CN103721296A - Tissue regeneration-guidable biomembrane and preparation method thereof - Google Patents
Tissue regeneration-guidable biomembrane and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a tissue regeneration-guidable biomembrane and a preparation method thereof. The tissue regeneration guidable biomembrane adopts a collagen membrane as a support, and adopts a natural three-dimensional space structure; the thickness of a protection layer formed by aluminium oxide or zirconium oxide granules arranged on one surface of the collagen membrane is 0.01-0.5 mum; the thickness of a hydroxyapatite strengthening layer arranged on the other surface of the collagen membrane is 0.01-0.1 mm. Compared with the tensile failure strength detection of the conventional pure collagen membrane, the mechanical strength of the biomembrane provided by the invention is raised by 7.5 times on average; animal reparation experiment effects have shown that the degradation time of the pure collagen membrane inside an animal body is 3-4 weeks, while the degradation time of the biomembrane provided by the invention inside the animal body can last for 12 weeks without collapsing; the biomembrane is fit closely on the reparation area through the strengthening layer without displacement and can ensure space for new tissues inside the animal body for a long period, and has good mechanical strength, can provide a good environment for growth of new bones to a greater extent, and the length of an newly-grown bone is the same as that of the original bone, so that an obviously optimal reparation effect compared with the pure collagen membrane is obtained.
Description
Technical field
The invention belongs to tissue engineering technical field of biomedical materials, be specifically related to biomembrane of a kind of bootable tissue regeneration and preparation method thereof.
Background technology
The biomembrane of bootable tissue regeneration is mainly used in the tissue regeneration art of repair tissue when damaged; with it as physical barriers; can stop perienchyma's defect area of growing into, as a kind of protecting film, defect area be protected, be the tissue growth headspace of defective region simultaneously.
Early stage guide tissue regeneration film is nonabsorable material (take polyethylene as representative), needs second operation to take out during this class materials'use, to patient, has increased misery.Develop subsequently the absorbable material (as degradable polymers such as polylactic acid, aliphatic polyesters) of synthetic, it is acid that its catabolite is, and can cause serious inflammatory reaction, is unfavorable for organization healing, the final effect that impact is repaired.In addition, also have some natural polymerses (regeneration membrane of preparing as chitosan and chitosan), its mechanical performance and water repelling property are poor, and degradation in vivo is very fast, can not coordinate well the growth of cambium.For addressing these problems, there is the degradability that adopts the method for chemical crosslinking to improve material, conventional cross-linking agent is glutaraldehyde, but the remaining toxicity that increases membrane material of reagent after crosslinked.
At present, people concentrate on xenogenesis natural biologic material emphasis.This class material source, in mammiferous membrane tissue, is prepared into laminar film through going antigen to process, and main component is collagen protein, therefore also claims collagem membrane.Collagem membrane degradation speed is very fast, in use along with the carrying out of degrading, its toughness drop, and to defect area, subside gradually, cannot effectively keep damaged space, affect growth and the final repairing effect of defective region cambium, especially particularly serious in damaged larger situation.
In US Patent No. 058372, disclose a kind of double-deck collagem membrane that has, adopted cross-linking method to delay the degraded of film, and improve the mechanical performance of film.Because chemical crosslinking meeting increase reagent is residual, and physical crosslinking (as heat cross-linking) is difficult to make collagem membrane to reach desirable mechanical performance, therefore still cannot solve collagem membrane degradation time too fast, easily forms the problem of subsiding.In Chinese patent application 201110145229.0, adopt the compactness extent of acetone raising film compacted zone, also fail to solve the mechanical performance problems of film.
Research discovery, whether film and defect can fully fit, and directly affect final skeletonization effect, fit more abundant, and growing amount and the Maturity of new bone are better.There is clinical report, due to film smooth surface, make film fixing insufficient and film displacement occurs cause infecting, finally treat unsuccessfully.And membrane material is in use, external environment can not be exposed to, otherwise the accelerated degradation of material can be caused, increase the risk that under film, cambium infects.
Through retrieval, find no the report about film and the abundant coating technique measure of defect, report that adopting multilayer film to cover prevents, this means the increase of surgery cost, patient is caused to economy and mental pressure.
Summary of the invention
The object of this invention is to provide biomembrane of a kind of bootable tissue regeneration and preparation method thereof, this biomembrane has natural three-D space structure and good mechanical performance, can provide sufficient space for cambium, in use be difficult for displacement phenomenon occurs, and can induce osteanagenesis.
