CN111494720A

CN111494720A - Function-integrated absorbable guided tissue regeneration membrane and preparation method thereof

Info

Publication number: CN111494720A
Application number: CN202010245659.9A
Authority: CN
Inventors: 莫秀梅; 刘学哲
Original assignee: Donghua University
Current assignee: Donghua University; National Dong Hwa University
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2020-08-07

Abstract

The invention relates to a function-integrated absorbable guide tissue regeneration membrane and a preparation method thereof. The regeneration membrane component comprises degradable artificially synthesized high molecular materials, natural degradable high molecular polymers and inorganic nano particles. The method comprises the following steps: dissolving degradable artificially synthesized high molecular material and natural degradable high molecular polymer in organic solvent, stirring, adding inorganic nanometer particles, stirring, ultrasonic treating and electrostatic spinning. The regeneration membrane has good mechanical property, biocompatibility, antibacterial property and bone promoting activity, and has obvious periodontal repair effect. The method has the advantages of wide material source, low preparation cost and potential clinical application prospect.

Description

Function-integrated absorbable guided tissue regeneration membrane and preparation method thereof

Technical Field

The invention belongs to the field of guided tissue regeneration membranes and preparation thereof, and particularly relates to a degradable periodontal guided tissue regeneration membrane with integrated antibacterial and osteogenesis functions and a preparation method thereof.

Background

Periodontitis is one of a common class of oral diseases in humans, affecting approximately 50-90% of the population worldwide, depending on its precise definition. Periodontitis is a type of chronic inflammatory disease caused by bacteria, and if treatment is not appropriate, inflammation is further aggravated and spreads deep in periodontal tissues, resulting in loss and irreversible restoration of connective tissues and alveolar bones around teeth, and finally tooth loss, which causes great inconvenience in daily life. Clinically traditional methods of periodontal disease treatment focus primarily on controlling or arresting the progression of periodontitis and are not effective in regenerating the lost periodontal tissue.

Guided Tissue Regeneration (GTR) is a new method for regenerating periodontal Tissue proposed in the last 80-90 th century because of its low technical barrier, simple operation and excellent effect, and has been widely used for periodontal Regeneration treatment. The basic principle is that a membranous material is placed between periodontal connective tissue and tooth root as a barrier to prevent epithelial tissue and connective tissue with over-high proliferation and migration rates from entering into the periodontal defect area below, so that a space is selectively created for the reattachment of periodontal membranous cells and osteoblasts with lower migration rates to the root surface to perform bone reconstruction, and the real regeneration of periodontal tissue is realized.

In GTR, tissue regeneration-guided membranes are a key role in this technology, and GTR membranes that are commonly used clinically are classified into degradable membranes and non-degradable membranes according to their degradability. Currently commercially available non-degradable membranes mainly comprise expanded polytetrafluoroethylene (e-PTFE, Gore-

) High density polytetrafluoroethylene (d-PTFE) and titanium-reinforced high density polytetrafluoroethylene (Ti-d-PTFE) films and the like^[1]. The e-PTFE membrane is recognized as the "gold standard" for GTR membrane materials because of its excellent clinical performance. The inert biomaterial has stable physicochemical properties, good biocompatibility and good mechanical property, can stably maintain the space required by the reconstruction of the lower bone tissue, effectively shield the fibrous tissue and the connective tissue from growing in, is convenient to operate and the like. However, their disadvantages are also evident in that such inert materials are generally hard and poorly integrated with the surrounding tissue, and may therefore cause the surrounding soft tissue in contact with the membrane to split open, thereby easily exposing the membrane and causing bacterial infection. Secondly, the non-degradable membranes require a second surgical removal, which may cause new trauma and damage to the delicate neonatal periodontal tissue, and also risks secondary infections, causing additional physiological and psychological distress to the patient, and thus the frequency of use of non-degradable membranes is gradually reduced clinically.

In addition, most of degradable membranes only exist as barrier membranes and lack the advantages of osteogenic activity and antibacterial property, and the like, and recently, Ali and the like utilize an electrostatic spinning technology to prepare a PC L/ZnO composite fiber membrane, the PC L/ZnO membrane has antibacterial and osteogenic effects, but the safety problem caused by the residual of heavy metals such as Zn in vivo is still not negligible^[2]. Therefore, the research and the preparation of a novel degradable functional GTR film material suitable for the Chinese situation are imperative.

