CN106693079B - Guided tissue regeneration membrane and preparation method thereof - Google Patents

Guided tissue regeneration membrane and preparation method thereof Download PDF

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CN106693079B
CN106693079B CN201510791576.9A CN201510791576A CN106693079B CN 106693079 B CN106693079 B CN 106693079B CN 201510791576 A CN201510791576 A CN 201510791576A CN 106693079 B CN106693079 B CN 106693079B
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tissue regeneration
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skin sheet
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CN106693079A (en
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李丽花
朱勇军
钟梅玲
佘振定
谭荣伟
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Shenzhen Lando Biomaterials Co ltd
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    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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Abstract

The invention discloses a method for guiding tissue reorganizationThe raw film and the preparation method thereof, the preparation method comprises the following steps: pretreating the skin of a mammal to obtain a dermis skin sheet; sequentially performing cell removal treatment, virus inactivation treatment and compact shrinkage treatment on the dermis skin sheet to obtain a precursor of the guided tissue regeneration membrane; and arranging the precursor of the guided tissue regeneration membrane in the strontium-doped hydroxyapatite dispersion liquid according to the mode that the rough surface faces upwards and the smooth surface faces downwards, shaking for 1-24 h at room temperature, taking out, cleaning, drying and sterilizing to obtain the guided tissue regeneration membrane. The preparation method of the guided tissue regeneration membrane comprises the steps of carrying out decellularization treatment, virus inactivation treatment and compact shrinkage treatment on a corium skin sheet to obtain a guided tissue regeneration membrane precursor which has a rough surface, wherein the rough surface is compounded with strontium-doped hydroxyapatite, and can slowly release Sr to peripheral defective bone tissues2+And Ca2+Has good osteoinduction and osteoconduction functions.

Description

Guided tissue regeneration membrane and preparation method thereof
Technical Field
The invention relates to the field of medical materials, in particular to a guided tissue regeneration membrane and a preparation method thereof.
Background
The Guided Tissue Regeneration (GTR) is the most advanced treatment method that can effectively treat periodontal disease at present, and the key point of the technology is to prevent the migration of gingival epithelial and alveolar Tissue cells to the root surface by using the space isolation effect of a biological barrier film, i.e. a Tissue Regeneration guide film, and simultaneously selectively adhere periodontal cells and alveolar bone cells to the root surface and differentiate to form periodontal new attachment. The role of the guide film is therefore of great importance. With the wider clinical application of GTR technology, the type and performance of the guide membrane has become a key factor in the development of the preparation thereof.
The traditional clinical guiding membranes are mainly classified into absorbable and non-absorbable. Since the non-absorbable guide membrane such as expanded polytetrafluoroethylene needs to be taken out by a secondary operation after being implanted, unnecessary pain and economic and psychological burden are brought to patients, so that the absorbable guide membrane gradually becomes a main trend of GTR development in the future. Collagen is the main component of the connective tissue, is used as a tissue regeneration guiding membrane, has incomparable bioactivity and biocompatibility of other absorbable materials, but has poor mechanical strength and excessively high in-vivo degradation speed, so that the application of the collagen is limited. With the rise and development of acellular biotechnology in recent years, animal acellular matrixes have the three-dimensional structure of natural tissues, good mechanical properties and the three-dimensional structure more suitable for tissue growth, so that the animal acellular matrixes become a new research hotspot for guiding the development of membrane technology.
With the continuous update of clinical technologies, the requirements for the function of the guided tissue regeneration membrane are also continuously improved. The tissue regeneration guiding film not only plays a role of barrier isolation, but also is a material for guiding bone tissue regeneration and repair. Therefore, the tissue regeneration guiding membrane is required to have a certain osteoinductive bone conduction function so as to promote the repair of the defective bone, shorten the treatment period and reduce the pain and the economic and psychological burden of the patient.
Disclosure of Invention
Based on the above, there is a need for a guided tissue regeneration membrane having an osteoinductive function and a method for preparing the same.
