CN111265722B

CN111265722B - Double-layer structured periosteum for diabetic bone repair and preparation method thereof

Info

Publication number: CN111265722B
Application number: CN202010259170.7A
Authority: CN
Inventors: 张佩华; 高原; 付少举; 郭虹; 孙玮; 沈为; 王峰
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2020-04-03
Filing date: 2020-04-03
Publication date: 2021-08-03
Anticipated expiration: 2040-04-03
Also published as: CN111265722A

Abstract

The invention relates to a double-layer structured periosteum for repairing diabetic bones and a preparation method thereof, belonging to the field of biomedical materials. The inner layer of the double-layer structured periosteum is a spongy nanofiber layer with a porous structure, which is formed by blending chitosan/polyvinyl alcohol/calycosin according to a certain proportion, and the outer layer of the double-layer structured periosteum is a poly (p-dioxanone) nanofiber layer, and the double-layer structured periosteum is prepared by compounding through an electrostatic spinning process. The outer layer of the double-layer structure periosteum has proper mechanical properties, can effectively support a bone defect part and prevent other soft tissues from growing in; the inner layer is loose and porous, is suitable for the growth, the attachment and the propagation of osteoblasts, and can play the roles of resisting and inhibiting bacteria, reducing blood sugar and the like. The double-layer periosteum has the composite functions of the inner layer and the outer layer, is beneficial to the growth of osteocytes and the repair of bone tissues, and has the characteristics of simple preparation process and convenience in operation.

Description

Double-layer structured periosteum for diabetic bone repair and preparation method thereof

Technical Field

The invention relates to a double-layer structured periosteum for repairing diabetic bones and a preparation method thereof, belonging to the field of medical materials.

Background

At present, diabetes is one of the most frequent chronic diseases, and patients with diabetes with bone defects caused by trauma, inflammation and the like are increasing. Bone defects are caused by trauma, infection or tumor, surgical debridement from osteomyelitis, and various congenital diseases. Bone defects are more common clinically. Due to the phenomenon of aging of people, which is increasingly aggravated in recent years, and sports injury, congenital malformation or accident wound and the like, large-area bone defect can be caused in China. The prevalence of osteopenia and osteoporosis in diabetic patients is as high as 48-72%. Hyperglycemia can change the biomineralization process of osteoblasts and enhance mineralization, so that the osteoblasts are over-expressed, and differentiation, maturation and function regulation of the osteoblasts and osteoclasts are influenced. Therefore, the search for suitable bone tissue repair materials for diabetic patients has become one of the most troublesome problems facing the field at present.

Clinically, the problems are generally treated by means of medicines, autologous or allogeneic bone transplantation and the like. Little attention is paid to coating a layer of periosteum around the implant material, and then the promotion effect of the periosteum on the bone regeneration microenvironment is discussed. Periosteum is a strong connective tissue envelope that coats the surface of bone except for the joint. Traditionally, the periosteum is divided into a superficial fibrous layer and a deep bone layer, and the fibrous layer is thicker and plays a role in fixing the periosteum and ligaments; the bone layer is closely adjacent to the outer surface of the bone, is arranged loosely and has osteogenesis capacity.

Electrospinning is currently the simplest and most efficient method for preparing nanofibers, but further applications are limited due to the structure of the material itself. At present, a plurality of biological and polymer material combinations have been applied to bone repair materials, such as small intestine submucosa (rich in active factors) stents, silk fibroin and polycaprolactone stents, collagen-coated polycaprolactone films and the like, and the combinations are proved to have certain bone regeneration promoting effects, but lack the effect of realizing periosteum regeneration from the structural and functional aspects.

The periosteum for repairing the diabetic bone needs to have good biocompatibility and bioactivity, also needs to have good mechanical support and isolation effects, is beneficial to differentiation and formation of osteoblasts at the contact surface with the bone, and can promote the climbing growth of osteoblasts with larger diameters. In order to solve the problem, the invention provides a double-layer structure periosteum and a preparation method thereof, which have good mechanical support and are beneficial to the growth of osteoblasts in a climbing manner.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the existing periosteum scaffold material only has certain effect of promoting bone regeneration and lacks the structure and function required by periosteum regeneration.

