CN106693080A - Guided tissue regeneration membrane and preparation method thereof - Google Patents
Guided tissue regeneration membrane and preparation method thereof Download PDFInfo
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- CN106693080A CN106693080A CN201510797128.XA CN201510797128A CN106693080A CN 106693080 A CN106693080 A CN 106693080A CN 201510797128 A CN201510797128 A CN 201510797128A CN 106693080 A CN106693080 A CN 106693080A
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Abstract
The invention discloses a guided tissue regeneration membrane and a preparation method thereof. The preparation method comprises the steps of pretreating mammalian skin to obtain corium layer skin graft; sequentially performing decellularized treatment, virus inactivation treatment and dense shrinkage treatment on the corium layer skin graft to obtain a guided tissue regeneration membrane precursor; arranging the guided tissue regeneration membrane precursor in solution of soluble strontium salt in a mode of making the rough surface upward and making the smooth surface downward to perform activation, transferring the precursor to simulated body fluid to perform mineralization, and performing precooling and freeze drying to obtain the guided tissue regeneration membrane. According to the method for preparing the guided tissue regeneration membrane, by performing decellularized treatment, virus inactivation treatment and dense shrinkage treatment on the corium layer skin graft, the obtained guided tissue regeneration membrane precursor has the rough surface, the rough surface is mineralized in the simulated body fluid after being compounded with strontium element, and the obtained guided tissue regeneration membrane can slowly release Sr<2+>and Ca<2+> to peripheral defective bone tissues and has good bone induction and bone conduction functions.
Description
Technical field
The present invention relates to medical material field, more particularly to a kind of guide tissue regeneration film and preparation method thereof.
Background technology
Guide tissue regeneration technology (Guided Tissue Regeneration, GTR) is can effectively to control at present
A kind of the most advanced treatment method of periodontosis is treated, its key problem in technology is organized again using biological barrier film
The space buffer action of raw guiding film, prevents gingival epithelium and knot hoof histocyte from being migrated to root face, while choosing
Make to selecting property periodontal cell and alveolar bone cell adherence in root face and be differentiated to form periodontal new attachment.Therefore guide
The effect of film is most important.With GTR technologies clinic be increasingly widely applied, the type of guiding film and
Performance has turned into the key factor for preparing its development.
Guiding film used by traditional clinic is broadly divided into the absorbable and class of nonabsorable two.Due to nonabsorable
Need second operation to take out after guiding film such as expanded polytetrafluoroethylsealing implantation, unnecessary pain is brought to patient
With economic psychological burden, therefore absorbable guiding film is increasingly becoming the main trend of GTR development from now on.Collagen
It is the main component for tying hoof tissue, it is incomparable with other absorbable materials as guide tissue regeneration film
Bioactivity and biocompatibility, but its mechanical strength is poor, internal degradation speed is too fast as being restriction
The a great problem of its application.Recently as the rise and development of de- cellular biological technique, the de- cell base of animal
Matter has the three-dimensional structure of natural tissues, and with good mechanical property and with tissue growth preferably three
Dimension structure, thus as the new study hotspot of guiding film technological development.
With the continuous renewal of clinical technology, the requirement to guide tissue regeneration film function is also constantly lifted.Draw
Lead the effect that tissue regeneration membrane not only only serves barrier isolation, or a kind of inducting osseous tissue regeneration and reparation
Material.Thus, it is desirable to guide tissue regeneration film has certain osteoinductive bone conduction function, to promote defect
The reparation of bone, shortens the painful and economic psychological burden that treatment cycle also reduces patient simultaneously.
The content of the invention
Based on this, it is necessary to provide a kind of guide tissue regeneration film with self-bone grafting function and preparation method thereof.
A kind of preparation method of guide tissue regeneration film, comprises the following steps：
Skin corium skin graft is obtained after being pre-processed to the skin of mammal；
Carry out de- cell treatment, viral inactivation treatment and fine and close shrink process successively to the skin corium skin graft,
Guide tissue regeneration film precursor is obtained, the guide tissue regeneration film precursor has the two of shiny surface and mat surface
Face structure, wherein, the operation of the de- cell treatment is：The skin corium skin graft is carried out successively hypotonic-high
Ooze salt treatment, ferment treatment and detergent-treatment；And
By the guide tissue regeneration film precursor according to it is coarse face up, shiny surface mode directed downwardly is arranged on matter
During amount percentage concentration is 0.5%~5% solution of soluble strontium salt, taken after being activated 1 day~3 days at 4 DEG C~8 DEG C
Go out, then continue at mineralising 3 days~7 days in the simulated body fluid that pH is 7~8.5, cleaned after taking-up, it is cold after precooling
Jelly is dried to obtain guide tissue regeneration film.
