CN103656749B - A kind of compound degradable antibacterial artificial dura mater and preparation method thereof - Google Patents

Abstract

A composite degradable antibacterial artificial dura mater, including vancomycin or ofloxacin hydrochloride injection, chitosan and sodium β-glycerophosphate. Collagen biofilm scaffolds were mixed with 2% (w/v) chitosan solution and 56% (w/v) β-glycerophosphate sodium solution, according to the complex of chitosan and β-glycerophosphate sodium solution at 37 To study the temperature-sensitive characteristics of hydrogel formation, add vancomycin or ofloxacin hydrochloride injection into the complex solution of chitosan and β-glycerophosphate sodium solution into the prepared spongy collagen biofilm scaffold The suture-free dura mater was placed in a 37°C incubator for 10 minutes, and the hydrogel complex was evenly immersed in the spongy collagen biofilm scaffold to form an absorbable artificial dura mater with antibacterial activity. And provide the preparation method. It has the characteristics of convenient production, easy film formation, low cost and convenient operation.

Description

A composite degradable antibacterial artificial dura mater and its preparation method

technical field

The invention belongs to the technical field of biomaterials, and belongs to the biomaterials available to the public, in particular to a composite degradable antibacterial artificial dura mater and a preparation method thereof.

Background technique

Chitosan, English chitosan, is a copolymer of p(1-4)-2-acetylamino-2-deoxy-D dextran and 2-amino-2-deoxy-D dextran. Biodimers are mainly obtained by deacetylating chitin. Due to the wide range of sources, non-toxicity, good tissue compatibility, biodegradability and adhesion, the material has been deeply studied and widely used in the fields of medicine and biology. Chenite et al. (See CheniteA, ChaputC, WangD, et al.Novelinjectableneutralsolutionsofchitosanformbiodegradablegelsinsitu[J].Biomaterials, 2000,21(21):2155-2161.) After neutralizing chitosan with sodium glycerophosphate, neutral pH value and Chitosan/β, which can remain liquid for a long time at room temperature, forms a hydrogel, and has the characteristics of non-carcinogenic, non-teratogenic, and non-mutagenic. After sterilization, it has become an injectable non-intravenous drug and is widely used in Tissue engineering has become a drug carrier and cell scaffold material.

As a dura mater substitute material, biological substitute materials have the advantages of safe and effective use, easy operation, flexibility, wide range of sources, low price, etc., and good biological adaptability. Bovine tendon, pig pericardium, bovine pericardium, sheep pericardium, bovine peritoneum, porcine peritoneum, and mesentery are often used instead of dura mater. But at the same time, it has the disadvantages of causing adhesion, not easy to preserve, not easy to disinfect, and possible immune reaction.

An ideal artificial dura mater should have the following characteristics: ①No immune rejection, no toxic reaction, no carcinogenic and teratogenic effects; ②Extensible, flexible, and able to prevent and treat infection; ③Suture or paste can prevent cerebrospinal fluid leakage , effectively protect the brain tissue, without adhesion to the brain tissue, and without scarring; ④After the formation of new meningoid-like tissue, it can be completely absorbed by the body; ⑤The material source is convenient and easy to store; ⑥The price is relatively moderate.

The artificial dura mater with many clinical applications is mostly imported, such as DuraMax, NormalGEN, DuraGen, and Durascaffold, which are expensive and there are many Cases of intracranial infection and rejection. There are also many patients with cerebrospinal fluid leakage, all of which cannot fully meet the above requirements.

The present invention aims to develop a dura mater substitute material more suitable for clinical needs.

Contents of the invention

The purpose of the present invention is to substantially improve and innovate the prior art, to provide a composite degradable antibacterial artificial dura mater, and to provide a preparation method thereof. The preparation method has the characteristics of convenient production, easy film formation, low cost, convenient operation and the like. The prepared composite degradable antibacterial artificial dura mater provides a spongy collagen biofilm scaffold for the dura mater defect site, which is conducive to the growth of fibroblasts and the development of new dura mater tissue. Star’s temperature-sensitive chitosan/β-sodium glycerophosphate complex is combined at 37°C to prepare a compound with good tissue compatibility and degradability, which can effectively reduce intracranial infection and cerebrospinal fluid leakage, and at the same time A new composite artificial dura mater that also reduces scarring between brain tissue and adjacent tissue.