In order to solve the problems, the technical scheme of the invention is to provide a preparation method of a double-layer periosteum for diabetic bone repair, which is characterized by comprising the following steps:

step 1, preparing an inner layer, which specifically comprises the following steps:

mixing chitosan and polyvinyl alcohol in a mass percentage of 10-30: adding 90-70 parts of the mixture into 20% acetic acid solution to prepare 10% solution, stirring the solution in a water bath at the temperature of 90 ℃ for more than 12 hours, standing and cooling the obtained solution, adding 5-9 uL of calycosin, and stirring the solution at room temperature for 6 hours to obtain inner-layer electrostatic spinning solution;

an electrostatic spinning device is adopted, a receiving distance is controlled to be 16-18 cm, the voltage is controlled to be 18-20 KV, the liquid flow rate is controlled to be 0.3-0.6 mL/h, a 20G flat needle head is adopted, and the electrostatic spinning solution is subjected to electrostatic spinning under the condition that the relative humidity is less than 50% to obtain an inner-layer nanofiber membrane;

and (2) selecting a glutaraldehyde solution with the concentration of 25% to perform crosslinking treatment on the obtained nanofiber membrane for 1h, then drying the nanofiber membrane for 30min at the temperature of 50 ℃ by using vacuum oven equipment, shearing the crosslinked nanofiber membrane, and adding a solvent with the volume ratio of 20: 80 tert-butanol: in a solvent of water, the nanofiber membrane is controlled: the mass-volume ratio of the tertiary butanol aqueous solution is 1: 200, obtaining a fiber dispersion liquid;

homogenizing the fiber dispersion liquid by a homogenizer for 10min, stirring for 3h at room temperature, transferring the solution into a culture dish, controlling the thickness of the fiber dispersion liquid to be 4-6 mm, pre-freezing for 24h at the temperature of-30 to-20 ℃, and then performing vacuum drying for 48h at the temperature of-10 to 0 ℃ to obtain an inner-layer porous structure spongy nanofiber layer;

step 2, preparing the outer layer and the double-layer structure periosteum:

adding 2.9-3.3 g of poly (p-dioxanone) into 7.1-6.7 mL of dimethylformamide, adding 0.01-0.015 g of sodium dodecyl sulfate, and fully stirring to obtain an outer layer electrostatic spinning solution with the concentration of 29-33%; fixing the inner-layer porous-structure spongy nanofiber layer obtained in the step 1 on a collector of an electrostatic spinning device, coating medical glue on the inner-layer porous-structure spongy nanofiber layer, and performing electrostatic spinning on an outer-layer electrostatic spinning solution by using the electrostatic spinning device under the conditions that the voltage is 18-20 KV, the receiving distance is 16-18 cm, the flow rate of an injection pump is 0.6-0.8mL/h, a 19G needle head and the relative humidity is less than 40% to obtain an outer-layer nanofiber layer, so as to form a double-layer-structure periosteum.

Preferably, the concentration of calycosin is 40 mmol/L.

The invention also provides a double-layer structured periosteum for repairing diabetic bones, which is characterized by comprising an inner layer contacted with a human body bone repairing material and an outer layer compounded with the inner layer through medical glue and an electrostatic spinning process, wherein the inner layer is a spongy nanofiber layer with a porous structure and prepared by blending chitosan/polyvinyl alcohol/calycosin according to a certain proportion, and the outer layer is a poly (p-dioxanone) nanofiber layer.

Preferably, the thickness range of the inner layer is 4-6 mm, and the fineness range of the nano-fibers is 0.27-0.31 μm.

Preferably, the thickness of the outer layer ranges from 0.17 mm to 0.2mm, and the fineness of the nano-fibers ranges from 0.27 μm to 0.34 μm.

