CN104474589A - Guided tissue regeneration membrane as well as preparation method and application thereof - Google Patents
Guided tissue regeneration membrane as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a guided tissue regeneration membrane. The membrane adopts a double-layer membrane structure and is characterized in that one layer is a compact layer prepared from biological fibers with longer degradation time, and the other layer is a potential loose layer prepared through mixing of biological fibers with different degradation time. After the potentialloose layer is implanted in a body, part of the biological fibers degrades quickly, so that the pore diameter of the membrane is increased, and ingrowth of cells is facilitated. The compact layer and the potential loose layer are connected through the same biological fibers with the longer degradation time, so that the double-layer membrane is free of splitting and fractures. The invention further discloses a preparation method of the guided tissue regeneration membrane as well as an application of the guided tissue regeneration membrane as a tissue repair product to tissue repair.
Description
Technical field
The invention belongs to biomedical materials field, relate to a kind of guide tissue regeneration film and preparation method thereof, and as the application of tissue repair goods in tissue repair.
Background technology
The blank of guide tissue regeneration technology starts from a kind of semi permeability filter membrane treatment periodontal disease of nineteen eighty-two Nyman, guide tissue regeneration technology is applied to Immediate implant placement by Dahlin in 1989 etc., guide bone regeneration around implant osteanagenesis with it, achieve desirable osteanagenesis effect.Now, guide tissue regeneration technology has been widely used in alveolus surgery, periodontal section and the field such as tooth-planting, orthopaedics.
Current useful clinically guide tissue regeneration film kind is a lot, according to whether being divided into two large classes by absorbing: Absorbable membrane and nonabsorable film; Nonabsorable film take poly tetrafluoroethylene as representative, and required second operation takes out in use, brings considerable distress, and because of its inactive, can not be used for the tissue regeneration of larger defect area, limit the scope at Clinical practice to patient.Do not need after absorbability degradative membrane implants to take out, decrease misery and the operating difficulty of patient, therefore obtain applying more widely.The degradable biological film of current bibliographical information mainly contains two kinds: a kind of collagem membrane made for animal collagen, the another kind of synthetic membrane for making with the synthetic such as polylactic acid, poly (lactic acid-glycolic acid) macromolecular material.
But although collagem membrane has excellent biocompatibility, it is expensive, bad mechanical property, degradation time are too fast, although can improve its performance by chemical crosslinking, also there is the hidden danger of residual cell toxicity; Polylactic acid-based synthetic membrane has hydrophobicity in addition, and surface lacks the functional group that can supply cell recognition factors or other biological bioactive molecule, what cause with Cell binding is indifferent, and the acid product in degradation process may cause inflammatory reaction, makes it apply and is restricted.The guide tissue regeneration film of document or patent report is designed to the form of duplicature mostly at present in addition, but easily there is the problem of fracture layering in double-deck guide tissue regeneration film, duplicature bonding is not tight, compacted zone in use procedure is caused to depart from weaker zone, for the operation avoiding this defect to need the complexity such as mechanical press, affect microstructure and the result of use of fiber.
Summary of the invention
The present invention is directed to the above-mentioned problems that current guide tissue regeneration film exists, by electrostatic spinning technique, raw materials different for degradation time in vivo is passed through electrostatic spinning technique co-blended spinning, obtain the duplicature with compacted zone and " potential weaker zone ", thus obtain all good timbering materials of a kind of mechanical property, degradation property and biocompatibility.
A kind of guide tissue regeneration film provided by the present invention is double membrane structure, it is characterized in that: one deck is by the obtained compacted zone of the biological fiber that degradation time is longer, and another layer is mix obtained potential weaker zone by the biological fiber that degradation time is different.After described potential weaker zone implants, part biological fiber fast degradation, makes the aperture of this film increase, is beneficial to growing into of cell.Preferably, compacted zone is connected by the biological fiber that same degradation time is longer with potential weaker zone, guarantees that duplicature there will not be division tomography at the degraded initial stage that implants.
The raw material of described biological fiber is selected from one or more synthesis resorbable polymeric materials, one or more natural macromolecular materials, or its combination.Synthesis resorbable polymeric materials comprises polylactic acid, polyglycolic acid, polycaprolactone, PLGA, polylactic acid caprolactone copolymer, ethylene glycol copolymer.Natural macromolecular material comprises gelatin, collagen, fibroin albumen, chitosan, modification of chitosan, hyaluronic acid, alginate, fibrin, cellulose.Preferably, synthesis resorbable polymeric materials selects polylactic acid or PLGA, and natural macromolecular material selects collagen or fibroin albumen, further, natural macromolecular material selects the type i collagen deriving from fish skin, reduces the risk infecting zoonosis.
Described duplicature is obtained by electrostatic spinning technique, and the method comprised the following steps can be adopted to obtain:
(1) the longer spinning liquid Z of degradation time and the shorter spinning liquid Q of degradation time is prepared;
(2) spinning liquid Z obtains compacted zone through electrostatic spinning;
(3) on step (2) gained compacted zone, spinning liquid Z and spinning liquid Q is adopted to carry out electrostatic spinning respectively, the obtained biological fiber potential weaker zone different containing two kinds of degradation times.
