CN109125812A - A kind of composite membrane and preparation method thereof for Guided Bone Regeneration - Google Patents
A kind of composite membrane and preparation method thereof for Guided Bone Regeneration Download PDFInfo
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Abstract
The present invention discloses a kind of composite membrane and preparation method thereof for Guided Bone Regeneration.The preparation method of the composite membrane includes following operating procedure: pig jejunum handles to obtain submucous layer of small intestine through mechanical means, multilayer transverse and longitudinal is superimposed to obtain compacted zone after the processes such as acid, detergent and alkali process, its spinning is equably obtained composite membrane through vacuum freeze drying after compacted zone is compound using electrostatic spinning technique by the collagen solution of preparation.There is excellent biocompatibility and bioactivity with composite membrane prepared by this method, easily it is absorbed by tissue, the compacted zone property of can choose in structure prevents soft tissue from growing into bone defect region, weaker zone is loose and porous structure, utilizes electrostatic spinning technique, weaker zone large specific surface area, be conducive to the adherency of the combination and cell between two membranes and grown into hole, it can be combined with bioactie agent or drug, support new regeneration, promote wound healing.The combination of the composite membrane makes it obtain excellent mechanical performance, can be in the stabilization of certain time holding regenerative space.
Description
Technical field
The present invention relates to biomedical materials fields, and in particular to a kind of natural composite membrane of bilayer of Guided Bone Regeneration is used
In the reparation of bone tissue and periodontium.
Background technique
Inducting osseous tissue regeneration art is at present clinically to the optimal selection of Repair of tissue defect, and principle is to pass through regeneration
The effects of physical barriers of film, prevents soft tissue from entering bone uptake area, is that the new bone growth in bone defect region reserves regenerative space,
And skeletonization class cell is guided to grow into new bone in bone defect area, to achieve the purpose that bony union.
Early stage for Guided Bone Regeneration the first generation regeneration membrane material be with polytetrafluoroethylene (PTFE) (e-PTFE) be representative not
Degradation material, such material need second operation to take out, increase wound due to that cannot be absorbed by tissue after placing 4-6 weeks
Chance, operation may cause to damage to tissue again, and generate destruction to the cell that newly adheres to and tissue, also, pass
The regeneration membrane material shortage antibacterial ability of system often results in repairing failure.
Biodegradable material is that current Guided Bone Regeneration film studies more a kind of material.Due to such material biofacies
Capacitive is good, and catabolite is nontoxic, easy to form and processing, has certain intensity with space needed for maintaining hyperblastosis,
After regeneration, material is degradable or is absorbed by tissue, and is a kind of material with favourable prospects.Biodegradable material
It is divided into artificial synthesized high molecular material and natural macromolecular material.Wherein with the artificial synthesized macromolecule material of polylactic acid-based equal representatives
Material has excellent mechanical property and biocompatibility, 201010105573.2 He of Chinese Patent Application No.
201310586143.0 preparing guide tissue regeneration film with polylactic acid material.But due to making after the fracture of polylactic acid hydrolysis of ester bonds with
Carboxyl, which appears increase, causes local ph to decline, and easily leads to patient and aseptic inflammation reaction occurs.Using collagen as the natural of representative
Macromolecule regeneration membrane has antigenic low, good biocompatibility, and has hemoglutination, may participate in tissue healing process, degradation speed
The advantages that rate can be adjusted as needed is current most commonly used guide tissue regeneration film both at home and abroad, as the country has listed
Bio-Gide collagem membrane, sea oral restoration film difficult to understand, rich special medical collagen membrane etc..But the simple of current wide clinical application comes from
The collagen of pig collagen, ox-hide or beef tendon has the following deficiencies: as guide tissue regeneration film first is that degradation time in vivo is short；
Second is that the requirement relative to regeneration membrane, the tension stress of collagem membrane is smaller, is easy to happen in application process and collapses and lose space
Maintenance ability；Third is that collagem membrane is easy to cause local chronic inflammatory to react.Before studies have shown that being exposed to intraoral collagen
Film (Bio-Gide collagem membrane and Hai Ao oral restoration film) meeting accelerated degradation, increases the risk of infection.(Bai Peng, Ye Ping, Wu Run
Hair, two kinds of collagem membranes of Dai Yongzhong are exposed to the comparative study China oral implantology magazine of degradation in oral environment,
2011,16 (01): 56-57.) different crosslinking technologicals and processing technology reduction glue can be passed through in the preparation process of collagem membrane
Former antigenicity improves biocompatibility and adjusts its degradation speed, increases its mechanical strength.Rich spy's medical collagen repair membrane is just
The white plates porous membrane being processed into is crosslinked for aldehydes.Chinese patent CN201710126662.7 and
CN201710124188.4 is all made of glutaraldehyde as cross linker to reach the effect for improving mechanical performance and reducing degradation speed, but
The introducing of the introducing of crosslinking agent, especially aldehyde crosslinking agent can generate potential toxic side effect to human body and find in the long run
It is a kind of without add crosslinking agent and be able to satisfy clinical demand guide tissue regeneration film it is imperative.
