CN113082294A

CN113082294A - Preparation method of acellular matrix scaffold and acellular matrix scaffold obtained by same

Info

Publication number: CN113082294A
Application number: CN202110342912.7A
Authority: CN
Inventors: 凌友; 陈俊霖; 梁志豪; 黄斯坦
Original assignee: Guanhao Biotech Co ltd
Current assignee: Guanhao Biotech Co ltd
Priority date: 2021-03-30
Filing date: 2021-03-30
Publication date: 2021-07-09

Abstract

The invention discloses a preparation method of a acellular matrix scaffold and the acellular matrix scaffold obtained by the method, wherein the preparation method comprises the following steps: a crosslinking step: injecting the natural cartilage matrix subjected to acellular treatment into a mold, freeze-drying, performing ultraviolet crosslinking for 4-8h, adding a mixed solution of EDC and NHS, performing crosslinking for 20-24h, cleaning, freeze-drying, and sterilizing to obtain an acellular matrix scaffold; the acellular matrix scaffold is prepared by the preparation method; the method adopts a double-crosslinking mode of combining ultraviolet crosslinking with mixed solution of EDC and NHS and a freeze-drying process, so that the uniformity and compactness of pores on the surface of the scaffold are improved, and the adhesion and growth conditions of cells on the surface of a material can be improved.

Description

Preparation method of acellular matrix scaffold and acellular matrix scaffold obtained by same

Technical Field

The invention relates to a preparation method of a acellular matrix scaffold and the acellular matrix scaffold obtained by the method, and belongs to the technical field of biological materials.

Background

Cartilage tissue is composed of collagen tissue, a few cells, and 60-80% of water, and adult cartilage tissue has no blood vessels or nerves, so that the cartilage tissue has a limited ability to repair itself after injury.

The advent of cartilage tissue engineering has enabled human beings to construct hyaline cartilage in vivo and in vitro by artificial means, providing a promising approach for the repair of articular cartilage defects. The articular cartilage damage repairing mode is that various scaffolds are prepared by some natural materials and synthetic materials and used for cartilage tissue repair, but the natural materials have low biomechanical strength and the synthetic materials have poor biocompatibility. Furthermore, the heterogeneous acellular matrix scaffold is more preferably used, and the preparation method mainly comprises the steps of carrying out acellular molding by using a chemical method, but the method has a general acellular effect and low extraction degree of collagen, and the prepared scaffold still has defects in the aspects of cartilage differentiation, maintenance of the structure and microenvironment with stable hyaline cartilage phenotype.

Disclosure of Invention

In order to overcome the defects of the prior art, the first object of the invention is to provide a preparation method of a acellular matrix scaffold, which adopts a double-crosslinking mode of combining ultraviolet crosslinking with a mixed solution of EDC and NHS and is matched with a freeze-drying process, so that the uniformity and compactness of pores on the surface of the scaffold are improved, and the adhesion and growth conditions of cells on the surface of a material can be improved.

The second purpose of the invention is to provide the acellular matrix scaffold obtained by the preparation method, wherein the acellular matrix scaffold has a good space bionic structure and can well promote cell growth.

The first purpose of the invention can be achieved by adopting the following technical scheme: a method of preparing an acellular matrix scaffold comprising:

a crosslinking step: injecting the natural cartilage matrix subjected to the decellularization treatment into a mold, freeze-drying, performing ultraviolet crosslinking for 4-8h, adding a mixed solution of EDC and NHS, performing crosslinking for 20-24h, cleaning, freeze-drying, and sterilizing to obtain the decellularized matrix scaffold.

Further, in the crosslinking step, the conditions of freeze drying twice are all at the temperature of-60 ℃ for 24-48 h.

Further, in the crosslinking step, a mixed solution of EDC and NHS was used as a solvent with 95 vt% ethanol solution, and the concentration of EDC and NHS was 2 mol/L.

Further, in the crosslinking step, sterilization is performed by irradiation sterilization with cobalt 60.