Disclosure of Invention

The invention aims to solve the technical problem of providing a function-integrated absorbable guided tissue regeneration membrane and a preparation method thereof, and overcoming the defects of poor mechanical property, uncontrollable degradation period, low biological activity, and lack of osteogenic activity and antibacterial property of a GTR degradable membrane in the prior art.

The invention provides a function-integrated absorbable guide tissue regeneration membrane, which comprises degradable artificially synthesized high molecular materials, natural degradable high molecular polymers and inorganic nanoparticles, wherein the mass ratio of the degradable artificially synthesized high molecular materials to the natural degradable high molecular polymers is 3: 1-5: 1, and the mass of the inorganic nanoparticles is 0.05-0.2 of the total mass of the degradable artificially synthesized high molecular materials and the natural degradable high molecular polymers.

The degradable artificially synthesized high molecular material includes one or several of polylactic acid P L A, polycaprolactone PC L, polylactic acid-glycolic acid copolymer P L GA and poly β -hydroxybutyric acid PHB.

The natural degradable high molecular polymer comprises one or more of collagen, gelatin and fibroin.

The inorganic nano particles comprise one or more of nano magnesium oxide particles and cobalt-substituted hydroxyapatite, and the size of the nano magnesium oxide particles is not more than 100 nm.

The thickness of the regeneration film is 200-300 mu m.

The invention also provides a preparation method of the functional integrated absorbable guide tissue regeneration membrane, which comprises the following steps:

(1) dissolving degradable artificially synthesized high molecular materials and natural degradable high molecular polymers in an organic solvent according to a mass ratio of 3: 1-5: 1, and stirring to obtain a spinning solution with a solute mass fraction of 8-10%;

(2) adding inorganic nano particles into the spinning solution obtained in the step (1), and stirring to obtain a spinning solution containing 0.5-3% of inorganic nano particles;

(3) and (3) performing ultrasonic treatment on the spinning solution in the step (2), then performing electrostatic spinning, and performing freeze drying to obtain the functional integrated absorbable guide tissue regeneration membrane.

In the step (1), the organic solvent is one or more of hexafluoroisopropanol HFIP, Dichloromethane (DCM), trifluoroacetic acid (TFA), Tetrahydrofuran (THF) and N, N-Dimethylformamide (DMF), and preferably hexafluoroisopropanol HFIP.

The stirring in the step (1) is as follows: stir at room temperature overnight.

And (3) in the step (2), the stirring temperature is room temperature, and the stirring time is 4-6 h.

And (4) the ultrasonic treatment time in the step (3) is 30-60 min.

The electrostatic spinning in the step (3) comprises the following technological parameters: the receiving device is a non-woven fabric with the size of 30x30cm, the spinning voltage is 10-15kV, the flow rate of the spinning solution is 1.0-3.0ml/h, the spinning distance is 10-15cm, and the spinning time is 1-2.5 h.

The freeze-drying time in the step (3) is one night, and the vacuum degree is 1 MPa.

And (4) packaging and sterilizing after freeze-drying in the step (3).

The invention also provides application of the function-integrated absorbable guide tissue regeneration membrane in preparation of a regeneration membrane for treating periodontal defects.

The regeneration membrane is prepared by an electrostatic spinning technology, the structure of the regeneration membrane is formed by stacking random continuous fibers, the regeneration membrane has a large pore specific surface area, the exchange of nutrient substances at a wound and the discharge of wastes are facilitated, and pores formed by fiber bridging (the pore diameter of a compact fiber membrane is less than 10 mu m) are far smaller than the size of cells, so that the rapid downward migration of epithelial cells to the defect can be effectively prevented. The membrane takes synthetic polymer materials as main components, provides sufficient mechanical properties, simultaneously adds natural polymer materials with proper proportion, improves the biocompatibility of the membrane and accelerates the degradation rate of the membrane, and finally compounds inorganic nano particles with antibacterial activity and osteogenic activity with proper proportion to endow the membrane with osteogenic property and antibacterial property.

Advantageous effects

(1) The GTR membrane prepared by the invention is a micro-nano-scale fiber structure on the microcosmic scale, structurally simulates a nanofiber structure of extracellular matrix, has high porosity and large specific surface area of pores, is beneficial to nutrient exchange and waste transportation at wounds, and meanwhile, the diameter of the pores formed by fiber stacking is far smaller than the diameter of cells, so that the excessive growth of epithelial fiber cells can be effectively prevented, and meanwhile, the GTR membrane is used as a help for the migration and proliferation of periodontal membrane cells below a bracket to the root of a tooth.