The biomembrane of the bootable tissue regeneration that the present invention proposes, is to take collagem membrane as support, and described collagem membrane is derived from animal membrane tissue, has natural three-D space structure, can provide sufficient space for cambium; The protective layer that has aluminium oxide or zirconia particles formation in the one side of collagem membrane, thickness is 0.01 ~ 0.5 μ m; Another side has hydroxyapatite back-up coat, and thickness is 0.01 ~ 0.1mm.The effect of protective layer is to avoid collagem membrane to be directly exposed under external environment, to cause accelerated degradation; The effect of hydroxyapatite back-up coat is that hydroxyapatite can be combined closely with the osseous tissue formation chemical bond of defect area, makes collagem membrane be fixed on osseous tissue place, is not easy to be shifted; Meanwhile, protective layer and back-up coat all have the effect that improves collagem membrane mechanical performance.
The biomembranous preparation method of the bootable tissue regeneration of the present invention, is characterized in that, animal membrane tissue is obtained having the collagem membrane of n cell epimatrix structure through pretreatment; Adopt the method for electrophoresis to be evenly laid on a surface of collagem membrane zirconium oxide or alumina particle, form protective layer; By mixed solution electrolysis under pulse current effect of anhydrous calcium chloride and sodium dihydrogen phosphate dihydrate and ethanol, react with NaOH again, another surface in collagem membrane forms hydroxyapatite back-up coat, finally by obtaining the biomembrane of bootable tissue regeneration after dry, packing, sterilizing.Concrete steps comprise:
The pretreatment of step 1, film: after adopting pure water to clean up in animal membrane tissue, put into normal saline or PBS buffer (phosphate buffer) oscillation cleaning, the speed with 0.5 ℃ ∕ min in 4 ℃ of environment of inserting is again cooled to-80 ℃, keep 12 hours, after taking-up, insert in the aqueous slkali of pH 7.5 ~ 9.0 and vibrate and thaw, concussion speed 80 ~ 100rpm; After cleaning, insert in the alcoholic solution of 75% volume ratio and soak 2 hours, then clean with pure water, obtain membrane material;
Wherein, the freeze thawing treatment to animal membrane tissue, makes to form ice crystal in cell, and membranolysis, facilitates cell debris stripping, improves the effect of follow-up de-cell and defat; By controlling the rate of temperature fall of refrigerating process, can avoid quick freezing to cause ice crystal extracellular matrix structural damage in cell.
The preparation of step 2, collagem membrane
Ungrease treatment: the membrane material of acquisition is placed in to extraction vessel with absorbent cotton parcel, take acetone as defat reagent, heat and control temperature at 80 ~ 100 ℃, condensing reflux extracting 2 ~ 3 hours; Membrane material after defat is cleaned 4 ~ 8 times with pure water;
De-cell is processed: the membrane material after cleaning is inserted to (glucosan concentration is 4 ~ 10% in mass ratio) in the PBS buffer containing glucosan, with plasticity bag, seal, insert in cold isobaric static pressure machine, controlling temperature is 10 ~ 30 ℃, speed with 20Mpa/min is progressively forced into 500MPa, keep 5min, then decompression at the same rate, membrane material PBS buffer solution for cleaning taken out; The mixed solution being placed in again containing the NaOH of 0.1 ~ 0.25M and the NaCl of 1M soaks 1h, proceeds in pure water and soaks 1h, 2 times so repeatedly; Finally use pure water rinsing, obtain collagem membrane; Wherein, if membrane tissue cattle source property needs first to soak in the NaOH of 1M solution 1h and removes virus, then proceed in the PBS buffer containing glucosan and soak;
Defat adopts Soxhlet extraction process, with defat reagent, to the continuous extracting processing of the liposoluble substance in membrane material, can at short notice fat in material be controlled at below 1%; In addition, this method has been saved degreasing time, and can reclaim reagent, has reduced defat cost.
De-cell is exerted pressure to membrane tissue by the method for liquid pressing, the direct cell membrane of disorganize cell, and release cellular component, separated with extracellular matrix; It is reported; in being greater than the pressure environment of 220MPa; pressurization can make the freezing point of water rise; now in cell, can generate ice crystal (http://zhidao.***.com/question/118684849.html qq-pf-to=pcqq.c2c); thereby destruction cellularity; adjustment by pressure, temperature and intermediate medium concentration, had both reached de-cell effect, natural structure that can also Cell protection epimatrix.
