CN114288473A - Preparation method of acellular small intestine submucosa composite bone scaffold with antibacterial function - Google Patents
Preparation method of acellular small intestine submucosa composite bone scaffold with antibacterial function Download PDFInfo
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- CN114288473A CN114288473A CN202210142830.2A CN202210142830A CN114288473A CN 114288473 A CN114288473 A CN 114288473A CN 202210142830 A CN202210142830 A CN 202210142830A CN 114288473 A CN114288473 A CN 114288473A
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Abstract
The invention provides a preparation method of a decellularized small intestine submucosa composite bone scaffold with a bacteriostatic function. The preparation method comprises the following steps: (1) taking fresh jejunum, cutting into several segments with length of about 10-18cm, and repeatedly wiping to remove mucosa, muscle and serosa layer of small intestine. (2) After treatment, the cells were soaked in 0.25% trypsin solution and digested at 37 ℃ for 24 hours, and rinsed with distilled water. (3) Then, the cells were removed by immersing in 0.5% SDS at 37 ℃ with continuous shaking for 24 hours, and rinsed with distilled water. (4) Then placing the mixture in an ethanol solution of peroxyacetic acid for soaking for 8 hours, rinsing the mixture by using distilled water, freezing and drying the rinsed mixture at a low temperature, and sterilizing the rinsed mixture by using ethylene oxide to obtain a bio-matrix membrane SIS; (5) and (3) placing the biological matrix membrane SIS in a honey solution overnight, and then freeze-drying to obtain the acellular small intestine submucosa nanofiber composite bone scaffold. The bone scaffold is soaked in honey, so that the bone scaffold not only has antibacterial performance, but also has a three-dimensional network structure, and has biomineralization, degradation activity and cell compatibility.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a preparation method of a decellularized small intestine submucosa composite bone scaffold with a bacteriostatic function.
Background
With the continuous and intensive research on tissue engineering bone, the preparation of extracellular matrix to simulate a microenvironment similar to that of autologous bone is gradually paid attention by tissue engineering researchers. The small intestinal submucosa SIS is a completely decellularized matrix membrane, the main component of which is collagen, which has the common characteristic of most collagen products, namely, stronger toughness. The collagen simulates the shape and molecular structure of ossein and mainly consists of I-type collagen fibers which are orderly arranged, and the three-dimensional nano network structure is similar to bone 3D extracellular matrix protein. Mucopolysaccharides, proteoglycans, glycoproteins, heparin, fibronectin and various functional growth factors including basic fibroblast growth factor (bFGF), Epidermal Growth Factor (EGF), transforming growth factor-beta (TGF-beta), Vascular Endothelial Growth Factor (VEGF), insulin-like growth factor-1, nerve growth factor, etc. are distributed thereon, and SIS implanted into a human body has almost no immune rejection. Therefore, the SIS used as a tissue engineering substitute material not only can play a supporting role, but also can regulate and control various vital activities of cells and repair and regeneration of tissues in vivo through a plurality of biological information contained in the SIS.
SIS is used as a biological template for synthesizing Hydroxyapatite (HA), and can rapidly induce the synthesis of the HA in simulated body fluid. The synthesized hydroxyapatite has natural osteoconductivity and osseointegration capability, the osteoconductivity provides a scaffold for the ingrowth of blood vessels and the formation of new bones, the osseointegration characteristic realizes the direct contact of the scaffold and a fibrous connective tissue interface layer of the bone tissue scaffold, and the scaffold cannot loosen and lose efficacy after being implanted. These characteristics indicate that it is a biological material with wide application prospect in bone tissue engineering. There have been some reports on using SIS materials as bone tissue engineering skeleton materials at home and abroad, but a key problem causes that the application of SIS is limited. SIS has a limited natural antibacterial capacity, is often associated with infection after being implanted into a body, causes inflammation, and can cause implantation failure if an effective antibacterial means is not adopted.