Or the method for following steps obtains: the tandem of step (2) and step (3) can be put upside down, namely spinning liquid Z and spinning liquid Q is first adopted to carry out electrostatic spinning respectively, the obtained biological fiber potential weaker zone different containing two kinds of degradation times, then obtains compacted zone with spinning liquid Z electrostatic spinning on potential weaker zone.
Described guide tissue regeneration film, can add one or more activating agents by electrostatic spinning technique in its biological fiber.Activating agent comprises antibiotic, anti-inflammatory drug, analgesic, somatomedin, amelogenin, hydroxyapatite, tricalcium phosphate, and preferably, activating agent is bone morphogenetic protein, hydroxyapatite.
The invention discloses the method for guide tissue regeneration film, preparation method comprises electrostatic spinning step.Can comprise the following steps:
(1) the longer spinning liquid Z of degradation time and the shorter spinning liquid Q of degradation time is prepared;
(2) spinning liquid Z obtains compacted zone through electrostatic spinning;
(3) on step (2) gained compacted zone, spinning liquid Z and spinning liquid Q is adopted to carry out electrostatic spinning respectively, the obtained biological fiber potential weaker zone different containing two kinds of degradation times.
Or the tandem of described step (2) and step (3) can be put upside down, namely spinning liquid Z and spinning liquid Q is first adopted to carry out electrostatic spinning respectively, the obtained biological fiber potential weaker zone different containing two kinds of degradation times, then obtains compacted zone with spinning liquid Z electrostatic spinning on potential weaker zone.
In said method, can also following steps be adopted, the guide tissue regeneration film of preparation containing activating agent:
(1) the shorter spinning liquid Q of the longer spinning liquid Z of degradation time, degradation time, spinning liquid Y containing activating agent is prepared;
(2) spinning liquid Z obtains compacted zone through electrostatic spinning;
(3) on step (2) gained compacted zone, employing spinning liquid Z is shell solution, spinning liquid Y is that sandwich layer solution carries out coaxial electrostatic spinning, another shower nozzle of spinning liquid Q is carried out electrostatic spinning, the obtained potential weaker zone containing activating agent simultaneously.
Or the tandem of step (2) and step (3) can be put upside down, namely first adopt that spinning liquid Z is shell solution, spinning liquid Y is that sandwich layer solution carries out coaxial electrostatic spinning, another shower nozzle of spinning liquid Q is carried out electrostatic spinning, the obtained potential weaker zone containing activating agent simultaneously; Then on potential weaker zone, compacted zone is obtained with spinning liquid Z electrostatic spinning.
In said method, when spinning potential weaker zone, spinning liquid Q is regulated to be 50:1-1:1 with the ratio of the spinning speed of spinning liquid Z, fiber that in potential weaker zone, degradation time is shorter and the longer proportion of fibers of degradation time is made to be 50:1-1:1, preferably, regulate spinning liquid Q to be 10:1-2:1 with the ratio of the spinning speed of spinning liquid Z, make fiber that in potential weaker zone, degradation time is shorter and the longer proportion of fibers of degradation time be 10:1-2:1.
In said method, in described step (1), the solid-liquid ratio of spinning liquid Z or spinning liquid Q is 1:5-1:50 (w/v), and preferably, the solid-liquid ratio of spinning liquid Z or spinning liquid Q is 1:10-1:20 (w/v); In the electrostatic spinning of described step (2) or step (3), the speed regulating micro-injection pump is 0.1-10ml/ hour, and the voltage regulating high tension generator is 10-30KV, and the receiving range regulating receiving system is 10-30cm.Preferably, the speed regulating micro-injection pump is 0.5 ~ 5ml/ hour, and the voltage regulating high tension generator is 10-20KV, and the receiving range regulating receiving system is 10-20cm.
In said method, the solvent of described spinning liquid is selected from hexafluoroisopropanol, trifluoroethanol, dichloromethane, chloroform, oxolane, formic acid, water, or its combination, and preferably, the solvent of described spinning liquid is hexafluoroisopropanol, trifluoroethanol.
In said method, the key component of the spinning liquid Z that degradation time is longer is synthesis resorbable polymeric materials or fibroin albumen, and auxiliary element is the natural macromolecular material except fibroin albumen, and the mass ratio of key component and auxiliary element is 10:0-5:5.Preferably, the key component of the spinning liquid Z that degradation time is longer is polylactic acid or fibroin albumen, and auxiliary element is collagen or gelatin, and the mass ratio of key component and auxiliary element is 10:1-2:1.
In said method, the key component of the spinning liquid Q that degradation time is shorter is the natural macromolecular material except fibroin albumen, and auxiliary element is synthesis resorbable polymeric materials or fibroin albumen, and the mass ratio of key component and auxiliary element is 10:0-5:5.Preferably, the key component of the spinning liquid Q that degradation time is shorter is collagen or gelatin, and auxiliary element is polylactic acid or fibroin albumen, and the mass ratio of key component and auxiliary element is 10:1-2:1.
