CN113230454A - Biological membrane capable of inducing bone regeneration and preparation method and application thereof - Google Patents
Biological membrane capable of inducing bone regeneration and preparation method and application thereof Download PDFInfo
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- CN113230454A CN113230454A CN202110476780.7A CN202110476780A CN113230454A CN 113230454 A CN113230454 A CN 113230454A CN 202110476780 A CN202110476780 A CN 202110476780A CN 113230454 A CN113230454 A CN 113230454A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
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Abstract
The invention provides a biomembrane capable of inducing bone regeneration and a preparation method and application thereof, belonging to the technical field of medical biomaterials. The biofilm is obtained from the peritoneum of a mammal by a series of immunogenic material removal treatments. The biomembrane capable of inducing bone regeneration provided by the invention has a smooth surface/hair surface double-sided structure, can simultaneously play a role of tissue barrier and promote the regeneration function of bone defect tissues, eliminates the immunogenicity of peritoneal tissues of xenogeneic animals, furthest retains the activity of mechanisms and components of tissue extracellular matrix, and provides a structural and functional foundation for inducing bone regeneration. The preparation process is simple and easy to implement and environment-friendly, and the harmless discharge of the waste reagent can be realized through inorganic acid-base neutralization, so that the method has good practical application value.
Description
The present application claims priority to a previously filed patent application having application number CN202010361418.0 and application date of 30/04 of 2020, entitled "a biofilm capable of inducing bone regeneration and a method for preparing the same". The entire contents of the above-referenced application are hereby incorporated by reference.
Technical Field
The invention belongs to the technical field of medical biomaterials, particularly relates to preparation of a human tissue regeneration medical material, and particularly relates to a biomembrane capable of inducing bone regeneration as well as a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The membrane-guided bone tissue regeneration technology is a new biological concept, originally generated in the treatment of periodontal disease, and the theoretical basis thereof is as follows: in the process of repairing bone wounds, different tissue cells grow and migrate into the wounds at different speeds, and a mechanical barrier is formed by placing a membrane to prevent other tissue cells from growing in, so that a guide space is provided for the growth of the bone cells, the regeneration capacity of the bone tissues is enhanced, the metabolism of the bone tissues is accelerated, the fiber healing of bone defects is effectively avoided, the bone healing is promoted, and the purposes of ideal tissue regeneration and repair are achieved.
The guiding membrane plays a key role in guiding bone tissue regeneration technology, and the current membrane materials can be generally divided into two categories, namely non-degradable materials and degradable materials, wherein the non-degradable materials cannot be absorbed by tissues, need to be taken out through secondary operation and are basically not used clinically. The degradable membrane material is mostly collagen material, has the advantages of good histocompatibility and no need of secondary operation, and can play a good role of isolation barrier. However, pure collagen materials are difficult to induce bone tissue regeneration due to their structure and material composition.
The ECM (extracellular matrix) biological scaffold obtained by the decellularization technology is widely applied to human tissue reconstruction, such as heart valve, skin, tendon, dura and the like, the three-dimensional structure of the ECM biological scaffold is close to the natural environment of in-vivo cell growth, the ECM biological scaffold not only can play a role of a scaffold material, but also contains various growth factors, and has an important promotion role in tissue repair and reconstruction. However, since xenogenic or xenogenic cellular antigens are considered as exosomes by the host, they induce inflammatory reactions and immune-mediated rejection reactions of the host, and thus must be completely removed before application, while the removal of immunogenic substances inevitably destroys the three-dimensional structure of the guide membrane, resulting in a great loss of the function of membrane-guided tissue regeneration.
There are many methods for decellularizing extracellular matrices, including physical, chemical and biological methods, but each of these methods alters the ECM components, causing various degrees of ultrastructural damage that affect the human body's response to the implanted matrix material. Therefore, it is necessary to study the extracellular matrix obtained by mild decellularization method, and to retain the components and native structure of tissue ECM to the maximum.
