阅读说明：本技术 一种共组装人工骨膜及其制备方法 (Co-assembled artificial periosteum and preparation method thereof ) 是由 毛杰 周雄 杜莹莹 王一帆 王江林 于 2020-06-05 设计创作，主要内容包括：本发明提供了一种共组装人工骨膜及其制备方法,所述共组装人工骨膜包括：由胶原蛋白介导的掺硅羟基磷灰石溶胶和丝素蛋白共组装制备得到的丝素蛋白/掺硅羟基磷灰石/胶原蛋白共组装人工骨膜；和/或：由丝素蛋白介导的掺硅羟基磷灰石溶胶和胶原蛋白共组装制备得到的胶原蛋白/掺硅羟基磷灰石/丝素蛋白共组装人工骨膜。本发明通过胶原蛋白和丝素蛋白的共组装改变丝素蛋白二级结构,使得该共组装人工骨膜具备良好的力学性能,生物相容性以及有序可控的降解性能,且含硅羟基磷灰石具备较低的结晶度,可提高成骨细胞活性,刺激干细胞的成骨分化,且解决了含硅羟基磷灰石,丝素蛋白,胶原蛋白三者共混后不易成型难以共组装缺点。(The invention provides a co-assembled artificial periosteum and a preparation method thereof, wherein the co-assembled artificial periosteum comprises the following components in parts by weight: the silk fibroin/silicon-doped hydroxyapatite/collagen co-assembled artificial periosteum is prepared by co-assembling collagen-mediated silicon-doped hydroxyapatite sol and silk fibroin; and/or: the collagen/silicon-doped hydroxyapatite/silk fibroin co-assembled artificial periosteum is prepared by co-assembling silk fibroin mediated silicon-doped hydroxyapatite sol and collagen. The invention changes the secondary structure of silk fibroin by the co-assembly of collagen and silk fibroin, so that the co-assembled artificial periosteum has good mechanical property, biocompatibility and orderly controllable degradation property, and the silicon-containing hydroxyapatite has lower crystallinity, can improve the activity of osteoblasts and stimulate the osteogenic differentiation of stem cells, and solves the defect that the co-assembly of the silicon-containing hydroxyapatite, the silk fibroin and the collagen is difficult to form after being blended.)

1. A co-assembled artificial periosteum, comprising:

the collagen/silicon-doped hydroxyapatite/silk fibroin co-assembled artificial periosteum is prepared by co-assembling collagen-mediated silicon-doped hydroxyapatite sol and silk fibroin;

and/or:

the silk fibroin/silicon-doped hydroxyapatite/collagen protein co-assembled artificial periosteum is prepared by co-assembling silk fibroin-mediated silicon-doped hydroxyapatite sol and collagen protein.

2. A co-assembled artificial periosteum according to claim 1, wherein the composition of the co-assembled artificial periosteum comprises: silk fibroin, silicon-doped hydroxyapatite and collagen, wherein the mass ratio of the silk fibroin to the silicon-doped hydroxyapatite to the collagen is (1-4): 2-4: 1 to 4.

3. The co-assembled artificial periosteum according to claim 1 or 2, wherein the silicon mass in the silicon-doped hydroxyapatite accounts for 0.4-1.6% of the mass of the silicon-doped hydroxyapatite.

4. The co-assembled artificial periosteum according to claim 1, wherein the thickness of the co-assembled artificial periosteum is 200 um-1000 um, and the porosity is 50-80%.

5. A method of preparing a co-assembled artificial periosteum according to claims 1-4, comprising:

obtaining collagen-mediated silicon-doped hydroxyapatite sol and silk fibroin-mediated silicon-doped hydroxyapatite sol;

obtaining silk fibroin and preparing the silk fibroin into a silk fibroin solution; obtaining collagen and preparing the collagen into a collagen solution;

obtaining a co-assembly solution comprising: uniformly mixing the collagen-mediated silicon-doped hydroxyapatite sol and the silk fibroin solution to obtain a first co-assembly solution; uniformly mixing the silk fibroin-mediated silicon-doped hydroxyapatite sol and a collagen solution to obtain a second co-assembly solution;

freezing and freeze-drying the co-assembly solution to obtain a co-assembly membrane;

crosslinking the co-assembled membrane to obtain a crosslinked substance;

and (3) drying and compressing the cross-linked product in vacuum to obtain the co-assembled artificial periosteum.