Further, the method also comprises a cell removing step before the crosslinking step:

pulverizing natural articular cartilage, adding HCl solution, shaking at 4-26 deg.C, mixing, collecting precipitate, and cleaning the precipitate; adding protease solution into the precipitate, stirring at 4-26 deg.C for 96-120h, adding NaOH solution, adjusting to neutral, centrifuging, and collecting supernatant; adding NaCl solution into the supernatant, performing differential centrifugation, removing part of supernatant after each centrifugation, removing all supernatant after the last centrifugation, and taking precipitate; dialyzing the precipitate to obtain natural cartilage matrix after cell removal treatment.

Further, in the cell removing step, the concentration of the HCl solution is 2-4 mol/L; shaking and uniformly mixing for 24-48 h; the concentration of the protease solution is 1-3 g/L; the concentration of the NaOH solution is 1 mol/L; the concentration of the NaCl solution is 1-4 mol/L.

Further, in the cell removal step, the differential centrifugation is the first centrifugation with the rotating speed of 2000-3000r/min, and 50% of the supernatant is removed after the centrifugation; the rotating speed of the second centrifugation is 4000-; the rotating speed of the third centrifugation is 6000-7000r/min, and 50 percent of supernatant is removed after the centrifugation; the rotation speed of the fourth centrifugation is 9000-.

Further, in the step of decellularization, the precipitate is placed into a dialysis bag with the cut-off molecular weight of 8000-14000d for dialysis, and is placed into 20-30 times of water for injection for dialysis for 48-96h, and the water is replaced every 12-24h, so that the natural cartilage matrix after the decellularization treatment is obtained.

The second purpose of the invention can be achieved by adopting the following technical scheme: an acellular matrix scaffold, which is obtained by the preparation method.

Further, the acellular matrix scaffold is circular or polygonal; the support is a porous structure, the aperture is 30-300 μm, and the maximum thickness is 2-3 mm.

Compared with the prior art, the invention has the beneficial effects that:

1. according to the preparation method, the uniformity and compactness of pores on the surface of the scaffold are improved by combining a double-crosslinking mode of a mixed solution of EDC and NHS through ultraviolet crosslinking and matching with a freeze-drying process, the scaffold has a good space bionic structure and good cell compatibility, and the adhesion and growth conditions of cells on the surface of a material can be improved;

2. the preparation method of the invention solves the problems of yellow appearance and crisp physical property of the bracket material for simple ultraviolet crosslinking, and simultaneously solves the problem of incomplete crosslinking of the ultraviolet crosslinking in the material, thereby improving the overall performance of the bracket;

3. the preparation method has high production efficiency, high yield and little damage to the collagen structure, and improves the retention of the total amount of collagen;

4. the acellular matrix scaffold prepared by the preparation method has a good space bionic structure, and can well promote cell growth.

Drawings

FIG. 1 is a surface electron micrograph of a substrate semi-finished product of comparative example 1 magnified 50 times;

FIG. 2 is an electron micrograph of the surface of the substrate holder after UV-crosslinking according to comparative example 1, magnified 50 times;

FIG. 3 is a cross-sectional view of a semi-finished substrate of comparative example 1, enlarged 50 times;

FIG. 4 is a cross-sectional view of the substrate scaffold after UV crosslinking according to comparative example 1, magnified 50 times;

FIG. 5 is a view showing staining of living cells of comparative example 2;

FIG. 6 is a view showing staining of dead cells of comparative example 2;

FIGS. 7-8 are views of the completed acellular matrix scaffolds of example 1;

FIG. 9 is an electron micrograph of the surface of the matrix support of example 1 magnified 50 times;

FIG. 10 is an electron micrograph of the surface of the matrix support of example 1 magnified 100 times;

FIG. 11 is a 20-fold enlarged cross-sectional view of the matrix scaffold of example 1;

FIG. 12 is a view of the completed acellular matrix scaffold of example 2;

FIG. 13 is an electron micrograph of the surface of the matrix support of example 2 magnified 100 times;

FIG. 14 is a cross-sectional view of the matrix scaffold of example 2 enlarged 50 times;

FIG. 15 is a view of the completed acellular matrix scaffold of example 3;

FIG. 16 is an electron micrograph of the surface of the matrix support of example 3 magnified 50 times;

FIG. 17 is a cross-sectional view of the matrix scaffold of example 3 magnified 50 times;

FIG. 18 is a circular dichroism spectrum of examples 1-3;

FIG. 19 is a DNA content detection chart;

FIG. 20 is a view showing staining of cultured living cells in example 1;

FIG. 21 is a view showing staining of dead cells in culture according to example 1;

FIG. 22 is a view showing staining of cultured living cells in example 2;

FIG. 23 is a view showing staining of dead cells in culture according to example 2;

FIG. 24 is a view showing staining of cultured living cells in example 3;

FIG. 25 is a view showing staining of dead cells in culture according to example 3;

FIG. 26 is a 100-fold enlarged view of negative cells;

FIG. 27 is a 100-fold magnified view of positive cells;

FIGS. 28 to 30 are views of cultured cells of examples 1 to 3.