Finally with the mixed solution of NaOH and NaCl, cell is fully dewatered, then be placed in pure water cell swelling is broken, the thoroughly cell debris in eluting tissue circulates 2 times.
Conventionally, use collagem membrane to seal reparation to wound, if occur, bad sealing or wound inflammation etc. cause wound dehiscence, make collagem membrane be exposed to external environment condition, can accelerate the degraded of collagem membrane; And protective layer makes collagem membrane and external environment condition isolation, avoid accelerated degradation, reduced the infection risk of cambium simultaneously; Zirconium oxide and aluminium oxide belong to bio-inert material, and stable in properties has good comprcssive strength, has also improved to a certain extent the mechanical performance of collagem membrane.
Step 4, prepare back-up coat: it is negative pole that the one side of the collagem membrane matcoveredn that step 3 is obtained is laid in steel plate, carbon-point connects positive source, and electrolyte is anhydrous calcium chloride and the sodium dihydrogen phosphate dihydrate of 0.003～0.25mol/L and the mixed solution of the ethanol that volume ratio is 20% that contains 0.004～0.42mol/L; Connect pulse direct current, electric current density is 2mA/cm
2, voltage is 1～100mV, the power-off again 1 minute of switching on after 1 minute 5 ~ 50 times so repeatedly, forms calcium phosphate coating on collagem membrane surface; Again after pure water cleans, be placed in mass volume ratio and be 0.4%～4% NaOH solution and soak 1 ~ 3h, on collagem membrane, form hydroxyapatite back-up coat; After cleaning with pure water, through vacuum drying, obtain the biomembrane of bootable tissue regeneration; Concentration of electrolyte is larger, voltage is higher, the cycle times of power on/off is more, and the thickness of back-up coat is just larger.
The present invention prepares back-up coat at the another side of collagem membrane, its objective is: hydroxyapatite has certain comprcssive strength, can improve the mechanical performance of collagem membrane; Secondly, hydroxyapatite can be degraded in vivo, the calcium of generation, phosphonium ion can with surrounding bone tissue in calcium, phosphonium ion form stable chemical bond, therefore, back-up coat can make collagem membrane and osseous tissue fit tightly, difficult displacement; In addition, calcium, phosphonium ion are the main components of skeleton and tooth, can be absorbed by body, inducting osseous tissue regeneration.
Bootable tissue regeneration biomembrane prepared by the present invention; for existing collagen membrane material bad mechanical property, easily subside, easily displacement and can not be exposed to the shortcomings such as external environment; by the surface at collagem membrane, form the coating of zirconium oxide or aluminium oxide bio-inert material; not only can protect collagem membrane to avoid the impact of external environment condition, also largely improve the mechanical performance of collagem membrane.By the another side at collagem membrane, form hydroxyapatite back-up coat, the Calcium and phosphorous component in hydroxyapatite dissolves rear and osseous tissue formation chemical bond, can make collagem membrane and osseous tissue combine closely, difficult displacement; Meanwhile, hydroxyapatite can be induced the growth of osseous tissue alive, accelerates reparation process.
By bootable tissue regeneration biomembrane and existing simple collagen film prepared by the present invention, carry out tensile break strength detection, comparing result demonstration, the biomembranous mechanical strength of the present invention on average improves approximately 7.5 times.And by these two kinds of films carry out that image processing, rabbit Ulna injuries, dog exodontia nest are filled, the animal repairing effect of lower bag of Os Canitis contrasts, find that the simple collagem membrane degradation in vivo time is 3～4w, the biomembrane degradation in vivo time of the present invention reaches 12w, and do not subside, biomembrane can fit tightly at restoring area by back-up coat, be not shifted, can guarantee for a long time in vivo the space of cambium; There is data to show that the mankind are 17w in the osteoplastic normal physiological cycle, visible biomembrane of the present invention has good mechanical strength, can be to a greater degree for new bone growth provides good environment, new osteogenetic height is consistent with normal bone, is obviously better than the repairing effect of contrast.