6. The preparation method of claim 5, wherein the mass concentration of collagen in the collagen solution is 0.5-5%, and the mass concentration of silk fibroin in the silk fibroin solution is 10-30%.

7. The method of claim 5, wherein the co-assembly solution comprises:

mixing the silk fibroin-mediated silicon-doped hydroxyapatite sol with the collagen solution, adjusting the pH to 6.5-7.5, and stirring and uniformly mixing to obtain a first co-assembled solution;

or mixing the collagen-mediated silicon-doped hydroxyapatite sol with the silk fibroin solution, adjusting the pH to 6.5-7.5, and stirring and uniformly mixing to obtain a second co-assembly solution.

8. The method for preparing the membrane according to claim 5, wherein the co-assembly solution is frozen and freeze-dried to obtain a co-assembly membrane, and the method comprises the following steps:

freezing the co-assembly solution at-20 to-60 ℃ for 2 to 4 hours; then freeze-drying for 24-48 h under the conditions that the vacuum degree is 1-50 Pa and the temperature is-40-60 ℃.

9. The method according to claim 5, wherein the crosslinking the co-assembled film to obtain a crosslinked product comprises:

and (3) carrying out gas crosslinking on the co-assembled membrane for 2-8 h by taking glutaraldehyde as a crosslinking agent under the conditions that the temperature is 30-37 ℃ and the concentration of glutaraldehyde steam is 2-10%, so as to obtain a crosslinked substance.

10. The preparation method according to claim 5, wherein the conditions for stirring and mixing are as follows: the stirring speed is 50rpm to 200rpm, the temperature is 20 ℃ to 40 ℃, and the stirring time is 6h to 12 h; the vacuum drying conditions are as follows: the vacuum degree is 10Pa to 100Pa, the temperature is 20 ℃ to 40 ℃, and the time is 12h to 24 h; the compression conditions are as follows: the pressure is 10 Mpa-40 Mpa, and the time is 10 s-30 s.

Technical Field

The invention relates to the technical field of artificial periosteum, in particular to a co-assembled artificial periosteum and a preparation method thereof.

Background

Periosteum plays an important role in maintaining the integrity of the bone structure, and comprises an outer fibrous layer containing collagen, fibroblasts, elastin, blood vessels and neural networks, and an inner forming layer containing adult mesenchymal progenitor cells, osteoprogenitor cells, osteoblasts, capillaries, etc. which promote bone healing. The periosteum formed in the healing area plays an important role in bone repair and reconstruction in the bone repair process, the mesenchymal stem cells in the periosteum cambium are differentiated to promote osteogenesis and cortical bone formation, and the bone marrow stem cells are mainly differentiated into bone marrow bone tissues. At present, the bone repair is clinically carried out by adopting a periosteum induction mode, the effect is good, but the clinically usable artificial periosteum is few, the artificial periosteum is mainly a mineralized collagen membrane, and the mineralized collagen membrane has the problems of poor mechanical property and obviously changed mechanical property after being wetted, so that the risks of the collapse in the operation period, the excessive degradation rate and other adverse effects are caused. The existing artificial periosteum HAs higher crystallinity, and the artificial periosteum with low crystallinity is beneficial to the degradation of HA in vivo, can improve the activity of osteoblasts and stimulate the osteogenic differentiation of stem cells.

Therefore, how to prepare a co-assembled artificial periosteum with moderate degradation rate, good biocompatibility and low crystallinity becomes a technical problem to be solved urgently.