In one embodiment, the skin to mammal obtains skin corium skin graft after pre-processing
Operate and be：Preliminary degreasing and defeathering are carried out to the skin of the mammal with Mechanical Method, dermatome is then used
The skin of the mammal is removed and be made thickness after the epidermis of 0.1mm~0.4mm and subcutaneus adipose tissue and be
The skin corium skin graft of 0.2mm~1mm.
In one embodiment, the skin of the mammal is the skin of pig, ox, sheep or horse.
In one embodiment, is additionally included in and the skin corium skin graft is carried out successively de- cell treatment, virus
Before the operation of inactivation treatment and fine and close shrink process, the skin corium skin graft is immersed in 0 DEG C~8 DEG C of antibiosis
Operation in plain solution overnight；
The solute of the antibiotic solution is Pen .- Strep, gentamicin, TOB, vancomycin
Or teicoplanin；
The mass percentage concentration of the antibiotic solution is 0.001%~0.1%.
In one embodiment, de- cell treatment, viral inactivation treatment are carried out successively to the skin corium skin graft
In operation with fine and close shrink process, the operation of the de- cell treatment is specially：By the skin corium skin graft
Respectively in mass percentage concentration for 0.1%~0.9% sodium chloride and mass percentage concentration are that 1.0%~10% sodium chloride is molten
Circulation immersion 1 time~3 times in liquid, the time of immersion is 4h~12h every time, then the skin corium skin graft is soaked
In the trypsin solution or neutral enzymatic solution that mass concentration is 0.1%~1.0%, 5 DEG C~37 DEG C shaking tables are placed in
Middle digestion 12h~48h, then by the skin corium skin graft in the detergent solution that mass concentration is 0.1%~1%
In 5 DEG C~37 DEG C wash 2 times~8 times, every time washing time be 2h~10h, finally with PBS or physiology
Salt solution cleans the skin corium skin graft 4 times~10 times, and the time of cleaning is 2h~10h every time；Wherein, it is described
Detergent is dodecyl sodium sulfate, Qula is logical or 3- [(3- cholesterol aminopropyl) dimethylamino] -1- propane sulfonic acid.
In one embodiment, de- cell treatment, viral inactivation treatment are carried out successively to the skin corium skin graft
In operation with fine and close shrink process, the operation of the viral inactivation treatment is specially：By the skin corium skin
Piece immersion mass percentage concentration is the ethanol solution or the ethanol solution of Peracetic acid of 0.5%~5% hydrogen peroxide
In, 30min~480min is soaked at room temperature, then cleaned with pure water, and by the skin corium skin graft according to upper
Epidermis upward, lower epidermis skin corium mode directed downwardly fix after -80 DEG C~-20 DEG C precooling 2h~12h, it is last cold
Dry 24h~48h is freezed, wherein, the peroxide is hydrogen peroxide or Peracetic acid.
In one embodiment, the operation of the viral inactivation treatment is specially：The skin corium skin graft is pressed
According to upper epidermal layer upward, lower epidermis skin corium mode directed downwardly fix after -80 DEG C~-20 DEG C precooling 2h~12h, connect
Freeze-drying 24h~48h, finally to freeze-drying after the skin corium skin graft carry out xeothermic inactivation, it is described
It is 50 DEG C~120 DEG C to do heat-inactivated temperature, described to do the heat-inactivated time for 3h~48h.
In one embodiment, de- cell treatment, viral inactivation treatment are carried out successively to the skin corium skin graft
In operation with fine and close shrink process, the operation of the fine and close shrink process is specially：By the skin corium skin
Piece according to upper epidermal layer upward, lower epidermis skin corium mode directed downwardly place after with pressure as 5MPa~50MPa
Pressurize 12h~48h, then submerges 15min~600min in acetone by the skin corium skin, then dries.
In one embodiment, the soluble strontium salt is strontium chloride, strontium nitrate or strontium hydroxide；
The simulated body fluid is prepared by following operation：By NaCl, 0.32g of 7.7g~8.3g~0.4g's
NaHCO3, 0.2g~0.25g KCl, 0.2g~0.25g K2HPO4·3H2O, 0.28g~0.34g's
MgCl2·6H2The CaCl of O, 0.27g~0.31g2, 0.068g~0.076g Na2SO4And 5.8g~6.4g
Tris dissolvings after to adjust pH with HCl solution and Tris be 7~8.5, be finally settled to 1L, simulated
Body fluid.