The patent application No. 201310041671.8 discloses a tissue regeneration guiding membrane and a preparation method thereof, the preparation method comprises: a pretreatment step, collecting the peritoneum of the mammal, washing and unhairing by flowing water, and preliminarily degreasing for later use; antigen removing treatment, namely further degreasing the standby peritoneum by adopting an enzyme treatment process, an alkali treatment process, an acid treatment process and a hypertonic treatment process in sequence to remove immunogenicity; a collagen fiber contraction treatment step, in which the peritoneum after antigen removal treatment is sequentially dehydrated, non-hydrophilic substances are removed and washed, so that the collagen fibers of the peritoneum are contracted; and (3) a drying step, namely spreading the peritoneum after the contraction treatment on the surface of a culture dish, enabling the dense layer of the peritoneum to contact the culture dish, quickly transferring the peritoneum to a refrigerator at the temperature of between 60 ℃ below zero and 80 ℃ below zero for pre-freezing for 1 to 8 hours, and then putting the culture dish into a freeze dryer for freeze-drying for 24 to 36 hours to obtain the collagen membrane capable of guiding tissue regeneration. The guide membrane provided by the invention has a compact layer and a loose layer, and also has good biocompatibility, hydrophilicity and osteoinduction capability, and long in-vivo degradation time. However, this method is cumbersome to handle and causes a great structural damage to the tissue material. The patent with application number 201510256512.9 discloses a preparation method and application of a guided tissue regeneration membrane, and the inventive method comprises the following steps: (1) soaking and treating the skin of the isolated animal by using a surfactant solution; (2) soaking the product of the step (1) by using an alkali solution; (3) soaking and treating the product of the step (2) by using a peroxide solution; (4) treating the product of step (3) with a solution of a radiation protection agent; (5) soaking and treating the product of the step (4) by using a buffer solution; (6) and (5) sequentially carrying out freeze drying and irradiation sterilization on the product obtained in the step (5) to obtain the guided tissue regeneration membrane. This method is highly destructive to the structure of the biological tissue and may risk incomplete removal of the immunogenic substances.
The ideal biomembrane for guiding bone regeneration not only plays a barrier role, but also can simulate the physiological environment of human tissues, provide a cell growth scaffold for tissue regeneration, induce the regeneration and differentiation of tissue cells and promote the regeneration and repair of damaged bone tissues.
Disclosure of Invention
The invention provides a biomembrane capable of inducing bone regeneration and a preparation method thereof aiming at the limitation of the preparation of the prior bone-guided regeneration membrane material, which effectively eliminates the immunogenicity of the prepared biomembrane and reduces the immunological rejection risk in clinical use by taking the peritoneal tissue of mammals and using a divalent metal chelating agent and acid-base treatment with different osmotic pressures, and simultaneously, the treatment in the process of removing immunogen substances fully considers the maintenance of the three-dimensional structure of the biomembrane, more importantly, after the treatment by the method provided by the invention, the biomembrane capable of inducing bone regeneration with a smooth surface/hair surface double-sided structure can be obtained, the smooth surface structure can play the barrier function of the membrane, prevent other tissue cells from growing into bone wounds, provide a good environment for bone regeneration, and the hair surface structure can provide a support structure for bone cell proliferation, promote the rapid proliferation of osteocyte, thus realizing the high-efficiency and rapid healing of defect.
The strategy for preparing the biomembrane capable of inducing bone regeneration comprises the following steps: taking fresh peritoneal tissue of mammal, repeatedly freezing and thawing to break cells, removing partial immunogen substance, and removing fat and redundant serosa on peritoneal fat surface with sodium bicarbonate powder to obtain uniform hair surface structure. And (3) sequentially and repeatedly using a divalent metal ion chelating agent, a hypertonic alkali solution, a hypotonic alkali solution and a hypertonic acid solution to remove immunogen substances, removing lipid, cell structures and various DNA and RNA components in peritoneal tissues, neutralizing, cleaning, and performing irradiation sterilization to obtain the biomembrane capable of inducing bone regeneration and applied to clinic.
The invention has the following beneficial effects:
1. the biomembrane capable of inducing bone regeneration, which is obtained by the preparation method provided by the invention, has a uniform smooth surface/hair surface double-sided structure, the smooth surface structure can play a role of a barrier of the membrane, other tissue cells are prevented from growing into a bone wound, a good environment is provided for bone regeneration, the hair surface structure can provide a support structure for bone cell proliferation, and the bone cell rapid proliferation is promoted, so that the efficient and rapid healing of the defect is realized.
