High-strength biological scaffold material and preparation method thereof

Abstract

A high-strength biological scaffold and preparation method thereof. The biological scaffold is comprised of a framework of boiled-off silk woven material, the form of the framework is determined by actual needs; the framework surface is coated with a layer of silk protein scaffold material, fibroin protein/gelatin biological scaffold material or fibroin protein/collagen biological scaffold material having a thickness of 100 micrometers to 5 centimeters. The high-strength biological scaffold material has high tear resistance strength and mechanical strength and good biocompatibility, has a porous structure suitable for tissue regeneration, and can be used for preparing anal fistula repair plugs.

Claims

1. A method for preparing a high-strength biological scaffold material, characterized in that, the method comprises the following steps: boiled-off silk obtained by degumming silk is woven into a woven material by a textile machine to form a framework according to the desired shape, and then the framework is placed into a mold; a solution containing silk fibroin is injected into the mold having the framework placed therein, and subjected to freezing and vacuum treatment; thereby a coating layer having a thickness of 100 microns to 5 cm is formed on the surface of the framework, and the high-strength biological scaffold material is prepared, wherein said solution containing silk fibroin is one selected from the group consisting of: an aqueous silk fibroin solution, a mixed solution of silk fibroin and gelatin, and a mixed solution of silk fibroin and collagen; the weave density of the woven material is such that the distance between adjacent boiled-off silks is between 0.5 mm and 3 mm, and the material of the coating layer is selected from the group consisting of: silk protein scaffold material, silk fibroin/gelatin biological scaffold material or silk fibroin/collagen biological scaffold material; wherein the aqueous silk fibroin solution is prepared by: subjecting silk to degumming, dissolution, and dialysis to obtain a silk fibroin solution, the aqueous silk fibroin solution having a mass concentration of 0.1 to 20%; leaving the aqueous silk fibroin solution at 0 to 80 C. for 1 to 48 hours; and wherein the vacuum treatment is performed in a vacuum dryer containing water, aqueous methanol or aqueous ethanol at the bottom thereof.

2. The method for preparing a high-strength biological scaffold material according to claim 1, characterized in that, the step of coating the surface of the framework with a coating layer comprises the specific steps of: 1) preparing a solution containing silk fibroin, the solution containing silk fibroin being one selected from the group consisting of: an aqueous silk fibroin solution, a mixed solution of silk fibroin and gelatin, and a mixed solution of silk fibroin and collagen; injecting the solution containing silk fibroin into the mold having the framework placed therein, and subjecting it to freezing under a low temperature of 10 to 80 C. for 1 to 24 hours to obtain frozen crystals; freeze-drying the frozen crystals to obtain a composite material, the composite material having boiled-off silk woven material as a framework and a layer of soluble silk fibroin layer/silk fibroin and gelatin composite layer/silk fibroin and collagen composite layer coated on the framework; 2) placing the composite material obtained in step 1) into a vacuum dryer and performing a vacuum treatment for 20 minutes to 24 hours to obtain a water-insoluble composite high-strength biological scaffold material, the composite high-strength biological scaffold material having boiled-off silk woven material as a framework and a coating layer provided on the framework, the material of the coating layer being selected from the group consisting of: silk protein scaffold material, silk fibroin/gelatin biological scaffold material or silk fibroin/collagen biological scaffold material; and the vacuum dryer containing water, aqueous methanol or aqueous ethanol at the bottom thereof; wherein the mixed solution of silk fibroin and gelatin is prepared by: preparing an aqueous gelatin solution by adding medical gelatin into distilled water, heating to obtain an aqueous gelatin solution, and leaving the solution at 0 to 10 C. for 30 minutes to 2 hours, the aqueous gelatin solution having a mass concentration of 0.01 to 20%; then mixing an aqueous silk fibroin solution with the aqueous gelatin solution at a mass ratio of silk fibroin to gelatin of 100:2 to 20, to obtain the mixed solution of silk fibroin and gelatin; the mixed solution of silk fibroin and collagen is prepared by: preparing an aqueous silk fibroin solution by subjecting silk to degumming, dissolution, and dialysis to obtain a silk fibroin solution, the aqueous silk fibroin solution having a mass concentration of 0.1 to 20%; leaving the aqueous silk fibroin solution at 0 to 80 C. for 1 to 48 hours; preparing a solution of collagen in acetic acid at a concentration of 0.01% to 2%, and leaving the solution at 0 to 10 C. for 30 minutes to 2 hours; then mixing the aqueous silk fibroin solution with the solution of collagen in acetic acid at a mass ratio of silk fibroin to collagen of 100:2 to 20, to obtain the mixed solution of silk fibroin and collagen.