The object of the present invention is achieved by the following technical solutions: a composite degradable antibacterial artificial dura mater, comprising vancomycin or ofloxacin hydrochloride injection, chitosan and β-sodium glycerophosphate, its characteristics Yes: make a sponge-like collagen biofilm scaffold from decellularized membranous bio-derived materials, apply 8ml of 2% (w/v) chitosan solution and 1ml of 56% (w/v) sodium β-glycerophosphate solution, according to Chitosan and β-sodium glycerophosphate solution complexes form the temperature-sensitive characteristic of hydrogel at 37 ℃, with 0.1g vancomycin or 0.1g ofloxacin hydrochloride injection 1ml, add chitosan and The solution of β-glycerophosphate sodium solution complex is immersed in the suture-free dura mater of the prepared spongy collagen biofilm scaffold, and placed in a 37°C incubator for 10 minutes, and the hydrogel composite is evenly immersed in the spongy collagen biofilm scaffold to form Absorbable artificial dura mater with antimicrobial activity.

The decellularized membranous bio-derived material includes any one of bovine tendon, porcine pericardium, bovine pericardium, sheep pericardium, bovine peritoneum, porcine peritoneum, bovine intestine, pig intestine and sheep intestine.

A preparation method of a composite degradable antibacterial artificial dura mater is characterized in that it comprises the following steps:

1) Preparation of sponge-like collagen biofilm scaffolds; prepared by freeze-drying collagen membranes, with a porous and loose structure, good penetration and water absorption capabilities, using decellularized membrane-like bio-derived materials placed in 75% ethanol solution and over Soak it in oxyacetic acid for 2 hours, rinse it repeatedly with phosphate buffer for 3 times; soak it in sodium hydroxide solution for 2 hours, then rinse it with phosphate buffer until pH7.4; put it in -80℃ cryogenic solution Freeze in nitrogen for 24 hours, freeze-dry in a vacuum freeze dryer for 24 hours, crush in a liquid nitrogen freezer for 40 minutes, take collagen particles with a particle size of 80 μm or less through a No. 9 sieve, and mix collagen particles with 0.5% trypsin and 0.2% Mix acetic acid and EDTA at a volume ratio of 1:5, oscillate in a refrigerator at 4°C for 24 hours, centrifuge at 5000 r/min for 20 minutes with a centrifugal radius of 8 cm, and discard the supernatant; wash repeatedly with phosphate buffer for 3 times, and discard the supernatant after centrifugation as above After centrifugation, mix the collagen microparticles with 1mol/L glacial acetic acid in a volume ratio of 1:5, and mix the collagen microparticles with pepsin in a mass ratio of 100:1, and shake continuously in a refrigerator at 4°C for 24 hours to remove the non-helical regions of collagen molecules in the collagen microparticles. , adjust the pH value to 7.4 with 1mol/L sodium hydroxide, centrifuge at 5000r/min for 20min with a centrifugal radius of 8cm; discard the supernatant liquid, wash with triple distilled water repeatedly for 3 times, and centrifuge with a centrifugal radius of 8cm for 20min at 5000r/min; Discard the supernatant liquid, and place the precipitate in a special cylindrical mold with specifications of 6cm×6cm; 8cm×8cm; 10cm×10cm; 12cm×120cm, with a thickness of 2-4 mm, freeze-dried with a vacuum freeze dryer, -80 After freezing in liquid nitrogen at low temperature for 24 hours, place it in a freeze dryer for 24 hours and take it out to obtain acellular sponge-like collagen biofilm, vacuum pack it, sterilize it with ethylene oxide, and store it in a refrigerator at 4°C for later use;

2) Preparation of chitosan/β-sodium glycerophosphate complex: take 0.43ml of 36% concentrated hydrochloric acid, add three-distilled water to a total liquid volume of 50ml, take 0.43ml of hydrochloric acid and add three-distilled water to a 50ml volumetric flask, add The volume of three distilled water is 50 ml, and it is prepared into 0.1M dilute hydrochloric acid. The specifications of chitosan are: viscosity 150mPa. dilute hydrochloric acid, fully stirred with a magnetic stirrer at room temperature at 23°C for 30 minutes, at a speed of 450r/min, and mixed to make a total of 100ml solution, prepared as a 2% (w/v) chitosan solution, and sterilized under high pressure at 121°C for 30min , Store in refrigerator at 4°C for later use, prepare 56g of β-glycerophosphate sodium solution with 56% (w/v) sodium β-glycerophosphate solution, add triple distilled water to dissolve completely, set the volume to 100ml, filter and sterilize, autoclave at 121°C for 30 minutes, 4 Store in the refrigerator at ℃ for later use, prepare 2% (w/v) chitosan and 56% (w/v) sodium β-glycerophosphate at a ratio of 8:1, the pH value is 7.25, and the time to become a hydrogel in a 37°C water bath is 6 minutes;