Disclosure of Invention

The invention aims to provide a co-assembled artificial periosteum and a preparation method thereof, the co-assembled artificial periosteum has good biocompatibility, excellent mechanical property and orderly and controllable degradation performance, and the silicon-containing hydroxyapatite has lower crystallinity, can improve the activity of osteoblasts and stimulate the osteogenic differentiation of stem cells.

In order to achieve the above objects, one object of the present invention is to provide a co-assembled artificial periosteum, which includes:

the collagen/silicon-doped hydroxyapatite/silk fibroin co-assembled artificial periosteum is prepared by co-assembling collagen-mediated silicon-doped hydroxyapatite sol and silk fibroin;

and/or:

the silk fibroin/silicon-doped hydroxyapatite/collagen protein co-assembled artificial periosteum is prepared by co-assembling silk fibroin-mediated silicon-doped hydroxyapatite sol and collagen protein.

Further, the composition of the co-assembled artificial periosteum comprises the following components: silk fibroin, silicon-doped hydroxyapatite and collagen, wherein the mass ratio of the silk fibroin to the silicon-doped hydroxyapatite to the collagen is (1-4): 2-4: 1 to 4.

Further, the mass of silicon in the silicon-doped hydroxyapatite accounts for 0.4-1.6% of the mass of the silicon-doped hydroxyapatite.

Further, the thickness of the co-assembled artificial periosteum is 200 um-1000 um, and the porosity is 50% -80%.

The invention also aims to provide a preparation method of the co-assembled artificial periosteum, which comprises the following steps:

obtaining collagen-mediated silicon-doped hydroxyapatite sol and silk fibroin-mediated silicon-doped hydroxyapatite sol;

obtaining silk fibroin and preparing the silk fibroin into a silk fibroin solution; obtaining collagen and preparing the collagen into a collagen solution;

obtaining a co-assembly solution comprising: uniformly mixing the collagen-mediated silicon-doped hydroxyapatite sol and the silk fibroin solution to obtain a first co-assembly solution; uniformly mixing the silk fibroin-mediated silicon-doped hydroxyapatite sol and a collagen solution to obtain a second co-assembly solution;

freezing and freeze-drying the co-assembly solution to obtain a co-assembly membrane;

crosslinking the co-assembled membrane to obtain a crosslinked substance;

and (3) drying and compressing the cross-linked product in vacuum to obtain the co-assembled artificial periosteum.

Further, the mass concentration of collagen in the collagen solution is 0.5-10%, and the mass concentration of silk fibroin in the silk fibroin solution is 10-20%.

Further, the co-assembly solution comprises:

mixing the silk fibroin-mediated silicon-doped hydroxyapatite sol with the collagen solution, adjusting the pH to 6.5-7.5, and stirring and uniformly mixing to obtain a first co-assembled solution;

or mixing the collagen-mediated silicon-doped hydroxyapatite sol with the silk fibroin solution, adjusting the pH to 6.5-7.5, and stirring and uniformly mixing to obtain a second co-assembly solution.

Further, the obtaining of the silk fibroin-mediated silica-doped hydroxyapatite sol comprises:

obtaining silk fibroin solution, phosphorus source solution, calcium source solution and silicon source;

mixing the silk fibroin solution with a phosphorus source solution to obtain a silk fibroin-phosphorus-containing solution;

mixing the calcium source solution with the silicon source to obtain a calcium-silicon solution;

and dripping the silk fibroin-phosphorus-containing solution into the calcium-silicon solution, adjusting the pH value to 7.4, continuously reacting for a period of time, stopping stirring, standing, collecting precipitates, washing and centrifuging to obtain the silk fibroin-mediated silicon-doped hydroxyapatite sol.

Further, the obtaining of the collagen-mediated silica-doped hydroxyapatite sol comprises the following steps:

obtaining collagen solution, calcium source solution, silicon source and phosphorus source solution;

mixing the collagen solution with the calcium source solution to obtain a collagen-calcium containing solution;

mixing the collagen-calcium-containing solution with a silicon source to obtain a collagen-calcium-silicon solution;

and dropwise adding the phosphorus source solution into the collagen-calcium-silicon solution, adjusting the pH value to 7.4, continuously reacting for a period of time, stopping stirring, standing, collecting precipitate, washing, and centrifuging to obtain the collagen-mediated silicon-doped hydroxyapatite sol.