In said method, described synthesis resorbable polymeric materials comprises polylactic acid, polyglycolic acid, polycaprolactone, PLGA, polylactic acid caprolactone copolymer, ethylene glycol copolymer; Described natural macromolecular material comprises gelatin, collagen, fibroin albumen, chitosan, modification of chitosan, hyaluronic acid, alginate, fibrin, cellulose.Preferably, synthesis resorbable polymeric materials selects polylactic acid or PLGA, and natural macromolecular material selects collagen or fibroin albumen, further, natural macromolecular material selects the type i collagen deriving from fish skin, reduces the risk infecting zoonosis.
In said method, the production of guide tissue regeneration film also comprises post-processing step, comprises vacuum lyophilization, packaging and sterilizing, and described sterilization steps can adopt γ irradiation sterilization or ethylene oxide sterilizing, and preferably, sterilization method adopts γ irradiation sterilization.
The application of the guide tissue regeneration film developed by the present invention in preparation tissue repair goods also belongs to protection scope of the present invention.Described tissue repair goods comprise artificial tooth's film, Bone Defect Repari film, postoperative anti-adhesion membrane, hernia paster, fistula reparation sticking patch, pelvic floor repairing patch, cerebral dura mater sticking patch, spinal meninges sticking patch, artificial skin, artificial blood vessel, artificial nerve catheter, artificial ligament, artificial heel string.Preferably, described tissue repair goods are artificial tooth's film, Bone Defect Repari film, hernia paster, postoperative anti-adhesion membrane.
In above-mentioned application, the Cranial defect after the bone after the bone while the clinical practice of described artificial tooth's film comprises Dental implantion after application bone regeneration, exodontia around immediate implant body increases art, exodontia around extension implant body increases art, bone splits damage place guided bone regeneration art, local alveolar ridge augmentation, alveolar ridge expansion art, radectomy, cystectomy and undesirable root arrachement is filled, periodontal bone defects is filled.Preferably, the clinical practice of artificial tooth's film is that the bone that the bone after exodontia around immediate implant body increases around art, the rear extension implant body of exodontia increases art.
In above-mentioned application, the clinical practice of described Bone Defect Repari film is the reparation of non-weight bearing area Cranial defect, and preferably, Bone Defect Repari film is applied to the reparation of the decorative sursery upper jaw or mandibular defect.Described hernia paster is used for the reparation of abdominal hernia, inguinal hernia, hiatal hernia.
Guide tissue regeneration film provided by the invention, has following innovative point and advantage compared with common guide tissue regeneration film:
(1) theory of " potential weaker zone " is proposed innovatively, by raw materials different for degradation time in vivo by electrostatic spinning technique co-blended spinning, obtain " the potential weaker zone " containing multiple fiber, fiber faster of degrading in " potential weaker zone " after implanting is degraded rapidly and fibrous membrane aperture is increased, formed " weaker zone " with large aperture three dimensional structure, this weaker zone is conducive to cell and grows into, promotes that cambium generates; Fiber faster of simultaneously degrading in " potential weaker zone " contains natural macromolecular material, and as collagen, gelatin etc., the product of its solvent components or degraded can the regeneration of inducing peripheral cambium, and the structural representation of duplicature as shown in Figure 1;
(2) compacted zone of duplicature is formed by multiple material mixing spinnings such as collagen, polylactic acid or fibroin albumens, good mechanical property, degradation property and biocompatibility can be had concurrently, such as collagen (or fibroin) and polylactic acid-based synthetic material co-blended spinning can alleviate the acid inflammatory reaction of polylactic acid, improve hydrophilic and the biocompatibility of polylactic acid membrane; Or collagen and fibroin co-blended spinning can solve the technological deficiency that collagem membrane bad mechanical property easily subsides, vivo degradation is too fast;
(3) production of duplicature is continuous print process, and two-layer fibrous membrane is connected by same fiber, ensure that not easily apportion tomography and shrinkage of subsiding in the highest quality of duplicature, use;
(4) fibroin used in duplicature gives timbering material certain bacteriostatic activity, even if product is exposed in external environment condition also can resist certain antibacterial erosion; Utilize coaxial electrostatic spinning technology to add in timbering material by bioactie agent or the medicine such as antibacterial, the bioactive bracket material with medicament slow release effect can be obtained;
(5) the potential weaker zone of duplicature can be used as the stent-induced cambium regeneration that cell is grown into, and compacted zone cell is not easily grown into, anti effect can be played, therefore the clinical practice of product of the present invention is extensive, Various Tissues can be developed as and repair reuse artifacts, such as artificial tooth's film, Bone Defect Repari film, postoperative anti-adhesion membrane, hernia paster, fistula repair sticking patch, pelvic floor repairing patch, cerebral dura mater sticking patch, spinal meninges sticking patch, artificial skin, artificial blood vessel, artificial nerve catheter, artificial ligament, artificial heel string, can bring huge Social benefit and economic benefit.