3) Take 8ml of 2% (w/v) chitosan solution: 1ml of 56% (w/v) sodium β-glycerophosphate solution: 0.1g vancomycin or 0.1g ofloxacin hydrochloride injection lml, the three After uniform mixing at 4°C, the decellularized sponge-like collagen biofilm was immersed in the above mixed solution, and placed in a 37°C incubator for 10 minutes, and the mixed solution formed a hydrogel and integrated with the collagen biofilm.

A composite degradable antibacterial artificial dura mater of the present invention is formed by evenly soaking the spongy collagen biofilm with a hydrogel compound, because there is no need for suturing during the operation due to the presence of the hydrogel, and it has a good connection with the tissue Compatibility, experiments have proved that no cerebrospinal fluid leakage occurs; the vancomycin or ofloxacin hydrogel in the hydrogel is slowly released during the gradual degradation of the hydrogel, achieving the purpose of preventing and treating intracranial infection; the degradation speed of the spongy collagen membrane Similar to the growth rate of the human body's own dura mater, it has good absorbability and histological compatibility; the material has a wide range of sources, low price, safe use, and long life.

The preparation method of the composite degradable antibacterial artificial dura mater of the present invention is scientific and reasonable, the preparation process is easy to control, the film forming quality is good, and the biological characteristics are good.

detailed description

The present invention will be further described below in conjunction with embodiment.

A composite degradable antibacterial artificial dura mater, including vancomycin or ofloxacin hydrochloride injection, chitosan and sodium β-glycerophosphate, characterized in that the decellularized membrane-like biological derivative material is made into a sponge Collagen biofilm scaffold, apply 2% (w/v) chitosan solution 8ml, 56% (w/v) β-sodium glycerophosphate solution 1ml mixes, according to chitosan and β-sodium glycerophosphate solution complex in The temperature sensitivity of hydrogel formation at 37°C, 0.1g vancomycin or 0.1g ofloxacin hydrochloride injection 1ml is added to the solution of chitosan and β-sodium glycerophosphate solution and immersed in the prepared The suture-free dura mater of the spongy collagen biofilm scaffold was placed in a 37°C incubator for 10 minutes, and the hydrogel complex was evenly immersed in the spongy collagen biofilm scaffold to form an absorbable artificial dura mater with antibacterial activity.

The decellularized membranous bio-derived material includes any one of bovine tendon, porcine pericardium, bovine pericardium, sheep pericardium, bovine peritoneum, porcine peritoneum, bovine intestine, pig intestine and sheep intestine.

Through the specific implementation of 9 embodiments, the present invention uses bovine tendon, porcine pericardium, bovine pericardium, sheep pericardium, bovine peritoneum, porcine peritoneum, bovine intestine, pig intestine and sheep intestine to treat the composite degradable antibacterial artificial dura mater. Preparations were all successful.

Example 1: Taking bovine tendon as one of the decellularized membranous biologically derived materials as an example to illustrate its specific composite degradable antibacterial artificial dura mater preparation method, the other decellularized membranous biologically derived materials are the same as in Example 1 : Include the following steps:

