A tissue engineering oriented scaffold and its preparation method and application

Abstract

The invention belongs to the field of biomedical engineering, and discloses a tissue engineering oriented scaffold and its preparation method and application. The tissue engineering oriented scaffold is prepared from chitin whiskers, silk fibroin and carboxymethyl chitosan as main raw materials, and the prepared tissue engineering oriented scaffold has a well-oriented pore structure, and has good dimensional stability and biocompatibility, can be used for tissue repair, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering, and in particular relates to a tissue engineering oriented scaffold and a preparation method and application thereof. Background Art

[0002] There are many tissues with oriented structures in the human body. For example, endothelial cells in the human body can be arranged parallel to the direction of blood flow; myocytes in skeletal muscle are highly parallel to provide mechanical support for tissue movement; when peripheral nerves are injured, Schwann cells will extend longitudinally in the endoneurium to form Büngner bands to help guide axon regeneration. That is, the surface micro-nanostructure of biomaterials can regulate cell adhesion, growth, morphogenesis and functional differentiation through "contact guidance".

[0003] Compared with biochemical methods, the method of regulating cell behavior by using oriented scaffolds constructed by tissue engineering is more stable. Tissue engineering scaffolds with oriented structures can guide cell orientation and further induce cell functionalization, thereby achieving effective tissue repair. However, the preparation of tissue engineering scaffolds with oriented structures, certain dimensional stability and good biocompatibility still faces huge challenges. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a tissue engineering oriented scaffold and a preparation method and application thereof. The tissue engineering oriented scaffold has a well-oriented pore structure, and has good dimensional stability and biocompatibility, can be used for tissue repair, and has broad application prospects.

[0005] The invention provides a tissue engineering oriented scaffold, which is prepared from chitin whiskers, silk fibroin and carboxymethyl chitosan as main raw materials.

[0006] Among the above-mentioned raw materials, silk fibroin can provide good mechanical properties and biocompatibility; one-dimensional nanoparticle chitosan whiskers prepared from chitosan are also often used to improve the mechanical properties of the scaffold; and carboxymethyl chitosan has good biocompatibility, giving the tissue engineering scaffold good biodegradability. The present invention uses the above three raw materials as the main raw materials, and the prepared tissue engineering oriented scaffold not only has good biocompatibility and degradability, but also has good dimensional stability and an oriented pore structure with a pore size of 200-500μm, which is conducive to the exchange of nutrients.

[0007] Preferably, the mass ratio of the chitin whiskers, silk fibroin and carboxymethyl chitosan is 0.5:(1-5):(1-5).

[0008] More preferably, the mass ratio of the chitin whiskers, silk fibroin and carboxymethyl chitosan is 0.5:3:3.

[0009] The present invention also provides a method for preparing the above-mentioned tissue engineering oriented scaffold, comprising the following steps:

[0010] Silk fibroin, chitin whiskers and carboxymethyl chitosan were added to water to prepare a mixed solution; the solution was sequentially subjected to directional crystallization, primary freeze drying, Ca 2+ Cross-linking, glutaraldehyde cross-linking, and secondary freeze-drying to obtain the tissue engineering oriented scaffold.

[0011] The invention realizes the preparation of oriented scaffolds by guiding the directional growth of ice crystals through temperature gradient and freeze drying, and the method is simple and the requirements for equipment are relatively simple; in addition, Ca 2+ Mixed cross-linking with glutaraldehyde can also effectively improve the water resistance of the oriented scaffold and enhance its performance.

[0012] Preferably, the preparation method of the chitosan whisker is: chitosan and hydrochloric acid are mixed, subjected to oil bath heating reaction, acid hydrolyzed and centrifuged, washed with water, and finally dialyzed and freeze-dried to obtain the chitosan whisker.

[0013] More preferably, the oil bath is heated at a temperature of 100-120°C.

[0014] More preferably, the concentration of the hydrochloric acid is 3-5 mol / L.

[0015] More preferably, the molecular weight cut-off during dialysis is 3500Da.

[0016] Preferably, the preparation method of the silk fibroin is: cutting the cocoons into small pieces, degumming the silk, placing the silk in an ethanol aqueous solution containing CaCl2, dialyzing, drying and concentrating, to obtain the silk fibroin.

[0017] More preferably, the degumming treatment method is: adding the silk cocoons into a sodium carbonate solution and heating to perform degumming.

[0018] More preferably, the molecular weight cut-off during dialysis is 3500Da.

[0019] Preferably, the total mass concentration of silk fibroin, chitin whiskers and carboxymethyl chitosan in the mixed solution is 2%-8%.

[0020] Preferably, the Ca 2+ The mass concentration is 0.5%-3%, and the cross-linking time is 2-20min.