In addition, the guide tissue regeneration film reported at present is designed to the form of duplicature or multilayer film mostly, but it is easy
Appearance is layered and tears problem.In order to avoid multilayer film bonding is not close, the complicated procedures of forming such as mechanical press are needed, and are affected again
The microcosmic result and repairing effect of filming.Multilayer complex films room machine intensity is inhomogenous simultaneously, and the structure of MULTILAYER COMPOSITE to plant
There is foreign body sensation after entering, and clinical application effect is bad.A kind of periosteum described in Chinese Patent Application No. CN201710126698.5
Preparation method, although can overcome disadvantages mentioned above, its cumbersome preparation process is unfavorable for actual production from now on very much.
Therefore, clinically it is badly in need of a kind of good biocompatibility, can induce that regeneration, degradation time be suitable, mechanical strength
Inducting osseous tissue regeneration film that is good and being not easy initiation infection, and the production method of this kind of film should be suitble to industrialization production.
Summary of the invention
Good, drop that it is an object of the invention to overcome the deficiencies of the prior art and provide a kind of biocompatibilities and mechanical strength
The solution time is suitable and is not easy to cause infection, while being able to guide the composite membrane and preparation method thereof of osteanagenesis again.
A kind of preparation method of above-mentioned composite membrane for Guided Bone Regeneration, the specific steps are as follows:
(1) preparation of composite membrane compacted zone
It takes the chitterlings tissue wash of fresh slaughtered animals clean, is placed in 0.5% acetum and impregnates 30min, chitterlings and acetic acid
The ratio of solution is 1:5, scrapes mucous layer, muscle layer, placenta percreta, the lymph node that division removes chitterlings jejunum, separation using physics
Submucosa (SIS) out, is cut into segment, is rinsed 3 times using purified water；
Using the mixed aqueous solution containing 1.0% Peracetic acid and 15% ethyl alcohol, the ratio of SIS material and mixed aqueous solution is 1:
10, under ultrasound condition, soaking at room temperature 100min is virus inactivated.It is cleaned by ultrasonic 3 times using purified water later；
The ethyl alcohol for the use of concentration being 95%, the ratio of SIS material and ethyl alcohol are 1:10, under ultrasound condition, soak at room temperature 2h.Later
It is cleaned by ultrasonic 3 times using water for injection；
Using containing 0.02% trypsase and containing the mixed aqueous solution of 0.02%EDTA, SIS material and trypsase/EDTA solution
Ratio be 1:5, under ultrasound condition, impregnate 30min under the conditions of 37 DEG C.It is cleaned by ultrasonic 3 times using PBS later；
Using the aqueous solution of the enzyme of DNA containing 5U/ml, the ratio of SIS material and DNA enzymatic solution is 1:5, under ultrasound condition, in 37
20min is impregnated under the conditions of DEG C.Later using PBS drift ultrasonic cleaning 3 times；
Using the aqueous solution of the alpha-galactosidase containing 5U/ml, the ratio of SIS material and alpha-galactoside enzyme solutions is 1:5, is surpassed
Under the conditions of sound, 20min is impregnated under the conditions of 30 DEG C.It is cleaned by ultrasonic 3 times using PBS later；
The NaOH aqueous solution for the use of concentration being 25mM, the ratio of SIS material and NaOH solution are 1:20, under ultrasound condition, room temperature
Impregnate 50min.Later using PBS ultrasonic cleaning until neutral；
By de- cell treated single layer trees-Osima jacoti, Osima excavata drawout, 2 ~ 10 are superimposed according to horizontal and vertical alternate mode
Layer, is laid on mold, and freeze-drying is spare, and the electron scanning micrograph of composite membrane compacted zone is shown in Fig. 1；
(2) preparation of collagen solution
With organic acid dissolution collagen, collagen solution is formed；The mass volume ratio concentration range of collagen solution is 0.05 ~ 8%, viscosity
Coefficient is 1000 ~ 4000cp.The good collagen solution of stirring and dissolving, standing and defoaming processing；
(3) preparation of composite membrane
The densification that (1) obtains is placed in mold, the collagen solution that (2) prepare equably is spun by electrostatic spinning technique
On compacted zone, then demoulded after vacuum freeze drying, the electron scanning micrograph of composite membrane weaker zone.
