue regeneration guide membrane according to claim 23, wherein the first spinning solution contains the polymer substance in an amount of 5 to 30 wt%.

25. The method for preparing a guiding membrane for bone tissue regeneration according to claim 22, wherein the step of obtaining the permselective disordered fibrous membrane layer comprises the steps of:

adding a high molecular substance into an organic solvent to obtain a second spinning solution;

and performing disordered electrostatic spinning on the second spinning solution to obtain the selective permeation disordered fiber film layer.

26. The method of preparing a bone tissue regeneration guide membrane according to claim 25, wherein the second spinning solution contains the polymer substance in an amount of 5 to 30 wt%.

27. The method for preparing a guiding membrane for bone tissue regeneration according to claim 22, wherein the operation of connecting the growth guiding oriented fiber membrane layer and the selective permeation disordered fiber membrane layer comprises the following steps:

carrying out microetching operation on the guided growth oriented fiber film layer or the selective permeation disordered fiber film layer so as to form a microetching interface on the guided growth oriented fiber film layer or the selective permeation disordered fiber film layer;

and mixing the selective permeation disordered fiber film layer with the microetching interface of the guided growth oriented fiber film layer, or mixing the guided growth oriented fiber film layer with the microetching interface of the selective permeation disordered fiber film layer, so that the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer are combined together.

28. The method for preparing a guiding membrane for bone tissue regeneration according to claim 27, wherein after the step of mixing the selectively permeable disordered fiber membrane layer with the microetched interface of the guided growth oriented fiber membrane layer or mixing the guided growth oriented fiber membrane layer with the microetched interface of the selectively permeable disordered fiber membrane layer, the method for preparing a guiding membrane for bone tissue regeneration further comprises the steps of:

and drying and shaping the mixed and dissolved selective permeation disordered fiber film layer and the guided growth oriented fiber film layer so as to enable the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer to be connected in a curing manner.

29. The method for preparing a guiding membrane for bone tissue regeneration as defined in claim 28, wherein the drying and shaping time for drying and shaping the mixed and dissolved selective permeable disordered fiber membrane layer and the guided growth oriented fiber membrane layer is 5-50 h.

30. The method for preparing a guiding membrane for bone tissue regeneration according to claim 27, wherein an organic solvent is used to microetch the guided growth oriented fiber membrane layer or the selectively permeable disordered fiber membrane layer, so as to form a microetched interface on the guided growth oriented fiber membrane layer or the selectively permeable disordered fiber membrane layer.

31. The method for preparing a guiding membrane for bone tissue regeneration according to claim 27, wherein the selective transmission disordered fiber membrane layer and the guided growth oriented fiber membrane layer are mixed and dissolved at the microetched interface by a pressing process, or the guided growth oriented fiber membrane layer and the selective transmission disordered fiber membrane layer are mixed and dissolved at the microetched interface by a pressing process, so that the guided growth oriented fiber membrane layer and the selective transmission disordered fiber membrane layer are combined together.

32. The method for preparing a guiding membrane for bone tissue regeneration according to claim 27, wherein the micro-etching operation on the growth guiding orientation fiber membrane layer or the selective permeation disordered fiber membrane layer is to use a spraying process or a painting process to attach an organic solvent on the growth guiding orientation fiber membrane layer or the selective permeation disordered fiber membrane layer, so that a micro-etched interface is formed on the growth guiding orientation fiber membrane layer or the selective permeation disordered fiber membrane layer.

33. The method of manufacturing a bone tissue regeneration guide membrane according to claim 22, wherein the method of manufacturing a bone tissue regeneration guide membrane is specifically a method of forming the selectively permeable disordered fiber membrane layer by performing a spinning attachment operation of a spinning solution on the obtained growth guide oriented fiber membrane layer through electrostatic spinning, and an organic solvent contained in the spinning solution microetches the growth guide oriented fiber membrane layer so that a microetched interface is formed on the growth guide oriented fiber membrane layer and is combined with the selectively permeable disordered fiber membrane layer.

34. The method of manufacturing a bone tissue regeneration guide membrane according to claim 22, wherein the bone tissue regeneration guide membrane is manufactured by forming the growth guide orientation fiber membrane layer by performing a spinning attachment operation of a spinning solution on the obtained permselective disordered fiber membrane layer through electrostatic spinning, and an organic solvent contained in the spinning solution microetches the permselective disordered fiber membrane layer to form a microetched interface on the permselective disordered fiber membrane layer and combine the microetched interface with the growth guide orientation fiber membrane layer.

35. The method for preparing a bone tissue regeneration guide membrane according to claim 22, wherein the method for preparing a bone tissue regeneration guide membrane specifically comprises the steps of:

dissolving a high molecular substance in an organic solvent to prepare a first spinning solution;

dissolving a high molecular substance and a bioactive substance in an organic solvent to prepare a second spinning solution;

carrying out electrostatic spinning by adopting a first spinning solution to obtain the oriented fiber film layer for guided growth;

and coating the growth-guiding oriented fiber film layer on a roller to serve as a receiving substrate, and performing electrostatic spinning by adopting a second spinning solution to obtain the combined growth-guiding oriented fiber film layer and the selective permeation disordered fiber film layer.

36. The method of preparing a bone tissue regeneration guide membrane according to claim 23, 25, 33, 34, or 35, wherein the electrospinning is performed by using an electrospinning machine in which a dope extrusion speed is set to 0.05mL/min to 0.4 mL/min.

37. The method for preparing a bone tissue regeneration guide membrane according to claim 23, 25, 33, 34 or 35, wherein the electrospinning is performed by an electrospinning machine having a set voltage of 8kV to 25 kV.

38. The method of preparing a bone tissue regeneration guide membrane according to claim 23, 25, 33, 34 or 35, wherein the electrospinning is performed by using an electrospinning machine in which a distance between the spinning solution nozzle and the receiving substrate is set to be 5cm to 30 cm.

39. The method for preparing a guiding membrane for bone tissue regeneration according to claim 23, 25, 30, 32, 33, 34 or 35, wherein the organic solvent is at least one of chloroform, dichloromethane, hexafluoroisopropanol and acetone.

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a bone tissue regeneration guiding membrane and a preparation method thereof.

Background

When bone defect occurs, the growth speed of soft tissue is faster than that of bone tissue, so that the soft tissue is easy to grow into the bone defect area to fill the bone defect part, the growth and healing of new bone tissue are seriously hindered, the bone defect part is recovered slowly, and even the bone defect part is difficult to heal to cause bone nonunion disease. To solve this problem, a concept of guiding bone regeneration was proposed in 1993, in which a bone regeneration guiding membrane is used to separate soft tissue from a bone defect region, physically block the soft tissue from entering the bone defect region, and provide a sufficient time for the growth of new bone, and the bone regeneration guiding membrane can also serve as a scaffold for bone regeneration to guide the adhesion, migration, and growth of bone cells.

However, the existing bone tissue regeneration guiding membrane generally only provides a porous structure for adhesion and migration for the growth of bone cells, but does not have the function of directionally guiding the growth and migration of bone cells, i.e., cannot guide the directional growth and migration of bone cells, thereby limiting the application range of the bone tissue regeneration guiding membrane.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a bone tissue regeneration guiding membrane capable of directionally guiding the growth and migration of bone tissues and a preparation method thereof.

The purpose of the invention is realized by the following technical scheme:

a bone tissue regeneration guiding membrane comprises a growth guiding directional fiber membrane layer and a selective permeation disordered fiber membrane layer, wherein the growth guiding directional fiber membrane layer is connected with the selective permeation disordered fiber membrane layer.

In one embodiment, the selectively permeable disordered fibrous membrane layer is miscible with the microetched growth directing fibrous membrane layer interface to bond the growth directing oriented fibrous membrane layer and the selectively permeable disordered fibrous membrane layer together.

In one embodiment, the growth-directing oriented fiber film layer is miscible with the microetched selectively permeable disordered fiber film layer at its interface to bond the growth-directing oriented fiber film layer and the selectively permeable disordered fiber film layer together.

In one embodiment, the guided growth oriented fiber membrane layer comprises the following components:

high molecular substances and bioactive substances.

In one embodiment, the guided growth oriented fiber film layer comprises the following components in parts by mass:

20-99 parts of high molecular substance;

1-80 parts of bioactive substances.

In one embodiment, the guided growth oriented fiber film layer comprises the following components in parts by mass:

80-90 parts of high molecular substance;

10-20 parts of bioactive substances.

In one embodiment, the polymer in the guided growth oriented fiber membrane layer is at least one of collagen, bone matrix gelatin, silk fibroin, chitosan, polylactic acid, polycaprolactone, polyamide, poly-L-lactic acid, polyglycolic acid, and polylactic-polyglycolic acid copolymer.

In one embodiment, the polylactic acid has an average molecular weight of 100000-1200000.

In one embodiment, the bioactive substance in the guided growth oriented fiber membrane layer is at least one of a bio-organic active substance, a bioactive inorganic ceramic, and a bioactive inorganic glass.

In one embodiment, the bioactive inorganic ceramic is at least one of a silicate bioactive ceramic, a borate bioactive ceramic, and a phosphate bioactive ceramic.

In one embodiment, the silicate bioactive ceramic contains dicalcium silicate and/or tricalcium silicate.

In one embodiment, the phosphate bioactive ceramic contains hydroxyapatite and/or β -tricalcium phosphate.

In one embodiment, the bioactive inorganic glass is at least one of a silicate bioactive glass, a borate bioactive glass, and a phosphate bioactive glass.

In one embodiment, the bioactive inorganic glass comprises silicon dioxide, calcium oxide, phosphorus pentoxide and sodium oxide as main components, and at least one of potassium oxide, magnesium oxide, aluminum oxide and calcium fluoride.

In one embodiment, the bioactive substance in the growth-directing oriented fiber membrane layer is granular and has a particle size distribution of 0.3 μm to 30 μm.

In one embodiment, the growth-directing oriented fiber membrane layer is formed by oriented stacking arrangement of fiber filaments.

In one embodiment, the selectively permeable disordered fibrous membrane layer is formed by a disordered interweaving or random packing arrangement of fiber filaments.

In one embodiment, the fiber filaments have a diameter of 100nm to 150 μm.

In one embodiment, the permselective disordered fiber film layer comprises the following components:

a high molecular substance.

In one embodiment, the polymer material selectively permeating through the disordered fiber membrane layer is at least one of collagen, bone matrix gelatin, silk fibroin, chitosan, polylactic acid, polycaprolactone, polyamide, poly-L-lactic acid, polyglycolic acid, and polylactic-polyglycolic acid copolymer.

In one embodiment, the polylactic acid has an average molecular weight of 100000-1200000.

A preparation method of a bone tissue regeneration guiding membrane comprises the following steps:

obtaining a guided growth oriented fiber film layer and a selective permeation disordered fiber film layer;

and connecting the guided growth oriented fiber membrane layer and the selective permeation disordered fiber membrane layer to obtain the bone tissue regeneration guiding membrane.

In one embodiment, the obtaining the guided growth oriented fiber membrane layer includes the following steps:

adding a high molecular substance and a bioactive substance into an organic solvent to obtain a first spinning solution;

and carrying out directional electrostatic spinning on the first spinning solution to obtain the oriented fiber film layer for guided growth.

In one embodiment, the mass percentage of the polymer substance in the first spinning solution is 5 wt% to 30 wt%.

In one embodiment, the obtaining the permselective disordered fibrous film layer comprises the steps of:

adding a high molecular substance into an organic solvent to obtain a second spinning solution;

and performing disordered electrostatic spinning on the second spinning solution to obtain the selective permeation disordered fiber film layer.

In one embodiment, the second spinning solution contains 5 wt% to 30 wt% of the polymer substance.

In one embodiment, the connecting operation of the guided growth oriented fiber film layer and the selective transmission disordered fiber film layer comprises the following steps:

carrying out microetching operation on the guided growth oriented fiber film layer or the selective permeation disordered fiber film layer so as to form a microetching interface on the guided growth oriented fiber film layer or the selective permeation disordered fiber film layer;

and mixing the selective permeation disordered fiber film layer with the microetching interface of the guided growth oriented fiber film layer, or mixing the guided growth oriented fiber film layer with the microetching interface of the selective permeation disordered fiber film layer, so that the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer are combined together.

In one embodiment, after the step of mixing the selective permeation disordered fiber membrane layer with the microetched growth-directing fiber membrane layer interface or mixing the growth-directing fiber membrane layer with the microetched selective permeation disordered fiber membrane layer interface, the method for preparing the bone tissue regeneration directing membrane further comprises the following steps:

and drying and shaping the mixed and dissolved selective permeation disordered fiber film layer and the guided growth oriented fiber film layer so as to enable the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer to be connected in a curing manner.

