CN114796607A - Biomaterial filled based on collagen nanofibers, preform, preparation method and application thereof - Google Patents
Biomaterial filled based on collagen nanofibers, preform, preparation method and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/24—Heart valves ; Vascular valves, e.g. venous valves; Heart implants, e.g. passive devices for improving the function of the native valve or the heart muscle; Transmyocardial revascularisation [TMR] devices; Valves implantable in the body
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Botany (AREA)
- Cardiology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
- Materials For Medical Uses (AREA)
Abstract
The application discloses a biomaterial prefabricated product and biomaterial based on collagen nanofiber filling, the biomaterial comprises a biomaterial matrix and collagen filled in the biomaterial matrix and subjected to cross-linking treatment, and the biomaterial absorbs water again after being folded without obvious crease; the application also discloses a biomaterial prefabricated product and a preparation method of the biomaterial, wherein the biomaterial is exposed in a collagen-containing solution for pretreatment, and collagen is dispersed in the biomaterial firstly and then self-assembled into nano collagen fibers to obtain the prefabricated product; and exposing the prefabricated product to a cross-linking agent, and carrying out cross-linking treatment to obtain the biological material. The present application also discloses a biomaterial-based heart valve and interventional system. This application is through filling collagen to realize its self-assembly in biological valve and become nanofiber structure, further fix through glutaraldehyde cross-linking, solve the recovery problem after the biological valve compression of dehydration and avoided the introduction of a large amount of new materials simultaneously, reduced biological valve's immunogenicity.
Description
Technical Field
The application relates to the technical field of modification of interventional biomaterials, in particular to a cross-linked modified biomaterial based on collagen nanofiber filling, a modification method and application.
Background
In recent years, interventional biomaterials such as interventional valve replacement surgery have gained more and more extensive clinical applications due to their convenient operation and greatly reduced secondary damage to patients. At the present stage, the interventional valves are all stored in glutaraldehyde solution; when the device is used in a surgical field, repeated cleaning and loading are needed, so that the time and additional risks of the surgery are increased; secondly, residual glutaraldehyde in the biological valve is easy to increase calcification and toxicity of the biological valve.
Biological valves perfectly solve the above problems by means of dehydration and preloading. However, this method requires the biological valve to have better elasticity and toughness, so that the compressed biological valve can recover better after absorbing water. For solving the problems of elasticity and toughness, the main solution in the present stage is to fill the biological valve with a synthetic polymer material and a hydrophilic natural polymer material to increase the elasticity. The introduction of a large amount of new materials undoubtedly increases the risk of immunogenicity, possibly forms new calcification points, and increases the risk of valve damage; on the other hand, hydrophilic natural polymer materials tend to have a limited effect of enhancing the elasticity of the membrane.
Disclosure of Invention
The application provides a biomaterial and a crosslinking modification method based on collagen nanofiber filling, which are characterized in that monomolecular collagen is filled, self-assembly of the collagen into a nanofiber structure in the biomaterial is realized, and further glutaraldehyde crosslinking fixation is performed, so that the recovery problem of the dehydrated biomaterial after compression is solved, and simultaneously, the introduction of a large amount of new materials is avoided.
A biomaterial comprises a biomaterial matrix and cross-linked collagen filled in the biomaterial matrix.
Optionally, the collagen is self-assembled in a monomolecular manner in the filling process, and then the cross-linking treatment is performed.
Optionally, the single molecule is collagen with the molecular weight of 100-400 Kda.
Optionally, wherein the collagen and the biomatrix material are of the same or different biological origin.
Optionally, the maximum breaking force N of the biomaterial is 25-35N.
Optionally, the method for detecting the maximum breaking force includes: after the dry film of the biomaterial was hydrated, it was cut into 1 × 5cm film strips, measured by a universal stretcher. The dry film hydration is understood to mean that the dry film of the biomaterial is reabsorbed, for example by soaking in an aqueous physiological saline solution.
Optionally, the dried film of biomaterial is folded and compressed into a 1ml syringe cylinder in 3 x 3cm film, and released into physiological saline solution after being placed in an oven at 40 ℃ for 3 days without significant folds. The condition that the dry film after folding and compression is released into the normal saline solution and the folded part after reabsorbing water has no trace can be understood as the condition that the whole film is unfolded and is in accordance with the state after hydration before the dry film is folded.
A preparation method of a biomaterial based on collagen nanofiber filling comprises the following steps:
exposing the biological matrix material to a collagen-containing solution for pretreatment to obtain a preform; and exposing the prefabricated product to a cross-linking agent, and carrying out cross-linking treatment to obtain the biomaterial.
Optionally, in the biological matrix material, the collagen content is 60-90%.
Optionally, the biological source of the biological matrix material comprises swine, cattle, horses or sheep.
Optionally, the source of the biomatrix comprises pericardium, heart valve, blood vessel, ligament, muscle, intestine, or skin.
Further optionally, the biological matrix material is porcine pericardium or bovine pericardium.
