CN107080861B - High-induction-activity repair material, preparation method and application - Google Patents
High-induction-activity repair material, preparation method and application Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—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
- 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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Abstract
The invention provides a repair material with high induction activity, a preparation method and application, which is characterized in that: the material consists of a natural polymer material with a three-dimensional reticular porous structure, and comprises a three-layer structure consisting of a middle layer, an upper surface layer and a lower surface layer; the middle layer comprises collagen, polysaccharide substances, active factors and growth factors, the upper surface layer and the lower surface layer comprise collagen, polysaccharide substances and active factors, and the repairing material can be degraded in vivo. One or more of the following growth factors may be included: the follicle-inhibiting protein, nerve growth factor, platelet-derived growth factor, epithelial growth factor, matrix gamma-carboxyglutamic acid protein and the like are applied to the growth and regulation of tissues such as cardiac muscle, nerve, smooth muscle, epithelium and the like, or the calcification of matrix materials removed by immunogen after implantation is prevented.
Description
Technical Field
The invention relates to the technical field of medical biomaterials, in particular to a repair material with high induction activity, a preparation method and application thereof.
Background
The immunogen Small Intestinal Submucosa (SIS) removed material is a biomedical material which is widely applied to repair and reinforcement of soft tissue defects such as abdominal wall, urinary tract, dura mater, cardiovascular repair and the like in clinic, and is a material which has no cells, no immunogenicity, biodegradability and good fiber elasticity.
The invention patent (103272278A) discloses a preparation method of animal derived implantable medical biomaterial, which introduces medical products with different sizes, thicknesses and mechanical strengths obtained by pretreatment separation, initial washing, virus inactivation, immunogen removal, sodium chloride treatment, molding, packaging and sterilization of animal tissue materials. The immunogen removal technology digests and lyses living cells of animal tissues or organs, removes cell surface antigens that cause immune reactions, and obtains extracellular matrix (ECM) that retains regulatory factors such as growth factors. The immunogen removing matrix is composed of various macromolecular substances such as collagen I, non-collagen glycoprotein, aminoglycan, proteoglycan, growth factors and the like, forms a porous three-dimensional structure, provides a proper growth microenvironment for various cells, and can regulate the attachment, the shape, the migration, the metabolism, the proliferation and the differentiation of various cells. The immunogen removal matrix is used as a natural biomedical material for tissue and organ transplantation, can induce the cells of a patient to grow into the immunogen removal matrix after being implanted into the body, provides a template for the cells to reconstruct damaged tissues, and repairs the damaged tissues into vascularized and functionalized tissues or organs, the immunogen removal matrix can be gradually degraded and basically synchronized with the process of reconstructing tissue regeneration, and finally the immunogen removal matrix patch is completely replaced by host tissues.
The immunogen removal SIS matrix patch contains aminoglycan, proteoglycan and growth factors, the aminoglycan and the proteoglycan are beneficial to the fixation and chemotaxis of the growth factors, and the growth factors comprise VEGF, TGF beta 1 and bFGF, so that the immunogen removal SIS matrix patch has obvious effects on the repair and vascularization of soft tissue defects and can well remodel and regenerate damaged tissues.
However, when the immunogen-removed SIS matrix patch is used for repairing tissues such as cardiac muscle, nerve, smooth muscle, epithelium and the like, the immunogen-removed SIS matrix patch has a limited induction effect on functional reconstruction of the tissues, mainly plays a role of physical barrier, forms a tissue repairing microenvironment, and prevents peripheral tissues from growing into damaged parts to form adhesion and scars. In addition, the immunogen removal SIS matrix patch is applied to a vascular valve, and the calcification prevention function of the patch is also considered, and the patch is added with growth factors for preventing calcification.
In order to apply the immunogen-removed SIS matrix patch to organs such as heart, blood vessel, nerve, bladder, esophagus, uterus and the like to realize the enhancement and repair of tissue functions, the immunogen-removed SIS matrix patch can be compounded with a single or multiple growth factors to promote the induction regulation and control effect of the immunogen-removed SIS matrix patch on specific tissues and organ cells. However, the half-life of growth factors in vivo is short, and thus the release behavior in vivo needs to be further addressed.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a repair material which is compounded with growth factors in an immunogen removing matrix and can enhance the high induction activity of the material on the specific tissue induction repair action.