Further, the co-assembly solution is frozen and freeze-dried to obtain a co-assembly membrane, which comprises:

freezing the co-assembly solution at-20 to-60 ℃ for 2 to 4 hours; then freeze-drying for 24-48 h under the conditions that the vacuum degree is 1-50 Pa and the temperature is-40-60 ℃.

Further, the cross-linking the co-assembled membrane to obtain a cross-linked product includes:

and (3) carrying out gas crosslinking on the co-assembled membrane for 2-8 h by taking glutaraldehyde as a crosslinking agent under the conditions that the temperature is 30-37 ℃ and the concentration of glutaraldehyde steam is 2-10%, so as to obtain a crosslinked substance.

Further, the conditions of stirring and uniformly mixing are as follows: the stirring speed is 50rpm to 200rpm, the temperature is 20 ℃ to 40 ℃, and the stirring time is 6h to 12 h; the vacuum drying conditions are as follows: the vacuum degree is 10Pa to 100Pa, the temperature is 20 ℃ to 40 ℃, and the time is 12h to 24 h; the compression conditions are as follows: the pressure is 10 Mpa-40 Mpa, and the time is 10 s-30 s.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

1. the invention provides a co-assembled artificial periosteum, which comprises:

the crystallinity of the collagen/silicon-doped hydroxyapatite/silk fibroin Col-SiHAp-SF prepared by adding the silk fibroin SF on the basis of the collagen-mediated silicon-doped hydroxyapatite sol Col-SiHAp and assembling is lower than that of the collagen-mediated silicon-doped hydroxyapatite Col-SiHAp;

the silk fibroin/silicon-doped hydroxyapatite/collagen co-assembled artificial periosteum SF-SiHAp-Col prepared by adding collagen Col on the basis of the silk fibroin-mediated silicon-doped hydroxyapatite sol SF-SiHAp is lower in crystallinity than the silk fibroin-mediated silicon-doped hydroxyapatite sol SF-SiHAp;

the low crystallinity is beneficial to the degradation of HA in vivo, can improve the activity of osteoblasts and stimulate the osteogenic differentiation of stem cells.

2. The invention provides a co-assembled artificial periosteum, which is a silk fibroin/silicon-doped hydroxyapatite/collagen co-assembled artificial periosteum, wherein the secondary structure of the collagen-doped hydroxyapatite is changed by adding silk fibroin to the collagen-mediated silicon-doped hydroxyapatite for co-assembly, the mechanical property, the degradation time and the like of the collagen-silicon-doped hydroxyapatite are improved, and the co-assembled artificial periosteum has an ordered porous space structure and is a preferred bone tissue defect repairing material; or collagen is added into the fibroin-mediated silicon-doped hydroxyapatite for co-assembly to change the secondary structure of the fibroin-mediated silicon-doped hydroxyapatite, improve the mechanical property of the fibroin-silicon-doped hydroxyapatite, degrade the time and the like, and have an ordered porous space structure.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to derive other drawings without creative efforts.

FIG. 1 is a pictorial view of a co-assembled artificial periosteum of the present invention;

FIG. 2 is a SEM image of a co-assembled artificial periosteum (SF-SiHAp-Col or Col-SiHAp-SF) of the present invention;

FIG. 3 shows SF-SiHAp bone powder prepared in comparative example 2;

FIG. 4 shows Col-SiHAp bone powder prepared in comparative example 1;

FIG. 5 shows the periosteum of SF-SiHAp-Col prepared in example 2;

FIG. 6 shows the Col-SiHAp-SF periosteum prepared in example 1;

figure 7 is an XRD pattern of periosteum of example 1, example 2, comparative example 1, comparative example 2, and silicon-doped hydroxyapatite.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

In order to solve the technical problems, the embodiment of the invention provides the following general ideas:

to achieve the above object, the present embodiment provides a co-assembled artificial periosteum, including:

the silk fibroin/silicon-doped hydroxyapatite/collagen co-assembled artificial periosteum is prepared by co-assembling collagen-mediated silicon-doped hydroxyapatite sol and silk fibroin;

and/or:

the collagen/silicon-doped hydroxyapatite/silk fibroin co-assembled artificial periosteum is prepared by co-assembling silk fibroin mediated silicon-doped hydroxyapatite sol and collagen.

