CN114081996A

CN114081996A - High-mineral-content induced bone regeneration fibrous membrane

Info

Publication number: CN114081996A
Application number: CN202111355762.XA
Authority: CN
Inventors: 赵勇刚; 王秀梅
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-11-16
Filing date: 2021-11-16
Publication date: 2022-02-25

Abstract

The application discloses high mineral content induced bone regeneration fibrous membrane, the fibrous membrane includes: mineralized collagen nanofibers doped with active elements; the mineralized collagen nano fiber doped with the active elements contains hydroxyapatite crystals; the calcium element in the crystal lattice of the hydroxyapatite is replaced by a cation containing the active element, and/or the phosphate group in the crystal lattice of the hydroxyapatite is replaced by an anion containing the active element. The fiber membrane provided by the application is far higher than other similar products in mineral content, strength, toughness, porosity and the like. And has the functions of guiding high-quality and rapid reconstruction of bone tissues, inducing rapid repair and regeneration of bones, promoting vascularization and neurogenesis, accelerating the bone healing speed and the like.

Description

High-mineral-content induced bone regeneration fibrous membrane

Technical Field

The invention relates to but is not limited to the field of biomedical materials, in particular to but not limited to a high mineral content induced bone regeneration fibrous membrane.

Background

The existing bone tissue engineering material is implanted at a bone defect, and risks of soft tissue ingrowth and the like exist. The new generation of bone tissue engineering material should have good bone compatibility, bone conductivity and good osteogenesis inducing ability. In addition, the new generation of bone tissue engineering material can not only realize the combination of the implanted material and the original bone tissue, but also collect the bone formation related cells and promote the differentiation of the bone formation related cells to the bone formation direction, thereby realizing the bone regeneration. In order to achieve good osteogenic properties, bone materials can be designed and prepared from a variety of aspects.

Modeling natural bone in terms of structure and composition is a recent trend in research. Compared with natural bone materials, the bionic material prepared in vitro has the advantages of sufficient sources and low immunogenicity. Calcium and phosphorus elements are selected as main components to simulate natural bone components, and even the bone materials which simulate the minimum structural units of the natural bone by an in vitro mineralization method are more, so that a better osteogenesis induction effect can be achieved. However, in clinical needs, the repair of bone defects not only needs to consider the bone regeneration performance, but also has the key of having targeted additional functions in combination with the reason for causing bone defects and the postoperative repair needs. Besides the main components of calcium and phosphorus, natural bone also contains various trace elements, such as magnesium, silicon, zinc, etc. The existing bone tissue engineering materials generally do not comprise the trace elements, and even if the trace elements are introduced, the corresponding effects are difficult to play.

Disclosure of Invention

The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the present application.

The present application provides a high mineral content (hydroxyapatite) fibrous membrane comprising: mineralized collagen nanofibers doped with active elements;

the mineralized collagen nano fiber doped with the active elements contains hydroxyapatite crystals;

part of the calcium element in the crystal lattice of the hydroxyapatite can be replaced by cations containing the active element,

and/or, part of the phosphate groups in the crystal lattice of the hydroxyapatite are replaced by anions containing the active element; the anion of the active element can be an oxyacid group of the active element anion.

The active element comprises any one or more of silicon, selenium, magnesium, zinc, strontium, silver, iron (e.g. ferric iron) and copper (e.g. cupric copper);

the ratio of the stoichiometric number of the calcium element to the sum of the active element-containing cation and the phosphate radical and the active element-containing anion is (1.45:1) to (1.80: 1);

contains the molar ratio of active element cation to calcium ion (5 to 8) to (92 to 95); contains the molar ratio of the active element anion to the phosphate radical (5 to 7) to (93 to 95).

In one embodiment provided herein, the active element may be incorporated in an amount of 0.

In one embodiment provided herein, the mineralized collagen nanofibers doped with active elements have an average length of 250nm to 300 nm;

in one embodiment provided herein, the mineralized collagen nanofibers doped with active elements have an average diameter of 5nm to 7 nm.

In another aspect, the present application provides a method for preparing the above mineralized collagen nanofibers doped with active elements, comprising: preparing the mineralized collagen nano fiber doped with the active element by using an in-situ co-assembly method, and adding cations containing the active element while adding calcium salt ions and/or adding anions containing the active element while adding phosphate ions.