In order to solve the technical problems, the invention adopts the technical scheme that: a repair material with high induction activity is characterized in that: the material consists of a natural polymer material with a three-dimensional reticular porous structure, and comprises a three-layer structure consisting of a middle layer, an upper surface layer and a lower surface layer; the middle layer comprises collagen, polysaccharide substances, active factors and growth factors, the upper surface layer and the lower surface layer comprise collagen, polysaccharide substances and active factors, and the repairing material can be degraded in vivo.
The repair material is made of animal small intestine submucosa tissue material.
The animal is a mammal, preferably a pig or a cow.
The collagen is a composition containing type I, type III, type IV and type VI collagen.
The polysaccharide substance is a composition containing chondroitin sulfate and hyaluronic acid.
The active factor is a composition containing fibronectin, laminin, integrin and a ligand thereof.
The growth factor includes but is not limited to follicle-inhibiting protein, nerve growth factor, platelet derived growth factor, epithelial growth factor, matrix gamma carboxy glutamic acid protein and one or a plurality of growth factor compositions.
The time of in vivo degradation is 3-6 months.
The porosity of the three-dimensional mesh porous structure of the upper surface layer and the lower surface layer is 45-70%.
The porosity of the three-dimensional mesh porous structure of the middle layer is 75-90%.
The specific porosity of the invention can control the release of growth factors, and a plurality of growth factors can be selected.
The high-induction-activity repair material is 1-20cm long, 1-10cm wide and 1-4mm thick.
The invention also provides a preparation method of the repair material with high induction activity, which adopts the animal small intestine submucosa tissue as a raw material, and obtains the repair material which eliminates animal-derived virus risk, cell components, DNA components and alpha-Gal antigen and retains the high induction activity of extracellular matrix components through the steps of tissue pretreatment, virus inactivation, immunogen removal, drying, slurry preparation, molding and freeze drying.
The preparation method of the repair material with high induction activity comprises the following specific steps:
(1) tissue pretreatment: taking animal small intestine submucosa tissue material, cleaning and draining water;
(2) virus inactivation: soaking animal small intestine submucosa tissue material in peroxyacetic acid-ethanol solution for virus inactivation; then processing in an ultrasonic device;
(3) immunogen removal: the immunogen removing solution adopts PBS solution containing trypsin and EDTA, and the immunogen removing process is carried out in an ultrasonic cleaning machine; then cleaning in an ultrasonic device to obtain a small intestine submucosa matrix material;
(4) and (3) drying: fixing the immunogen-removed matrix material obtained in the step (3) on a mould, and then putting the mould and the matrix material together in an oven for drying;
(5) preparing slurry: cutting the matrix material cleaned in the step (3), and grinding and crushing the matrix material by using a liquid nitrogen freezing and crushing device to obtain particles; adding the granules into an acetic acid solution, drying in vacuum and crushing; then adding a growth factor solution into the particles to form slurry;
(6) molding: taking the dried small intestine submucosa matrix material film obtained in the step (4) as an upper surface layer and a lower surface layer, adding the slurry obtained in the step (5) between the two surface layers, and paving the mixture on a mold for molding;
(7) and (3) freeze drying: and (3) placing the formed material in a vacuum freeze dryer for vacuum freeze drying.
In the step (2), the volume percentage concentration of the peroxyacetic acid is 0.1-5%, the volume percentage concentration of the ethanol is 5-40% (the ethanol is prepared into the solution by water), the volume ratio of the peroxyacetic acid-ethanol solution to the small intestine submucosa tissue material is (3-20): 1, the inactivation time is 2-4 hours, and the temperature range is 10-40 ℃. The ultrasonic device in the step (2) comprises the following steps: ultrasonically treating small intestine submucosa tissue material with PBS solution with pH of 7.0 at 20 deg.C, wherein the ratio (volume ratio) of PBS solution to small intestine submucosa tissue material is 30: 1, preferably 3 times, each for 20 min; cleaning with 20 deg.C purified water at a ratio of purified water to small intestine submucosa tissue material of 30: 1 until the detected conductivity is below 10 μ S/cm; the cleaning process is carried out in an ultrasonic cleaning machine, the frequency is preferably 40kHz, and the power is preferably more than 3000W.
