CN111569149B

CN111569149B - Co-assembled artificial periosteum and preparation method thereof

Info

Publication number: CN111569149B
Application number: CN202010503883.3A
Authority: CN
Inventors: 毛杰; 周雄; 杜莹莹; 王一帆; 王江林
Original assignee: Asia Biomaterials Wuhan Co ltd
Current assignee: Asia Biomaterials Wuhan Co ltd
Priority date: 2020-06-05
Filing date: 2020-06-05
Publication date: 2022-06-17
Anticipated expiration: 2040-06-05
Also published as: CN111569149A

Abstract

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

Description

Co-assembled artificial periosteum and preparation method thereof

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 components of the co-assembled artificial periosteum comprise: 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-assembled 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-assembly 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 method for obtaining the silk fibroin-mediated silicon-doped hydroxyapatite sol comprises the following steps:

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 50 rpm-200 rpm, the temperature is 20 ℃ to 40 ℃, and the stirring time is 6 h-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-silicon-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-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, which includes:

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 has low reactivity with natural bone tissue and bone forming cells, resulting in a slow rate of osteointegration and sufficient activity to be imparted upon the addition of silicon.

Preferably, the silicon content in the silicon-doped hydroxyapatite is 0.4-1.6%; the silicon mass in the silicon-doped hydroxyapatite accounts for 0.4-1.6% of the mass of the silicon-doped hydroxyapatite and is based on a unit cell parameterConsidering the number and biological activity, Si is SiO₄ ^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, in order to ensure the molding of the membrane, simultaneously, 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:

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;

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 potential 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.

Example 1Col-SiHAp-SF Co-Assembly Artificial periosteum

Step 1, weighing 1g of ateloceptide I type collagen, completely dissolving the ateloceptide I type collagen in 0.2L0.5mol/L acetic acid solution, and carrying out water bath at 37 ℃; adding 0.11g of silicon orthoacetate solid, stirring and hydrolyzing for 4 h; weighing 3.52g of calcium nitrate tetrahydrate, dissolving in the Col-silicon hydrolysis solution, mixing for 3h at the stirring speed of 100rpm, and adjusting the pH of the solution to be about 7.4 to prepare a collagen-calcium-silicon solution (Col-Ca-Si); weighing 1.12g of diammonium hydrogen phosphate, dissolving in 0.2L of purified water, slowly dropwise adding a diammonium hydrogen phosphate solution into the Col-Ca-Si solution, keeping the pH of the system to be about 7.4, controlling the temperature of the reaction system at 37 ℃ through a water bath, controlling the drop acceleration of a monoammonium phosphate collagen solution to be 2-3 ml/min, and stirring at the speed of 100 rpm; continuously stirring for 3h after the addition of the phosphonium salt is finished, keeping the pH value of the system to be about 7.4, and standing and aging for 4h at 37 ℃; after aging, the supernatant is poured off, the precipitate is washed to be neutral, and the Col-SiHAp sol is obtained by centrifugation at 4000 rpm.

Step 2, preparing 10 mass percent silk fibroin Solution (SF) from 1g of silk fibroin;

step 3, mixing Col-SiHAp sol with 10ml of 10% (wt) SF solution, adjusting the pH to 7, mixing at 50rpm for 3 hours, and standing for 12 hours;

step 4, pouring the blended and standing Col-SiHAp-SF solution into a square plate, wherein the thickness of the liquid is about 0.8cm, and pre-freezing the liquid in an ultra-low temperature refrigerator at minus 40 ℃ for 2 hours;

step 5, carrying out freeze drying on the pre-frozen Col-SiHAp-SF, wherein the temperature of a cold trap is-40 to-60 ℃, the vacuum degree is less than 50Pa, and the freeze drying time is 36 hours;

step 6, carrying out gaseous crosslinking on the freeze-dried Col-SiHAp-SF, wherein a crosslinking agent is a glutaraldehyde solution with the concentration of 4%, and the crosslinking conditions are as follows: crosslinking for 2 hours at 37 ℃;

step 7, vacuum drying the cross-linked Col-SiHAp-SF co-assembled artificial periosteum to remove residual glutaraldehyde, wherein the vacuum drying conditions are as follows: 30-50 Pa at 37 ℃ and drying time of 48 h. Compressing for 20s under 20MPa to obtain Col-SiHAp-SF co-assembled artificial periosteum with the thickness of about 200 um.

