CN117752873A - Preparation method and application of bionic remineralized collagen membrane - Google Patents
Preparation method and application of bionic remineralized collagen membrane Download PDFInfo
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- Medicinal Preparation (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention provides a preparation method and application of a biomimetic remineralized collagen membrane, comprising the steps of dissolving polyamide-amine PAMAM with modified functional groups in PB buffer solution with pH of 5.0-7.0 to prepare PAMAM solution with concentration of 0.01-10%; the oral cavity absorbable collagen film and mineralized solution are soaked in the mineralized solution according to the ratio of 5-30, and the biomimetic remineralized oral cavity absorbable collagen film is obtained after shaking for 5-60 min at room temperature. The invention adopts in-vivo mineralization technology, can deposit and mineralize free mineralization ions in the environment through modification of surface functional groups, prolongs the degradation time of the product, and simultaneously induces repair and regeneration of bone defects, thus being an active repair mode. Has good mineralization capability, can induce the repair of bone defects, greatly improves the success rate of GBR operation and improves the satisfaction degree of patients.
Description
Technical Field
The invention belongs to the technical field of implantable degradable medical devices, and particularly relates to a preparation method and application of a biomimetic remineralized collagen membrane.
Background
An oral absorbable collagen film belongs to an implantable degradable medical device material and is widely applied to GBR (gigabit receptor) operation. It is commonly used in combination with bio-inorganic bone and serves to isolate the soft and hard tissue interface, prevent soft tissue intrusion into the bone defect repair, and provide sufficient support space for bone regeneration in the defect area. The oral absorbable collagen membrane has good biocompatibility and degradability, avoids the risk of secondary operation, and is deeply favored by doctors and patients.
At present, the main product functions of the oral absorbable collagen membrane on the market provide space for bone tissue regeneration, and have good barrier property, degradability and biocompatibility. It can be seen that the existing oral absorbable collagen membrane is more passively repaired, so that the degradation performance of the collagen membrane is required to be consistent with the bone defect repair speed, and if the degradation speed of the collagen membrane is faster than the bone defect repair time, early exposure of the bone defect part can be caused, so that soft tissues with higher growth speed grow into the bone defect area, and the overall bone defect repair effect is affected. However, the bone defect repair process is a very complex process that is affected by many factors such as the effect of the surgery, the maintenance after surgery, the condition of the patient, and the oral environment. Thus, in clinical practice, the repair time of bone defects is not consistent. It is therefore a difficult matter to truly achieve the same rate of degradation of the orally absorbable collagen membrane as the repair of bone defects. Aiming at the problems, various production enterprises reduce the degradation speed of respective products and prolong the degradation time so as to ensure enough bone repair time.
Patent CN 112206359A discloses a self-mineralizing collagen membrane, a preparation method and application, the principle is that polyanion is crosslinked on GBR collagen membrane, then calcium and phosphorus ions in body fluid are spontaneously enriched by polyanion and mineralization in fiber is gradually induced, so that the collagen membrane is converted from ductile material to rigid material in vivo. It uses a polyacrylic acid with a molecular weight of 450kDa as the polyanion.
Patent CN 114470326A discloses a preparation method of a biomimetic mineralized collagen-glycosaminoglycan material, which comprises the main process of pretreating the collagen-glycosaminoglycan material and then soaking the pretreated collagen-glycosaminoglycan material in mineralized liquid to obtain the biomimetic mineralized collagen-glycosaminoglycan material. The mineralizing liquid is a solution containing cerium acetylacetonate, the pretreatment comprises pretreatment soaking in a pretreatment liquid I and a pretreatment liquid II in sequence, the pretreatment liquid I is prepared by collagenase or mercaptopropionic acid, and the pretreatment liquid II is prepared by polyacrylic acid or ammonium polyphosphate with the molecular weight of 450 kDa.
Patent CN 106492283A discloses a mineralized guided tissue regeneration membrane, a preparation method and application thereof, and the product structure comprises a loose layer and a compact layer, wherein the loose layer is formed by compounding type I collagen and nano hydroxyapatite; a dense layer on the porous layer, the dense layer being composed of type i collagen.