2. The preparation method of the biomembrane capable of inducing bone regeneration provided by the invention is simple and effective, the used reagent is environment-friendly, the waste reagent can be treated by simple acid-base neutralization, the method is simple and easy to implement, the preparation process does not use a surfactant, the difficulty in removing the surfactant is avoided, and a cross-linking agent and a synthetic material are not introduced, so that the biomembrane has no potential cytotoxicity, and the reactions such as fibrosis, chronic inflammation and the like are not caused.
3. The preparation method of the biomembrane capable of inducing bone regeneration provided by the invention has small damage to components and three-dimensional structures of the extracellular matrix, retains the biological activity of effective components of the extracellular matrix to the maximum extent, and has strong tissue repair capability.
4. The preparation method of the biomembrane capable of inducing bone regeneration provided by the invention can thoroughly remove immunogenic substances in tissues, such as lipid, cell components, hybrid protein, various DNA and RNA components and the like.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a biofilm smooth surface capable of inducing bone regeneration prepared in example 1 of the present invention;
FIG. 2 is a biofilm hair side capable of inducing bone regeneration prepared in example 1 of the present invention;
FIG. 3 is HE staining, 20X, of a biofilm capable of inducing bone regeneration, prepared according to example 1 of the present invention;
FIG. 4 is raw HE staining, 20X, untreated;
FIG. 5 is a scanning electron microscope (smooth surface) of a biological membrane capable of inducing bone regeneration, prepared in example 1 of the present invention;
FIG. 6 is a scanning electron microscope (hair side) of a biological membrane capable of inducing bone regeneration prepared in example 1 of the present invention;
FIG. 7 is an infrared spectrum of a biofilm capable of inducing bone regeneration prepared in example 1 of the present invention;
FIG. 8 is a diagram showing the culture of osteoblast bodies outside the hair side of the product of example 1 of the present invention;
FIG. 9 is a photograph showing the outside of the optical surface of the product of example 1 of the present invention;
FIG. 10 is blank defect group, HE staining, 7E 4X;
FIG. 11 is the defect repair group of example 1, HE stained, 11A 4X.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.
As previously mentioned, there are many decellularization methods available for the preparation of extracellular matrices, but each of these decellularization methods can alter the ECM components, causing varying degrees of ultrastructural damage that can affect the human body's response to the implanted matrix material.
In view of the above, in an exemplary embodiment of the present invention, there is provided a biofilm for inducing bone regeneration, the biofilm being prepared from mammalian peritoneal tissue and having a shiny/matte double-sided structure, wherein the matte side can induce cell adhesion growth and promote cell proliferation and functional differentiation, and the shiny side can prevent cell adhesion growth and tissue adhesion; meanwhile, the biological membrane is high in safety, free of immune toxicity and cell nucleus residue, the fat content is lower than 1%, the DNA residue is lower than 5ng/mg of the dry weight of a sample and is far lower than the cell-free matrix DNA residue prepared in literature reports, the total sugar content is lower than 0.3%, the alpha-Gal antigen clearance rate is higher than 95%, the biological membrane is in a porous structure, and cells and cell debris residue are avoided; the collagen fiber is continuous without fracture, the three-strand helical structure of the collagen is completely preserved, the effective components of the extracellular matrix are retained to the maximum extent, and the collagen has good repairing function. Meanwhile, the porosity of the biological membrane is about 85%, so that the biological membrane can successfully block permeation of fibroblasts on the basis of keeping good air permeability, and the barrier effect of the product is exerted.