In one embodiment provided herein, a method for preparing mineralized collagen nanofibers doped with active elements comprises the following steps:

(a) mixing the type I collagen sponge with a phosphoric acid solution to completely dissolve the type I collagen sponge to obtain a collagen template solution for later use;

when the active element is doped in the form of anion, firstly, the salt containing the anion is mixed with the phosphoric acid solution until the salt is completely dissolved, and then, the salt is mixed with the type I collagen sponge;

(b) preparing a calcium salt solution, wherein the calcium salt solution and the collagen template solution have the same volume;

when the active element is doped in the form of cation, firstly, the salt containing the cation is mixed with the calcium salt solution until the salt is completely dissolved, and then the salt is mixed with the collagen template solution;

(c) preparing a buffer solution;

(d) titrating the collagen template solution and the calcium salt solution in a buffer solution to obtain a mixed solution after titration is finished; during the titration, the pH value of the mixed solution is maintained between 8 and 10;

(e) carrying out high-speed centrifugation on the mixed solution obtained in the step (d), replacing the supernatant obtained by the centrifugation with ultrapure water with the same volume until the pH value of the measured supernatant is about 7-8, and stopping the high-speed centrifugation;

(f) removing water from the precipitate obtained in the step (e) to obtain mineralized collagen nanofibers doped with active elements;

alternatively, the preparation method consists of the above.

In one embodiment provided herein, the molar ratio of active element-containing cations to calcium ions is (5 to 10): (95 to 90);

in one embodiment provided herein, the molar ratio of active element-containing anions to calcium ions is (5 to 10): (95 to 90);

in one embodiment provided herein, the fibers are prepared in a process wherein the ratio of the stoichiometric numbers of (calcium ions + active element cations)/(phosphate ions + active element anions) is from (1.65:1) to (1.8: 1);

in one embodiment provided herein, the concentration of the phosphoric acid solution of step (a) is 0.5 to 2mol/L, and the ratio of the mass of the type I collagen sponge to the volume of the phosphoric acid solution is 0.1 to 10 g/L;

in one embodiment provided herein, the mixing of step (a) is for a mixing time of 4 to 12 hours; optionally, the mixing temperature of the mixing is 35 ℃ to 38 ℃.

In one embodiment provided herein, the calcium salt solution in step (c) further comprises hydroxide, and the molar ratio of the sum of the calcium ions and the active element cations to the hydroxide is 2: 1.

In one embodiment provided herein, the buffer solution of step (c) is selected from any one or more of Tris-HCl and phosphate buffer solutions.

In one embodiment provided herein, the flow rate of the titration of step (d) is from 400ml/h to 500 ml/h;

in one embodiment provided herein, after the titration in step (d) is completed, stirring for 12 to 36 hours; optionally, the reaction temperature is controlled at 30 ℃ to 37 ℃ after the titration is completed.

In one embodiment provided herein, the centrifugation of step (e) is at a speed of 3000 rpm to 17000 rpm.

In one embodiment provided herein, the removing of water in step (f) is lyophilization, comprising: drying the precipitate at 4-8 ℃, and then removing residual water by adopting freeze drying; optionally, the freeze dryer has a cold well temperature of between 2 ℃ and 4 ℃, a vacuum degree of between 10Pa and 30Pa, and a freeze drying duration of between 24h and 72 h.

In one embodiment provided herein, the resulting mixture is thoroughly mixed using a magnetic stirrer for agitation.

In one embodiment provided herein, the fibrous membrane further comprises a biodegradable polymer comprising any one or more of poly-epsilon-caprolactone, poly-L-lactic acid, poly-caprolactone-co-L-lactic acid, poly-lactic acid-co-glycolic acid, collagen, gelatin, silk fibroin, fibrinogen, cellulose, chitosan;

in the fibrous membrane, the mass of inorganic matters in the mineralized collagen nano fibers doped with the active elements accounts for 8-32% of the whole fibrous membrane;

in one embodiment provided herein, the porosity of the membrane is from 70% to 85%;

in one embodiment provided herein, the film has a density of 1.2g/cm³To 1.5g/cm³。

In another aspect, the present application provides a method for preparing the above fiber membrane, comprising the steps of:

(1) selecting the mineralized collagen nanofiber doped with the active elements, selecting a biodegradable polymer and a solvent, and uniformly mixing the raw materials to obtain a mixture;

the mass ratio of the mineralized collagen nano fiber doped with the active element, the biodegradable polymer and the solvent is (0.3-1.8): (0.6-0.9): (10-20);

(2) and (2) preparing a continuous collagen fiber membrane with a certain thickness by using the mixture obtained in the step (1) through one or two of electrostatic spinning or gas spinning processes, and drying the obtained membrane to obtain the fiber membrane.