1) Preparation of sponge-like collagen biofilm scaffolds; prepared by freeze-drying collagen membranes, with a porous and loose structure, and good penetration and water absorption capabilities. Bovine tendon, one of the decellularized membrane-like bio-derived materials, was placed at 75 % ethanol solution and peracetic acid for 2 hours, rinsed repeatedly with phosphate buffer for 3 times; soaked in sodium hydroxide solution for 2 hours, then rinsed with phosphate buffer until pH7.4; Freeze in low-temperature liquid nitrogen at -80°C for 24 hours, freeze-dry in a vacuum freeze dryer for 24 hours, pulverize with a liquid nitrogen freezer pulverizer for 40 minutes, take collagen particles with a particle size of 80 μm or less through a No. 9 sieve, mix collagen particles with 0.5% trypsin, 0.2 %ethylenediaminetetraacetic acid (EDTA) was mixed at a volume ratio of 1:5, oscillated in a refrigerator at 4°C for 24 hours, centrifuged at 5000r/min for 20min with a centrifugal radius of 8cm, discarded the supernatant liquid; washed repeatedly with phosphate buffer for 3 times, and centrifuged as above Discard the supernatant, mix the centrifuged collagen microparticles with 1mol/L glacial acetic acid at a volume ratio of 1:5, and mix the collagen microparticles with pepsin at a mass ratio of 100:1, and shake continuously at 4°C for 24 hours to remove the collagen in the collagen microparticles. For the non-helical region of the molecule, adjust the pH value to 7.4 with 1mol/L sodium hydroxide, centrifuge at 5000r/min for 20min with a centrifugal radius of 8cm; /min centrifuge for 20min; discard the supernatant, and place the sediment in a special cylindrical mold with specifications of 6cm×6cm; 8cm×8cm; 10cm×10cm; 12cm×120cm, with a thickness of 2-4mm. Freeze-dried, frozen in -80°C low-temperature liquid nitrogen for 24 hours, placed in a freeze dryer for 24 hours and taken out to obtain acellular sponge-like collagen biofilm, vacuum-packed, ethylene oxide sterilized, and stored in a 4°C refrigerator for later use;

2) Preparation of chitosan/β-sodium glycerophosphate complex: take 0.43ml of 36% concentrated hydrochloric acid, add three-distilled water to a total liquid volume of 50ml, take 0.43ml of hydrochloric acid and add three-distilled water to a 50ml volumetric flask, add The volume of three distilled water is 50 ml, and it is prepared into 0.1M dilute hydrochloric acid. The specifications of chitosan are: viscosity 150mPa. dilute hydrochloric acid, fully stirred with a magnetic stirrer at room temperature at 23°C for 30 minutes, at a speed of 450r/min, and mixed to make a total of 100ml solution, prepared as a 2% (w/v) chitosan solution, and sterilized under high pressure at 121°C for 30min , Store in refrigerator at 4°C for later use, prepare 56g of β-glycerophosphate sodium solution with 56% (w/v) sodium β-glycerophosphate solution, add triple distilled water to dissolve completely, set the volume to 100ml, filter and sterilize, autoclave at 121°C for 30 minutes, 4 Store in the refrigerator at ℃ for later use, prepare 2% (w/v) chitosan and 56% (w/v) sodium β-glycerophosphate at a ratio of 8:1, the pH value is 7.25, and the time to become a hydrogel in a 37°C water bath is 6 minutes;

3) Take 8ml of 2% (w/v) chitosan solution: 1ml of 56% (w/v) sodium β-glycerophosphate solution: 0.1g vancomycin or 0.1g ofloxacin hydrochloride injection lml, the three After uniform mixing at 4°C, the decellularized sponge-like collagen biofilm was immersed in the above mixed solution, and placed in a 37°C incubator for 10 minutes, and the mixed solution formed a hydrogel and integrated with the collagen biofilm.

The composite degradable antibacterial artificial dura mater made by a kind of composite degradable antibacterial artificial dura mater preparation method of the present invention, after 1-10 month's use of 30 cases groups, the effect is good, has realized the purpose of the present invention and achieved the stated effect.

Claims (3)