The preparation method comprises the following steps: taking fresh peritoneal tissue of mammal, repeatedly freezing and thawing to break cells, removing partial immunogen substance, and removing fat and redundant serosa on peritoneal fat surface with sodium bicarbonate powder to obtain uniform hair surface structure. And (3) sequentially and repeatedly using a divalent metal ion chelating agent, a hypertonic alkali solution, a hypotonic alkali solution and a hypertonic acid solution to remove immunogen substances, removing lipid, cell structures and various DNA and RNA components in peritoneal tissues, neutralizing, cleaning, and performing irradiation sterilization to obtain the composition. In the process of preparing the biological membrane, the invention does not use a surfactant, avoids the difficulty of removing the surfactant, does not introduce a cross-linking agent and a synthetic material, has no potential cytotoxicity, does not cause reactions such as fibrosis, chronic inflammation and the like, has mild reaction conditions, has small damage to components and three-dimensional structures of the extracellular matrix, retains the biological activity of effective components of the extracellular matrix to the maximum extent, and has strong tissue repairing capability.
In still another embodiment of the present invention, the prepared biological membrane capable of inducing bone regeneration has a smooth/hairy double-sided structure.
In yet another embodiment of the present invention, the mammal includes pig, cow, dog, sheep, rabbit and mouse, preferably calf.
In yet another embodiment of the present invention, the divalent metal ion chelating agent is EDTA, and the concentration of the EDTA solution is 0.1mol/L to 0.5mol/L, preferably 0.5 mol/L;
in another embodiment of the present invention, the hypertonic alkali solution is a high concentration NaOH solution, preferably a 2% to 5% NaOH solution, and more preferably a 2% NaOH solution;
in another embodiment of the present invention, the hypotonic alkali solution is a low concentration NaOH solution, preferably 0.1% to 0.5% NaOH solution, and more preferably 0.5% NaOH solution.
In another embodiment of the present invention, the hypertonic acid solution is an HCl-NaCl solution, and preferably, the HCl-NaCl solution is prepared by: NaCl was added to a 0.5% HCl solution at a concentration of 1%.
In yet another embodiment of the present invention, the radiation sterilization is a cobalt-60 sterilization treatment.
In another embodiment of the present invention, there is provided a method for preparing a biofilm capable of inducing bone regeneration, comprising:
1) taking fresh mammal peritoneal tissue, placing at-20 deg.C, repeatedly freezing and thawing for 3-5 times, taking out, flattening the peritoneum, placing on ice bag with fat face facing upward, uniformly sprinkling sodium bicarbonate powder on the fat face side, scrubbing, removing fat and excessive serosa, and rinsing with running water for 12 h.
2) Putting the membrane treated in the step 1) into an EDTA solution, shaking for 5-20h at 15-25 ℃, transferring into a NaOH solution, and shaking for 2h at room temperature.
3) Transferring the membrane treated in the step 2) into an EDTA solution, shaking for 2-5h at 15-25 ℃, transferring into an NaOH solution, and shaking for 2h at room temperature.
4) And (3) transferring the membrane treated in the step 3) into a NaOH solution, and shaking, wherein the NaOH solution is replaced for 3 times, and shaking is carried out for 30min each time.
5) Repeating the steps 2) to 4) for 3 to 5 times.
6) And (3) putting the membrane treated in the step 5) into an HCl-NaCl solution for treatment for 3-5 times, and neutralizing by using a 1% sodium bicarbonate solution until the pH value is neutral, wherein each time is 2 hours.
7) Washing the peritoneum treated in step 6) with purified water or water for injection until the conductivity of the washing solution is reduced to below 60us/cm, and lyophilizing.
8) Subjecting the peritoneum treated in step 7) to cobalt-60 sterilization.
In still another embodiment of the present invention, the mammals in step 1) include pigs, cows, dogs, sheep, rabbits and mice, preferably calves.
In still another embodiment of the present invention, the concentration of the EDTA solution in the step 2) and the step 3) is 0.1mol/L to 0.5mol/L, preferably 0.5mol/L, and the concentration of the NaOH solution is 2% to 5% NaOH solution, preferably 2% NaOH solution.
In still another embodiment of the present invention, the concentration of the NaOH solution in the step 4) is 0.1% to 0.5% NaOH solution, preferably 0.5% NaOH solution.
In another embodiment of the present invention, the preparation method of the HCl — NaCl solution in step 6) is: NaCl was added to a 0.5% HCl solution at a concentration of 1%.
In another embodiment of the present invention, there is provided a biofilm capable of inducing bone regeneration, which is prepared by the above-mentioned preparation method.
In another embodiment of the present invention, the biofilm capable of inducing bone regeneration has a smooth/hairy double-sided structure.