(2) The GTR membrane prepared by the invention has good mechanical property and biocompatibility, and can also promote the proliferation and proliferation of mesenchymal stem cells.

(3) The degradability of the GTR film prepared by the invention is controllable, and the degradation rate of the GTR film can be regulated and controlled by changing the molecular weight of the artificially synthesized polymer matrix serving as the main component of the GTR film, so that the GTR film is suitable for different application scenes.

(4) The GTR membrane prepared by the invention has strong antibacterial property, and can obviously inhibit the activity of gram-positive bacteria staphylococcus aureus and gram-negative bacteria escherichia coli.

(5) The GTR membrane prepared by the invention has bone promoting activity and can promote the A L P activity of rat bone marrow mesenchymal stem cells.

(6) The GTR film prepared by the invention has obvious periodontal repair effect and obviously improves the recovery condition of alveolar bone defect of rats.

(7) The material used in the invention has wide source and low preparation cost, and has potential clinical application prospect.

Drawings

FIG. 1 is a graph showing the morphology of electrospun fiber membranes in examples 1-4 and comparative example 1, wherein A is SEM and B and C are respectively energy spectrum scans;

FIG. 2 is a stress-strain plot of electrospun fiber membranes of examples 1-4 and comparative example 1;

FIG. 3 is a graph showing the degradation profiles of the electrospun fiber membranes of examples 1-4 and comparative example 1;

FIG. 4 is a cell proliferation map of the electrospun fibrous membranes of examples 1-4 and comparative example 1;

FIG. 5 is a graph comparing the antibacterial effects of the electrospun fiber membranes of examples 1 to 4 and comparative example 1, wherein A is a digital photograph of an agar plate and B is the survival rate of bacteria calculated from CFU;

FIG. 6 is a graph showing the activity of A L P of the electrospun fiber membranes of examples 1-4 and comparative example 1.

FIG. 7 is a graph showing the repair effect of periodontal defects in vivo by electrospun fibrous membranes in examples 1-3 and comparative example 1, wherein A is a model view of periodontal defects, B is a view of surgical procedure, C is a 3D reconstructed view (left) and a Micro-CT cross-sectional view (right) of defect site, D CEJ-ABC distance, BV/TV, BMD, Tb.N, Tb.Th and other parameters related to alveolar bone are quantitatively analyzed.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

0.8g of P L A (molecular weight: 10 ten thousand, Jinandai handle) and 0.2g of gelatin (25000-50000, Sigma) were weighed out and added to 10ml of hexafluoroisopropanol solution, and stirred overnight on a magnetic stirrer to prepare a 10% polymer solution A.

Weighing nano magnesium oxide particles (50nm, Aladdin) with the mass of 0.05g, adding the nano magnesium oxide particles into the polymer solution A, stirring the mixture for 4 hours on a magnetic stirrer, carrying out ultrasonic oscillation for 60 minutes, then carrying out electrostatic spinning, taking non-woven fabrics with the size of 30x30cm as a receiving device, setting the voltage to be 15kV, setting the spinning speed to be 3ml/h, spinning for 2 hours, and setting the receiving distance to be 15cm to obtain an electro-spun fiber membrane with the thickness of about 300 mu m,

the electrospun fiber membrane is placed in a refrigerator at the temperature of minus 80 ℃ for 30min, and then is placed in a freeze dryer for drying overnight, so that no residual solvent exists in the fiber membrane. The fiber membrane was designated as nMgO-0.5 (indicating that the ratio of the mass of nano-magnesium oxide contained in the fiber membrane to the total mass of polylactic acid-gelatin was 5%).

Example 2

According to example 1, the mass of the nano magnesium oxide particles was changed to 0.1g, and the rest was the same as example 1, so as to obtain an electrospun fiber membrane with a thickness of about 300 μm, and the freeze-drying was the same as example 1. The fiber membrane was designated as nMgO-1 (indicating that the ratio of the mass of nano-magnesium oxide contained in the fiber membrane to the total mass of polylactic acid-gelatin was 10%).

Example 3

According to example 1, the mass of the nano magnesium oxide particles was changed to 0.15g, and the rest was the same as example 1, so as to obtain an electrospun fiber membrane with a thickness of about 300 μm, and the freeze-drying was the same as example 1. The fiber membrane was designated as nMgO-1.5 (indicating that the mass ratio of the nano-magnesia-containing mass to the total mass of the polylactic acid-gelatin in the fiber membrane was 15%).

