CN110787323A

CN110787323A - HAp-CSA-SF composite gel material and preparation method and application thereof

Info

Publication number: CN110787323A
Application number: CN201911119640.3A
Authority: CN
Inventors: 葛少华; 马保金; 商玲玲; 刘宏
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2020-02-14
Anticipated expiration: 2039-11-15
Also published as: CN110787323B

Abstract

The invention provides an HAp-CSA-SF composite gel material, which comprises a chondroitin sulfate-silk fibroin composite gel layer and a hydroxyapatite rich layer arranged on at least one end face of the chondroitin sulfate-silk fibroin composite gel layer. The hydroxyapatite-rich layer is used for repairing damaged inner bone tissues on one hand and enhancing the bonding strength between cartilage and adjacent bones on the other hand so as to ensure that the repaired cartilage can normally perform functions; and the chondroitin-silk fibroin composite gel layer is an integral gel formed by mixing chondroitin sulfate and silk fibroin. With the degradation of silk fibroin, chondroitin sulfate can be released at the same time, the regeneration of cartilage is further promoted, and the repair of the cartilage is realized.

Description

HAp-CSA-SF composite gel material and preparation method and application thereof

Technical Field

The invention belongs to the technical field of cartilage tissue repair, and particularly relates to an HAp-CSA-SF composite gel material, and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Cartilage tissue engineering is one of the important directions of tissue engineering. Cartilage has important functions in the human body, such as load bearing, force transmission, and cushioning. Cartilage in joints is the basis for maintaining normal movement of the human body. However, wear of cartilage and damage of cartilage due to external force occur in some cases, and problems such as inflammation, pain, and dyskinesia are caused. How to repair damaged cartilage tissue efficiently, rapidly and accurately becomes an important problem to be solved urgently in cartilage tissue engineering.

The traditional cartilage tissue repair material has poor toughness, and cannot effectively play the functions of bearing, buffering and the like in time after being implanted. The main components of cartilage tissue include collagen and chondroitin sulfate. Studies show that Silk Fibroin (SF) with high biocompatibility and good degradability is used as a protein source, and can effectively promote cartilage regeneration. However, the existing SF stent material has poor toughness and low support performance. In addition, in the current repair process, the transition between cartilage and bone tissue is often ignored, resulting in a weak bond between cartilage and bone after repair.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a hydroxyapatite-chondroitin sulfate-silk fibroin (HAp-CSA-SF) composite gel material, and a preparation method and application thereof. The present invention forms an integral gel by uniformly mixing CSA and SF. As the SF is degraded, CSA can be released at the same time, and the regeneration of cartilage is further promoted. The prepared SF solution has viscosity, can adhere HAp and form an HAp layer on one surface of gel, thereby promoting the close combination between cartilage and bone and promoting the accurate bionic repair in the cartilage repair process, and having good value of practical application.

One of the purposes of the invention is to provide a HAp-CSA-SF composite gel material.

The invention also aims to provide a preparation method of the HAp-CSA-SF composite gel material.

The invention also aims to provide application of the HAp-CSA-SF composite gel material.

In order to achieve the purpose, the invention relates to the following technical scheme:

the invention provides a HAp-CSA-SF composite gel material, which comprises a chondroitin sulfate-silk fibroin composite gel layer and a hydroxyapatite rich layer arranged on at least one end face of the chondroitin sulfate-silk fibroin composite gel layer, wherein the hydroxyapatite can be used for repairing damaged inner bone tissues on one hand and effectively enhancing the bonding strength between cartilage and adjacent bones on the other hand so as to ensure that the repaired cartilage can normally perform functions; and the chondroitin-silk fibroin composite gel layer is an integral gel formed by mixing chondroitin sulfate and silk fibroin. With the degradation of silk fibroin, chondroitin sulfate can be released at the same time, the regeneration of cartilage is further promoted, and the repair of the cartilage is realized.

The hydroxyapatite layer is composed of hydroxyapatite with a nano structure, and the nano hydroxyapatite can be granular, rod-shaped or linear, such as hydroxyapatite nanoparticles, hydroxyapatite nanorods, hydroxyapatite short nanowires, hydroxyapatite long nanowires and the like.

In a second aspect of the present invention, there is provided a method for preparing the HAp-CSA-SF composite gel material, the method comprises using an acid-soluble alcohol-solid method to prepare the HAp-CSA-SF composite gel material, and specifically comprises:

preparation of HAp: mixing oleic acid and ethanol, adding calcium salt solution, adding alkali liquor, adding sodium hydrogen phosphate solution, stirring, and reacting at high temperature.

