CN110882424B - Oral cavity guided bone regeneration barrier membrane - Google Patents
Oral cavity guided bone regeneration barrier membrane Download PDFInfo
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Abstract
The invention relates to an oral material, in particular to an oral cavity guided bone regeneration barrier membrane. The oral cavity guided bone regeneration barrier film is composed of a magnesium alloy material in a film shape and a coating coated on the surface of the magnesium alloy material, wherein a plurality of holes are formed in the barrier film and are in a net structure, and the magnesium alloy material comprises the following components by taking the total weight of the magnesium alloy as 100 percent: aluminum: 10-20%, zinc: 10-30%, manganese: 0.5-10%, silver: 0.01-2%, calcium: 0-10%, titanium dioxide: 1-10% and the balance of Mg. The coating is a mussel mucin BMP-2 coating. The application adds the mussel mucin BMP-2 coating, utilizes the strong biological viscosity and good biocompatibility of mussel protein, and mussel protein wraps BMP-2 to form microspheres and is strongly adsorbed on the surface of the magnesium alloy to form a coating capable of promoting and inducing bone growth.
Description
Technical Field
The invention relates to an oral material, in particular to an oral cavity guided bone regeneration barrier membrane.
Background
With the development and the wide application of the oral implanting technology, the repairing problem of tooth loss is successfully solved. However, the failure of dental implantation due to insufficient alveolar bone mass after the tooth loss has once been an obstacle to the application of the oral implantation technique. To solve this problem, a guided bone regeneration technique that has been recently developed provides an osteogenic space in a bone surface closed bone defect region using a barrier membrane material to increase alveolar bone mass. The technology basically eliminates the problems of alveolar process and insufficient bone mass of the implant, and also provides a good remedy for implant failure.
Barrier membranes currently used in clinical applications are of many types and are generally classified into non-absorbable membrane materials and absorbable membrane materials. The non-absorbable film material has the following advantages: (1) the barrier effect of the membrane material is stable; (2) the mechanical property and the maintenance capability of a regeneration space are strong; (3) the retention time of the medicine in the body can be adjusted at will and the medicine can be taken out at any time. The application time of the material is the most common at the earliest, but the material has a plurality of defects, so that the material tends to be replaced by absorbable membrane material, (1) the material is hard and difficult to shape; (2) the compatibility of the organism is poor, the organism is easy to crack in the early stage, and the osteogenesis effect is not ideal after exposure; (3) the body cannot absorb the medicine, so the medicine needs to be taken out through a secondary operation. The absorbable membrane material has the greatest advantages that the absorbable membrane material is not required to be taken out after a secondary operation, the degradation products in vivo of the absorbable membrane material can be absorbed by organisms, the absorbable membrane material has good histocompatibility, the organisms have no rejection reaction, and the epithelial movement can be effectively inhibited. At present, the absorbable membrane materials mainly comprise collagen membranes and synthetic polymer membranes. However, the existing absorbable membrane material also has the defects of poor supporting capability, poor antibacterial performance and the like.
Disclosure of Invention
The invention aims to provide a barrier membrane which has strong supporting capability, good antibacterial performance, is degradable in vivo and can promote bone growth and guide bone regeneration in the oral cavity.
The oral cavity guided bone regeneration barrier film is composed of a magnesium alloy material in a film shape and a coating coated on the surface of the magnesium alloy material, wherein a plurality of holes are formed in the barrier film and are in a net structure, and the magnesium alloy material comprises the following components by taking the total weight of the magnesium alloy as 100 percent: aluminum: 10-20%, zinc: 10-30%, manganese: 0.5-10%, silver: 0.01-2%, calcium: 0-10%, titanium dioxide: 1-10% and the balance of Mg.
Further, the coating is a silver-loaded hydroxyapatite coating.
Further, the coating is a mussel mucin BMP-2 coating.
Further, the magnesium alloy material comprises aluminum: 10-20%, zinc: 10-30%, manganese: 0.5-10%, silver: 0.01-2%, calcium: 0-10%, titanium dioxide: 4-6% and the balance of Mg.
Further, the magnesium alloy material comprises aluminum: 10-20%, zinc: 10-30%, manganese: 0.5-10%, silver: 0.01-2%, calcium: 0-10%, titanium dioxide: 5.5 percent, and the balance being Mg.