In the step (3), the immunogen removing solution is PBS solution containing trypsin and EDTA, and the mass percent concentration of the trypsin in the immunogen removing solution is 0.01-0.2%, preferably 0.02-0.05%; the concentration of EDTA is 0.1-1mmol/L, preferably 0.4-0.8 mmol/L; the pH value of the immunogen removing solution is 7.0-8.0, preferably 7.2-7.5; the volume ratio of the immunogen removing liquid to the small intestine submucosa tissue material is (20-40): 1, and the immunogen removing process is carried out in an ultrasonic cleaning machine at least comprising two ultrasonic frequencies, wherein the low-frequency range is 20-40KHz, the high-frequency range is 60-90KHz, the low-frequency treatment is 5-40min, the high-frequency treatment is 5-40min, and the temperature range is 20-35 ℃. Using trypsin and EDTA to break the connection between the cells and the extracellular matrix; and the cells are crushed by adopting low-frequency ultrasound, and simultaneously, the high-frequency ultrasound acts on the crushed cells and the extracellular matrix, so that the cells are further separated from the extracellular matrix, and the purpose of removing immunogen is achieved. By adopting the mode, all steps in the whole process of separating the cells from the matrix are strengthened, so that the cells are completely separated from the matrix, and the optimal immunogen removing effect is achieved. Then respectively treating the mixture with PBS solution and cooling injection water in an ultrasonic cleaning machine to obtain a small intestine submucosa matrix material; the ratio of PBS solution with pH value of 7.2-7.4 to small intestine submucosa tissue material is (20-40): 1, the ratio of water for injection to small intestine submucosa tissue material is (20-40): 1, and the water for injection is treated until the difference of the conductivity of the water for injection before and after detection and cleaning is less than 1 muS/cm; the ultrasonic frequency is preferably 40kHz, and the power is preferably more than 3000W.
The step (4) of the invention comprises the following steps: placing the immunogen tissue-removing material in an environment with the temperature of 25-40 ℃ and the time of 8-16 hours to form an upper surface layer or a lower surface layer.
The slurry in the step (5) of the invention is prepared by: shearing the matrix material cleaned in the step (3), grinding and crushing the matrix material by using a liquid nitrogen freezing and crushing device, crushing the sheared matrix material, and screening out particles with the particle size of below 250 micrometers, preferably below 150 micrometers through a screen (for controlling the uniformity of a loose structure, the smaller the particles are, the more uniform the loose structure is formed); adding 0.3 percent by mass of acetic acid solution into the particles, drying in vacuum and crushing; then adding growth factor aqueous solution into the particles according to the mass ratio of the growth factor to the particles of (1-50):10000 to form slurry for preparing an intermediate layer (active layer).
The freeze-drying of the present invention (7) is: freeze-drying the surface layer and the active layer, pre-freezing to-45 deg.C, maintaining for 1-2 hr, adjusting temperature to-15 deg.C, maintaining for 5-7 hr, adjusting temperature to 0 deg.C, maintaining for 2 hr, adjusting temperature to 25 deg.C, and maintaining for 4 hr to obtain the composite structure.
The invention further provides an application of the repair material with high induction activity, which comprises the following specific steps: the highly inducible repair material may comprise one or more of the following growth factors: follicle-inhibiting protein, nerve growth factor, platelet-derived growth factor, epithelial growth factor, matrix gamma-carboxyglutamic acid protein, and the like.
The application of a repair material with high induction activity can be applied to repair of the tissue injuries of cardiac muscle, nerve, smooth muscle, epithelium and the like.
The application of the repairing material with high induction activity can be fixed at the position of damaged tissues, provide mechanical support and isolation protection, slowly release growth factors, regulate and control the regeneration and repair processes of the tissues with high activity, and prevent calcification of the material after long-term implantation.
Compared with the prior art, the invention has the following remarkable advantages and beneficial effects:
(1) using trypsin and EDTA to break the connection between the cells and the extracellular matrix; the cells are crushed by adopting low-frequency ultrasound, and simultaneously, the high-frequency ultrasound acts on the crushed cells and the extracellular matrix, so that the cells are further separated from the extracellular matrix, and the purpose of removing the cells is achieved. In the above manner, the whole process of separating the cells from the matrix is reinforced, so that the cells are completely separated from the matrix. The optimal immunogen removing effect is achieved.
(2) The high-efficiency crushing technology comprises the following steps: the low-temperature ultrahigh-speed shearing and crushing technology protects collagen and biological factor components from being damaged;
(3) degradable absorption: the degradable in vivo, the degradation process is controllable, and the action period is long;
(4) multilayer sealing film: the three-layer structure is characterized in that the upper surface layer and the lower surface layer are complete immunogen removing matrix layers, the middle layer is a growth factor containing layer, a sandwich structure is formed, and the material degradation and growth factor release processes are controlled;
(5) multiple growth factors can be applied to functional reconstitution of various tissues of different cells: a highly inducible repair material may comprise one or more of the following growth factors: the follicle-inhibiting protein, nerve growth factor, platelet-derived growth factor, epithelial growth factor, matrix gamma-carboxyglutamic acid protein and the like are applied to the growth and regulation of tissues such as cardiac muscle, nerve, smooth muscle, epithelium and the like, or the calcification of matrix materials removed by immunogen after implantation is prevented.