Preparing a CSA-SF composite acid solution: preparing a silk fibroin formic acid solution, and adding chondroitin sulfate into the silk fibroin formic acid solution.

The preparation method also comprises the steps of putting the CSA-SF composite mixed acid solution into a mold carrying the HAp, and volatilizing and cooling the acid solution to obtain the HAp-CSA-SF composite gel material.

Further, the HAp-CSA-SF composite gel material is soaked in absolute ethyl alcohol and solidified to obtain the HAp-CSA-SF composite gel material.

In a third aspect of the invention, the application of the HAp-CSA-SF composite gel material in preparing a bone repair material is provided. The bone repair material is further a cartilage repair material.

The bone repair material has at least one or more of the following uses:

(a) promoting bone/cartilage production;

(b) promoting the reconstruction of subchondral bone tissues;

(c) increasing bone volume fraction (BV/TV);

(d) increasing trabecular bone thickness (tb.th);

(e) increasing trabecular number (tb.n);

(f) the trabecular compartment (tb.sp) is reduced.

The invention has the beneficial technical effects that:

the invention provides an HAp-CSA-SF composite gel material, which comprises a chondroitin sulfate-silk fibroin composite gel layer and a hydroxyapatite rich layer arranged on at least one end face of the chondroitin sulfate-silk fibroin composite gel layer. The hydroxyapatite repairs damaged inner bone tissues on one hand, and enhances the bonding strength between the cartilage and adjacent bones on the other hand so as to ensure that the repaired cartilage can normally perform functions; and the chondroitin-silk fibroin composite gel layer is an integral gel formed by mixing chondroitin sulfate and silk fibroin. With the degradation of silk fibroin, chondroitin sulfate can be released at the same time, the regeneration of cartilage is further promoted, and the repair of the cartilage is realized.

Tests prove that the HAp-CSA-SF composite gel material has high toughness and good effect of promoting bone to form cartilage, thereby having good value of practical application.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a representation of hydroxyapatite nanowires in an embodiment of the invention; wherein a and b are SEM images of the hydroxyapatite nanowires under different times; c, TEM image of hydroxyapatite nano-wire; and d, HRTEM image of hydroxyapatite nano-wires.

FIG. 2 is a representation of an SF plane in accordance with an embodiment of the present invention; wherein a and b are SEM images of SF planes under different multiples; c and d are SEM images of the HAp surface under different magnifications, and e is a sectional view; f is a macroscopic view.

FIG. 3 is an X-ray diffraction (XRD) of SF in an example of the present invention; tensile rupture test stress-strain curve; fourier Infrared Spectroscopy (FTIR) plots.

FIG. 4 shows a CCK-8 cell proliferation assay in an example of the present invention; a-f are graphs of viable and dead cell staining of cells on control TCP, SF gel, and SF gel + 8% CSA.

FIG. 5 is a graph relating to osteogenic differentiation of BMSCs on HAp/Silk composite gel scaffolds in accordance with the present invention, a-f, cytoskeleton and immunofluorescence staining showing the expression of OPN and OCN at the protein level; i and j, qRT-PCR showed expression of OCN and OPN at the gene level.

FIG. 6 is a graph showing cartilage differentiation of MSCs on a CSA-Silk composite gel scaffold according to an embodiment of the present invention; wherein a-f are toluidine blue staining of different materials; g-i is qRT-PCR showing the expression pattern of COL2A, ACAN and SOX9 at the gene level.

FIG. 7 is a graph of gross observations and the International society for cartilage repair (ICRS) scores for examples of the invention.

FIG. 8 is a diagram of Micro-CT analysis of the reconstruction of subchondral bone in an embodiment of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; the present invention is not particularly limited, and may be commercially available.

As introduced in the background art, the existing SF stent material has poor toughness and low support performance. In addition, in the current repair process, the transition between cartilage and bone tissue is often ignored, resulting in a weak bond between cartilage and bone after repair.

In view of the above, in one embodiment of the present invention, there is provided a HAp-CSA-SF composite gel material comprising a chondroitin sulfate-silk fibroin composite gel layer, and a hydroxyapatite-rich layer disposed on at least one end surface of the chondroitin sulfate-silk fibroin composite gel layer.

In one or more embodiments of the present invention, the nanoscale hydroxyapatite may be in a particle shape, a rod shape, or a linear shape, such as hydroxyapatite nanoparticles, hydroxyapatite nanorods, hydroxyapatite short nanowires, and hydroxyapatite long nanowires, and by forming an HAp layer on one side of the chondroitin sulfate-silk fibroin composite gel, in a cartilage repair process, tight bonding between cartilage and bone is promoted, thereby promoting accurate biomimetic repair.

