CN103705542A - Decellularized blood vessel matrix gel, preparation method therefor and applications thereof - Google Patents

Abstract

The invention relates to a decellularized vascular matrix gel and its preparation method and application. It is prepared by the following method: a) After removing the adventitia and fascia components of blood vessels, it is cut into small pieces of blood vessels, decellularized in TritonX-100 solution, then freeze-dried, crushed into powder, and filtered through a 60-mesh sieve to obtain decellularized blood vessels. Cellular vascular matrix powder, b) Place the decellularized vascular matrix powder in a hydrochloric acid solution containing 0.09-0.11% pepsin for digestion for 66-78 hours, add sodium hydroxide solution to neutralize the hydrochloric acid, and then add PBS to counter ion concentration , and finally put the gel solution into a 37°C incubator to form a gel. The decellularized vascular matrix gel has good plasticity and mechanical properties, is loose inside, has a certain porosity, has a suitable diameter of collagen fibers, and has good stability. Scaffold material and/or cell culture requirements.

Description

A kind of decellularized vascular matrix gel and its preparation method and application

technical field

The invention relates to the technical field of tissue engineering gel, in particular to a decellularized vascular matrix gel and its preparation method and application.

Background technique

Fibrin gel is a good scaffold material and has always been one of the focuses of tissue engineering research. Fibrin gel is mainly composed of natural extracellular matrix components, has good biocompatibility, effective bioactivity and biodegradability, and also has a three-dimensional porous structure and good plasticity. In recent years, fibrin gel has been widely used in tissue engineering research as a scaffold material, which plays an important role in the formation of muscle tissue, bone and cartilage tissue, epithelial tissue, etc. Sufficient mechanical strength.

Acellular vascular matrix gel is a kind of tissue engineering gel, which can be used in the following fields: 1) It is used for injection to directly treat ischemic diseases: such as lower limb ischemia, myocardial ischemia, etc.; 2) As a cell therapy The carrier and gel are mixed with cells and injected locally, which can locally restrict the flow and loss of cells; 3) It is used as a layer in the in vitro culture of endothelial cells and smooth muscle cells, so as to help cell adhesion, maintain cell shape and 4) It can be used as a carrier for three-dimensional cell culture and induce cell differentiation.

The Chinese journal "Chinese Medical Journal", No. 09, 2004, published a paper "Experimental Research on Constructing Tissue-Engineered Myocardial Tissue Using Liquid Collagen as a Scaffold", which combined liquid type I collagen with matrix factors and isolated and cultured neonatal Rat cardiomyocytes were mixed and annular cardiomyocyte/collagen strips were cast in annular grooves. The Chinese journal "Chinese Tissue Engineering Research and Clinical Rehabilitation", Issue 49, 2008, published a paper "Extracellular matrix gel scaffold mixed with human umbilical vein-derived endothelial cells to construct tissue-engineered endothelial cell sheets", which first took newborn Type I liquid collagen solution was prepared from the tail of SD rats, and then mixed with human umbilical vein endothelial cells to observe the growth process and development of endothelial cells in extracellular matrix gel. Chinese patent document CN200310118986.4, published on November 17, 2004, the title of the invention is "a method for constructing tissue engineered cartilage with bone matrix gel", which first induces cartilage by separating and culturing chondrocytes or stromal stem cells The cells are to obtain seed cells, and then take New Zealand rabbit or human embryo long bone or metaphyseal cancellous bone to construct bone matrix gel BMG, and finally inoculate the seed cells on cortical bone or cancellous bone BMG for in vitro culture to construct tissue engineered cartilage. There are also some reports that obtain fibrinogen and thrombin from the plasma obtained from autologous sources through cryopreservation, centrifugation, precipitation, ion exchange, dialysis, etc. Polymerization occurs, transforms into fibrin, cross-links into a three-dimensional network structure, and forms fibrin gel.

But there is no report about the decellularized vascular matrix gel and its preparation method.

Contents of the invention

The object of the present invention is to provide a decellularized vascular matrix gel aiming at the deficiencies in the prior art.

Another object of the present invention is to provide a preparation method of decellularized vascular matrix gel.

Another object of the present invention is to provide the use of the above-mentioned decellularized vascular matrix gel.