A preparation method of a guided tissue regeneration membrane comprises the following steps:
pretreating the skin of a mammal to obtain a dermis skin sheet;
sequentially performing cell removal treatment, virus inactivation treatment and dense shrinkage treatment on the dermis skin sheet to obtain a tissue regeneration guiding membrane precursor, wherein the tissue regeneration guiding membrane precursor has a two-sided structure of a smooth surface and a rough surface, and the operation of the cell removal treatment comprises the following steps: sequentially carrying out hypotonic-hypertonic salt treatment, enzyme treatment and detergent treatment on the dermis leather sheet; and
arranging the precursor of the guided tissue regeneration film in a strontium-doped hydroxyapatite dispersion liquid with the mass percentage concentration of 0.01-0.5% according to the mode that the rough surface faces upwards and the smooth surface faces downwards, vibrating for 1-24 h at room temperature, taking out, cleaning, drying and sterilizing to obtain the guided tissue regeneration film, wherein the atomic ratio of Sr/[ Ca + Sr ] in the strontium-doped hydroxyapatite is 0.01-0.1: 1.
in one embodiment, the operation of obtaining the dermis skin sheet after the pretreatment of the skin of the mammal is as follows: the skin of the mammal is preliminarily degreased and dehaired by a mechanical method, and then 0.1mm to 0.4mm of epidermis and subcutaneous adipose tissue of the skin of the mammal are removed by a skin taking machine to prepare a dermis slice with the thickness of 0.2mm to 1 mm.
In one embodiment, the skin of the mammal is a pig, cow, sheep or horse skin.
In one embodiment, the method further comprises the operation of soaking the corium skin piece in an antibiotic solution at 0-8 ℃ overnight before the operations of the decellularization treatment, the virus inactivation treatment and the dense shrinkage treatment are sequentially carried out on the corium skin piece;
the solute of the antibiotic solution is penicillin-streptomycin, gentamicin, tobramycin, vancomycin or teicoplanin;
the mass percentage concentration of the antibiotic solution is 0.001-0.1%.
In one embodiment, the operations of performing the decellularization treatment, the virus inactivation treatment and the dense shrinkage treatment on the dermis skin sheet sequentially include: the corium skin piece is respectively soaked in sodium chloride solution with the mass percentage concentration of 0.1-0.9% and sodium chloride solution with the mass percentage concentration of 1.0-10% for 1-3 times in a circulating way, the soaking time is 4-12 hours each time, then the corium skin piece is soaked in trypsin solution or neutral enzyme solution with the mass concentration of 0.1-1.0%, the corium skin piece is placed in a shaking table with the temperature of 5-37 ℃ for shaking digestion for 12-48 hours, then the corium skin piece is washed in detergent solution with the mass concentration of 0.1-1% for 2-8 times with the temperature of 5-37 ℃ and the washing time of 2-10 hours each time, and finally the corium skin piece is washed 4-10 times by PBS or physiological saline, and the washing time of 2-10 hours each time; wherein the detergent is sodium dodecyl sulfate, triton or 3- [ (3-cholesterol aminopropyl) dimethylamino ] -1-propanesulfonic acid.
In one embodiment, in the sequentially performing the cell-removing treatment, the virus inactivation treatment and the dense shrinkage treatment on the dermis, the operation of the virus inactivation treatment specifically includes: soaking the leather skin sheet in 0.5-5 wt% ethanol solution of hydrogen peroxide or peracetic acid for 30-480 min at room temperature, washing with pure water, fixing the leather skin sheet in a manner that the upper surface skin layer faces upwards and the lower surface skin layer faces downwards, pre-cooling for 2-12 h at-80-20 ℃, and finally freeze-drying for 24-48 h, wherein the peroxide is hydrogen peroxide or peracetic acid.
In one embodiment, the operation of the virus inactivation treatment is specifically: fixing the leather skin sheet according to the mode that the upper surface layer faces upwards and the lower surface layer faces downwards, pre-cooling for 2-12 h at-80-20 ℃, then freeze-drying for 24-48 h, and finally performing dry heat inactivation on the freeze-dried leather sheet, wherein the dry heat inactivation temperature is 50-120 ℃, and the dry heat inactivation time is 3-48 h.
In one embodiment, in the sequentially performing the decellularization treatment, the virus inactivation treatment and the dense shrinkage treatment on the dermis, the operation of the dense shrinkage treatment is specifically: and placing the corium layer sheet according to the mode that the upper surface layer faces upwards and the lower surface corium layer faces downwards, keeping the pressure for 12-48 h at the pressure of 5-50 MPa, then immersing the corium layer in acetone for 15-600 min, and drying.