Patent CN 115518206A discloses a self-mineralized GBR membrane and a preparation method thereof, comprising a metal bracket and a self-mineralized collagen membrane arranged on the surface of the metal bracket, wherein the preparation method is to place the metal bracket deposited with collagen into mineralized precursor solution for culture, complete the self-assembly process of collagen fiber net, and enable the self-mineralized collagen membrane to be formed on the surface of the metal bracket, thus obtaining the self-mineralized GBR membrane. The mineralized precursor solution is prepared by mixing calcium salt inorganic ions with a polymer. The polymer is a polyelectrolyte and/or a polyacid molecule; any one of polyacrylic acid, polyaspartic acid, polyallylamine and casein phosphopeptide.
Patent CN 113018519B discloses a mineralized guided tissue regeneration membrane, comprising a dense layer and a porous layer. The compact layer is formed by co-assembling type I collagen and silk fibroin; the loose layer is formed by mineralizing nano hydroxyapatite on a type I collagen and silk fibroin double-molecule template.
Patent CN 110420359A discloses a guided tissue regeneration membrane and a method for preparing the same, comprising a dense layer facing the periosteum and a loose layer facing the bone defect area; the compact layer is composed of I-type collagen, and the surface is smooth; the loose layer is formed by compounding type I collagen and mineralized collagen nano particles, and the inside of the loose layer is of a porous structure. After the compact layer and the loose layer are compounded, the compounded material is further subjected to roll forming and crosslinking treatment in sequence.
Patent CN 110898253B discloses a preparation method of a bionic mineralized collagen membrane in fiber capable of promoting cell osteogenic differentiation, firstly synthesizing a bionic mineralized solution containing amorphous calcium phosphate, and further preparing the bionic mineralized collagen membrane in fiber by using the bionic mineralized solution. Wherein the biomimetic mineralizing liquid of amorphous calcium phosphate comprises calcium ions, phosphorus ions, glutamic acid and polyacrylic acid. The preparation method of the intra-fiber biomimetic mineralized collagen membrane comprises the steps of preparing collagen gel by crosslinking a collagen solution, placing the collagen gel into biomimetic mineralized solution for culture, and finally obtaining the intra-fiber biomimetic mineralized collagen membrane through freeze drying.
Patent CN 114949364B discloses a multilayer tissue engineering bionic periosteum scaffold and a preparation method and application, which comprises a mineralized nanofiber membrane-cell composite layer which is overlapped layer by layer, wherein the mineralized nanofiber membrane-cell composite layer comprises a mineralized nanofiber membrane and stem cells inoculated on the mineralized nanofiber membrane, wherein mineralization is to coat the surface of the prepared nanofiber membrane with three layers of collagen-PSS-collagen, and then the three layers of coatings are placed in simulated body fluid for incubation, so that calcium phosphate is orderly deposited on the nanofiber membrane, and the mineralized nanofiber membrane is obtained.
Patent CN 105688288B discloses a collagen-based composite oral cavity repairing film material and a preparation method thereof, wherein oxidized carboxymethyl chitosan is used for carrying out crosslinking modification on type I collagen, a thermal driving method is adopted for promoting self-assembly of the collagen at the same time, then the collagen-based material is subjected to further crosslinking and pre-calcification treatment successively, finally biomimetic mineralization is carried out in SBF solution which is 1-10 times of that of the micro PAA solution, and the collagen-based composite oral cavity repairing film material is obtained after freeze drying and sterilization treatment of the product.
Combining the above similar protocol techniques, it is mainly to use polyanions, such as polyacrylic acid (PAA), in combination with collagen membranes or by mixing collagen slurries with mineralized substances to prepare biomimetic remineralised collagen membranes. The combination mode comprises chemical crosslinking, physical mixing and covalent reaction, the whole process is complex, the process condition is harsh, and industrialization is difficult to realize.
Disclosure of Invention
The invention aims to provide a bionic remineralizing collagen membrane, which is implanted into a defect part to deposit minerals such as calcium ions in the oral cavity environment, so that the product is mineralized, the degradation time of the product is prolonged, and the bone defect can be induced to repair and regenerate, thereby being an active repair mode, having simple process and being suitable for industrialized application.
In order to achieve the above purpose, the invention provides a preparation method of a biomimetic remineralizing collagen membrane, comprising the following steps:
step 1, preparing mineralized solution:
the mineralized solution is a solution of dendritic macromolecule polyamide-amine PAMAM with modified functional groups, and the specific preparation process is as follows: dissolving polyamide-amine PAMAM with modified functional groups in PB buffer solution with pH of 5.0-7.0 to prepare PAMAM solution with concentration of 0.01-10%, so as to obtain mineralized solution;
and 2, soaking the oral cavity absorbable collagen film in a mineralized solution according to the ratio of 5-30, and oscillating for 5-60 min at room temperature to obtain the biomimetic remineralized oral cavity absorbable collagen film.