Detailed Description

The invention will be further described with reference to the accompanying drawings and the detailed description below:

a method of preparing an acellular matrix scaffold comprising:

a cell removing step: cleaning and crushing fresh natural articular cartilage, adding 2-4mol/L HCl solution, shaking and uniformly mixing for 24-48h at the temperature of 4-26 ℃, taking precipitate, and cleaning the precipitate with water for injection; adding 1-3g/L protease solution into the precipitate, stirring for 96-120h at 4-26 ℃, then adding 1mol/L NaOH solution to adjust to neutrality, centrifuging at 2000r/min of 1000-; adding 1-4mol/L NaCl solution into the supernatant, wherein the NaCl solution with the concentration has the highest salting-out efficiency, and the problems of poor local salting-out effect, collagen denaturation and the like can be caused by overhigh salt concentration; performing differential centrifugation, wherein the differential centrifugation is the first centrifugation at the rotating speed of 2000-3000r/min, and removing 50% of supernatant after centrifugation; the rotating speed of the second centrifugation is 4000-; the rotating speed of the third centrifugation is 6000-7000r/min, and 50 percent of supernatant is removed after the centrifugation; the rotating speed of the fourth centrifugation is 9000-; dialyzing the precipitate, wherein the dialysis is to put the precipitate into a dialysis bag with the cut-off molecular weight of 8000-14000d for dialysis, put 20-30 times of water for injection for dialysis for 48-96h, and replace the water every 12-24h to obtain the natural cartilage matrix after the cell removal treatment;

through the cell removing step, the extraction degree of the collagen is greatly improved, the production time is shortened, and the purity of the collagen is high;

a crosslinking step: diluting the natural cartilage matrix subjected to the decellularization treatment with water for injection, injecting the diluted natural cartilage matrix into a mold, carrying out ultraviolet crosslinking for 4-8h after freeze drying at the temperature of-60 ℃ for 24-48h, then adding a mixed solution of EDC and NHS, and then carrying out crosslinking for 20-24h, wherein the crosslinking time is controlled to be 20-24h, so that the condition of pore closure caused by excessive crosslinking can be avoided; cleaning, freeze-drying at-60 deg.C for 24-48h, and sterilizing with cobalt 60 by irradiation to obtain acellular matrix scaffold.

Wherein the natural articular cartilage is mammalian articular cartilage, preferably pig articular cartilage.

The mixed solution of EDC and NHS takes 95 vt% ethanol solution as solvent, the concentration of EDC and NHS is 2mol/L, the material obtained by matching with ultraviolet crosslinking at the concentration has moderate performance, and has better pores under the condition of ensuring physical performance;

the acellular matrix scaffold obtained by the method is circular or polygonal; the scaffold has porous structure, pore diameter of 30-300 μm, maximum thickness of 2-3mm, DNA residue less than 100ng/mg, no cytotoxicity, and complete collagen structure of extracellular matrix main component type II.

The problem of uneven pores can be solved by combining ultraviolet crosslinking with double crosslinking of a mixed solution of EDC and NHS, wherein the basic physical properties of the material are ensured by the ultraviolet crosslinking, the defect that the chemical crosslinking of the mixed solution of low-dose EDC and NHS cannot provide enough physical properties is overcome, the defect that the ultraviolet crosslinking cannot act on the inside is overcome by the mixed solution of low-dose EDC and NHS, the criss-cross pore structure in the matrix scaffold is ensured, and the biocompatibility of the material is not reduced; in general, the combination of the two crosslinking modes can reduce the dosage of the two crosslinking modes and ensure the crosslinking quality.