Accompanying drawing explanation
Fig. 1 is the double membrane structure schematic diagram of guide tissue regeneration film of the present invention, and one deck is by the obtained compacted zone of the biological fiber that degradation time is longer, and another layer is mix obtained potential weaker zone by the biological fiber that degradation time is different.Compacted zone is connected by the biological fiber that same degradation time is longer with potential weaker zone, guarantees that duplicature there will not be division tomography.The schematic diagram only structure composition of directviewing description duplicature and fiber distributes, and can not reflect true aperture and the fiber concrete form of fibrous membrane.
Stereoscan photograph when Fig. 2 is embodiment 7 fibrous membrane potential weaker zone external degradation 0 week (before degraded);
Stereoscan photograph when Fig. 3 is the potential weaker zone external degradation of embodiment 7 fibrous membrane 4 weeks;
Fig. 4 is the HE staining tissue slides that embodiment 8 fibrous membrane compacted zone cultivates people's Gingival Fibroblasts;
Fig. 5 is the HE staining tissue slides that the potential weaker zone of embodiment 8 fibrous membrane cultivates people's Gingival Fibroblasts;
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method; Material used, reagent etc., if no special instructions, all can buy from commercial channels.Unless there are specified otherwise, the solution concentration prepared with liquid volume ratio represents (v/v), and the solution concentration prepared with solid w/v represents (w/v).
The preparation method of embodiment 1 one kinds of guide tissue regeneration films
1, dosing
Collagen and polylactic acid (PLA) are dissolved in hexafluoroisopropanol (HFIP) according to the ratio of mass ratio 1:9, solid-liquid ratio is 1:10, is stirred to dissolve completely to obtain the longer spinning liquid of homogeneous degradation time (Z);
Collagen and polylactic acid (PLA) are dissolved in hexafluoroisopropanol (HFIP) according to the ratio of mass ratio 9:1, solid-liquid ratio is 1:10, is stirred to dissolve completely to obtain the shorter spinning liquid of homogeneous degradation time (Q).
2, compacted zone is spinned
Above-mentioned spinning liquid Z is loaded electrostatic spinning syringe, and the speed regulating micro-injection pump is 5ml/ hour, and the voltage of electrostatic spinning machine is 20KV, and the receiving range regulating receiving system is 20cm, stops spinning, enter next step rapidly after reaching 0.1mm thickness;
3, potential weaker zone is spinned
Above-mentioned spinning liquid Z and spinning liquid Q is respectively charged in electrostatic spinning syringe Z and Q, the injection flow velocity regulating syringe Z is 0.2ml/ hour, and regulate syringe Q to be 19:1 with the ratio of the injection flow velocity of syringe Z, namely the fiber that the degradation time that spins for spinning liquid Z of fibrous membrane 5% is longer, the fiber that 95% degradation time spinned for spinning solution Q is shorter.The voltage regulating electrostatic spinning machine is 20KV, and the receiving range of receiving system is 20cm, by preparing the composite fibre film of two kinds of degraded different in kinds to spinning while bi-material, stops spinning after reaching 0.2mm thickness.
4, post processing
The fibrous membrane of spinning is put into vacuum freeze drier vacuum drying, and the demoulding can obtain the guide tissue regeneration film with two-layer composite, and one deck is compacted zone, and another layer is potential weaker zone.Pack be cut into the bulk of suitable size according to clinical requirement after, ethane via epoxyethane sterilization is for subsequent use.
The preparation method of embodiment 2 one kinds of guide tissue regeneration films
1, dosing
Be dissolved in trifluoroethanol by gelatin and fibroin albumen according to the ratio of mass ratio 2:8, solid-liquid ratio is 1:20, is stirred to dissolve completely to obtain the longer spinning liquid of homogeneous degradation time (Z);
Be dissolved in trifluoroethanol by gelatin and fibroin albumen according to the ratio of mass ratio 8:2, solid-liquid ratio is 1:20, is stirred to dissolve completely to obtain the shorter spinning liquid of homogeneous degradation time (Q).
2, compacted zone is spinned
Above-mentioned spinning liquid Z is loaded electrostatic spinning syringe, and the speed regulating micro-injection pump is 0.5ml/ hour, and the voltage of electrostatic spinning machine is 10KV, and the receiving range regulating receiving system is 10cm, stops spinning, enter next step rapidly after reaching 0.1mm thickness;
3, potential weaker zone is spinned
Above-mentioned spinning liquid Z and spinning liquid Q is respectively charged in electrostatic spinning syringe Z and Q, the injection flow velocity regulating syringe Z is 2ml/ hour, and regulate syringe Q to be 1:1 with the ratio of the injection flow velocity of syringe Z, namely the fiber that the degradation time that spins for spinning liquid Z of fibrous membrane 50% is longer, the fiber that 50% degradation time spinned for spinning liquid Q is shorter.The voltage regulating electrostatic spinning machine is 10KV, and the receiving range of receiving system is 10cm, by preparing the composite fibre film of two kinds of degraded different in kinds to spinning while bi-material, stops spinning after reaching 0.2mm thickness.