The mass ratio of the hydroxyapatite rich layer to the chondroitin sulfate-silk fibroin composite gel layer can be adjusted according to the cartilage thickness of the area to be repaired, the injury degree of the inner layer bone and the like, so that a foundation is laid for personalized bone tissue repair, and in one or more specific embodiments of the invention, the mass ratio of the hydroxyapatite rich layer to the chondroitin sulfate-silk fibroin composite gel layer is 0.5-2: 1-50, such as 1:2, 1:5, 1:10, 1:20, 1:25, 1:30, 1:40, 1:50, 1:100 and the like.

Researches find that chondroitin sulfate and silk fibroin can more effectively promote chondrogenesis and improve the cartilage repair efficiency under the condition of proper mass ratio. Thus, in one or more embodiments of the present invention, the mass ratio of chondroitin sulfate to silk fibroin is 100: 0.1-20, such as 100:1, 100:4, 100:8, 100:16, 100: 20; more preferably 100: 8.

In one or more embodiments of the present invention, a preparation method of the HAp-CSA-SF composite gel material is provided, wherein the preparation method comprises an acid-soluble alcohol-solid method, and specifically comprises:

In one or more specific embodiments of the present invention, the preparation method further includes placing the CSA-SF composite mixed acid solution into a mold carrying HAp, and obtaining the HAp-CSA-SF composite gel material after the acid solution is volatilized and cooled.

In one or more specific embodiments of the present invention, the preparation method further includes soaking the HAp-CSA-SF composite gel material prepared as described above in absolute ethanol to perform a curing treatment.

In one or more embodiments of the invention, the calcium salt is calcium chloride and the lye is sodium hydroxide solution; therefore, the volume-mass ratio of the oleic acid to the ethanol to the calcium chloride to the sodium hydroxide to the sodium hydrogen phosphate is 5-10 ml: 8-15 ml: 0.1-0.5 g: 0.2-1 g: 0.1 to 0.5g (preferably 8 ml: 10 ml: 0.147 g: 0.5 g: 0.156 g).

In one or more specific embodiments of the present invention, the high temperature reaction strip is reacted at 160-200 ℃ for 5-20 h (preferably at 180 ℃ for 12 h); the morphology of the HAp can be regulated by controlling the addition amount of raw materials and reaction conditions, under the preferred condition of the invention, the HAp is in a short nanowire shape, the length of the HAp is about 5 mu m, the width of the HAp is about 15nm, and the TEM result shows that the lattice width of the HAp is about 0.817 nm. The SF surface in the gel composite material has certain fluctuation and is relatively compact, thereby laying a foundation for the high toughness of the composite material.

In one or more embodiments of the present invention, a method for preparing a fibroin formic acid solution is provided: dissolving calcium salt in formic acid solution, and adding silk into the above solution.

Wherein the calcium salt is calcium chloride, and the formic acid is 88% (mass fraction) formic acid solution; the mass ratio of the calcium chloride to the silk is 0.2-1: 1-2. The silk is obtained by degumming silkworm cocoons.

In one or more specific embodiments of the present invention, the mass ratio of chondroitin sulfate to silk fibroin is 100: 0.1-20, such as 100:1, 100:4, 100:8, 100:16, 100: 20; more preferably 100: 8.

In one or more embodiments of the invention, the HAp-CSA-SF composite gel material is used for preparing bone repair materials. The bone repair material is further a cartilage repair material.

The bone repair material has at least one or more of the following uses:

(a) promoting bone/cartilage production;

(b) promoting the reconstruction of subchondral bone tissues;

(c) increasing bone volume fraction (BV/TV);

(d) increasing trabecular bone thickness (tb.th);

(e) increasing trabecular number (tb.n);

(f) the trabecular compartment (tb.sp) is reduced.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Examples

Experimental materials methods:

preparation and characterization of HAp-CSA-SF high-toughness gel material

1. Degumming 2.55g of anhydrous NaCO₃Adding the mixture into boiled deionized water, pouring 3g of cut silkworm shells after the silkworm shells are completely dissolved, stirring and heating for 30min, taking out the degummed silk, washing with the deionized water for 2-3 times, soaking overnight, taking out, and airing at room temperature.