Comparative example 1:

the conventional matrix scaffold is subjected to ultraviolet crosslinking only once, if the dosage is too large, most acidic or basic side chains on the ablated collagen fibers exist, the proinflammatory peptides are possibly released to trigger inflammation and potential collagen denaturation, and the material is yellow and brittle; while crosslinking with a high dose of a mixed solution of EDC and NHS can provide good physical properties without denaturing collagen, chemical crosslinking also affects collagen groups, thereby reducing cell attachment to the material surface.

FIG. 1 is an electron microscope image of the surface of a matrix semi-finished product after freeze-drying by a mold, which is magnified by 50 times, FIG. 2 is an electron microscope image of the surface of a matrix scaffold after only one UV-crosslinking, which shows that a non-uniform pore structure is formed after the UV-crosslinking; fig. 3 is a sectional view of a matrix semi-finished product after mold freeze-drying, which is enlarged by 50 times, fig. 4 is a sectional view of a matrix support after only one-time ultraviolet crosslinking, which is enlarged by 50 times, and fig. 3-4 show that the middle part of the matrix support is not well crosslinked because the matrix support is thick and the ultraviolet crosslinking only affects the surface, and the ultraviolet crosslinking does not work well for the internal structure.

Comparative example 2:

after single EDC and NHS mixed solution is crosslinked for 48h, cell live and dead staining observation is carried out after 72h of planting cells on the surface of the matrix scaffold, wherein green is live cell staining and red is dead cell staining, and from figures 5-6, the matrix scaffold treated by the comparative example 2 has almost no live cells on the surface and most of the cells are dead, so that the crosslinking only by using EDC and NHS mixed solution is known to cause the reduction of the biocompatibility of the material.

Example 1:

a method of preparing an acellular matrix scaffold comprising:

a cell removing step: cleaning and crushing fresh pig articular cartilage, adding 3mol/L HCl solution, shaking and uniformly mixing for 25h at the temperature of 5 ℃, taking a precipitate, and cleaning the precipitate with water for injection; adding 2g/L protease solution into the precipitate, stirring at 5 deg.C for 97h, adding 1mol/L NaOH solution, adjusting to neutrality, centrifuging at 1500r/min, and collecting supernatant; adding 2mol/L NaCl solution into the supernatant; performing differential centrifugation, wherein the differential centrifugation is the first centrifugation at the rotating speed of 2500r/min, and removing 50% of supernatant after centrifugation; the rotating speed of the second centrifugation is 4500r/min, and 50% of supernatant is removed after the centrifugation; the rotating speed of the third centrifugation is 6500r/min, and 50% of supernatant is removed after the centrifugation; the rotating speed of the fourth centrifugation is 9500r/min, all supernatant liquid is removed after the centrifugation, and sediment is taken; dialyzing the precipitate, namely putting the precipitate into a dialysis bag with the cut-off molecular weight of 8500d for dialysis, putting 25 times of water for injection for dialysis for 90h, and replacing water every 12h to obtain natural cartilage matrix subjected to cell removal treatment;

a crosslinking step: diluting the natural cartilage matrix subjected to the decellularization treatment with water for injection, injecting the diluted natural cartilage matrix into a mold, freeze-drying the diluted natural cartilage matrix at the temperature of 60 ℃ below zero for 40 hours, performing ultraviolet crosslinking for 8 hours (performing ultraviolet crosslinking for 4 hours on the front side and the back side respectively), adding a mixed solution of EDC and NHS, and performing crosslinking for 20 hours (performing crosslinking for 10 hours on the front side and the back side respectively); cleaning, freeze-drying at-60 deg.C for 40h, and sterilizing with cobalt 60 by irradiation to obtain acellular matrix scaffold.

The acellular matrix scaffold obtained in example 1 can be prepared into a square or round shape as shown in FIGS. 7-8, and it can be seen that the scaffold has a white color and no yellowing phenomenon; FIGS. 9 to 10 are the surface electron micrographs of the substrate scaffold magnified 50 times and 100 times, respectively; FIG. 11 is a cross-sectional view of the matrix scaffold magnified 20 times and having uniform pore distribution and pore size.