4, post processing
The fibrous membrane of spinning is put into vacuum freeze drier vacuum drying, and the demoulding can obtain the guide tissue regeneration film with two-layer composite, and one deck is compacted zone, and another layer is potential weaker zone.Pack be cut into the bulk of suitable size according to clinical requirement after, for subsequent use through gamma-radiation irradiation sterilization.
The preparation method of embodiment 3 one kinds of guide tissue regeneration films
1, dosing
Be dissolved in formic acid by chitosan and poly (lactic acid-glycolic acid) (PLGA) according to the ratio of mass ratio 3:7, solid-liquid ratio is 1:5, is stirred to dissolve completely to obtain the longer spinning liquid of homogeneous degradation time (Z);
Be dissolved in formic acid by chitosan and poly (lactic acid-glycolic acid) (PLGA) according to the ratio of mass ratio 7:3, solid-liquid ratio is 1:5, is stirred to dissolve completely to obtain the shorter spinning liquid of homogeneous degradation time (Q).
2, potential weaker zone is spinned
Above-mentioned spinning liquid Z and spinning liquid Q is respectively charged in electrostatic spinning syringe Z and Q, the injection flow velocity regulating syringe Z is 0.1ml/ hour, and regulate syringe Q to be 49:1 with the ratio of the injection flow velocity of syringe Z, namely the fiber that the degradation time that spins for spinning liquid Z of fibrous membrane 2% is longer, the fiber that 98% degradation time spinned for spinning liquid Q is shorter.The voltage regulating electrostatic spinning machine is 30KV, and the receiving range of receiving system is 30cm, by preparing the composite fibre film of two kinds of degraded different in kinds to spinning while bi-material, stops spinning, enter next step rapidly after reaching 0.2mm thickness.
3, compacted zone is spinned
Above-mentioned spinning liquid Z is loaded electrostatic spinning syringe, and the speed regulating micro-injection pump is 10ml/ hour, and the voltage of electrostatic spinning machine is 30KV, and the receiving range regulating receiving system is 30cm, stops spinning after reaching 0.1mm thickness.
4, post processing
The fibrous membrane of spinning is put into vacuum freeze drier vacuum drying, and the demoulding can obtain the guide tissue regeneration film with two-layer composite, and one deck is compacted zone, and another layer is potential weaker zone.Pack be cut into the bulk of suitable size according to clinical requirement after, ethane via epoxyethane sterilization is for subsequent use.
Embodiment 4 preparation has the guide tissue regeneration film of growth factor slow-release function
1, dosing
Be dissolved in dichloromethane by hyaluronic acid and polylactic acid caprolactone (PLA-CL) according to the ratio of mass ratio 4:6, solid-liquid ratio is 1:15, is stirred to dissolve completely to obtain the longer spinning liquid of homogeneous degradation time (Z);
Be dissolved in dichloromethane by hyaluronic acid and polylactic acid caprolactone (PLA-CL) according to the ratio of mass ratio 6:4, solid-liquid ratio is 1:15, is stirred to dissolve completely to obtain the shorter spinning liquid of homogeneous degradation time (Q).
Recombinant human bone morphogenetic protein (rhBMP-2) is dissolved in pure water, makes the final concentration of rhBMP-2 reach 20 μ g/ml, be stirred to dissolving completely and obtain homogeneous medicament slow release spinning sandwich layer solution (Y).
2, compacted zone is spinned
Above-mentioned spinning liquid Z is loaded electrostatic spinning syringe, and the speed regulating micro-injection pump is 1ml/ hour, and the voltage of electrostatic spinning machine is 15KV, and the receiving range regulating receiving system is 15cm, stops spinning, enter next step rapidly after reaching 0.1mm thickness;
3, spinning carries the potential weaker zone of medicine
Using the sandwich layer solution of above-mentioned spinning solution Y as coaxial electrically spun, spinning liquid Z is as the shell solution of coaxial electrically spun, be respectively charged in electrostatic spinning syringe Y and Z, the shower nozzle that above-mentioned two syringes are corresponding is coaxial spinning nozzle, in addition spinning liquid Q is loaded in spinning syringe Q, the shower nozzle of its correspondence with coaxial spinning nozzle side by side above receiving system.The injection flow velocity regulating syringe Z is 1ml/ hour, and syringe Y is 1:9 with the ratio of the injection flow velocity of syringe Z, then the injection flow velocity of coaxial spinning nozzle is syringe Y and syringe Z flow velocity sum.Regulate syringe Q to be 4:1 with the ratio of the injection flow velocity of coaxial spinning nozzle, namely fibrous membrane 20% be the longer fiber of the degradation time of spinning liquid Z and Y spinning, and 80% is the shorter fiber of the degradation time of spinning liquid Q spinning.The voltage regulating electrostatic spinning machine is 15KV, and the receiving range of receiving system is 15cm, by preparing the composite fibre film of two kinds of degraded different in kinds to spinning while bi-material, stops spinning after reaching 0.2mm thickness.