3. The method for preparing a high-strength biological scaffold material according to claim 2, wherein the material is in a shape of an anal fistula repair plug, characterized in that, the method comprises the following steps: 1) preparing a solution containing silk fibroin, the solution containing silk fibroin being one selected from the group consisting of: an aqueous silk fibroin solution, a mixed solution of silk fibroin and gelatin, and a mixed solution of silk fibroin and collagen; 2) weaving degummed boiled-off silk into a tubular material to provide a framework and placing the tubular material into a mold for preparing anal fistula repair plug, the weave density of the tubular material being such that the distance between adjacent boiled-off silks is between 0.5 mm and 3 mm; 3) injecting the solution containing silk fibroin into the mold having the tubular material placed therein, and subjecting it to freezing under a low temperature of 10 to 30 C. for 20 to 24 hours to obtain a frozen body; freeze-drying the frozen body to obtain a soluble composite anal fistula repair plug, the soluble composite anal fistula repair plug having the tubular material woven from boiled-off silk as a framework and a layer of soluble silk fibroin layer/silk fibroin and gelatin composite layer/silk fibroin and collagen composite layer coated on the framework; 4) placing the soluble composite anal fistula repair plug into a vacuum dryer and performing a vacuum treatment for 4 hours or more to obtain a water-insoluble composite anal fistula repair plug, the water-insoluble composite anal fistula repair plug having the tubular material woven from boiled-off silk as a framework and a biological scaffold material as a coating layer, the biological scaffold material being selected from the group consisting of: silk protein biological scaffold material, silk fibroin/gelatin biological scaffold material or silk fibroin/collagen biological scaffold material, the framework being embedded inside the coating layer; wherein in step 1), the aqueous silk fibroin solution is prepared by: preparing an aqueous silk fibroin solution having a mass concentration of 0.5% to 5% by a conventional method, and leaving the aqueous silk fibroin solution at 0 to 10 C. for 30 minutes or more; the mixed solution of silk fibroin and gelatin is prepared by: preparing an aqueous gelatin solution having a mass concentration of 0.05% to 1%, and leaving the solution at 0 to 10 C. for 30 minutes to 2 hours; mixing an aqueous silk fibroin solution with the aqueous gelatin solution uniformly such that the mass concentration of silk fibroin is 0.2% to 3% and the mass concentration of gelatin is 0.02% to 0.2% upon mixing; and leaving the mixture stand for 4 to 10 hours to obtain the mixed solution of silk fibroin and gelatin; the mixed solution of silk fibroin and collagen is prepared by: preparing a solution of collagen in acetic acid at a concentration of 0.05% to 1%, and leaving the solution at 0 to 10 C. for 30 minutes to 2 hours; mixing an aqueous silk fibroin solution with the solution of collagen in acetic acid uniformly at 0 to 10 C. such that the mass concentration of silk fibroin is 0.2% to 3% and the mass concentration of collagen is 0.02% to 0.2% upon mixing; and leaving the mixture stand for 4 to 10 hours to obtain the mixed solution of silk fibroin and collagen.

4. The method for preparing the high-strength anal fistula repair plug according to claim 3, characterized in that, in step 4), the coating layer has a thickness of 100 microns to 5 mm; and the biological scaffold material has a pore size of 200 to 400 microns, and a porosity of 80% or more.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a photograph of a tubular material woven from boiled-off silk used in Example 1;

(2) FIG. 2 is a photograph of the silk fibroin/gelatin anal fistula repair plug reinforced by boiled-off silk prepared in Example 1;

(3) FIG. 3 is the micro porous structure of the silk fibroin/gelatin anal fistula repair plug reinforced by boiled-off silk prepared in Example 1;

(4) FIG. 4 is the IR spectra result of the silk fibroin/gelatin anal fistula repair plug reinforced by boiled-off silk prepared in Example 1.