Drawings
FIG. 1 is a schematic representation of a protein-loaded xenogeneic immunogen removal matrix material of the present invention with high induction activity;
FIG. 2 is a SEM image showing the microstructure of different layers of the xenogeneic immunogen removal matrix material of the highly inducible protein carrier of the present invention, wherein the left image is the immunogen removal matrix layer and the right image is the growth factor layer;
FIG. 3 is a graph showing the release profile of the active factor from the matrix material for the removal of xenogeneic immunogens with a highly inducible protein carrier according to the present invention;
FIG. 4 shows the effect of the removal of matrix material from the protein-loaded xenogeneic immunogen of the present invention on the survival of nerve cells.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but is not limited thereto.
The small intestine submucosa tissue material of the present invention is derived from a mammal, and either porcine small intestine submucosa tissue material (SIS) or bovine small intestine submucosa tissue material is suitable for use in embodiments of the present invention.
Example 1:
the preparation method of the animal-derived implantable bioprosthesis patch comprises the following operation steps:
(1) tissue pretreatment:
cutting small intestine submucosa tissue material into specific size of 10cm wide and 15cm long, removing lymphoid tissue, washing with tap water for 3 times, washing with purified water until the surface is free of stain, placing the washed small intestine submucosa tissue material on a filter screen or other water filtering device, standing for more than 5min, and filtering to dry.
(2) Virus inactivation:
the virus inactivation is carried out by soaking small intestine submucosa tissue material in peroxyacetic acid-ethanol solution, and the process can be carried out in a stainless steel barrel. The volume percentage concentration of the peroxyacetic acid in the peroxyacetic acid-ethanol solution is 2 percent, the volume percentage concentration of the ethanol is 20 percent, the ratio (volume ratio) of the peroxyacetic acid-ethanol solution to the small intestine submucosa tissue material is 5: 1, the inactivation time is 2 hours, and the temperature range is 20 ℃; then respectively treating the mixture with PBS and purified water in an ultrasonic device; wherein the small intestine submucosa tissue material is ultrasonically treated by PBS (phosphate buffer solution) with pH value of 7.2-7.4, the temperature of the solution is 20 ℃, the volume ratio of the PBS solution to the small intestine submucosa tissue material is 30: 1, and the treatment is preferably carried out for 3 times, and each time lasts 20 minutes; cleaning with 20 deg.C purified water at a ratio of purified water to small intestine submucosa tissue material of 30: 1 until the detected conductivity is below 10 μ S/cm; the cleaning process is carried out in an ultrasonic cleaning machine, the frequency is 40kHz, and the power is more than 3000W.
(3) Immunogen removal:
the immunogen removing liquid is PBS (phosphate buffer solution) with the pH value of 7 and containing 0.02 mass percent of trypsin and 0.5mmol/L of EDTA, the mixing ratio (volume ratio) of the immunogen removing liquid to the small intestine submucosa tissue material is 30: 1, the immunogen removing process is carried out in a multi-frequency ultrasonic device, and the ultrasonic power is more than 5000W; the multifrequency ultrasound at least comprises two ultrasound frequencies, wherein the low frequency range is 35KHz, the high frequency range is 70KHz, the low frequency treatment is 5min, the high frequency treatment is 10min, and the temperature is 22 ℃. Then respectively treating the mixture with PBS solution and purified water in an ultrasonic cleaning machine to obtain a small intestine submucosa matrix material; treating PBS solution with pH of 7.2-7.4 and small intestine submucosa tissue material at a ratio of 30: 1 and purified water at a ratio of 30: 1 with injection water at 24 deg.C until the difference between the conductivity of the injection water before and after cleaning is 1 μ S/cm; the ultrasonic frequency is preferably 40kHz, and the power is preferably more than 3000W.
(4) And (3) drying:
and (3) in a thermal cycling hundred-grade oven, preheating the oven to 40 ℃, fixing the immunogen-removed matrix material obtained in the step (3) on a mold, and putting the mold and the matrix material in the oven for drying for 16 hours.