A kind of guide tissue regeneration film, is prepared using the preparation method of above-mentioned guide tissue regeneration film.
The preparation method of this guide tissue regeneration film is by carrying out de- cell treatment, virus to skin corium skin graft
Inactivation treatment and fine and close shrink process, the guide tissue regeneration film precursor for obtaining have shiny surface and mat surface,
Shiny surface can effectively stop that gingival epithelium and knot hoof are organized in root and look unfamiliar length, and mat surface by with solubility
Strontium element in the solution of strontium salt is combined, the guide tissue regeneration film for then being obtained after mineralising in simulated body fluid
With stronger mechanical performance, meanwhile, being compounded with the guide tissue regeneration film after strontium element and mineralising can be to
The bone tissue of surrounding defect slowly discharges Sr2+And Ca2+, with good self-bone grafting and bone conduction function, so that
The reparation and regeneration of defect bone tissue around can be effectively facilitated.
Brief description of the drawings
Fig. 1 is the flow chart of the preparation method of the guide tissue regeneration film of an implementation method；
Fig. 2 is the scanning electron microscopy of the obtained shiny surface for preparing guide tissue regeneration film precursor of embodiment 1
Mirror photo；
Fig. 3 is the scanning electron microscopy of the obtained mat surface for preparing guide tissue regeneration film precursor of embodiment 1
Mirror photo.
Specific embodiment
Further is made to guide tissue regeneration film and preparation method thereof mainly in combination with drawings and the specific embodiments below
Detailed description.
The preparation method of guide tissue regeneration film as shown in Figure 1, comprises the following steps：
S10, the skin to mammal obtain skin corium skin graft after pre-processing.
The operation that skin corium skin graft is obtained after being pre-processed to the skin of mammal is：With Mechanical Method to feeding
The skin of newborn animal carries out preliminary degreasing and defeathering, is then removed the skin of mammal with dermatome
The skin corium skin that thickness is 0.2mm~1mm is made after the epidermis of 0.1mm~0.4mm and subcutaneus adipose tissue
Piece.
General, skin corium skin graft can be cut into the skin graft of 3cm × 6cm~5cm~10cm sizes.
The skin of mammal includes the fur, epidermis, skin corium and the hypodermis that stack gradually.
Specifically, skin of the skin of mammal for pig, ox, sheep or horse.Preferably, mammal
Skin is the skin of abdomen of pig, ox, sheep or horse.
The skin corium skin graft for obtaining can be standby with low-temperature storage.Specifically, the skin corium skin graft for obtaining can be stored up
In the presence of standby at -20 DEG C.
S10 also includes for the pretreated skin corium skin graft that obtains being immersed in mistake in 0 DEG C~8 DEG C of antibiotic solution
The operation at night.
The solute of antibiotic solution is Pen .- Strep, gentamicin, TOB, vancomycin or replaces
Koala is peaceful.Wherein, Pen .- Strep is that penicillin and streptomysin are 3 according to mass ratio：5 mixing for being formed
Thing.
The mass percentage concentration of antibiotic solution is 0.001%~0.1%.
S20, the skin corium skin graft obtained to S10 carry out de- cell treatment, viral inactivation treatment and densification successively
Shrink process, obtains guide tissue regeneration film precursor.
The guide tissue regeneration film precursor for obtaining has the two sides structure of shiny surface and mat surface.
The operation of de- cell treatment is：Skin corium skin graft is carried out successively hypotonic-hypertonic salt treatment, ferment treatment with
And detergent-treatment.
Specifically, the operation of de- cell treatment is：It is in mass percentage concentration respectively by skin corium skin graft
0.1%~0.9% sodium chloride and mass percentage concentration are circulation immersion 1 time~3 in 1.0%~10% sodium chloride solution
Secondary, the time of immersion is 4h~12h every time, then it is 0.1%~1.0% that skin corium skin graft is immersed in into mass concentration
Trypsin solution or neutral enzymatic solution in, be placed in 5 DEG C~37 DEG C shaking tables digestion 12h~48h, then will
Skin corium skin graft in the detergent solution that mass concentration is 0.1%~1% 5 DEG C~37 DEG C wash 2 times~4 times, often
The time of secondary washing is 2h~4h, skin corium skin graft is finally cleaned with PBS or physiological saline 4 times~10 times,
The time of cleaning is 2h~6h every time.Wherein, detergent is that dodecyl sodium sulfate, Qula be logical or 3- [(3- courages
Sterol aminopropyl) dimethylamino] -1- propane sulfonic acid.