Accompanying drawing explanation
Accompanying drawing 1 is the tensile break strength testing result comparison diagram of the bootable tissue regeneration biomembrane of the present invention and existing simple collagen film.The testing result that in figure, a is collagem membrane, in figure, b is the biomembranous testing result of the present invention.The biomembranous mechanical strength of the present invention has improved 7.5 times than existing collagem membrane.
Accompanying drawing 2 is histology's result contrast photo that the bootable tissue regeneration biomembrane of the present invention and existing simple collagen film are repaired for image processing.The repairing effect photo that figure a is collagem membrane, can find out that depression appears in defect area, and the degradable collagem membrane of recess, illustrates that simple collagem membrane degraded is too fast, can not support defect area top, and subside to defect area, has affected area of new bone generation; Figure b is the biomembranous repairing effect photo of the present invention; its defect area has been full of area of new bone fibrous tissue, without depressed phenomenon, illustrates that biomembrane of the present invention can stably support the top of defect area; defective region is played a protective role, guarantee the Fast Growth of new bone.
Accompanying drawing 4 is the X-ray film observed result contrast photo that the bootable tissue regeneration biomembrane of the present invention and existing simple collagen film are filled for dog exodontia nest.The repairing effect that figure a is collagem membrane, can find out that there is obvious depression in exodontia region; Figure b is the biomembranous repairing effect of the present invention, and the height of its area of new bone reaches the level of normal bone substantially.Simple too fast the subsiding of causing of collagem membrane degraded is described, is important influence factor to new bone growth.
Accompanying drawing 5 is histology's result contrast photo that the bootable tissue regeneration biomembrane of the present invention and existing simple collagen film are repaired for lower bag of Os Canitis.The repairing effect that figure a is collagem membrane, can find out, clear area has appearred in defect area, its top has a large amount of connective tissues to invade, and illustrates that too fast the causing of degraded of simple collagem membrane subsides, and has affected new osteogenesis, also can lose inhibition, cause connective tissue to be invaded, form long junctional epithelium; Figure b is the biomembranous repairing effect of the present invention, can find out, area of new bone has been filled up defective region, and repairing effect is good, proves that biomembrane of the present invention can guarantee the Fast Growth of new bone, basically identical with normal bone.
The specific embodiment
Below in conjunction with example, technical solution of the present invention and effect are described further.The animal membrane tissue adopting in example is purchased from slaughterhouse, after removing the subsidiary impurity such as fat of membrane tissue, adopts PBS buffer (phosphate buffer) or normal saline to clean and removes bloodstain, then clean with pure water.
The cold isobaric static pressure machine adopting in example is manufactured by Taiyuan City Zhong Ping Science and Technology Ltd., the size 230 * 300mm of operating room, and maximum working pressure 600MPa, dwell time Pressure Drop (in 300s) is no more than 5%; Electrophoresis tank is that Liuyi Instruments Plant, Beijing produces, and model is DYCZ-21; Electrolysis bath is that Henan Pu Le education and science company produces, and model is 26023.
Take bovine pericardium as raw material, prepare according to the following steps guide tissue regeneration biomembrane.
The pretreatment of step 1, film: after bovine pericardium is cleaned with pure water, put into normal saline oscillation cleaning, then the speed with 0.5 ℃ ∕ min in 4 ℃ of environment of inserting is cooled to-80 ℃, maintenance 12h, after taking-up, insert in the sodium carbonate liquor of pH8 and vibrate and thaw, concussion speed is 90rpm; After cleaning, insert in the alcoholic solution of 75% volume ratio and soak 2h, then clean with pure water, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: the membrane material of acquisition is placed in to extraction vessel with absorbent cotton parcel, take acetone as defat reagent, heat and control temperature at 85 ℃; Condensing reflux extracting 2h; Take out the membrane material after defat, with pure water, clean 4 times;
De-cell is processed: the membrane material after cleaning is first soaked to 1h in the NaOH of 1M solution, insert again (glucosan concentration is 10% in mass ratio) in the PBS buffer containing glucosan, with plasticity bag, seal, insert in cold isobaric static pressure machine, control temperature at 10 ℃, with the speed of 20Mpa/min, be progressively forced into 500MPa, keep 5min, decompression at the same rate, takes out membrane material PBS buffer solution for cleaning again; Then the mixed solution being placed in containing the NaOH of 0.25M and the NaCl of 1M soaks 1h, proceeds in pure water and soaks 1h, 2 times so repeatedly; Finally with pure water, clean, obtain collagem membrane;
Step 4, prepare back-up coat: it is negative pole that the one side of the collagem membrane matcoveredn that step 3 is obtained is laid in steel plate, collagem membrane upward, carbon-point connects positive source, and electrolyte is the anhydrous calcium chloride that contains 0.008mol/L and the mixed solution of the sodium dihydrogen phosphate dihydrate of 0.005mol/L and the ethanol of volume ratio 20%; Connect pulse direct current, electric current density 2mA/cm
2, voltage 2mV, each 1 minute is a cycle for power on/off, 50 times repeatedly, on collagem membrane surface, forms calcium phosphate (chemical formula CaHPO
42H
2o) coating, then after pure water cleans, is placed in mass volume ratio and is 0.8% NaOH solution and soak 1h, forms hydroxyapatite (chemical formula Ca on collagem membrane
10(PO
4)
6(OH)
2) back-up coat (thickness 0.015mm), after cleaning with pure water, through vacuum drying, obtain the biomembrane of bootable tissue regeneration.