For realizing above-mentioned object, the technical scheme that the present invention takes is:

A decellularized vascular matrix gel, which is prepared by the following method:

a) Preparation of decellularized vascular matrix powder: remove the adventitia and fascia components, cut into small pieces of blood vessels, place in 0.9-1.1% Triton X-100 solution, shake and decellularize, wash with water every other day and replace 0.9- 1.1% Triton X-100 solution; then place the decellularized blood vessel pieces in a -80°C refrigerator for overnight freeze-drying in a vacuum freeze-drying oven until the blood vessel pieces become dry and hard; break into powder and filter through a 60-mesh sieve ;

b) Take the decellularized vascular matrix powder prepared in step a) and place it in 0.009-0.011mol/L hydrochloric acid solution containing pepsin at a concentration of 0.09-0.11%, digest it with magnetic stirring for 66-78 hours, and add it to the sodium hydroxide solution and hydrochloric acid, then add PBS to balance the ion concentration, and finally put the gel solution into a 37°C incubator to form a gel; wherein, the ratio of the decellularized vascular matrix powder to the hydrochloric acid solution is 5-15mg/ml, and the The molar weight of sodium hydroxide in the sodium hydroxide solution is equal to the molar weight of hydrochloric acid in the hydrochloric acid solution, and the ratio of the PBS to the hydrochloric acid solution is 1: (850-950) mol/mL.

Preferably, the acellular vascular matrix gel is prepared by the following method:

a) Preparation of decellularized vascular matrix powder: remove the adventitia and fascia components, cut into small pieces of blood vessels, place them in 1% Triton X-100 solution, shake and decellularize, wash with water every other day and replace with 1% Triton X -100 solution; then freeze the decellularized blood vessel pieces in a -80°C refrigerator overnight, and then freeze-dry them in a vacuum freeze-drying box until the blood vessel pieces become dry and hard; crush them into powder, and filter through a 60-mesh sieve;

b) Place the decellularized vascular matrix powder prepared in step a) in a 0.01mol/L hydrochloric acid solution containing 0.1% pepsin, digest with magnetic stirring for 72 hours, add sodium hydroxide solution to neutralize the hydrochloric acid, and then add PBS Balance the ion concentration, and finally put the gel solution into a 37°C incubator to form a gel; wherein, the ratio of the decellularized vascular matrix powder to the hydrochloric acid solution is 5-15mg/ml, and the hydrogen in the sodium hydroxide solution The molar amount of sodium oxide is equal to the molar amount of hydrochloric acid in the hydrochloric acid solution, and the ratio of the PBS to the hydrochloric acid solution is 1:900mol/mL.

Preferably, the shaking decellularization treatment is specifically performed on a shaker at 37° C. at 200 rpm for 6-7 days.

Preferably, said putting the gel solution into a 37°C incubator to form a gel is specifically putting the gel solution into a 37°C incubator for at least 1 hour.

For realizing above-mentioned second purpose, the technical scheme that the present invention takes is:

A preparation method of decellularized vascular matrix gel, which comprises the following steps:

a) Preparation of decellularized vascular matrix powder: remove the adventitia and fascia components, cut into small pieces of blood vessels, place in 0.9-1.1% Triton X-100 solution, shake and decellularize, wash with water every other day and replace 0.9- 1.1% Triton X-100 solution; then place the decellularized blood vessel pieces in a -80°C refrigerator for overnight freeze-drying in a vacuum freeze-drying oven until the blood vessel pieces become dry and hard; break into powder and filter through a 60-mesh sieve ;

b) Take the decellularized vascular matrix powder prepared in step a) and place it in 0.009-0.011mol/L hydrochloric acid solution containing pepsin at a concentration of 0.09-0.11%, digest it with magnetic stirring for 66-78 hours, and add it to the sodium hydroxide solution and hydrochloric acid, then add PBS to balance the ion concentration, and finally put the gel solution into a 37°C incubator to form a gel; wherein, the ratio of the decellularized vascular matrix powder to the hydrochloric acid solution is 5-15mg/ml, and the The molar weight of sodium hydroxide in the sodium hydroxide solution is equal to the molar weight of hydrochloric acid in the hydrochloric acid solution, and the ratio of the PBS to the hydrochloric acid solution is 1: (850-950) mol/mL.

Preferably, the preparation method of the decellularized vascular matrix gel comprises the following steps:

a) Preparation of decellularized vascular matrix powder: remove the adventitia and fascia components, cut into small pieces of blood vessels, place them in 1% Triton X-100 solution, shake and decellularize, wash with water every other day and replace with 1% Triton X -100 solution; then freeze the decellularized blood vessel pieces in a -80°C refrigerator overnight, and then freeze-dry them in a vacuum freeze-drying box until the blood vessel pieces become dry and hard; crush them into powder, and filter through a 60-mesh sieve;

b) Place the decellularized vascular matrix powder prepared in step a) in a 0.01mol/L hydrochloric acid solution containing 0.1% pepsin, digest with magnetic stirring for 72 hours, add sodium hydroxide solution to neutralize the hydrochloric acid, and then add PBS Balance the ion concentration, and finally put the gel solution into a 37°C incubator to form a gel; wherein, the ratio of the decellularized vascular matrix powder to the hydrochloric acid solution is 5-15mg/ml, and the hydrogen in the sodium hydroxide solution The molar amount of sodium oxide is equal to the molar amount of hydrochloric acid in the hydrochloric acid solution, and the ratio of the PBS to the hydrochloric acid solution is 1:900mol/mL.