The operation of de- cell treatment is：After carrying out inactivation treatment to skin corium skin graft, by by inactivation treatment
Skin corium skin graft according to upper epidermal layer upward, lower epidermis skin corium mode directed downwardly be placed in precooling in low temperature refrigerator
Freeze-drying afterwards.
Specifically, the operation of viral inactivation treatment is：It is by skin corium skin graft immersion mass percentage concentration
In the ethanol solution of 0.5%~5% hydrogen peroxide or the ethanol solution of Peracetic acid, soak at room temperature
30min~480min, is then cleaned with pure water, and by skin corium skin graft according to upper epidermal layer upward, lower epidermis
Skin corium mode directed downwardly fix after -80 DEG C~-20 DEG C precooling 2h~12h, last freeze-drying 24h~48h.Its
In, peroxide is hydrogen peroxide or Peracetic acid.
Or, the operation of viral inactivation treatment is specially：By skin corium skin graft according to upper epidermal layer towards upper and lower
Epidermis dermis layer mode directed downwardly fix after -80 DEG C~-20 DEG C precooling 2h~12h, then freeze-drying 24h~48h,
Xeothermic inactivation finally is carried out to the skin corium skin graft after freeze-drying.Wherein, it is 50 DEG C to do heat-inactivated temperature
~120 DEG C, the heat-inactivated time is done for 3h~48h.
Specifically, the operation of fine and close shrink process is：By skin corium skin graft according to upper epidermal layer upward, following table
Skin skin corium mode directed downwardly place after with pressure as 5MPa~50MPa pressurizes 12h~36h, then by corium
Layer skin submerges 15min~600min in acetone, finally dries.
S30, the guide tissue regeneration film precursor for obtaining S20 according to it is coarse face up, shiny surface side directed downwardly
Formula is arranged in the solution of soluble strontium salt that mass percentage concentration is 0.5%~5%, is activated 1 day at 4 DEG C~8 DEG C
Taken out after~3 days, then continue at mineralising 3 days~7 days in the simulated body fluid that pH is 7~8.5, cleaned after taking-up,
Freeze-drying obtains guide tissue regeneration film after precooling.
Soluble strontium salt can be strontium chloride, strontium nitrate or strontium hydroxide.
Simulated body fluid (SBF) is prepared by following operation：By NaCl, 0.32g of 7.7g~8.3g~0.4g's
NaHCO3, 0.2g~0.25g KCl, 0.2g~0.25g K2HPO4·3H2O, 0.28g~0.34g's
MgCl2·6H2The CaCl of O, 0.27g~0.31g2, 0.068g~0.076g Na2SO4And 5.8g~6.4g
Tris dissolvings after to adjust pH with HCl solution and Tris be 7~8.5, be finally settled to 1L, simulated
Body fluid.
Preferably, simulated body fluid (SBF) is prepared by following operation：By NaCl, 0.355g of 8.035g
NaHCO3, 0.225g KCl, 0.231g K2HPO4·3H2The MgCl of O, 0.311g2·6H2O、
The CaCl of 0.292g2, 0.072g Na2SO4And adjust pH with HCl solution after the Tris dissolvings of 6.118g
It is 7.4, is finally settled to 1L, obtains simulated body fluid.
In S30, the operation of freeze-drying is after precooling：- 80 DEG C~-20 DEG C precooling 2h~12h, then freeze dry
Dry 24h~48h.
The preparation method of this guide tissue regeneration film is by carrying out de- cell treatment, virus to skin corium skin graft
Inactivation treatment and fine and close shrink process, the guide tissue regeneration film precursor for obtaining have shiny surface and mat surface,
Shiny surface can effectively stop that gingival epithelium and knot hoof are organized in root and look unfamiliar length, and mat surface by with solubility
Strontium element in the solution of strontium salt is combined, the guide tissue regeneration film for then being obtained after mineralising in simulated body fluid
With stronger mechanical performance, meanwhile, being compounded with the guide tissue regeneration film after strontium element and mineralising can be to
The bone tissue of surrounding defect slowly discharges Sr2+And Ca2+, with good self-bone grafting and bone conduction function, so that
The reparation and regeneration of defect bone tissue around can be effectively facilitated.
The preparation method of this guide tissue regeneration film uses hypotonic-hypertonic joint enzyme treatment process, with de- thin
Born of the same parents are thoroughly and the infringement to tissue extracelluar matrix natural structure is light, maintain the complete of collagenous fibres support
Property, be conducive to the adhesion and growth of later stage cell.