Example 2SF-SiHAp-Col Co-Assembly Artificial periosteum

Step 1, weighing 1g of fibroin, completely dissolving in 18mL9.3mol/L lithium bromide solution, dialyzing, and centrifuging; taking a small amount of supernatant, drying and calculating the concentration to be 8%; taking 12.5g of the SF solution, and adding 0.1L of water for dilution; weighing 1.12g of diammonium phosphate to dissolve in the SF solution to prepare a phosphorus-fibroin (P-SF) solution; weighing 0.11g of silicon orthoacetate solid, stirring and hydrolyzing in 0.2L of purified water for 3-5 h, adding 3.52g of calcium nitrate tetrahydrate, continuously stirring for 3h, and adjusting the pH of the solution to about 7.4 to prepare a calcium-silicon solution (Ca-Si); controlling the temperature of the P-SF solution at 37 ℃ through a water bath, stirring at 100rpm, dropwise adding the Ca-Si solution into the P-SF solution at a speed of 2-3 ml/min, and keeping the pH value of the system to be about 7.4; after the dropwise addition, continuously stirring for 3h, keeping the pH of the system to be about 7.4, and standing and aging for 4h at 37 ℃; after the aging is finished, pouring out the supernatant, washing the precipitate to be neutral, and centrifuging at 4000rpm to obtain SF-SiHAp sol;

step 2, preparing a collagen solution (Col) with the mass concentration of 1% from 1g of collagen, and adjusting the pH value to 7;

step 3, mixing the SF-SiHAp sol with 100ml of 1% (wt) Col solution, adjusting the pH to 7, mixing at 50rpm for 3 hours, and standing for 12 hours;

step 4, pouring the blended and standing SF-SiHAp-Col solution into a square plate, wherein the thickness of the liquid is about 0.8cm, and pre-freezing the liquid in an ultra-low temperature refrigerator at minus 40 ℃ for 2 hours;

step 5, carrying out freeze drying on the pre-frozen SF-SiHAp-Col at a cold trap temperature of-40 to-60 ℃, a vacuum degree of less than 50Pa and freeze-drying time of 36 h;

step 6, carrying out gaseous crosslinking on the freeze-dried SF-SiHAp-Col, wherein a crosslinking agent is a glutaraldehyde solution with the concentration of 10%, and the crosslinking conditions are as follows: crosslinking for 4 hours at 37 ℃;

step 7, carrying out vacuum drying on the cross-linked SF-SiHAp-Col co-assembled artificial periosteum to remove residual glutaraldehyde, wherein the vacuum drying conditions are as follows: and (4) drying at 37 ℃ under 30-50 Pa for 48 h. Compressing for 20s under 20MPa to obtain Col-SiHAp-SF co-assembled artificial periosteum with the thickness of about 200 um.

Example 3 to example 4

In this example, the steps are the same as in example 1 except that the mass ratio of silk fibroin, silica-doped hydroxyapatite, and collagen is different, and are specifically shown in table 1.

Comparative example 1Col-SiHAp bone powder

Weighing 1g of atelocladin I type collagen, completely dissolving the atelocladin I type collagen in 0.2L0.5mol/L acetic acid solution, and carrying out water bath at 37 ℃; adding 0.11g of silicon orthoacetate solid, stirring and hydrolyzing for 4 h; weighing 3.52g of calcium nitrate tetrahydrate, dissolving in the Col-silicon hydrolysis solution, mixing for 3h at the stirring speed of 100rpm, and adjusting the pH of the solution to be about 7.4 to prepare a collagen-calcium-silicon solution (Col-Ca-Si); weighing 1.12g of diammonium hydrogen phosphate, dissolving in 0.2L of purified water, slowly dropwise adding a diammonium hydrogen phosphate solution into the Col-Ca-Si solution, keeping the pH of the system to be about 7.4, controlling the temperature of the reaction system at 37 ℃ through a water bath, controlling the drop acceleration of a monoammonium phosphate collagen solution to be 2-3 ml/min, and stirring at the speed of 100 rpm; continuously stirring for 3h after the addition of the phosphonium salt is finished, keeping the pH value of the system to be about 7.4, and standing and aging for 4h at 37 ℃; after aging, the supernatant is poured off, the precipitate is washed to be neutral, and the Col-SiHAp sol is obtained by centrifugation at 4000 rpm. Then freeze-drying the obtained powder to obtain Col-SiHAp powder.