In one embodiment provided herein, the mixing in step (1) is stirring for 28 to 50 hours;

in one embodiment provided herein, the solvent in step (1) is selected from any one or more of hexafluoroisopropanol and trifluoroethanol.

In one embodiment provided herein, the drying in step (2) is performed at 25 ℃ to 35 ℃ for 24h to 36 h;

in one embodiment provided herein, multiple layers of the fibrous membrane produced in step (2) are stacked to produce a fibrous membrane of a specified thickness.

In one embodiment provided herein, preparing a fibrous membrane of a particular thickness further comprises the steps of:

(a) superposing the fiber membranes in multiple layers, and pressing for 5min to 15min under the pressure of 1MPa to 10 MPa;

(b) crosslinking the multi-layer superposed fiber membrane pressed in the step (a) by using a crosslinking agent solution;

(c) and (c) removing the cross-linking agent in the multilayer film obtained in the step (b) to obtain the fiber film with the specific thickness.

In one embodiment provided herein, the solvent that may remain in the fiber membrane may be removed by drying the fiber membrane or the like.

In one embodiment provided herein, the method of removing the cross-linking agent may include vacuum drying after washing with alcohol, etc.;

in one embodiment provided herein, the cross-linking agent is selected from any one or more of carbonyldiimine, N-hydroxysuccinimide, and genipin;

in one embodiment provided herein, the solvent of the crosslinker solution comprises either or both of water and ethanol; the final concentration of each cross-linking agent is within the range of 5mol/L to 20 mol/L.

In one embodiment provided herein, the temperature of the crosslinking is from 0 ℃ to 4 ℃ (water at 0 ℃ does not freeze); the crosslinking time is 0.5h to 8 h.

In yet another aspect, the present application provides the use of the fibrous membrane described above in osteogenic repair.

In a further aspect, the present application provides the use of the fibrous membrane described above in nerve conduits, artificial biomimetic periosteum, induced bone regeneration membrane (GBR membrane) and guided tissue regeneration membrane (GTR membrane).

The method can be particularly applied to: the application provides a fibrous membrane with the function consistent with the GBR (induced bone regeneration membrane) function when used for bone repair, and can be applied to serial applications such as fracture, bone defect, oral cavity and the like. Although the mineralized collagen nanofiber doped fibrous membrane without other active elements has very light inductive effect, the bionic effect is more obvious, the structure is higher, and the mineral content is higher. After the fiber membrane provided by the application is added with the active elements, the bionic effect and the higher structural effect are still achieved, and the osteogenesis induction capability can be obviously improved.

Compared with the traditional collagen-based/levorotatory polylactic acid (PLLA)/Polycaprolactone (PCL)/gelatin film mixed with beta calcium phosphate (beta-TCP) or Hydroxyapatite (HA) and other guided tissue regeneration films (GTR) and induced bone regeneration films (GBR), the fibrous film provided by the application HAs better toughness, strength, suture property, orientation, high porosity, uniformity and high mineral content.

The application is based on a fiber membrane structure obtained by electrospinning/air spinning, is obtained by pressing under proper pressure, such as 1MPa to 10MPa, and maintains micron-scale pore characteristics. The mineralized collagen nano fiber doped with the doped active elements can meet the cell microenvironment for inhibiting tumor, resisting infection and regulating immunity, which is provided by material degradation in the bone repair process, through a plurality of active element doping methods, and the elements are uniformly distributed. The fibrous membrane has good lasting isolation efficacy, bone tissue regeneration promotion function, complete degradability and absorbability, doping of various active elements and the like as a GBR membrane. The content of the doped inorganic minerals is uniform and reaches up to 35 percent, and the composite material is prepared by an electrospinning/gas spinning one-step method. The mineral content, strength, toughness, porosity and the like of the product are far higher than similar products obtained by other processes. The modified fiber membrane is subjected to low-temperature 4 ℃ crosslinking for 0.5-8h by an EDC-NHS crosslinking agent, and the initial pressing thickness and the crosslinking time are adjusted to regulate the in vivo degradation rate to be matched with the new bone regeneration rate. Guiding high-quality and rapid reconstruction of bone tissues, inducing rapid repair and regeneration of bones, promoting vascularization and neurogenesis and accelerating the bone healing speed. In addition, the fiber membrane provided by the application does not shield X-rays.