In one embodiment, in the drying after washing and sterilizing operation, the drying is freeze drying, and the sterilizing is cobalt 60 or high-energy electron irradiation sterilizing.
The guided tissue regeneration membrane is prepared by adopting the preparation method of the guided tissue regeneration membrane.
The preparation method of the guided tissue regeneration membrane comprises the steps of carrying out decellularization treatment, virus inactivation treatment and dense shrinkage treatment on a corium skin sheet to obtain a guided tissue regeneration membrane precursor which has a smooth surface and a rough surface, wherein the smooth surface can effectively prevent gingival epithelium and crusted tissuesThe strontium-doped hydroxyapatite is compounded with the rough surface to obtain the guided tissue regeneration membrane with strong mechanical property, and the guided tissue regeneration membrane compounded with the strontium-doped hydroxyapatite can slowly release Sr to the peripheral defective bone tissue2+And Ca2+Has good bone induction and bone conduction functions, thereby effectively promoting the repair and regeneration of peripheral defective bone tissues.
Drawings
FIG. 1 is a flow chart of a method for preparing a guided tissue regeneration membrane according to one embodiment;
fig. 2 is a scanning electron microscope photograph of the smooth side of the prepared guided tissue regeneration membrane precursor of example 1;
fig. 3 is a scanning electron microscope photograph of the rough surface of the precursor for preparing a guided tissue regeneration film prepared in example 1.
Detailed Description
The following will mainly refer to the accompanying drawings and the specific embodiments to further explain the guided tissue regeneration membrane and the preparation method thereof in detail.
The preparation method of the guided tissue regeneration membrane shown in fig. 1 comprises the following steps:
s10, obtaining the dermis leather skin sheet after the skin of the mammal is pretreated.
The operation of obtaining the dermis skin sheet after the skin of the mammal is pretreated is as follows: the skin of the mammal is preliminarily degreased and dehaired by a mechanical method, and then the epidermis and subcutaneous adipose tissue of 0.1mm to 0.4mm of the skin of the mammal are removed by a skin taking machine to prepare the dermis layer skin sheet with the thickness of 0.2mm to 1 mm.
Generally, the real leather layer leather sheet can be cut into leather sheets with the size of 3cm multiplied by 6cm to 5cm to 10 cm.
Mammalian skin includes a fur layer, an epidermal layer, a dermal layer, and a subcutaneous tissue, which are sequentially stacked.
Specifically, the skin of the mammal is the skin of pig, cattle, sheep or horse. Preferably, the mammalian skin is the abdominal skin of a pig, cow, sheep or horse.
The obtained corium skin sheet can be stored at low temperature for later use. Specifically, the obtained dermal skin sheets can be stored at-20 deg.C for use.
S10 further comprises the operation of soaking the obtained corium skin piece after pretreatment in the antibiotic solution at 0-8 ℃ overnight.
The solute of the antibiotic solution is penicillin-streptomycin, gentamicin, tobramycin, vancomycin or teicoplanin. Wherein, the penicillin-streptomycin is penicillin and streptomycin according to the mass ratio of 3: 5 of the mixture formed.
The mass percentage concentration of the antibiotic solution is 0.001-0.1%.
And S20, sequentially performing cell removal treatment, virus inactivation treatment and dense shrinkage treatment on the dermis cortical slice obtained in the step S10 to obtain the precursor of the guided tissue regeneration membrane.
The obtained precursor of the guided tissue regeneration membrane has a two-sided structure of a smooth side and a rough side.
The operation of the decellularization treatment is as follows: hypo-hypertonic salt treatment, enzyme treatment and detergent treatment are sequentially carried out on the dermis leather sheet.
Specifically, the operation of the decellularization treatment is as follows: the method comprises the steps of circularly soaking a corium skin sheet in sodium chloride solution with the mass percentage concentration of 0.1-0.9% and sodium chloride solution with the mass percentage concentration of 1.0-10% for 1-3 times, wherein the soaking time is 4-12 hours each time, soaking the corium skin sheet in trypsin solution or neutral enzyme solution with the mass concentration of 0.1-1.0%, placing the corium skin sheet in a shaking table with the temperature of 5-37 ℃ for 12-48 hours, washing the corium skin sheet in detergent solution with the mass concentration of 0.1-1% for 2-4 times at the temperature of 5-37 ℃, wherein the washing time is 2-4 hours each time, and finally washing the corium skin sheet with PBS or physiological saline for 4-10 times, wherein the washing time is 2-6 hours each time. Wherein the detergent is sodium dodecyl sulfate, triton or 3- [ (3-cholesterol aminopropyl) dimethylamino ] -1-propanesulfonic acid.