Worldwide, fractures are susceptible to bacterial infection during healing, causing inflammation and placing a significant burden on patients and medical systems. In addition, bacterially induced infection hinders re-epithelialization and collagen synthesis, thereby challenging the bone healing process. By 2021, the global anti-infective market is estimated to reach $ 204 billion. However, many bacteria develop resistant strains due to abuse and abuse of antibiotics, and the frequency of occurrence has exceeded the speed of development of new antibiotics. This causes great difficulty in the treatment of the disease. Although the antibacterial peptide is a peptide active substance which is generated by a host nonspecific immune defense system and is used for resisting exogenous pathogens, the antibacterial peptide is an effector molecule of natural immunity, is not easy to induce the generation of drug-resistant strains, and has wide antibacterial spectrum. Has no harm to higher animal cells and no residue in vivo, but the antibacterial activity of the antibiotic is far lower than that of the traditional antibiotic. Nanoparticles have suitable antimicrobial properties and they can also act as a diffusion into the body of a patient causing small size and serious health problems.
Disclosure of Invention
The invention aims to solve the problem of a preparation method of a decellularized small intestine submucosa SIS/HONEY composite bone scaffold which is green and synthesized, has a simple operation process, a three-dimensional network structure, bioactivity and a bacteriostatic function, and aims to prepare a degradable mineralized decellularized small intestine submucosa SIS/HONEY composite bone scaffold with a nanofiber network structure and a bacteriostatic function in a mode of compounding natural HONEY and the decellularized small intestine submucosa SIS.
The invention firstly provides a preparation method of a decellularized small intestine submucosa composite bone scaffold with a bacteriostatic function, which comprises the following steps:
(1) fresh jejunum of health adult livestock subjected to quarantine within 4h slaughtering is taken, washed clean by clear water repeatedly, and then is cut into a plurality of intestine sections with the length of about 10-18cm by selecting the parts with relatively uniform pipe cavities, no obvious damage to pipe walls and no lymph node attachment, and the intestine sections are longitudinally split and laid flat, and the mucous membrane layer of the small intestine is removed by repeated wiping. Turning over the small intestine, removing the muscular layer and serosal layer by the same method, and repeatedly rinsing with distilled water to remove the residual tissue on the submucosa of the small intestine;
(2) mechanically prepared intestinal submucosa is soaked in 0.25% trypsin solution and digested at 37 deg.C for 24 hr, and repeatedly rinsed with distilled water.
(3) Immersing the small intestine submucosa digested by trypsin and rinsed by distilled water into 0.5% SDS, shaking continuously at 37 ℃ for decellularizing for 24h, and rinsing repeatedly by distilled water.
(4) Then, the small intestine submucosa tissue is placed in an ethanol solution of peroxyacetic acid for soaking for 8 hours, and is subjected to low-temperature freeze drying after being repeatedly rinsed by distilled water and ethylene oxide sterilization to obtain a bio-matrix membrane SIS;
(5) and (3) placing the biological matrix membrane SIS in a honey solution overnight, and then freeze-drying to obtain the acellular small intestine submucosa nanofiber composite bone scaffold.
Preferably, the honey is manuka honey.
Preferably, the ethanol solution of peroxyacetic acid is a 20% ethanol solution containing 0.1% peroxyacetic acid.
Preferably, the small intestine is a porcine, bovine, ovine or equine small intestine.
Preferably, the honey solution has a weight/volume concentration of 1% to 100%, more preferably the honey solution has a weight/volume concentration of 10% to 100%.
Preferably, the ethanol solution of peroxyacetic acid in step (4) is a 20% ethanol solution containing 0.1% peroxyacetic acid.
Preferably, the freeze-drying process in the step (5) is carried out under the conditions of-50 ℃, vacuum degree of 5-20 and time of 12 h.
The second technical scheme provided by the invention is as follows: the decellularized small intestine submucosa composite bone scaffold with the bacteriostatic function is prepared by the preparation method.
The third technical scheme provided by the invention is as follows: the application of the decellularized small intestine submucosa composite bone scaffold with the bacteriostatic function prepared by the preparation method in biomedical materials.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the natural broad-spectrum antibacterial agent honey and the SIS matrix membrane are compounded to prepare the bone scaffold, the scaffold HAs a three-dimensional nano network structure similar to bone and can induce the synthesis of Hydroxyapatite (HA), the natural honey with undeniable antibacterial advantage is added into the scaffold to prepare the acellular small intestine submucosa SIS nano fiber composite bone scaffold, and the scaffold HAs a good application prospect in bone substitute materials.