1. A compound degradable antibacterial artificial dura mater, comprising vancomycin or ofloxacin hydrochloride injection, chitosan and sodium β-glycerophosphate, characterized in that: the decellularized membrane-like biological derivative material is made Spongy collagen biofilm scaffold, mixed with 8ml of 2% (w/v) chitosan solution and 1ml of 56% (w/v) β-glycerophosphate sodium solution, according to the compounding of chitosan and β-glycerophosphate sodium solution The temperature sensitivity characteristics of the hydrogel formed by the substance at 37°C, 0.1g vancomycin or 0.1g ofloxacin hydrochloride injection 1ml was added to the solution of chitosan and β-sodium glycerophosphate solution complex and immersed in the already The suture-free dura mater of the prepared spongy collagen biofilm scaffold was placed in a 37°C incubator for 10 minutes, and the hydrogel complex was evenly immersed in the spongy collagen biofilm scaffold to form an absorbable artificial dura mater with antibacterial activity. 2. A kind of composite degradable antibacterial artificial dura mater according to claim 1, characterized in that: the decellularized membranous biological derivative material comprises bovine tendon, pig pericardium, bovine pericardium, sheep pericardium, bovine peritoneum, porcine peritoneum, Any one of beef intestines, pig intestines and sheep intestines. 3. the preparation method of a kind of composite degradable antibacterial artificial dura mater according to claim 1, is characterized in that, it comprises the following steps: 1) Preparation of sponge-like collagen biofilm scaffolds; prepared by freeze-drying collagen membranes, with a porous and loose structure, good penetration and water absorption capabilities, using decellularized membrane-like bio-derived materials placed in 75% ethanol solution and over Soak it in oxyacetic acid for 2 hours, rinse it repeatedly with phosphate buffer for 3 times; soak it in sodium hydroxide solution for 2 hours, then rinse it with phosphate buffer until pH7.4; put it in -80℃ cryogenic solution Freeze in nitrogen for 24 hours, freeze-dry in a vacuum freeze dryer for 24 hours, crush in a liquid nitrogen freezer for 40 minutes, take collagen particles with a particle size of 80 μm or less through a No. 9 sieve, and mix collagen particles with 0.5% trypsin and 0.2% Mix acetic acid and EDTA at a volume ratio of 1:5, oscillate in a refrigerator at 4°C for 24 hours, centrifuge at 5000 r/min for 20 minutes with a centrifugal radius of 8 cm, and discard the supernatant; wash repeatedly with phosphate buffer for 3 times, and discard the supernatant after centrifugation as above After centrifugation, mix the collagen microparticles with 1mol/L glacial acetic acid in a volume ratio of 1:5, and mix the collagen microparticles with pepsin in a mass ratio of 100:1, and shake continuously in a refrigerator at 4°C for 24 hours to remove the non-helical regions of collagen molecules in the collagen microparticles. , adjust the pH value to 7.4 with 1mol/L sodium hydroxide, centrifuge at 5000r/min for 20min with a centrifugal radius of 8cm; discard the supernatant liquid, wash with triple distilled water repeatedly for 3 times, and centrifuge with a centrifugal radius of 8cm for 20min at 5000r/min; Discard the supernatant liquid, and place the precipitate in a special cylindrical mold with specifications of 6cm×6cm; 8cm×8cm; 10cm×10cm; 12cm×120cm, with a thickness of 2-4 mm, freeze-dried with a vacuum freeze dryer, -80 After freezing in liquid nitrogen at low temperature for 24 hours, place it in a freeze dryer for 24 hours and take it out to obtain acellular sponge-like collagen biofilm, vacuum pack it, sterilize it with ethylene oxide, and store it in a refrigerator at 4°C for later use; 2) Preparation of chitosan/β-sodium glycerophosphate complex: take 0.43ml of 36% concentrated hydrochloric acid, add three-distilled water to a total liquid volume of 50ml, take 0.43ml of hydrochloric acid and add three-distilled water to a 50ml volumetric flask, add The volume of three distilled water is 50 milliliters, and it is prepared into 0.1M dilute hydrochloric acid. The specifications of chitosan are: viscosity 150mPa. Dilute hydrochloric acid, fully stir with a magnetic stirrer at room temperature at 23°C for 30 minutes, and rotate at a speed of 450r/min, mix to make a total of 100ml solution, prepare a 2% (w/v) chitosan solution, and sterilize under high pressure at 121°C for 30 minutes, Store in the refrigerator at 4°C for later use, prepare 56% (w/v) sodium β-glycerophosphate solution, add 56g sodium glycerophosphate, add triple distilled water to dissolve completely, set the volume to 100ml, sterilize by filtration, and sterilize under high pressure at 121°C for 30 minutes, 4 Store in the refrigerator at ℃ for later use, prepare 2% (w/v) chitosan and 56% (w/v) sodium β-glycerophosphate at a ratio of 8:1, the pH value is 7.25, and the time to become a hydrogel in a water bath at 37°C is 6 minutes; 3) Take 8ml of 2% (w/v) chitosan solution: 1ml of 56% (w/v) sodium β-glycerophosphate solution: 0.1g vancomycin or 0.1g ofloxacin hydrochloride injection lml, the three After uniform mixing at 4°C, the decellularized sponge-like collagen biofilm was immersed in the above mixed solution, and placed in a 37°C incubator for 10 minutes, and the mixed solution formed a hydrogel and integrated with the collagen biofilm.