(5) Preparing slurry:
shearing the matrix material cleaned in the step (3), grinding and crushing by using a liquid nitrogen freezing and crushing device, feeding the sheared matrix material and liquid nitrogen into the device for crushing, and screening out particles with the particle size of below 150 mu m by using a screen; adding 0.3% (mass percent) acetic acid solution into the granules, wherein the mass ratio of the granules to the acetic acid solution is (0.5-2):1, and crushing the granules into granules after vacuum drying; then adding growth factor (such as epithelial growth factor) water solution into the granules according to the mass ratio of the growth factor to the granules of (1-50):10000, for example, adding the growth factor water solution at the ratio of 20:10000, and forming slurry. Determining the quality of the growth factor according to the quality and the proportional relation of the particles, preparing the growth factor and water into a solution, wherein the mass ratio of the growth factor to the water is (1-50):1000, and then dripping the solution into the particles and uniformly mixing; purified water can be further added to adjust the viscosity of the slurry; water may be added in an amount of 5 to 50 times the mass of the granules, preferably 30 times the mass of the granules.
(6) And (3) forming control:
taking the dried small intestine submucosa matrix material film obtained in the step (4) as a surface layer, adding the slurry obtained in the step (5) between the two surface layers, and flatly paving the mixture on a mold.
(7) And (3) freeze drying:
and (3) paving the molded material in a vacuum freeze dryer, closing a door of a freeze drying chamber, opening a circulating pump for about 1min, starting a compressor to refrigerate the freeze drying box, prefreezing the mold and the material in the step (6) to-45 ℃, preserving heat for 2 hours, then starting a vacuum pump, regulating the temperature to-15 ℃, preserving heat for 6 hours, regulating the temperature to 0 ℃, preserving heat for 2 hours, finally regulating the temperature to 25 ℃, preserving heat for 4 hours, and completing vacuum freeze drying.
The preparation method in this embodiment may further include the steps of:
(8) sterilization and resolution:
adopting ethylene oxide for sterilization, wherein the sterilization conditions are as follows: firstly, preserving the heat for 4 hours at the temperature of 40 ℃ and the humidity of 70 percent, then introducing epoxy ethane with the concentration of 500mg/L, and sterilizing for 6 hours; the analysis process was carried out in a ventilated analysis chamber, the temperature was controlled between 20 ℃ and the time 14 d.
The structure of the product obtained by the invention is shown in figure 1: including upper and lower surface layers (upper and lower surface layers) and an intermediate layer, the microstructure is shown in fig. 2.
The chemical compositions of the materials obtained by the invention are detected as shown in the following table 1:
TABLE 1
| Protein (%) | Carbohydrate (%) | Lipids (%) | Moisture content | Ash content | Growth factor |
| 75％-85％ | 15％-25％ | <1％ | <5％ | <1％ | 0.01-2％ |
Example 2: performance detection
1) The substrate material for removing the heterogeneous immunogen with high induced activity and protein carrying capacity is of a three-layer structure, the upper surface layer and the lower surface layer are complete surface layers, the middle layer is a layer containing a growth factor to form a sandwich structure, the surface layers are compact, the degradation behavior of the material and the release speed of the growth factor can be controlled, and the growth factor layer is a porous structure layer and is beneficial to storing the growth factor. The porosity of the surface layer is 67.4 +/-5.2%, and the porosity of the growth factor layer is 90.5 +/-7.0%.
2) The mechanical property detection shows that the tensile breaking strength of the sample reaches 105N/cm, and the suture holding force is greater than 21N.
3) According to the method for detecting the residual DNA of the biological preparation, namely the fourth part of the 2015 edition, the residual DNA of the sample provided by the example 1 is detected by a fluorescent staining method, wherein the residual DNA of the sample provided by the example 1 is 2.56 +/-0.14 ng/mg.
4) The DNA copy number of the virus was detected by real-time quantitative PCR and 3 batches of samples were tested. As a result: the viral DNA copy number is 0.
5) According to GB/T14233.2-2005 part 2 of the test method of medical transfusion, blood transfusion and injection apparatus: biological test method "the samples provided in example 1 were tested, and the results were as follows: the bacterial endotoxicity is 20 EU/set.
6) Galactosidase (. alpha. -Gal) Clearance Material before immunogen removal treatment Gal value 23.41. + -. 2.34X 1014Mg, Gal value of the sample in example 1 is 0.13. + -. 0.02X 1014The clearance rate of galactosidase (alpha-Gal) is more than 99.40 percent.
7) The type I, III, IV and VI collagen protein is detected by an immunohistochemical staining method and is positive. The content detection of polysaccharide substances shows that the average value of the chondroitin sulfate content in the sample is 5687 +/-201 mu g/g, and the average value of the retention amount of Hyaluronic Acid (HA) is 354 +/-35 mu g/g.