Compared with prior art, the present invention have following remarkable advantage and the utility model has the advantages that
A kind of composite membrane for for Guided Bone Regeneration provided by the invention, biocompatibility and mechanical strength are good, degrade
Time is suitable and is not easy to cause infection, while being able to guide osteanagenesis again, after implanting, can obtain it is following the utility model has the advantages that
1. Guided Bone Regeneration composite membrane provided by the invention, when there is excellent mechanical performance and suitable degradation due to compacted zone
Between, the property of can choose prevents unwanted soft tissue from growing into bone defect area, and keep and extend regenerative space maintenance ability and
Stability；
2. Guided Bone Regeneration composite membrane provided by the invention, since weaker zone is prepared using electrostatic spinning technique, institute
The advantage that collagen fiber layer has large specific surface area is obtained, is conducive to enhance the combination dynamics between compacted zone and weaker zone, favorably
In the ability that raising cell is grown in the adhesive capacity and cell of weaker zone into hole, be conducive to the regeneration of new bone；
3. in the preparation method of Guided Bone Regeneration composite membrane provided by the invention, electrostatic spinning compared with coating, can be more convenient and
Exactly by adjusting process parameter and time, to control aperture and the thickness of collagem membrane, it is suitable for continuous production；
4. Guided Bone Regeneration composite membrane provided by the invention has preferable adhesion and flexibility, not tearable broken, it is conducive to
Surgical procedure；
5. the polypeptide moiety of the compacted zone of composite membrane provided by the invention, catabolite has anti-microbial property, can further drop
The incidence of inflammation and infection after low implantation；
6. the weaker zone of composite membrane provided by the invention can add different pharmaceutical or growth factor, to obtain preferably bone tissue
Regeneration effect and acceleration wound healing；Promoting healing substance or antibiosis can also be loaded by immersion way before implanting
Element, to further promote Wound healing and reduce infection rate；
7. the compacted zone of composite membrane provided by the invention can overcome one direction mechanical strength using the alternate mode of transverse and longitudinal
Deficiency achievees the purpose that each mechanical property of material is uniform, avoids the case where material is torn in operation.
Detailed description of the invention
Fig. 1 is the electron scanning micrograph of composite membrane compacted zone of the present invention；
Fig. 2 is the electron scanning micrograph of composite membrane weaker zone of the present invention；
Fig. 3 is the anti-microbial property testing result of the embodiment of the present invention 3；
Fig. 4 is the scanning result of the zoopery Micro CT of embodiment 5；
Fig. 5 is the zoopery defective region bone rege-neration (BV/TV) of embodiment 5；
Fig. 6 is growing state of the 6 porphyromonas single cell colonies of embodiment in composite membrane；
Fig. 7 is growing state of 6 periodontal ligament stem cell of embodiment in composite membrane.
Specific embodiment
Below by specific embodiment, technical scheme is described further, but these specific embodiments are not
The protection scope limiting the invention in any way.