4, post processing
The fibrous membrane of spinning is put into vacuum freeze drier vacuum drying, and the demoulding can obtain the guide tissue regeneration film with two-layer composite, and one deck is compacted zone, and another layer is for carrying the potential weaker zone of rhBMP-2.Pack be cut into the bulk of suitable size according to clinical requirement after, for subsequent use through gamma-radiation irradiation sterilization.
Embodiment 5 preparation has the guide tissue regeneration film of antibacterials slow-release function
1, dosing
Be dissolved in the aqueous formic acid of 10% by fibrin and fibroin albumen according to the ratio of mass ratio 0:10, solid-liquid ratio is 1:50, is stirred to dissolve completely to obtain the longer spinning liquid of homogeneous degradation time (Z);
Be dissolved in the aqueous formic acid of 10% by fibrin and fibroin albumen according to the ratio of mass ratio 10:0, solid-liquid ratio is 1:50, is stirred to dissolve completely to obtain the shorter spinning liquid of homogeneous degradation time (Q).
Metronidazole is dissolved in pure water, makes the final concentration of metronidazole reach 40 μ g/ml, be stirred to dissolving completely and obtain homogeneous medicament slow release spinning sandwich layer solution (Y).
2, spinning carries the potential weaker zone of medicine
Using the sandwich layer solution of above-mentioned spinning solution Y as coaxial electrically spun, spinning liquid Z is as the shell solution of coaxial electrically spun, be respectively charged in electrostatic spinning syringe Y and Z, the shower nozzle that above-mentioned two syringes are corresponding is coaxial spinning nozzle, in addition spinning liquid Q is loaded in spinning syringe Q, the shower nozzle of its correspondence with coaxial spinning nozzle side by side above receiving system.The injection flow velocity regulating syringe Z is 2ml/ hour, and syringe Y is 1:5 with the ratio of the injection flow velocity of syringe Z, then the injection flow velocity of coaxial spinning nozzle is syringe Y and syringe Z flow velocity sum.Regulate syringe Q to be 3:1 with the ratio of the injection flow velocity of coaxial spinning nozzle, namely fibrous membrane 25% be the longer fiber of the degradation time of spinning liquid Z and Y spinning, and 75% is the shorter fiber of the degradation time of spinning liquid Q spinning.The voltage regulating electrostatic spinning machine is 25KV, and the receiving range of receiving system is 25cm, by preparing the composite fibre film of two kinds of degraded different in kinds to spinning while bi-material, stops spinning, enter next step rapidly after reaching 0.2mm thickness.
3, compacted zone is spinned
Above-mentioned spinning liquid Z is loaded electrostatic spinning syringe, and the speed regulating micro-injection pump is 6ml/ hour, and the voltage of electrostatic spinning machine is 25KV, and the receiving range regulating receiving system is 25cm, stops spinning after reaching 0.1mm thickness.
4, post processing
The fibrous membrane of spinning is put into vacuum freeze drier vacuum drying, and the demoulding can obtain the guide tissue regeneration film with two-layer composite, and one deck is compacted zone, and another layer is for carrying the potential weaker zone of metronidazole.Pack be cut into the bulk of suitable size according to clinical requirement after, ethane via epoxyethane sterilization is for subsequent use.
The bacteriostatic activity test of the guide tissue regeneration film of embodiment 6 containing fibroin
First embodiment 2 gained guide tissue regeneration film is cut into by sterile working the disk that diameter is about 0.5cm, and using the aseptic filter paper sheet of equal size as negative control group, disk is inserted overnight incubation on the flat board being coated with escherichia coli or staphylococcus aureus, then colony growth situation both observing, result of the test is in table 1, and the sample of result display obtained by embodiment 1 has certain bacteriostasis.
The bacteriostatic activity test of table 1 guide tissue regeneration film
The degradation property test of embodiment 7 guide tissue regeneration film
By aseptic technique 37 DEG C by embodiment 1 sample culturing in DMEM culture medium, carry out external degradation test.Within 2 weeks, 4 weeks, 8 weeks, sample respectively in 0 week (before cultivation) and cultivation, dry laggard line scanning electron microscopic observation and hot strength test.Result of the test in table 2, Fig. 2 and Fig. 3 be fibrous membrane degraded 0 week and 4 weeks time potential weaker zone stereoscan photograph.Can find out the increase along with the time, the aperture of potential weaker zone constantly increases, and reach maximum 30-50 μm to when 4 weeks, residual fiber when 8 weeks in fibrous membrane obviously expands, and aperture is slightly reduced.Fibrous membrane 8 weeks after-drawing intensity of degrading still is greater than 5MPa in addition, meets the requirement of mechanical strength of guide tissue regeneration film.
The degradation property test of table 1 guide tissue regeneration film
The cell compatibility test of embodiment 8 guide tissue regeneration film
The compacted zone and the potential weaker zone that people's Gingival Fibroblasts are inoculated into the fibrous membrane of embodiment 1 are cultivated, with the upgrowth situation of HE dyeing observation of cell on membrane material after 14 days.Result shows: 1. people's Gingival Fibroblasts is only in the superficial growth of fibrous membrane compacted zone, shows that compacted zone can play good barrier cell effect, as shown in Figure 4; 2. people's Gingival Fibroblasts potential loose tunic of can growing into is inner, and show that potential weaker zone can as the support of Growth of Cells, induction regenerative cell is grown into, as shown in Figure 5.
