CN103721296B - Tissue regeneration-guidable biomembrane and preparation method thereof - Google Patents
Tissue regeneration-guidable biomembrane and preparation method thereof Download PDFInfo
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- Materials For Medical Uses (AREA)
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 tissue regeneration art during repair tissue defect; with it as physical barriers; perienchyma can be stoped to grow into defect area, and defect area protected as a kind of protecting film, be the tissue growth headspace of defective region simultaneously.
Early stage guide tissue regeneration film is nonabsorable material (taking polyethylene as representative), needs second operation to take out, add misery to patient during this kind of materials'use.Develop the absorbable material (as the degradable polymer such as polylactic acid, aliphatic polyester) of synthetic subsequently, its catabolite, in acid, can cause serious inflammatory reaction, be unfavorable for organization healing, the final effect that impact is repaired.In addition, also have some natural polymerses (as regeneration membrane prepared by chitosan and chitosan), its mechanical performance and water repelling property poor, degradation in vivo is very fast, can not coordinate the growth of cambium well.For addressing these problems, have and adopt the method for chemical crosslinking to improve the degradability of material, conventional cross-linking agent is glutaraldehyde, but the remaining toxicity adding membrane material of crosslinked rear reagent.
At present, people concentrate on xenogenesis natural biologic material emphasis.This kind of material source is in mammiferous membrane tissue, and through going antigen process to be prepared into laminar film, main component is collagen protein, therefore also claims collagem membrane.Collagem membrane degradation speed is very fast, and in use along with the carrying out of degraded, its toughness reduces, and subside to defect area gradually, defect space be cannot effectively keep, the growth of defective region cambium and final repairing effect have impact on, especially particularly serious in the larger situation of defect.
Disclose one in US Patent No. 058372 and there is double-deck collagem membrane, adopt cross-linking method to delay the degraded of film, and improve the mechanical performance of film.Remain because chemical crosslinking can increase reagent, and physical crosslinking (as heat cross-linking) is difficult to the mechanical performance that makes collagem membrane reach desirable, therefore still cannot solve collagem membrane degradation time too fast, easily form the problem of subsiding.Adopt acetone to improve the compactness extent of film compacted zone in Chinese patent application 201110145229.0, also fail to solve the mechanical performance problems of film.
Research finds, whether film and defect can fully fit, and directly affects final skeletonization effect, fits more abundant, the growing amount of new bone and Maturity better.Have clinical report, due to film smooth surface, make film fix insufficient and occur film displacement cause infect, final Endodontic failure.And membrane material in use, can not external environment be exposed to, otherwise the accelerated degradation of material can be caused, the risk that under increase 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, and this means the increase of surgery cost, causes economy and mental pressure to patient.
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, sufficient space can be provided for cambium, in use not easily there is shifting phenomena, and can osteanagenesis be induced.
The biomembrane of the bootable tissue regeneration that the present invention proposes, be take collagem membrane as support, described collagem membrane is derived from animal membrane tissue, has natural three-D space structure, can provide sufficient space for cambium; At the protective layer that the one side of collagem membrane has aluminium oxide or zirconia particles to be formed, thickness is 0.01 ~ 0.5 μm; Another side has hydroxyapatite back-up coat, and thickness is 0.01 ~ 0.1mm.The effect of protective layer causes accelerated degradation under avoiding collagem membrane to be directly exposed to external environment; The effect of hydroxyapatite back-up coat is that hydroxyapatite can form chemical bond with the osseous tissue of defect area and combines closely, and makes collagem membrane be fixed on osseous tissue place, displacement is less likely to occur; Meanwhile, protective layer and back-up coat all have the effect improving 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 the collagem membrane with natural extracellular matrix 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, hydroxyapatite back-up coat is formed, finally by the biomembrane obtaining bootable tissue regeneration after dry, packaging, sterilizing in another surface of collagem membrane.Concrete steps comprise:
The pretreatment of step one, film: after adopting pure water to clean up in animal membrane tissue, put into normal saline or PBS buffer (phosphate buffer) oscillation cleaning, insert again in 4 DEG C of environment and be cooled to-80 DEG C with the speed of 0.5 DEG C ∕ min, keep 12 hours, insert vibration in the aqueous slkali of pH 7.5 ~ 9.0 after taking-up to thaw, concussion speed 80 ~ 100rpm; Insert after cleaning in the alcoholic solution of 75% volume ratio and soak 2 hours, then with pure water cleaning, obtain membrane material;
Wherein, to the freeze thawing treatment of animal membrane tissue, make to form ice crystal in cell, 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, quick freezing can be avoided to cause ice crystal extracellular matrix structural damage in cell.