The characteristics and preparation method of the mineralized collagen nanofiber doped with active elements in the fibrous membrane are shown as follows:

the application provides an active element doped mineralized collagen nanofiber, which consists of collagen fibers and needle-shaped hydroxyapatite crystals;

the calcium element in the crystal lattice of the hydroxyapatite is replaced by the cation containing the active element,

and/or the phosphate groups in the crystal lattice of the hydroxyapatite are replaced by anions containing the active element;

the active element comprises any one or more of silicon, selenium, magnesium, zinc, strontium, silver, iron (ferric iron) and copper (cupric copper);

the ratio of the stoichiometric number of the calcium element to the sum of the active element-containing cation and the phosphate radical and the active element-containing anion is (1.45:1) to (1.8: 1);

containing the molar ratio of the active element cation to the calcium ion (5 to 8) to (92 to 95); contains the molar ratio of the active element anion to the phosphate radical (5 to 7) to (93 to 95).

In another aspect, the present application provides a method for preparing the above mineralized collagen nanofibers doped with active elements, comprising: the mineralized collagen nano fiber doped with active elements and prepared by an in-situ co-assembly method is added with cations containing the active elements when calcium salt ions are added, and/or added with anions containing the active elements when phosphate ions are added.

(1) mixing the type I collagen sponge with a phosphoric acid solution to completely dissolve the type I collagen sponge to obtain a collagen template solution for later use;

(2) preparing a calcium salt solution, wherein the calcium salt solution and the collagen template solution have the same volume;

when the active element is incorporated in the form of cations, first the salt containing the cations is mixed with the calcium salt solution until complete dissolution, in which the collagen template solution is mixed;

(3) preparing a buffer solution;

(4) titrating the collagen template solution and the calcium salt solution in a buffer solution to obtain a mixed solution after titration is finished; during the titration, the pH value of the mixed solution is maintained between 8 and 10;

(5) carrying out high-speed centrifugation on the mixed solution obtained in the step (4), replacing the supernatant obtained by the centrifugation with ultrapure water with the same volume until the pH value of the measured supernatant is about 7-8, and stopping the high-speed centrifugation;

(6) removing water from the precipitate obtained in the step (5) to obtain mineralized collagen nanofibers doped with active elements;

alternatively, the preparation method consists of the above.

in one embodiment provided herein, the stoichiometric ratio of (calcium ions + active element cations)/(phosphate ions + active element anions) is from 1.65 to 1.8;

in one embodiment provided herein, the concentration of the phosphoric acid solution of step (1) is 0.5 to 2mol/L, and the volume ratio of the mass of the type I collagen sponge to the phosphoric acid solution is 0.1 to 10 g/L;

in one embodiment provided herein, the mixing time for the mixing of step (1) is 4 to 12 hours; optionally, the mixing temperature of the mixing is 35 ℃ to 38 ℃.

In one embodiment provided herein, the calcium salt solution in step (2) further comprises hydroxide, and the molar ratio of the sum of the calcium ions and the active element cations to the hydroxide is 2: 1.

In one embodiment provided herein, the buffer solution of step (3) is selected from any one or more of Tris-HCl and phosphate buffer solutions.

In one embodiment provided herein, the flow rate of the titration of step (4) is 400ml/h to 500 ml/h;

in one embodiment provided herein, after the titration in step (4) is completed, stirring for 12 to 36 hours; optionally, the reaction temperature is controlled at 30 ℃ to 37 ℃ after the titration is completed.

In one embodiment provided herein, the centrifugation of step (5) is performed at a speed of 3000 rpm to 17000 rpm.

In one embodiment provided herein, the removing of water in step (6) is low temperature lyophilization, comprising: drying the precipitate at 4-8 ℃, and then removing residual water by adopting freeze drying; optionally, the temperature of a cold well of the freeze dryer is between 2 and 4 ℃, the vacuum degree is between 10 and 30Pa, and the freeze drying duration is between 24 and 72 hours.

Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the application. Other advantages of the present application may be realized and attained by the invention in its aspects as described in the specification.

Drawings

The accompanying drawings are included to provide an understanding of the present disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the examples serve to explain the principles of the disclosure and not to limit the disclosure.