Namely, the co-assembled artificial periosteum is sequentially connected with two types of a silk fibroin/silicon-doped hydroxyapatite/collagen co-assembled artificial periosteum (SF-SiHAp-Col) and a collagen/silicon-doped hydroxyapatite/silk fibroin co-assembled artificial periosteum (Col-SiHAp-SF). The Col-SiHAp-SF (Col-SiHAp-SF) in the Col-SiHAp-SF co-assembled artificial periosteum has a stable protein secondary structure. FIG. 5 shows the periosteum of SF-SiHAp-Col prepared in example 2; FIG. 6 shows the Col-SiHAp-SF periosteum prepared in example 1; the basic characteristics prove that the film forming effect of Col-SiHAp-SF is better than that of SF-SiHAp-Col.

The applicant finds that compared with SF-SiHAp-Col co-assembled artificial periosteum or Col-SiHAp-SF co-assembled artificial periosteum, compared with silk fibroin mediated silicon-doped hydroxyapatite SF-SiHAp or collagen mediated silicon-doped hydroxyapatite Col-SiHAp, the crystallinity of the hydroxyapatite is greatly reduced, the crystallinity of the hydroxyapatite is reduced, the hydroxyapatite is beneficial to being degraded in vivo after being implanted, osteoblast activity can be promoted, and osteogenic differentiation of stem cells is stimulated.

The co-assembled artificial periosteum is a silk fibroin/silicon-doped hydroxyapatite/collagen co-assembled artificial periosteum, the secondary structure of the collagen-doped hydroxyapatite is changed by adding silk fibroin into the collagen-mediated silicon-doped hydroxyapatite for co-assembly, the mechanical property, the degradation time and the like of the collagen-doped hydroxyapatite are improved, and the collagen-doped hydroxyapatite has an ordered porous space structure and is a preferred bone tissue defect repairing material; or collagen is added into the fibroin-mediated silicon-doped hydroxyapatite for co-assembly to change the secondary structure of the fibroin-mediated silicon-doped hydroxyapatite, improve the mechanical property of the fibroin-silicon-doped hydroxyapatite, degrade the time and the like, and have an ordered porous space structure.

(1) The artificial periosteum has a structure similar to a natural periosteum, has good mechanical property and biocompatibility, can be assembled together to form a stable protein secondary structure and is fixed in a cross-linking way by Col-SF, so that the mechanical property can be effectively improved, the artificial periosteum can be prevented from being degraded too fast, and the artificial periosteum can realize ordered and controllable degradation regulation; the bionic composition and structure of the human periosteum and the addition of active silicon elements cover the defect area to improve the integrity, improve the bone bioactivity of the bone defect area and promote the repair of the periosteum of the bone defect area; the cross-linking process improves the degradation rate and the mechanical property, can also effectively reduce the immunogenicity and the virus removal rate of animal-derived raw materials, and can improve the use safety of animal-derived implanted products; (2) the silicon-containing hydroxyapatite HAs lower crystallinity, is beneficial to the degradation of the silicon-containing hydroxyapatite HA in vivo, can improve the activity of osteoblasts and stimulate the osteogenic differentiation of stem cells; (3) the method for preparing collagen-silicon-doped hydroxyapatite sol or silk fibroin-silicon-doped hydroxyapatite sol overcomes the defects that the mixture of silicon-containing hydroxyapatite, silk fibroin and collagen is not easy to form, difficult to assemble and the like.