Example 4

According to example 1, the mass of the nano magnesium oxide particles was changed to 0.2g, and the rest was the same as example 1, so as to obtain an electrospun fiber membrane with a thickness of about 300 μm, and the freeze-drying was the same as example 1. The fiber membrane was designated as nMgO-2 (indicating that the ratio of the mass of nano-magnesium oxide contained in the fiber membrane to the total mass of polylactic acid-gelatin was 20%).

Comparative example 1

The polymer solution A was electrospun, and the configuration and spinning conditions of the polymer solution A were the same as those in example 1, to obtain an electrospun fiber membrane having a thickness of about 300 μm, and freeze-dried in the same manner as in example 1. The fiber membrane was designated as nMgO-0 (indicating that the ratio of the mass of nano-magnesium oxide contained in the fiber membrane to the total mass of polylactic acid-gelatin was 0%).

Experimental example 5

The films obtained in examples 1 to 4 and comparative example 1 were cut into a strip of 5X 1cm in size, and the tensile properties thereof were measured by a universal material tensile tester. The tensile rate was set to 1mm/min, and the results of the stress-strain curves of the above 5 films are shown in fig. 2, and it can be found that the maximum tensile strengths of the films of examples 1 to 4 were all more than 1MPa and at most 3.6MPa of example 1, indicating that the obtained GTR film has good mechanical properties and that the mechanical properties can be changed by changing the nMgO content.

Experimental example 6

The films obtained in examples 1 to 4 and comparative example 1 were cut out into circular film pieces 14mm in diameter, each tested for their mass with a punch, designated a0, and then falling-film placed in sterile centrifuge tubes containing 5ml of artificial saliva, then transferred to a 37 ℃ incubator, the material was removed at intervals and rinsed with PBS, freeze-dried to remove moisture, and weighed as a 1. The remaining mass percentage is calculated according to the following formula: the result of the remainded mass (%) being a1/a 0x 100 is shown in fig. 3, and it was found that the degradation rate of the membrane gradually increased with the increase of the nMgO content in the membrane, suggesting that it is possible to prepare a GTR membrane matching the defect healing cycle by changing the nMgO content.

Experimental example 7

The membranes obtained in examples 1 to 4 and comparative example 1 were cut into circular membranes having a diameter of 14mm, respectively, using a punch, sterilized under an ultraviolet lamp for 30 minutes and sterilized with 70% alcohol for 1 hour, followed by washing with sterile PBS for several times and incubating with incomplete medium for 2 hours, digesting mesenchymal stem cells in logarithmic growth phase and incubating with 1X10⁵Membrane density it was planted on round membrane plates, supplemented with complete medium to 1ml per well, and replaced with fresh medium every 2 days. The medium was aspirated at predetermined time points (1 day, 3 days, 5 points), the cell-seeded membranes were incubated with 0.1ml of 10% CCK-8 medium for 90 minutes, and the OD was measured using a microplate reader₄₅₀The value is obtained. The results of CCK-8 of the above 5 membranes are shown in FIG. 4, and all the membranes containing nMgO (examples 1-3) significantly promoted the proliferation of mesenchymal stem cells except the membrane of example 4 (nMgO-2), indicating more excellent cell compatibility.

Experimental example 8

The membranes obtained in examples 1 to 4 and comparative example 1 were cut into circular membrane pieces having a diameter of 14mm using a punch, sterilized with 70% alcohol for 1 hour, and washed with PBS several times to remove excess alcohol. 0.2ml of each of Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) strains (1X 10) in an amplified state was taken⁷CFU/ml) were uniformly planted on different membrane materials, sealed, and then cultured in an incubator, after 24 hours the membranes were transferred to a centrifuge tube containing 10ml of pbs, and the bacteria on the membranes were eluted by shaking, and diluted 10000 times, then 0.1ml of the eluate was uniformly smeared on an agar plate, cultured overnight in the incubator, and the next day by a colony counter technique, and the results are shown in fig. 5. We found that the number of colonies on agar plates decreased gradually with increasing nMgO content, indicating that the GTR films obtained according to the invention had a significant antibacterial effect against both typical bacteria.