In a further embodiment of the invention, there is provided the use of a biofilm as described above in any one or more of:
1) guiding bone tissue regeneration and/or preparing a guiding bone tissue regeneration material;
2) inducing bone tissue defect repair and/or preparing a material for inducing bone tissue defect repair;
3) isolating tissue surrounding the area of bone tissue repair to prevent tissue adhesion and/or preparing an isolated tissue surrounding the area of bone tissue repair to prevent tissue adhesion material.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
Preparation of a calf peritoneal biomembrane capable of inducing bone regeneration:
taking fresh calf peritoneal tissue, placing the tissue at the temperature of minus 20 ℃, repeatedly freezing and thawing for 3 times, taking out, flattening the peritoneum, placing the flattened peritoneum on an ice bag, enabling the fat surface to face upwards, adding 0.5 g of sodium bicarbonate powder on the fat surface side according to the proportion of adding 0.5 g per square centimeter, rubbing and washing, removing fat and redundant serosa, and washing for 12 hours by running water, wherein the pretreatment step is carried out. Transferring the membrane into 0.5mol/L EDTA solution, shaking for 15h at 25 ℃, transferring into 3% NaOH solution, shaking for 2h at room temperature, transferring the membrane into 0.5mol/L EDTA solution, shaking for 2h at 25 ℃, transferring into 3% NaOH solution, shaking for 2h at room temperature, transferring the membrane into 0.1% NaOH solution, shaking for 3 times during the process, and shaking for 30min each time. The above pretreatment steps were repeated 3 times. And (3) treating the treated membrane in 0.5% HCl-1% NaCl solution for 3 times, 2h each time, neutralizing with 1% sodium bicarbonate solution until the pH value is neutral, washing with purified water until the conductivity of the washing liquor is reduced to 60us/cm, freeze-drying, and performing irradiation sterilization for later use.
Example 2
A preparation method of pig peritoneal biomembrane capable of inducing bone regeneration comprises the following steps:
taking fresh porcine peritoneal tissue, placing the tissue at the temperature of-20 ℃, repeatedly freezing and thawing for 3 times, taking out, flattening the peritoneum, placing the peritoneum on an ice bag, enabling the fat surface to face upwards, adding 0.5 g of sodium bicarbonate powder to the fat surface side according to the proportion of adding 0.5 g per square centimeter, rubbing and washing, removing fat and redundant serosa, and washing for 12 hours by running water, wherein the pretreatment step is carried out. Transferring the membrane into 0.5mol/L EDTA solution, shaking for 15h at 25 ℃, transferring into 2% NaOH solution, shaking for 2h at room temperature, transferring the membrane into 0.5mol/L EDTA solution, shaking for 2h at 25 ℃, transferring into 2% NaOH solution, shaking for 2h at room temperature, transferring the membrane into 0.5% NaOH solution, shaking for 3 times during the process, and shaking for 30min each time. The above pretreatment steps were repeated 3 times. And (3) treating the treated membrane in 0.5% HCl-1% NaCl solution for 3 times, 2h each time, neutralizing with 1% sodium bicarbonate solution until the pH value is neutral, washing with purified water until the conductivity of the washing liquor is reduced to 60us/cm, freeze-drying, and performing irradiation sterilization for later use.
Example 3
For safety of the samples, the samples prepared in examples 1 and 2 were subjected to detection of an immunogenic substance.
(1) The cell residue detection method comprises the following steps: fixing biological material and skin tissue with 10% formaldehyde, collecting material, dehydrating, embedding in paraffin, slicing, performing HE staining and Masson staining, and observing cell nucleus residue and matrix fiber structure under microscope, as shown in FIG. 3 and FIG. 4.
(2) The DNA residue detection method comprises the following steps: tissue engineering medical products according to YY/T0606.25-2014 part 25: animal-derived biomaterial DNA residue assay: the requirement of fluorescent staining method.
(3) The fat content detection method comprises the following steps: the determination was carried out according to Soxhlet extraction method, the first method in the determination of fat in food products of national standard for food safety of GB 5009.6-2016.