The operation of the decellularization treatment is as follows: after the leather skin sheet is subjected to inactivation treatment, the leather skin sheet subjected to inactivation treatment is placed in a low-temperature refrigerator according to the mode that the upper surface leather layer faces upwards and the lower surface leather layer faces downwards, and then the mixture is pre-cooled and freeze-dried.
Specifically, the operation of the virus inactivation treatment is as follows: soaking the leather skin sheet in 0.5-5 wt% concentration ethanol solution of hydrogen peroxide or peracetic acid solution at room temperature for 30-480 min, washing with pure water, fixing the leather skin sheet with the upper surface layer facing upwards and the lower surface layer facing downwards, pre-cooling at-80-20 deg.c for 2-12 hr, and final freeze drying for 24-48 hr. Wherein the peroxide is hydrogen peroxide or peroxyacetic acid.
Or, the operation of virus inactivation treatment specifically comprises: fixing the leather skin sheet according to the mode that the upper surface skin layer faces upwards and the lower surface skin layer faces downwards, pre-cooling for 2-12 h at the temperature of minus 80-minus 20 ℃, then freeze-drying for 24-48 h, and finally performing dry heat inactivation on the freeze-dried leather skin sheet. Wherein the temperature of the dry heat inactivation is 50-120 ℃, and the time of the dry heat inactivation is 3-48 h.
Specifically, the operation of the densification shrinkage treatment is as follows: placing the leather sheet with the upper leather layer facing upwards and the lower leather layer facing downwards, keeping the pressure at 5-50 MPa for 12-36 h, then immersing the leather sheet in acetone for 15-600 min, and finally drying.
And S30, arranging the precursor of the guided tissue regeneration membrane obtained in the step S20 in a strontium-doped hydroxyapatite dispersion liquid with the mass percentage concentration of 0.01-0.5% in a mode that the rough surface faces upwards and the smooth surface faces downwards, shaking for 1-24 h at room temperature, taking out, washing, drying and sterilizing to obtain the guided tissue regeneration membrane.
The atomic ratio of Sr/[ Ca + Sr ] in the strontium-doped hydroxyapatite is 0.01-0.1: 1.
cleaning, drying, and sterilizing by cobalt 60 or high-energy electron irradiation.
Hydroxyapatite (HAP) is the major constituent of vertebrate bone and teeth, with a hydroxyapatite content of about 96 Wt.% in human enamel. The hydroxyapatite has excellent biocompatibility and bioactivity, can be used as an induction factor of bones or teeth, and has better remineralization, desensitization and whitening effects on the teeth in the field of oral health care. Strontium (Sr) is a trace element necessary for human bodies, and the content of strontium (Sr) in bones accounts for about 0.01 percent of the mass of the bones. The trace strontium element has the functions of guiding the formation of new bone tissues and inhibiting the absorption of bones. After the strontium element is introduced into the hydroxyapatite, the performance is improved, so that the hydroxyapatite has the dual activities of the hydroxyapatite and the strontium element, and has better biocompatibility and bioactivity in the field of oral health care. After strontium is doped into hydroxyapatite, the solubility and the degradation performance are improved, the local concentration of calcium and phosphorus ions can be increased in the oral cavity, and the effect on mineralization of enamel and repair of damaged enamel is better than that of hydroxyapatite.
The preparation method of the guided tissue regeneration membrane comprises the steps of carrying out decellularization treatment, virus inactivation treatment and compact shrinkage treatment on a corium epidermis sheet to obtain a guided tissue regeneration membrane precursor which has a smooth surface and a rough surface, wherein the smooth surface can effectively prevent gingival epithelium and a crusted tissue from growing on the root surface, the rough surface is compounded with strontium-doped hydroxyapatite, the obtained guided tissue regeneration membrane has strong mechanical property, and meanwhile, the guided tissue regeneration membrane compounded with the strontium-doped hydroxyapatite can slowly release Sr to peripheral defective bone tissues2+And Ca2+Has good bone induction and bone conduction functions, thereby effectively promoting the repair and regeneration of peripheral defective bone tissues.