FIGS. 1a and 1b are the structural features of the fibrous membrane prepared in example 6;

FIGS. 2a and 2b are TEM images of mineralized collagen nanofibers doped with active elements in the fibrous membrane prepared in example 6 of the present application;

3a, 3b, 3c, 3d, 3e, 3f and 3g show the surface morphology and Si/Mg element distribution of the fiber membrane prepared in example 6;

FIG. 4 is a schematic diagram of the distribution of elements in the fiber membrane prepared in example 6;

fig. 5 is a stress-strain curve of the fiber membrane prepared in example 6 and the fiber membrane prepared in comparative example 2. As can be seen from the figure, the strength is significantly improved;

fig. 6 is a schematic diagram of a rat cranioplasty animal experiment using strontium-doped mineralized collagen nanofiber membranes provided in example 3 of the present application (the dimensions of each set of membranes are the same). In the figure, contrast is a control group, collagen membrane is a normal collagen membrane (a collagen fiber membrane prepared by electrospinning), BLB is a fiber membrane prepared in comparative example 2, Sr substistuted BLB is a fiber membrane prepared in example 3, 2W is two weeks, and 3W is three weeks; it can be seen from the figure that the fibrous structure prepared in example 1 can promote bone repair by mixing with other high molecular structure materials. The addition of Sr element can promote the bone repair process.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, embodiments of the present application are described in detail below. It should be noted that the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict.

In the examples provided herein, the raw materials were sourced as follows: sodium silicate (Na)₂SiO₃Analytically pure), anhydrous calcium chloride (CaCl)₂Analytically pure), sodium hydroxide (NaOH, analytically pure) and ammonia (NH)₄OH analytical pure) was purchased from pharmaceutical chemicals, ltd; strontium chloride hexahydrate (SrCl)₂6H2O, 99.5%), hexafluoroisopropanol (HFIP, 99.5%), carbodiimides (EDC), N-hydroxysuccinimide (NHS) from Shanghai Arlatin Biotech Ltd; collagen type I sponge (atelocollagen from oxtail skin, MW 300,000 oxtail, Hebei KagakuForest company); phosphoric acid (H)₃PO₄Analytically pure, Shanghai Tantake Technique, Inc.); Tris-HCl (C)₄H₁₁NO₃HCl, analytical pure, beijing baidi biotechnology limited).

Example 1

(1) Dissolving the type I collagen sponge in phosphoric acid ultrapure water solution, wherein the concentration of the phosphoric acid solution is 0.5mol/L, and the volume ratio of the mass of the type I collagen sponge to the phosphoric acid solution is 0.1g: 1L. And fully mixing the obtained mixture by using a magnetic stirrer for 6 hours to fully dissolve the added I-type collagen template, and marking as an anionic solution for later use. The temperature of the system was maintained between 35 ℃ and 38 ℃ during stirring.

(2) Preparing calcium chloride ultrapure water solution, adding CaCl according to the preset molar relative percentage of doped cation salt (Sr) of 9%₂The solution is stirred and dissolved uniformly (i.e. the molar ratio of strontium ions to calcium ions is 9: 91).

Preparing an ultrapure aqueous NaOH solution, and ensuring that the molar ratio of (calcium ions + strontium ions)/OH is 2. Adding NaOH solution into calcium-containing strontium cation solution, and recording as cation solution for standby.

The volume ratio of the prepared cation solution to the solution prepared in the step (1) is 1: 1. The total amount ensured that the stoichiometric ratio of (calcium ions + strontium ions)/(phosphate ions) was 1.65.

(3) Tris-HCl buffer solution with the solubility of 1mol/L is prepared, namely 0.2mol of Tris-HCl is weighed and dissolved in 200ml of ultrapure water to be used as reaction kettle liquid. And adjusting the pH value of the reaction kettle liquid to be between 8 and 10 by using 0.5mol/L ammonia water and HCl solution.

(4) Simultaneously titrating the anion solution and the cation solution in the reaction kettle liquid, controlling the flow rate to be maintained at 500ml/h, ensuring that the pH value of the reaction kettle liquid is maintained between 8 and 10 in the titration process, controlling the reaction temperature to be 37 ℃ after the titration is finished, and fully stirring for 16h in a dark place. And finishing titration of the anion solution and the cation solution with equal volumes.

(5) And (5) transferring the system in the step (4) into a centrifugal tube for high-speed centrifugation. And after each centrifugation, collecting the supernatant, measuring the pH value of the supernatant, adding ultrapure water with the same volume as the supernatant into the centrifuge tube, stirring the precipitate by using a glass rod to make the precipitate fully contact with the ultrapure water so as to clean the precipitate, and centrifuging again. This was repeated two to three times (3000 rpm) until the pH of the eluate was measured at around 7 to 8; finally 17000 r/min to obtain the cleaned precipitate.