And 3, placing the biomimetic remineralizing oral absorbable collagen membrane in a cleaning agent with a feed-liquid ratio of 5-30 for cleaning, wherein the cleaning agent is any one or two of purified water or water for injection.
And 4, spreading the cleaned bionic remineralized oral absorbable collagen film on a freeze-drying mold, and drying in a freeze-drying agent.
And 5, sterilizing the freeze-dried biomimetic remineralized oral absorbable collagen by adopting cobalt-60 gamma rays or ethylene oxide, wherein the sterilization dose of the cobalt-60 gamma rays is 15-30 KGy.
The polyamide-amine PAMAM is a G3-G10 dendritic polyamide-amine modified with any one of carboxyl, hydroxyl, amino, phosphorylated or acetamido groups.
The biomimetic remineralizing oral absorbable collagen membrane is applied to repairing soft and hard tissue defects in oral GBR operation, and is mainly used for physically isolating and inducing calcium and phosphorus atom combination in oral environment, promoting remineralization of bone defect areas and inducing new bone growth.
The beneficial effects of the invention are as follows: the technology core is dendritic polymer material polyamide-amine (PAMAM) which can be modified by various functional groups and has good biocompatibility, can be subjected to covalent reaction with collagen fibers under simple and mild conditions, and is tightly combined. The self spherical mesh structure has strong adsorption effect, can be used as an initial crystal nucleus in the mineralization process, can deposit and mineralize free mineralized ions in the environment through modification of surface functional groups, can induce repair and regeneration of bone defects while prolonging the degradation time of the product, and is an active repair mode. Has good mineralization capability, can induce the repair of bone defects, greatly improves the success rate of GBR operation and improves the satisfaction degree of patients.
The present invention will be described in further detail with reference to the accompanying drawings.
Drawings
FIG. 1 is a flow chart of a process for preparing a biomimetic remineralizing collagen membrane.
Detailed Description
The following detailed description, structural features and functions of the present invention are provided with reference to the accompanying drawings and examples in order to further illustrate the technical means and effects of the present invention to achieve the predetermined objects.
The orally absorbable collagen film is mainly divided into animal-derived tissue collagen film and microbial fermentation collagen film, and its main component is type I collagen and contains a small amount of type III collagen. The product has rich pore structure and good hydrophilicity. Collagen itself has a complex spatial structure and chemical composition, with diverse physical and chemical properties. Such as collagen amphiphilicity, thermal denaturation, colloidality, and the like. The collagen can be modified with pertinence based on a full understanding of its properties.
Polyamide-amine (PAMAM) dendrimers are novel polymers, and are star-burst-shaped dendrimers synthesized for the first time by Tomalia subject group. The three-dimensional hyperbranched highly ordered space geometrical symmetry structure consists of a central core, an internal cavity, rich branched structures and modifiable surface functional groups. PAMAM has the characteristics of controllable molecular weight, multifunction, good solubility, biocompatibility and the like, and is widely applied to the fields of biology, medicine and health, industrial production and the like.
The amino-terminal PAMAM can chelate and deposit calcium and phosphorus ions in artificial saliva while adsorbing on the tooth surface. PAMAM-NH2 is used as a cationic polymer, and is acted on a negatively charged bacterial envelope through static electricity, so that the envelope is damaged, intracellular substances leak out, bacterial death is finally caused, and the bacteria are not induced to generate drug resistance, so that the PAMAM-NH2 is a good antibacterial substance. In addition, PAMAM has good adsorption capacity with tooth hard tissue interfaces such as dentin, enamel and the like, can be firmly combined on the surface of the tooth hard tissue interface by specific adsorption with charged tooth hard tissue, prolongs the retention time and can play a role in a longer period.
Dendrimers can theoretically act as both three-dimensional collagen scaffolds and non-collagen analogues (NCPs) that play a decisive role in the formation of collagen-based biominerals, while their excellent chemical and biological properties and availability make it possible to induce biomimetic remineralization using them. Current related studies indicate that type I collagen fibers enrich the positively charged e-segment, PAMAM can enter the interior of collagen fibers by the action of charge attraction, bind to specific sites of type I collagen fibers at the e-segment and have superior binding capacity, thereby inducing nucleation and further mineralization growth of amorphous calcium phosphate at the binding sites within the collagen fibers.