The biofilm thickness that this example makes is 0.12mm, and tensile break strength is 190～210MPa, compares with simple collagen membrane material, and mechanical performance improves approximately 7.5 times (referring to accompanying drawings 1).It is damaged that this material is applicable to the osseous tissue among a small circle that a variety of causes causes, in repair process, can stably cover above defective region, and phenomenon can not be shifted.
Take pig peritoneum as raw material, prepare according to the following steps guide tissue regeneration biomembrane.
The pretreatment of step 1, film: after pig peritoneum is cleaned up with pure water, put into normal saline oscillation cleaning; The speed with 0.5 ℃ of ∕ min in 4 ℃ of environment of inserting is again cooled to-80 ℃, keeps 12h, inserts in the sodium hydroxide solution of pH8.5 and vibrate and thaw after taking-up, and concussion speed is 80rpm; After cleaning, insert in the alcoholic solution of 75% volume ratio and soak 2h, then clean with pure water, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: the membrane material of acquisition is placed in to extraction vessel with absorbent cotton parcel, take acetone as defat reagent, heat and control temperature at 90 ℃; Condensing reflux extracting 3h; Take out the membrane material after defat, with pure water, clean 8 times;
De-cell is processed: the membrane material after cleaning is inserted to (glucosan concentration is 6% in mass ratio) in the PBS buffer containing glucosan, with plasticity bag, seal, insert in cold isobaric static pressure machine, control temperature at 20 ℃, speed with 20Mpa/min is progressively forced into 500MPa, keep 5min, then decompression at the same rate, membrane material PBS buffer solution for cleaning taken out; Then the mixed solution being placed in containing the NaOH of 0.15M and the NaCl of 1M soaks 1h, proceeds in pure water and soaks 1h, 2 times so repeatedly; Finally with pure water, clean, obtain collagem membrane;
Step 4, prepare back-up coat: it is negative pole that the one side of the collagem membrane matcoveredn that step 3 is obtained is laid in steel plate, collagem membrane upward, carbon-point connects positive source, and electrolyte is anhydrous calcium chloride and the sodium dihydrogen phosphate dihydrate of 0.013mol/L and the mixed solution of the ethanol that volume ratio is 20% that contains 0.021mol/L; Connect pulse direct current, electric current density is 2mA/cm
2voltage is 5mV, each 1 minute is a cycle for power on/off, 40 times repeatedly, on collagem membrane surface, form calcium phosphate coating, then after pure water cleans, be placed in mass volume ratio and be 2% NaOH solution and soak 1h, on collagem membrane, form hydroxyapatite back-up coat (thickness 0.05mm), after cleaning with pure water, through vacuum drying, obtain the biomembrane of bootable tissue regeneration.
The biofilm thickness that this example makes is 0.15mm.Its enough thick protective layer increased the stability of material when exposing, be very suitable for applying in oral cavity.This material is filled reparative experiment for dog exodontia nest, when sewing up, takes gingiva the mode of not exclusively sealing, fraction membrane material is exposed in oral cavity, the observation of drawing materials after January, the area of new bone height of finding this material group still can reach the effect consistent with normal bone, and simple collagen membrane material group can be observed obvious local bone amount growth not enough (referring to accompanying drawing 2) at field of operation.
Take Cor Sus domestica peplos as raw material, prepare according to the following steps guide tissue regeneration biomembrane.