Preferably, the shaking decellularization treatment is specifically performed on a shaker at 37° C. at 200 rpm for 6-7 days.

Preferably, said putting the gel solution into a 37°C incubator to form a gel is specifically putting the gel solution into a 37°C incubator for at least 1 hour.

For realizing above-mentioned 3rd purpose, the technical scheme that the present invention takes is:

The use of the decellularized vascular matrix gel as described above, the use is selected from:

a) for the preparation of medicaments for the treatment of ischemic diseases,

b) as a scaffold material for tissue engineering, and/or

c) Preparation of medium for cell culture.

The ischemic disease refers to the disease of organ or tissue ischemia, such as lower extremity ischemia, myocardial ischemia, etc.; the scaffold material used as tissue engineering refers to the carrier used as cell therapy, which is mixed with cells It is used for local injection treatment; the medium for preparing cell culture can be used as a layer in the in vitro culture of endothelial cells and smooth muscle cells, so as to help cell adhesion, maintain cell shape and function, and promote proliferation, etc. , can also be used as a carrier for three-dimensional cell culture, induce cell differentiation, etc., but not limited thereto.

It should be noted that for the above-mentioned "until the blood vessel piece becomes dry and hard", the method is preferably to freeze the decellularized blood vessel piece in a -80°C refrigerator overnight, and then put it in a vacuum freeze-drying box and freeze it at a temperature of about -50°C. Dry around 46-50 hours. For the above pepsin digestion process, magnetic stirring is preferably carried out at 37° C. and 340-360 rpm.

The present invention has the advantage that:

The invention successfully prepares the decellularized vascular matrix gel. The preparation method uses Triton X-100 solution for decellularization, the decellularization effect is thorough, and pure matrix components can be obtained. The concentration of pepsin and the treatment time are reasonable, which can ensure the decellularized blood vessels The protein components in the matrix powder are fully digested to ensure the uniformity of the gel, and the balance ion concentration is appropriate to ensure that the gel can be formed quickly after 1 hour. The prepared acellular vascular matrix gel has good shaping ability, maintains certain mechanical properties, and ensures internal looseness, has certain porosity, suitable diameter of collagen fibers, and good gelation stability, which can meet the requirements of preparation Drugs for the treatment of ischemic diseases, scaffold materials for tissue engineering and/or cell culture requirements.

Description of drawings

Accompanying drawing 1 is the preparation schematic drawing of acellular vascular matrix gel.

Accompanying drawing 2 is the state of decellularized vascular matrix powder during the preparation process of decellularized vascular matrix gel.

Accompanying drawing 3 is the identification of decellularized vascular matrix gel (5mg/ml).

Accompanying drawing 4 is the identification of decellularized vascular matrix gel (10mg/ml).

Accompanying drawing 5 is the identification of decellularized vascular matrix gel (15mg/ml).

Accompanying drawing 6 is the collagen fiber diameter analysis result of decellularized vascular matrix gel.

Accompanying drawing 7 is the change situation of OD value of gel within 60 minutes.

Detailed ways

The specific embodiments provided by the present invention will be described in detail below in conjunction with the accompanying drawings.

Herein, the PBS buffer solution has a pH of 7.2-7.4 and contains the following components: NaCl 137mmol/L, KCl 2.7mmol/L, Na ₂ HPO ₄ 10mmol/L, KH ₂ PO4 2mmol/L. 10×PBS buffer is 0.1M PBS.

Example 1 Preparation and detection of acellular vascular matrix gel (1)

1. Preparation of acellular vascular matrix gel

1. Preparation of acellular vascular matrix powder

Purchase the aorta of adult pigs (about 120kg) from the slaughterhouse, remove the adventitia and fascia, cut it into irregular small pieces of blood vessels, place them in 1% Triton X-100 solution, shake at 37°C, and shake at 200rpm to decellularize After 6.5 days of treatment, wash with water every other day and replace with 1% Triton X-100 solution until the blood vessels turn white. The decellularized blood vessel slices were frozen overnight in a -80°C refrigerator, and then placed in a vacuum freeze-drying oven (Labconco Triad 2.5L, the United States) for freeze-drying at a temperature of -50°C for about 48 hours until the blood vessel slices became dry. Dry and hard. Then use a small pulverizer to crush it into powder, and filter through a 60-mesh sieve.

2. Preparation of acellular vascular matrix gel

Weigh 50, 100, and 150 mg of the decellularized vascular matrix powder prepared in step 1 above and place them in 10 ml of 0.01 mol/L hydrochloric acid solution containing pepsin (P7012-1G, Sigma) at a mass volume concentration of 0.1%, at 37°C , 350 rpm magnetic stirring and digestion for 72 hours, then add 1/10 volume (ie 1ml) of 0.1mol/L sodium hydroxide solution to neutralize hydrochloric acid, then add 1/9 volume (ie 1.111ml) of 10×PBS counter ion concentration. Draw 500 μl of the gel solution into a 2ml syringe with the head cut off, and put it in a 37°C incubator for 1 hour to form the acellular vascular matrix gel.