In one embodiment, the drying and shaping time for drying and shaping the mixed and dissolved selective permeation disordered fiber film layer and the guided growth oriented fiber film layer is 5-50 hours.

In one embodiment, the guided growth oriented fiber film layer or the selective transmission disordered fiber film layer is microetched by using an organic solvent, so that a microetched interface is formed on the guided growth oriented fiber film layer or the selective transmission disordered fiber film layer.

In one embodiment, the selective permeation disordered fiber film layer and the selective permeation disordered fiber film layer are mixed and dissolved at the microetching interface by a pressing process, or the selective permeation disordered fiber film layer and the selective permeation disordered fiber film layer are mixed and dissolved at the microetching interface by a pressing process, so that the selective permeation disordered fiber film layer and the selective permeation disordered fiber film layer are combined together.

In one embodiment, the microetching of the growth-directing oriented fiber film layer or the selective permeation disordered fiber film layer is performed by attaching an organic solvent to the growth-directing oriented fiber film layer or the selective permeation disordered fiber film layer by a spraying process or a brushing process, so that a microetched interface is formed on the growth-directing oriented fiber film layer or the selective permeation disordered fiber film layer.

In one embodiment, the preparation method of the bone tissue regeneration guide membrane is specifically to perform a spinning attachment operation of a spinning solution on the obtained growth guide oriented fiber membrane layer through electrostatic spinning to form the selective permeation disordered fiber membrane layer, wherein an organic solvent contained in the spinning solution microetches the growth guide oriented fiber membrane layer, so that a microetching interface is formed on the growth guide oriented fiber membrane layer and is combined with the selective permeation disordered fiber membrane layer.

In one embodiment, the preparation method of the bone tissue regeneration guide membrane is specifically to perform a spinning attachment operation of a spinning solution on the obtained permselective disordered fiber membrane layer through electrostatic spinning to form the growth guide oriented fiber membrane layer, wherein an organic solvent contained in the spinning solution microetches the permselective disordered fiber membrane layer, so that a microetching interface is formed on the permselective disordered fiber membrane layer and is combined with the growth guide oriented fiber membrane layer.

In one embodiment, the preparation method of the bone tissue regeneration guiding membrane specifically comprises the following steps:

dissolving a high molecular substance in an organic solvent to prepare a first spinning solution;

dissolving a high molecular substance and a bioactive substance in an organic solvent to prepare a second spinning solution;

carrying out electrostatic spinning by adopting a first spinning solution to obtain the oriented fiber film layer for guided growth;

and coating the growth-guiding oriented fiber film layer on a roller to serve as a receiving substrate, and performing electrostatic spinning by adopting a second spinning solution to obtain the combined growth-guiding oriented fiber film layer and the selective permeation disordered fiber film layer.

In one embodiment, the electrospinning is performed using an electrospinning machine in which the extrusion speed of the spinning solution is set to 0.05mL/min to 0.4 mL/min.

In one embodiment, the electrostatic spinning is performed by using an electrostatic spinning machine with a set voltage of 8kV to 25 kV.

In one embodiment, the electrospinning is performed using an electrospinning machine in which the distance between the spinning solution nozzle and the receiving substrate is set to be 5cm to 30 cm.

In one embodiment, the organic solvent is at least one of chloroform, dichloromethane, hexafluoroisopropanol, and acetone.

Compared with the prior art, the invention has at least the following advantages:

1. in the bone tissue regeneration guide membrane, the oriented fiber membrane layer for guiding growth is formed by oriented stacking and arranging fiber filaments, so that the differentiation of osteocytes can be promoted, the oriented growth migration of derived cells for guiding the osteocytes can be better realized, the oriented growth migration guide membrane has the functions of oriented growth migration guide and growth migration promotion on the osteocytes, the oriented repair of bone defects is better realized, and the application range of the bone tissue regeneration guide membrane is improved;

2. in the bone tissue regeneration guide membrane, the guide growth directional fiber membrane layer is formed by directional stacking and arranging fiber filaments, the porosity is low, and the bone tissue regeneration guide membrane has high mechanical property and deformation resistance, namely good structural strength;

3. in the bone tissue regeneration guiding membrane, the selective permeation disordered fiber membrane layer is formed by disorderly and randomly arranging the fiber filaments, so that the porosity is high, the fibroblast is more prone to differentiation, proliferation, migration and growth in the selective permeation disordered fiber membrane layer, the effect of blocking the growth of the fibroblast into the growth guiding oriented fiber membrane layer is achieved, namely, the fibroblast is prevented from growing into a bone defect part, and the rapid oriented repair and healing of the bone defect part are facilitated. In addition, the selective permeation disordered fiber film layer has higher porosity, can support the input of nutrient elements in human tissue fluid and the exchange of metabolites at the bone defect, and shows that fibroblasts are selectively prevented from growing into the bone defect to allow the uninterrupted transmission of the nutrient elements in the human tissue fluid and the exchange of the metabolites at the bone defect, so that nutrient substances in the human body can reach the bone defect to promote the directional repair and healing of the bone defect;

4. in the preparation method of the bone tissue regeneration guiding membrane, the obtained oriented fiber membrane layer for guiding growth and the disordered fiber membrane layer are connected, so that the bone tissue regeneration guiding membrane has a good oriented repair and healing effect on bone defects, and the bone tissue regeneration guiding membrane allows the uninterrupted delivery of nutrient elements in human tissue fluid and the exchange of metabolites at the bone defect positions, so that nutrient substances in the human body can reach the bone defect positions to promote the oriented repair and healing of the bone defects;

5. the preparation method of the bone tissue regeneration guide membrane has simple operation steps and effectively improves the production efficiency of the bone tissue regeneration guide membrane.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is an electron microscope image of the guided growth oriented fiber membrane layer of the guided growth bone tissue regeneration guiding membrane of the present invention;

FIG. 2 is another electron microscope image of the guided growth oriented fiber membrane layer of the guided bone tissue regeneration guiding membrane of the present invention;

FIG. 3 is an electron microscope image of a bone tissue regeneration guide membrane according to the present invention;

FIG. 4 is an electron microscope image of a selectively permeable disordered fibrous membrane layer of the bone tissue regeneration guiding membrane of the present invention;

FIG. 5 is another electron micrograph of a selectively permeable disordered fibrous membrane layer of the guided membrane for bone tissue regeneration according to the present invention;

FIG. 6 is another electron micrograph of a guide membrane for bone tissue regeneration according to the present invention;

FIG. 7 is another electron micrograph of a bone tissue regeneration guide membrane according to the present invention;

FIG. 8 is a flowchart illustrating a method for preparing a bone tissue regeneration guiding membrane according to an embodiment of the present invention;

FIG. 9 is a cell proliferation test chart of fibroblasts cultured on the surface of the bone tissue regeneration guiding membrane selectively permeating the disordered fibrous membrane layer for 1, 3 and 7 days;

FIG. 10 shows cell proliferation test of rabbit bone marrow mesenchymal stem cells (hereinafter, rabbit bone marrow mesenchymal stem cells are abbreviated as rBMSC) after culturing on the surface of oriented fiber membrane layer for guided growth of bone tissue regeneration guiding membrane for 1, 3 and 7 days;

FIG. 11 is a confocal micrograph of fibroblasts after 3 days of culture on the surface of a guiding membrane for bone tissue regeneration, which selectively permeates a disordered fibrous membrane layer;

FIG. 12 is an optical micrograph of the guided growth oriented fiber membrane layer of the bone tissue regeneration guide membrane;

FIG. 13 is an electron microscope image of the growth-guiding oriented fiber membrane layer of the bone tissue regeneration guiding membrane;

FIG. 14 is a photograph of a bilateral radius of a rabbit in a bilateral radius fracture model experiment.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The present application provides a bone tissue regeneration guiding membrane. The bone tissue regeneration guiding membrane comprises a growth guiding directional fiber membrane layer and a selective permeation disordered fiber membrane layer, wherein the growth guiding directional fiber membrane layer is connected with the selective permeation disordered fiber membrane layer.

In the bone tissue guide membrane, the oriented fiber membrane layer for guiding growth is formed by oriented stacking and arranging fiber filaments, so that not only can the differentiation of osteocytes be promoted, but also the oriented growth and migration of derivative cells for guiding the osteocytes can be better realized, and the oriented growth and migration guide membrane has the functions of oriented growth and migration guide and growth and migration promotion on the osteocytes, further the oriented repair of bone defects is better realized, and the application range of the bone tissue regeneration guide membrane is improved; the growth-oriented fiber film layer is formed by oriented stacking arrangement of fiber filaments, the porosity is low, and the fiber film layer has high mechanical property and deformation resistance, namely good structural strength; the selective permeation disordered fiber membrane layer is formed by disordered random arrangement of fiber filaments, has higher porosity, further ensures that fibroblasts tend to differentiate, proliferate, migrate and grow in the selective permeation disordered fiber membrane layer, has the effect of blocking the growth of the fibroblasts in the growth-guiding oriented fiber membrane layer, namely blocks the fibroblasts from growing into the bone defect part, and is beneficial to rapid oriented repair and healing of the bone defect part. In addition, the selective permeation disordered fiber film layer has high porosity, can support the input of nutrient elements in human tissue fluid and the exchange of metabolites at the bone defect, and selectively prevents fibroblasts from growing into the bone defect to allow the uninterrupted transmission of the nutrient elements in the human tissue fluid and the exchange of the metabolites at the bone defect, so that nutrient substances in the human body can reach the bone defect to promote the directional repair and healing of the bone defect.

It should be noted that, referring to fig. 1, fig. 2 and fig. 3 together, the oriented fiber film layer for guiding growth is a fiber film formed by oriented stacking arrangement of fiber filaments, and the oriented stacking arrangement of fiber filaments is not a close stacking arrangement, so that the oriented fiber film layer for guiding growth has a certain porosity and is greatly different from a dense film, but the porosity of the oriented fiber film layer for guiding growth is lower than that of the unordered fiber film layer for selective permeation, and the oriented stacking arrangement of fiber filaments enables the oriented fiber film layer for guiding growth to have higher mechanical properties and deformation resistance, i.e. better structural strength.

It should be further noted that the fiber structure of the fiber filaments is very similar to the extracellular matrix in human tissue in microscopic morphology, and can provide an ideal differentiation, proliferation, migration and growth place for cells, so as to accelerate the regeneration of damaged tissues, and guide the fiber filaments in the growth-oriented fiber membrane layer to be directionally stacked and arranged, so that not only can the differentiation of bone cells be promoted, but also the oriented growth and migration of derived cells of bone cells can be better guided, and the effect of directionally guiding growth and migration and promoting growth and migration of bone cells is shown, so that the oriented repair of bone defects is better realized, and the application range of the bone tissue regeneration guide membrane is improved. In addition, the guided growth oriented fiber membrane layer has higher mechanical property and deformation resistance, so that the bone tissue regeneration guiding membrane with a double-layer membrane structure formed by connecting the guided growth oriented fiber membrane layer and the selective permeation disordered fiber membrane layer has higher mechanical property and deformation resistance.

It should be further noted that, referring to fig. 4, fig. 5 and fig. 6 together, the selective permeation disordered fiber film layer is a three-dimensional reticular fiber film formed by disordered interweaving or random stacking arrangement of fiber filaments, so that the selective permeation disordered fiber film layer has higher porosity, and the selective permeation disordered fiber film layer with higher porosity can better provide an ideal differentiation, proliferation, migration and growth place for fibroblasts compared with the directed growth oriented fiber film layer, so that the fibroblasts are more prone to differentiate, proliferate, migrate and grow in the selective permeation disordered fiber film layer, and when the fibroblast is fully filled with the selective permeation disordered fiber film layer, the osteocyte is fully differentiated, proliferated, migrated and grown in the directed growth oriented fiber film layer, even if a part of the fibroblasts overflow the selective permeation disordered fiber film layer to grow into the directed growth oriented fiber film layer, the effect of inhibiting the growth of osteocytes fully differentiated, proliferated, migrated and grown in the guided growth oriented fiber membranous layer is difficult to generate, the phenomenon that the fibroblast grows into the guided growth oriented fiber membranous layer through the disordered fiber membranous layer and plays a role in blocking the fibroblast growth is provided for the fibroblast while an ideal differentiation, proliferation, migration and growth place is provided for the fibroblast through the disordered fiber membranous layer, namely, the fibroblast is prevented from growing into a bone defect part, and the rapid oriented repair and healing of the bone defect part are facilitated.