The preparation method of this guide tissue regeneration film is using fine and close shrink process technique, the guiding for preparing
Film has the two sides structure of shiny surface and mat surface, with natural three-D space structure and good mechanicalness
Can, the space of abundance can be provided for newborn periodontium.
The guide tissue regeneration film of one implementation method, using the preparation method system of above-mentioned guide tissue regeneration film
It is standby to obtain.
This guide tissue regeneration film is prepared from by above-mentioned method, and with shiny surface and mat surface two
Face structure；By the way that using enzyme and surfactant alternate treatment, the supporting structure in processing procedure to corium breaks
It is bad smaller, can well ensure the integrality of fibrous framework, be conducive to the adhesion and growth of later stage cell.
This guide tissue regeneration film is prepared from by above-mentioned method, with shiny surface and mat surface, light
Sliding surface can effectively stop that gingival epithelium and knot hoof are organized in root and look unfamiliar length, and mat surface by with soluble strontium
Strontium element in the solution of salt is combined, the guide tissue regeneration film tool for then being obtained after mineralising in simulated body fluid
There is stronger mechanical performance, meanwhile, the guide tissue regeneration film being compounded with after strontium element and mineralising can be to week
The bone tissue for enclosing defect slowly discharges Sr2+And Ca2+, with good self-bone grafting and bone conduction function, so as to
Enough it is effectively facilitated the reparation and regeneration of defect bone tissue around.
It is below specific embodiment.
The simulated body fluid occurred in embodiment 1~3 is prepared by following operation：By NaCl, 0.355g of 8.035g
NaHCO3, 0.225g KCl, 0.231g K2HPO4·3H2The MgCl of O, 0.311g2·6H2O、
The CaCl of 0.292g2, 0.072g Na2SO4And adjust pH with HCl solution after the Tris dissolvings of 6.118g
It is 7.4, is finally settled to 1L, obtains simulated body fluid.
Embodiment 1
(1) preparation of skin corium skin graft
Whole belly skin of health pig is carried out into preliminary degreasing with Mechanical Method, hair is rejected clean with tweezers, used
Electric dermatome first removes the subcutaneous fat of pigskin, then removes epidermis 0.4mm, is made thickness for 0.3mm
Skin corium skin graft, then be immersed in the aqueous solution of the Pen .- Strep that mass concentration is 0.01%, insert
- 25 DEG C of low temperature refrigerator freezen protectives are transferred to after 4 DEG C of refrigerator overnights standby.
(2) preparation of guide tissue regeneration film precursor
De- cell treatment：It is successively that 0.1% sodium chloride is (hypotonic with mass concentration by the skin corium skin graft after defrosting
Salt) and mass concentration be 1.0% sodium chloride (hypertonic salt) solution circulation immersion 2 times, 6h/ times, by hypotonic
Solution treatment promotes tissue water swelling, so that the cell rupture in tissue, while the collagen in tissue is fine
Dimension structure is unclamped, so that collagen beam becomes loose, then the DNA of smudge cells is processed by hypertonic salt solution
Separated from histone；Skin corium skin graft is immersed in the trypsase that mass concentration is 0.25% again molten
In liquid, it is placed in 30 DEG C of shaking tables and digests 24h, washes cell fragment that tissue residue is removed in tissue and thin
Karyon；The subsequent sodium dodecyl sulfate solution by skin corium skin graft 0.1%, 35 DEG C are washed 3 times, 2h/
It is secondary, to remove the cell and nucleus that remain in tissue, the immunogenicity of tissue is thoroughly dispelled, while can be with
Reach the effect of fat in certain removal tissue；6 times, 2h/ times are finally washed with PBS.
Viral inactivation treatment：It is 2% Peracetic acid by the skin corium skin graft immersion mass concentration after the treatment of de- cell
Ethanol solution in, 30min is soaked at room temperature, then cleaned with pure water to PH7.4, finally by skin corium skin
Piece is laid in Teflon mould, and wherein upward, lower epidermis skin corium down, is placed in -80 DEG C to upper epidermal layer
It is transferred to immediately after low temperature refrigerator precooling 4h in freeze drier and is dried 24h.
Fine and close shrink process：Skin corium skin graft after will be lyophilized presses upper epidermal layer upward, lower epidermis skin corium court
Under be laid on stainless steel flat plate, be placed in tablet press machine with pressure 20MPa pressurize 24h, then will molding after
Guiding film first submerge 30min in acetone, then dry remove residual organic solvent.