The application of embodiment 9 guide tissue regeneration film in Dental implantion Cranial defect is filled
4 adult Beagle dogs, pull out dog bilateral lower jaw P2, P3, P4 tooth, at once Implant, the mesial wall of implantation body's distance mesial root teeth socked has the Cranial defect gap of 2-3mm, adopt half mouthful of own control, immediate implant+covering embodiment 1 fibrous membrane (experimental group) is carried out in side, opposite side carries out immediate implant+covering Bio-Gide collagem membrane (matched group), 3 months after operation puts to death animal, take out and carry out histological observation with the mandibular bone of implantation body, and statistical analysis is carried out to the bone contact ratio of two groups of bone regeneration around implant and New born formation rate.All by new bone filling when result display experimental group and matched group Cranial defect place 3 months, the bone contact ratio of two groups of bone regeneration around implant and New born formation rate no significant difference, this shows that the fibrous membrane that the present invention develops and commercially available Bio-Gide collagem membrane have suitable Guided Bone Regeneration effect.
The application of embodiment 10 guide tissue regeneration film in non-weight bearing area bone defect healing
6 adult Beagle dogs, are divided into 2 groups at random: experimental group 3, matched group 3.Cut palatine mucosa after general anesthesia, remove high 4cm, the triangle hone lamella of wide 1.5cm, cuts nasal side mucoperiosteum.Experimental group embodiment 1 gained fibrous membrane covers Cranial defect district, and is interrupted tuberosity stitching mucoperiosteum with No. 1 silk thread, and matched group is directly interrupted tuberosity with No. 1 silk thread and sews up mucoperiosteum.Within postoperative 6 months, put to death animal, carry out x-ray CT palate plate coronal scan observe upper jaw bone, the new bone formation of result display experimental group is better than matched group, the new osteanagenesis of the fibrous membrane that the present invention develops bootable non-weight bearing area Cranial defect.
The application of embodiment 11 guide tissue regeneration film in abdominal hernia is repaired
8 rabbit, body weight 2.5 ± 0.5kg, female male each 4.Shear 2cm × 2cm size wound at abdominal part after general anesthesia, deeply reach abdominal cavity, wound surface uses gauze capping immediately, sews up fixing, and obtained abdominal hernia animal model carries out repairing test after 24 hours.Be divided into 2 groups at random: experimental group 4, matched group 4.Two groups of all first debridements, experimental group is repaired with embodiment 1 gained fibrous membrane, and matched group is repaired with propene polymer patch.Repair materials to be placed between peritoneum and muscle fixing, drainage from indwelling Tube Drain, skin closure external application gauze is sewed up fixing.Draw materials when 3 months after operation and carry out gross examination of skeletal muscle.Result shows two treated animals all without wound dehiscence, and experimental group does not find wound surface and visceral adhesion, and matched group all sticks together.Result shows that fibrous membrane that the present invention develops can be used for the reparation of abdominal hernia, and its curative effect is better than propene polymer patch, effectively can prevent the generation of visceral adhesion.
Claims (21)
1. a guide tissue regeneration film, this film is double membrane structure, it is characterized in that: one deck is by the obtained compacted zone of the biological fiber that degradation time is longer, another layer is mix obtained potential weaker zone by the biological fiber that degradation time is different, after potential weaker zone implants, part biological fiber fast degradation, makes the aperture of this film increase, is beneficial to growing into of cell.
2. guide tissue regeneration film according to claim 1, is characterized in that: described compacted zone is connected by the biological fiber that same degradation time is longer with potential weaker zone, guarantees that duplicature there will not be division tomography.
3. guide tissue regeneration film according to claim 1, is characterized in that: the raw material of described biological fiber is selected from one or more synthesis resorbable polymeric materials, one or more natural macromolecular materials, or its combination.
4. guide tissue regeneration film according to claim 3, is characterized in that: described synthesis resorbable polymeric materials comprises polylactic acid, polyglycolic acid, polycaprolactone, PLGA, polylactic acid caprolactone copolymer, ethylene glycol copolymer.
5. guide tissue regeneration film according to claim 3, is characterized in that: described natural macromolecular material comprises gelatin, collagen, fibroin albumen, chitosan, modification of chitosan, hyaluronic acid, alginate, fibrin, cellulose.
6. guide tissue regeneration film according to claim 1, is characterized in that: described duplicature is obtained by electrostatic spinning technique.
7. guide tissue regeneration film according to claim 1, can add one or more activating agents by electrostatic spinning technique in its biological fiber.
8. guide tissue regeneration film according to claim 7, is characterized in that: described activating agent comprises antibiotic, anti-inflammatory drug, analgesic, somatomedin, amelogenin, hydroxyapatite, tricalcium phosphate.