It should be further noted that when the guiding membrane for bone tissue regeneration is placed at the bone defect, the selectively permeable disordered fiber membrane layer has a high porosity, so that the selectively permeable disordered fiber membrane layer can prevent fibroblasts from growing into the bone defect, and simultaneously can support the input of nutrient elements in human tissue fluid and the exchange of metabolites at the bone defect, which means that the fibroblast growth into the bone defect is selectively blocked, the uninterrupted transmission of the nutrient elements in the human tissue fluid and the exchange of the metabolites at the bone defect are allowed, and further, the nutrient substances in the human body can reach the bone defect, and the directional repair and healing of the bone defect are effectively promoted.

It should be further noted that the bone tissue regeneration guiding membrane with a double-layer membrane structure formed by connecting the guided growth oriented fiber membrane layer and the selective permeation disordered fiber membrane layer is not only suitable for local damage or defect of general bones, but also suitable for treatment after fracture and before fracture of bones, the bone tissue regeneration guiding membrane is wound at the fracture position, the guided growth oriented fiber membrane layer of the bone tissue regeneration guiding membrane conducts oriented guided growth migration on bone cells so as to enable derived cells of the bone cells to be directionally arranged and connected between the bones, and nutrient substances required for the bone cells to uninterruptedly select permeation growth through the disordered fiber membrane layer are selected through the disordered fiber membrane layer, so that repair and healing of the fracture between the bones are accelerated. In addition, the bone tissue regeneration guiding membrane is also suitable for spinal fusion, in the spinal fusion operation, as the spinous processes of the spine are difficult to be completely and seamlessly implanted with bones, the phenomenon of incomplete posterior fusion caused by insufficient bone grafting amount often occurs, the bone tissue regeneration guiding membrane is wound on the vertebrae needing to be fused, the growth guiding directional fiber membrane layer of the bone tissue regeneration guiding membrane guides the growth and migration of bone cells in a directional way, so that derived cells of the bone cells are directionally arranged and connected between the implanted bones and the vertebrae, and nutrient substances required by the growth of the bone cells are uninterruptedly selected and penetrated through the disordered fiber membrane layer by selecting and penetrating through the disordered fiber membrane layer, thereby effectively accelerating the fusion between the vertebrae.

In one embodiment, the selectively permeable disordered fibrous membrane layer is miscible with the microetched growth directing fibrous membrane layer interface so that the growth directing fibrous membrane layer is bonded to the selectively permeable disordered fibrous membrane layer. It can be understood that the selective permeation disordered fiber film layer and the microetching interface on the growth guiding oriented fiber film layer are mixed and combined together, and the microetching occurs at the contact part of the selective permeation disordered fiber film layer and the growth guiding oriented fiber film layer, so that the selective permeation disordered fiber film layer and the growth guiding oriented fiber film layer are mixed and combined into a whole, the selective permeation disordered fiber film layer and the growth guiding oriented fiber film layer are firmly combined, the structures of the selective permeation disordered fiber film layer and the growth guiding oriented fiber film layer are not damaged, the triple functions of shielding fibroblast, uninterruptedly conveying nutrient elements and exchanging metabolites of the bone tissue regeneration guiding film and directionally guiding the growth and migration of bone cells are ensured, and the repairing and healing effects of the bone tissue regeneration guiding film on bone defects are further ensured.

In one embodiment, the guided growth oriented fiber membrane layer is miscible with the microetched, interface of the selectively permeable disordered fiber membrane layer such that the guided growth oriented fiber membrane layer is bonded to the selectively permeable disordered fiber membrane layer. The method can be understood that the interface of the guided growth oriented fiber membrane layer and the selective permeation disordered fiber membrane layer is mixed and dissolved together, and the micro corrosion occurs at the contact part of the selective permeation disordered fiber membrane layer and the guided growth oriented fiber membrane layer, so that the selective permeation disordered fiber membrane layer and the guided growth oriented fiber membrane layer are mixed and dissolved into a whole, the selective permeation disordered fiber membrane layer and the guided growth oriented fiber membrane layer are firmly combined, the structures of the selective permeation disordered fiber membrane layer and the guided growth oriented fiber membrane layer are not damaged, the triple functions of shielding fibroblast, uninterruptedly conveying nutrient elements and exchanging metabolites and directionally guiding the growth and migration of bone cells of the bone tissue regeneration guiding membrane are ensured, and the repairing and healing effects of the bone tissue regeneration guiding membrane on bone defects are further ensured.

It can be understood that the mixing and dissolving emphasizes the micro-molecular compatibility, which is characterized in that micro-phase separation is carried out at the contact part of the guided growth oriented fiber film layer and the selective permeation unordered fiber film layer without macro-phase separation, that is, the contact part of the guided growth oriented fiber film layer and the selective permeation unordered fiber film layer in the bone tissue regeneration guiding film does not show phase separation macroscopically, and the existence of a two-phase structure can be observed actually microscopically, that is, the guided growth oriented fiber film layer is phase separated microscopically to form a microetching interface, so that when the selective permeation unordered fiber film layer is contacted with the microetching interface, the selective permeation unordered fiber film layer surface is also phase separated to form a microetching interface, the selective permeation unordered fiber film layer surface is microetching interface and the guided growth oriented fiber film layer surface are compatible on micro-molecules, and then combined together, or the selective permeation disordered fiber film layer is microscopically phase-separated to form a microetching interface, so that when the guided growth oriented fiber film layer is contacted with the microetching interface, the surface of the guided growth oriented fiber film layer is also microscopically phase-separated to form the microetching interface, the microetching interface on the surface of the selective permeation disordered fiber film layer and the microetching interface on the surface of the guided growth oriented fiber film layer are microscopically compatible and combined together, and the mixed dissolution combination of the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer through the microetching interface does not damage the structures of the guided growth oriented fiber film layer and the selective permeation unordered fiber film layer, thereby ensuring the triple functions of shielding fibroblasts, uninterruptedly conveying nutrient elements and exchanging metabolites and directionally guiding the growth and migration of bone cells of the bone tissue regeneration guiding film, thereby ensuring the repairing and healing effects of the bone tissue regeneration guide membrane on the bone defect.

It is also understood that the microetched interface is a microetched interface or a microetched interface. The microetching interface is formed at the contact part of the growth-guiding oriented fiber film layer and the selective permeation disordered fiber film layer, only physical change is generated on the microetching interface, and chemical change is not generated, and specifically, the microetching interface is an interface which is formed by microscopic phase separation on the surface of the growth-guiding oriented fiber film layer and can be mixed with the selective permeation disordered fiber film layer, or an interface which is formed by microscopic phase separation on the surface of the selective permeation disordered fiber film layer and can be mixed with the growth-guiding oriented fiber film layer.

It can also be understood that the combination of the growth-directing oriented fiber membrane layer and the selective permeation disordered fiber membrane layer is not firm or easily breaks the structure of the growth-directing oriented fiber membrane layer and the selective permeation disordered fiber membrane layer, the method for combining the oriented fiber film layer with the selective permeation disordered fiber film layer is a hot pressing method, the hot pressing method needs to accurately control parameters such as hot pressing temperature, pressing time and the like, the difficulty of controlling the hot pressing temperature and the pressing time is high, the bone tissue regeneration guiding film with a damaged hole structure and an oriented structure is easy to obtain, the hot pressing temperature is too low, and the pressing time is too short, the fusion effect is poor, so that the combination of the oriented fiber membrane layer for guiding growth and the disordered fiber membrane layer for selective permeation is not firm; the temperature is higher, when the laminating time is longer, the guide growth oriented fiber film layer and the selective permeation disordered fiber film layer can be melted and deformed, the pore structures of the guide growth oriented fiber film layer and the selective permeation disordered fiber film layer can be damaged, when the temperature is higher, the guide growth oriented fiber film layer and the selective permeation disordered fiber film layer can be laminated into a firm compact film, and the pore structures of the guide growth oriented fiber film layer and the selective permeation disordered fiber film layer can be completely damaged, and the directional structure of the guided growth directional fiber membrane layer is destroyed, thus completely losing the original advantages of the guided growth directional fiber membrane layer and the selective permeation disordered fiber membrane layer, failing to realize the shielding of fibroblast, uninterrupted delivery of nutrient elements and exchange of metabolites of the bone tissue regeneration guiding membrane, and the three functions of directionally guiding the growth and migration of bone cells, so that the bone tissue regeneration guiding membrane has poor repairing and healing effects on bone defects. .

Therefore, in order to solve the problem that the technological parameters of the hot pressing method are difficult to accurately control, the guiding growth oriented fiber film layer and the selective permeation disordered fiber film layer are provided, the bone tissue regeneration guiding film is ensured to have a porous shielding structure for shielding the growth of the fibroblast and a porous oriented structure for the growth of the osteocyte and the angioblast, the requirements of the mechanical strength and the mechanical property of the bone tissue regeneration guiding film are ensured, the guiding growth oriented fiber film layer and the selective permeation disordered fiber film layer are mixed and dissolved through the microetching of the guiding growth oriented fiber film layer or the selective permeation disordered fiber film layer, the guiding growth oriented fiber film layer and the selective permeation disordered fiber film layer are combined together, the problem that the structures of the guiding growth oriented fiber film layer and the selective permeation disordered fiber film layer are damaged is avoided, and the bone tissue regeneration guiding film has the dual functions of oriented bone formation guiding and oriented bone formation, thereby better achieving the purpose of accelerating the healing of the bone defect.

In one embodiment, the growth-directing oriented fiber membrane layer comprises the following components: high molecular substances and bioactive substances. It can be understood that the high molecular substance has better biocompatibility and degradability, so that the in-vivo degradability and nontoxicity of the oriented fiber membrane layer guided to grow are better realized, and the active ingredients in the bioactive substance are favorable for promoting the proliferation and differentiation of bone cells, further favorable for promoting the repair of bone defects and accelerating the repair and healing of the bone defects.

In one embodiment, the oriented fiber film layer for guided growth comprises the following components in parts by mass: 20-99 parts of high molecular substance; 1-80 parts of bioactive substances. It is understood that although nutrients in human tissue fluid can be continuously delivered to the bone defect through the disordered fibrous membrane layer, nutrients which can be provided in the tissue fluid of the human body are limited, and thus, a bioactive substance needs to be further provided to the bone defect to promote rapid repair and healing of the bone defect. In the oriented fiber membrane layer for guided growth, if the dosage of the high molecular substance is too large, the healing and repairing effects of the bone defect area are poor; if the amount of the high molecular substance is too small, the mechanical strength and the mechanical property of the guided growth oriented fiber film layer are poor, and the combination stability of the high molecular substance and the bioactive substance cannot be realized, so that the guided growth oriented fiber film layer is easy to shed powder or tear, and therefore, in the application, the guided growth oriented fiber film layer comprises the following components in parts by mass: 20 to 99 parts of high molecular substance and 1 to 80 parts of bioactive substance, thereby ensuring the structural stability, mechanical strength and mechanical property of the oriented fiber film layer for guided growth.

In one embodiment, the oriented fiber film layer for guided growth comprises the following components in parts by mass: 80-90 parts of high molecular substance; 10-20 parts of bioactive substances. It can be understood that the oriented fiber membrane layer for guided growth comprises the following components in parts by mass: 80-90 parts of high molecular substance; 10 to 20 portions of bioactive substances better ensure the structural stability, mechanical strength and mechanical property of the oriented fiber membrane layer for guided growth.

It can be understood that the guided growth oriented fiber membrane layer takes high molecular substances and bioactive substances as main components, and can also comprise biodegradable substances with medium molecular weight or low molecular weight, so that the biocompatibility, biodegradability and cytotoxicity-free property of the guided growth oriented fiber membrane layer are ensured.

In one embodiment, the high molecular substance in the growth-oriented fiber membrane layer is at least one of collagen, bone matrix gelatin, silk fibroin, chitosan, polylactic acid, polycaprolactone, polyamide, poly-L-lactic acid, polyglycolic acid, and polylactic-polyglycolic acid copolymer. It can be understood that the guided growth oriented fiber membrane layer prepared from at least one of collagen, bone matrix gelatin, silk fibroin, chitosan, polylactic acid, polycaprolactone, polyamide, poly-L-lactic acid, polyglycolic acid and polylactic acid-polyglycolic acid copolymer has good biodegradability speed, high mechanical strength and mechanical property, and further ensures the timeliness of the guided bone tissue regeneration membrane.