Further, the preparation method of the mussel mucin BMP-2 coating comprises the following steps: (1) pretreating the surface of the magnesium alloy; (2) preparing a coating dipping solution: dissolving the modified mussel mucin in a disodium hydrogen phosphate and sodium dihydrogen phosphate buffer solution, adding rhBMP-2 polypeptide, mixing and stirring uniformly until the solution is soaked; (3) and (3) dipping the pretreated magnesium alloy material in a solution to be dipped for 10min, stirring in the dipping process, taking out, washing with distilled water, blow-drying, and preparing the mussel mucin BMP-2 coating on the surface of the magnesium alloy.
The mussel mucin is glycosyl modified mussel mucin.
The preparation method of the magnesium alloy comprises the following steps: placing a proper amount of titanium dioxide powder and a magnesium alloy to be processed into a ball mill according to a proportion, carrying out high-speed ball milling under a protective atmosphere to obtain mixed powder, and then carrying out selective laser melting under the protective atmosphere to prepare a magnesium alloy melt with an antibacterial function; rapidly adding the dried pure calcium into the pure magnesium melt, continuously stirring, and smelting for 1-1.5 hours to obtain a casting-state magnesium alloy containing calcium; placing the cast magnesium alloy in a vacuum tube furnace, and carrying out solid solution treatment for 10-30 hours at the temperature of 280-500 ℃; and aging at 220-480 deg.C for 20-60 hr.
The oral cavity guided bone regeneration barrier film is made of magnesium alloy materials, has the advantages of strong support, good antibacterial performance, in-vivo degradability and the like, can improve the column modeling performance and biocompatibility of the materials by adding silver element into the adopted magnesium alloy, and can regulate and control the microstructure of the materials through heat treatment at different time, thereby regulating and controlling the degradation rate of the materials.
The addition of calcium element can refine the crystal grains of the magnesium alloy, achieve the effect of fine grain strengthening and obviously improve the formability and the strength of the magnesium alloy. Meanwhile, the calcium element can also inhibit the oxidation of molten metal in the smelting process of the magnesium alloy, and part of Mg2Ca is precipitated on grain boundaries, so that the formability and the strength of the magnesium alloy are improved. And internal defects of the cast ingot are reduced. The calcium can reduce the micro-battery effect of the magnesium alloy and improve the corrosion resistance of the magnesium alloy.
The condition of bacterial infection of wounds frequently occurs in the bone grafting and repairing process, which easily causes the failure of bone repair, and although the alkaline product after the biological magnesium alloy is degraded has certain antibacterial activity, the effect is very limited. Therefore, the invention adds antibacterial titanium dioxide into the magnesium alloy to effectively improve the regeneration effect.
The mussel mucin BMP-2 coating is added, and by utilizing the strong biological viscosity and good biocompatibility of the mussel protein, the mussel protein wraps the BMP-2 to form microspheres which are strongly adsorbed on the surface of the magnesium alloy to form a coating capable of promoting and inducing bone growth.
The mussel mucin has adhesive protein of a series of basic proteins, the amino acid containing hydroxyl is up to 60-70%, wherein most Pro is modified into Hyn by post-translation hydroxylation, and the effects between polyphenol proteins and collagen molecules are stabilized; the phenol group of the polyphenol has strong metal chelating capacity, forms an organic metal complex on the surface of a magnesium alloy material, and can form strong hydrogen bonds with polar polymers such as protein and the like, so that the BMP2 can be stably adhered to the surface of the magnesium alloy barrier film. With the barrier membrane of the present invention, BMP2 can be continuously stabilized to induce alveolar bone regeneration during alveolar bone growth.
Drawings
FIG. 1: the growth condition of cells in the barrier membrane material under an electron microscope;
FIG. 2: calcium nodule Von Kossa staining pattern.
Detailed Description
Example 1: preparation of magnesium alloy material
The magnesium alloy material of the barrier film comprises the following components by taking the total weight of the magnesium alloy material as 100 percent: aluminum: 10%, zinc: 15%, manganese: 0.8%, silver: 0.05%, calcium: 8%, titanium dioxide: 7 percent and the balance of Mg.