Optionally, the biomatrix material is subjected to conventional pretreatment before exposure to a collagen-containing solution; the conventional pretreatment comprises acid-base soaking and decellularization, wherein the acid-base soaking comprises acid soaking and/or alkali soaking; one of the three processing modes can be selected, any two combinations of the three processing modes can be selected, or the combination of the three processing modes can be selected; the sequence of acid-base soaking can be alternated, but not performed simultaneously.
The acid-base soaking can improve the microstructure of the biological matrix material, so that the collagen fibers in the biological matrix material are looser. Optionally, in the acid soaking treatment, the fresh biological matrix material is soaked in an aqueous acid solution; acids include, but are not limited to, hydrochloric acid, acetic acid, oxalic acid, citric acid; the concentration of the acid solution is 2-10 mM; the soaking time is 5-120 min.
Optionally, in the alkali soaking treatment, the fresh biological matrix material is soaked in an alkali aqueous solution; bases include, but are not limited to, sodium hydroxide or sodium carbonate; the concentration of the alkali solution is 2-10 mM; the soaking time is 5-120 min.
The decellularization process can wash away the immunogen from the biomatrix material. Optionally, in the cell removal treatment, the fresh biological matrix material is soaked in a cell removal solution; the cell removal liquid is an aqueous solution of a combination of sodium dodecyl sulfate and Triton X-100, the mass percentage concentration of the cell removal liquid is 0.1-5%, and the cell removal time is 2-72 h.
Optionally, the mass percentage concentration of the collagen in the collagen-containing solution is 0.01-10%. The mass percent concentration refers to the concentration during the initial reaction; the solvent in the solution is purified water.
Further optionally, the collagen-containing solution has a collagen concentration of 1-5% by mass.
Optionally, the molecular weight range of the collagen in the collagen-containing solution is 100 KDa-400 KDa.
Optionally, the collagen is from the same or different biological source as the biomatrix material.
The same biological source may be understood as being derived from different tissue parts of the same organism or the same tissue part of the same organism, for example the biomaterial used for the preparation of the biomaterial is porcine pericardium, the collagen being also extracted from porcine pericardium.
In the pretreatment process, unimolecular collagen molecules are firstly dispersed in the biological material and then self-assembled to form the nano collagen fibers. The biological matrix material is exposed in a collagen-containing solution, and collagen is dispersed in the biological matrix material by controlling different reaction conditions and then self-assembled into the nano collagen fiber.
In an optional preprocessing mode, the preprocessing sequentially includes:
in the first stage, the pH value of the collagen-containing solution is weakly acidic;
in the second stage, the pH value of the collagen-containing solution is weakly alkaline.
When the pretreatment mode is adopted:
optionally, in the first stage, the pH of the collagen-containing solution is 3 to 5.
Optionally, the treatment time of the first stage is 0.5 h-36 h.
Optionally, in the second stage, the pH value of the collagen-containing solution is 7-8.
Further optionally, in the second stage, the pH of the collagen-containing solution is 7 to 7.5.
Optionally, the second stage treatment time is 0.5 h-36 h.
In another optional pretreatment mode, the pretreatment sequentially comprises:
in the first stage, the pH value of the collagen-containing solution is weakly acidic;
in the second stage, the solvent composition in the reaction solution is changed relative to the first stage.
When the pretreatment mode is adopted:
optionally, in the first stage, the pH of the collagen-containing solution is 3 to 5.
Optionally, the treatment time of the first stage is 0.5 h-36 h.
Optionally, in the second stage, the volume of the organic solvent in the reaction solution accounts for 40-60% of the total volume of the reaction solution. Taking the case that the volume of the organic solvent accounts for 50% of the total volume of the reaction solution, the volume ratio of the added volume of the organic solvent to the weakly acidic collagen-containing solution is 1: 1.
Optionally, in the second stage, the solvent component in the reaction solution is changed by adding an organic solvent, wherein the added organic solvent is isopropanol or ethanol.
Optionally, the second stage treatment time is 0.5 h-36 h.
Under the weak acidic condition, unimolecular collagen is filled into a biological matrix material, a collagen-containing solution is adjusted to be alkalescent, and is continuously exposed in the alkalescent collagen solution for 0.5-36 h, or an organic solvent is added into the weakly acidic collagen-containing solution until the volume of the organic solvent accounts for 40-60% of the total volume of the reaction solution, and is continuously exposed in a collagen organic solvent mixed solution for 0.5-36 h, and the unimolecular collagen entering the biological matrix material is assembled to form the nano collagen fiber.
Optionally, during the pretreatment, the biological matrix material is exposed in the collagen solution in a static contact or dynamic contact manner.
Static contact is understood to mean that the biomatrix and the collagen solution are kept relatively still, e.g. soaked, during the treatment of the collagen solution. Dynamic contact is understood to mean relative movement between the biomatrix and the collagen solution during the treatment of the collagen solution, for example by cyclically spraying the collagen solution onto the biomatrix or by stirring or shaking the reaction system while the biomatrix is immersed in the collagen solution. The following description will be made in terms of static contact or dynamic contact, unless otherwise specified.