In one embodiment, the average molecular weight of the polylactic acid is 100000-1200000, so that the oriented fiber film layer for guiding growth has better mechanical strength and mechanical property.

In one embodiment, the bioactive substance in the guided growth oriented fiber membrane layer is at least one of a bio-organic active substance, a bioactive inorganic ceramic, and a bioactive inorganic glass. It can be understood that the bioactive organic substance, the bioactive inorganic ceramic and the bioactive inorganic glass have the same elements with the components of the skeleton, can provide corresponding nutrient substances for the growth and differentiation of bone cells, effectively promote the growth and differentiation of the bone cells, and further effectively promote the healing and repair of the bone defect area.

In one embodiment, the bioactive inorganic ceramic is at least one of a silicate bioactive ceramic, a borate bioactive ceramic, and a phosphate bioactive ceramic. It can be understood that the nutrients for the growth and differentiation of osteoblasts, such as silicon, calcium and phosphorus, of the silicate bioactive ceramics, the borate bioactive ceramics and the phosphate bioactive ceramics, after biodegradation, effectively promote the growth and differentiation of osteoblasts, and further effectively promote the healing and repair of bone defect areas.

In one embodiment, the silicate bioactive ceramic comprises dicalcium silicate and/or tricalcium silicate. It can be understood that the dicalcium silicate or tricalcium silicate provides a large amount of required silicon and calcium for the growth and differentiation of bone cells after biodegradation, effectively promotes the growth and differentiation of the bone cells, and further promotes the healing and repair of the bone defect area.

In one embodiment, the phosphate bioactive ceramic contains hydroxyapatite and/or β -tricalcium phosphate. It can be understood that the hydroxyapatite or the beta-tricalcium phosphate provides a large amount of required silicon, calcium and phosphorus for the growth and differentiation of bone cells after biodegradation, effectively promotes the growth and differentiation of the bone cells, and further promotes the healing and repair of a bone defect area.

In one embodiment, the bioactive inorganic glass is at least one of a silicate bioactive glass, a borate bioactive glass, and a phosphate bioactive glass. It can be understood that the growth and differentiation nutrients of the osteoblasts, such as silicon, calcium and phosphorus, of the silicate bioactive glass, the borate bioactive glass and the phosphate bioactive glass, after biodegradation, effectively promote the growth and differentiation of the osteoblasts, and further effectively promote the healing and repair of the bone defect area.

In one embodiment, the bioactive inorganic glass comprises silicon dioxide, calcium oxide, phosphorus pentoxide and sodium oxide as main components, and further comprises at least one of potassium oxide, magnesium oxide, aluminum oxide and calcium fluoride. It can be understood that the above-mentioned substances in the bioactive inorganic glass provide a large amount of required silicon, calcium, magnesium, sodium, potassium and phosphorus for the growth and differentiation of bone cells after biodegradation, effectively promote the growth and differentiation of bone cells, and further promote the healing and repair of bone defect area.

In one embodiment, the bioactive substance is at least one of type 45S5 bioglass, type 58S bioglass, or type A-W bioglass.

In one embodiment, the bioactive substance in the growth-directing oriented fiber membrane layer is granular and has a particle size distribution of 0.3 μm to 30 μm. It can be understood that the bioactive substance is in the form of particles, which ensures that the bioactive substance and the polymer substance form a co-embedded structure, and the particle size of the bioactive substance is required to be sufficient for embedding the polymer substance or for the connecting group of the bioactive substance and the polymer substance to be sufficiently contacted. When the particle size of the bioactive substance is 0.3-30 μm, the bioactive substance and the high molecular substance form a co-embedded structure with stable combination. If the particle size of the bioactive substance is too large, the polymer substance is not enough to support the bioactive substance, the combination stability of the bioactive substance and the polymer substance is reduced, the guided growth oriented fiber membrane layer is easy to shed powder or tear, the degradation speed of the bioactive substance is low, and the guided growth oriented fiber membrane layer cannot timely and effectively promote the healing and repairing of a bone defect area. If the particle size of the bioactive substance is too small, the bioactive substance is easy to accumulate locally, the dispersibility of the bioactive substance and the macromolecular substance is reduced, and the mechanical strength and the mechanical property of the guided growth oriented fiber membrane layer are poor.

Referring to fig. 1, 2 and 3, in one embodiment, the growth-inducing oriented fiber membrane layer is formed by oriented stacking arrangement of fiber filaments. The guided growth oriented fiber membrane layer is formed by oriented stacking and arranging of the fiber filaments, so that the differentiation of osteocytes can be promoted, the oriented growth migration of derived cells of the guided osteocytes can be better realized, the guided growth oriented fiber membrane layer has the functions of oriented guided growth migration and growth migration promotion on the osteocytes, further, the oriented repair of bone defects is better realized, the application range of the bone tissue regeneration guide membrane is improved, in addition, the guided growth oriented fiber membrane layer is formed by oriented stacking and arranging of the fiber filaments, the porosity is lower, and the guided growth oriented fiber membrane layer has higher mechanical property and deformation resistance, namely, better structural strength.

Referring to fig. 4, 5 and 6, in one embodiment, the selectively permeable random fiber membrane layer is formed by randomly interlacing or stacking fiber filaments. The selective permeation disordered fiber membrane layer is a three-dimensional reticular fiber membrane formed by disorderly interweaving or randomly stacking and arranging fiber filaments, so that the selective permeation disordered fiber membrane layer has higher porosity, and compared with the guided growth oriented fiber membrane layer, the selective permeation disordered fiber membrane layer with higher porosity can better provide an ideal differentiation, proliferation, migration and growth place for fibroblasts, further the fibroblasts are more prone to be differentiated, proliferated, migrated and grown in the selective permeation disordered fiber membrane layer, when the selective permeation disordered fiber membrane layer is fully filled with the fibroblasts, the osteocytes are also fully differentiated, proliferated, migrated and grown in the guided growth oriented fiber membrane layer, at the moment, even if part of the fibroblasts overflow the selective permeation disordered fiber membrane layer and grow in the guided growth oriented fiber membrane layer, the effect of inhibiting the growth of osteocytes fully differentiated, proliferated, migrated and grown in the guided growth oriented fiber membranous layer is difficult to generate, the phenomenon that the fibroblast grows into the guided growth oriented fiber membranous layer through the disordered fiber membranous layer and plays a role in blocking the fibroblast growth is provided for the fibroblast while an ideal differentiation, proliferation, migration and growth place is provided for the fibroblast through the disordered fiber membranous layer, namely, the fibroblast is prevented from growing into a bone defect part, and the rapid oriented repair and healing of the bone defect part are facilitated.

In one embodiment, the filaments have a diameter of 100nm to 150 μm. It can be understood that the diameter of the fiber filament selectively permeating the disordered fiber film layer is 100 nm-150 μm, so that the high porosity and specific surface area of the selectively permeating disordered fiber film layer are further ensured, the growth of the fibroblast in the selectively permeating disordered fiber film layer is facilitated, an ideal differentiation, proliferation, migration and growth place is provided for the fibroblast, the growth speed of the fibroblast into the growth-guiding oriented fiber film layer is further reduced, the fibroblast is prevented from growing into the bone defect part, and the rapid oriented repair and healing of the bone defect part are further facilitated. In addition, the diameter of the fiber filament of the guided growth oriented fiber membrane layer is 100 nm-150 μm, so that the guided growth oriented fiber membrane layer is further ensured to have better mechanical property and deformation resistance, the porosity and the specific surface area of the guided growth oriented fiber membrane layer are facilitated, the guided growth and migration of bone cells in the guided growth oriented fiber membrane layer are facilitated, and the rapid oriented repair and healing of the bone defect are facilitated.

In one embodiment, the permselective disordered fibrous membrane layer comprises the following components: a high molecular substance. It can be understood that the permselective disordered fiber film layer formed by the high molecular substances has good biocompatibility, biodegradability and no cytotoxicity, so that degradation products of the permselective disordered fiber film layer are nontoxic and can be normally metabolized by a human body, and the high molecular substance degradation products can directly participate in tissue repair without secondary taking out, thereby reducing secondary damage to the human body and effectively avoiding postoperative infection.

It can be understood that the selective permeation disordered fiber membrane layer takes a high molecular substance as a main component, and can also comprise a biodegradable substance with medium molecular weight or low molecular weight, so that the biocompatibility, biodegradability and cytotoxicity of the selective permeation disordered fiber membrane layer are ensured. In one embodiment, the polymer material selectively permeating through the disordered fiber membrane layer is at least one of collagen, bone matrix gelatin, silk fibroin, chitosan, polylactic acid, polycaprolactone, polyamide, poly-L-lactic acid, polyglycolic acid, and polylactic-polyglycolic acid copolymer. It can be understood that collagen, bone matrix gelatin, silk fibroin, chitosan, polylactic acid, polycaprolactone, polyamide, poly-L-lactic acid, polyglycolic acid and polylactic acid polyglycolic acid copolymer have better biocompatibility and degradability, and the in vivo degradability and nontoxicity of the selective permeation disordered fiber membrane layer are better realized.

In one embodiment, the polylactic acid has an average molecular weight of 100000-1200000, and better ensures that the selective permeable disordered fiber film layer has better mechanical strength and mechanical property.

The application also provides a preparation method of the bone tissue regeneration guiding membrane. The preparation method of the bone tissue regeneration guide membrane comprises the following steps: obtaining a guided growth oriented fiber film layer and a selective permeation disordered fiber film layer; and connecting the oriented fiber membrane layer for guided growth and the disordered fiber membrane layer for selective permeation to obtain the bone tissue regeneration guiding membrane.

In the preparation method of the bone tissue regeneration guiding membrane, the obtained growth guiding directional fiber membrane layer and the selective permeation disordered fiber membrane layer are connected, so that the bone tissue regeneration guiding membrane has a good directional repair and healing effect on bone defects, and the bone tissue regeneration guiding membrane allows the uninterrupted delivery of nutrient elements in human tissue fluid and the exchange of metabolites at the bone defect positions, so that nutrient substances in the human body can reach the bone defect positions to promote the directional repair and healing of the bone defects. In addition, the preparation method of the bone tissue regeneration guiding membrane has simple operation steps, and effectively improves the production efficiency of the bone tissue regeneration guiding membrane.

Referring to fig. 7 and 8 together, in order to better understand the method for preparing the bone tissue regeneration guiding membrane of the present application, the method for preparing the bone tissue regeneration guiding membrane of the present application is further explained below, and the method for preparing the bone tissue regeneration guiding membrane of one embodiment includes the following steps:

s100, obtaining a guided growth oriented fiber film layer and a selective permeation disordered fiber film layer. The obtained oriented fiber membrane layer for guiding growth is formed by oriented stacking and arranging of the fiber filaments, so that the differentiation of osteocytes can be promoted, the oriented growth migration of derived cells of the guided osteocytes can be better realized, the oriented growth migration guiding and promoting effects on the osteocytes are shown, the oriented repair of bone defects is better realized, and the application range of the bone tissue regeneration guiding membrane is widened. The obtained selective permeation disordered fiber membrane layer is formed by disorderly and randomly arranging fiber filaments, has higher porosity, further ensures that fibroblasts tend to differentiate, proliferate, migrate and grow in the selective permeation disordered fiber membrane layer, and has the effect of blocking the growth of the fibroblasts into the growth-guiding directional fiber membrane layer, namely blocking the fibroblasts from growing into the bone defect part, and is beneficial to the rapid directional repair and healing of the bone defect part. In addition, the selective permeation disordered fiber film layer has high porosity, can support the input of nutrient elements in human tissue fluid and the exchange of metabolites at the bone defect, and selectively prevents fibroblasts from growing into the bone defect to allow the uninterrupted transmission of the nutrient elements in the human tissue fluid and the exchange of the metabolites at the bone defect, so that nutrient substances in the human body can reach the bone defect to promote the directional repair and healing of the bone defect.

S200, carrying out connection operation on the oriented fiber membrane layer for guiding growth and the disordered fiber membrane layer for selective permeation to obtain the bone tissue regeneration guiding membrane. The bone tissue regeneration guide membrane is formed by connecting a growth oriented fiber membrane and a selective permeation disordered fiber membrane, so that the bone tissue regeneration guide membrane has a good oriented repair and healing effect on bone defects, and the bone tissue regeneration guide membrane allows the uninterrupted delivery of nutrient elements in human tissue fluid and the exchange of metabolites at the bone defect positions, so that nutrient substances in the human body can reach the bone defect positions to promote the oriented repair and healing of the bone defects.