The adopted preparation method comprises the following steps:
preparing titanium dioxide and magnesium alloy matrix powder according to a proportion; mixing the powder, putting the mixture into a ball mill, and grinding the powder to be uniform under a protective atmosphere; under the protection atmosphere, obtaining a magnesium solution containing titanium dioxide by adopting a selective laser melting process; rapidly adding the dried pure calcium into the magnesium melt according to the proportion, stirring the mixture from time to time, and smelting the mixture for 1.5 hours to obtain a casting-state magnesium alloy containing calcium; placing the casting magnesium alloy containing calcium into a vacuum tube furnace, and carrying out solution treatment for 20 hours at 350 ℃; and aging at 400 deg.C for 48 hr.
Example 2: mussel mucin BMP-2 coating on magnesium alloy material surface
Preparation of sugar-based modified mussel mucin:
preparing CELL-TAK natural mussel mucin mussel into 0.5% suspension by using double distilled water, adding D-galactosamine with the mass 5 times that of the mussel mucin and glutamine transaminase of 20U/g mussel mucin into the suspension, adding NaOH 1M to adjust the pH value to 7.0 for direct reaction (the pH value of the suspension is close to 7 in certain batches), and reacting for 4 hours in a water bath at 40 ℃; inactivating glutamine transaminase for 5min in water bath at 80 deg.C; dialyzing the double distilled water for 24h by using a 1000Da membrane to remove the redundant D-galactosamine (changing the solution for 3 times); and (5) freeze-drying to obtain the modified mussel mucin.
The magnesium alloy material obtained in example 1 was coated with a coating by the following process:
(1) removing foreign matters on the surface of the magnesium alloy material, ultrasonically cleaning in an ethanol solution, and airing;
(2) putting the magnesium alloy material treated in the step (1) into a sodium hydroxide aqueous solution with the concentration of 90g/L, degreasing for 20min at 50 ℃, taking out, cleaning with distilled water, and blow-drying for later use;
(3) placing the magnesium alloy material treated in the step (2) into a phosphoric acid aqueous solution with the concentration of 60 wt%, corroding for 5s at 25 ℃, and keeping the ratio of the volume of the phosphoric acid aqueous solution to the surface area of the magnesium alloy to be pickled to be not less than 100mL/cm2 in the pickling process;
(4) soaking the magnesium alloy material treated in the step (3) in an activating solution for activating treatment, wherein the activating solution is an NH4F aqueous solution with the concentration of 80g/L, and the temperature of the activating treatment is 30 ℃ and the time is 0.5 min;
(5) dissolving the modified mussel mucin in a disodium hydrogen phosphate and sodium dihydrogen phosphate buffer solution, adding 2% rhBMP-2 polypeptide, mixing and stirring uniformly, and waiting for dipping the solution;
(6) and (3) dipping the magnesium alloy material treated in the step (4) in a solution to be dipped for 10min, stirring in the dipping process, taking out, washing with distilled water, drying by blowing, and preparing the mussel mucin BMP-2 coating on the surface of the magnesium alloy.
The magnesium alloy material of the mussel mucin BMP-2 coating is obtained, cut to a proper size according to the requirement, and prepared into the barrier film with a reticular hole structure for alveolar bone guided regeneration.
The mussel mucin has adhesive protein of a series of basic proteins, the amino acid containing hydroxyl is up to 60-70%, wherein most Pro is modified into Hyn by post-translation hydroxylation, and the effects between polyphenol proteins and collagen molecules are stabilized; the phenol group of the polyphenol has strong metal chelating capacity, forms an organic metal complex on the surface of a magnesium alloy material, and can form strong hydrogen bonds with polar polymers such as protein and the like, so that the BMP2 can be stably adhered to the surface of the magnesium alloy barrier film. With the barrier membrane of the present invention, BMP2 can be continuously stabilized to induce alveolar bone regeneration during alveolar bone growth.
Example 3: stability test
The regenerated barrier membrane coated with the mussel mucin BMP2 obtained in example 2 was allowed to stand at 25 ℃. + -. 2 ℃ and humidity 60%. + -. 10% for 6 months, and samples were taken at 0 month, 1 month, 3 months and 6 months for in vitro release assay, and the cumulative release percentages were compared for 12h, 24h, 120h, 240h, 480h and 720 h.