The pretreatment of step 1, film: after Cor Sus domestica peplos is cleaned up with pure water, put into normal saline oscillation cleaning, then the speed with 0.5 ℃ ∕ min in 4 ℃ of environment of inserting is cooled to-80 ℃, maintenance 12h, after taking-up, insert in the solution of potassium carbonate of pH8.5 and vibrate and thaw, concussion speed is 85rpm; After cleaning, insert in the alcoholic solution of 75% volume ratio and soak 2h, then clean with pure water, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: the membrane material of acquisition is wrapped up and is placed in extraction vessel with absorbent cotton, take acetone as defat reagent, heat and control temperature at 90 ℃; Condensing reflux extracting 2.5h; Take out the membrane material after defat, with pure water, clean 8 times;
De-cell is processed: the membrane material after cleaning is inserted to (glucosan concentration is 5% in mass ratio) in the PBS buffer containing glucosan, with plasticity bag, seal, insert in cold isobaric static pressure machine, control temperature at 30 ℃, speed with 20Mpa/min is progressively forced into 500MPa, keep 5min, then decompression at the same rate, membrane material PBS buffer solution for cleaning taken out; Then the mixed solution being placed in containing the NaOH of 0.2M and the NaCl of 1M soaks 1h, proceeds in pure water and soaks 1h, 2 times so repeatedly; Finally use pure water rinsing, obtain collagem membrane;
Step 4, prepare back-up coat: it is negative pole that the one side of the collagem membrane matcoveredn that step 3 is obtained is laid in steel plate, collagem membrane upward, carbon-point connects positive source, and electrolyte is anhydrous calcium chloride and the sodium dihydrogen phosphate dihydrate of 0.081mol/L and the mixed solution of the ethanol that volume ratio is 20% that contains 0.136mol/L; Connect pulse current, electric current density is 2mA/cm
2voltage is 30mV, each 1 minute is a cycle for power on/off, 8 times repeatedly, on the surface of collagem membrane, form calcium phosphate coating, then after pure water cleans, be placed in mass volume ratio and be 2% NaOH solution and soak 2h, on collagem membrane, form hydroxyapatite back-up coat (thickness 0.03mm), after cleaning with pure water, through vacuum drying, obtain the biomembrane of bootable tissue regeneration.
The biofilm thickness that this example makes is 0.15mm, is applicable to shallow-layer osseous tissue defect repair, and protective layer and back-up coat have increased stability and the plasticity of material, can make material settling out and be supported on defective region.This material is repaired for rabbit Ulna injuries, and the area of new bone height after January is consistent with normal bone, and good with surrounding bone organizational integration, and simple collagen membrane material group is repaired district and occurred blank, this for material subside due to (referring to accompanying drawing 3).
embodiment 4,
Take cattle peritoneum as raw material, prepare according to the following steps guide tissue regeneration biomembrane.
The pretreatment of step 1, film: after cattle peritoneum is cleaned with pure water, put into PBS buffer oscillation cleaning; The speed with 0.5 ℃ of ∕ min in 4 ℃ of environment of inserting is again cooled to-80 ℃, keeps 12h, inserts in the potassium bicarbonate solution of pH8 and vibrate and thaw after taking-up, and concussion speed is 100rpm; After cleaning, insert in the alcoholic solution of 75% volume ratio and soak 2h, then clean with pure water, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: the membrane material of acquisition is placed in to extraction vessel with absorbent cotton parcel, take acetone as defat reagent, heat and control temperature at 95 ℃; Condensing reflux extracting 2h; Take out the membrane material after defat, with pure water, clean 5 times;
De-cell is processed: the membrane material after cleaning is soaked to 1h in the NaOH of 1M aqueous solution, then insert (glucosan concentration is 4% in mass ratio) in the PBS buffer containing glucosan, with plasticity bag, seal, insert in cold isobaric static pressure machine, control temperature at 15 ℃, with the speed of 20Mpa/min, be progressively forced into 500MPa, keep 5min, decompression at the same rate, takes out membrane material PBS buffer solution for cleaning again; Then the mixed solution being placed in containing the NaOH of 0.15M and the NaCl of 1M soaks 1h, proceeds in pure water and soaks 1h, 2 times so repeatedly; Finally use pure water rinsing, obtain collagem membrane;
Step 4, prepare back-up coat: it is negative pole that the one side of the collagem membrane matcoveredn that step 3 is obtained is laid in steel plate, collagem membrane upward, carbon-point connects positive source, and electrolyte is anhydrous calcium chloride and the sodium dihydrogen phosphate dihydrate of 0.125mol/L and the mixed solution of the ethanol that volume ratio is 20% that contains 0.21mol/L; Connect pulse direct current, electric current density 2mA/cm
2voltage 50mV, each 1 minute is a cycle for power on/off, 11 times repeatedly, on collagem membrane surface, form calcium phosphate coating, then after pure water cleans, be placed in mass volume ratio and be 3% NaOH solution and soak 2.5h, on collagem membrane, form hydroxyapatite back-up coat (thickness 0.08mm), after cleaning with pure water, through vacuum drying, obtain the biomembrane of bootable tissue regeneration.