Optionally, the cross-linking agent is glutaraldehyde; exposing the prefabricated product to a glutaraldehyde solution, wherein the mass percentage concentration of glutaraldehyde in the glutaraldehyde solution is 0.05-25%, and the solvent is a conventional solvent, and can be selected from water, normal saline or buffer solution; the crosslinking time is 6h to 3 weeks; the crosslinking temperature is 0-37 ℃.
Optionally, in the step of crosslinking, the preform is in static or dynamic contact with a solution of the crosslinking agent.
Optionally, the preparation method further comprises post-treatment, specifically comprising: the crosslinked biomaterial is again exposed to a solution containing gelatin. The treatment step can seal residual aldehyde groups on the surface of the biological material on one hand, and on the other hand, the gelatin protein has smaller molecular weight and better solubility and can enhance the elasticity of the biological material.
Optionally, in the post-treatment, the mass percentage concentration of the gelatin in the gelatin-containing solution is 0.01-5%, and the solvent is a conventional solvent, for example, the solvent can be selected from water, normal saline or buffer solution; the exposure time in the solution containing the gelatin protein is 0.5h to one week.
Optionally, in the post-treatment step, the biological material is in static or dynamic contact with the solution comprising the gelatin protein.
Optionally, the preparation method further comprises a softening treatment: comprising exposing the finished post-treated biomaterial to a softening agent.
Optionally, the softener comprises a first component, a second component and a solvent;
the first component is glycerol, and the content of the first component is 5-50 wt%;
the second component is one or the combination of any more of histidine, glycine, lysine, sodium hyaluronate, mannitol, sorbitol, stearic acid, glyceryl stearate and polyglycerol, and the content of the second component is 1-30 wt%;
the solvent is one or a mixed solvent of water, ethanol and isopropanol;
the exposure time in the softener is 0.5h to 3 weeks.
Optionally, in the step of the softening treatment, the biological material and the softener are in static contact or dynamic contact.
Optionally, the preparation method further comprises drying the biological material subjected to the softening treatment; the drying process comprises the following steps: drying for 2-72 h at room temperature, and then drying by air blowing or vacuum drying for 1-5 days at the temperature of 15-100 ℃.
Optionally, the gelatin protein is from the same or different biological source as the biological matrix material.
Optionally, the collagen and the gelatin are both self-extracted from a biomatrix material of the same or different biological origin as the biomatrix material; the extraction process of the collagen sequentially comprises the steps of cell removal, organic solvent soaking, acid soaking, homogenate, enzymolysis and purification; the extraction process of the gelatin protein sequentially comprises the steps of cell removal, organic solvent soaking, acid soaking, homogenate, enzymolysis, degradation and purification; in the degradation treatment process, the degradation time is controlled to be 1-60min, and the degradation temperature is controlled to be 40-80 ℃.
The self-extraction process of collagen and gelatin protein comprises the processes of pretreatment of biological materials, homogenate, enzymolysis, post-treatment and the like; the pretreatment process is the same, and comprises cell removal, organic solvent soaking and acid soaking which are sequentially carried out; the post-treatment of collagen comprises purification; the post-treatment of the gelatin protein extraction comprises degradation and purification which are carried out in sequence; the purified collagen or gelatin can be used for modification filling of biological materials, and can also be subjected to drying treatment.
Optionally, in the treatment process of cell removal, the fresh biological matrix material is soaked in a cell removal solution; the cell removal solution is an aqueous solution formed by combining sodium dodecyl sulfate and Triton X-100, the mass percentage concentration of the cell removal solution is 0.1-5%, and the cell removal time is 2-72 h.
Optionally, in the organic solvent soaking process, the biological matrix material is soaked in an organic solvent, the organic solvent includes but is not limited to ethanol or isopropanol, and the soaking time is 1-24 h.
Optionally, in the acid soaking treatment process, the biological matrix material is soaked in an aqueous solution of an acid, wherein the acid includes, but is not limited to, hydrochloric acid, acetic acid, oxalic acid or citric acid; the concentration of the acid solution is 0.001-0.5M; the soaking time is 5-120 min.
Optionally, in the homogenization treatment process, the acid-soaked pericardium is cut into small pieces of about 3 × 3cm, and the homogenization is performed at different rotation speeds and for different time until the material is completely broken.
Optionally, in the enzymolysis treatment process, pepsin is added for enzymolysis, the enzyme concentration (namely the mass percentage concentration of the added enzyme in the reaction system) is 0.1-10%, the time is 4-24h, and the temperature is 4-37 ℃.
Optionally, in the degradation process of the post-treatment, the heating time is controlled to be 1-60min, and the heating temperature is controlled to be 40-80 ℃.
Optionally, in the purification process of the post-treatment, one or more combination modes of centrifugation, NaCl salting-out washing, ethanol sedimentation washing and dialysis are adopted.
Optionally, in the drying process of the post-treatment, the drying mainly comprises drying and freeze-drying; the drying conditions are conventional conditions for protein drying.