Experimental example 9

By means of separate punchesThe membranes obtained in examples 1 to 4 and comparative example 1 were cut into circular membrane pieces having a diameter of 14mm, sterilized with 70% ethanol for 1 hour, and then washed with PBS several times to remove excess ethanol. The third generation of bone marrow mesenchymal stem cells in logarithmic proliferation is processed at 5x10⁴The membrane density is planted on different fiber membranes, the culture is carried out for one day by using a complete culture medium and then replaced by an osteogenic induction culture medium (sulfolobus sp.), then a fresh osteogenic induction culture medium is replaced every 3 days, the volume of the replaced osteogenic induction culture medium is 1 ml/membrane, after the culture is carried out for 7 days and 14 days, the osteogenic induction culture medium is sucked out and washed for 3 times by PBS, and the A L P activity of the mesenchymal stem cells on different membranes is quantitatively measured by an A L P kit (Biyun day) and a BCA kit (Biyun day) according to the instruction, and the result is shown in figure 6.

Experimental example 10

The periodontal guided tissue regeneration effect of the membrane was evaluated by an in vivo periodontal defect model by first anesthetizing SD rats with sodium pentobarbital, then incising the biting muscles and periosteum outside the first molars of the rats and continuously rinsing with saline, followed by exposing the alveolar bone and creating a defect of about 3x 1.5x 2mm with a dental drill. Subsequently, the film of examples 1 to 3 and comparative example 1 cut in advance was covered on the defect site, and the wound site was sutured. The animals were sacrificed after six weeks of surgery with excess anesthesia, the maxilla was collected and fixed with 4% paraformaldehyde, and the recovery of the defect sites was calculated by Micro-CT scanning, the results of which are shown in fig. 7. We have found that the films obtained in examples 1-3(nMgO-0.5, nMgO-1, nMgO-1.5) have more remarkable repairing effect on alveolar bone defect of rat as the nMgO content in the film increases, and they are all better than the results of the film obtained in comparative example 1(nMgO-0), indicating that the GTR film of the present invention can remarkably improve periodontal regeneration of rat.

The present invention relates to the following citations:

[1]G.Sam,B.R.Pillai,Evolution of barrier membranes in periodontalregeneration-"are the third generation membranes really here？",J Clin DiagnRes 8(12)(2014)ZE14-7.

[2]A.Nasajpour,S.Ansari,C.Rinoldi,A.S.Rad,T.Aghaloo,S.R.Shin,Y.K.Mishra,R. Adelung,W.Swieszkowski,N.Annabi,A.Khademhosseini,A.Moshaverinia,A.Tamayol, A multifunctional polymeric periodontal membranewith osteogenic and antibacterial。 characteristics,Advanced FunctionalMaterials 28(3)(2018).

Claims

1. the function-integrated absorbable guided tissue regeneration membrane is characterized by comprising a degradable artificially synthesized high molecular material, a natural degradable high molecular polymer and inorganic nanoparticles, wherein the mass ratio of the degradable artificially synthesized high molecular material to the natural degradable high molecular polymer is 3: 1-5: 1, and the mass of the inorganic nanoparticles is 0.05-0.2 of the total mass of the degradable artificially synthesized high molecular material and the natural degradable high molecular polymer.

2. The regeneration membrane of claim 1, wherein the degradable synthetic polymer material comprises one or more of polylactic acid P L A, polycaprolactone PC L, polylactic acid-glycolic acid copolymer P L GA and poly β -hydroxybutyrate PHB, and the natural degradable polymer material comprises one or more of collagen, gelatin and fibroin.

3. The regeneration membrane according to claim 1, wherein the inorganic nanoparticles comprise one or more of nano magnesium oxide particles, cobalt-substituted hydroxyapatite and nano zinc oxide particles, and the size of the nano magnesium oxide particles is not more than 100 nm; the thickness of the regenerated film is 200-300 μm.

4. A preparation method of a functional integrated absorbable guide tissue regeneration membrane comprises the following steps:

5. The method according to claim 4, wherein the organic solvent in step (1) is one or more of Hexafluoroisopropanol (HFIP), dichloromethane, trifluoroacetic acid, tetrahydrofuran and N, N-dimethylformamide; stirring is as follows: stir at room temperature overnight.

6. The method according to claim 4, wherein the stirring temperature in the step (2) is room temperature, and the stirring time is 4-6 h.

7. The method according to claim 4, wherein the ultrasound time in the step (3) is 30-60 min; the technological parameters of electrostatic spinning are as follows: the receiving device is a non-woven fabric with the size of 30x30cm, the spinning voltage is 10-15kV, the flow rate of the spinning solution is 1.0-3.0ml/h, the spinning distance is 10-15cm, and the spinning time is 1-2.5 h.

8. Use of the regenerative membrane according to claim 1 for preparing a regenerative membrane for treating periodontal defects.