Compared with the prior art, the invention has the beneficial effects that:

(1) the inner layer porous structure spongy nanofiber layer has the advantages that: the chitosan can adjust the pH value in vivo to make it reach alkalescence, which effectively improves the utilization rate of insulin and improves the comprehensive performance of the stent by compounding with polyvinyl alcohol. Meanwhile, the calycosin is carried, so that the immunity function can be improved, and the effects of resisting bacteria and inhibiting viruses can be achieved. The scaffold has good biocompatibility and effectively promotes the proliferation and differentiation of osteoblasts. The scaffold has good drug slow-release capacity and antibacterial capacity, overcomes the defects of a single high polymer material sponge scaffold, and the layer has large pores, thereby being beneficial to the growth and climbing of osteoblasts with large diameters.

(2) The outer nanofiber membrane has the advantages that: has excellent degradability, biocompatibility and absorbability, and the degradation product has no toxic or side effect. The rigidity of the mask is similar to that of the cartilage of a human body, and the mask can effectively shield external stress. The aperture is smaller than that of the spongy nanofiber layer of the inner layer, so that the inner layer can be protected to a certain extent while the supporting effect is achieved, and the growth of other soft tissues is prevented.

In conclusion, the double-layer structured periosteum has excellent biodegradability and absorbability, can effectively regulate blood sugar of a focus part and promote growth, differentiation and propagation of osteoblasts, and has good biocompatibility.

Drawings

FIG. 1 is a schematic view of a two-layer periosteal structure of the present invention;

FIG. 2 is a microscopic electron micrograph of the periosteal lining of the bilayer structure;

FIG. 3 is a microscopic electron micrograph of the outer periosteal layer of the bilayer structure.

Reference numerals: 1 — an inner layer; 2-outer layer.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings:

as shown in figure 1, the periosteum of the double-layer structure for repairing the diabetic bone has an inner layer and an outer layer; the inner layer 1 which is contacted with the human bone repair material is a spongy nanofiber layer with a porous structure and formed by blending chitosan/polyvinyl alcohol/calycosin according to a certain proportion; the outer layer 2 is a poly (p-dioxanone) nanofiber layer, and the inner layer 1 and the outer layer 2 are compounded by medical adhesive and an electrostatic spinning process. The preparation method comprises the following steps which are respectively provided according to the embodiments.

Example 1

Step 1: preparation of the inner layer:

mixing Chitosan (CS) and polyvinyl alcohol (PVA) according to the mass percentage of 10: adding 90 into 20% acetic acid solution to obtain 10% solution, stirring in 90 deg.C water bath for more than 12 hr, standing, cooling, adding 5 μ L of 40mmol/L calycosin, and stirring at room temperature for 6 hr to obtain inner layer electrostatic spinning solution;

adopting an electrostatic spinning device, controlling a receiving distance of 16cm, a voltage of 18KV, a liquid flow rate of 0.3ml/h and a 20G flat needle head, and carrying out electrostatic spinning under the condition of relative humidity of 30% to obtain an inner-layer nanofiber membrane;

and (2) selecting a 25% glutaraldehyde solution to perform crosslinking treatment on the obtained nanofiber membrane for 1h, then performing drying treatment for 30min at 50 ℃ through a vacuum oven, shearing the crosslinked nanofiber membrane, and adding a solvent with a volume ratio of 20: 80 tert-butanol: in a solvent of water, the nanofiber membrane is controlled: the mass volume ratio of the tertiary butanol aqueous solution is 1: 200, obtaining a fiber dispersion liquid;

homogenizing the fiber dispersion liquid by a homogenizer for 10min, stirring for 3h at room temperature, transferring the solution into a culture dish, controlling the thickness of the fiber dispersion liquid to be 4mm, pre-freezing for 24h at-30 ℃, and then performing vacuum drying for 48h at-10 ℃ to obtain an inner-layer porous structure spongy nanofiber layer;