2. Detection of acellular vascular matrix gel

1. HE staining

The decellularized blood vessel pieces and the decellularized vascular matrix gel formed in the above 2ml syringe were fixed in 4% paraformaldehyde and embedded in paraffin. The sections were dewaxed and hydrated, stained with hematoxylin for 5 minutes, rinsed and differentiated with hydrochloric acid alcohol for a few seconds. Wash with tap water to turn blue for 30 minutes, stain with eosin overnight, dehydrate with ethanol, clear with xylene, and seal with neutral resin.

2. Scanning electron microscope

The acellular vascular matrix gel formed in the above 2ml syringe was fixed with cold 2.5% glutaraldehyde for 24 hours, and then rinsed three times with 1×PBS for 30 minutes each. Then go through gradient ethanol dehydration (30, 50, 70, 90, 100), 30 minutes each time, and finally place in 100% ethanol at 4°C overnight, and rinse with 100% ethanol three times, 30 minutes each time. After drying in a vacuum oven, spray gold with an ion sprayer, then observe and take pictures with a scanning electron microscope (FEI QUANTA 250).

3. Analysis of gel collagen fiber diameter

When calculating the diameter of collagen fibers in the decellularized vascular matrix gel, 100 fibers were randomly selected on the scanning electron microscope image of the decellularized vascular matrix gel, and their diameters were measured by Image software. Finally, the average value and standard deviation were calculated, and Statistical Analysis.

4. Gel absorbance test

Put the decellularized vascular matrix gel samples on ice (including the three kinds of decellularized vascular matrix gels prepared by the above steps, the ratios of decellularized vascular matrix powder and hydrochloric acid solution are 5mg/ml, 10mg/ml, 15mg/ml respectively ) each pipette 10 μl into a 96-well plate, repeat 6 wells for each sample, put it into a multi-functional microplate reader preheated to 37°C for detection (SYNERGY/2 MICROPLATE READER, BioTek), and detect the data under the absorbance of 405nm , read every 2 minutes for 60 minutes.

3. Experimental results

1. Preparation of decellularized vascular matrix powder

The acellular vascular matrix powder was prepared according to the method in the preparation diagram of the acellular vascular matrix gel (Figure 1). The fresh porcine aorta was cut into red (Figure 2A), and the appearance was white after decellularization (Figure 2B). , HE staining showed that the cells were removed completely, and the matrix components remained (Figure 2C). After vacuum freeze-drying, the blood vessels became hard and dry (Figure 2D), and after crushing with a small grinder, they became a uniform white powder (Figure 2E).

2. Formation of acellular vascular matrix gel

Dissolve the above crushed acellular vascular matrix powder in pepsin solution and digest it for 72 hours, and then it becomes milky white and viscous (Figure 2F). Neutralize with sodium hydroxide, adjust the ion concentration and put it in a 37°C incubator for 1 Hours later, a translucent jelly-like semi-solid formed, with good shaping ability (Fig. 3A, 3B, 4A, 4B, 5A, 5B).

3. Gel HE staining

HE staining shows that a network connection with extracellular matrix as the main component is formed inside the acellular vascular matrix gel. This structure not only maintains certain mechanical properties, but also ensures internal looseness and certain porosity (Figure 3C, 4C , 5C).

4. Gel scanning electron microscope

The results of scanning electron microscopy showed that the surface of the acellular vascular matrix gel had a good collagen fiber structure, which was arranged in a disordered grid pattern, and the rest of the matrix components except collagen were distributed in it or attached to the surface of the collagen fibers (Figure 3D, 4D, 5D ).

5. Analysis of gel collagen fiber diameter

According to the statistics, the collagen fiber diameter of 5mg/ml acellular vascular matrix gel is 5.82037nm±1.39185nm, and the diameter of 10mg/ml gel collagen fiber is 5.89368nm±1.25852nm, there is no significant statistical difference between the two (P =0.3532), while the collagen fiber diameter of 15mg/ml gel was 96.27796nm±19.78365nm, which was significantly statistically different from the former two (P=4.15593E-68 and 1.48086E-68) (Figure 6).

6. Detection of gel absorbance at 405nm

According to the absorbance value of the acellular vascular matrix gel at 405nm at 37°C, the acellular matrix gradually becomes turbid and gelatinous, and becomes stable in about 20-30 minutes, and its OD value remains inconvenient until 60 minutes (Fig. 7), indicating good gelation stability.