The preparation method of the guided tissue regeneration membrane adopts a hypotonic-hypertonic salt synthase treatment process, has thorough decellularization and light damage to the natural structure of the extracellular matrix of the tissue, maintains the integrity of the collagen fiber scaffold, and is beneficial to the adhesion and growth of cells in the later period.
The preparation method of the guide tissue regeneration membrane adopts a compact shrinkage treatment process, and the prepared guide membrane has a two-sided structure of a smooth surface and a rough surface, has a natural three-dimensional space structure and good mechanical properties, and can provide sufficient space for the newborn periodontal tissue.
The guided tissue regeneration membrane according to an embodiment is produced by the above-described method for producing a guided tissue regeneration membrane.
The guided tissue regeneration membrane is prepared by the method and has a two-sided structure of a smooth surface and a rough surface; by adopting the alternative treatment of enzyme and surfactant, the damage to the bracket structure of the dermis is small in the treatment process, the integrity of the fibrous bracket can be well ensured, and the adhesion and the growth of cells in the later period are facilitated.
The guided tissue regeneration membrane is prepared by the method, has a two-sided structure of a smooth surface and a rough surface, the smooth surface can effectively prevent gingival epithelium and a crusted tissue from growing on the root surface, the rough surface is compounded with strontium-doped hydroxyapatite, the obtained guided tissue regeneration membrane has strong mechanical property, and meanwhile, the guided tissue regeneration membrane compounded with the strontium-doped hydroxyapatite can slowly release Sr to peripheral defective bone tissues2+And Ca2+Has good bone induction and bone conduction functions, thereby effectively promoting the repair and regeneration of peripheral defective bone tissues.
The following are specific examples.
Example 1
(1) Preparation of dermis leather sheet
The whole belly skin of a healthy pig is preliminarily degreased by a mechanical method, hairs are removed by tweezers, subcutaneous tissues of the pig skin are removed by an electric skin taking machine, then the skin is removed by 0.4mm to prepare a corium skin sheet with the thickness of 0.3mm, the corium skin sheet is soaked in an aqueous solution of penicillin-streptomycin with the mass concentration of 0.01 percent and is placed in a refrigerator at 4 ℃ for overnight and then is transferred to a refrigerator at the low temperature of 25 ℃ below zero for freezing and storage for later use.
(2) Preparation of guided tissue regeneration membrane precursor
And (3) cell removal treatment: the thawed dermis skin sheet is sequentially soaked in sodium chloride (hypotonic salt) with the mass concentration of 0.1% and sodium chloride (hypertonic salt) with the mass concentration of 1.0% for 2 times and 6 hours/time in a circulating manner, the tissue is promoted to absorb water and swell through the treatment of the hypotonic solution, so that cells in the tissue are ruptured, meanwhile, the collagen fiber structure in the tissue is loosened, so that collagen bundles become loose, and DNA of the ruptured cells is separated from tissue protein through the treatment of the hypertonic salt solution; soaking the dermis in 0.25% trypsin solution, digesting in a shaker at 30 deg.C for 24 hr, and cleaning to remove residual cell debris and cell nucleus from the tissue; then, the dermis layer skin sheet is washed for 3 times and 2 hours/time at 35 ℃ in 0.1 percent sodium dodecyl sulfate solution to remove residual cells and cell nucleuses in the tissue, completely remove the immunogenicity of the tissue and simultaneously achieve a certain effect of removing fat in the tissue; finally wash 6 times with PBS 2 h/time.
Virus inactivation treatment: soaking the acellular dermal layer skin sheet in an ethanol solution with the mass concentration of 2% of peroxyacetic acid for 30min at room temperature, then washing the acellular dermal layer skin sheet to the pH value of 7.4 by using pure water, finally, flatly paving the dermal layer skin sheet in a polytetrafluoroethylene mold, wherein the upper dermal layer faces upwards, the lower dermal layer faces downwards, placing the mold in a low-temperature refrigerator at minus 80 ℃ for precooling for 4h, and immediately transferring the mold into a freeze dryer for drying for 24 h.
And (3) dense shrinkage treatment: and (3) flatly paving the freeze-dried dermis sheet on a stainless steel flat plate with the upper epidermis layer facing upwards and the lower epidermis layer facing downwards, placing the flat plate in a tablet machine, maintaining the pressure for 24 hours at the pressure of 20MPa, then immersing the die-pressed guide membrane in acetone for 30min, and then drying to remove the residual organic solvent.