(6) Drying the precipitate obtained after cleaning at 4 deg.C, and removing residual water by freeze dryer with freeze well temperature of 4 deg.C, vacuum degree of 30Pa, and freeze drying duration of 50 hr or more. And drying to obtain the strontium-doped mineralized collagen nanofiber.

Calcium ions in crystal lattices of hydroxyapatite in the strontium-doped mineralized collagen nanofibers prepared by the embodiment are replaced by Sr element.

As shown in table 1, the ratio of the sum of the stoichiometric numbers of calcium ions and strontium ions to the stoichiometric number of phosphate anions in the strontium-doped mineralized collagen nanofibers prepared in this example was 1.80: 1.

As shown in table 1, the molar ratio of strontium ions to the calcium ions in the strontium-doped mineralized collagen nanofibers prepared in this example was 6: 94.

The strontium-doped mineralized collagen nanofibers prepared by the present example had an average length of 250nm to 300nm and an average diameter of 5nm to 7 nm.

Table 1 elemental distribution of strontium-doped mineralized collagen nanofibers obtained in example 1

Element(s)	Mass percentage of	Atomic number percentage
			C	12.64	24.35
O	45.13	55.06
			Sr	1.62	0.79
P	15.06	7.36
			Ca	25.55	12.44
Total of	100.00	100

Example 2

This example differs from example 1 only in that:

comprising Mg cation doping and Si silicate anion doping.

In the embodiment, step (1) introduces silicon element: (1) doping with anion Salts (SiO) according to a predetermined value₃) Adding the phosphoric acid into the phosphoric acid ultrapure water solution according to the relative mol percentage of 6 percent, and uniformly stirring. And dissolving the I-type collagen sponge in the phosphoric acid ultrapure water solution, wherein the concentration of the phosphoric acid solution is 0.5mol/L, and the volume ratio of the mass of the I-type collagen sponge to the phosphoric acid solution is 0.1g: 1L. And fully mixing the obtained mixture by using a magnetic stirrer for 6 hours to fully dissolve the added I-type collagen template, and marking as an anionic solution for later use. Maintaining during the stirring processThe temperature of the system is between 35 ℃ and 38 ℃.

In the present embodiment, step (2) introduces magnesium ions: (2) preparing calcium chloride ultrapure water solution, adding CaCl according to the preset doped cation salt (Mg) molar relative percentage of 9%₂The solution is stirred and dissolved uniformly.

Preparing an ultrapure aqueous NaOH solution, and ensuring that the molar ratio of (calcium ions + magnesium ions)/OH is 2. Adding NaOH solution into calcium-containing magnesium cation solution, and recording as cation solution for standby.

As shown in table 2, the stoichiometric ratio of (calcium ion + magnesium ion)/(phosphate ion + silicate ion) in the mg-si doped mineralized collagen nanofibers prepared in this example was 1.45.

As shown in table 2, the molar ratio of silicate to phosphate in the mineralized collagen nanofibers doped with magnesium and silicon prepared in this example is 5: 95;

as shown in table 2, the molar ratio of magnesium ions to calcium ions in the mineralized collagen nanofibers doped with magnesium and silicon prepared in this example is 7.8: 92.2;

the mineralized collagen nanofibers doped with magnesium and silicon prepared by the embodiment have the average length of 250nm to 300nm and the average diameter of 5nm to 7 nm.

Table 2 distribution of element content in mg-si doped mineralized collagen nanofibers prepared in example 2

Comparative example 1

This comparative example differs from example 1 only in that:

this comparative example does not relate to Sr element, and other raw materials and preparation methods are the same as those of example 1.

Example 3

In this embodiment, the preparation of the fibrous membrane using the strontium-doped mineralized collagen nanofibers prepared in example 1 as a raw material comprises the following steps:

(1) the strontium-doped mineralized collagen nanofibers prepared in example 1 were taken, mixed with collagen and hexafluoroisopropanol, and continuously stirred vigorously for 28h to ensure thorough and uniform mixing. The mass ratio of the strontium doped mineralized collagen nanofibers, collagen and hexafluoroisopropanol solution prepared in example 1 was 1.0:0.6: 10.