In the process of soaking the collagen solution before crosslinking, firstly, the collagen solution is adjusted to be weakly acidic, and monomolecular collagen enters the biological matrix material and is uniformly dispersed in the biological matrix material; then by adjusting the collagen solution to be alkalescent or slowly adding an organic solvent such as isopropanol into the weakly acidic collagen solution, the monomolecular collagen dispersed in the biological matrix material is self-assembled and arranged in the biological matrix material according to a certain rule. The self-assembly process of the monomolecular collagen dispersed in the biological matrix material and in the biological matrix material is physical combination. In the next step of crosslinking, the filled collagen nanofibers and the collagen of the biomaterial are mutually crosslinked and fixed through a crosslinking agent.
The collagen used to fill the biomatrix in this application may be of the same or different biological origin as the biomatrix used to cross-link. Homologous collagen is extracted from biological matrix materials of the same biological source, self-assembly of the homologous collagen into a nanofiber structure in the biological matrix materials is realized, and the recovery problem of the dehydrated biological materials after compression is solved by further crosslinking and fixing glutaraldehyde, so that the introduction of a large amount of new materials is avoided, and the immunogenicity of the biological valve is reduced.
A biomaterial prepared as described above.
The biomaterials prepared herein may be used to access biological valves, such as by minimally invasive access, and may also be used to surgically implant biological valves, such as by surgical implantation.
A method for preparing a biomaterial preform based on collagen nanofiber filling, comprising:
exposing the biological matrix material to a collagen-containing solution for pretreatment to obtain a preform; the collagen is from the same or different biological source as the biological matrix material. The pretreatment process is the same as the pretreatment step in the biomaterial preparation method described above.
A biomaterial preform prepared as described above.
A biological valve comprising a stent and a valve disposed on the stent, the valve being a biological material as described herein. The biological valve may be an interventional valve through minimally invasive intervention or an implanted valve through surgical implantation.
Optionally, the biological valve is a heart valve; comprises a net cylinder shaped bracket and a valve arranged in the bracket.
An interventional system comprising a heart valve and a delivery catheter, the heart valve being delivered by the delivery catheter after folding, the heart valve being as described above.
Compared with the prior art, the application has at least one of the following beneficial effects:
(1) the recovery problem of the dehydrated biological material after compression is solved by filling monomolecular collagen, realizing self-assembly of the monomolecular collagen into a nanofiber structure in the biological material and further cross-linking and fixing the monomolecular collagen by glutaraldehyde;
(2) the homologous collagen is extracted from the biological materials with the same biological source, self-assembly of the homologous collagen into a nanofiber structure in the biological materials is realized, and the recovery problem of the dehydrated biological materials after compression is solved by further crosslinking and fixing the homologous collagen by glutaraldehyde, so that the introduction of a large amount of new materials is avoided, and the immunogenicity of the biological valves is reduced.
(3) During the extraction process of the gelatin protein, proteins with different molecular weights can be obtained by controlling different degradation times and different degradation temperatures, and the gelatin protein can be used for post-treatment of biological materials, so that the elasticity of the biological materials can be further improved.
(4) After the dry film of the biological material is hydrated, the biological material is cut into 1 x 5cm film strips, and the maximum breaking force n can reach 25-35 measured by a universal stretching machine.
(5) Folding the dry film of the biomaterial with 3 × 3cm membrane, compressing into a 1ml syringe cylinder, and placing in an oven at 40 deg.C for 3 days; and then releasing the membrane into a physiological saline solution, observing the flattening condition of the membrane, and after releasing the soaked water, no obvious crease is visible.
Drawings
FIG. 1 is a flow chart of an embodiment of the cross-linking modification process of collagen-filled biomaterial according to the present application.
FIG. 2 is a flow chart of an embodiment of the collagen self-extraction process of the present application.
FIG. 3 is a flow chart of an embodiment of the present invention for gelatin protein self-extraction.
FIG. 4 is a graph showing the results of comparing the maximum breaking force of the biomaterial of example 1 of the present application with that of a conventional glutaraldehyde-crosslinked membrane.
FIG. 5 is a graph showing the dry film water absorption flattening results of the biomaterial of example 1 of the present application.
FIG. 6 is a graph showing the results of comparing the maximum breaking force of the biomaterial of example 3 of the present application with that of a conventional glutaraldehyde-crosslinked membrane.
FIG. 7 is a graph showing the dry film water absorption flattening results of the biomaterial according to example 3 of the present application.
FIG. 8 is a graph showing the results of comparing the maximum breaking force of the biomaterial of example 4 of the present application with that of a conventional glutaraldehyde-crosslinked membrane.
FIG. 9 is a graph showing the dry film water absorption flattening results of the biomaterial of example 4 of the present application.
FIG. 10 is a graph showing the results of comparing the maximum breaking force of the biomaterial of example 5 of the present application with that of a conventional glutaraldehyde-crosslinked membrane.
FIG. 11 is a graph showing the dry film water absorption flattening results of the biomaterial of example 5 of the present application.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
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. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
As shown in fig. 1, the preparation of the biomaterial preform and the cross-linked modified biomaterial may be implemented by performing the corresponding steps in the flowchart, and the preparation of the two in the same flowchart is merely for convenience of comparison and is not limited additionally. In general terms. The process sequentially comprises the steps of conventional pretreatment, crosslinking, soaking of gelatin protein solution, soaking of softener and drying of the biological matrix material. The conventional pretreatment process includes a step selected from acid-base soaking and decellularization, but is not the focus of the present application, and is merely used as a conventional pretreatment means for ensuring the reaction effect.