(3) preparation of guide tissue regeneration film
Guide tissue regeneration film precursor prepared by (2) is faced down by smooth, coarse facing up is put into mould
Fix, then it is immersed together in the solution of soluble strontium salt that mass percentage concentration is 2.5%, it is living at 6 DEG C
Taken out after changing 2 days, then continue at mineralising 5 days in the simulated body fluid that pH is 7.4, cleaned after taking-up, precooling
Freeze-drying afterwards obtains guide tissue regeneration film.
Guide tissue regeneration film precursor obtained in embodiment 1 is observed under SEM (SEM),
Obtain Fig. 2 and Fig. 3.
Guide tissue regeneration film precursor has good as obtained in Fig. 2 and Fig. 3 can be seen that embodiment 1
Collagenous fibres supporting structure, shiny surface is fine and close, and mat surface is porous fibrous structure, is conducive to later stage skeletonization thin
Adhesion and growth of the born of the same parents in mat surface.
Embodiment 2
(1) preparation of skin corium skin graft
Whole belly skin of health pig is carried out into preliminary degreasing with Mechanical Method, hair is rejected clean with tweezers, used
Electric dermatome first removes the hypodermis of pigskin, then removes epidermis 0.1mm, is made thickness for 0.9mm
Skin corium skin graft, then be immersed in the aqueous solution of the gentamicin that mass concentration is 0.1%, insert 4 DEG C of ice
It is standby that case is overnight transferred to -25 DEG C of low temperature refrigerator freezen protectives afterwards.
(2) preparation of guide tissue regeneration film precursor
De- cell treatment：It is successively that 0.9% sodium chloride is (hypotonic with mass concentration by the skin corium skin graft after defrosting
Salt) and mass concentration be 9.0% sodium chloride (hypertonic salt) solution circulation immersion 3 times, 4h/ times, by hypotonic
Solution treatment promotes tissue water swelling, so that the cell rupture in tissue, while the collagen in tissue is fine
Dimension structure is unclamped, so that collagen beam becomes loose, then the DNA of smudge cells is processed by hypertonic salt solution
Separated from histone；Skin corium skin graft is immersed in the neutral enzymatic solution that mass concentration is 0.1% again
In, it is placed in 35 DEG C of shaking tables and digests 24h, wash the cell fragment and cell of removal tissue residue in tissue
Core；The subsequent logical solution of Qula by skin corium skin graft 0.1%, 35 DEG C are washed 4 times, 4h/ times, to remove group
The cell and nucleus of middle residual are knitted, the immunogenicity of tissue is thoroughly dispelled, certain going is can be simultaneously reached
Except the effect of fat in tissue；Finally use brine 4 times, 6h/ times.
Viral inactivation treatment：It is 0.5% peroxide second by the skin corium skin graft immersion mass concentration after the treatment of de- cell
In the ethanol solution of acid, 240min is soaked at room temperature, be laid in skin corium skin graft after then being cleaned with pure water
In Teflon mould, wherein upward, lower epidermis skin corium down, is placed in -80 DEG C of Low-temperature Ices to upper epidermal layer
It is transferred to immediately after case precooling 4h in freeze drier and is dried 24h.
Fine and close shrink process：Skin corium skin graft after will be lyophilized presses upper epidermal layer upward, lower epidermis skin corium court
Under be laid on stainless steel flat plate, be placed in tablet press machine with pressure 5MPa pressurize 48h, then by after molding
Guiding film first submerges 300min in acetone, finally dries the organic solvent for removing residual.
(3) preparation of guide tissue regeneration film
Guide tissue regeneration film precursor prepared by (2) is faced down by smooth, coarse facing up is put into mould
Fix, then it is immersed together in the solution of soluble strontium salt that mass percentage concentration is 0.5%, it is living at 8 DEG C
Taken out after changing 3 days, then continue at mineralising 7 days in the simulated body fluid that pH is 7.4, cleaned after taking-up, precooling
Freeze-drying afterwards obtains guide tissue regeneration film.
Embodiment 3
(1) preparation of skin corium skin graft.
Whole belly skin of health pig is carried out into preliminary degreasing with Mechanical Method, hair is rejected clean with tweezers, used
Electric dermatome first removes the hypodermis of pigskin, then removes epidermis 0.2mm, is made thickness for 0.5mm
Skin corium skin graft, then be immersed in the aqueous solution of the TOB that mass concentration is 0.1%, insert 4 DEG C of ice
It is standby that case is overnight transferred to -25 DEG C of low temperature refrigerator freezen protectives afterwards.