Example 2 Preparation and detection of acellular vascular matrix gel (2)

1. Preparation of acellular vascular matrix gel

1. Preparation of acellular vascular matrix powder

Purchase the aorta of adult pigs (about 120kg) from the slaughterhouse, remove the adventitia and fascia, cut it into irregular small pieces of blood vessels, place them in 0.9% Triton X-100 solution, shake at 37°C, and shake at 200rpm to decellularize After 7 days of treatment, wash with water and replace with 0.9% Triton X-100 solution every other day until the blood vessel pieces turn white. The decellularized blood vessel slices were frozen overnight in a -80°C refrigerator, and then placed in a vacuum freeze-drying oven (Labconco Triad 2.5L, the United States) for freeze-drying at a temperature of -50°C for about 46 hours until the blood vessel slices became dry. Dry and hard. Then use a small pulverizer to crush it into powder, and filter through a 60-mesh sieve.

2. Preparation of acellular vascular matrix gel

Weigh 50, 100, and 150 mg of the decellularized vascular matrix powder prepared in step 1 above and place them in 10 ml of 0.009 mol/L hydrochloric acid solution containing pepsin (P7012-1G, Sigma) at a mass volume concentration of 0.09%, at 37°C , 360 rpm magnetic stirring and digestion for 78 hours, then add 1/10 volume (ie 1ml) of 0.09mol/L sodium hydroxide solution to neutralize hydrochloric acid, then add 1/8.5 volume (ie 1.176ml) of 10×PBS counter ion concentration. Draw 500 μl of the gel solution into a 2ml syringe with the head cut off, and put it in a 37°C incubator for 1 hour to form the acellular vascular matrix gel.

2. Detection of acellular vascular matrix gel

The detection method is the same as in Example 1.

3. Experimental results

1. Preparation of decellularized vascular matrix powder

The fresh porcine aorta is red after being chopped, and white after removing the cells. HE staining shows that the cells are removed completely, and the matrix components are left. Uniform white powder.

2. Formation of acellular vascular matrix gel

Dissolve the above crushed acellular vascular matrix powder in pepsin solution and digest it for 78 hours, then it becomes milky white and viscous, neutralize it with sodium hydroxide, adjust the ion concentration and put it in a 37°C incubator for 1 hour, then it will be half visible Transparent jelly-like semi-solid form, with good shaping ability.

3. Gel HE staining

HE staining showed that a network connection with extracellular matrix as the main component was formed inside the acellular vascular matrix gel, which had a certain porosity.

4. Gel scanning electron microscope

The results of scanning electron microscopy showed that the surface of the acellular vascular matrix gel had a good collagen fiber structure, which was arranged in a disordered grid pattern, and the rest of the matrix components except collagen were distributed in it or attached to the surface of the collagen fibers.

5. Analysis of gel collagen fiber diameter

According to statistics, the diameter of collagen fibers in 5mg/ml acellular vascular matrix gel is 5.85068nm±1.27134nm, and the diameter of 10mg/ml gel collagen fibers is 5.91672nm±1.30861nm, there is no significant statistical difference between the two, while The diameter of collagen fibers in 15mg/ml gel was 95.87289nm±20.34718nm, which was significantly statistically different from the former two.

6. Detection of gel absorbance at 405nm

According to the absorbance value of the acellular vascular matrix gel at 405nm at 37°C, the acellular matrix gradually becomes turbid and gelatinous, and becomes stable in about 20-30 minutes, and its OD value remains inconvenient until 60 minutes, indicating that The gelling stability is good.

Example 3 Preparation and detection of acellular vascular matrix gel (3)

1. Preparation of acellular vascular matrix gel

1. Preparation of acellular vascular matrix powder

Purchase the aorta of adult pigs (about 120kg) from the slaughterhouse, remove the adventitia and fascia, cut into small pieces of irregular blood vessels, place them in 1.1% Triton X-100 solution, shake at 37°C, and shake at 200rpm to decellularize After 6 days of treatment, wash with water and replace with 1.1% Triton X-100 solution every other day until the blood vessel pieces turn white. The decellularized blood vessel slices were frozen overnight in a -80°C refrigerator, and then placed in a vacuum freeze-drying oven (Labconco Triad 2.5L, the United States) for freeze-drying at a temperature of -50°C for about 50 hours until the blood vessel slices became dry. Dry and hard. Then use a small pulverizer to crush it into powder, and filter through a 60-mesh sieve.

2. Preparation of acellular vascular matrix gel

Weigh 50, 100, and 150 mg of the decellularized vascular matrix powder prepared in step 1 above and place them in 10 ml of 0.011 mol/L hydrochloric acid solution containing pepsin (P7012-1G, Sigma) with a mass volume concentration of 0.11%, at 37 °C , 340 rev/min magnetic stirring for 66 hours after digestion, add 1/10 volume (ie 1ml) of 0.11mol/L sodium hydroxide solution to neutralize hydrochloric acid, then add 1/9.5 volume (ie 1.053ml) of 10×PBS counter ion concentration. Draw 500 μl of the gel solution into a 2ml syringe with the head cut off, and put it in a 37°C incubator for 1.1 hours to form the acellular vascular matrix gel.