DETAILED EMBODIMENTS

(5) Next, the present invention is further described by the aid of drawings and examples.

Example 1

(6) In this example, the silk fibroin porous scaffold is prepared according to the following steps: 1) 5 g of cocoons are added to 2 liters of a sodium carbonate solution having a mass concentration of 0.5%, treated at 98 to 100 C. for 40 min to remove the external sericin, and sufficiently washed to obtain pure silk fibroin. 2) The degummed silk fibroin (also referred to as boiled-off silk) is woven into a tubular material having a diameter of 4 mm by a textile machine to form a framework, the weave density of the material is such that the distance between adjacent warp boiled-off silks is about 1 mm, and the distance between adjacent weft boiled-off silks is about 1 mm. The tubular material is placed into a repair plug mold having a diameter of 5 mm. 3) The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromide solution at about 60 C., to obtain a fibroin mixed solution. The fibroin mixed solution is added to a cellulose dialysis membrane, and dialysized with deionized water to remove lithium bromide. Thus, a pure aqueous silk fibroin solution is obtained. 4) The mass concentration of the silk fibroin is adjusted to 4%, and the aqueous solution is placed at 4 C. for 1 hour, such that the solution temperature is stably maintained at 4 C. 5) A gelatin solution is diluted to 1%. 6) The silk fibroin solution is mixed with the gelatin solution in equal volume at 4 C., and is allowed to stand for 6 hours. 7) The mixed solution is injected into the mold having boiled-off silk tube placed therein, and frozen at 20 C. for 24 hours to obtain a silk fibroin/gelatin frozen body. 8) The frozen silk fibroin/gelatin mixture is placed into a freeze dryer, and subjected to a freeze-drying process for 48 hours to obtain a soluble silk fibroin/gelatin porous repair plug reinforced by boiled-off silk having a thickness of 5 mm. 9) The porous repair plug is placed into a vacuum dryer having water provided at the bottom, and subjected to a vacuum treatment for 6 hours to obtain a water-insoluble silk fibroin/gelatin anal fistula repair plug reinforced by boiled-off silk.

(7) FIGS. 1 and 2 show a boiled-off silk tube having a diameter of 4 mm, and a silk fibroin/gelatin anal fistula repair plug compounded with the boiled-off silk tube, prepared according to the above method. It can be seen, the boiled-off silk is well compounded with the silk fibroin/gelatin porous material.

(8) FIG. 3 is a scanning electron micrograph of a silk fibroin/gelatin porous anal fistula repair plug prepared according to the above method. It can be seen, a good porous structure is formed in the material which has a pore diameter of 200-400 microns.

(9) FIG. 4 is the IR spectra of the silk fibroin/gelatin porous anal fistula repair plug reinforced by boiled-off silk prepared according to the above method. It can be seen, the characteristic absorption peak of the silk fibroin/gelatin porous repair plug is at 1630 cm.sup.1 and has a distinct shoulder peak at 1650 cm.sup.1, which indicates that both silk I and silk II crystalline structures exist.

Example 2

(10) 5 g of cocoons are added to 2 liters of a sodium carbonate solution having a mass concentration of 0.5%, treated at 98 to 100 C. for 40 min to remove the external sericin, and sufficiently washed to obtain pure silk fibroin.

(11) The degummed silk fibroin (also referred to as boiled-off silk) is woven into a tubular material having a diameter of 3 mm by a textile machine. The tubular material is placed into a conical repair plug mold having an upper diameter of 3 mm and a lower diameter of 5 mm.

(12) The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromide solution at about 60 C., to obtain a fibroin mixed solution. The fibroin mixed solution is added to a cellulose dialysis membrane, and dialysized with deionized water to remove lithium bromide. Thus, a pure aqueous silk fibroin solution is obtained.

(13) The mass concentration of the silk fibroin is adjusted to 4%, and the aqueous solution is placed at 4 C. for 1 hour, such that the solution temperature is stably maintained at 4 C.