Conventional pretreatment: the conventional pretreatment adopts acid-base soaking or cell removal or the combination of the acid-base soaking and the cell removal; the acid-base soaking comprises acid soaking and/or alkali soaking, and when the acid soaking and the alkali soaking are adopted simultaneously, the acid soaking and the alkali soaking can be replaced in sequence but cannot be carried out simultaneously.
In the acid soaking treatment, fresh biological matrix materials are soaked in an acid aqueous solution; acids include, but are not limited to, hydrochloric acid, acetic acid, oxalic acid, citric acid; the concentration of the acid solution is 2-10 mM; the soaking time is 5-120 min.
In the alkali soaking treatment, fresh matrix biological materials are soaked in an alkali aqueous solution; bases include, but are not limited to, sodium hydroxide, sodium carbonate; the concentration of the alkali solution is 2-10 mM; the soaking time is 5-120 min.
In the cell removal treatment, fresh biological matrix materials are soaked in cell removal liquid; the cell removal liquid is a water solution of the combination of sodium dodecyl sulfate and Triton X-100, the mass percentage concentration of the cell removal liquid is 0.1-5%, and the cell removal time is 2-72 h.
In the conventional pretreatment process, after each step of treatment, one or at least two mixed cleaning solutions of PBS, normal saline and ethanol are adopted for cleaning for 2-4 times, and each time lasts for 10-20 min.
The biological matrix material can be selected from porcine pericardium or bovine pericardium.
Pretreatment (collagen nanofiber filling): dissolving collagen in a weakly acidic solution or dissolved water and adjusting the pH value of the solution to be weakly acidic, wherein the pH value is in a range of 3.0-5.0, soaking the pretreated biological matrix material such as pig heart envelope or bovine pericardium in the collagen-containing solution for 0.5-36 h (soaking time before self-assembly is initiated), and under the weakly acidic condition, the molecular weight of monomolecular collagen in the collagen-containing solution is in a range of 100-400 KDa; the mass percentage concentration range of the collagen in the collagen-containing solution is 0.01-10%.
After the weakly acidic collagen solution is soaked for 0.5 to 36 hours, slowly adjusting the pH value of the collagen-containing solution to be alkalescent, for example, about 7.4, continuously soaking for 0.5 to 36 hours, and performing self-assembly on monomolecular collagen in the biological material; or slowly adding an organic solvent such as isopropanol into the collagen-containing solution until the addition amount of the isopropanol accounts for about 50% of the volume of the mixed solution containing the collagen and the organic solvent, continuously soaking for 0.5-36 h, and carrying out self-assembly on the monomolecular collagen in the biological material.
After the step of filling the collagen nanofibers is completed, the monomolecular collagen is dispersed and self-assembled in the biomaterial to obtain the biomaterial preform.
And (3) crosslinking: in the cross-linking step, the filled collagen nanofibers and the collagen of the biomaterial are cross-linked and fixed with each other through a cross-linking agent.
One embodiment of the cross-linking process is glutaraldehyde cross-linking, the concentration of the glutaraldehyde is 0.05% -25%, the cross-linking time is 6 h-3 weeks, and the cross-linking temperature is 0-37 ℃.
The cross-linking treatment is optionally followed by a post-treatment, which may be selected from one or a combination of any one of gelatin solution soaking, softener soaking and drying.
Soaking in gelatin protein solution: optionally soaking in gelatin protein solution after crosslinking treatment; the protein concentration range is 0.01-5%, and the soaking time is 0.5 h-1 week.
Soaking a softener: optionally, a softener soaking treatment may be performed after the crosslinking treatment.
The softener comprises a first component, a second component and a solvent. The first component is glycerol; the second component is 1 or more of histidine, glycine, lysine, sodium hyaluronate, mannitol, sorbitol, stearic acid, glyceryl stearate and polyglycerol; the solvent is 1 or mixed solvent of water, ethanol and isopropanol. The mass percentage of the first component is 5-50%; the mass percentage content of the second component is 0.1-30%; the soaking time is 0.5h to 3 weeks.
And (3) drying: optionally, a drying process is performed to produce a dry film that facilitates pre-loading for use in the interventional system.
The drying process is drying for 2-72 h at room temperature, and then drying for 1-5 days by air blowing or vacuum drying at 15-100 ℃.