The preparation of step 2, collagem membrane
Ungrease treatment: by the membrane material absorbent cotton of acquisition parcel be placed in extraction vessel, take acetone as degreasing agent, heating and control temperature at 80 ~ 100 DEG C, condensing reflux extracting 2 ~ 3 hours; By the pure water cleaning 4 ~ 8 times of the membrane material after defat;
De-cell process: the membrane material after cleaning is inserted in the PBS buffer containing glucosan (glucosan concentration is 4 ~ 10% in mass ratio), seal with plasticity bag, insert in cold isobaric static pressure machine, control temperature is 10 ~ 30 DEG C, progressively 500MPa is forced into the speed of 20Mpa/min, keep 5min, then reduce pressure at the same rate, take out membrane material PBS buffer solution for cleaning; The mixed solution being placed in the NaCl of NaOH and 1M containing 0.1 ~ 0.25M again 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, need in the NaOH solution of 1M, first soak 1h and remove virus, then proceed in the PBS buffer containing glucosan and soak;
Defat adopts soxhlet extraction methods, with degreasing agent to the continuous extracting process of the liposoluble substance in membrane material, can control below 1% by fat in material at short notice; In addition, this method has saved degreasing time, and can reclaim reagent, reduces defat cost.
De-cell applies pressure by the method for liquid pressing to membrane tissue, can the cell membrane of direct disorganize cell, and release cellular component, is separated with extracellular matrix; It is reported; in the pressure environment being greater than 220MPa; pressurization can make the freezing point of water rise; now can generate ice crystal (http://zhidao.baidu.com/question/118684849.html qq-pf-to=pcqq.c2c) in cell; thus destruction cellularity; by the adjustment of pressure, temperature and intermediate medium concentration, both reached de-cell effect, can also the natural structure of Cell protection epimatrix.
Finally with the mixed solution of NaOH and NaCl, cell is fully dewatered, then be placed in pure water and make that cell is swelling to break, circulating 2 times can cell debris thoroughly in eluting tissue.
Step 3, prepare protective layer: collagem membrane step 2 obtained is laid on steel plate as electrophoresis negative electrode, zirconium oxide or alumina particle is mixed with the mass ratio of deionized water by 1 ︰ 3 ~ 15, using ultrasonic disperse evenly after as electrophoresis liquid; Carry out electrophoresis (DC voltage 0.5 ~ 10V), form zirconium oxide or protective layer of alumina on collagem membrane surface; Its thickness realizes by the adjustment of voltage and electrophoretic liquid concentration, voltage and electrophoretic liquid concentration higher, the thickness of protective layer is larger;
Usually, use collagem membrane to carry out closing 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 isolate, avoid accelerated degradation, reduce the infection risk of cambium simultaneously; Zirconium oxide and aluminium oxide belong to bio-inert material, stable in properties, have good comprcssive strength, also improve the mechanical performance of collagem membrane to a certain extent.