(4) And (3) total sugar content determination: the determination is carried out according to the I type collagen characterization method of the YY/T1453-2016 tissue engineering medical instrument product, appendix E total sugar content determination.
(5) alpha-Gal antigen clearance assay: according to the industry standard YY/T1465.5-2016 section 5 of the method for evaluating immunogenicity of medical devices: the M86 antibody was used to determine alpha-Gal antigen clearance in animal derived medical devices by the inhibition ELISA assay specified in alpha-Gal antigen clearance.
(6) Immunotoxicity testing: according to the principle of GB/T16886.20-2015 'principle and method for medical device biological evaluation part 20 of medical device immunotoxicology test', Balb/C mice are used as experimental animals, products with different doses are contacted with the animals in a back subcutaneous implantation mode, changes of indexes such as humoral immunity and cellular immunity of the mice are detected after 4 weeks of immunization, the immunotoxicity of the mice is evaluated, and experimental basis is provided for safety risk analysis of the products. The result shows that the product has no immune toxicity. The results are shown in the following table.
Example 4
The samples prepared in example 1 and example 2 were tested for biological properties, histological examination, bacterial endotoxin, and osteoblast in vitro culture assay.
(1) Biological Performance testing
The method comprises the following steps: the test was carried out with reference to the GB/T16886 series of methods.
As a result: no pyrogenicity; the hemolysis rate of the samples prepared in the embodiment 1 and the embodiment 2 is 0%, and the negative control and the positive control both meet the requirements, so that no hemolysis reaction exists; no acute systemic toxic reaction; the relative proliferation rate of the L929 cells is 91%, the cytotoxicity reaction is graded as 1 grade, and the experimental results of the negative control and the positive control are consistent with the expected results; no guinea pig skin sensitization reaction; the intracutaneous reaction shows that the difference between the final scores of the intracutaneous reaction of the test sample and the solvent control rabbit is not more than 1.0; the result of the genotoxicity test shows that the salmonella typhimurium beads TA97, TA98, TA100 and TA102 have no mutagenicity, and the lymphoma test of the mice has negative reaction and no chromosome teratogenesis;
(2) histological examination
1) Observation with an optical microscope
The method comprises the following steps: fixing biological material and skin tissue with 10% formaldehyde, taking materials, dehydrating, embedding in paraffin, slicing, performing HE staining and Masson staining, and observing cell nucleus residual condition and matrix fiber structure under microscope.
As a result: no cells and cell debris remain; the collagen fibers were continuous without breaks as shown in fig. 3 and 4.
2) Ultrastructural observation
The method comprises the following steps: and scanning the smooth surface and the rough surface of the product by using a scanning electron microscope.
As a result: the material has a porous structure, and collagen fibers are not broken, as shown in fig. 5 and 6.
3) Infrared spectroscopic analysis
The method comprises the following steps: the 3mg sample was ground to a powder and tabletted with potassium bromide. The solid sample can be tabletted after being ground for 5-15 min, and the pressure of the oil press is usually 8000-15000 kg/cm2And keeping the pressurizing time for at least 1min to obtain a transparent ingot. Then putting the sample into an in-situ infrared spectrometer, and selecting 4000-500 cm-1Infrared spectral absorption scans were performed over the range.
As a result: the material contains characteristic peaks of amide A band, amide I band and amide III band, and is consistent with the characteristic peaks of collagen, which fully indicates that the triple helix structure of the collagen in the material is completely preserved, and the bioactivity of the effective components of the extracellular matrix is retained to the maximum extent as shown in figure 7.
(3) Bacterial endotoxins
The method comprises the following steps: the measurement was carried out according to the gel method in pharmacopoeia of the people's republic of China (2020 edition, four departments) 1143.
As a result: are all less than 0.5 EU/mL.
(4) In vitro culture test of osteoblasts
The method comprises the following steps: osteoblasts of newborn SPF-grade SD rat were isolated and purified and prepared to 105In 96 ofThe wells were co-incubated with the samples.