2. Detection of acellular vascular matrix gel

The detection method is the same as in Example 1.

3. Experimental results

1. Preparation of decellularized vascular matrix powder

Fresh porcine aorta is red after being chopped, and white after removing the cells. HE staining shows that the cells are removed completely, and the matrix components are left. After vacuum freeze-drying, the blood vessels become hard and dry. Uniform white powder.

2. Formation of acellular vascular matrix gel

Dissolve the above crushed acellular vascular matrix powder in pepsin solution and digest it for 66 hours, then it becomes milky white and viscous, neutralize it with sodium hydroxide, adjust the ion concentration and put it in a 37°C incubator for 1.1 hours. Transparent jelly-like semi-solid form, with good shaping ability.

3. Gel HE staining

HE staining showed that a network connection with extracellular matrix as the main component was formed inside the acellular vascular matrix gel, which had a certain porosity.

4. Gel scanning electron microscope

The results of scanning electron microscopy showed that the surface of the acellular vascular matrix gel had a good collagen fiber structure, which was arranged in a disordered grid pattern, and the rest of the matrix components except collagen were distributed in it or attached to the surface of the collagen fibers.

5. Analysis of gel collagen fiber diameter

According to statistics, the diameter of collagen fibers in 5mg/ml acellular vascular matrix gel is 5.97253nm±1.28240nm, and the diameter of 10mg/ml gel collagen fibers is 5.86259nm±1.29851nm, there is no significant statistical difference between the two, while The diameter of collagen fibers in 15mg/ml gel was 94.99804nm±21.01755nm, which was significantly statistically different from the former two.

6. Detection of gel absorbance at 405nm

According to the absorbance value of the acellular vascular matrix gel at 405nm at 37°C, the acellular matrix gradually becomes turbid and gelatinous, and becomes stable in about 20-30 minutes, and its OD value remains inconvenient until 60 minutes, indicating that The gelling stability is good.

Comparative example 1

1. Preparation of acellular vascular matrix gel

1. Preparation of acellular vascular matrix powder

Purchase the aorta of an adult pig (about 120kg) from a slaughterhouse, remove the outer membrane and fascia, cut it into small pieces of irregular blood vessels, place it in 0.85% Triton X-100 solution, shake at 37°C, shake at 200rpm to decellularize After 7 days of treatment, wash with water every other day and replace with 0.85% Triton X-100 solution until the blood vessel pieces turn white. The decellularized blood vessel slices were frozen overnight in a -80°C refrigerator, and then placed in a vacuum freeze-drying oven (Labconco Triad 2.5L, the United States) for freeze-drying at a temperature of -50°C for about 46 hours until the blood vessel slices became dry. Dry and hard. Then use a small pulverizer to crush it into powder, and filter through a 60-mesh sieve.

2. Preparation of acellular vascular matrix gel

Weigh 50, 100, and 150 mg of the decellularized vascular matrix powder prepared in step 1 above and place them in 10 ml of 0.0009 mol/L hydrochloric acid solution containing pepsin (P7012-1G, Sigma) at a mass volume concentration of 0.09%, at 37°C , 360 rpm magnetic stirring and digestion for 78 hours, then add 1/10 volume (ie 1ml) of 0.009mol/L sodium hydroxide solution to neutralize hydrochloric acid, then add 1/8.5 volume (ie 1.176ml) of 10×PBS counter ion concentration. Draw 500 μl of the gel solution into a 2ml syringe with the head cut off, and put it in a 37°C incubator for 1 hour to form the acellular vascular matrix gel.

2. Detection of acellular vascular matrix gel

The detection method is the same as in Example 1.

3. Experimental results

1. Preparation of decellularized vascular matrix powder

HE staining showed that the cells were not completely removed, and cells were mixed in the matrix components.

Comparative example 2

1. Preparation of acellular vascular matrix gel

1. Preparation of acellular vascular matrix powder

Purchase the aorta of adult pigs (about 120kg) from the slaughterhouse, remove the adventitia and fascia, cut it into irregular small pieces of blood vessels, place them in 1% Triton X-100 solution, shake at 37°C, and shake at 200rpm to decellularize After 6.5 days of treatment, wash with water every other day and replace with 1% Triton X-100 solution until the blood vessels turn white. The decellularized blood vessel slices were frozen overnight in a -80°C refrigerator, and then placed in a vacuum freeze-drying oven (Labconco Triad 2.5L, the United States) for freeze-drying at a temperature of -50°C for about 48 hours until the blood vessel slices became dry. Dry and hard. Then use a small pulverizer to crush it into powder, and filter through a 60-mesh sieve.