Step 4, prepare back-up coat: it is negative pole that the one side of collagem membrane matcoveredn step 3 obtained is laid in steel plate, carbon-point connects positive source, and electrolyte is contain the mixed solution that the anhydrous calcium chloride of 0.004 ~ 0.42mol/L and the sodium dihydrogen phosphate dihydrate of 0.003 ~ 0.25mol/L and volume ratio are the ethanol of 20%; Connect pulse direct current, electric current density is 2mA/cm
2, voltage is 1 ~ 100mV, and be energized after 1 minute power-off again 1 minute, 5 ~ 50 times so repeatedly, forms calcium phosphate coating on collagem membrane surface; Again after pure water cleaning, be placed in NaOH solution immersion 1 ~ 3h that mass volume ratio is 0.4% ~ 4%, collagem membrane is formed hydroxyapatite back-up coat; After pure water cleaning, obtain the biomembrane of bootable tissue regeneration through vacuum drying; 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 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, and calcium, the phosphonium ion of generation can form stable chemical bond with the calcium in surrounding bone tissue, phosphonium ion, and therefore, back-up coat can make collagem membrane and osseous tissue fit tightly, and is not easily shifted; In addition, calcium, phosphonium ion are the main components of skeleton and tooth, can be absorbed, inducting osseous tissue regeneration by body.
Bootable tissue regeneration biomembrane prepared by the present invention; for existing collagen membrane material bad mechanical property, easily subside, easily displacement and can not the shortcomings such as external environment be exposed to; by forming the coating of zirconium oxide or aluminium oxide bio-inert material on the surface of collagem membrane; collagem membrane can not only be protected from the impact of external environment condition, also largely improve the mechanical performance of collagem membrane.Form hydroxyapatite back-up coat by the another side at collagem membrane, the Calcium and phosphorous component in hydroxyapatite dissolves afterwards and osseous tissue forms chemical bond, collagem membrane and osseous tissue can be made to combine closely, be not easily shifted; Meanwhile, hydroxyapatite can induce the growth of osseous tissue alive, accelerates reparation process.
Carry out tensile break strength detection by the bootable tissue regeneration biomembrane prepared the present invention and existing simple collagen film, comparing result shows, and the biomembranous mechanical strength of the present invention on average improves about 7.5 times.And the animal repairing effect of bag contrasts under these two kinds of films being carried out image processing, rabbit Ulna injuries, the filling of dog teeth socked, Os Canitis, 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 subside, biomembrane can be fitted tightly at restoring area by back-up coat, be shifted, the space of cambium can be ensured in vivo for a long time; Having data to show the mankind is 17w in the osteoplastic normal physiological cycle, visible biomembrane of the present invention has good mechanical strength, can to a greater degree for new bone growth provide good environment, the height of New born formation is consistent with normal bone, is obviously better than the repairing effect contrasted.
Accompanying drawing explanation
Accompanying drawing 1 is the tensile break strength testing result comparison diagram of the present invention's bootable tissue regeneration biomembrane and existing simple collagen film.In figure, a is the testing result of collagem membrane, and in figure, b is the biomembranous testing result of the present invention.The biomembranous mechanical strength of the present invention improves 7.5 times than existing collagem membrane.
Accompanying drawing 2 is that the Histological results that the present invention's bootable tissue regeneration biomembrane and existing simple collagen film are repaired for image processing contrasts photo.Figure a is the repairing effect photo of collagem membrane, can find out that depression appears in defect area, and the collagem membrane that recess is degradable, illustrates that simple collagem membrane degraded is too fast, can not support above defect area, and subside to defect area, have impact on area of new bone and generate; Figure b is the biomembranous repairing effect photo of the present invention; its defect area is filled with area of new bone fibrous tissue, without depressed phenomenon, illustrates that biomembrane of the present invention stably can support the top of defect area; defective region is played a protective role, ensures the quick growth of new bone.