The molecular weight range of collagen adopted in the filling of the collagen nanofiber is 100 KDa-400 KDa; the collagen can be self-extracted from a biological matrix material with the same biological source as the modified biological material, can also be self-extracted from a biological material with a biological source different from the modified biological material, and can also adopt other collagen meeting the molecular weight requirement, in one embodiment, the self-extraction process flow of the collagen is shown in figure 2, the self-extraction process flow of the gelatin is shown in figure 3 (the extraction process of the collagen reduces degradation steps compared with the extraction process of the gelatin, and other steps are consistent), and the self-extraction process of the collagen and the gelatin comprises the processes of pretreatment, homogenization, enzymolysis, post-treatment and the like of a homogeneous biological matrix material; the pretreatment processes of collagen and gelatin are the same, and the pretreatment process comprises cell removal, organic solvent soaking and acid soaking which are sequentially carried out; the post-treatment process is different from the treatment of the collagen and the gelatin, and the post-treatment of the collagen comprises purification; the post-treatment of the gelatin protein comprises degradation and purification which are carried out in sequence; the purified collagen or gelatin can be used for modification filling of biological matrix materials, and can also be subjected to drying treatment.
And (3) cell removal: in the process of cell removal, fresh biological matrix material pig heart envelope or bovine pericardium is soaked in cell removal liquid; the cell removal solution is an aqueous solution of a combination of sodium dodecyl sulfate and Triton X-100, the mass percentage concentration of the cell removal solution is 0.1-5%, and the cell removal time is 2-72 h.
Soaking in an organic solvent: in the organic solvent soaking process, the biological matrix material is soaked in an organic solvent, wherein the organic solvent includes but is not limited to ethanol or isopropanol, and the soaking time is 1-24 h.
Acid soaking: in the acid soaking treatment process, the biological matrix material is soaked in an aqueous solution of acid, wherein the acid comprises but is not limited to hydrochloric acid, acetic acid, oxalic acid or citric acid; the concentration of the acid solution is 0.001-0.5M; the soaking time is 5-120 min.
Homogenizing: during the homogenization treatment, the acid-soaked pericardium is cut into small blocks of about 3X 3cm, and the rotating speed and the time are adjusted to homogenize until the material is completely broken.
Enzymolysis: in the enzymolysis treatment process, pepsin is added for enzymolysis, the concentration of enzyme in an enzymolysis system is 0.1-10 wt%, the time is 4-24h, and the temperature is 4-37 ℃.
And (3) degradation: in the degradation process of the post-treatment, the mixture is heated for different time (1-60min) and at different temperature (40-80℃)
And (3) purification: in the extraction of collagen, direct purification is carried out after enzymolysis, in the extraction of gelatin, degradation and purification are carried out after enzymolysis, and in the purification process, one or more combination modes of centrifugation, NaCl salting-out washing, ethanol sedimentation washing and dialysis are carried out.
And (3) drying: in the drying process of the post-treatment, the drying mainly comprises drying and freeze-drying; the drying conditions are conventional conditions for protein drying, drying is an optional step, and the purified collagen can be directly used in the biomaterial filling step.
The following is a description of specific examples:
the concentrations in the following examples are given as percentages by mass unless otherwise specified.
Example 1
Soaking porcine pericardium with 1% triton and 1% SDS solution at 37 deg.C for 12 h; then, soaking in isopropanol solution for 12h to remove grease; soaking in 0.5M acetic acid solution for 2 h; cutting into small pieces, adding 2 times of 0.5M acetic acid solution, homogenizing at 15000rpm for 2min for 3 times intermittently; adding 2% pepsin, and performing enzymolysis at 4 deg.C for 12 hr; centrifuging to remove insoluble substances, and dialyzing in a 10KDa dialysis bag for two days; freeze-drying to obtain collagen freeze-dried powder for self-assembly.
Heating the sample subjected to enzymolysis for 20min at 60 ℃ for degradation; centrifuging to remove insoluble substances, salting out with 0.2M NaCl solution, washing for 3 times, and dialyzing in 10KDa dialysis bag for two days; freeze drying to obtain gelatin protein freeze-dried powder.
Soaking fresh pig heart capsule with 0.5% sodium dodecyl sulfate and 0.5% triton X-100 for 8 hr, and washing for 3 times; then soaking in 2% collagen solution (the collagen lyophilized powder is dissolved in water and the pH value of the water solution is adjusted to 4.5) with pH of 4.5 for 2 h; then gradually adjusting the pH value of the reaction solution to 7.4 and continuously standing for 12 hours; then, the mixture is crosslinked in 0.05 percent glutaraldehyde aqueous solution for 6 hours and washed clean by physiological saline. Soaking in 25% glycerol for 0.5 h. Then dried for 2h at room temperature and dried by blowing at 15 ℃ for 1 day.
Example 2
Soaking porcine pericardium with 1% triton and 1% SDS solution at 37 deg.C for 12 h; then, soaking in isopropanol solution for 12h to remove grease; soaking in 0.02M hydrochloric acid solution for 2 h; cutting into small pieces, adding 2 times of 0.02M hydrochloric acid solution, homogenizing at 15000rpm for 2min, and intermittently homogenizing for 3 times; adding 2% pepsin, and performing enzymolysis at 37 deg.C for 12 hr; centrifuging to remove insoluble substances, and dialyzing in a 10KDa dialysis bag for two days; and (5) freeze-drying to obtain collagen freeze-dried powder.
Heating the sample subjected to enzymolysis for 5min at 80 ℃ to degrade collagen; centrifuging to remove insoluble substances, salting out with 0.2M NaCl solution, washing for 3 times, and dialyzing in 10KDa dialysis bag for two days; freeze drying to obtain gelatin protein freeze-dried powder.