step 2: preparing an outer layer and a double-layer structure periosteum:

adding 2.9G of poly (p-dioxanone) (PPDO) into 7.1mL of dimethyl formamide (DMF), adding 0.01G of Sodium Dodecyl Sulfate (SDS) and fully stirring to obtain 29% outer-layer electrostatic spinning solution, fixing the inner-layer porous-structure spongy nanofiber layer obtained in the step 1 on a collector, coating medical glue on the inner-layer porous-structure spongy nanofiber layer, and performing electrostatic spinning under the conditions that the voltage is 18KV, the receiving distance is 16cm, the injection pump flow rate is 0.6mL/h, the 19G needle head and the relative humidity is 30% to obtain the double-layer-structure periosteum.

The thickness of the periosteum inner layer of the double-layer structure is 4mm, and the fineness of the nano-fiber is 0.295 mu m; the thickness of the outer layer is 0.196mm, the fineness of the nano-fiber is 0.258 μm, and the breaking stress is 4.19 MPa.

Example 2

Step 1: preparation of the inner layer:

mixing Chitosan (CS) and polyvinyl alcohol (PVA) in a mass percentage of 20: adding 80 into 20% acetic acid solution to obtain 10% solution, stirring in 90 deg.C water bath for more than 12 hr, standing, cooling, adding 7uL of 40mmol/L calycosin, and stirring at room temperature for 6 hr to obtain inner layer electrostatic spinning solution;

adopting an electrostatic spinning device, controlling a receiving distance of 17cm, a voltage of 19KV, a liquid flow rate of 0.45mL/h and a 20G flat needle head, and carrying out electrostatic spinning under the condition of relative humidity of 30% to obtain an inner-layer nanofiber membrane;

and (2) selecting a glutaraldehyde solution with the concentration of 25% to perform crosslinking treatment on the obtained nanofiber membrane for 1h, then performing drying treatment on the nanofiber membrane for 30min at the temperature of 50 ℃ by using vacuum oven equipment, shearing the crosslinked nanofiber membrane, and adding a solvent with the volume ratio of 20: 80 tert-butanol: in a solvent of water, the nanofiber membrane is controlled: the mass-volume ratio of the tertiary butanol solution is 1: 200, obtaining a fiber dispersion liquid;

homogenizing the fiber dispersion liquid by a homogenizer for 10min, stirring for 3h at room temperature, transferring the solution into a culture dish, controlling the thickness of the fiber dispersion liquid to be 5mm, pre-freezing for 24h at-25 ℃, and then performing vacuum drying for 48h at-5 ℃ to obtain an inner-layer porous structure spongy nanofiber layer;

step 2: preparing an outer layer and a double-layer structure periosteum:

adding 3.1G of poly (p-dioxanone) (PPDO) into 6.9mL of dimethyl formamide (DMF), adding 0.012G of Sodium Dodecyl Sulfate (SDS) and fully stirring to obtain an outer-layer electrostatic spinning solution with the concentration of 31%, fixing the inner-layer porous-structure spongy nanofiber layer obtained in the step 1 on a collector, smearing medical glue on the inner-layer porous-structure spongy nanofiber layer, and then performing electrostatic spinning under the conditions that the voltage is 19KV, the receiving distance is 17cm, the injection pump flow rate is 0.7mL/h, the 19G needle head and the relative humidity is 30% to obtain the double-layer-structure periosteum.

The thickness of the periosteum inner layer of the double-layer structure is 5mm, and the fineness of the nano-fiber is 0.309 mu m; the thickness of the outer layer is 0.189, the fineness of the nano-fiber is 0.286 mu m, and the breaking stress is 4.3 MPa.