(14) A collagen solution in acetic acid is diluted to 0.5%.

(15) The silk fibroin solution is mixed with the collagen solution in equal volume at 4 C., and is allowed to stand for 6 hours.

(16) The mixed solution is injected into the mold having boiled-off silk tube placed therein, and frozen at 20 C. for 24 hours to obtain a silk fibroin/collagen frozen body.

(17) The frozen silk fibroin/collagen mixture is placed into a freeze dryer, and subjected to a freeze-drying process for 48 hours to obtain a soluble silk fibroin/collagen porous repair plug reinforced by boiled-off silk having an upper diameter of 3 mm and a lower diameter of 5 mm.

(18) The porous repair plug is placed into a vacuum dryer having water provided at the bottom, and subjected to a vacuum treatment for 6 hours to obtain a water-insoluble silk fibroin/collagen anal fistula repair plug reinforced by boiled-off silk.

Example 3

(19) 5 g of cocoons are added to 2 liters of a sodium carbonate solution having a mass concentration of 0.5%, treated at 98 to 100 C. for 40 min to remove the external sericin, and sufficiently washed to obtain pure silk fibroin.

(20) The degummed silk fibroin (also referred to as boiled-off silk) is woven into a tubular material having a diameter of 4 mm by a textile machine. The tubular material is placed into a conical repair plug mold.

(21) The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromide solution at about 60 C., to obtain a fibroin mixed solution. The fibroin mixed solution is added to a cellulose dialysis membrane, and dialysized with deionized water to remove lithium bromide. Thus, a pure aqueous silk fibroin solution is obtained.

(22) The mass concentration of the silk fibroin is adjusted to 4%, and the aqueous solution is placed at 4 C. for 1 hour, such that the solution temperature is stably maintained at 4 C.

(23) A collagen solution in acetic acid is diluted to 0.5%.

(24) The silk fibroin solution is mixed with the collagen solution in equal volume at 4 C., and is allowed to stand for 6 hours.

(25) The mixed solution is injected into the mold having boiled-off silk tube placed therein, and frozen at 20 C. for 24 hours to obtain a silk fibroin/collagen frozen body.

(26) The frozen silk fibroin/collagen mixture is placed into a freeze dryer, and subjected to a freeze-drying process for 48 hours to obtain a soluble silk fibroin/collagen porous repair plug reinforced by boiled-off silk.

(27) The porous repair plug is placed into a vacuum dryer having an 80% ethanol solution provided at the bottom, and subjected to a vacuum treatment for 6 hours to obtain a water-insoluble silk fibroin/collagen porous anal fistula repair plug reinforced by boiled-off silk. This porous scaffold has a pore diameter of 200 microns or more, and a porosity of 90% or more. The main crystal structure is silk II.

Example 4

(28) 5 g of cocoons are added to 2 liters of a sodium carbonate solution having a mass concentration of 0.5%, treated at 98 to 100 C. for 40 min to remove the external sericin, and sufficiently washed to obtain pure silk fibroin.

(29) The degummed silk fibroin (also referred to as boiled-off silk) is woven into a tubular material having a diameter of 4 mm by a textile machine. The tubular material is placed into a conical repair plug mold.

(30) The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromide solution at about 60 C., to obtain a fibroin mixed solution. The fibroin mixed solution is added to a cellulose dialysis membrane, and dialysized with deionized water to remove lithium bromide. Thus, a pure aqueous silk fibroin solution is obtained.

(31) The mass concentration of the silk fibroin is adjusted to 4%, and the aqueous solution is placed at 4 C. for 1 hour, such that the solution temperature is stably maintained at 4 C.

(32) An aqueous gelatin solution is diluted to 0.5%.

(33) The silk fibroin solution is mixed with the gelatin solution in equal volume at 4 C., and is allowed to stand for 6 hours.

(34) The mixed solution is injected into the mold having boiled-off silk tube placed therein, and frozen at 20 C. for 24 hours to obtain a silk fibroin/gelatin frozen body.

(35) The frozen silk fibroin/gelatin mixture is placed into a freeze dryer, and subjected to a freeze-drying process for 48 hours to obtain a soluble silk fibroin/gelatin porous repair plug reinforced by boiled-off silk.