2. The preparation process is green and environment-friendly, is simple to operate, does not need large-scale equipment, and has low energy consumption. Low production cost and easy realization of industrialized production of finished products.
Drawings
Fig. 1 is a schematic diagram of preparation of a decellularized small intestine submucosa composite bone scaffold with a bacteriostatic function.
FIG. 2 shows the bacteriostatic results of the decellularized small intestine submucosa composite bone scaffold with bacteriostatic function on Escherichia coli and Staphylococcus aureus.
FIG. 3 is SEM morphology of the three-dimensional network structure of the decellularized small intestine submucosa composite bone scaffold with bacteriostatic function in example 1
FIG. 4 shows FITR results of the decellularized small intestine submucosa composite bone scaffold having bacteriostatic function of example 2.
FIG. 5 shows the mineralization and degradation properties of the decellularized intestinal submucosa composite bone scaffold having bacteriostatic function in example 2 (a, c-mineralization and degradation SEM results; b, d-changes of scaffold quality in different time periods of mineralization and degradation)
FIG. 6 shows the results of MSC immunofluorescence staining of mesenchymal stem cells cultured for 3d on decellularized small intestine submucosa complex bone scaffolds with bacteriostatic function of example 3 (a-complex scaffold; b-control (no scaffold)).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit and scope of the appended claims. It is to be understood that the scope of the invention is not limited to the procedures, properties, or components defined, as these embodiments, as well as others described, are intended to be merely illustrative of particular aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be covered by the scope of the appended claims.
Example 1
Preparing a decellularized small intestine submucosa SIS/HONEY bone scaffold with an antibacterial function:
(1) fresh jejunum of health adult livestock subjected to quarantine within 4h slaughtering is taken, washed clean by clear water repeatedly, and then is cut into a plurality of intestine sections with the length of about 10-18cm by selecting the parts with relatively uniform pipe cavities, no obvious damage to pipe walls and no lymph node attachment, and the intestine sections are longitudinally split and laid flat, and the mucous membrane layer of the small intestine is removed by repeated wiping. The small intestine was inverted, the muscular layer and serosal layer were removed in the same manner, and the remaining tissue on the submucosa of the small intestine was removed by repeated rinsing with distilled water. (ii) a
(2) Mechanically prepared intestinal submucosa is soaked in 0.25% trypsin solution and digested at 37 deg.C for 24 hr, and repeatedly rinsed with distilled water.
(3) Immersing the small intestine submucosa digested by trypsin and rinsed by distilled water into 0.5% SDS, shaking continuously at 37 ℃ for decellularizing for 24h, and rinsing repeatedly by distilled water.
(4) Then, the small intestine submucosa tissue is placed in an ethanol solution of peroxyacetic acid for soaking for 8 hours, and is subjected to low-temperature freeze drying after being repeatedly rinsed by distilled water and ethylene oxide sterilization to obtain a bio-matrix membrane SIS;
(5) and (3) placing the SIS matrix membrane obtained in the step (4) in 10% (volume percentage) HONEY solution for 12h, and then carrying out freeze drying at the freeze drying temperature of-50 ℃ and the vacuum degree of 10 for 12h to obtain the acellular small intestine submucosa SIS/HONEY composite bone scaffold with the bacteriostatic function.
Respectively mixing the Escherichia coli of the gram-positive bacterium and the staphylococcus aureus of the gram-negative bacterium at the density of 1 multiplied by 106the/mL was inoculated on MH plates, then the SIS/HONEY composite bone scaffolds were placed on the plated plates, and the bacteriostatic effect was observed overnight. FIG. 2 is the results of the bacteriostasis of E.coli and S.aureus by the decellularized small intestine submucosa SIS/HONEY composite bone scaffold including the bacteriostasis function of example 1. For two representative strains, the antibacterial performance is more obvious along with the increase of the honey concentration.
FIG. 3 is SEM image of the three-dimensional network structure of the acellular small intestine submucosa SIS/HONEY composite bone scaffold with bacteriostatic function in example 1.