Example 2:

a method of preparing an acellular matrix scaffold comprising:

a cell removing step: cleaning and crushing fresh pig articular cartilage, adding 3mol/L HCl solution, shaking and uniformly mixing for 25h at the temperature of 15 ℃, taking a precipitate, and cleaning the precipitate with water for injection; adding 2g/L protease solution into the precipitate, stirring at 15 deg.C for 96-120h, adding 1mol/L NaOH solution, adjusting to neutrality, centrifuging at 1950r/min, and collecting supernatant; adding 2mol/L NaCl solution into the supernatant; performing differential centrifugation, wherein the differential centrifugation is the first centrifugation at the rotating speed of 2950r/min, and removing 50% of supernatant after centrifugation; the rotation speed of the second centrifugation is 4950r/min, and 50% of supernatant is removed after the centrifugation; the rotating speed of the third centrifugation is 6950r/min, and 50% of supernatant is removed after the centrifugation; centrifuging for the fourth time at 10000r/min, removing all supernatant, and collecting precipitate; dialyzing the precipitate, namely putting the precipitate into a dialysis bag with the cut-off molecular weight of 8500d for dialysis, putting 20-30 times of water for injection for dialysis for 48h, and replacing water every 12-24h to obtain natural cartilage matrix subjected to cell removal treatment;

a crosslinking step: diluting the natural cartilage matrix subjected to the decellularization treatment with water for injection, injecting the diluted natural cartilage matrix into a mold, freeze-drying the diluted natural cartilage matrix at the temperature of 60 ℃ below zero for 45 hours, performing ultraviolet crosslinking for 8 hours (performing ultraviolet crosslinking for 4 hours on the front side and the back side respectively), adding a mixed solution of EDC and NHS, and performing crosslinking for 22 hours (performing crosslinking for 11 hours on the front side and the back side respectively); cleaning, freeze-drying at-60 deg.C for 30h, and sterilizing with cobalt 60 by irradiation to obtain acellular matrix scaffold.

The acellular matrix scaffold obtained in example 2 is shown in FIG. 12, which shows that the scaffold is white in color and has no yellowing phenomenon; FIG. 13 is an electron micrograph of the surface of the stromal scaffold magnified 100 times; FIG. 14 is a cross-sectional view of the matrix scaffold magnified 50 times and the pore size and pore distribution of the matrix scaffold are uniform.

Example 3:

a method of preparing an acellular matrix scaffold comprising:

a cell removing step: cleaning and crushing fresh pig articular cartilage, adding 3mol/L HCl solution, shaking and uniformly mixing for 46h at 25 ℃, taking a precipitate, and cleaning the precipitate with water for injection; adding 2g/L protease solution into the precipitate, stirring at 25 deg.C for 115h, adding 1mol/L NaOH solution, adjusting to neutrality, centrifuging at 1950r/min, and collecting supernatant; adding 2mol/L NaCl solution into the supernatant; performing differential centrifugation, wherein the differential centrifugation is the first centrifugation at the rotating speed of 2050r/min, and removing 50% of supernatant after centrifugation; the rotation speed of the second centrifugation is 4950r/min, and 50% of supernatant is removed after the centrifugation; the rotating speed of the third centrifugation is 6950r/min, and 50% of supernatant is removed after the centrifugation; the rotating speed of the fourth centrifugation is 11500r/min, all supernatant is removed after the centrifugation, and sediment is taken; dialyzing the precipitate, namely filling the precipitate into a dialysis bag with the cut-off molecular weight of 13000d for dialysis, putting 20-30 times of water for injection for dialysis for 95h, and replacing water every 24h to obtain the natural cartilage matrix subjected to cell removal treatment;

a crosslinking step: diluting the natural cartilage matrix subjected to the decellularization treatment with water for injection, injecting the diluted natural cartilage matrix into a mold, freeze-drying the diluted natural cartilage matrix at the temperature of 60 ℃ below zero for 46 hours, performing ultraviolet crosslinking for 8 hours (performing ultraviolet crosslinking for 4 hours on the front side and the back side respectively), adding a mixed solution of EDC and NHS, and performing crosslinking for 22 hours (performing crosslinking for 11 hours on the front side and the back side respectively); cleaning, freeze-drying at-60 deg.C for 48h, and sterilizing with cobalt 60 by irradiation to obtain acellular matrix scaffold.