(3) Preparation of guided tissue regeneration membrane
And (3) putting the precursor of the guide tissue regeneration membrane prepared in the step (2) into a mould for fixing according to the smooth surface facing downwards and the rough surface facing upwards, immersing the precursor of the guide tissue regeneration membrane into strontium-doped hydroxyapatite dispersion liquid with the Sr/[ Ca + Sr ] atomic ratio of 0.05 and the mass percentage concentration of 0.1%, shaking for 12 hours at room temperature, taking out, washing with clear water, freeze-drying, and sterilizing with cobalt 60 to obtain the guide tissue regeneration membrane.
The guided tissue regeneration membrane precursor prepared in example 1 was observed under a Scanning Electron Microscope (SEM), and fig. 2 and 3 were obtained.
As can be seen from fig. 2 and 3, the precursor of the guided tissue regeneration membrane prepared in example 1 has a good collagen fiber scaffold structure, a smooth surface is dense, and a rough surface is a porous fiber structure, which is beneficial to the adhesion and growth of osteoblasts on the rough surface in the later period.
Example 2
(1) Preparation of dermis leather sheet
The whole belly skin of a healthy pig is preliminarily degreased by a mechanical method, hairs are removed by tweezers, subcutaneous tissues of the pig skin are removed by an electric skin taking machine, then the skin is removed by 0.1mm to prepare a corium skin sheet with the thickness of 0.9mm, the corium skin sheet is soaked in a gentamicin water solution with the mass concentration of 0.1 percent and is placed in a refrigerator at 4 ℃ for overnight and then is transferred to a refrigerator at the low temperature of 25 ℃ for freezing and storage for later use.
(2) Preparation of guided tissue regeneration membrane precursor
And (3) cell removal treatment: the thawed dermis skin sheet is sequentially soaked in sodium chloride (hypotonic salt) with the mass concentration of 0.9 percent and sodium chloride (hypertonic salt) with the mass concentration of 9.0 percent for 3 times and 4 hours/time in a circulating manner, the tissue is promoted to absorb water and swell through the treatment of the hypotonic solution, so that cells in the tissue are ruptured, meanwhile, the collagen fiber structure in the tissue is loosened, so that collagen bundles become loose, and DNA of the ruptured cells is separated from tissue protein through the treatment of the hypertonic salt solution; soaking the corium skin sheet in neutral enzyme solution with the mass concentration of 0.1%, placing the corium skin sheet in a shaking table at 35 ℃ for digestion for 24 hours, and cleaning the tissue to remove cell fragments and cell nucleuses remained in the tissue; then, the dermis skin sheet is washed for 4 times and 4 hours/time at 35 ℃ in 0.1 percent triton solution to remove residual cells and cell nucleuses in the tissue, completely remove the immunogenicity of the tissue and simultaneously achieve a certain effect of removing fat in the tissue; finally, the mixture was washed 4 times with physiological saline for 6 hours.
Virus inactivation treatment: soaking the acellular dermis in an ethanol solution of peroxyacetic acid with the mass concentration of 0.5% at room temperature for 240min, then cleaning with pure water, spreading the dermis in a polytetrafluoroethylene mold, placing the mold in a low-temperature refrigerator at minus 80 ℃ for precooling for 4h, and immediately transferring the mold to a freeze dryer for drying for 24 h.
And (3) dense shrinkage treatment: and (3) flatly paving the freeze-dried dermis sheet on a stainless steel flat plate according to the condition that the upper epidermis layer faces upwards and the lower epidermis layer faces downwards, maintaining the pressure in a tablet machine for 48 hours at the pressure of 5MPa, then immersing the die-pressed guide membrane in acetone for 300min, and finally drying to remove the residual organic solvent.
(3) Preparation of guided tissue regeneration membrane
And (3) putting the precursor of the guide tissue regeneration membrane prepared in the step (2) into a mould for fixing according to the smooth surface facing downwards and the rough surface facing upwards, immersing the precursor of the guide tissue regeneration membrane into strontium-doped hydroxyapatite dispersion liquid with the Sr/[ Ca + Sr ] atomic ratio of 0.10 mass percent concentration of 0.01%, shaking the dispersion liquid at room temperature for 24 hours, taking the dispersion liquid out, washing the dispersion liquid with clear water, freeze-drying the dispersion liquid, and sterilizing the dispersion liquid with cobalt 60 to obtain the guide tissue regeneration membrane.