8) The active factors such as fibronectin, laminin and integrin are detected to be positive by an immunohistochemical staining method.
Example 3: detection of active factor Release Profile
High inducing Activity protein-loaded xenogeneic immunogen removal matrix material samples were prepared as in example 1 and placed in 12 well plates with 2 ml PBS (pH 7.4) per well. 5% CO at 37 ℃ of saturated humidity2Atmospheric CO2Incubate 1, 3, 5, 7, 14, 21, 28d (days) in the incubator, transfer the PBS to a centrifuge tube, detect the content of platelet-derived growth factor M1 in the PBS solution, add the initial addition amount as denominator M0, and calculate the growth factor release rate as M1/M0. As shown in FIG. 3, the growth factorThe release is stable and controllable, the release rate of growth solids on day 1 is 19.92 +/-1.96%, the release rate on day 3 is 46.32 +/-3.36%, the release rate on day 7 is 64.16 +/-2.50%, and the total release rate on day 28 reaches 86.56 +/-3.42%. Therefore, the growth factor can last for more than 28 days, has no burst release phenomenon, and embodies high induction activity. Example 4: effect of nerve growth factor addition on nerve cell survival
A sample was prepared as in example 1, in which the added growth factor was replaced with nerve growth factor, the prepared sample was cut into small samples, which were placed in 96 wells for nerve cell culture, and the survival rate of nerve cells was measured by MTT, and only the medium was added to the blank control group. The structure is shown in figure 4, when the culture is carried out for 3 days, the cell survival rate of the test group is 29.62 +/-2.66 percent higher than that of the blank control group, when the culture is carried out for 5 days, the cell survival rate of the test group is 50.04 +/-12.69 percent higher than that of the blank control group, and when the culture is carried out for 7 days, the cell survival rate of the test group is 54.87 +/-9.47 percent higher than that of the blank control group, so the nerve cell survival rate is obviously improved after the matrix material of the heterogeneous immunogen with high induced activity protein is removed and the nerve growth factor is added.
In summary, the present invention provides a repair material with high inducing activity:
(1) the DNA residue can reach below 10ng/mg, is 30-50ng/mg lower than similar products, has higher galactosidase removal rate and can reach more than 99 percent;
(2) reducing the risk of causing allergy, infection, inflammation, granuloma, local abscess;
(3) retaining active growth factors in the extracellular matrix;
(4) a plurality of beneficial growth factors are anchored and added by utilizing amino glycan and proteoglycan;
(5) the in vivo degradation, controllable release of growth factors, long action cycle and high induced activity;
(6) the mechanical property can be controlled, and the release and the mechanical property of the growth factor can be controlled by different layers of sealing films;
(7) the production process can realize standardization and industrialization.
The above-described embodiments of the present invention are illustrative of the present invention and are not intended to be limiting, and any changes within the meaning and scope equivalent to the claims of the present invention are intended to be included within the scope of the claims.
Claims (11)
1. A repair material with high induction activity is characterized in that: the material consists of a natural polymer material with a three-dimensional reticular porous structure, and comprises a three-layer structure consisting of a middle layer, an upper surface layer and a lower surface layer; the middle layer comprises collagen, polysaccharide substances, active factors and growth factors, the upper surface layer and the lower surface layer comprise collagen, polysaccharide substances and active factors, and the repair material can be degraded in vivo;
the high-inductivity repair material is prepared by the following method:
(1) tissue pretreatment: taking animal small intestine submucosa tissue material, cleaning and draining water;
(2) virus inactivation: soaking animal small intestine submucosa tissue material in peroxyacetic acid-ethanol solution for virus inactivation; then cleaning in an ultrasonic device;
(3) immunogen removal: the immunogen removing solution adopts PBS solution containing trypsin and EDTA, and the immunogen removing process is carried out in a multi-frequency ultrasonic device; then cleaning in an ultrasonic cleaning machine to obtain the small intestine submucosa matrix material;
the mass percentage concentration of trypsin in the immunogen removing solution is 0.01-0.2%, the concentration of EDTA is 0.1-1mmol/L, and the pH value of the immunogen removing solution is 7.0-8.0; the volume ratio of the immunogen removing liquid to the small intestine submucosa tissue material is (20-40): 1, and the immunogen removing process is carried out in an ultrasonic cleaning machine at least comprising two ultrasonic frequencies, wherein the low-frequency range is 20-40KHz, the high-frequency range is 60-90KHz, the low-frequency treatment is 5-40min, the high-frequency treatment is 5-40min, and the temperature range is 20-35 ℃; then respectively treating the mixture with PBS solution and cooling injection water in an ultrasonic cleaning machine to obtain a small intestine submucosa matrix material; the ratio of PBS solution with pH value of 7.2-7.4 to small intestine submucosa tissue material is (20-40): 1, the ratio of water for injection to small intestine submucosa tissue material is (20-40): 1, and the water for injection is treated until the difference of the conductivity of the water for injection before and after detection and cleaning is less than 1 muS/cm; ultrasonic frequency is 40kHz, and power is more than 3000W;