Comparative example 2SF-SiHAp bone powder

Weighing 2g of silk fibroin, completely dissolving the silk fibroin in 18mL9.3mol/L lithium bromide solution, dialyzing and centrifuging; taking a small amount of supernatant, drying and calculating the concentration to be 8%; taking 12.5g of the SF solution, and adding 0.1L of water for dilution; weighing 1.12g of diammonium phosphate to dissolve in the SF solution to prepare a phosphorus-fibroin (P-SF) solution; weighing 0.11g of silicon orthoacetate solid, stirring and hydrolyzing in 0.2L of purified water for 3-5 h, adding 3.52g of calcium nitrate tetrahydrate, continuously stirring for 3h, and adjusting the pH of the solution to about 7.4 to prepare a calcium-silicon solution (Ca-Si); controlling the temperature of the P-SF solution at 37 ℃ through a water bath, stirring at 100rpm, dropwise adding the Ca-Si solution into the P-SF solution at a speed of 2-3 ml/min, and keeping the pH value of the system to be about 7.4; after the dropwise addition, continuously stirring for 3h, keeping the pH of the system to be about 7.4, and standing and aging for 4h at 37 ℃; after the aging is finished, pouring out the supernatant, washing the precipitate to be neutral, and centrifuging at 4000rpm to obtain SF-SiHAp sol; and freeze-drying to obtain SF-SiHAp powder.

Comparative examples 3 to 4

The comparative examples 3 to 4 are the same as example 1 except that the mass ratio of silk fibroin, silica-doped hydroxyapatite and collagen is different, and are specifically shown in table 1.

Comparative example 5

The comparative example is a commercial sized, mineralized periosteum.

Comparative example 6

The comparative example directly blends the silicon-containing hydroxyapatite, the silk fibroin and the collagen, and as a result, the components cannot be assembled together and are difficult to form.

Test example 1

The detection of SEM, FT-IR and XRD proves that the SF-SiHAp-Col (Col-SiHAp-SF) periosteum in the embodiment 1 and 2 has a micropore structure, a protein stable structure-beta-sheet exists, and 1641cm of infrared spectrum^-1，1531cm^-1Two peaks (alpha-helix, random coil infrared signature) were converted to 1621cm^-11517cm-1 (. beta. -fold infrared characteristic peak), and all have a silicate characteristic absorption peak (875 cm)^-1) Description of SiO₄ ^-Entering into hydroxyapatite crystal lattice; the XRD shows that the crystallinity of the hydroxyapatite is lower due to the influence of the collagen and the silk fibroin; after sintering, the content of SF-SiHAp-Col (Col-SiHAp-SF) periosteum Si-HAp is 49 percent and 48 percent, which accords with the expected addition value. Specifically, the method comprises the following steps:

fig. 1 is a diagram of a co-assembled artificial periosteum in example 1/example 2 of the present invention, and it can be seen from fig. 1 that the co-assembled artificial periosteum is successfully prepared by the present invention.

Fig. 2 is an SEM image of periosteum in example 1/example 2 of the present invention, and it can be seen from fig. 2 that the periosteum surface is smooth and has a porous structure, and the porosity is 70%.

FIG. 3 is a view showing the SF-SiHAp periosteum prepared in comparative example 2; FIG. 4 is a Col-SiHAp periosteum 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; it was found that the Col-SiHAp-SF periosteum of example 1 and the SF-SiHAp-Col periosteum of example 2 had a microporous structure, and both had a protein stabilizing structure- -beta-sheet, and they were found to have a size of 1641cm in the infrared spectrum^-1，1531cm^-1Two peaks (alpha-helix, random coil infrared signature) were converted to 1621cm^-1，1517cm^-1(beta-sheet infrared characteristic peak) and all have a silicate characteristic absorption peak (875 cm)^-1) Description of SiO₄ ^-Entering into hydroxyapatite crystal lattices, which shows that the co-assembled artificial periosteum is successfully prepared in the embodiments 1-2 of the invention.