(36) The porous repair plug is placed into a vacuum dryer having an 80% methanol solution provided at the bottom, and subjected to a vacuum treatment for 6 hours to obtain a water-insoluble silk fibroin/collagen porous anal fistula repair plug reinforced by boiled-off silk. This porous scaffold has a pore diameter of 200 microns or more, and a porosity of 90% or more. The main crystal structure is silk II.

Example 5

(37) 5 g of cocoons are added to 2 liters of a sodium carbonate solution having a mass concentration of 0.5%, treated at 98 to 100 C. for 40 min to remove the external sericin, and sufficiently washed to obtain pure silk fibroin.

(38) The degummed silk fibroin (also referred to as boiled-off silk) is woven into a tubular material having a diameter of 3 mm by a textile machine. The tubular material is placed into a repair plug mold having an inner diameter of 2.9 mm and an outer diameter of 3.2 mm.

(39) The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromide solution at about 60 C., to obtain a fibroin mixed solution. The fibroin mixed solution is added to a cellulose dialysis membrane, and dialysized with deionized water to remove lithium bromide. Thus, a pure aqueous silk fibroin solution is obtained.

(40) The mass concentration of the silk fibroin is adjusted to 4%, and the aqueous solution is placed at 4 C. for 1 hour, such that the solution temperature is stably maintained at 4 C.

(41) A collagen solution in acetic acid is diluted to 0.5%.

(42) The silk fibroin solution is mixed with the collagen solution in equal volume at 4 C., and is allowed to stand for 6 hours.

(43) The mixed solution is injected into the mold having boiled-off silk tube placed therein, and frozen at 20 C. for 24 hours to obtain a silk fibroin/collagen frozen body having a thickness of 300 microns.

(44) The frozen silk fibroin/collagen mixture is placed into a freeze dryer, and subjected to a freeze-drying process for 48 hours to obtain a soluble silk fibroin/collagen porous repair plug reinforced by boiled-off silk.

(45) The porous repair plug is placed into a vacuum dryer having an 80% ethanol solution provided at the bottom, and subjected to a vacuum treatment for 6 hours to obtain a water-insoluble silk fibroin/collagen porous anal fistula repair plug reinforced by boiled-off silk. This porous scaffold has a pore diameter of 200 microns or more, and a porosity of 90% or more. The main crystal structure is silk II.

Example 6

(46) 5 g of cocoons are added to 2 liters of a sodium carbonate solution having a mass concentration of 0.5%, treated at 98 to 100 C. for 40 min to remove the external sericin, and sufficiently washed to obtain pure silk fibroin.

(47) The degummed silk fibroin (also referred to as boiled-off silk) is woven into a sheet material having an area of 44 cm. The sheet material is placed into a mold having a height of 4 cm.

(48) The dried silk fibroin is dissolved into a 9.3 mol/L lithium bromide solution at about 60 C., to obtain a fibroin mixed solution. The fibroin mixed solution is added to a cellulose dialysis membrane, and dialysized with deionized water to remove lithium bromide. Thus, a pure aqueous silk fibroin solution is obtained.

(49) The mass concentration of the silk fibroin is adjusted to 4%, and the aqueous solution is placed at 4 C. for 1 hour, such that the solution temperature is stably maintained at 4 C. The silk fibroin solution is slowly concentrated to 30%, placed at 4 C. for 3 days, and re-diluted to 4%.

(50) The diluted silk fibroin solution is injected into the mold having boiled-off silk sheet placed therein, and frozen at 20 C. for 24 hours to obtain a silk fibroin frozen body having a height of 4 cm and an area of 44 cm.

(51) The frozen body is placed into a freeze dryer, and subjected to a freeze-drying process for 48 hours to obtain a soluble silk fibroin bulk material reinforced by boiled-off silk.

(52) The bulk material is placed into a vacuum dryer having an 80% methanol solution provided at the bottom, and subjected to a vacuum treatment for 6 hours to obtain a water-insoluble silk fibroin repair sheet material. This porous repair plug has a pore diameter of 200 microns or more, and a porosity of 90% or more. The main crystal structure is silk II.