In vitro release assay: samples for 0 month, 1 month, 3 months and 6 months were taken and placed in dialysis bags, which were placed in 20ml of PBS buffer containing 0.2% sodium azide and having a pH of 7.0. After standing at 37 ℃ and sampling 1ml of each of 12h, 24h, 120h, 240h, 480h, 720h and 960h (followed by 1ml of PBS buffer containing 0.2% sodium azide and having pH 7.0), the BMP2 concentration was measured by ELISA according to the kit instructions and the cumulative percentage release was calculated as a conversion. The results are shown in the following table.
As can be seen from the above table, the regeneration barrier membrane coated with the mussel mucin BMP2 obtained in example 2 maintained substantially stable in vitro release after 6 months of stability, sustained release of BMP2 was maintained for 960h, and alveolar bone regeneration was continuously induced.
Example 4: osteogenesis induction experiment of alveolar bone guided regeneration barrier membrane material on alveolar-derived bone marrow stromal cells
The purpose of the test is as follows: the alveolar bone marrow mesenchymal cells are taken as seed cells, the seed cells and the regeneration barrier membrane material are compositely cultured, the growth condition of the cells on the material and the osteogenic induction capability of the material on the alveolar bone mesenchymal cells are observed, so as to discuss the influence of the regeneration barrier membrane material on the growth and differentiation of the alveolar bone progenitor cells.
The test method comprises the following steps: the method comprises the steps of extracting bone marrow at a tooth wound part of a patient with wisdom tooth extraction, obtaining alveolar bone marrow stromal cells by a gradient centrifugation method, carrying out in-vitro culture and passage cell counting, detecting a cell proliferation cycle by a flow cytometer, measuring surface molecules and carrying out cytochemical staining to know the biological characteristics of alveolar bone, randomly dividing cultured primary cells into two groups, inoculating one group of the cells into a regeneration barrier membrane material, carrying out in-vitro co-culture to obtain an experimental group, observing the composite condition of the cells at a scanning electron microscope, culturing the other group of culture solution to obtain a control group, and comparing the difference of osteogenic characteristics of the two groups of cells.
Test materials and instruments:
regeneration of barrier membrane material: the mussel mucin BMP-2 coated magnesium alloy material obtained in example 1;
alveolar bone marrow source: healthy male beagle dogs, 9-11kg of weight, 9-12 months of age and 9-11kg of weight, are raised conventionally in animal centers of military medical academy of sciences. 1ml/kg of 3% sodium pentobarbital, lying on the side after intravenous injection of general anesthesia on beagle dogs, removing hair in the anterior superior iliac spine region, disinfecting and paving a towel, supporting a puncture needle against the iliac spine for vertical needle insertion, replacing a needle core with a 20ml syringe (2 ml of heparin anticoagulant is extracted in advance) after the empty feeling, pushing in a small amount of anticoagulant, and extracting about 10ml of bone marrow.
The main reagents are as follows: trypsin powder, EDTA powder, dexamethasone, indomethacin, 3-isobutyric acid-1-methylxanthine (BMX) were purchased from Sigma, Phosphate Buffered Saline (PBS), Fetal Bovine Serum (FBS) from Gibico;
the main apparatus is as follows: nikon4500 inverted phase contrast microscope (Nikon corporation), carbon dioxide cell culture incubator (Harris corporation), flow cytometer (EFACS Calibur, Becton Dickinson corporation), GL-16 low speed centrifuge (Zhuhai Hema corporation), YG-875B clean bench (Suzhou medical facilities).
The first experiment method comprises the following steps:
1. alveolar bone marrow stromal cell culture
Carefully superposing the bone marrow to a 15ml centrifuge tube containing 6ml of 1.077g/l Percoll lymphocyte separating medium (1: 1 of bone marrow and lymphocyte separating medium), centrifuging at 1800r/min for 20min, carefully sucking the middle milky interface layer, adding 5 times of normal saline, mixing, centrifuging at 1500 r for 10min, and washing to remove residual Percoll component. Inoculating the strain into DMEM culture solution containing 10% FBS, 100u/L penicillin and 100mg/L streptomycin at the density of 2.0 x 108L-1, putting the DMEM culture solution into a carbon dioxide cell culture incubator with the volume fraction of 0.05 at 37 ℃ for culture, replacing the culture solution for 4d, removing nonadherent cells, collecting the cells in a long fusiform shape by using a cell scraper, and performing bottle separation culture to form clones.