Embodiment 1
Composite regenerated film compacted zone preparation
(1) it pre-processes
It takes the chitterlings tissue wash of fresh slaughtered animals clean, is placed in 0.5% acetum and impregnates 30min, chitterlings and acetic acid
The ratio of solution is 1:5, scrapes mucous layer, muscle layer, placenta percreta, the lymph node that division removes chitterlings jejunum, separation using physics
Submucosa out is cut into segment, is rinsed 3 times using purified water；
(2) inactivation of virus
Using the mixed aqueous solution containing 1.0% Peracetic acid and 15% ethyl alcohol, the ratio of SIS material and mixed aqueous solution is 1:
10, under ultrasound condition, soaking at room temperature 100min is virus inactivated.It is cleaned by ultrasonic 3 times using purified water later；
(3) immunogenic substances are removed
The ethyl alcohol for the use of concentration being 95%, the ratio of SIS material and ethyl alcohol are 1:10, under ultrasound condition, soak at room temperature 2h.Later
It is cleaned by ultrasonic 3 times using water for injection；
Using containing 0.02% trypsase and containing the mixed aqueous solution of 0.02%EDTA, SIS material and trypsase/EDTA solution
Ratio be 1:5, under ultrasound condition, impregnate 30min under the conditions of 37 DEG C.It is cleaned by ultrasonic 3 times using PBS later；
Using the aqueous solution of the enzyme of DNA containing 5U/ml, the ratio of SIS material and DNA enzymatic solution is 1:5, under ultrasound condition, in 37
20min is impregnated under the conditions of DEG C.Later using PBS drift ultrasonic cleaning 3 times；
Using the aqueous solution of the alpha-galactosidase containing 5U/ml, the ratio of SIS material and alpha-galactoside enzyme solutions is 1:5, is surpassed
Under the conditions of sound, 20min is impregnated under the conditions of 30 DEG C.It is cleaned by ultrasonic 3 times using PBS later；
The NaOH aqueous solution for the use of concentration being 25mM, the ratio of SIS material and NaOH solution are 1:20, under ultrasound condition, room temperature
Impregnate 50min.Later using PBS ultrasonic cleaning until neutral；
(4) it is lyophilized
By de- cell treated single layer trees-Osima jacoti, Osima excavata drawout, 6 layers are superimposed according to horizontal and vertical alternate mode,
It is laid on freeze-drying mold and is lyophilized, for use.
Embodiment 2
Immunogenic substance detection is carried out to the sample that embodiment 1 is prepared
(1) cell residue quantity measuring method: being fixed, paraffin embedding with 10% neutral formalin, is cut into 0.4 micron of thin slice, warp
Dimethylbenzene dewaxing, serial dehydration of alcohol, hematoxylin-eosin stains, microscopically observation cell residue situation and matrix fiber knot
Structure；
(2) DNA content detection method: according to YY/T 0606.25-2014 " the animal derived biomaterial DNA determination of residual amount
Method: fluorescence colour " it is detected；
(3) α-Gal antigenic content detection method: after sample is fixed with paraformaldehyde, routine paraffin wax embedded section, piece thickness is 3 micro-
Rice.Immunohistochemical reaction is carried out using the special affinity characteristic of biotin labeling BSI-B4 and α-Gal antigen.Coloration result determines:
Dark brown yellow particle is strong positive (+++), and brown yellow granule is positive (++), and yellow particle is weakly positive (+), has no coloring
For negative (-)；
(4) it lipid content detection method: is surveyed referring to soxhlet extraction methods in " fatty measurement in 5009.6 food of GB/T "
It is fixed；
As the result is shown: residual cell amount is 0/mirror downward view (400 ×), DNA residual quantity is 80 ± 12pg/g, α-Gal anti-
It originally was negative, lipid content 0.8%.