(2) By electrospinning, a continuous fiber membrane having a certain thickness (the thickness may be 20 μm to 200 μm as needed) is formed. The electrospinning process can be referred to as "[ 1] Limonite, Huang Gong Ming. electrospinning of polymers [ J ]. macromolecules bulletin, 2006(05): 12-19" or as conventional in the art electrospinning process.

(3) And (3) drying the membrane prepared in the step (2) in a drying oven at 25 ℃ for 24 hours to ensure that residual trace hexafluoroisopropanol is removed. And then overlapping the multilayer films, and performing isostatic pressing at 3MPa for 5min by using a tablet press to obtain the modified fiber film with the multilayer structure.

(4) The method for preparing the EDC-NHS crosslinking solution by crosslinking a film with a certain thickness obtained under a certain pressure by using EDC-NHS comprises the following steps: EDS with the final concentration of 5mol/L and EDS with the final concentration of 10mol/LNHS are prepared and dissolved in a solvent, the solvent consists of 90% alcohol and 10% ultrapure water, the crosslinking is carried out at the low temperature of 4 ℃ for 2h, and the crosslinked membrane can not be layered.

(5) And cleaning the crosslinked modified fiber membrane by using alcohol to remove unreacted EDC, NHS and other impurity phases. And then the mixture is dried and stored in vacuum at 25 ℃, and finally the fibrous membrane of the strontium-doped mineralized collagen nanofiber is obtained.

In the fibrous membrane prepared in this example, the inorganic substance in the mineralized collagen nanofibers doped with strontium accounts for 24.5% by mass of the entire fibrous membrane.

The porosity of the fiber membrane prepared in the embodiment is 80%;

the density of the fiber film obtained in this example was 1.3g/cm³。

Example 4

This example 4 differs from example 3 only in that: the amount ratio of the strontium doped mineralized collagen nanofibers, collagen and hexafluoroisopropanol solution prepared in example 1 was different from that of example 3 and the spinning method was different.

In step (1) of this example, the mass ratio of the strontium-doped mineralized collagen nanofibers prepared in example 1 to the collagen and hexafluoroisopropanol solution was 1:0.6: 15.

In the present example, an air-jet spinning process is used, which may be referred to as "Song J, Li Z, Wu H. Blowspinning" a new choice for nanofibers [ J ]. ACS Applied Materials & Interfaces,2020,12(30): 33447-.

Other preparation processes, raw materials and dosage are the same as those of example 3.

In the fibrous membrane prepared in the embodiment, the mass of inorganic matters in the strontium-doped mineralized collagen nanofibers accounts for 26.7% of the total fibrous membrane;

the porosity of the fiber membrane prepared in the embodiment is 80%;

the density of the fiber film obtained in this example was 1.3g/cm³。

Example 5

This example 5 differs from example 3 only in that: the dosage ratio of the strontium doped mineralized collagen nanofibers, collagen and hexafluoroisopropanol solution prepared in example 1 was different from that of example 3.

In step (1) of this example, the mass ratio of the strontium-doped mineralized collagen nanofibers prepared in example 1 to the collagen and hexafluoroisopropanol solution was 1.5:0.8: 15.

The mass ratio of the strontium-doped mineralized collagen nanofibers to the biodegradable polymer in the fibrous membrane prepared by the embodiment is 30%;

the porosity of the fiber membrane prepared in the embodiment is 80%;

the density of the fiber film obtained in this example was 1.3g/cm³。

Example 6

This example 6 differs from example 3 only in that: the mineralized collagen nanofibers doped with magnesium and silicon prepared in example 2 were used. Other preparation processes, raw materials and dosage are the same as those of example 3.

In the fibrous membrane prepared by the embodiment, the mass of inorganic matters in the mineralized collagen nano fibers doped with magnesium and silicon accounts for 24.5 percent of the whole fibrous membrane;

the porosity of the fiber membrane prepared in the embodiment is 80%;

the density of the fiber film obtained in this example was 1.3g/cm³。

The molar ratio of the sum of the calcium element and the magnesium element to the sum of the phosphorus element and the silicon element in the fiber film is 1.64: 1.

Table 3 distribution of element content in mg-si doped mineralized collagen nanofiber membranes prepared in example 6

Element(s)	Mass percentage of	Atomic number percentage
			C	55.12	70.02
O	26.89	23.67
			Mg	0.78	0.27
Si	0.57	0.09
			Ca	10.39	3.65
P	6.25	2.3
			Total of	100	100

Comparative example 2

The comparative example uses the mineralized collagen nanofibers doped with inactive elements prepared in comparative example 1 to prepare a fibrous membrane, and the preparation process, other raw materials, the use amount and the like are the same as those of example 3.