Fresh pig heart envelope is soaked in 2% collagen solution (lyophilized collagen powder dissolved in water and pH adjusted to 4.5) with pH of 4.5 for 0.5 h. Isopropanol was added slowly until 50% (i.e. isopropanol to protein solution volume ratio 1: 1) and allowed to stand for 2 h. Crosslinking in 0.05% glutaraldehyde solution for 6h, and cleaning with normal saline. Soaking in 25% glycerol for 0.5 h. Then dried for 2h at 4 ℃ and dried by blowing air at 15 ℃ for 1 day.
Example 3
Soaking porcine pericardium with 1% triton and 1% SDS solution at 37 deg.C for 12 h; subsequently, the mixture was immersed in an isopropyl alcohol solution for 12 hours to remove fats and oils. Soaking in 0.02M hydrochloric acid solution for 2 h; cutting into small pieces, adding 2 times of 0.02M hydrochloric acid solution, homogenizing at 15000rpm for 2min, and intermittently homogenizing for 3 times; adding 2% pepsin, and performing enzymolysis at 37 deg.C for 12 hr; centrifuging to remove insoluble substances, and dialyzing in a 10KDa dialysis bag for two days; and (5) freeze-drying to obtain collagen freeze-dried powder.
Heating the sample subjected to enzymolysis at 80 ℃ for 5min to degrade collagen; centrifuging to remove insoluble substances, salting out with 0.2M NaCl solution, washing for 3 times, and dialyzing in 10KDa dialysis bag for two days; freeze drying to obtain gelatin protein freeze-dried powder.
Soaking fresh pig heart capsule in 10mM acetic acid for 120min, cleaning with purified water, and soaking with 5% sodium dodecyl sulfate and 5% Triton X-100 for 72 h; soaking the membrane in collagen solution (pH 4.5 and 5% of collagen lyophilized powder dissolved in water and adjusting pH of the water solution to 4.5) for 12 hr, adjusting pH to 7.4, and standing for 12 hr; crosslinking in 25% glutaraldehyde solution for 3 weeks, and cleaning with normal saline; soaking in 15% glycerol and 5% glyceryl stearate mixture for 0.5 h. And then freeze-dried.
Example 4
Soaking porcine pericardium with 1% triton and 1% SDS solution at 37 deg.C for 12 h; subsequently, the mixture was immersed in an isopropyl alcohol solution for 12 hours to remove fats and oils. Soaking in 0.02M hydrochloric acid solution for 2 h; cutting into small pieces, adding 2 times of 0.02M hydrochloric acid solution, homogenizing at 15000rpm for 2min, and intermittently homogenizing for 3 times; adding 2% pepsin, and performing enzymolysis at 37 deg.C for 12 hr; centrifuging to remove insoluble substances, and dialyzing in a 10KDa dialysis bag for two days; and (5) freeze-drying to obtain collagen freeze-dried powder.
Heating the sample after enzymolysis for 5min at 80 ℃ to degrade collagen; centrifuging to remove insoluble substances, salting out with 0.2M NaCl solution, washing for 3 times, and dialyzing in 10KDa dialysis bag for two days; freeze drying to obtain gelatin protein freeze-dried powder.
Soaking fresh pig heart capsule in 10mM acetic acid for 120min, cleaning with purified water, and soaking with 5% sodium dodecyl sulfate and 5% Triton X-100 for 72 h; soaking the membrane in collagen solution (pH 4.5 and 5% of collagen lyophilized powder dissolved in water and adjusting pH of the water solution to 4.5) for 12 hr, adjusting pH to 7.4, and standing for 12 hr; crosslinking in 25% glutaraldehyde solution for 3 weeks, and cleaning with normal saline; soaking the membrane in 5% gelatin protein solution (dissolving gelatin protein lyophilized powder in water as described above), standing for 2 hr, and cleaning with physiological saline; soaking in 15% glycerol and 5% glyceryl stearate mixture for 0.5 h. And then air-dried.
Example 5
Soaking fresh bovine pericardium in 1% triton and 1% SDS solution at 37 deg.C for 12 hr; subsequently, the mixture was immersed in an isopropyl alcohol solution for 12 hours to remove fats and oils. Soaking in 0.02M hydrochloric acid solution for 2 h; cutting into small pieces, adding 2 times of 0.02M hydrochloric acid solution, homogenizing at 15000rpm for 2min, and intermittently homogenizing for 3 times; adding 2% pepsin, and performing enzymolysis at 37 deg.C for 12 hr; centrifuging to remove insoluble substances, salting out with 0.2M NaCl solution, washing for 3 times, and dialyzing in 10KDa dialysis bag for two days; freeze drying to obtain bovine collagen freeze-dried powder.
Heating the sample after enzymolysis for 5min at 80 ℃ to degrade collagen; centrifuging to remove insoluble substances, and dialyzing in a 10KDa dialysis bag for two days; freeze drying to obtain bovine gelatin protein freeze-dried powder.