Accompanying drawing 3 is that the X-ray film observed result that the present invention's bootable tissue regeneration biomembrane and existing simple collagen film are repaired for rabbit Ulna injuries contrasts photo.Figure a is the effect photo that collagem membrane is repaired, and can find out that defect area has significant depressions with surrounding normal bone photo ratio, proves that simple collagem membrane degraded is too fast further, can not support above defect area, and subside to defect area, have impact on the generation of area of new bone; Figure b is the effect photo of Biomembran repair of the present invention, and the height of its defect area and surrounding normal bone is basically identical, proves that biomembrane can stably support above defect area, and for the generation of new bone provides adequate space, height and the normal bone of new bone are basically identical.
Accompanying drawing 4 is that the X-ray film observed result that the present invention's bootable tissue regeneration biomembrane and existing simple collagen film are filled for dog teeth socked contrasts photo.Figure a is the repairing effect of 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.Illustrate the degraded of simple collagem membrane too fast cause subside, be important influence factor to new bone growth.
Accompanying drawing 5 is that the Histological results that the present invention's bootable tissue regeneration biomembrane and existing simple collagen film are repaired for bag under Os Canitis contrasts photo.Figure a is the repairing effect of collagem membrane, and can find out, clear area has appearred in defect area, there is a large amount of connective tissues to invade above it, illustrate that the degraded of simple collagem membrane too fast causing subsides, have impact on New born formation, also can lose inhibition, cause connective tissue to invade, form long junctional epithelium; Figure b is the biomembranous repairing effect of the present invention, and can find out, area of new bone fills up defective region, and repairing effect is good, proves that biomembrane of the present invention can ensure the quick growth of new bone, basically identical with normal bone.
Detailed description of the invention
Below in conjunction with example, technical solution of the present invention and effect are described further.The animal membrane tissue adopted in example is purchased from slaughterhouse, after removing the impurity such as the subsidiary fat of membrane tissue, adopts PBS buffer (phosphate buffer) or normal saline cleaning removing bloodstain, then cleans with pure water.
The cold isobaric static pressure machine adopted in example is manufactured by Zhong Ping Science and Technology Ltd. of Taiyuan City, operating room size 230 × 300mm, maximum working pressure 600MPa, and 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.
embodiment 1,
Take bovine pericardium as raw material, prepare guide tissue regeneration biomembrane according to the following steps.
The pretreatment of step one, film: by bovine pericardium with after pure water cleaning, put into normal saline oscillation cleaning, then insert in 4 DEG C of environment and be cooled to-80 DEG C with the speed of 0.5 DEG C ∕ min, keep 12h, insert vibration in the sodium carbonate liquor of pH8 after taking-up to thaw, concussion speed is 90rpm; Insert after cleaning in the alcoholic solution of 75% volume ratio and soak 2h, then with pure water cleaning, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: by the membrane material absorbent cotton of acquisition parcel be placed in extraction vessel, take acetone as degreasing agent, heating and control temperature at 85 DEG C; Condensing reflux extracting 2h; Take out the membrane material after defat, clean 4 times with pure water;
De-cell process: the membrane material after cleaning is first soaked 1h in the NaOH solution of 1M, insert again (glucosan concentration is 10% in mass ratio) in the PBS buffer containing glucosan, seal with plasticity bag, insert in cold isobaric static pressure machine, control temperature, at 10 DEG C, is progressively forced into 500MPa with the speed of 20Mpa/min, keeps 5min, reduce pressure at the same rate again, take out membrane material PBS buffer solution for cleaning; Then the mixed solution being placed in the NaCl of NaOH and 1M containing 0.25M soaks 1h, proceeds in pure water and soaks 1h, 2 times so repeatedly; Finally with pure water cleaning, obtain collagem membrane;
Step 3, prepare protective layer: the collagem membrane obtained is laid on steel plate as electrophoresis negative electrode, is mixed with the mass ratio of deionized water by 1 ︰ 15 by zirconia particles, using ultrasonic disperse evenly after as electrophoresis liquid; Carry out electrophoresis (DC voltage 1.0V), in electrophoresis liquid, zirconia particles is gradually to movable cathode, is evenly laid in the surface of collagem membrane, forms zirconium oxide protective layer (thickness 0.05 μm);
Step 4, prepare back-up coat: it is negative pole that the one side of collagem membrane matcoveredn step 3 obtained is laid in steel plate, collagem membrane upward, carbon-point connects positive source, and electrolyte is the mixed solution of anhydrous calcium chloride containing 0.008mol/L and 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, power on/off is a cycle in each 1 minute, 50 times repeatedly, forms calcium phosphate (chemical formula CaHPO on collagem membrane surface
42H
2o) coating, then through pure water cleaning after, be placed in mass volume ratio be 0.8% NaOH solution soak 1h, collagem membrane is formed hydroxyapatite (chemical formula Ca
10(PO
4)
6(OH)
2) back-up coat (thickness 0.015mm), after pure water cleaning, obtain the biomembrane of bootable tissue regeneration through vacuum drying.