Embodiment 3
Anti-microbial property detection is carried out to the sample that embodiment 1 is prepared
Sample obtained by embodiment 1 is ground in the hydrochloric acid of 0.01M with grinding rod, until being visible by naked eyes particle, and is adjusted
Its concentration is 100mg/10mL.Pepsin digestion is added, pepsin: sample ratio is 1:10.It is persistently stirred at 25 DEG C
48h, after be cooled to 4 DEG C, the 0.1 M sodium hydroxide that 1/10 volume is added adjusts PH to 7.2-7.4；
Hybrid NC machine tool base plate is prepared, picks them separately a little cultured staphylococcus aureus and Escherichia coli with oese
Bacteria suspension is made in sterile saline 5ml in inclined-plane culture substratess.Take bacteria suspension 1.0ml and the above-mentioned sample through degrading
1ml is added in the culture dish of sterilizing-drying, and addition is cooled to 50 DEG C or so of ordinary nutritional broth agar culture medium, is shaken up,
Spare after sufficiently condensing, 35~37 DEG C of inversions are cultivated 24 hours, observe bacterial growth situation；Increase simultaneously and does not have to antibacterial material
Material and 5 μ g/mL antibacterial peptides of addition compare, and as a result see Fig. 3.
Embodiment 4
Mechanics properties testing is carried out to the sample that embodiment 1 is prepared
(1) suture strength
Method: with the non-absorbing suture of 3-0 in sample both sides center far from being sutured at edge 2mm, by the suture other end and sample
The other end be separately fixed on tensiometer both ends, stretched with the speed of 20mm/min, until stitch points are torn, record
Maximal force；
As a result: suture strength is in 3N-5N range；
(2) tensile strength
Method: it is 10mm shape that sample is cut to width respectively in both directions；In relative humidity 40%-60%, temperature after cutting
It is tested after being placed 2 hours in 22 ± 2 DEG C of environment of degree.Fixture spacing is 25mm, and sample both ends are fixed on cupping machine
Collet on, the maximal force with the speed tensile of 100mm/min, when record fracture；
As a result: tensile strength is in 30N-35N range；
(3) bursting strength
Method: according to the measurement side of " YY 0500-2004 cardiovascular implant artificial blood vessel " 8.3.3.2 probe rupture strength
Method is chosen 9.5mm diameter probe and is detected；
As a result: bursting strength is in 85N-90N range.
Embodiment 5
(1) it is used for the composite membrane of bone tissue regeneration
With acetate dissolution collagen, collagen solution is formed, viscosity coefficient is 1800 cp.Bones morphology is added in collagen solution
Protein growth factors, content are 5.0 μ g/mL, after mixing standing and defoaming；
The compacted zone that embodiment 1 obtains is cut into rectangular die size, is placed in mold, the collagen solution prepared is adopted
With electrostatic spinning technique equably by its spinning on compacted zone, at -40 DEG C after 8 h of pre-freeze, be transferred in freeze drier -
Dry 48h at 50 DEG C.Demoulding can be obtained the composite regenerated film of inducting osseous tissue regeneration；
(2) preparation of lyophilized collagen
Collagen solution prepared by (1) at -40 DEG C after 8 h of pre-freeze, is transferred in freeze drier at -50 DEG C dry 48h.
Demoulding can be obtained the lyophilized collagen of the growth factor containing bone morphogenetic protein；
(3) zoopery
Method: using embodiment 1 obtain fine and close sticking patch layer, embodiment 5(2) obtain lyophilized collagen and embodiment 5(1) obtain
Composite membrane be respectively used to the dog mandibular defect area Beagle covering oral cavity repairing research；
After implantation 3 months, Micro CT scanning result is shown in Fig. 4, defective region bone rege-neration diaphysis fraction (BV/TV) table
Show and see Fig. 5, the results show that the complex repairation effect of composite membrane is good more than single fine and close sticking patch layer or lyophilized collagen layer very much.