In the preparation method of the bone tissue regeneration guiding membrane, the obtained growth guiding directional fiber membrane layer and the selective permeation disordered fiber membrane layer are connected, so that the bone tissue regeneration guiding membrane has a good directional repair and healing effect on bone defects, and the bone tissue regeneration guiding membrane allows the uninterrupted delivery of nutrient elements in human tissue fluid and the exchange of metabolites at the bone defect positions, so that nutrient substances in the human body can reach the bone defect positions to promote the directional repair and healing of the bone defects. In addition, the preparation method of the bone tissue regeneration guiding membrane has simple operation steps, and effectively improves the production efficiency of the bone tissue regeneration guiding membrane.

In one embodiment, obtaining the guided growth oriented fiber membrane layer comprises the following steps:

adding a high molecular substance and a bioactive substance into an organic solvent to obtain a first spinning solution. It can be understood that, in order to obtain a uniform guided growth oriented fiber film layer, the first spinning solution should have certain fluidity, and the high molecular substance and the bioactive substance need to be uniformly dispersed before electrostatic spinning, and the high molecular substance and the bioactive substance are added into the organic solvent, so that the uniformity of the dispersion of the high molecular substance and the bioactive substance in the organic solvent is ensured, and further, the uniformity of the guided growth oriented fiber film layer is ensured.

Further, the first spinning solution is subjected to directional electrostatic spinning to obtain a directional fiber film layer for guiding growth. It can be understood that the oriented fiber filaments of the oriented fiber membrane layer for guiding growth obtained by the oriented electrostatic spinning operation are oriented, stacked and arranged, have a porous structure and a large specific surface area, are beneficial to providing an ideal site for oriented differentiation, proliferation, migration and growth for bone cells, and accelerate the repair and healing of bone defects, wherein the pore structure of the oriented fiber membrane layer for guiding growth can be obviously distinguished by an electron microscope.

In the step of obtaining the guided growth oriented fiber membrane layer, the polymer material and the bioactive material are dispersed in the organic solvent, so that the uniformity and the fluidity of the polymer material and the bioactive material in the guided growth oriented fiber membrane layer and the organic solvent are ensured, the guided growth oriented fiber membrane layer with certain porosity and oriented stacking arrangement of fiber filaments can be obtained by oriented electrostatic spinning, the guided growth oriented fiber membrane layer is close to the microscopic form of extracellular matrix in the tissue, an ideal site for oriented differentiation, proliferation, migration and growth is effectively provided for bone cells, and the repair and healing of bone defects are accelerated.

In one embodiment, the mass percentage of the high molecular substance in the first spinning solution is 5 wt% to 30 wt%. It can be understood that the content of the high molecular substance in the first spinning solution is too low, the first spinning solution cannot effectively form charged jet flow to form a growth-directing oriented fiber membrane layer with certain porosity and large specific surface area, the content of the high molecular substance is too high, and the first spinning solution has poor fluidity and cannot be subjected to electrostatic spinning to form the growth-directing oriented fiber membrane layer. Therefore, in the first spinning solution, the mass percentage of the high molecular substance is 5 wt% -30 wt%, the first spinning solution is ensured to be capable of carrying out electrostatic spinning, the obtained growth-guiding oriented fiber film layer is ensured to have a certain porosity and a larger specific surface area, the mechanical property and the anti-deformation capability of the fiber yarn forming the growth-guiding oriented fiber film layer are ensured, and the mechanical property and the anti-deformation capability of the growth-guiding oriented fiber film layer are further ensured.

In one embodiment, obtaining a permselective disordered fibrous membrane layer comprises the steps of:

and adding the high molecular substance into the organic solvent to obtain a second spinning solution. It can be understood that, in order to obtain a uniform permselective disordered fiber film layer, the second spinning solution should have certain fluidity, and the high molecular substance needs to be uniformly dispersed before electrostatic spinning, and the high molecular substance is added into the organic solvent, so that the uniformity of the dispersion of the high molecular substance in the solvent is ensured, and further, the uniformity of the permselective disordered fiber film layer is ensured.

Further, the second spinning solution is subjected to disordered electrostatic spinning to obtain a selective permeation disordered fiber film layer. It can be understood that the filaments of the fibers selectively permeating the disordered fiber film layer obtained by the disordered electrostatic spinning operation are disordered and interwoven or randomly stacked and arranged, the porosity is high, the specific surface area is large, an ideal differentiation, proliferation, migration and growth place is provided for fibroblasts, and then the growth of the fibroblasts into the growth-guiding directional fiber film layer is blocked, namely the fibroblasts are prevented from growing into the bone defect part, the rapid repair and healing of the bone defect part are facilitated, and the pore structure selectively permeating the disordered fiber film layer can be obviously distinguished by an electron microscope.

In the step of obtaining the selective permeation disordered fiber film layer, the polymer material is dispersed in the organic solvent, so that the uniformity and the fluidity of the polymer material in the solvent in the selective permeation disordered fiber film layer are ensured, disordered electrostatic spinning is facilitated to obtain the selective permeation disordered fiber film layer with disordered interweaving or random stacking arrangement of the fiber yarns and high porosity, the selective permeation disordered fiber film layer is close to the microscopic form of extracellular matrix in the tissue, an ideal differentiation, proliferation, migration and growth place is effectively provided for fibroblasts, and then the effect of blocking the growth of the fibroblasts into the oriented growth fiber film layer is achieved, namely, the fibroblasts are prevented from growing into the bone defect part, and the rapid repair and healing of the bone defect part are facilitated.

In one embodiment, the second spinning solution contains 5 wt% to 30 wt% of the polymer. It can be understood that in the second spinning solution, the content of the high molecular substance is too low, the second spinning solution cannot effectively form an electrified jet flow to form a permselective disordered fiber film layer with certain porosity and a large specific surface area, the content of the high molecular substance is too high, and the second spinning solution is poor in fluidity and cannot be subjected to electrostatic spinning to form the permselective disordered fiber film layer. Therefore, in the second spinning solution, the mass percentage of the high molecular substance is 5 wt% -30 wt%, the second spinning solution can be used for carrying out electrostatic spinning, the obtained selective permeation disordered fiber film layer is ensured to have higher porosity and larger specific surface area, the mechanical property and the anti-deformation capability of the fiber yarns forming the selective permeation disordered fiber film layer are ensured, and the mechanical property and the anti-deformation capability of the selective permeation disordered fiber film layer are further ensured.

In one embodiment, the connecting operation of the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer comprises the following steps: and carrying out microetching operation on the guide growth oriented fiber film layer or the selective permeation disordered fiber film layer so as to form a microetching interface on the guide growth oriented fiber film layer or the selective permeation disordered fiber film layer. It can be understood that the purpose of the microetching operation is to form a microetching interface, ensure that the combination of the growth-guiding oriented fiber membrane layer or the selective permeation disordered fiber membrane layer does not damage the structure of the growth-guiding oriented fiber membrane layer or the selective permeation disordered fiber membrane layer, ensure that the bone tissue regeneration guiding membrane has triple functions of shielding fibroblasts, uninterruptedly conveying nutrient elements and exchanging metabolites, and directionally guiding the growth and migration of bone cells, and further ensure the repair and healing effects of the bone tissue regeneration guiding membrane on bone defects. In addition, the micro-etching operation is carried out on the guide growth oriented fiber film layer or the selective permeation disordered fiber film layer, so that the guide growth oriented fiber film layer or the selective permeation disordered fiber film layer is subjected to micro-phase separation, but the guide growth oriented fiber film layer or the selective permeation disordered fiber film layer is not subjected to macro-phase separation, and the combination stability of the guide growth oriented fiber film layer and the selective permeation disordered fiber film layer is improved.

Further, the selective permeation disordered fiber film layer and the interface for guiding the microetching of the growth oriented fiber film layer are mixed and dissolved, or the interface for guiding the microetching of the growth oriented fiber film layer and the interface for guiding the microetching of the selective permeation disordered fiber film layer are mixed and dissolved, so that the guide growth oriented fiber film layer and the selective permeation disordered fiber film layer are combined together. It can be understood that the mixing and dissolving emphasizes the micro-molecular compatibility, which is characterized in that micro-phase separation and macro-phase separation do not occur at the contact part of the selective permeation disordered fiber film layer and the growth guiding oriented fiber film layer, that is, the contact part of the selective permeation disordered fiber film layer and the growth guiding oriented fiber film layer in the bone tissue regeneration guiding film does not show phase separation macroscopically, and the existence of a two-phase structure can be observed microscopically, the structure of the selective permeation disordered fiber film layer and the growth guiding oriented fiber film layer can not be damaged by mixing and dissolving the selective permeation disordered fiber film layer and the growth guiding oriented fiber film layer together through a microetching interface, the combination stability of the selective permeation disordered fiber film layer and the growth guiding oriented fiber film layer is improved, and the shielding fibroblast, uninterrupted nutrient element delivery and metabolic product exchange of the bone tissue regeneration guiding film are ensured, and the three functions of directionally guiding the growth and migration of bone cells, thereby ensuring the effect of the bone tissue regeneration guiding membrane on the repair and healing of bone defects.

In one embodiment, after the step of mixing the selective permeation disordered fiber membrane layer with the microetching interface for guiding the growth oriented fiber membrane layer, or mixing the growth oriented fiber membrane layer with the microetching interface for guiding the growth oriented fiber membrane layer, the method for preparing the bone tissue regeneration guiding membrane further comprises the following steps: and drying and shaping the mixed and dissolved selective permeation disordered fiber film layer and the guided growth oriented fiber film layer so as to enable the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer to be connected in a curing manner. It can be understood that the selective permeation disordered fiber film layer and the guided growth oriented fiber film layer which are mixed and dissolved together also contain residual organic solvent, so that the selective permeation disordered fiber film layer and the guided growth oriented fiber film layer which are mixed and dissolved together are dried and shaped, the residual organic solvent is reduced, on one hand, the combination stability of the bone tissue regeneration guiding film is improved, and on the other hand, the biological irritation of the bone tissue regeneration guiding film is reduced.

In one embodiment, the drying and shaping time for drying and shaping the mixed and dissolved selective permeation disordered fiber membrane layer and the guided growth oriented fiber membrane layer is 5-50 h. It can be understood that the drying and shaping time of the drying and shaping operation is 5-50 h, the organic solvent is effectively removed, and further the combination stability of the bone tissue regeneration guiding membrane is improved and the biological irritation of the bone tissue regeneration guiding membrane is reduced.

In one embodiment, the drying and shaping operation is carried out in a room temperature ventilation place, the drying and shaping time of the drying and shaping operation is 45-50 h, organic solvents are effectively removed, and therefore the combination stability of the bone tissue regeneration guiding membrane is improved, and the biological irritation of the bone tissue regeneration guiding membrane is reduced.

In one embodiment, the drying and shaping operation is carried out in an oven at 50-60 ℃, the drying and shaping time is 5-10 h, organic solvents are effectively removed, and therefore the combination stability of the bone tissue regeneration guiding membrane is improved and the biostimulation of the bone tissue regeneration guiding membrane is reduced.

In one embodiment, the guided growth oriented fiber membrane layer is microetched using an organic solvent to form a microetched interface on the guided growth oriented fiber membrane layer or selectively through the disordered fiber membrane layer. It can be understood that the microetching operation achieves the effect of leading the surface of the growth oriented fiber film layer to form micro-phase separation without generating macro-phase separation, the organic solvent is adopted to act on the surface of the growth oriented fiber film layer, the micro-phase separation and the macro-phase separation are effectively formed on the surface of the growth oriented fiber film layer, the microetching interface formed by the organic solvent on the surface of the growth oriented fiber film layer leads the mixing and dissolving effect of the growth oriented fiber film layer and the selective permeation unordered fiber film layer to be better, the combination stability of the growth oriented fiber film layer and the selective permeation unordered fiber film layer is improved, and the structure of the growth oriented fiber film layer and the selective permeation unordered fiber film layer cannot be damaged in the process of mixing and dissolving the growth oriented fiber film layer and the selective permeation unordered fiber film layer to form the bone tissue regeneration guide film through the microetching interface, the three functions of shielding fibroblasts, uninterruptedly conveying nutrient elements and exchanging metabolites and directionally guiding the growth and migration of osteocytes of the bone tissue regeneration guiding membrane are ensured, and the repairing and healing effects of the bone tissue regeneration guiding membrane on bone defects are further ensured.