[0021] Preferably, the mass concentration of the glutaraldehyde is 0.25%-3%, and the cross-linking time is 8-32 hours.

[0022] The present invention also provides the use of the tissue engineering oriented scaffold in preparing tissue repair materials.

[0023] Preferably, the tissue comprises bone tissue or muscle tissue.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The present invention uses silk fibroin, chitin whiskers and carboxymethyl chitosan as the main raw materials for preparing tissue engineering oriented scaffolds, giving them good mechanical properties, biocompatibility and biodegradability. In addition, the tissue engineering oriented scaffold also has good dimensional stability and an oriented pore structure with a pore size of 200-500 μm, which is conducive to the exchange of nutrients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a scanning electron microscope image of chitin whiskers.

[0027] Figure 2 This is a scanning electron micrograph of the cross section of the tissue engineering oriented scaffold in Example 1.

[0028] Figure 3 This is a scanning electron micrograph of the longitudinal section of the tissue engineering oriented scaffold in Example 1.

[0029] Figure 4 The product pictures of different tissue engineering oriented scaffolds; among them, a is the tissue engineering oriented scaffold in Example 1, b is the tissue engineering oriented scaffold in Comparative Example 1, and c is the tissue engineering oriented scaffold in Comparative Example 2. DETAILED DESCRIPTION

[0030] In order to make those skilled in the art more clearly understand the technical solution of the present invention, the following examples are listed for illustration. It should be pointed out that the following examples are only preferred embodiments of the present invention and do not constitute a limitation on the protection scope of the present invention. Any modification, substitution, and combination made without violating the spirit and principle of the present invention are included in the protection scope of the present invention.

[0031] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0032] Example 1

[0033] This embodiment provides a tissue engineering oriented scaffold, which is prepared from chitosan whiskers, silk fibroin and carboxymethyl chitosan as main raw materials. The specific preparation method includes the following steps:

[0034] 1. Preparation of chitosan whisker: 1g chitosan is added to 30mL of 3mol / L HCl solution, reacted for 3 hours at 104 ℃, wait to cool slightly, pour out the supernatant HCl solution, add a little ultrapure water to transfer precipitation to 50mL centrifuge tube, after centrifugal 10 minutes (4000rpm), add deionized water to wash suspended matter and remove unnecessary HCl solution, repeat three times. Centrifugal chitosan is added with a little deionized water and the precipitation is mixed, poured in the dialysis bag, dialyzed for three days, changed water three times every day. After the dialysis, be positioned over-80 degrees Celsius refrigerator freezing (preferably spending the night), put into freeze drying instrument again and dry, namely obtain chitosan whisker powder. Figure 1 The scanning electron microscope image of the prepared chitosan whisker shows that the chitosan whisker is a one-dimensional nanoparticle with a relatively high aspect ratio.

[0035] 2. Preparation of silk fibroin: Weigh 7.5g of silk cocoon, cut it into one-fourth the size of a fingernail, soak it in a beaker containing 400mL of 1.1g / L sodium carbonate solution, and heat it in a water bath at 100°C for 1 hour; pour out the liquid in the beaker, prepare a new 400mL of 0.4g / L sodium carbonate solution and continue to heat it in boiling water for 30 minutes; pour out the liquid in the beaker, rinse it with clean water 2-3 times, drain the water roughly, freeze it in a -80°C refrigerator (preferably overnight), and then dry it in a freeze dryer for 12 hours.

[0036] Weigh 1.5g of degummed silkworm cocoons and soak them in the prepared ternary system (the molar ratio of CaCl2: ethanol: H2O is 1:2:8). Seal the mouth of the beaker with plastic wrap, put it into a water bath at 70°C, heat and stir to dissolve it; take out the beaker and wait for the liquid to cool slightly, then pour it into a dialysis bag and soak it in ultrapure water for three days, changing the water every day; then pour it into a beaker and concentrate it in a forced air drying oven for 12 hours.

[0037] 3. Scaffold preparation: mixed solution of silk fibroin / carboxymethyl chitosan / chitin whiskers: prepare a mixed solution of silk fibroin, carboxymethyl chitosan and chitosan whiskers, with the mass fractions of silk fibroin, carboxymethyl chitosan and chitosan whiskers being 3%, 3% and 0.5% respectively;

[0038] Use a pipette to draw the mixed solution into the mold; then put it into a thermos bottle filled with liquid nitrogen for about 1 hour, move it to a -80 degrees Celsius refrigerator (preferably overnight) for directional crystallization, so that the ice crystals grow along the temperature gradient between liquid nitrogen and air; after freezing, put it in a freeze dryer for freeze drying.