The biofilm thickness 0.2mm that this example makes, is applicable to the damaged reparation of osseous tissue on a large scale, between its back-up coat and osseous tissue surface, forms sufficient chemical bond, can be close to top, defective region, guarantees the growing space of new bone.This material is used for to image processing reparation, after January, draw materials through histological observation, its material still has complete structure, and area of new bone height is consistent with normal bone, and simple collagen membrane material is because fixing insufficient causing subsides, bone amount growth not enough (referring to accompanying drawing 4).
embodiment 5,
Take trees-Osima jacoti, Osima excavata as raw material, prepare according to the following steps guide tissue regeneration biomembrane.
The pretreatment of step 1, film: after trees-Osima jacoti, Osima excavata is cleaned with pure water, put into PBS buffer oscillation cleaning; The speed with 0.5 ℃ of ∕ min in 4 ℃ of environment of inserting is again cooled to-80 ℃, keeps 12h, inserts in the sodium radio-phosphate,P-32 solution of pH8.5 and vibrate and thaw after taking-up, and concussion speed is 85rpm; After cleaning, insert in the alcoholic solution of 75% volume ratio and soak 2h, then clean with pure water, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: the membrane material of acquisition is placed in to extraction vessel with absorbent cotton parcel, take acetone as defat reagent, heat and control temperature at 90 ℃; Condensing reflux extracting 3h; Take out the membrane material after defat, with pure water, clean 8 times;
De-cell is processed: the membrane material after cleaning is inserted to (glucosan concentration is 5% in mass ratio) in the PBS buffer containing glucosan, with plasticity bag, seal, insert in cold isobaric static pressure machine, control temperature at 30 ℃, speed with 20Mpa/min is progressively forced into 500MPa, keep 5min, then decompression at the same rate, membrane material PBS buffer solution for cleaning taken out; Then the mixed solution being placed in containing the NaOH of 0.1M and the NaCl of 1M soaks 1h, proceeds in pure water and soaks 1h, 2 times so repeatedly; Finally use pure water rinsing, obtain collagem membrane;
Step 4, prepare back-up coat: it is negative pole that the one side of the collagem membrane matcoveredn that step 3 is obtained is laid in steel plate, collagem membrane upward, carbon-point connects positive source, and electrolyte is anhydrous calcium chloride and the sodium dihydrogen phosphate dihydrate of 0.25mol/L and the mixed solution of the ethanol that volume ratio is 20% that contains 0.42mol/L; Connect pulse direct current, electric current density is 2mA/cm
2voltage is 100mV, each 1 minute is a cycle for power on/off, 7 times repeatedly, on collagem membrane surface, form calcium phosphate coating, then after pure water cleans, be placed in mass volume ratio and be 4% NaOH solution and soak 3h, on collagem membrane, form hydroxyapatite back-up coat (thickness 0.1mm), after cleaning with pure water, through vacuum drying, obtain the biomembrane of bootable tissue regeneration.
The biofilm thickness that this example makes is 0.15mm, is applicable to deep layer osseous tissue defect repair, and calcium, phosphonium ion that back-up coat degraded produces are distributed in defect area, for the regeneration of freshman bone tissue, play guiding function, can accelerate the repair process that osseous tissue is damaged.This material is used for to the lower bag reparative experiment of Os Canitis, the discovery of drawing materials after January, area of new bone and normal bone are basically identical, and simple collagen membrane material has the intrusion of a large amount of connective tissues in bone defective region, affect the growth (referring to accompanying drawing 5) of new bone.