After the primary cells are cloned, observing the growth condition of the cells under an inverted phase contrast microscope, carrying out passage when the cells are contacted with each other and grow and the bottom of a culture bottle is covered by more than 80%, removing pancreatin, adding 1ml of newborn calf serum to stop digestion, carrying out passage inoculation in DMEM culture solution according to the ratio of 1:3, changing the solution every other day until adherent cells are close to each other and fused, and repeating the operation when 80% of the bottom of the bottle is fully paved. The experiment was performed using 3-5 generations of cells.
2. In-vitro composite culture of regenerative barrier membrane material and bone marrow stromal cells
Digesting the 3 rd to 5 th generation bone marrow stromal cells after the marrow stromal cells are paved at the bottom of the bottle, collecting cell suspension, and preparing the cell suspension into the cell concentration 1 x 106And/ml, randomly dividing the culture solution into two groups A and B, adding a regeneration barrier membrane material (sterilized by ethylene oxide and soaked in a culture medium-free medium) into the culture solution of the group A, and adding a scaffold material into the culture solution of the group B to obtain a blank control group.
3. Observation by scanning electron microscope
(1) At 3d of the culture of the experimental group A, the bone marrow stromal cells and the regeneration barrier membrane material complex are carefully taken out and rinsed twice with PBS;
(2) soaking and fixing a sample in mixed fixing solution of 5% of glutaraldehyde and 4% of paraformaldehyde by volume fraction at 4 ℃ for 24 hours;
(3) rinsing with 0.1mol/L phosphate buffer solution for 2 h;
(4) performing gradient dehydration with ethanol and acetone of different concentrations, wherein the concentration gradient is 30%, 50%, 70%, 90% ethanol, 90%, 100% (3 times) acetone in sequence, each time for 15 min;
(5) after dehydration and natural drying, the sample is pasted on a metal sample table by silver powder mixed with low-resistance resin liquid;
(6) and (5) coating the film on the vacuum spraying box, and observing under a scanning electron microscope.
And (4) observing results:
the surface of the control group is of a regular porous net structure, the pore diameters of large pores are basically consistent, the arrangement is uniform, the sizes of small pores are not uniform, the interval thickness is not uniform, and the control group is arranged in a disordered way, which is shown in the figure 1a and the figure b; the experimental group of cells are inoculated on the surface of the regeneration barrier membrane material, a large amount of granular and flaky cell matrixes on the surface of the material can be seen, the cell protrusions are formed by inserting the populus pseudopoda among the cell matrixes, the surface of the regeneration barrier membrane material can be seen with rich cells and cell matrixes, the cell pseudopoda protrusions are stretched in a cord shape, and the cell matrixes are scab-shaped or granular and almost cover the surface and gaps of the material, as shown in fig. 1c and d.
4. Calcium nodule Von Kossa staining
Two groups of the third generation bone marrow stromal cells are taken and 5 x 105The cell concentration per ml is inoculated on a culture dish of 3.5cm, and the growth condition of the cells is observed under an inverted microscope, and the cell can be identified by the formation of nodules. After the cells of the test group are subjected to induction culture for 9 days, calcium nodules begin to form on the cells, the calcium nodules gradually increase and aggregate along with the prolonging of the culture time, a plurality of round opaque mineralized nodules exist in the cells, and the Von Kossa staining shows a black strong positive reaction, which is shown in figure 2; the control group had a cell mass but no calcification.
5. ALP Activity assay
Cells were expressed as 2 x 103The concentration of each well is inoculated in a 24-well plate, two groups are divided into 12 wells, and two groups of generation 3 cells are respectively cultured for 24And h, removing a supernatant, washing with 0.04% PBS for 3 times, adding 50ul of 10.1% TritonX-100, placing in a refrigerator at 4 ℃ overnight, repeatedly blowing, adding a newly configured substrate in an ALP kit, incubating for 30min, adding 0.2nolNaOH to terminate the reaction, and measuring the light absorption value (410 wavelength) on an enzyme-linked immunosorbent assay instrument to obtain the OD value. The secretion amount of ALP in the cell supernatant of the experimental group was 3.312 + -0.007 u/ml, the content of ALP in the control group was 2.485 + -0.013 u/ml, and the difference between the two was statistically significant (p)<0.01) Table 1: comparison of alkaline phosphatase (ALP) content in two groups of cells
6. Discussion of results
The alveolar bone marrow stromal cells have better in-vitro proliferation and osteogenesis differentiation capacity, and the bone regeneration barrier membrane consists of a magnesium alloy material and a mussel mucin BMP2 coating coated on the surface of the magnesium alloy material, can effectively promote osteogenesis differentiation of the alveolar bone marrow stromal cells, and has good osteogenesis induction capacity.