The porosity of the fibrous membrane prepared in this comparative example was 80%;

the density of the fiber membrane prepared by the application example is 1.3g/cm³。

As can be seen from fig. 5 and 6, the mineralized collagen nanofibers doped with active elements provided by the present application can improve osteogenesis inducing ability of the material, provide a multi-element strengthening effect in the bone repair field, and can significantly increase toughness and strength of the material. In addition, the doping scheme of multiple active elements can also meet the requirement of providing a cell microenvironment for inhibiting tumor, resisting infection and immunoregulation when the material is degraded in the bone repair process; and adjusting the crystallinity of the mineralized collagen nanofibers and maintaining the fiber structure of 250nm to 300nm to the maximum extent. The crystallinity state is adjusted to meet the requirement that the degradation rate of the bone repair scaffold material in vivo is matched with the growth speed of new bone.

Although the embodiments disclosed in the present application are described above, the descriptions are only for the convenience of understanding the present application, and are not intended to limit the present application. 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 disclosure as defined by the appended claims.

Claims

1. A fibrous membrane comprising: mineralized collagen nanofibers doped with active elements;

optionally, the active element comprises any one or more of silicon, selenium, magnesium, zinc, strontium, silver, iron and copper;

optionally, the ratio of elemental calcium to the stoichiometric number containing the active element cation to the stoichiometric number of the sum of the phosphate and the active element-containing anion is from (1.45:1) to (1.80: 1);

optionally, a molar ratio (5 to 8) of active element cations to calcium ions (92 to 95); contains the molar ratio of the active element anion to the phosphate radical (5 to 7) to (93 to 95).

2. The fibrous membrane of claim 1, wherein the fibrous membrane further comprises a biodegradable polymer comprising any one or more of poly-epsilon-caprolactone, poly-L-lactic acid, poly-caprolactone-co-L-lactic acid, poly-lactic acid-co-glycolic acid, collagen, gelatin, silk fibroin, fibrinogen, cellulose, chitosan;

in the fibrous membrane, the mass of inorganic matters in the mineralized collagen nano fibers doped with the active elements accounts for 8-35% of the whole fibrous membrane;

optionally, the membrane has a porosity of 70% to 85%;

alternatively, the film has a density of 1.2g/cm³To 1.5g/cm³。

3. The fibrous membrane of claim 1, wherein the active element-doped mineralized collagen nanofibers have an average length of 250nm to 300 nm;

optionally, the active element-doped mineralized collagen nanofibers have an average diameter of 5nm to 7 nm.

4. The method for producing a fiber membrane according to claim 2 or 3, comprising the steps of:

(2) and (2) preparing the mixture obtained in the step (1) into a film through one or two of electrostatic spinning or gas spinning processes, and drying the obtained film to obtain the fiber film.

5. The method for producing a fiber membrane according to claim 4, wherein the mixing in step (1) is stirring for 28 to 50 hours;

optionally, the solvent in the step (1) is selected from any one or two of hexafluoroisopropanol and trifluoroethanol.

6. The method for preparing a fiber membrane according to claim 4, wherein the drying condition is drying at 25 ℃ to 35 ℃ for 24h to 36 h;

optionally, a plurality of layers of the fiber membranes prepared in the step (2) are superposed to prepare the fiber membranes with specific thickness.

7. The method of producing a fiber membrane according to any one of claims 4 to 6, wherein producing a fiber membrane of a specific thickness further comprises the steps of:

(a) stacking the fibrous membranes of any of claims 4 to 6 in layers, pressing at a pressure of 1 to 10MPa for 5 to 15 min;

8. The method for producing a fiber membrane according to claim 7, wherein the crosslinking agent is selected from any one or more of carbonyldiimine, N-hydroxysuccinimide, and genipin;

optionally, the solvent of the crosslinker solution comprises any one or more of water and ethanol; optionally, the temperature of the crosslinking is from 0 ℃ to 4 ℃; the crosslinking time is 0.5h to 8 h.

9. Use of a fibrous membrane according to any one of claims 1 to 3 in osteogenic repair.

10. Use of the fibrous membrane of any one of claims 1 to 3 in nerve conduits, artificial biomimetic periosteum, induced bone regeneration membrane and guided tissue regeneration membrane.