2. Preparation of acellular vascular matrix gel

Weigh 45mg and 155mg of the decellularized vascular matrix powder prepared in the above step 1 and place them in 10ml of 0.001mol/L hydrochloric acid solution containing pepsin (P7012-1G, Sigma) with a mass volume concentration of 0.1%, at 37°C, 350 After 72 hours of digestion with magnetic stirring at rpm, add 1/10 volume (ie 1ml) of 0.01mol/L sodium hydroxide solution to neutralize hydrochloric acid, then add 1/9 volume (ie 1.111ml) of 10×PBS counter ion concentration. Draw 500 μl of the gel solution into a 2ml syringe with the head cut off, and put it into a 37°C incubator to form a gel.

2. Detection of acellular vascular matrix gel

The detection method is the same as in Example 1.

3. Experimental results

1. Formation of acellular vascular matrix gel

The above crushed acellular vascular matrix powder was crushed and dissolved in pepsin solution to digest for 72 hours, and after 72 hours of digestion, it became milky white and viscous. Neutralized with sodium hydroxide, adjusted the ion concentration and put it in a 37°C incubator to form a gel. After 5 hours, the treatment with a concentration of 4.5mg/ml still cannot form a jelly-like semi-solid; after 1 hour, the treatment with a concentration of 15.5mg/ml can be seen to form a translucent jelly-like semi-solid, which has good plasticity.

2. Gel HE staining

HE staining showed that the 15.5 mg/ml acellular vascular matrix gel had a large number of particles dispersed inside, indicating that the acellular vascular matrix powder was not completely digested.

Comparative example 3

1. Preparation of acellular vascular matrix gel

1. Preparation of acellular vascular matrix powder

Purchase the aorta of adult pigs (about 120kg) from the slaughterhouse, remove the adventitia and fascia, cut into small pieces of irregular blood vessels, place them in 1.1% Triton X-100 solution, shake at 37°C, and shake at 200rpm to decellularize After 6 days of treatment, wash with water and replace with 1.1% Triton X-100 solution every other day until the blood vessel pieces turn white. The decellularized blood vessel slices were frozen overnight in a -80°C refrigerator, and then placed in a vacuum freeze-drying oven (Labconco Triad 2.5L, the United States) for freeze-drying at a temperature of -50°C for about 50 hours until the blood vessel slices became dry. Dry and hard. Then use a small pulverizer to crush it into powder, and filter through a 60-mesh sieve.

2. Preparation of acellular vascular matrix gel

Weigh 50, 100, and 150 mg of the decellularized vascular matrix powder prepared in step 1 above and place them in 10 ml of 0.011 mol/L hydrochloric acid solution containing pepsin (P7012-1G, Sigma) with a mass volume concentration of 0.11%, at 37 °C , 340 rpm magnetic stirring digestion for 66 hours, then add 1/10 volume (ie 1ml) of 0.11mol/L sodium hydroxide solution to neutralize hydrochloric acid, then add 1/8.4 volume (ie 1.190ml) and 1/9.7 volume respectively (i.e. 1.031ml) of 10×PBS counter ion concentration. Draw 500 μl of the gel solution into a 2ml syringe with the head cut off, and put it into a 37°C incubator to form a gel.

2. Detection of acellular vascular matrix gel

The detection method is the same as in Example 1.

3. Experimental results

1. Formation of acellular vascular matrix gel

After the above treatment was placed in a 37°C incubator for 3 hours, no translucent jelly-like semi-solid was formed, which shows that its shaping ability is poor.

2. Gel scanning electron microscope

The results of scanning electron microscopy showed that the collagen fibers on the surface of the acellular vascular matrix gel were cross-arranged, but there was aggregation, and the distribution of other matrix components except collagen also appeared aggregation.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, some improvements and supplements can also be made, and these improvements and supplements should also be considered Be the protection scope of the present invention.

Claims (9)