[0039] The freeze-dried scaffold was pre-cross-linked for 5 minutes using a solution consisting of 2 mL of deionized water, 0.04 g of CaCl2 and 4 mL of 95% ethanol; after the pre-cross-linking, the scaffold was chemically cross-linked with 2.5% glutaraldehyde for 24 hours to improve the stability of the scaffold; the cross-linked scaffold was taken out and freeze-dried in a freeze dryer for 12 hours to obtain a tissue engineering oriented scaffold.

[0040] Figure 2-3 The scanning electron micrographs of the cross section and longitudinal section of the prepared tissue engineering oriented scaffold are shown respectively. It can be seen from the figures that it has a well-oriented pore structure with a pore size of 200-500μm, which is conducive to the exchange of nutrients and can be used for the repair of damaged tissues.

[0041] Comparative Example 1

[0042] This comparative example provides a tissue engineering oriented scaffold, and its preparation method is basically the same as that of Example 1, except that: it is prepared from carboxymethyl chitosan and chitosan whiskers as main raw materials, and the mass fractions of carboxymethyl chitosan and chitosan whiskers in the mixed solution of carboxymethyl chitosan / chitosan whiskers are 6% and 0.5% respectively.

[0043] Comparative Example 2

[0044] This comparative example provides a tissue engineering oriented scaffold, and its preparation method is basically the same as that of Example 1, except that: it is prepared from silk fibroin and chitosan whiskers as main raw materials, and the mass fractions of silk fibroin / chitosan whiskers and chitosan whiskers in the mixed solution are 6% and 0.5% respectively.

[0045] Dimensional stability test

[0046] like Figure 4 As shown, compared with comparative examples 1-2, the tissue engineering oriented scaffold prepared in Example 1 after cross-linking and freeze-drying has a smaller deformation degree and exhibits better dimensional stability.

[0047] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A tissue engineering oriented scaffold, characterized in that: It is made of chitin whiskers, silk fibroin and carboxymethyl chitosan as main raw materials; The mass ratio of the chitin whiskers, silk fibroin and carboxymethyl chitosan is 0.5:(1-5):(1-5); The tissue engineering oriented scaffold is prepared by the following preparation method: Silk fibroin, chitin whiskers and carboxymethyl chitosan were added to water to prepare a mixed solution; the solution was successively subjected to directional crystallization, primary freeze drying, Ca 2+ cross-linking, glutaraldehyde cross-linking, and secondary freeze-drying to obtain the tissue engineering oriented scaffold; The directional crystallization is guided by the temperature gradient, specifically comprising: placing the mixed solution in a thermos bottle filled with liquid nitrogen for 1 hour, and then moving it to a -80 degrees Celsius refrigerator for directional crystallization, so that ice crystals grow directionally along the temperature gradient between liquid nitrogen and air.

2. The method for preparing the tissue engineering oriented scaffold according to claim 1, characterized in that: The following steps are involved: Silk fibroin, chitin whiskers and carboxymethyl chitosan were added to water to prepare a mixed solution; the solution was successively subjected to directional crystallization, primary freeze drying, Ca 2+ cross-linking, glutaraldehyde cross-linking, and secondary freeze-drying to obtain the tissue engineering oriented scaffold; The directional crystallization is guided by the temperature gradient, specifically comprising: placing the mixed solution in a thermos bottle filled with liquid nitrogen for 1 hour, and then moving it to a -80 degrees Celsius refrigerator for directional crystallization, so that ice crystals grow directionally along the temperature gradient between liquid nitrogen and air.

3. The preparation method according to claim 2, characterized in that: The preparation method of the chitosan whisker comprises the following steps: mixing chitosan and hydrochloric acid, heating in an oil bath for reaction, acid hydrolyzing and centrifuging, washing with water, and finally dialyzing and freeze-drying to obtain the chitosan whisker.

4. The preparation method according to claim 2, characterized in that: The preparation method of the silk fibroin is as follows: the silk cocoons are cut into small pieces, after degumming treatment, the silk is placed in an ethanol aqueous solution containing CaCl2, and then dialyzed, dried and concentrated to obtain the silk fibroin.

5. The preparation method according to claim 2, characterized in that: The total mass concentration of silk fibroin, chitin whiskers and carboxymethyl chitosan in the mixed solution is 2%-8%.

6. The preparation method according to claim 2, characterized in that: The Ca 2+ The mass concentration is 0.5%-3%, and the cross-linking time is 2-20min.

7. The preparation method according to claim 2, characterized in that: The mass concentration of the glutaraldehyde is 0.25%-3%, and the cross-linking time is 8-32h.

8. Use of the tissue engineering oriented scaffold according to claim 1 in preparing tissue repair materials.

9. The use according to claim 8, characterized in that: The tissue includes bone tissue or muscle tissue.

Images