(4) and (3) drying: fixing the immunogen-removed matrix material obtained in the step (3) on a mould, and putting the mould and the matrix material in an oven for drying;
(5) preparing slurry: cutting the matrix material cleaned in the step (3), and grinding and crushing the matrix material by using a liquid nitrogen freezing and crushing device to obtain particles; adding acetic acid solution into the granules, drying in vacuum and crushing into granules; then adding a growth factor solution into the particles to form slurry;
(6) molding: taking the dried small intestine submucosa matrix material film obtained in the step (4) as a first surface layer and a second surface layer, adding the slurry obtained in the step (5) between the two surface layers, and flatly paving the mixture on a mold;
(7) and (3) freeze drying: and (3) placing the formed material in a vacuum freeze dryer for vacuum freeze drying.
2. The repair material with high inducing activity according to claim 1, wherein: the repair material is made of animal small intestine submucosa tissue material.
3. The repair material with high inducing activity according to claim 2, wherein: the animal is a mammal.
4. The repair material with high inducing activity according to claim 3, wherein: the mammal is pig or cattle.
5. The repair material with high inducing activity according to claim 1, wherein: the collagen is a composition containing type I, type III, type IV and type VI collagen; the polysaccharide substance is a composition containing chondroitin sulfate and hyaluronic acid; the active factor is a composition containing fibronectin, laminin, integrin and a ligand thereof; the growth factor comprises one or a combination of follicle-inhibiting protein, nerve growth factor, platelet-derived growth factor, epithelial growth factor and matrix gamma-carboxyglutamic acid protein.
6. The repair material with high inducing activity according to claim 1, wherein: the time of in vivo degradation is 3-6 months.
7. The repair material with high inducing activity according to claim 1, wherein the porosity of the three-dimensional network porous structure of the upper surface layer and the lower surface layer is 45 ~ 70%, the porosity of the three-dimensional network porous structure of the middle layer is 75 ~ 90%, and the repair material has a length of 1-20cm, a width of 1-10cm and a thickness of 1-4 mm.
8. A preparation method of a repair material with high induction activity is characterized by comprising the following steps: animal small intestine submucosa tissues are used as raw materials, and the repairing materials for eliminating animal source virus risks, cell components, DNA components and alpha-Gal antigens and reserving extracellular matrix components are obtained through the steps of tissue pretreatment, virus inactivation, immunogen removal, drying, slurry preparation, molding and freeze drying;
wherein:
(1) tissue pretreatment: taking animal small intestine submucosa tissue material, cleaning and draining water;
(2) virus inactivation: soaking animal small intestine submucosa tissue material in peroxyacetic acid-ethanol solution for virus inactivation; then cleaning in an ultrasonic device;
(3) immunogen removal: the immunogen removing solution adopts PBS solution containing trypsin and EDTA, and the immunogen removing process is carried out in a multi-frequency ultrasonic device; then cleaning in an ultrasonic cleaning machine to obtain the small intestine submucosa matrix material;
the mass percentage concentration of trypsin in the immunogen removing solution is 0.01-0.2%, the concentration of EDTA is 0.1-1mmol/L, and the pH value of the immunogen removing solution is 7.0-8.0; the volume ratio of the immunogen removing liquid to the small intestine submucosa tissue material is (20-40): 1, and the immunogen removing process is carried out in an ultrasonic cleaning machine at least comprising two ultrasonic frequencies, wherein the low-frequency range is 20-40KHz, the high-frequency range is 60-90KHz, the low-frequency treatment is 5-40min, the high-frequency treatment is 5-40min, and the temperature range is 20-35 ℃; then respectively treating the mixture with PBS solution and cooling injection water in an ultrasonic cleaning machine to obtain a small intestine submucosa matrix material; the ratio of PBS solution with pH value of 7.2-7.4 to small intestine submucosa tissue material is (20-40): 1, the ratio of water for injection to small intestine submucosa tissue material is (20-40): 1, and the water for injection is treated until the difference of the conductivity of the water for injection before and after detection and cleaning is less than 1 muS/cm; ultrasonic frequency is 40kHz, and power is more than 3000W;
(4) and (3) drying: fixing the immunogen-removed matrix material obtained in the step (3) on a mould, and putting the mould and the matrix material in an oven for drying;
(5) preparing slurry: cutting the matrix material cleaned in the step (3), and grinding and crushing the matrix material by using a liquid nitrogen freezing and crushing device to obtain particles; adding acetic acid solution into the granules, drying in vacuum and crushing into granules; then adding a growth factor solution into the particles to form slurry;
(6) molding: taking the dried small intestine submucosa matrix material film obtained in the step (4) as a first surface layer and a second surface layer, adding the slurry obtained in the step (5) between the two surface layers, and flatly paving the mixture on a mold;
(7) and (3) freeze drying: and (3) placing the formed material in a vacuum freeze dryer for vacuum freeze drying.