Soaking fresh pig heart capsule in 10mM acetic acid for 120min, cleaning with purified water, and soaking with 5% sodium dodecyl sulfate and 5% Triton X-100 for 72 h; soaking the membrane in a bovine collagen solution (the lyophilized bovine collagen powder is dissolved in water and the pH value of the aqueous solution is adjusted to 4.5) with pH of 4.5 and concentration of 5% for 12h, adjusting the pH to 7.4 and standing for 12 h; crosslinking in 25% glutaraldehyde solution for 3 weeks, and cleaning with normal saline; soaking the membrane in 5% bovine gelatin protein solution (the above lyophilized bovine gelatin protein powder is dissolved in water), standing for 2 hr, and cleaning with physiological saline; soaking in 15% glycerol and 5% glyceryl stearate mixture for 0.5 h. And then air-dried.
The films prepared in example 1, example 3, example 4 and example 5 were subjected to a breaking force test and a water absorption flattening test, respectively.
Breaking force test: after the dry film is hydrated, cutting the film into 1 × 5cm film strips, and measuring the maximum breaking force by a universal stretching machine; a conventional glutaraldehyde cross-linked membrane was used as a control.
The result of the example 1 is shown in fig. 4, and the breaking force of the membrane prepared in the example 1 is 25-30N, which is obviously superior to that of the conventional glutaraldehyde cross-linked membrane.
The structure of example 3 is shown in fig. 6, and the breaking force of the membrane prepared in example 3 is 25-30N, which is obviously superior to that of the conventional glutaraldehyde cross-linked membrane.
The structure of example 4 is shown in fig. 8, and the breaking force of the membrane prepared in example 4 is 25-35N, which is obviously superior to that of the conventional glutaraldehyde cross-linked membrane.
The structure of example 5 is shown in fig. 10, and the breaking force of the membrane prepared in example 5 is 25-35N, which is obviously superior to that of the conventional glutaraldehyde cross-linked membrane.
Water absorption flattening experiment: 3 x 3cm dry film was folded and compressed into a 1ml syringe cylinder as shown (corresponding to a in the figure), and placed in an oven at 40 ℃ for 3 days; then released into physiological saline solution, and the flattening of the patch was observed.
The results of example 1 are shown in fig. 5B and C, where B is a conventional glutaraldehyde cross-linked dry film, and the film sheet has a large number of folds; c is a collagen nanofilled HE dry film (prepared in example 1) with no visible crease after release from immersion in water.
The results of example 3 are shown in fig. 7B and C, where B is a conventional glutaraldehyde cross-linked dry film, and the film sheet has a large number of folds; c was a collagen nano-filled HE dry film (prepared in example 3) with no visible crease after release soaking.
The results of example 4 are shown in fig. 9, B and C, where B is a conventional glutaraldehyde cross-linked dry film, the film sheet having a large number of folds; c is a collagen nano-filled HE dry film (prepared in example 4) with no visible crease after release soaking.
The results of example 5 are shown in fig. 11, B and C, where B is a conventional glutaraldehyde cross-linked dry film, the film sheet having a large number of folds; c was a collagen nano-filled HE dry film (prepared in example 5) with no visible crease after release soaking.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The biomaterial is characterized by comprising a biomaterial and collagen which is filled in the biomaterial and is subjected to cross-linking treatment.
2. The biomaterial according to claim 1, wherein the collagen is self-assembled by a unimolecular method during the filling process, and then the cross-linking treatment is performed; the single molecule is collagen with the molecular weight of 100 KDa-400 KDa; the collagen and the biological matrix material adopt the same or different biological sources; the maximum breaking force N of the biological material is 25-35N.
3. A preparation method of a biomaterial based on collagen nanofiber filling is characterized by comprising the following steps:
exposing the biological matrix material to a collagen-containing solution for pretreatment to obtain a preform; and exposing the prefabricated product to a cross-linking agent, and carrying out cross-linking treatment to obtain the biomaterial.
4. The method of claim 3, wherein the pre-treating comprises, in order:
in the first stage, the pH value of the collagen-containing solution is weakly acidic;
in the second stage, the pH value of the collagen-containing solution is alkalescent;
or
The pretreatment comprises the following steps in sequence:
in the first stage, the pH value of the collagen-containing solution is weakly acidic;
in the second stage, the solvent composition in the reaction solution is changed relative to the first stage.
5. The production method according to claim 3, further comprising a post-treatment, a softening treatment, and a drying treatment which are performed in this order; the post-treatment comprises the following steps: the crosslinked biomaterial is again exposed to a solution containing gelatin.
6. The biomaterial produced by the production method according to any one of claims 3 to 5.
7. A method for preparing a biomaterial preform based on collagen nanofiber filling, comprising:
the biomatrix is exposed to a collagen-containing solution for pretreatment to obtain a preform.
8. A biomaterial preform prepared by the method of claim 7.
9. A biological valve comprising a stent and a valve arranged on the stent, wherein the valve is the biological material as claimed in any one of claims 1 to 2 and 6.
10. An interventional system comprising a heart valve and a delivery catheter, the heart valve being delivered by the delivery catheter after folding, wherein the heart valve is the biological valve of claim 9.
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