Preparation of HAp short nanowires

8ml of oleic acid and 10ml of absolute ethanol are first mixed and 147mg of CaCl are then added₂·2H₂O aqueous solution (6ml), followed by addition of 0.5g of NaOH aqueous solution (8ml) and further 156mg of NaH₂PO₄·2H₂O aqueous solution (4ml), stirred for 10 min. Finally, the reaction is carried out for 12 hours in an oven at 180 ℃. And (4) cleaning the obtained product by using absolute ethyl alcohol to obtain the HAp short nanowire.

3. Dissolving and weighing 0.2-1 g of anhydrous calcium chloride, dissolving the anhydrous calcium chloride in 10-15 ml of 88% formic acid, adding 1-2 g of silk into the solution, stirring until the silk is completely dissolved, pouring the silk into a prepared mould, and placing the mould in a fume hood for 3-5 hours until the formic acid is completely volatilized.

4. And (3) curing, namely soaking the gel volatilizing the formic acid in deionized water for half an hour, and then soaking in absolute ethyl alcohol for curing. Preparing into silk fibroin gel.

Preparation of HAp-CSA-SF high-toughness gel Material A (CSA) fibroin formic acid solution with 0%, 4%, 8% and 16% chondroitin sulfate is respectively mixed, the mixed solution is poured into a mold with a prepared HAp short nanowire layer, and after formic acid volatilizes, the composite gel is prepared.

6. Characterization of the material the ultrastructure of the composite material was observed by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM); the physical and chemical properties of the materials were analyzed by XRD (X-ray diffraction), tensile fracture experiments and FTIR (fourier infrared spectroscopy).

(II) in vitro experimental study on the influence of CSA-loaded SF-HAp high-toughness gel material on the biological activity of BMSCs

1. Biocompatibility testing

(1) Bone marrow mesenchymal stem cells (BMSCs) are inoculated on the surface of SF material loaded with CSA with different concentrations, and the cell proliferation condition is detected by CCK-8.

(2) Cell growth on the material was detected by staining with live and dead cells.

2. Detection of osteogenic Properties

(1) BMSCs are respectively inoculated on the HAp face and the SF face of the composite material for mineralization induction, and the expression conditions of osteogenic indexes OPN and OCN are observed through immunofluorescence.

(2) The expression of OPN and OCN at the gene level was detected by qRT-PCR.

3. Detection of chondrogenic Performance

(1) Cells were seeded on SF surfaces loaded with CSA at different concentrations and stained with toluidine blue to observe their chondrogenic effect in vitro.

(2) The expression of cartilage indexes COL2A, ACAN and SOX9 at the gene level is detected by qRT-PCR.

(III) in vivo experiment detection of effects of CSA-loaded SF-HAp high-toughness gel material on chondrogenic differentiation, osteogenic differentiation and articular osteochondral regeneration

1. The animal experiments were divided into five groups, namely a blank control group (negative control), a single SF material (SF group), a CSA-loaded SF material (SF + CSA group), a CSA-free composite material group (SF + HAp) and a CSA-loaded composite material group (SF + CSA + HAp). A defect of 1.6mm in diameter and depth was created in the femoral trochlear of an 8 w-old male SD rat, and the above materials were placed in the defect, respectively. All experimental animals were sacrificed and harvested at 6 weeks post-surgery.

2. Gross specimen observation and evaluation of cartilage regeneration using the international cartilage tissue repair society (ICRS) score.

3. After the rats were sacrificed, the effect of the composite material on the reconstruction of subchondral bone caused by articular cartilage damage was observed by performing micro computed tomography (micro CT) on different groups of articular samples.

The experimental results are as follows:

(I) characterization of materials

SEM results show that the prepared HAp short nanowires are about 5 μm in length and about 15nm in width, and TEM results show that the crystal lattice width is about 0.817nm (FIG. 1). The SF surface of the composite material has certain fluctuation and is relatively compact, which lays a foundation for high toughness; moreover, the cross-sectional view can also show that SF and HAp are tightly and firmly combined; the HAp face of the material has the characteristics of the short nanowires; the macroscopic view of the material shows that the material can be of any size and shape (fig. 2).

XRD results indicate that the composite material shows a characteristic crystal image peak of simple HAp; the stretching-breaking experiment shows that the material is broken when the deformation amount of the material reaches 60 percent, and after HAp is compounded, the tensile force required for the material to generate the same deformation is larger, so that the material is prompted to have better toughness; the FTIR results indicated that the composite material had characteristic peaks for HAp as well as CSA (fig. 3).

(II) in vitro experiments

CCK-8 cell proliferation experiment results show that the SF material loaded with high-concentration (12% and 16%) CSA obviously inhibits cell proliferation; the staining results of the live and dead cells suggest that the SF material and the 8% CSA-SF material have good biocompatibility on the surface compared to the control TCP (fig. 4).