9. prepare the method for guide tissue regeneration film described in claim 1, it is characterized in that: comprise the following steps:
(1) the longer spinning liquid Z of degradation time and the shorter spinning liquid Q of degradation time is prepared;
(2) spinning liquid Z obtains compacted zone through electrostatic spinning;
(3) on step (2) gained compacted zone, spinning liquid Z and spinning liquid Q is adopted to carry out electrostatic spinning respectively, the obtained biological fiber potential weaker zone different containing two kinds of degradation times.
10. method according to claim 9, it is characterized in that: the tandem of described step (2) and step (3) can be put upside down, namely spinning liquid Z and spinning liquid Q is first adopted to carry out electrostatic spinning respectively, the obtained biological fiber potential weaker zone different containing two kinds of degradation times, then obtains compacted zone with spinning liquid Z electrostatic spinning on potential weaker zone.
11. methods preparing guide tissue regeneration film described in claim 7, is characterized in that: comprise the following steps:
(1) the shorter spinning liquid Q of the longer spinning liquid Z of degradation time, degradation time, spinning liquid Y containing activating agent is prepared;
(2) spinning liquid Z obtains compacted zone through electrostatic spinning;
(3) on step (2) gained compacted zone, employing spinning liquid Z is shell solution, spinning liquid Y is that sandwich layer solution carries out coaxial electrostatic spinning, another shower nozzle of spinning liquid Q is carried out electrostatic spinning, the obtained potential weaker zone containing activating agent simultaneously.
12. methods according to claim 11, it is characterized in that: the tandem of step (2) and step (3) can be put upside down, namely first adopt that spinning liquid Z is shell solution, spinning liquid Y is that sandwich layer solution carries out coaxial electrostatic spinning, another shower nozzle of spinning liquid Q is carried out electrostatic spinning, the obtained potential weaker zone containing activating agent simultaneously; Then on potential weaker zone, compacted zone is obtained with spinning liquid Z electrostatic spinning.
13. according to the arbitrary described method of claim 9 or 11, it is characterized in that: when spinning potential weaker zone, regulate spinning liquid Q to be 50:1-1:1 with the ratio of the spinning speed of spinning liquid Z, make fiber that in potential weaker zone, degradation time is shorter and the longer proportion of fibers of degradation time be 50:1-1:1.
14., according to the arbitrary described method of claim 9 or 11, is characterized in that: the solid-liquid ratio of described step (1) spinning liquid Z is the solid-liquid ratio of 1:5-1:50 (w/v), spinning liquid Q is 1:5-1:50 (w/v); In the electrostatic spinning of described step (2) or step (3), the speed regulating micro-injection pump is 0.1 ~ 10ml/ hour, and the voltage regulating high tension generator is 10 ~ 30KV, and the receiving range regulating receiving system is 10-30cm.
15., according to the arbitrary described method of claim 9 or 11, is characterized in that: the solvent of described spinning liquid is selected from hexafluoroisopropanol, trifluoroethanol, dichloromethane, chloroform, oxolane, formic acid, water, or its combination.
16. according to the arbitrary described method of claim 9 or 11, it is characterized in that: the key component of the spinning liquid Z that degradation time is longer is synthesis resorbable polymeric materials or fibroin albumen, auxiliary element is the natural macromolecular material except fibroin albumen, and the mass ratio of key component and auxiliary element is 10:0-5:5; The key component of the spinning liquid Q that degradation time is shorter is the natural macromolecular material except fibroin albumen, and auxiliary element is synthesis resorbable polymeric materials or fibroin albumen, and the mass ratio of key component and auxiliary element is 10:0-5:5.
17. methods according to claim 16, is characterized in that: described synthesis resorbable polymeric materials comprises polylactic acid, polyglycolic acid, polycaprolactone, PLGA, polylactic acid caprolactone copolymer, ethylene glycol copolymer; Described natural macromolecular material comprises gelatin, collagen, fibroin albumen, chitosan, modification of chitosan, hyaluronic acid, alginate, fibrin, cellulose.
18. 1 kinds of guide tissue regeneration films, it is prepared by the arbitrary described method of claim 9 or 11.
The application of 19. guide tissue regeneration films according to claim 1 in preparation tissue repair goods, is characterized in that described tissue repair goods comprise artificial tooth's film, Bone Defect Repari film, postoperative anti-adhesion membrane, hernia paster, fistula reparation sticking patch, pelvic floor repairing patch, cerebral dura mater sticking patch, spinal meninges sticking patch, artificial skin, artificial blood vessel, artificial nerve catheter, artificial ligament, artificial heel string.
20. application according to claim 19, is characterized in that: the Cranial defect after the bone after the bone after bone regeneration, exodontia are applied in the clinical practice of described artificial tooth's film while Dental implantion around immediate implant body increases art, exodontia around extension implant body increases art, bone splits damage place guided bone regeneration art, local alveolar ridge augmentation, alveolar ridge expansion art, radectomy, cystectomy and undesirable root arrachement is filled, periodontal bone defects is filled.
21. application according to claim 19, is characterized in that: the clinical practice of described Bone Defect Repari film is the reparation of non-weight bearing area Cranial defect; Described hernia paster is used for the reparation of abdominal hernia, inguinal hernia, hiatal hernia.
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