Claims (1)
1. A preparation method of an oral cavity guided bone regeneration barrier membrane is characterized by comprising the following steps:
preparation of magnesium alloy material
The magnesium alloy material of the barrier film comprises the following components by taking the total weight of the magnesium alloy material as 100 percent: aluminum: 10%, zinc: 15%, manganese: 0.8%, silver: 0.05%, calcium: 8%, titanium dioxide: 7 percent, and the balance being Mg;
the adopted preparation method comprises the following steps:
preparing titanium dioxide and magnesium alloy matrix powder according to a proportion; mixing the powder, putting the mixture into a ball mill, and grinding the powder to be uniform under a protective atmosphere; under the protection atmosphere, obtaining a magnesium solution containing titanium dioxide by adopting a selective laser melting process; rapidly adding the dried pure calcium into the magnesium melt according to the proportion, stirring the mixture from time to time, and smelting the mixture for 1.5 hours to obtain a casting-state magnesium alloy containing calcium; placing the casting magnesium alloy containing calcium into a vacuum tube furnace, and carrying out solution treatment for 20 hours at 350 ℃; and aging at 400 deg.C for 48 hr;
secondly, coating mussel mucin BMP-2 on the surface of the magnesium alloy material
2.1 preparation of sugar-based modified mussel mucin:
preparing CELL-TAK natural mussel mucin mussel into 0.5% suspension by using double distilled water, adding D-galactosamine with the mass 5 times that of the mussel mucin and glutamine transaminase of 20U/g mussel mucin into the suspension, adding NaOH 1M to adjust the pH value to 7.0, directly reacting, and reacting for 4 hours in a water bath at 40 ℃; inactivating glutamine transaminase for 5min in water bath at 80 deg.C; dialyzing the double distilled water for 24h by using a 1000Da membrane to remove redundant D-galactosamine, and changing the solution for 3 times; freeze-drying to obtain modified mussel mucin;
2.2 coating the surface of the magnesium alloy material obtained in the step one by adopting the following process:
(1) removing foreign matters on the surface of the magnesium alloy material, ultrasonically cleaning in an ethanol solution, and airing;
(2) putting the magnesium alloy material treated in the step (1) into a sodium hydroxide aqueous solution with the concentration of 90g/L, degreasing for 20min at 50 ℃, taking out, cleaning with distilled water, and blow-drying for later use;
(3) placing the magnesium alloy material treated in the step (2) into a phosphoric acid aqueous solution with the concentration of 60 wt%, corroding for 5s at 25 ℃, and keeping the ratio of the volume of the phosphoric acid aqueous solution to the surface area of the magnesium alloy to be pickled to be not less than 100mL/cm in the pickling process2；
(4) Soaking the magnesium alloy material treated in the step (3) in an activating solution for activating treatment, wherein the activating solution is NH with the concentration of 80g/L, and the temperature of the activating treatment is 30 ℃ and the time is 0.5min4F, water solution;
(5) dissolving the modified mussel mucin in a disodium hydrogen phosphate and sodium dihydrogen phosphate buffer solution, adding 2% rhBMP-2 polypeptide, mixing and stirring uniformly, and waiting for dipping the solution;
(6) soaking the magnesium alloy material treated in the step (4) in a solution to be soaked for 10min, stirring in the soaking process, taking out, washing with distilled water, blow-drying, and preparing a mussel mucin BMP-2 coating on the surface of the magnesium alloy;
and thirdly, cutting the magnesium alloy material with the mussel mucin BMP-2 coating into a proper size according to the requirement, and preparing the magnesium alloy material into the barrier film with a mesh hole structure for alveolar bone guided regeneration.
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