As a result: the product has better compatibility with osteoblasts, can induce the osteoblasts to grow pseudo feet from the edge of the product, and contrasts the smooth surface and the hair surface results, the hair surface cells are more in a radial shape, a large number of microfilaments are connected among the cells to form a nearly net-shaped structure, the cell nucleuses are more overlapped, the cell number is more (shown in figure 8), the smooth surface cells are less, the connection among the cells is not obvious, and the cells are more in an isolated state (shown in figure 9), so that the product can induce the cell adhesion growth on the hair surface, simultaneously plays a role in promoting the cell proliferation and functional differentiation, and the smooth surface can prevent the cell adhesion growth, and plays a role in preventing the tissue adhesion.
(5) Rabbit head skull defect repair test
The method comprises the following steps: before the operation, the operation is not performed for 4 hours, 20% of ethyl carbamate (normal saline preparation, anesthetic dose: 4ml/kg) is injected into ear margin vein, the ear margin is fixed on an operation table after anesthesia, the head top is prepared, alcohol is wiped for disinfection, a notch is made in the center of the skull, bone powder is added to the defect to cover the prepared sample, meanwhile, a blank defect group is arranged, gentamicin sulfate is dripped to prevent infection, then, the wound is sutured, and iodophor is disinfected. Can be fed with water after waking. Three days after the operation, penicillin sodium is continuously injected into muscles to prevent infection. And 6W and 12W after the operation are sacrificed to observe the repair condition, and pathological section detection is carried out.
As a result: in the blank defect group part defects, new bone cells are generated between the periosteum and the dura mater (black arrows in figure 10), bone repair occurs, but the amount of new bone is small, and the repair effect cannot be achieved. Compared with the blank defect group, the new bone and the original bone are directly connected (the left side of the black line in figure 11 is the original bone, the right side is the new bone), and the new bone quantity of the defect repair group is obviously increased.
Example 5
The physical properties of the samples prepared in example 1 and example 2 were measured.
(1) Porosity of the material
The method comprises the following steps: the porosity is the percentage of the volume of the internal pores of the material in the total volume of the material, and the measurement is mostly carried out by adopting a liquid discharge methodNamely, the gas in the inner pores of the material is exhausted by a circulating vacuum-pumping method, and the material is filled with liquid, and the weight of the liquid in the material is calculated to be the percentage of the total weight of the material. Weighing 0.2g (W) of dried sample, cutting into strips, placing into a volumetric flask with water, circularly vacuumizing until no air bubbles overflow on the surface of the sample and the sample sinks to the bottom, and weighing the volumetric flask containing the sample and water (W)2) Then, the sample containing water was taken out, and the remaining volume flask and the weight of water (W) were weighed3) The formula for calculating porosity is ═ W2-W3-W1)/(W2-W3) 100%, the results are given in the following table:
and (4) conclusion: the porosity of the sample in example 1 and the sample in example 2 is about 85%, which indicates that the product can successfully block the permeation of fibroblasts on the basis of maintaining good air permeability, and the product can play a barrier role.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A biological membrane capable of inducing bone regeneration, which is prepared from mammal peritoneal tissue and has a smooth/hairy double-sided structure, and the biological membrane has a porous structure and is free of cells and cell debris residues; the biological membrane collagen fiber is continuous without fracture, and the three-strand helical structure of the collagen is complete.
2. The biofilm capable of inducing bone regeneration of claim 1, wherein said biofilm is non-immunotoxic, free of nuclear residues, has a fat content of less than 1%, a DNA residue of less than 5ng/mg, a total sugar content of less than 0.3%, an α -Gal antigen clearance of greater than 95%, and a biofilm porosity of-85%.
3. A biofilm capable of inducing bone regeneration, which is prepared by the following steps: taking fresh peritoneal tissue of a mammal, repeatedly freezing and thawing to break cells, removing partial immunogen substances, and removing fat and redundant serosa on the fat surface of the peritoneal by using sodium bicarbonate powder to obtain a uniform hair surface structure; and (3) sequentially and repeatedly using a divalent metal ion chelating agent, a hypertonic alkali solution, a hypotonic alkali solution and a hypertonic acid solution to remove immunogen substances, removing lipid, cell structures and various DNA and RNA components in peritoneal tissues, neutralizing, cleaning, and performing irradiation sterilization to obtain the composition.