(2) preparation of guide tissue regeneration film precursor
De- cell treatment：It is successively that 0.5% sodium chloride is (hypotonic with mass concentration by the skin corium skin graft after defrosting
Salt) and mass concentration be 5.0% sodium chloride (hypertonic salt) solution circulation immersion 3 times, 12h/ times, by low
Osmometer solution treatment promotes tissue water swelling, so that the cell rupture in tissue, while the collagen in tissue
Fibre structure unclamps, so that collagen beam becomes loose, then the DNA of smudge cells is processed by hypertonic salt solution
Separated from histone；Skin corium skin graft is immersed in the trypsin solution that mass concentration is 1% again
In, it is placed in 25 DEG C of shaking tables and digests 36h, wash the cell fragment and cell of removal tissue residue in tissue
Core；Subsequent 3- [(3- cholesterol aminopropyl) dimethylamino] -1- propanesulfonic acid solutions by skin corium skin graft 1%,
35 DEG C are washed 3 times, 2h/ times, to remove the cell and nucleus that are remained in tissue, thoroughly dispel exempting from for tissue
Epidemic focus, can be simultaneously reached the effect of fat in certain removal tissue；Finally washed with PBS 6 times,
2h/ times.
Viral inactivation treatment：Skin corium skin graft after the treatment of de- cell is laid in Teflon mould,
Wherein upward, lower epidermis skin corium down, turns upper epidermal layer immediately after being placed in -20 DEG C of low temperature refrigerator precooling 12h
Enter and be dried 36h in freeze drier, then in 100 DEG C of hot air sterilization 12h.
Fine and close shrink process：Skin corium skin graft after will be lyophilized presses upper epidermal layer upward, lower epidermis skin corium court
Under be laid on stainless steel flat plate, be placed in tablet press machine with pressure 50MPa pressurize 12h, then will molding after
Guiding film first submerge 480min in acetone, finally vacuum drying remove residual organic solvent.
(3) preparation of guide tissue regeneration film
Guide tissue regeneration film precursor prepared by (2) is faced down by smooth, coarse facing up is put into mould
Fix, then it is immersed together in the solution of soluble strontium salt that mass percentage concentration is 5%, it is living at 4 DEG C
Taken out after changing 1 day, then continue at mineralising 3 days in the simulated body fluid that pH is 7.4, cleaned after taking-up, precooling
Freeze-drying afterwards obtains guide tissue regeneration film.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed,
But therefore can not be interpreted as the limitation to the scope of the claims of the present invention.It should be pointed out that for this area
Those of ordinary skill for, without departing from the inventive concept of the premise, can also make it is some deformation and
Improve, these belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be with appended
Claim is defined.
Claims (10)
1. a kind of preparation method of guide tissue regeneration film, it is characterised in that comprise the following steps：
Skin corium skin graft is obtained after being pre-processed to the skin of mammal；
Carry out de- cell treatment, viral inactivation treatment and fine and close shrink process successively to the skin corium skin graft,
Guide tissue regeneration film precursor is obtained, the guide tissue regeneration film precursor has the two of shiny surface and mat surface
Face structure, wherein, the operation of the de- cell treatment is：The skin corium skin graft is carried out successively hypotonic-high
Ooze salt treatment, ferment treatment and detergent-treatment；And
By the guide tissue regeneration film precursor according to it is coarse face up, shiny surface mode directed downwardly is arranged on matter
During amount percentage concentration is 0.5%~5% solution of soluble strontium salt, taken after being activated 1 day~3 days at 4 DEG C~8 DEG C
Go out, then continue at mineralising 3 days~7 days in the simulated body fluid that pH is 7.4, cleaned after taking-up, freezed after precooling
It is dried to obtain guide tissue regeneration film.
2. the preparation method of guide tissue regeneration film according to claim 1, it is characterised in that described
The operation that skin corium skin graft is obtained after being pre-processed to the skin of mammal is：With Mechanical Method to the food in one's mouth
The skin of newborn animal carries out preliminary degreasing and defeathering, is then removed the skin of the mammal with dermatome
The skin corium skin that thickness is 0.2mm~1mm is made after the epidermis of 0.1mm~0.4mm and subcutaneus adipose tissue
Piece.
3. the preparation method of guide tissue regeneration film according to claim 2, it is characterised in that described
The skin of mammal is the skin of pig, ox, sheep or horse.