Based on the above theoretical basis, this embodiment provides a biomimetic remineralizing collagen membrane, which adopts PAMAM as a core material for realizing biomimetic remineralizing of the oral cavity absorbable collagen membrane, and the biomimetic remineralizing oral cavity absorbable collagen membrane is obtained after the absorbable collagen membrane is compounded with a remineralizing solution, and then the biomimetic remineralizing oral cavity absorbable collagen membrane is obtained after cleaning treatment, drying treatment and sterilization treatment. The mineral substances such as calcium ions in the oral cavity environment can be deposited at the implantation defect part, so that the product is mineralized, the degradation time of the product is prolonged, and the repair and regeneration of the bone defect can be induced, thereby being an active repair mode. Has good mineralization capability, can induce the repair of bone defects, greatly improves the success rate of GBR operation and improves the satisfaction degree of patients.
As can be seen from fig. 1, the preparation method of the biomimetic remineralizing collagen membrane provided in this embodiment includes preparing mineralizing solution, mineralizing treatment, cleaning treatment, drying treatment and sterilizing treatment. Wherein the mineralization solution is PAMAM solution with modified functional groups, and the mineralization treatment is to soak the oral absorbable collagen membrane into the mineralization solution, and obtain the remineralized collagen membrane after a certain process condition treatment. And then cleaning, drying and sterilizing to obtain the final bionic remineralization oral absorbable collagen membrane. The preparation process is as follows:
step 1, preparing mineralized solution:
the mineralized solution is a solution of dendritic macromolecule polyamide-amine PAMAM with modified functional groups, and the specific preparation process is as follows: dissolving polyamide-amine PAMAM with modified functional groups in PB buffer solution with pH of 5.0-7.0 to prepare PAMAM solution with concentration of 0.01-10%, so as to obtain mineralized solution; the polyamide-amine PAMAM referred to herein is a G3 to G10 dendritic polyamide-amine modified with any one of carboxyl, hydroxyl, amino, phosphorylated or acetamido groups.
Step 2, soaking the oral cavity absorbable collagen film and mineralized solution according to the ratio of 5-30, and oscillating for 5-60 min at room temperature to obtain the bionic remineralization oral cavity absorbable collagen film which is a primary product and cannot be used in actual diagnosis and treatment, wherein the step 3 is cleaning, and the bionic remineralization oral cavity absorbable collagen film is placed in a cleaning agent with the ratio of 5-30 for cleaning, wherein the cleaning agent is one or two of purified water and water for injection.
And step 4, drying, namely spreading the cleaned bionic remineralization oral absorbable collagen film on a freeze-drying mold, closely attaching the smooth surface of the bionic remineralization oral absorbable collagen film to the freeze-drying mold, and placing the bionic remineralization oral absorbable collagen film in a freeze drying agent for drying treatment without air bubbles in the middle.
And 5, sterilizing, namely sterilizing the freeze-dried biomimetic remineralized oral absorbable collagen by adopting cobalt-60 gamma rays or ethylene oxide, wherein the sterilization dose of the cobalt-60 gamma rays is 15-30 KGy.
The bionic remineralization collagen membrane provided by the embodiment is mainly applied to soft and hard tissue defect repair treatment in oral cavity GBR operation, can play a physical isolation role in the repair treatment process, and meanwhile, the composite PAMAM molecules can effectively induce calcium and phosphorus atom combination in an oral cavity environment, promote remineralization of a bone defect area and induce new bone growth.