In one embodiment, the selectively permeable disordered fibrous membrane layer is microetched using an organic solvent such that a microetched interface is formed on the direct growth oriented fibrous membrane layer or on the selectively permeable disordered fibrous membrane layer. It can be understood that the microetching operation achieves the effect that microscopic phase separation is formed on the surface of the selective permeation disordered fiber film layer without generating macroscopic phase separation, the organic solvent is adopted to act on the surface of the selective permeation disordered fiber film layer, so that the microscopic phase separation is effectively formed on the surface of the selective permeation disordered fiber film layer without generating macroscopic phase separation, and the microetching interface formed on the surface of the selective permeation disordered fiber film layer by the organic solvent ensures that the mixing and dissolving effect of the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer is better, the combination stability of the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer is improved, and the structure of the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer cannot be damaged in the process of mixing and dissolving the guided growth oriented fiber film layer and the selective permeation disordered fiber film layer to form the bone tissue regeneration guide film by the microetching interface, the three functions of shielding fibroblasts, uninterruptedly conveying nutrient elements and exchanging metabolites and directionally guiding the growth and migration of osteocytes of the bone tissue regeneration guiding membrane are ensured, and the repairing and healing effects of the bone tissue regeneration guiding membrane on bone defects are further ensured.

In one embodiment, the selective transmission disordered fiber film layer and the microetched growth-directing oriented fiber film layer are mixed and dissolved at the interface by adopting a pressing process, so that the growth-directing oriented fiber film layer and the selective transmission disordered fiber film layer are combined together. It can be understood that if the spraying process or the brushing process is adopted to attach the organic solvent to the surface of the growth-guiding oriented fiber film layer for microetching operation, so that the growth-guiding oriented fiber film layer and the selective permeation disordered fiber film layer are mixed together, and the condition that the surface of the selective permeation disordered fiber film layer and the microetching interface of the growth-guiding oriented fiber film layer cannot be completely contacted exists, therefore, in the preparation method of the bone tissue regeneration guiding film, the selective permeation disordered fiber film layer is mixed and dissolved on the microetching interface of the growth-guiding oriented fiber film layer by adopting the pressing process, and the condition that the selective permeation disordered fiber film layer is completely mixed and dissolved to form the bone tissue regeneration guiding film which is stable in combination and complete in structure on the microetching interface of the growth-guiding oriented fiber film layer is ensured.

In one embodiment, the growth-directing oriented fiber film layer and the selective transmission disordered fiber film layer are mixed and dissolved at the microetched interface by adopting a pressing process, so that the growth-directing oriented fiber film layer and the selective transmission disordered fiber film layer are combined together. It can be understood that if the spraying process or the brushing process is adopted to attach the organic solvent to the surface of the selective permeable disordered fiber film layer for microetching operation, so that the guided growth oriented fiber film layer and the selective permeable disordered fiber film layer are mixed together, and the condition that the microetching interface of the guided growth oriented fiber film layer and the selective permeable disordered fiber film layer cannot be completely contacted exists, therefore, in the preparation method of the bone tissue regeneration guide film, the guided growth oriented fiber film layer is mixed and dissolved on the microetching interface of the selective permeable disordered fiber film layer by adopting the pressing process, and the condition that the guided growth oriented fiber film layer is completely mixed and dissolved to form the bone tissue regeneration guide film which is stable in combination and complete in structure on the microetching interface of the selective permeable disordered fiber film layer is ensured.

In one embodiment, the microetching of the selectively permeable disordered fibrous membrane layer is performed by attaching an organic solvent to the selectively permeable disordered fibrous membrane layer using a spraying process or a brushing process so that a microetched interface is formed on the selectively permeable disordered fibrous membrane layer. It can be understood that the selective permeation disordered fiber film layer and the guided growth oriented fiber film layer are obtained respectively, and the organic solvent is acted on the surface of the selective permeation disordered fiber film layer by adopting a spraying process or a brushing process, so that microscopic phase separation is effectively formed on the surface of the selective permeation disordered fiber film layer, macroscopic phase separation is not generated, and a bone tissue regeneration guide film which is stable in combination and complete in structure is formed when the guided growth oriented fiber film layer and the microetched interface of the selective permeation disordered fiber film layer are mixed and dissolved. The invention protects the operation of microetching on the selectively permeable disordered fiber film layer by adopting the spraying process or the brushing process, so that the microetching interface is formed on the selectively permeable disordered fiber film layer, and the use amount of the organic solvent in the spraying process or the brushing process is not limited.

In one embodiment, the microetching operation of the growth-directing oriented fiber film layer is to attach an organic solvent to the growth-directing oriented fiber film layer by using a spraying process or a brushing process so as to form a microetched interface on the growth-directing oriented fiber film layer. It can be understood that the selective permeation disordered fiber film layer and the guided growth oriented fiber film layer are obtained respectively, and the organic solvent is acted on the surface of the guided growth oriented fiber film layer by adopting a spraying process or a brushing process, so that the microscopic phase separation is effectively formed on the surface of the guided growth oriented fiber film layer, and the macroscopic phase separation is not generated, and a bone tissue regeneration guide film which is stable in combination and complete in structure is formed when the selective permeation disordered fiber film layer and the guided growth oriented fiber film layer are mixed and dissolved at the microetching interface. The invention protects the microetching operation of the growth-guiding oriented fiber film layer by adopting the spraying process or the brushing process, so that the microetching interface is formed on the surface of the growth-guiding oriented fiber film layer, and the use amount of the organic solvent in the spraying process or the brushing process is not limited.

In one embodiment, the preparation method of the bone tissue regeneration guiding membrane specifically comprises the steps of performing spinning attachment operation of spinning solution on the obtained growth guiding oriented fiber membrane layer through electrostatic spinning to form a selective permeation disordered fiber membrane layer, and microetching the growth guiding oriented fiber membrane layer by using an organic solvent contained in the spinning solution so as to form a microetching interface on the growth guiding oriented fiber membrane layer and combine the microetching interface with the selective permeation disordered fiber membrane layer, thereby effectively ensuring that the structures of the growth guiding oriented fiber membrane layer and the selective permeation disordered fiber membrane layer are not damaged in the process of mixing, dissolving and combining the growth guiding oriented fiber membrane layer and the selective permeation disordered fiber membrane layer to form the bone tissue regeneration guiding membrane, ensuring the triple functions of shielding fibroblasts, uninterruptedly conveying nutrient elements and exchanging metabolites and directionally guiding the growth and migration of bone cells of the bone tissue regeneration guiding membrane, thereby ensuring the repairing and healing effects of the bone tissue regeneration guide membrane on the bone defect.

In one embodiment, the preparation method of the bone tissue regeneration guiding membrane specifically comprises the steps of performing spinning attachment operation of spinning solution on the obtained selective permeation disordered fiber membrane layer through electrostatic spinning to form a growth guiding oriented fiber membrane layer, and microetching the selective permeation disordered fiber membrane layer by using an organic solvent contained in the spinning solution so as to form a microetching interface on the selective permeation disordered fiber membrane layer and combine the microetching interface with the growth guiding oriented fiber membrane layer, thereby effectively ensuring that the structures of the growth guiding oriented fiber membrane layer and the selective permeation unordered fiber membrane layer are not damaged in the process of mixing, dissolving and combining the growth guiding oriented fiber membrane layer and the selective permeation disordered fiber membrane layer to form the bone tissue regeneration guiding membrane, ensuring the triple functions of shielding fibroblasts, uninterruptedly conveying nutrient elements and exchanging metabolites and directionally guiding the growth and migration of bone cells of the bone tissue regeneration guiding membrane, thereby ensuring the repairing and healing effects of the bone tissue regeneration guide membrane on the bone defect.

In one embodiment, the preparation method of the bone tissue regeneration guiding membrane specifically comprises the following steps:

dissolving a high molecular substance in an organic solvent to prepare a first spinning solution;

dissolving a high molecular substance and a bioactive substance in an organic solvent to prepare a second spinning solution;

carrying out electrostatic spinning by adopting a first spinning solution to obtain a growth-guiding directional fiber film layer;

and coating the growth-guiding oriented fiber film layer on a roller as a receiving substrate, and performing electrostatic spinning by adopting a second spinning solution to obtain a combined growth-guiding oriented fiber film layer and a selective permeation disordered fiber film layer.

According to the preparation method of the bone tissue regeneration guide membrane, the growth guide directional fiber membrane layer is wrapped on the roller to serve as a receiving substrate, the second spinning solution is adopted for electrostatic spinning to obtain the combined growth guide directional fiber membrane layer and the selective permeation disordered fiber membrane layer, the structures of the growth guide directional fiber membrane layer and the selective permeation disordered fiber membrane layer are effectively guaranteed not to be damaged in the process of mixing, dissolving and combining the growth guide directional fiber membrane layer and the selective permeation disordered fiber membrane layer to form the bone tissue regeneration guide membrane, the triple effects of shielding fibroblasts, uninterruptedly conveying nutrient elements and exchanging metabolites and directionally guiding the growth and migration of bone cells of the bone tissue regeneration guide membrane are guaranteed, and the repairing and healing effects of the bone tissue regeneration guide membrane on bone defects are further guaranteed.

It should be noted that the electrostatic spinning machine comprises a liquid storage device, an injector, a spray head, a high-voltage power supply and a receiver. The liquid storage device is connected with the injector and provides spinning solution for the injector, the injector is connected with the spray head, the spray head is used for spraying the spinning solution, the spray head is connected with the high-voltage power supply, and the spray head and the injector are correspondingly arranged. The process of electrostatic spinning of the spinning solution comprises the steps that the spinning solution is stored in a liquid storage device, the spinning solution is extruded out from a nozzle, the injection speed of the spinning solution is controlled by an injector with a capillary tube, the spinning solution extruded from the nozzle is subjected to the combined action of an electric field force formed by a high-voltage power supply and an electrostatic repulsion force to form an electrified jet flow, the electrified jet flow is stretched and thinned under the action of the electric field force and then is accepted by a receiver, and the electrified jet flow is deposited on a receiving substrate of the receiver after the spinning solution is fixedly formed.

In one embodiment, electrostatic spinning is carried out by using an electrostatic spinning machine with the extrusion speed of the spinning solution set to be 0.05 mL/min-0.4 mL/min, so that the porosity and the specific surface area of the oriented fiber film layer and the random fiber film layer are ensured.

In one embodiment, the electrostatic spinning machine with the set voltage of 8kV to 25kV is used for electrostatic spinning, so that the porosity and the specific surface area of the oriented fiber film layer and the random fiber film layer which are guided to grow and selectively permeate are ensured.

In one embodiment, electrostatic spinning is carried out by using an electrostatic spinning machine with the distance between a spinning solution nozzle and a receiving substrate set to be 5 cm-30 cm, so that the porosity and the specific surface area of the oriented fiber film layer and the random fiber film layer are ensured to be guided to grow and selectively permeate.

In one embodiment, the organic solvent is at least one of chloroform, dichloromethane, hexafluoroisopropanol, and acetone. It can be understood that chloroform, dichloromethane, hexafluoroisopropanol and acetone have a large dissolving effect on high molecular substances, so that a microetching interface is easily formed on the surfaces of the oriented fiber film layer for guiding growth and the disordered fiber film layer for selective permeation, and the oriented fiber film layer for guiding growth and the disordered fiber film layer for selective permeation are effectively combined together after being mixed and dissolved.

The organic solvent used in the step of obtaining the oriented fiber film layer for guided growth, the organic solvent used in the step of obtaining the selectively permeable disordered fiber film layer, and the organic solvent used in the microetching treatment are independently at least one selected from chloroform, dichloromethane, hexafluoroisopropanol, and acetone, and the organic solvent used in the step of obtaining the oriented fiber film layer for guided growth, the organic solvent used in the step of obtaining the selectively permeable disordered fiber film layer, and the organic solvent used in the microetching treatment may be the same or different.