After sintering, the invention finds that the SF-SiHAp-Col (Col-SiHAp-SF) periosteum Si-HAp accounts for 49 percent and 48 percent, and accords with the expected addition value. Silicon can promote the synthesis of type I collagen and the differentiation of osteoblasts, and can also promote the bone repair of bone injury parts; small amounts of soluble silicon are required for bone mineralization in organisms; si deficiency may lead to the formation of abnormal bone; in bone and cartilage, Si may play an important role in the metabolism of connective tissue; pure hydroxyapatite is less reactive with natural bone tissue and bone forming cells, resulting in a slower rate of osteointegration and may confer sufficient activity upon the addition of silicon.

Preferably, the silicon content in the silicon-doped hydroxyapatite is 0.4-1.6%; the mass of silicon in the silicon-doped hydroxyapatite accounts for 0.4-1.6% of the mass of the silicon-doped hydroxyapatite, and the silicon is considered as SiO based on the unit cell parameters and the biological activity₄ ^4-Formal substitution of PO in hydroxyapatite₄ ^3-，SiO₄ ^4-Tetrahedral structure greater than PO₄ ³The space structure has certain influence on the hydroxyapatite.

Preferably, the mass ratio of the silk fibroin, the silicon-doped hydroxyapatite and the collagen is 1-4: 2-4: 1-4; and (2) performing thermal sintering on the obtained artificial periosteum, decomposing collagen or silk fibroin at 800 ℃, and controlling the mass ratio of the silk fibroin, the silicon-doped hydroxyapatite and the collagen to be 1-4: 2-4: 1-4, the forming of the membrane is ensured, the content of hydroxyapatite can be maximized, the use of silk fibroin and collagen is reduced, and the cost is reduced. The silk fibroin proportion is increased, and the periosteum is degraded slowly.

The preferred ratio ranges are: the mass ratio of the silk fibroin, the silicon-doped hydroxyapatite to the collagen is 1-2: 3-4: 2-4; the degradation rate is more moderate, and the degradation rate is 55 to 72 percent after one week.

Preferably, the thickness of the co-assembled artificial periosteum is 200 um-1000 um, and the porosity is 50% -80%.

The thickness of the co-assembled artificial periosteum is 200-1000 um, and the thickness is favorable for implantation and is matched with the thickness of the periosteum of a human body; the porosity of 50-80% is favorable for inputting nutrient substances in blood vessels.

In the embodiment of the invention, the collagen is ateloceptide I type collagen, and the silk fibroin is mulberry Silk Fibroin (SF).

The embodiment of the invention also provides a preparation method of the co-assembled artificial periosteum, which comprises the following steps:

step 1, obtaining a co-assembly solution, which specifically comprises two schemes:

the first scheme is as follows:

obtaining silk fibroin solution, phosphorus source solution, calcium source solution and silicon source;

mixing the silk fibroin solution with a phosphorus source solution to obtain a silk fibroin-phosphorus-containing solution;

mixing the calcium source solution with the silicon source to obtain a calcium-silicon solution;

dripping the silk fibroin-phosphorus-containing solution into the calcium-silicon solution, adjusting the pH value to 7.4, continuously reacting for a period of time, stopping stirring, standing, collecting precipitates, washing and centrifuging to obtain silk fibroin-mediated silicon-doped hydroxyapatite sol (SF-SiHAp sol for short);

and stirring and mixing the silk fibroin mediated silicon-doped hydroxyapatite sol and the collagen solution, and adjusting the pH to 6.5-7.5 to obtain a co-assembly solution (SF-SiHAp-Col co-assembly solution for short).

Scheme II: obtaining collagen solution, calcium source solution, silicon source and phosphorus source solution;

mixing the collagen solution with the calcium source solution to obtain a collagen-calcium containing solution;

mixing the collagen-calcium-containing solution with a silicon source to obtain a collagen-calcium-silicon solution;

dropwise adding the phosphorus source solution into the collagen-calcium-silicon solution, adjusting the pH value to 7.4, continuously reacting for a period of time, stopping stirring, standing, collecting precipitate, washing, and centrifuging to obtain collagen-mediated silicon-doped hydroxyapatite sol (Col-SiHAp sol for short);

and stirring and mixing the collagen-mediated silicon-doped hydroxyapatite sol and the silk fibroin solution, and adjusting the pH to 6.5-7.5 to obtain a co-assembly solution (Col-SiHAp-SF co-assembly solution for short).