2. The immunofluorescence staining result shows that the HAp surface can obviously promote the expression of OPN and OCN at the protein level; further qRT-PCR results found that HAp significantly promoted the expression of OPN and OCN at the gene level (FIG. 5).

3. Through toluidine blue staining, compared with a control group and other experimental groups, the SF material loaded with 8% chondroitin sulfate enables toluidine blue staining to be more obvious, and chondrogenic characteristics to be more obvious; qRT-PCR results show that compared with a control group, the SF material loaded with 8% chondroitin sulfate obviously promotes the expression of chondrogenic indexes COL2A, ACAN and SOX 9. (FIG. 6)

(III) in vivo experiments

1. Gross observations and ICRS scoring results indicated that SF + CSA + HAp comprised more pronounced cartilage effects than the other groups, with new cartilage filling the defect and integrating with surrounding tissues. (FIG. 7)

Micro-CT results show that the reconstruction effect of subchondral bone of the SF + CSA + HAp group is better, the bone volume fraction (BV/TV), the trabecular bone thickness (Tb.Th) and the trabecular bone number (Tb.N) are all obviously enhanced, and the trabecular bone spacing (Tb.sp) is obviously reduced. (FIG. 8)

In conclusion, the HAp-CSA-SF high-toughness gel material successfully prepared by the embodiment has the effect of osteogenic cartilage formation.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The HAp-CSA-SF composite gel material is characterized by comprising a chondroitin sulfate-silk fibroin composite gel layer and a hydroxyapatite rich layer arranged on at least one end face of the chondroitin sulfate-silk fibroin composite gel layer.

2. The HAp-CSA-SF composite gel material of claim 1, wherein the hydroxyapatite-rich layer is comprised of nano-sized hydroxyapatite, the nano-sized hydroxyapatite being in the form of particles, rods or wires.

3. The HAp-CSA-SF composite gel material of claim 1, wherein the mass ratio of the hydroxyapatite-rich layer to the chondroitin sulfate-silk fibroin composite gel layer is 0.5-2: 1-50.

4. The HAp-CSA-SF composite gel material of claim 1, wherein the mass ratio of chondroitin sulfate to silk fibroin is 100: 0.1 to 20.

5. The preparation method of the HAp-CSA-SF composite gel material of any one of claims 1 to 4, which comprises using an acid-soluble alcohol-solid method, and specifically comprises:

preparation of HAp: mixing oleic acid and ethanol, adding calcium salt solution, adding alkali liquor, adding sodium hydrogen phosphate solution, stirring, and reacting at high temperature to obtain the final product;

6. The preparation method of claim 5, further comprising placing the CSA-SF composite mixed acid solution into a mold carrying HAp, and obtaining the HAp-CSA-SF composite gel material after the acid solution is volatilized and cooled;

preferably, the preparation method further comprises the step of soaking the HAp-CSA-SF composite gel material prepared in the above way in absolute ethyl alcohol for curing treatment.

7. The method of claim 5, wherein the calcium salt is calcium chloride, the alkali solution is sodium hydroxide solution;

preferably, the volume mass ratio of the oleic acid to the ethanol to the calcium chloride to the sodium hydroxide to the sodium hydrogen phosphate is 5-10 ml: 8-15 ml: 0.1-0.5 g: 0.2-1 g: 0.1-0.5 g;

or, the high-temperature reaction strip is reacted for 5-20 hours at 160-200 ℃.

8. The method of claim 5, wherein the fibroin formic acid solution is prepared by: dissolving calcium salt in formic acid solution, and adding silk into the above solution;

preferably, the calcium salt is calcium chloride, and the formic acid is 88% (mass fraction) formic acid solution; the mass ratio of the calcium chloride to the silk is 0.2-1: 1-2; the silk is obtained by degumming silkworm cocoons.

9. Use of the HAp-CSA-SF composite gel material according to any one of claims 1 to 4 or the HAp-CSA-SF composite gel material prepared by the preparation method according to any one of claims 5 to 8 for the preparation of a bone repair material; preferably, the bone repair material is a cartilage repair material.

10. The use according to claim 9, wherein the bone repair material has any one or more of the following uses:

(a) promoting bone/cartilage production;

(b) promoting the reconstruction of subchondral bone tissues;

(c) increasing bone volume fraction;

(d) the thickness of the trabecula is improved;

(e) the number of trabeculae is increased;

(f) the trabecular bone spacing is reduced.