Compared with the prior art, the invention has at least the following advantages:

1. in the bone tissue regeneration guide membrane, the oriented fiber membrane layer for guiding growth is formed by oriented stacking and arranging fiber filaments, so that the differentiation of osteocytes can be promoted, the oriented growth migration of derived cells for guiding the osteocytes can be better realized, the oriented growth migration guide membrane has the functions of oriented growth migration guide and growth migration promotion on the osteocytes, the oriented repair of bone defects is better realized, and the application range of the bone tissue regeneration guide membrane is improved;

2. in the bone tissue regeneration guide membrane, the guide growth directional fiber membrane layer is formed by directional stacking and arranging fiber filaments, the porosity is low, and the bone tissue regeneration guide membrane has high mechanical property and deformation resistance, namely good structural strength;

3. in the bone tissue regeneration guiding membrane, the selective permeation disordered fiber membrane layer is formed by disorderly and randomly arranging the fiber filaments, so that the porosity is high, the fibroblast is more prone to differentiation, proliferation, migration and growth in the selective permeation disordered fiber membrane layer, the effect of blocking the growth of the fibroblast into the growth guiding oriented fiber membrane layer is achieved, namely, the fibroblast is prevented from growing into a bone defect part, and the rapid oriented repair and healing of the bone defect part are facilitated. In addition, the selective permeation disordered fiber film layer has higher porosity, can support the input of nutrient elements in human tissue fluid and the exchange of metabolites at the bone defect, and shows that fibroblasts are selectively prevented from growing into the bone defect to allow the uninterrupted transmission of the nutrient elements in the human tissue fluid and the exchange of the metabolites at the bone defect, so that nutrient substances in the human body can reach the bone defect to promote the directional repair and healing of the bone defect;

4. in the preparation method of the bone tissue regeneration guiding membrane, the obtained oriented fiber membrane layer for guiding growth and the disordered fiber membrane layer are connected, so that the bone tissue regeneration guiding membrane has a good oriented repair and healing effect on bone defects, and the bone tissue regeneration guiding membrane allows the uninterrupted delivery of nutrient elements in human tissue fluid and the exchange of metabolites at the bone defect positions, so that nutrient substances in the human body can reach the bone defect positions to promote the oriented repair and healing of the bone defects;

5. the preparation method of the bone tissue regeneration guide membrane has simple operation steps and effectively improves the production efficiency of the bone tissue regeneration guide membrane.

Some specific examples are illustrated below, and all references to% are by weight. It should be noted that the following examples are not intended to be exhaustive of all possible cases, and that the materials used in the following examples are commercially available without specific recitation.

Example 1

Dissolving 1kg of polylactic acid (hereinafter, polylactic acid is abbreviated as PLLA) in 10L of chloroform, and magnetically stirring until the polylactic acid is completely dissolved to obtain a spinning solution containing PLLA;

2/3 spinning solution containing PLLA and 0.29kg of pretreated bioactive inorganic glass are dissolved in chloroform, and are magnetically stirred until the mixture is completely dissolved, so that the spinning solution containing PLLA and bioactive inorganic glass is obtained;

injecting the residual 1/3 PLLA-containing spinning solution into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 10kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.3mL/min, and the distance between a spinning solution nozzle and a roller is 15cm, so that a smooth selective permeation disordered fiber film layer is obtained;

and taking the obtained selective permeation disordered fiber membrane layer as a receiving substrate, injecting a spinning solution containing PLLA and bioactive inorganic glass into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 10kV in the electrostatic spinning process, extruding the spinning solution by the injector at the speed of 0.3mL/min, wherein the distance between a nozzle of the spinning solution and a roller is 8cm, cutting a semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and standing the semi-finished product in a fume hood at normal temperature for 48 hours to fully volatilize the residual chloroform in the semi-finished product of the bone tissue regeneration guiding membrane, thereby obtaining the bone tissue regeneration guiding membrane.

Example 2

Dissolving 0.8kg of PLLA in 10L of chloroform, and magnetically stirring until the PLLA is completely dissolved to obtain a spinning solution containing the PLLA;

3/2 spinning solution containing PLLA and 0.36kg of hydroxyapatite (hereinafter hydroxyapatite is abbreviated as HAp) are dissolved in chloroform, and are magnetically stirred until the hydroxyapatite is completely dissolved to obtain spinning solution containing PLLA and HAp;

injecting the residual 1/3 PLLA-containing spinning solution into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 10kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.3mL/min, and the distance between a spinning solution nozzle and a roller is 15cm, so that a smooth selective permeation disordered fiber film layer is obtained;

and taking the obtained selective permeation disordered fiber membrane layer as a receiving substrate, injecting a spinning solution containing PLLA and HAp into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 15kV in the electrostatic spinning process, extruding the spinning solution by the injector at the speed of 0.2mL/min, wherein the distance between a nozzle of the spinning solution and a roller is 5cm, cutting the semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and standing the semi-finished product in a ventilation cabinet at normal temperature for 48 hours to fully volatilize the residual chloroform in the semi-finished product of the bone tissue regeneration guiding membrane, thereby obtaining the bone tissue regeneration guiding membrane.

Example 3

Dissolving 1.5kg of PLLA in 10L of chloroform, and magnetically stirring until the PLLA is completely dissolved to obtain a spinning solution containing the PLLA;

3/2 spinning solution containing PLLA and 1kg of beta-tricalcium phosphate (hereinafter, the beta-tricalcium phosphate is abbreviated as TCP) are dissolved in chloroform, and the mixture is magnetically stirred until the beta-tricalcium phosphate is completely dissolved to obtain the spinning solution containing PLLA and TCP;

injecting the residual 1/3 PLLA-containing spinning solution into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 20kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.3mL/min, and the distance between a spinning solution nozzle and a roller is 15cm, so that a smooth selective permeation disordered fiber film layer is obtained;

and taking the obtained selective permeation disordered fiber membrane layer as a receiving substrate, injecting a spinning solution containing PLLA and TCP into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 18kV in the electrostatic spinning process, extruding the spinning solution by the injector at the speed of 0.4mL/min, and adjusting the distance between a nozzle of the spinning solution and a roller to be 23cm, cutting the semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and standing the semi-finished product in a ventilation cabinet at normal temperature for 48 hours to fully volatilize the residual chloroform in the semi-finished product of the bone tissue regeneration guiding membrane, thereby obtaining the bone tissue regeneration guiding membrane.

Example 4

Dissolving 1kg of polylactic-co-glycolic acid (PLGA) in 12L hexafluoroisopropanol, and magnetically stirring until the PLGA is completely dissolved to obtain a PLGA-containing spinning solution;

3/2 spinning solution containing PLLA and 0.074kg of pretreated bioactive inorganic glass are dissolved in hexafluoroisopropanol, and the mixture is magnetically stirred until the mixture is completely dissolved to obtain spinning solution containing PLGA and bioactive inorganic glass;

injecting the residual 1/3 PLLA-containing spinning solution into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 12kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.3mL/min, and the distance between a spinning solution nozzle and a roller is 20cm, so that a smooth selective permeation disordered fiber film layer is obtained;

and taking the obtained selective permeation disordered fiber membrane layer as a receiving substrate, injecting a spinning solution containing PLGA and bioactive inorganic glass into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 13kV in the electrostatic process, extruding the spinning solution by the injector at the speed of 0.4mL/min, wherein the distance between a nozzle of the spinning solution and a roller is 22cm, cutting the semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and placing the semi-finished product in a ventilation cabinet at normal temperature for 48 hours to fully volatilize the hexafluoroisopropanol remained in the semi-finished product of the bone tissue regeneration guiding membrane so as to obtain the bone tissue regeneration guiding membrane.

Example 5

Dissolving 1.2kg of polylactic-co-glycolic acid (hereinafter, the polylactic-co-glycolic acid is abbreviated as PLGA) in 10L of dichloromethane, and magnetically stirring until the polylactic-co-glycolic acid is completely dissolved to obtain a PLGA-containing spinning solution;

2/3 spinning solution containing PLGA and 0.2kg of pretreated bioactive inorganic glass are dissolved in dichloromethane, and the spinning solution containing PLGA and bioactive inorganic glass is obtained after magnetic stirring until the spinning solution is completely dissolved;

injecting the residual 1/3 PLGA-containing spinning solution into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 13kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.2mL/min, and the distance between a spinning solution nozzle and a roller is 14cm, so that a smooth selective permeation disordered fiber film layer is obtained;

and taking the obtained selective permeation disordered fiber membrane layer as a receiving substrate, injecting spinning solution containing PLGA and bioactive inorganic glass into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 13kV in the electrostatic spinning process, extruding the spinning solution by the injector at the speed of 0.15mL/min, wherein the distance between a nozzle of the spinning solution and a roller is 8cm, cutting the semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and standing the semi-finished product in a ventilation cabinet at normal temperature for 48 hours to fully volatilize the dichloromethane remained in the semi-finished product of the bone tissue regeneration guiding membrane, thereby obtaining the bone tissue regeneration guiding membrane.

Example 6

Dissolving 1.2kg of silk fibroin in 10L of acetone, and magnetically stirring until the silk fibroin is completely dissolved to obtain a spinning solution containing the silk fibroin;

2/3 spinning solution containing silk fibroin and 0.2kg of pretreated bioactive inorganic ceramic are dissolved in dichloromethane, and the mixture is magnetically stirred until the mixture is completely dissolved to obtain spinning solution containing PLGA and bioactive inorganic ceramic;

injecting the residual 1/3 spinning solution containing silk fibroin into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 8kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.05mL/min, and the distance between a spinning solution nozzle and a roller is 5cm, so that a smooth selective permeation disordered fiber film layer is obtained;

and taking the obtained selective permeation disordered fiber membrane layer as a receiving substrate, injecting spinning solution containing silk fibroin and bioactive inorganic ceramic into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 13kV in the electrostatic spinning process, extruding the spinning solution by the injector at the speed of 0.15mL/min, wherein the distance between a nozzle of the spinning solution and a roller is 8cm, cutting the semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and standing the semi-finished product in a ventilation cabinet at normal temperature for 45 hours to fully volatilize the residual dichloromethane and acetone in the semi-finished product of the bone tissue regeneration guiding membrane so as to obtain the bone tissue regeneration guiding membrane.

Example 7

Dissolving 1.2kg of silk fibroin in 10L of hexafluoroisopropanol, and magnetically stirring until the silk fibroin is completely dissolved to obtain a spinning solution containing the silk fibroin;

2/3 spinning solution containing silk fibroin and 0.2kg of pretreated 45S5 type bioglass are dissolved in hexafluoroisopropanol, and the mixture is magnetically stirred until the mixture is completely dissolved to obtain spinning solution containing PLGA and bioactive inorganic ceramic;

injecting the residual 1/3 spinning solution containing silk fibroin into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 15kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.2mL/min, and the distance between a spinning solution nozzle and a roller is 18cm, so that a smooth selective permeation disordered fiber film layer is obtained;

and taking the obtained selective permeation disordered fiber membrane layer as a receiving substrate, injecting a spinning solution containing silk fibroin and bioactive inorganic ceramic into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 8kV in the electrostatic spinning process, extruding the spinning solution by the injector at the speed of 0.2mL/min, wherein the distance between a nozzle of the spinning solution and a roller is 20cm, cutting the semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and standing the semi-finished product in a ventilation cabinet at normal temperature for 48 hours to fully volatilize the residual hexafluoroisopropanol in the semi-finished product of the bone tissue regeneration guiding membrane, thereby obtaining the bone tissue regeneration guiding membrane.

Example 8

Dissolving 1.2kg of collagen in 10L of dichloromethane, and magnetically stirring until the collagen is completely dissolved to obtain a spinning solution containing the collagen;

2/3 spinning solution containing collagen and 0.2kg of pretreated 45S5 type bioglass are dissolved in dichloromethane and are magnetically stirred until the bioglass and the bioglass are completely dissolved to obtain spinning solution containing PLGA and bioactive inorganic ceramic;

injecting the residual 1/3 spinning solution containing collagen into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 20kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.3mL/min, and the distance between a spinning solution nozzle and a roller is 20cm, so that a smooth selective permeation disordered fiber film layer is obtained;

and taking the obtained selective permeation disordered fiber membrane layer as a receiving substrate, injecting spinning solution containing collagen and bioactive inorganic ceramic into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 25kV in the electrostatic spinning process, extruding the spinning solution by the injector at the speed of 0.4mL/min, adjusting the distance between a nozzle of the spinning solution and a roller to be 30cm, cutting the semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and standing the semi-finished product in a ventilation cabinet at normal temperature for 50h to fully volatilize the dichloromethane remained in the semi-finished product of the bone tissue regeneration guiding membrane so as to obtain the bone tissue regeneration guiding membrane.