Example 3
(1) Preparation of dermis leather sheet
The whole belly skin of a healthy pig is preliminarily degreased by a mechanical method, hairs are removed by tweezers, subcutaneous tissues of the pig skin are removed by an electric skin taking machine, then the skin is removed by 0.2mm to prepare a corium skin sheet with the thickness of 0.5mm, the corium skin sheet is soaked in tobramycin aqueous solution with the mass concentration of 0.1 percent, and the corium skin sheet is placed in a refrigerator at 4 ℃ for overnight and then is transferred to a refrigerator at the low temperature of-25 ℃ for freezing and storage for later use.
(2) And (3) guiding the preparation of the tissue regeneration membrane precursor.
And (3) cell removal treatment: the thawed dermis skin sheet is sequentially soaked in sodium chloride (hypotonic salt) with the mass concentration of 0.5% and sodium chloride (hypertonic salt) with the mass concentration of 5.0% for 3 times and 12 hours/time in a circulating manner, the tissue is promoted to absorb water and swell through the treatment of the hypotonic solution, so that cells in the tissue are ruptured, meanwhile, the collagen fiber structure in the tissue is loosened, so that collagen bundles become loose, and DNA of the ruptured cells is separated from tissue protein through the treatment of the hypertonic salt solution; soaking the dermis in 1% trypsin solution, digesting in a shaker at 25 deg.C for 36h, and cleaning to remove cell debris and cell nucleus remaining in the tissue; then, the dermis skin sheet is washed in 1 percent of 3- [ (3-cholesteryl aminopropyl) dimethylamino ] -1-propanesulfonic acid solution for 3 times and 2 hours/time at 35 ℃ to remove residual cells and cell nucleuses in the tissue, completely remove the immunogenicity of the tissue and simultaneously achieve certain effect of removing fat in the tissue; finally wash 6 times with PBS 2 h/time.
Virus inactivation treatment: and (3) flatly paving the corium sheet subjected to the cell removal treatment in a polytetrafluoroethylene mold, wherein the upper corium layer faces upwards, the lower corium layer faces downwards, placing the mold in a low-temperature refrigerator at the temperature of-20 ℃ for precooling for 12h, immediately transferring the mold into a freeze dryer for drying for 36h, and then performing dry heat sterilization at the temperature of 100 ℃ for 12 h.
And (3) dense shrinkage treatment: and (3) flatly paving the freeze-dried corium layer sheet on a stainless steel flat plate with the upper corium layer facing upwards and the lower corium layer facing downwards, placing the flat plate on a tablet machine, keeping the pressure for 12 hours at the pressure of 50MPa, then immersing the die-pressed guide membrane in acetone for 480min, and finally, carrying out vacuum drying to remove the residual organic solvent.
(3) Preparation of guided tissue regeneration membrane
And (3) putting the precursor of the guide tissue regeneration membrane prepared in the step (2) into a mould for fixing according to the smooth surface facing downwards and the rough surface facing upwards, immersing the precursor of the guide tissue regeneration membrane into strontium-doped hydroxyapatite dispersion liquid with the Sr/[ Ca + Sr ] atomic ratio of 0.01 and the mass percentage concentration of 0.5%, shaking for 4 hours at room temperature, taking out, washing with clear water, freeze-drying, and sterilizing by high-energy electron irradiation to obtain the guide tissue regeneration membrane.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (8)

1. A preparation method of a guided tissue regeneration membrane is characterized by comprising the following steps:
pretreating the skin of a mammal to obtain a dermis skin sheet, wherein the skin of the mammal is the skin of a pig;
sequentially performing cell removal treatment, virus inactivation treatment and dense shrinkage treatment on the dermis skin sheet to obtain a tissue regeneration guiding membrane precursor, wherein the tissue regeneration guiding membrane precursor has a two-sided structure of a smooth surface and a rough surface, and the operation of the cell removal treatment comprises the following steps: sequentially carrying out hypotonic-hypertonic salt treatment, enzyme treatment and detergent treatment on the dermis leather sheet; and
arranging the precursor of the guided tissue regeneration film in a strontium-doped hydroxyapatite dispersion liquid with the mass percentage concentration of 0.1% according to the mode that a rough surface faces upwards and a smooth surface faces downwards, shaking for 12 hours at room temperature, taking out, cleaning, drying and sterilizing to obtain the guided tissue regeneration film, wherein the atomic ratio of Sr/[ Ca + Sr ] in the strontium-doped hydroxyapatite is 0.05: 1;
the operation of the dense shrinkage treatment specifically comprises the following steps: and placing the leather skin sheet according to the mode that the upper surface skin layer faces upwards and the lower surface skin layer faces downwards, maintaining the pressure for 12-48 h at the pressure of 5-50 MPa, then immersing the leather skin sheet in acetone for 15-600 min, and then drying.