Example 2
Preparing a decellularized small intestine submucosa SIS/HONEY bone scaffold with an antibacterial function:
(1) fresh jejunum of health adult livestock subjected to quarantine within 4h slaughtering is taken, washed clean by clear water repeatedly, and then is cut into a plurality of intestinal sections with the length of about 10-18cm by selecting the parts with relatively uniform pipe cavities, no obvious damage to pipe walls and no lymph node attachment, and the intestinal sections are longitudinally split and laid flat, and the mucous membrane layer of the small intestine is removed by repeated wiping. Turning over the small intestine, removing the muscular layer and serosal layer by the same method, and repeatedly rinsing with distilled water to remove the residual tissue on the submucosa of the small intestine;
(2) mechanically prepared intestinal submucosa is soaked in 0.25% trypsin solution and digested at 37 deg.C for 24 hr, and repeatedly rinsed with distilled water.
(3) Immersing the small intestine submucosa digested by trypsin and rinsed by distilled water into 0.5% SDS, shaking continuously at 37 ℃ for decellularizing for 24h, and rinsing repeatedly by distilled water.
(4) Then, the small intestine submucosa tissue is placed in an ethanol solution of peroxyacetic acid for soaking for 8 hours, and is subjected to low-temperature freeze drying after being repeatedly rinsed by distilled water and ethylene oxide sterilization to obtain a bio-matrix membrane SIS;
(5) and (3) placing the obtained SIS matrix membrane into a HONEY solution with the volume percentage of 100% for 12h, and then carrying out freeze drying at the freeze drying temperature of-50 ℃ and the vacuum degree of 10 for 12h to obtain the acellular small intestine submucosa SIS/HONEY composite bone scaffold with the bacteriostatic function.
FIG. 4 is a FITR map of the decellularized small intestine submucosa SIS/HONEY composite bone scaffold of bacteriostatic function of example 2. By comparing the difference in FITR results for SIS, HONEY and SIS/HONEY composite bone scaffolds, it was confirmed that FITR of SIS/HONEY composite bone scaffold is the composite of HONEY and SIS matrix membrane.
The weight of the sample before soaking the stent prepared in example 2 was weighed as W0, and then 10 times of the simulated body fluid and 10 times of the phosphate buffer solution were soaked in 50g/mL for 1h, 2h and 6h, respectively, according to the weight of the stent, and the solution was changed every 2h for 3 samples in each time period. Samples were taken at time points, gently rinsed 3 times with distilled water, oven dried at 37 ℃ and weighed as W1. And (5) recording mineralization and degradation conditions. FIG. 5 shows the morphology and the change of mass of the decellularized small intestine submucosa SIS/HONEY composite bone scaffold with the bacteriostatic function in example 2 after being mineralized and degraded for 6 h. The results show that: the mineralized product assembled by the nano-shaped sheets is covered on the mineralized surface of the composite bracket, and the change of the mineralized quality is firstly rapid and then slowed down along with the prolonging of time; the degraded stent shows a clear three-dimensional network structure, and the degradation characteristic corresponds to mineralization and is firstly slowed down quickly.
Example 3
Preparing a decellularized small intestine submucosa SIS/HONEY bone scaffold with an antibacterial function:
(1) fresh jejunum of health adult livestock subjected to quarantine within 4h slaughtering is taken, washed clean by clear water repeatedly, and then is cut into a plurality of intestinal sections with the length of about 10-18cm by selecting the parts with relatively uniform pipe cavities, no obvious damage to pipe walls and no lymph node attachment, and the intestinal sections are longitudinally split and laid flat, and the mucous membrane layer of the small intestine is removed by repeated wiping. Turning over the small intestine, removing the muscular layer and serosal layer by the same method, and repeatedly rinsing with distilled water to remove the residual tissue on the submucosa of the small intestine;
(2) soaking small intestine submucosa prepared by mechanical method in 0.25% trypsin solution, digesting at 37 deg.C for 24 hr, and repeatedly rinsing with distilled water;
(3) immersing the small intestine submucosa digested by trypsin and rinsed by distilled water into 0.5% SDS, shaking continuously at 37 ℃ for decellularizing for 24h, and rinsing repeatedly by distilled water;
(4) then, the small intestine submucosa tissue is placed in an ethanol solution of peroxyacetic acid for soaking for 8 hours, and is subjected to low-temperature freeze drying after being repeatedly rinsed by distilled water and ethylene oxide sterilization to obtain a bio-matrix membrane SIS;
(5) and (3) placing the obtained SIS matrix membrane into a HONEY solution with the volume percentage of 100% for 12h, and then carrying out freeze drying at the freeze drying temperature of-50 ℃ and the vacuum degree of 10 for 12h to obtain the acellular small intestine submucosa SIS/HONEY composite bone scaffold with the bacteriostatic function.