Example 9

Dissolving 1.2kg of collagen in 10L of hexafluoroisopropanol, and magnetically stirring until the collagen is completely dissolved to obtain a spinning solution containing silk fibroin;

2/3 spinning solution containing silk fibroin and 0.2kg of pretreated 45S5 type bioglass are dissolved in dichloromethane and are magnetically stirred until the bioglass and the bioglass are completely dissolved to obtain spinning solution containing PLGA and bioactive inorganic ceramics;

injecting spinning solution containing silk fibroin and bioactive inorganic ceramic into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 8kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.2mL/min, and the distance between a spinning solution nozzle and a roller is 20cm, so that a flat directional fiber film layer for guiding growth is obtained;

and taking the obtained guided growth oriented fiber membrane layer as a receiving substrate, injecting the residual 1/3 spinning solution containing silk fibroin into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 15kV in the electrostatic spinning process, extruding the spinning solution by the injector at the speed of 0.2mL/min, wherein the distance between a nozzle of the spinning solution and a roller is 18cm, cutting the semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and placing the semi-finished product of the bone tissue regeneration guiding membrane in a 50 ℃ oven for 10 hours to fully volatilize the residual hexafluoroisopropanol in the semi-finished product of the bone tissue regeneration guiding membrane so as to obtain the bone tissue regeneration guiding membrane.

Example 10

Dissolving 1.2kg of silk fibroin in 10L of acetone, and magnetically stirring until the silk fibroin is completely dissolved to obtain a spinning solution containing the silk fibroin;

2/3 spinning solution containing silk fibroin and 0.2kg of pretreated bioactive inorganic ceramic are dissolved in dichloromethane, and the mixture is magnetically stirred until the mixture is completely dissolved to obtain spinning solution containing PLGA and bioactive inorganic ceramic;

injecting spinning solution containing silk fibroin and bioactive inorganic ceramic into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 13kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.15mL/min, and the distance between a spinning solution nozzle and a roller is 8cm, so that a flat directional fiber film layer for guiding growth is obtained;

and taking the obtained guided growth oriented fiber membrane layer as a receiving substrate, injecting the residual 1/3 spinning solution containing silk fibroin into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 8kV in the electrostatic spinning process, extruding the spinning solution by the injector at the speed of 0.05mL/min, wherein the distance between a nozzle of the spinning solution and a roller is 5cm, cutting the semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and placing the semi-finished product of the bone tissue regeneration guiding membrane in a 55 ℃ oven for 8 hours to fully volatilize the residual dichloromethane and acetone in the semi-finished product of the bone tissue regeneration guiding membrane, thereby obtaining the bone tissue regeneration guiding membrane.

Example 11

Dissolving 1.2kg of collagen in 10L of dichloromethane, and magnetically stirring until the collagen is completely dissolved to obtain a spinning solution containing the collagen;

2/3 spinning solution containing collagen and 0.2kg of pretreated 45S5 type bioglass are dissolved in dichloromethane and are magnetically stirred until the bioglass and the bioglass are completely dissolved to obtain spinning solution containing PLGA and bioactive inorganic ceramic;

injecting spinning solution containing collagen and bioactive inorganic ceramic into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 25kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.4mL/min, and the distance between a spinning solution nozzle and a roller is 30cm, so that a flat directional fiber film layer for guiding growth is obtained;

and taking the obtained oriented fiber membrane layer for guided growth as a receiving substrate, injecting the residual 1/3 spinning solution containing collagen into an injector for electrostatic spinning, adjusting the voltage of electrostatic spinning equipment to be 20kV in the electrostatic spinning process, extruding the spinning solution by the injector at the speed of 0.3mL/min, adjusting the distance between a nozzle of the spinning solution and a roller to be 20cm, cutting the semi-finished product of the bone tissue regeneration guiding membrane from the roller after the spinning is finished, and placing the semi-finished product of the bone tissue regeneration guiding membrane in an oven at 60 ℃ for 5 hours to fully volatilize the residual dichloromethane in the semi-finished product of the bone tissue regeneration guiding membrane so as to obtain the bone tissue regeneration guiding membrane.

Example 12

Dissolving 1.5kg of PLLA in 10L of chloroform, and magnetically stirring until the PLLA is completely dissolved to obtain a spinning solution containing the PLLA;

3/2 spinning solution containing PLLA and 1kg of TCP are dissolved in chloroform, and the mixture is magnetically stirred until the mixture is completely dissolved, so that the spinning solution containing PLLA and TCP is obtained;

injecting the residual 1/3 PLLA-containing spinning solution into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 20kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.3mL/min, and the distance between a spinning solution nozzle and a roller is 15cm, so that a smooth selective permeation disordered fiber film layer is obtained;

injecting a spinning solution containing PLLA and TCP into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 18kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.4mL/min, and the distance between a spinning solution nozzle and a roller is 23cm, so that a flat oriented fiber film layer for guiding growth is obtained;

spraying chloroform on the guided growth oriented fiber membrane layer, pressing the selective permeation disordered fiber membrane layer on the guided growth oriented fiber membrane layer, and pressing for 45h at a ventilation position to fully volatilize the residual chloroform in the semi-finished product of the bone tissue regeneration guided membrane, thus obtaining the bone tissue regeneration guided membrane.

Example 13

Dissolving 1.5kg of PLLA in 10L of chloroform, and magnetically stirring until the PLLA is completely dissolved to obtain a spinning solution containing the PLLA;

3/2 spinning solution containing PLLA and 1kg of TCP are dissolved in chloroform, and the mixture is magnetically stirred until the mixture is completely dissolved, so that the spinning solution containing PLLA and TCP is obtained;

injecting the residual 1/3 PLLA-containing spinning solution into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 20kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.3mL/min, and the distance between a spinning solution nozzle and a roller is 15cm, so that a smooth selective permeation disordered fiber film layer is obtained;

injecting a spinning solution containing PLLA and TCP into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 18kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.4mL/min, and the distance between a spinning solution nozzle and a roller is 23cm, so that a flat oriented fiber film layer for guiding growth is obtained;

spraying chloroform on the selective permeation disordered fiber film layer, pressing the guided growth oriented fiber film layer on the selective permeation disordered fiber film layer, and pressing for 50h at a ventilation position to fully volatilize the residual chloroform in the semi-finished product of the bone tissue regeneration guiding film, thus obtaining the bone tissue regeneration guiding film.

Example 14

Dissolving 1.5kg of PLLA in 10L of chloroform, and magnetically stirring until the PLLA is completely dissolved to obtain a spinning solution containing the PLLA;

3/2 spinning solution containing PLLA and 1kg of TCP are dissolved in chloroform, and the mixture is magnetically stirred until the mixture is completely dissolved, so that the spinning solution containing PLLA and TCP is obtained;

injecting the residual 1/3 PLLA-containing spinning solution into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 20kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.3mL/min, and the distance between a spinning solution nozzle and a roller is 15cm, so that a smooth selective permeation disordered fiber film layer is obtained;

injecting a spinning solution containing PLLA and TCP into an injector for electrostatic spinning, wherein the voltage of electrostatic spinning equipment is adjusted to be 18kV in the electrostatic spinning process, the speed of extruding the spinning solution by the injector is 0.4mL/min, and the distance between a spinning solution nozzle and a roller is 23cm, so that a flat oriented fiber film layer for guiding growth is obtained;

and brushing chloroform on the selective permeation disordered fiber film layer, pressing the guided growth oriented fiber film layer on the selective permeation disordered fiber film layer, and pressing for 50 hours in a ventilation position to fully volatilize the residual chloroform in the semi-finished product of the bone tissue regeneration guiding film, thus obtaining the bone tissue regeneration guiding film.

In the following, in vitro cell culture experiments were performed using examples 1, 2, 12, 13 and 14, with example 1, 2, 12, 13 and 14 corresponding to group 1, 2, 3, 4 and 5, respectively.

The following in vitro cell culture tests for 1, 3 and 7 days were performed on the bone tissue regeneration guiding membranes prepared in groups 1 to 5, and ex vivo human dermal fibroblasts were selected as proliferation cultured cells, so as to investigate the influence rule of the bone tissue regeneration guiding membrane on cell proliferation, and the experimental results are shown in fig. 9, which is the cell proliferation conditions of different groups obtained by measuring after culturing human dermal fibroblasts on the disordered fibrous membrane layer for 1, 3 and 7 days. The result shows that the cytotoxicity of the selective permeation disordered fiber membrane layer of the bone tissue regeneration guiding membrane is in a safe range, and the cells have a good proliferation effect on the surface of the selective permeation disordered fiber membrane layer.

In the following, in vitro cell culture tests for 1, 3 and 7 days are performed on the bone tissue regeneration guiding membranes prepared in the groups 1 to 5, and rBMSC is selected as a cell for proliferation culture, so as to explore the influence rule of the bone tissue regeneration guiding membrane on cell proliferation, and the experimental result is shown in FIG. 10, wherein FIG. 10 shows the cell proliferation conditions of different groups obtained by measurement after rBMSC is cultured on the guided growth oriented fiber membrane layer of the bone tissue regeneration guiding membrane for 1, 3 and 7 days. The result shows that the cytotoxicity of the guided growth oriented fiber membrane layer of the bone tissue regeneration guiding membrane is in a safe range, and the rBMSC has a good proliferation effect on the surface of the functional layer.

Respectively inoculating fibroblasts to the disordered fiber membrane selective permeation guide membrane prepared by the bone tissue regeneration guide membranes of groups 1-5, culturing for 3 days, fixing for 40min by 2.5% glutaraldehyde, washing for three times by PBS, adding fluorescein isothiocyanate labeled Phalloidin (FITC labeled Phalloidin) dye to dye the cytoskeleton for 20min, washing for 3 times by PBS, washing for 5min each time, then dyeing cell nuclei by DAPI for 5min, washing for 3 times by PBS, soaking in PBS, photographing by using a confocal microscope, observing the positions of the fibroblasts on the disordered fiber membrane selective permeation guide membrane, selecting a confocal micrograph of the bone tissue regeneration guide membrane of group 1 to illustrate to obtain a figure 11, and as can be seen from the figure 11, inoculating the fibroblasts to the disordered fiber membrane selective permeation guide membrane of the bone tissue regeneration guide membrane for 3 days, allowing the fibroblasts to still grow on the disordered fiber membrane selective permeation guide membrane, the bone tissue regeneration guiding membrane is characterized in that the bone tissue regeneration guiding membrane is a membrane layer which is formed by a plurality of fibrous membrane layers and has a certain barrier effect on fibroblasts, and the fibrous membrane layers are arranged on the surface of the bone tissue regeneration guiding membrane.

The guided growth oriented fiber membrane layers of the bone tissue regeneration guide membranes prepared in the groups 1-5 are respectively photographed in a transmission mode of an optical microscope, an optical microscopic picture of the guided growth oriented fiber membrane layer of the bone tissue regeneration guide membrane of the group 2 is selected for explanation, namely, the picture is shown in figure 12, as shown in figure 12, a lot of black granular substances are distributed in fiber filaments of the guided growth oriented fiber membrane layer, and as light cannot penetrate through inorganic particles, the black granular substances appearing in the fibers are inorganic particles doped in a high polymer material, so that the high polymer material and the inorganic material are proved to have good mixing effect.

Respectively cutting off the fiber filaments of the guided growth oriented fiber film layer of the bone tissue regeneration guide film prepared in the groups 1-5 to obtain a scanning electron microscope image, selecting the electron microscope image of the guided growth oriented fiber film layer of the bone tissue regeneration guide film of the group 3 to explain, namely, as shown in fig. 13, observing the morphology structure of the guided growth oriented fiber film layer under the scanning electron microscope as shown in fig. 13, and finding that the inorganic particles in the single fiber filament of the guided growth oriented fiber film layer are uniformly dispersed, thereby proving that the mixing effect of the high polymer material and the inorganic material is good.

In order to verify the repair effect of the prepared periosteum product on bone defects, a rabbit bilateral radius fracture model experiment is carried out, the left leg is an experimental group, bone tissue regeneration guide membranes prepared by groups 1-5 are adopted for auxiliary repair, and the right leg is a blank control. Raising for 2 weeks after operation and taking X-ray pictures. The X-ray photograph of the bilateral radius of the rabbit obtained after the left leg of the rabbit is repaired by the bone tissue regeneration guide membrane of the group 4 is selected for explanation, namely, the X-ray photograph of the group 14 shows that the bone repair effect of the left side radius of the rabbit is obviously better than that of the right side radius of the rabbit, and the fracture crack of the left side radius of the rabbit is basically healed, so that the bone tissue regeneration guide membrane has a better bone defect repair healing effect, and can promote the directional proliferation and differentiation of bone cells. The bone tissue regeneration guide membranes prepared in examples 1 to 14 were subjected to biodegradation tests, and the bone tissue regeneration guide membranes prepared in examples 1 to 14 were placed in a simulated human body external environment for biodegradation, and no residue was observed after the bone tissue regeneration guide membranes were degraded for 3 to 5 months.

The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.