2. The method for preparing a membrane for guiding tissue regeneration according to claim 1, wherein the pre-treating of the skin of the mammal to obtain the dermal skin is performed by: the skin of the mammal is preliminarily degreased and dehaired by a mechanical method, and then 0.1 mm-0.4 mm of epidermis and subcutaneous adipose tissue of the skin of the mammal are removed by a skin taking machine to prepare a dermis slice with the thickness of 0.2 mm-1 mm.
3. The method for preparing a membrane for tissue regeneration according to claim 1, further comprising the step of soaking the dermal skin in an antibiotic solution at 0-8 ℃ overnight before the steps of decellularizing, inactivating viruses and compacting and shrinking the dermal skin;
the solute of the antibiotic solution is penicillin-streptomycin, gentamicin, tobramycin, vancomycin or teicoplanin;
the mass percentage concentration of the antibiotic solution is 0.001% -0.1%.
4. The method for preparing a membrane for guiding tissue regeneration according to claim 1, wherein the operations of decellularizing, inactivating viruses and compacting and shrinking the dermis skin sheet are sequentially performed, and the operations of decellularizing are specifically as follows: the method comprises the following steps of circularly soaking the corium skin sheet in sodium chloride solution with the mass percentage concentration of 0.1-0.9% and sodium chloride solution with the mass percentage concentration of 1.0-10% for 1-3 times, wherein the soaking time is 4-12 hours each time, soaking the corium skin sheet in trypsin solution or neutral enzyme solution with the mass concentration of 0.1-1.0%, placing the corium skin sheet in a shaking table at 5-37 ℃ for shaking digestion for 12-48 hours, washing the corium skin sheet in detergent solution with the mass concentration of 0.1-1% at 5-37 ℃ for 2-8 times each time, wherein the washing time is 2-10 hours each time, and finally washing the corium skin sheet with PBS or physiological saline for 4-10 times, wherein the washing time is 2-10 hours each time; wherein the detergent is sodium dodecyl sulfate, triton or 3- [ (3-cholesterol aminopropyl) dimethylamino ] -1-propanesulfonic acid.
5. The method for preparing a membrane for guided tissue regeneration according to claim 1, wherein the operations of decellularizing, inactivating viruses and compacting and shrinking the dermis skin sheet sequentially comprise: soaking the leather skin sheet into 0.5-5% of ethanol solution of hydrogen peroxide or peracetic acid in percentage by mass for 30-480 min at room temperature, then washing with pure water, fixing the leather skin sheet in a manner that an upper surface skin layer faces upwards and a lower surface skin layer faces downwards, precooling for 2-12 h at-80-20 ℃, and finally freeze-drying for 24-48 h, wherein the peroxide is hydrogen peroxide or peracetic acid.
6. The method for preparing a membrane for guided tissue regeneration according to claim 1, wherein the virus inactivation treatment is specifically performed by: fixing the leather skin sheet according to the mode that the upper surface skin layer faces upwards and the lower surface skin layer faces downwards, pre-cooling for 2-12 h at-80-20 ℃, then freeze-drying for 24-48 h, and finally performing dry heat inactivation on the freeze-dried leather skin sheet, wherein the dry heat inactivation temperature is 50-120 ℃, and the dry heat inactivation time is 3-48 h.
7. The method for preparing a membrane for guided tissue regeneration according to claim 1, wherein the drying after washing is freeze-drying and the sterilization is cobalt 60 or high-energy electron irradiation sterilization.
8. A guided tissue regeneration membrane, which is prepared by the preparation method of the guided tissue regeneration membrane according to any one of claims 1 to 7.
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