Embodiment 6
Composite regenerated film for paradenlal tissue regeneration
With acetate dissolution collagen, the collagen solution of 0.5% mass volume ratio is formed.Metronidazole is added in collagen solution, concentration is
1.0 mg/mL, after mixing standing and defoaming；
The compacted zone that embodiment 1 obtains is cut into rectangular die size, is placed in mold, the collagen solution prepared is adopted
With electrostatic spinning technique equably by its spinning on compacted zone, at -20 DEG C after 20 h of pre-freeze, it is transferred to freeze drier
In at -54 DEG C it is dry for 24 hours.Demoulding can be obtained the composite regenerated film of Guide Periodontal Tissue Regeneration；
By the composite regenerated film containing metronidazole of above-mentioned acquisition, using periodontitis the main pathogenic fungi-unicellular bacterium of porphyromonas
Antibacterial activity in vitro experiment is carried out, the antibacterial effect of composite regenerated film is as a result can verify that, sees Fig. 6.Wherein Fig. 6 (left side), which is shown, is
The porphyromonas single cell colonies of growth, Fig. 6 (right side) display is the porphyromonas single cell colonies being killed.It can be seen that
Composite regenerated film containing metronidazole of the invention, can keep the drug effect of metronidazole, reach obvious and inhibit porphyromonas unicellular
The effect of bacterium growth；
By the composite regenerated film containing metronidazole of above-mentioned acquisition, cell in vitro adherency is carried out using periodontal stem cell and sprawls reality
It tests, as a result the susceptible of proof composite regenerated film has excellent promotion cell spreading ability, sees Fig. 7.
The above embodiment of the present invention is the description of the invention and cannot be used for the limitation present invention, with right of the invention
Any change in the comparable meaning and scope of claim, is all considered as being included within the scope of the claims.
Claims (9)
1. a kind of composite membrane for Guided Bone Regeneration, it is characterised in that: composite membrane includes compacted zone and weaker zone, the densification
Layer is made of trees-Osima jacoti, Osima excavata, and weaker zone is made of the collagem membrane prepared using electrostatic spinning technique.
2. composite membrane according to claim 1, it is characterised in that:
The trees-Osima jacoti, Osima excavata compacted zone passes through inactivation of virus, removal immunogenic substances, superposition, freeze-drying preparation process system
At；
The collagen weaker zone is made up of dissolution, load medicine, standing and defoaming, electrostatic spinning, freeze-drying preparation process.
3. composite membrane according to claim 1, it is characterised in that: the weaker zone can use electrostatic spinning technique, lead to
Technological parameter and time are overregulated to control aperture and the thickness of collagem membrane.
4. composite membrane according to claim 1, it is characterised in that: the compacted zone is uniform-distribution with mutual perforative hole,
30 μm of aperture <, trees-Osima jacoti, Osima excavata laterally, longitudinal alternately 2-10 layers of superposition, 100-1000 μm of thickness, the weaker zone is
Vacuum freeze drying is formed after collagen solution is formed a film by electrostatic spinning, 300-500 μm of aperture, and 100-500 μm of thickness, Static Spinning
The fibre diameter of silk is 100-350 nm.
5. composite membrane according to claim 1 or 2, it is characterised in that: the compacted zone immunogenic substance is extremely low, including
Residual cell amount is feminine gender no more than 120pg/g, α-Gal antigen no more than 1/mirror downward view (400 ×), DNA residual quantity
(immunofluorescence assay), lipid content are not more than 1%.
6. composite membrane according to claim 1 or 2, it is characterised in that: the compacted zone has certain mechanical properties, stretches
Intensity is not less than 2N not less than 2N, tearing strength not less than 10N, suture strength not less than 18N, bursting strength.
7. composite membrane according to claim 1 or 2, it is characterised in that: drug can be added in the weaker zone, drug can be with
For taxol, adriamycin, gentamicin, acetyl spiramycin, Amoxicillin, metronidazole, tetracycline, azithromycin, penicillin
One of or it is a variety of, the 0.1-20 mg/mL of the concentration of drug is added.
8. composite membrane according to claim 1 or 2, it is characterised in that: bioactie agent can be added in the weaker zone,
Growth factor can be one of bone morphogenetic protein, platelet derived growth factor, fibroblast growth factor or more
Kind, the content that growth factor is added is 2-10 μ g/mL.
9. composite membrane according to claim 1, it is characterised in that: the composite membrane has degradable in vivo, when degradation
Between be 8-20 week, with antibacterial action after degradation.
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