9. The method for preparing a repair material with high inducing activity according to claim 8, wherein:
in the step (2), the volume percentage concentration of the peroxyacetic acid is 0.1-5%, the volume percentage concentration of the ethanol is 5-40%, the volume ratio of the peroxyacetic acid-ethanol solution to the small intestine submucosa tissue material is (3-20): 1, the inactivation time is 2-4 hours, the temperature range is 10-40 ℃, and the ultrasonic power is more than 5000W; the ultrasonic device in the step (2) comprises the following steps: ultrasonically treating the small intestine submucosa tissue material by adopting a PBS solution with the pH value of 7.2-7.4, wherein the temperature of the solution is 20 ℃, the volume ratio of the PBS solution to the small intestine submucosa tissue material is 30: 1, and treating for 3 times, and each time lasts for 20 minutes; cleaning with 20 deg.C purified water at a ratio of purified water to small intestine submucosa tissue material of 30: 1 until the detected conductivity is below 10 μ S/cm; the cleaning process is carried out in an ultrasonic cleaning machine, the frequency is 40kHz, and the power is more than 3000W;
the drying in the step (4) comprises the following steps: placing the immunogen tissue-removing material in an environment with the temperature of 25-40 ℃ and the time of 8-16 hours to form an upper surface layer or a lower surface layer;
the slurry prepared in the step (5) is prepared by: cutting the substrate material cleaned in the step (3), grinding and crushing the substrate material by using a liquid nitrogen freezing and crushing device, crushing the cut substrate material, and screening the crushed substrate material by using a screen to obtain the substrate material with the particle size of below 250 micrometers; adding 0.3 percent by mass of acetic acid solution into the particles, drying in vacuum and crushing; then adding growth factor aqueous solution into the particles according to the mass ratio of the growth factor to the particles (1-50):10000 to form slurry for preparing an intermediate layer;
the freeze drying in the step (7) comprises the following steps: freeze-drying the surface layer and the active layer, pre-freezing to-45 deg.C, maintaining for 1-2 hr, adjusting temperature to-15 deg.C, maintaining for 5-7 hr, adjusting temperature to 0 deg.C, maintaining for 2 hr, adjusting temperature to 25 deg.C, and maintaining for 4 hr to obtain the composite structure.
10. The method for preparing a repair material with high inducing activity according to claim 9, wherein: the mass percentage concentration of the trypsin in the immunogen removing solution in the step (3) is 0.02-0.05%, the concentration of EDTA is 0.4-0.8mmol/L, and the pH value of the immunogen removing solution is 7.2-7.5.
11. Use of a highly inductive active prosthetic material according to any one of claims 1 to 7 for the preparation of a prosthetic implant device, characterized in that: the repairing implantation instrument is applied to repairing of myocardial, nerve, smooth muscle and epithelial tissue damage or is fixed at the position of the damaged tissue, provides mechanical support and isolation protection, and slowly releases growth factors.
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| WO2007011644A2 (en) * | 2005-07-15 | 2007-01-25 | Cormatrix Cardiovascular, Inc. | Compositions for regenerating defective or absent tissue |
| CN102743791A (en) * | 2011-08-01 | 2012-10-24 | 四川大学华西医院 | Tissue repair material, preparation method thereof and application |
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| WO2007011644A2 (en) * | 2005-07-15 | 2007-01-25 | Cormatrix Cardiovascular, Inc. | Compositions for regenerating defective or absent tissue |
| CN102743791A (en) * | 2011-08-01 | 2012-10-24 | 四川大学华西医院 | Tissue repair material, preparation method thereof and application |
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