How to co-assemble the collagen-mediated silicon-doped hydroxyapatite sol and the silk fibroin and how to co-assemble the silk fibroin-mediated silicon-doped hydroxyapatite sol and the collagen also become a big difficulty of the invention.

If only the siliceous hydroxyapatite, the silk fibroin and the collagen are simply blended, the molding is not easy and the co-assembly is difficult. The current silk fibroin research adopts a large amount of organic solvents to promote the molding of the silk fibroin and is difficult to assemble into an ordered spatial structure.

The inventor finds that firstly, the collagen-mediated silicon-doped hydroxyapatite sol or the silk fibroin-mediated silicon-doped hydroxyapatite sol is prepared; stirring and mixing the collagen-mediated silicon-doped hydroxyapatite sol and the silk fibroin solution, or stirring and mixing the silk fibroin-mediated silicon-doped hydroxyapatite sol and the collagen solution, adjusting the pH value to 6.5-7.5 to obtain a co-assembly solution, and assembling through the subsequent steps 2-4 to obtain a successfully-assembled artificial periosteum;

the pH value is controlled to be 6.5-7.5 in the steps so as to realize the co-assembly of the collagen-mediated silicon-doped hydroxyapatite sol and the collagen, wherein the collagen-mediated silicon-doped hydroxyapatite sol is decomposed if the pH value is less than 6.5, and the collagen is denatured if the pH value is more than 7.5.

Step 2, freezing the co-assembly solution at-20 to-60 ℃ for 2 to 4 hours; then freeze-drying for 24-48 h under the conditions that the temperature is-40 to-60 ℃ and the vacuum degree is 1Pa to 50 Pa;

under the condition, the final forming of the periosteum is facilitated, when the temperature is higher than-20 ℃, the freeze-dried sample can cause cracks after the periosteum is finally formed, and the condition lower than-60 ℃ is difficult to meet.

The freeze drying is carried out for 24 h-48 h under the conditions of-40 ℃ to-60 ℃ and the vacuum degree of 1 Pa-50 Pa, which is a common freeze drying and dewatering condition.

Step 3, crosslinking the co-assembled membrane in a gaseous state for 2 to 8 hours at the temperature of between 30 and 39 ℃ and under the condition that the concentration of glutaraldehyde steam is between 2 and 10 percent by taking glutaraldehyde as a crosslinking agent to obtain a crosslinked substance;

the method has the advantages of selecting gaseous crosslinking and glutaraldehyde as a crosslinking agent, and selecting the reason that the temperature is 37-52 ℃: wherein the gaseous cross-linking can (1) reduce the possible harm of glutaraldehyde to the operator; (2) reducing the residue of glutaraldehyde in periosteum after crosslinking; (3) glutaraldehyde is selected because glutaraldehyde is one of the safest cross-linking agents at present; the temperature is 30-39 ℃, which is favorable for the glutaraldehyde to form gas state and can ensure the collagen invariance (the collagen denaturation temperature is 40 ℃).

Step 4, drying and compressing the cross-linked product in vacuum to obtain the co-assembled artificial periosteum, wherein the vacuum drying conditions are as follows: the temperature is 20-40 ℃, the vacuum degree is 10-100 Pa, and the time is 12-24 h; the compression conditions were: the pressure is 10-40 Mpa and the time is 10-30 s.

The vacuum drying condition is selected because the condition can remove residual glutaraldehyde and ensure the invariance of collagen, and the compression condition is selected through experiments to find that the film thickness can meet the requirements under the condition.

A co-assembled artificial periosteum and a method for preparing the same according to the present application will be described in detail with reference to examples and experimental data.