4. the preparation method of guide tissue regeneration film according to claim 1, it is characterised in that also wrap
Include is carrying out de- cell treatment, viral inactivation treatment and fine and close shrink process to the skin corium skin graft successively
Before operation, the skin corium skin graft is immersed in the operation in 0 DEG C~8 DEG C of antibiotic solution overnight；
The solute of the antibiotic solution is Pen .- Strep, gentamicin, TOB, vancomycin
Or teicoplanin；
The mass percentage concentration of the antibiotic solution is 0.001%~0.1%.
5. the preparation method of guide tissue regeneration film according to claim 1, it is characterised in that to institute
In stating the operation that skin corium skin graft carries out de- cell treatment, viral inactivation treatment and fine and close shrink process successively,
The operation of the de- cell treatment is specially：It is in mass percentage concentration respectively by the skin corium skin graft
0.1%~0.9% sodium chloride and mass percentage concentration are circulation immersion 1 time~3 in 1.0%~10% sodium chloride solution
Secondary, the time of immersion is 4h~12h every time, then the skin corium skin graft is immersed in into mass concentration is
In 0.1%~1.0% trypsin solution or neutral enzymatic solution, it is placed in 5 DEG C~37 DEG C shaking tables and digests
12h~48h, then by the skin corium skin graft 5 DEG C in the detergent solution that mass concentration is 0.1%~1%
~37 DEG C are washed 2 times~8 times, and the time of washing is 2h~10h every time, finally clear with PBS or physiological saline
Wash the skin corium skin graft 4 times~10 times, the time of cleaning is 2h~10h every time；Wherein, the detergent
For dodecyl sodium sulfate, Qula are led to or 3- [(3- cholesterol aminopropyl) dimethylamino] -1- propane sulfonic acid.
6. the preparation method of guide tissue regeneration film according to claim 1, it is characterised in that to institute
In stating the operation that skin corium skin graft carries out de- cell treatment, viral inactivation treatment and fine and close shrink process successively,
The operation of the viral inactivation treatment is specially：It is by skin corium skin graft immersion mass percentage concentration
In the ethanol solution of 0.5%~5% hydrogen peroxide or the ethanol solution of Peracetic acid, soak at room temperature
30min~480min, is then cleaned with pure water, and by the skin corium skin graft according to upper epidermal layer towards upper and lower
Epidermis dermis layer mode directed downwardly fix after -80 DEG C~-20 DEG C precooling 2h~12h, last freeze-drying 24h~48h,
Wherein, the peroxide is hydrogen peroxide or Peracetic acid.
7. the preparation method of guide tissue regeneration film according to claim 1, it is characterised in that described
The operation of viral inactivation treatment is specially：By the skin corium skin graft according to upper epidermal layer upward, lower epidermis it is true
Cortex mode directed downwardly fix after -80 DEG C~-20 DEG C precooling 2h~12h, then freeze-drying 24h~48h, finally
The skin corium skin graft after to freeze-drying carries out xeothermic inactivation, described to do heat-inactivated temperature for 50 DEG C
It is~120 DEG C, described to do the heat-inactivated time for 3h~48h.
8. the preparation method of guide tissue regeneration film according to claim 1, it is characterised in that to institute
In stating the operation that skin corium skin graft carries out de- cell treatment, viral inactivation treatment and fine and close shrink process successively,
The operation of the fine and close shrink process is specially：By the skin corium skin graft according to upper epidermal layer upward, following table
Skin skin corium mode directed downwardly place after with pressure as 5MPa~50MPa pressurizes 12h~48h, then will be described
Skin corium skin submerges 15min~600min in acetone, then dries.
9. the preparation method of guide tissue regeneration film according to claim 1, it is characterised in that described
Soluble strontium salt is strontium chloride, strontium nitrate or strontium hydroxide；
The simulated body fluid is prepared by following operation：By NaCl, 0.32g of 7.7g~8.3g~0.4g's
NaHCO3, 0.2g~0.25g KCl, 0.2g~0.25g K2HPO4·3H2O, 0.28g~0.34g's
MgCl2·6H2The CaCl of O, 0.27g~0.31g2, 0.068g~0.076g Na2SO4And 5.8g~6.4g
Tris dissolvings after to adjust pH with HCl solution and Tris be 7.4, be finally settled to 1L, obtain simulated body fluid.
10. a kind of guide tissue regeneration film, it is characterised in that the guide tissue regeneration film is using such as right
It is required that the preparation method of the guide tissue regeneration film any one of 1~9 is prepared.
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| CN116617471B (en) * | 2023-07-24 | 2023-10-13 | 成都贝施美医疗科技股份有限公司 | Oral collagen membrane with double-layer structure and preparation method thereof |
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