Prepared in example 3
The scaffold was placed in a 6-well cell culture plate for use. Then configuring the density of cultured bone marrow Mesenchymal Stem Cells (MSC) to 5 × 105The cells at this density were seeded at 400. mu.L/well in 6-well cell culture plates, cultured for 4h, supplemented with 10% serum in 1.6mL of high-glucose DMEM medium, and immunofluorescent-stained after 3d of culture. FIG. 6 shows the MSCs immunofluorescence staining results of bone marrow mesenchymal stem cells cultured for 3d on the acellular small intestine submucosa SIS/HONEY composite bone scaffold with bacteriostatic function in example 3. Comparing the cell staining results of the control group (FIG. 6-a) and the composite scaffold group (FIG. 6-b), it was found that the cells on the composite scaffold grew well.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. A preparation method of a decellularized small intestine submucosa composite bone scaffold with a bacteriostatic function is characterized by comprising the following steps:
(1) fresh jejunum of health adult livestock subjected to quarantine within 4h slaughtering is taken, washed clean by clear water repeatedly, and then is cut into a plurality of intestine sections with the length of about 10-18cm by selecting the parts with relatively uniform pipe cavities, no obvious damage to pipe walls and no lymph node attachment, and the intestine sections are longitudinally split and laid flat, and the mucous membrane layer of the small intestine is removed by repeated wiping. The small intestine was inverted, the muscular layer and serosal layer were removed in the same manner, and the remaining tissue on the submucosa of the small intestine was removed by repeated rinsing with distilled water.
(2) Mechanically prepared intestinal submucosa is soaked in 0.25% trypsin solution and digested at 37 deg.C for 24 hr, and repeatedly rinsed with distilled water.
(3) Immersing the small intestine submucosa digested by trypsin and rinsed by distilled water into 0.5% SDS, shaking continuously at 37 ℃ for decellularizing for 24h, and rinsing repeatedly by distilled water.
(4) Then, the small intestine submucosa tissue is placed in an ethanol solution of peroxyacetic acid for soaking for 8 hours, and is subjected to low-temperature freeze drying after being repeatedly rinsed by distilled water and ethylene oxide sterilization to obtain a bio-matrix membrane SIS;
(5) and (3) placing the biological matrix membrane SIS in a honey solution overnight, and then freeze-drying to obtain the acellular small intestine submucosa nanofiber composite bone scaffold.
2. The production method according to claim 1,
the honey is Manuka honey.
3. The production method according to claim 1,
the weight/volume concentration of the honey solution is 1% -100%, and optionally the weight/volume concentration of the honey solution is 10% -100%.
4. The production method according to claim 1,
the small intestine is pig small intestine, cattle small intestine, sheep small intestine or horse small intestine.
5. The production method according to claim 1,
the ethanol solution of peroxyacetic acid in the step (4) is 20% ethanol solution, which contains 0.1% peroxyacetic acid.
6. The production method according to claim 1,
the freeze drying process in the step (3) is carried out under the conditions of-50 ℃, vacuum degree of 5-20 and time of 12 h.
7. The decellularized small intestine submucosa composite bone scaffold having a bacteriostatic function prepared by the preparation method according to any one of claims 1 to 7.
8. The use of the decellularized small intestine submucosa composite bone scaffold with bacteriostatic function according to any one of claims 1 to 7 in biomedical materials, which is prepared by the preparation method.
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Cited By (1)
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| CN115737939A (en) * | 2022-11-24 | 2023-03-07 | 四川大学 | SIS (styrene-isoprene-styrene) film based on hydroxyapatite coating as well as preparation method and application thereof |
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