4. The biofilm of claim 3, wherein said mammals include pigs, cattle, dogs, sheep, rabbits and mice, preferably calves.
5. The biofilm according to claim 3, wherein the divalent metal ion chelating agent is EDTA, the concentration of the EDTA solution being between 0.1mol/L and 0.5mol/L, preferably 0.5 mol/L; or the like, or, alternatively,
the hypertonic alkali solution is a high-concentration NaOH solution, preferably a 2-5% NaOH solution, and further preferably a 2% NaOH solution; or the like, or, alternatively,
the hypotonic alkali solution is a low-concentration NaOH solution, preferably a 0.1-0.5% NaOH solution, and further preferably a 0.5% NaOH solution; or the like, or, alternatively,
the hypertonic acid solution is HCl-NaCl solution; or the like, or, alternatively,
the irradiation sterilization adopts cobalt-60 sterilization treatment.
6. A method for preparing a biofilm capable of inducing bone regeneration, comprising:
1) taking fresh mammal peritoneal tissue, placing at-20 deg.C, repeatedly freezing and thawing for 3-5 times, taking out, flattening the peritoneum, placing on ice bag with fat surface facing upward, uniformly sprinkling sodium bicarbonate powder on the fat surface side, scrubbing, removing fat and excessive serosa, and flushing with running water for 12 h;
2) putting the membrane treated in the step 1) into an EDTA solution, shaking for 5-20h at 15-25 ℃, transferring into a NaOH solution, and shaking for 1-5h at room temperature;
3) transferring the membrane treated in the step 2) into an EDTA solution, shaking for 2 hours at the temperature of 15-25 ℃, transferring into an NaOH solution, and shaking for 2 hours at room temperature;
4) transferring the membrane treated in the step 3) into a NaOH solution, and shaking, wherein the NaOH solution is replaced for 3 times, and shaking is carried out for 30min each time;
5) repeating the step 2) to the step 4) for 3 to 5 times;
6) putting the membrane treated in the step 5) into HCl-NaCl solution for treatment for 3-5 times, each time for 2h, and neutralizing by using 1% sodium bicarbonate solution until the pH value is neutral;
7) washing the peritoneum treated in step 6) with purified water or water for injection until the conductivity of the washing solution is reduced to below 60us/cm, and lyophilizing;
8) subjecting the peritoneum treated in step 7) to cobalt-60 sterilization.
7. The method of claim 6, wherein the mammals in step 1) include pigs, cows, dogs, sheep, rabbits and mice, preferably calves; or the like, or, alternatively,
in the step 2) and the step 3), the concentration of the EDTA solution is 0.1-0.5 mol/L, preferably 0.5mol/L, and the concentration of the NaOH solution is 2-5% of NaOH solution, preferably 2% of NaOH solution; or the like, or, alternatively,
the concentration of the NaOH solution in the step 4) is 0.1-0.5% of NaOH solution, and 0.5% of NaOH solution is preferred; or the like, or, alternatively,
the preparation method of the HCl-NaCl solution in the step 6) comprises the following steps: NaCl was added to a 0.5% HCl solution at a concentration of 1%.
8. A biofilm capable of inducing bone regeneration, which is produced by the production method according to claim 6 or 7.
9. The biofilm of claim 8, wherein said biofilm has a shiny/matte duplex configuration.
10. Use of a biofilm according to any one of claims 1 to 5 or 8 in any one or more of:
1) guiding bone tissue regeneration and/or preparing a guiding bone tissue regeneration material;
2) inducing bone tissue defect repair and/or preparing a material for inducing bone tissue defect repair;
3) isolating tissue surrounding the area of bone tissue repair to prevent tissue adhesion and/or preparing an isolated tissue surrounding the area of bone tissue repair to prevent tissue adhesion material.
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| CN111420122A (en) * | 2020-04-30 | 2020-07-17 | 山东隽秀生物科技股份有限公司 | Biological membrane capable of inducing bone regeneration and preparation method thereof |
| CN111821523A (en) * | 2020-08-11 | 2020-10-27 | 上海市第六人民医院 | Aponeurosis stent and preparation method and application thereof |
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| CN111420122A (en) | 2020-07-17 |
| CN113230454B (en) | 2022-11-25 |
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