The prior art shows that the prior bionic remineralization oral absorbable collagen membrane can be roughly divided into in-vitro mineralization and in-vivo mineralization according to the mineralization mode, wherein the in-vitro mineralization is realized by combining collagen with mineral ions and then implanting the collagen into a body. In-vivo mineralization is to combine the oral absorbable collagen membrane with mineralization factors and then implant the collagen membrane into the body, and deposit mineral ions in the oral environment in the body by the mineralization factors, so as to realize the aim of biomimetic mineralization. In vitro mineralization is divided into two modes of combining collagen membrane with mineral ions and mixing collagen slurry with mineral ions. The technical problem of the maximum in-vitro mineralization is that the flexibility of the collagen membrane can be changed or lost, so that the collagen membrane becomes more rigid and more brittle, and the clinical application is difficult to realize. In order to reduce the influence of in-vitro mineralization on the flexibility of the collagen membrane, the mineralization condition needs to be strictly controlled, and the process requirement is harsh, thus being an obstacle for realizing large-scale industrialization. The original performance of the absorbable collagen membrane of the mineralized oral cavity in the body has lower influence, and the degradation time of the collagen membrane can be prolonged along with the continuous deposition of the mineral after implantation, so that the risk of early exposure of a bone repair area is avoided. Is a preferred route for biomimetic remineralization.
According to the biomimetic remineralization technology disclosed in patent CN 112206359A, the core is polyanion, polyacrylic acid (PAA). The main principle of remineralization is that polyanion electrolytes such as polyacrylic acid (PAA) can be used as substitutes of NCPs, and amorphous precursor (ACP) phases are stabilized in the form of high-negative charge nanoclusters, so that the mineralization process in fibers is regulated. The process firstly needs to construct a collagen model with positive/negative charges, and then the polyanion is grafted on the collagen surface through a covalent bonding method by a chemical crosslinking agent EDC/NHS. The whole process of the process has higher requirements, the chemical cross-linking agent has toxicity, the residue of the chemical cross-linking agent can influence the biocompatibility of the product, and the chemical cross-linking agent has certain safety risk. Also, the pretreatment and mineralization process of the CN 114470326A material uses a large amount of toxic reagents, and there is a safety risk of reagent residues. The biomimetic remineralizing oral absorbable collagen membrane provided by the embodiment adopts an in-vivo mineralizing technology, the technical core is dendritic polymer material polyamide-amine (PAMAM), the PAMAM can be modified by various functional groups, the biocompatibility is good, covalent reaction can be carried out with collagen fibers under simple and mild conditions, and the bonding is tight. The self spherical mesh structure has strong adsorption effect, can be used as an initial crystal nucleus of a mineralization process, and can deposit and mineralize free mineralization ions in the environment through modification of surface functional groups.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.
Claims (6)
1. A method for preparing a biomimetic remineralized collagen membrane, which is characterized by comprising the following steps:
step 1, preparing mineralized solution:
the mineralized solution is a solution of dendritic macromolecule polyamide-amine PAMAM with modified functional groups, and the specific preparation process is as follows: dissolving polyamide-amine PAMAM modified by functional group in
preparing a PAMAM solution with the concentration of 0.01-10% in a PB buffer solution with the pH of 5.0-7.0 to obtain the mineralized solution;
and 2, soaking the oral cavity absorbable collagen film in a mineralized solution according to the ratio of 5-30, and oscillating for 5-60 min at room temperature to obtain the biomimetic remineralized oral cavity absorbable collagen film.
2. The method for preparing the biomimetic remineralizing collagen membrane according to claim 1, further comprising the step 3 of placing the biomimetic remineralizing oral absorbable collagen membrane in a cleaning agent with a feed-liquid ratio of 5-30 for cleaning, wherein the cleaning agent is either or both of purified water and water for injection.
3. The method for preparing a biomimetic remineralizing collagen membrane according to claim 2, further comprising step 4, spreading the cleaned biomimetic remineralizing oral absorbable collagen membrane on a freeze-drying mold, and placing the freeze-drying mold in a freeze-drying agent for drying treatment.
4. The method for preparing a biomimetic remineralizing collagen membrane according to claim 3, further comprising step 5 of sterilizing the freeze-dried biomimetic remineralizing orally absorbable collagen by cobalt-60 gamma rays or ethylene oxide, wherein the sterilization dose of the cobalt-60 gamma rays is 15-30 KGy.
5. The method of preparing a biomimetic remineralizing collagen membrane according to claim 1 or 2 or 3 or 4, wherein the polyamidoamine PAMAM is a G3-G10 dendritic polyamidoamine modified with any one of carboxyl, hydroxyl, amino, phosphorylating or acetamido groups.
6. The biomimetic remineralizing collagen membrane according to claim 1 or 2 or 3 or 4, for use in repairing soft and hard tissue defects in oral GBR surgery, mainly for physically isolating and inducing calcium-phosphorus atom bonding in oral environment, promoting remineralization of bone defect areas and inducing new bone growth.
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