Example 3

Step 1: preparation of the inner layer:

mixing Chitosan (CS) and polyvinyl alcohol (PVA) in a mass percentage of 30: 70 adding into 20% acetic acid solution to obtain 10% solution, stirring in 90 deg.C water bath for more than 12 hr, standing, cooling, adding 9 μ L of 40mmol/L calycosin, stirring at room temperature for 6 hr to obtain inner layer electrostatic spinning solution;

adopting an electrostatic spinning device, controlling a receiving distance of 18cm, a voltage of 20KV, a liquid flow rate of 0.6ml/h and a 20G flat needle head, and carrying out electrostatic spinning under the condition of relative humidity of 30% to obtain an inner-layer nanofiber membrane;

and (2) selecting a glutaraldehyde solution with the concentration of 25% to perform crosslinking treatment on the obtained nanofiber membrane for 1h, then performing drying treatment for 30min at the temperature of 50 ℃ through a vacuum oven, shearing the crosslinked nanofiber membrane, and adding a solvent with the volume ratio of 20: 80 tert-butanol: in a solvent of water, the nanofiber membrane is controlled: the mass volume ratio of the tertiary butanol solution is 1: 200, obtaining a fiber dispersion liquid;

homogenizing the fiber dispersion liquid by a homogenizer for 10min, stirring for 3h at room temperature, transferring the solution into a culture dish, controlling the thickness of the fiber dispersion liquid to be 6mm, pre-freezing for 24h at-20 ℃, and then performing vacuum drying for 48h at 0 ℃ to obtain an inner-layer porous structure spongy nanofiber layer;

step 2: preparing an outer layer and a double-layer structure periosteum:

adding 3.3G of poly (p-dioxanone) (PPDO) into 6.7mL of dimethyl formamide (DMF), adding 0.015G of Sodium Dodecyl Sulfate (SDS) and fully stirring to obtain an outer layer electrostatic spinning solution with the concentration of 33%, fixing the inner layer porous structure spongy nanofiber layer obtained in the step 1 on a collector, smearing medical glue on the inner layer porous structure spongy nanofiber layer, and then performing electrostatic spinning under the conditions that the voltage is 20KV, the receiving distance is 18cm, the injection pump flow rate is 0.8mL/h, the 19G needle head and the relative humidity is 30% to obtain the double-layer structure periosteum.

The thickness of the periosteum inner layer of the double-layer structure is 6mm, and the fineness of the nano-fiber is 0.309 mu m; the thickness of the outer layer is 0.201mm, the fineness of the nano-fiber is 0.331 mu m, and the breaking stress is 4.51 MPa.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Claims

1. A preparation method of a double-layer structure periosteum for diabetic bone repair is characterized by comprising the following steps:

mixing chitosan and polyvinyl alcohol in a mass percentage of 10-30: adding 90-70 parts of the mixture into 20% acetic acid solution to prepare 10% solution, stirring the solution in a water bath at the temperature of 90 ℃ for more than 12 hours, standing and cooling the obtained solution, adding 5-9 uL of calycosin with the concentration of 40mmol/L, and stirring the solution at room temperature for 6 hours to obtain inner-layer electrostatic spinning solution;

step 2, preparing the outer layer and the double-layer structure periosteum:

2. A double-layer structured periosteum for diabetic bone repair, which is prepared by the method of claim 1, comprising an inner layer (1) contacting a human bone repair material, and an outer layer (2) compounded with the inner layer (1) through medical glue and an electrostatic spinning process, wherein the inner layer (1) is a spongy nanofiber layer with a porous structure and prepared by blending chitosan/polyvinyl alcohol/calycosin according to a certain proportion, and the outer layer (2) is a poly (p-dioxanone) nanofiber layer.

3. The double-layered periosteum for diabetic bone repair according to claim 2, wherein: the thickness range of the inner layer (1) is 4-6 mm, and the fineness range of the nano-fibers is 0.27-0.31 mu m.

4. The double-layered periosteum for diabetic bone repair according to claim 2, wherein: the thickness range of the outer layer (2) is 0.17-0.2 mm, and the fineness range of the nano-fibers is 0.27-0.34 mu m.