The acellular matrix scaffold obtained in example 3 is shown in FIG. 15, which shows that the scaffold is white in color and has no yellowing phenomenon; FIG. 16 is an electron micrograph of the surface of the stromal scaffold at 50 times magnification; FIG. 17 is a cross-sectional view of the matrix scaffold magnified 50 times and the pore size and pore distribution of the matrix scaffold were uniform.

Examples 1-3 show the same circular dichroism spectrum results, as shown in FIG. 18, the circular dichroism spectrum shows that the secondary structure of type II collagen in the matrix scaffold remains intact, thereby illustrating that examples 1-3 have no effect on type II collagen.

FIG. 19 is a graph showing DNA content detection of the raw material and the matrix scaffold of examples 1 to 3, and examples 1 to 3 all well removed DNA from the raw material. (represents P < 0.01)

The cells were stained after 72h of culture on the matrix scaffolds of examples 1-3, respectively, wherein green is staining of live cells and red is staining of dead cells; FIGS. 20-21 are staining of cells cultured on example 1, FIGS. 22-23 are staining of cells cultured on example 2, and FIGS. 24-25 are staining of cells cultured on example 3; the cells on the material are in a fusiform or oval shape for normal growth and reproduction, and few dead cells exist.

The substrate scaffolds of examples 1-3 were cultured in a complete medium of 3cm²PermL 5 vt% CO at 37 ℃²Placing in an incubator for 24h for leaching, and collecting supernatant for later use after leaching is finished. Cell morphology (bar 100 μm) was co-cultured with cells for 48 h. FIGS. 26 to 30 are views of negative cells, positive cells, and cells cultured in examples 1 to 3 magnified 100 times, respectively, and the matrix scaffolds obtained in examples 1 to 3 have no potential cytotoxicity and good biocompatibility.

Various other changes and modifications to the above-described embodiments and concepts will become apparent to those skilled in the art from the above description, and all such changes and modifications are intended to be included within the scope of the present invention as defined in the appended claims.

Claims

1. A method for preparing an acellular matrix scaffold, which is characterized by comprising the following steps:

2. The method for preparing an acellular matrix scaffold according to claim 1, wherein in the cross-linking step, the conditions of freeze-drying are both at a temperature of-60 ℃ for 24-48 h.

3. The method for preparing an acellular matrix scaffold according to claim 1, wherein the mixed solution of EDC and NHS in the crosslinking step is 95 vt% ethanol solution as a solvent, and the concentration of EDC and NHS is 2 mol/L.

4. The method for preparing an acellular matrix scaffold according to claim 1, wherein in the step of crosslinking, the sterilization is irradiation sterilization with cobalt 60.

5. The method of preparing an acellular matrix scaffold according to claim 1, wherein the step of cross-linking further comprises, prior to the step of decellularizing:

6. The method for preparing an acellular matrix scaffold according to claim 5, wherein in the acellular step, the concentration of the HCl solution is 2 to 4 mol/L; the shaking and uniform mixing time is 24-48 h; the concentration of the protease solution is 1-3 g/L; the concentration of the NaOH solution is 1 mol/L; the concentration of the NaCl solution is 1-4 mol/L.

7. The method for preparing a acellular matrix scaffold according to claim 5, wherein in the acellular step, the differential centrifugation is the first centrifugation at a rotation speed of 2000-3000r/min, and 50% of the supernatant is removed after the centrifugation; the rotating speed of the second centrifugation is 4000-; the rotating speed of the third centrifugation is 6000-7000r/min, and 50 percent of supernatant is removed after the centrifugation; the rotation speed of the fourth centrifugation is 9000-.

8. The method for preparing a acellular matrix scaffold according to claim 5, wherein in the step of acellular processing, the precipitate is placed into a dialysis bag with a cut-off molecular weight of 8000-14000d for dialysis, and then placed into 20-30 times of water for injection for dialysis for 48-96h, and the water is replaced every 12-24h to obtain the acellular processed natural cartilage matrix.

9. An acellular matrix scaffold, characterized in that it is obtained by the method of preparation according to claim 1.

10. The acellular matrix scaffold according to claim 9, wherein the acellular matrix scaffold is circular or polygonal; the support is of a porous structure, the aperture is 30-300 mu m, and the maximum thickness is 2-3 mm.