FIG. 7 is an XRD pattern of periosteum of example 1, example 2, comparative example 1 and comparative example 2 and hydroxyapatite doped with silicon, and it is found by XRD characterization that the periosteum of examples 1-2 of the present invention has lower crystallinity compared to the periosteum of comparative example 1-2 (generally, the lower the back in the XRD pattern, the higher the intensity of diffraction peak, the sharper the diffraction peak, i.e. the smaller the half height width, the better the crystallinity); and 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.

After sintering, Si-HAp in the Col-SiHAp-SF periosteum of example 1 and the SF-SiHAp-Col periosteum of example 2 was found to account for 49%, 48%, and was in accordance with the expected addition values.

Test example 2 influence of raw material quality ratio on Co-assembled Artificial periosteum

The mass ratio of silk fibroin, silicon-doped hydroxyapatite and collagen is set in different ranges, the rest steps are the same as the example 1, the degradation performance of the artificial periosteum assembled by the mass ratio of the silk fibroin, the silicon-doped hydroxyapatite and the collagen is explored, and the degradation performance is compared with that of the existing glue-coated original mineralized periosteum product on the market.

TABLE 1

Group of	The mass ratio of silk fibroin, silicon-doped hydroxyapatite and collagen	Speed of periosteal degradation
			Example 1	2：3：2	Degraded by 55 percent after one week
Example 2	2：3：2	Degraded by 57 percent after one week
			Example 3	1：4：4	72 percent of the material is degraded after one week
Example 4	4：2：1	35 percent of the material is degraded after one week
			Comparative example 3	5：2：1	26 percent of the material is degraded after one week
Comparative example 4	4：2：5	34 percent of the material is degraded after one week
			Comparative example 5	Collagen mineralized periosteum on market	Complete degradation after 2 days

From the data in table 2, it can be seen that:

in comparative example 3, the silk fibroin ratio was increased, resulting in slower periosteal degradation and a slower degradation rate, compared to example 4.

In comparative example 4, the collagen ratio was increased as compared with example 4, and the periosteum degradation rate was not changed much, but the strength was decreased.

In comparative example 5, the collagen mineralized periosteum on the market without adding the silk fibroin is completely degraded after 2 days, and the degradation speed is too high.

In the embodiment 1-4 of the invention, the mass ratio of silk fibroin, silicon-doped hydroxyapatite and collagen is controlled to be 1-4: 2-4: 1-4, the degradation performance of the artificial periosteum is moderate, the degradation performance is greatly improved, and orderly and controllable degradation can be realized.

In summary, the co-assembled artificial periosteum and the preparation method thereof provided by the invention have the advantages that the co-assembled artificial periosteum has good biocompatibility, can realize orderly and controllable degradation, has low crystallinity of silicon-containing hydroxyapatite, can improve the activity of osteoblasts and stimulate the osteogenic differentiation of stem cells, and in addition, the preparation method overcomes the defects that the silicon-containing hydroxyapatite, the silk fibroin and the collagen are not easy to form after being blended, are difficult to co-assemble and the like.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

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; alternatively, the first and second electrodes may be,

the silk fibroin/silicon-doped hydroxyapatite/collagen co-assembled artificial periosteum is prepared by co-assembling silk fibroin mediated silicon-doped hydroxyapatite sol and collagen, and 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 (1-2): 3-4: 2-4, the thickness of the co-assembled artificial periosteum is 200 um-1000 um, and the porosity is 50% -80%.

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

3. A method of preparing a co-assembled artificial periosteum according to claim 1 or 2, comprising:

obtaining collagen-mediated silicon-doped hydroxyapatite sol or fibroin-mediated silicon-doped hydroxyapatite sol;

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

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

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

crosslinking the first or second co-assembled membrane to obtain a crosslinked material;

4. The preparation method of claim 3, 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%.

5. The method of claim 3, 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;

6. The method of claim 3, wherein the freezing and freeze-drying the first or second co-assembly solution to obtain the first or second co-assembly membrane comprises:

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

7. The method of claim 3, wherein the cross-linking the first or second co-assembled membrane to obtain a cross-linked product comprises:

and (3) performing gas crosslinking on the first or second co-assembled membrane by using glutaraldehyde as a crosslinking agent for 2-8 h 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.

8. The preparation method according to claim 3, 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 10 Pa-100 Pa, the temperature is 20 ℃ -40 ℃, and the time is 12 h-24 h; the compression conditions are as follows: the pressure is 10 Mpa-40 Mpa, and the time is 10 s-30 s.