Claims (5)
1. a biomembrane for bootable tissue regeneration, is characterized in that take that collagem membrane is as support, and described collagem membrane is derived from animal membrane tissue, has natural three-D space structure; The protective layer that has aluminium oxide or zirconia particles formation in the one side of collagem membrane, thickness is 0.01 ~ 0.5 μ m; Another side has hydroxyapatite back-up coat, and thickness is 0.01 ~ 0.1mm.
2. the biomembranous method of preparation bootable tissue regeneration claimed in claim 1, is characterized in that, animal membrane tissue is obtained having the collagem membrane of n cell epimatrix structure through pretreatment; Adopt the method for electrophoresis to be evenly laid on a surface of collagem membrane zirconium oxide or alumina particle, form protective layer; By mixed solution electrolysis under pulse current effect of anhydrous calcium chloride and sodium dihydrogen phosphate dihydrate and ethanol, react with NaOH again, another surface in collagem membrane forms hydroxyapatite back-up coat, finally by obtaining the biomembrane of bootable tissue regeneration after dry, packing, sterilizing.
3. preparation method according to claim 2, is characterized in that, concrete steps comprise:
The pretreatment of step 1, film: after adopting pure water to clean up in animal membrane tissue, put into normal saline or PBS buffer oscillation cleaning, the speed with 0.5 ℃ ∕ min in 4 ℃ of environment of inserting is again cooled to-80 ℃, keep 12 hours, after taking-up, insert in the aqueous slkali of pH 7.5 ~ 9.0 and vibrate and thaw, concussion speed 80 ~ 100rpm; After cleaning, insert volumetric concentration and be in 75% alcoholic solution and soak 2 hours, then clean with pure water, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: the membrane material of acquisition is placed in to extraction vessel with absorbent cotton parcel, take acetone as defat reagent, heat and control temperature at 80 ~ 100 ℃, condensing reflux extracting 2 ~ 3 hours; Membrane material after defat is cleaned 4 ~ 8 times with pure water;
De-cell is processed: the membrane material after cleaning is inserted in the PBS buffer containing glucosan, with plasticity bag, seal, insert in cold isobaric static pressure machine, controlling temperature is 10 ~ 30 ℃, speed with 20Mpa/min is progressively forced into 500MPa, keep 5min, then decompression at the same rate, membrane material PBS buffer solution for cleaning taken out; The mixed solution being placed in again containing the NaOH of 0.1 ~ 0.25M and the NaCl of 1M soaks 1h, proceeds in pure water and soaks 1h, 2 times so repeatedly; Finally use pure water rinsing, obtain collagem membrane;
Step 3, prepare protective layer: the collagem membrane that step 2 is obtained is laid on steel plate as electrophoresis negative electrode, and zirconium oxide or alumina particle are mixed by the mass ratio of 1 ︰ 3 ~ 15 with deionized water, using after ultrasonic being uniformly dispersed as electrophoresis liquid; Carry out electrophoresis, on collagem membrane surface, form zirconium oxide or protective layer of alumina;
Step 4, prepare back-up coat: it is negative pole that the one side of the collagem membrane matcoveredn that step 3 is obtained is laid in steel plate, carbon-point connects positive source, and electrolyte is anhydrous calcium chloride and the sodium dihydrogen phosphate dihydrate of 0.003～0.25mol/L and the mixed solution of the ethanol that volume ratio is 20% that contains 0.004～0.42mol/L; Connect pulse direct current, electric current density is 2mA/cm
2, voltage is 1～100mV, the power-off again 1 minute of switching on after 1 minute 5 ~ 50 times so repeatedly, forms calcium phosphate coating on collagem membrane surface; Again after pure water cleans, be placed in mass volume ratio and be 0.4%～4% NaOH solution and soak 1 ~ 3h, on collagem membrane, form hydroxyapatite back-up coat; After cleaning with pure water, through vacuum drying, obtain the biomembrane of bootable tissue regeneration.
4. preparation method according to claim 3, is characterized in that, in the de-cell of step 2 is processed, the concentration of described glucosan is 4 ~ 10% in mass ratio.
5. preparation method according to claim 3, is characterized in that, if animal membrane is organized as cattle source property, in the de-cell of step 2 is processed, needs first in the NaOH of 1M solution, soak 1h and removes virus, then proceed in the PBS buffer containing glucosan and soak.
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