1. A decellularized vascular matrix gel, characterized in that it is prepared by the following method: a) Preparation of decellularized vascular matrix powder: remove the adventitia and fascia components, cut into small pieces of blood vessels, place in 0.9-1.1% Triton X-100 solution, shake and decellularize, wash with water every other day and replace 0.9- 1.1% Triton X-100 solution; then place the decellularized blood vessel pieces in a -80°C refrigerator for overnight freeze-drying in a vacuum freeze-drying oven until the blood vessel pieces become dry and hard; break into powder and filter through a 60-mesh sieve ; b) Take the decellularized vascular matrix powder prepared in step a) and place it in 0.009-0.011mol/L hydrochloric acid solution containing pepsin at a concentration of 0.09-0.11%, digest it with magnetic stirring for 66-78 hours, and add it to the sodium hydroxide solution and hydrochloric acid, then add PBS to balance the ion concentration, and finally put the gel solution into a 37°C incubator to form a gel; wherein, the ratio of the decellularized vascular matrix powder to the hydrochloric acid solution is 5-15mg/ml, and the The molar weight of sodium hydroxide in the sodium hydroxide solution is equal to the molar weight of hydrochloric acid in the hydrochloric acid solution, and the ratio of the PBS to the hydrochloric acid solution is 1: (850-950) mol/mL. 2. The decellularized vascular matrix gel according to claim 1, characterized in that it is prepared by the following method: a) Preparation of decellularized vascular matrix powder: remove the adventitia and fascia components, cut into small pieces of blood vessels, place them in 1% Triton X-100 solution, shake and decellularize, wash with water every other day and replace with 1% Triton X -100 solution; then freeze the decellularized blood vessel pieces in a -80°C refrigerator overnight, and then freeze-dry them in a vacuum freeze-drying box until the blood vessel pieces become dry and hard; crush them into powder, and filter through a 60-mesh sieve; b) Place the decellularized vascular matrix powder prepared in step a) in a 0.01mol/L hydrochloric acid solution containing 0.1% pepsin, digest with magnetic stirring for 72 hours, add sodium hydroxide solution to neutralize the hydrochloric acid, and then add PBS Balance the ion concentration, and finally put the gel solution into a 37°C incubator to form a gel; wherein, the ratio of the decellularized vascular matrix powder to the hydrochloric acid solution is 5-15mg/ml, and the hydrogen in the sodium hydroxide solution The molar amount of sodium oxide is equal to the molar amount of hydrochloric acid in the hydrochloric acid solution, and the ratio of the PBS to the hydrochloric acid solution is 1:900mol/mL. 3. The decellularized vascular matrix gel according to claim 1 or 2, characterized in that, the shaking decellularization treatment is specifically performed on a shaker at 37° C. at 200 rpm for 6-7 days. 4. The acellular vascular matrix gel according to claim 1 or 2, characterized in that putting the gel solution into a 37°C incubator to form a gel is specifically putting the gel solution into a 37°C incubator for at least 1 hour. 5. A preparation method of decellularized vascular matrix gel, characterized in that it comprises the following steps: a) Preparation of decellularized vascular matrix powder: remove the adventitia and fascia components, cut into small pieces of blood vessels, place in 0.9-1.1% Triton X-100 solution, shake and decellularize, wash with water every other day and replace 0.9- 1.1% Triton X-100 solution; then place the decellularized blood vessel pieces in a -80°C refrigerator for overnight freeze-drying in a vacuum freeze-drying oven until the blood vessel pieces become dry and hard; break into powder and filter through a 60-mesh sieve ; b) Take the decellularized vascular matrix powder prepared in step a) and place it in 0.009-0.011mol/L hydrochloric acid solution containing pepsin at a concentration of 0.09-0.11%, digest it with magnetic stirring for 66-78 hours, and add it to the sodium hydroxide solution and hydrochloric acid, then add PBS to balance the ion concentration, and finally put the gel solution into a 37°C incubator to form a gel; wherein, the ratio of the decellularized vascular matrix powder to the hydrochloric acid solution is 5-15mg/ml, and the The molar weight of sodium hydroxide in the sodium hydroxide solution is equal to the molar weight of hydrochloric acid in the hydrochloric acid solution, and the ratio of the PBS to the hydrochloric acid solution is 1: (850-950) mol/mL. 6. the preparation method according to claim 5, is characterized in that, it comprises the following steps: a) Preparation of decellularized vascular matrix powder: remove the adventitia and fascia components, cut into small pieces of blood vessels, place them in 1% Triton X-100 solution, shake and decellularize, wash with water every other day and replace with 1% Triton X -100 solution; then freeze the decellularized blood vessel pieces in a -80°C refrigerator overnight, and then freeze-dry them in a vacuum freeze-drying box until the blood vessel pieces become dry and hard; crush them into powder, and filter through a 60-mesh sieve; b) Place the decellularized vascular matrix powder prepared in step a) in a 0.01mol/L hydrochloric acid solution containing 0.1% pepsin, digest with magnetic stirring for 72 hours, add sodium hydroxide solution to neutralize the hydrochloric acid, and then add PBS Balance the ion concentration, and finally put the gel solution into a 37°C incubator to form a gel; wherein, the ratio of the decellularized vascular matrix powder to the hydrochloric acid solution is 5-15mg/ml, and the hydrogen in the sodium hydroxide solution The molar amount of sodium oxide is equal to the molar amount of hydrochloric acid in the hydrochloric acid solution, and the ratio of the PBS to the hydrochloric acid solution is 1:900mol/mL. 7. The preparation method according to claim 5 or 6, characterized in that, the shaking decellularization treatment is specifically performed on a shaker at 37° C. at 200 rpm for 6-7 days. 8. The preparation method according to claim 5 or 6, characterized in that, putting the gel solution into a 37°C incubator to form a gel is specifically putting the gel solution into a 37°C incubator for at least 1 Hour. 9. The application of the decellularized vascular matrix gel according to claim 1 or 2, characterized in that the application is selected from: a) for the preparation of medicaments for the treatment of ischemic diseases, b) as a scaffold material for tissue engineering, and/or c) Preparation of medium for cell culture.

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