The biofilm thickness that this example obtains is 0.12mm, and tensile break strength is 190 ~ 210MPa, and compared with simple collagen membrane material, mechanical performance improves about 7.5 times (see accompanying drawing 1).This material is applicable to the defect of osseous tissue among a small circle that a variety of causes causes, and can stably cover above defective region, shifting phenomena can not occur in repair process.
embodiment 2,
With pig peritoneum for raw material, prepare guide tissue regeneration biomembrane according to the following steps.
The pretreatment of step one, film: after being cleaned up by pig peritoneum pure water, put into normal saline oscillation cleaning; Insert in 4 DEG C of environment again and be cooled to-80 DEG C with the speed of 0.5 DEG C of ∕ min, keep 12h, insert vibration in the sodium hydroxide solution of pH8.5 after taking-up and thaw, concussion speed is 80rpm; Insert after cleaning in the alcoholic solution of 75% volume ratio and soak 2h, then with pure water cleaning, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: by the membrane material absorbent cotton of acquisition parcel be placed in extraction vessel, take acetone as degreasing agent, heating and control temperature at 90 DEG C; Condensing reflux extracting 3h; Take out the membrane material after defat, clean 8 times with pure water;
De-cell process: the membrane material after cleaning is inserted in the PBS buffer containing glucosan (glucosan concentration is 6% in mass ratio), seal with plasticity bag, insert in cold isobaric static pressure machine, control temperature is at 20 DEG C, progressively 500MPa is forced into the speed of 20Mpa/min, keep 5min, then reduce pressure at the same rate, take out membrane material PBS buffer solution for cleaning; Then the mixed solution being placed in the NaCl of NaOH and 1M containing 0.15M soaks 1h, proceeds in pure water and soaks 1h, 2 times so repeatedly; Finally with pure water cleaning, obtain collagem membrane;
Step 3, prepare protective layer: the collagem membrane obtained being laid on steel plate is electrophoresis negative electrode, is mixed by zirconia particles with the mass ratio of deionized water by 1 ︰ 10, using ultrasonic disperse evenly after as electrophoresis liquid; Carry out electrophoresis (DC voltage 5.0V), in electrophoresis liquid, zirconia particles is gradually to movable cathode, is evenly laid in the surface of collagem membrane, forms zirconium oxide protective layer (thickness 0.25 μm);
Step 4, prepare back-up coat: it is negative pole that the one side of collagem membrane matcoveredn step 3 obtained is laid in steel plate, collagem membrane upward, carbon-point connects positive source, the mixed solution of electrolyte to be the sodium dihydrogen phosphate dihydrate of anhydrous calcium chloride containing 0.021mol/L and 0.013mol/L and volume ratio the be ethanol of 20%; Connect pulse direct current, electric current density is 2mA/cm
2voltage is 5mV, power on/off is a cycle in each 1 minute, 40 times repeatedly, form calcium phosphate coating on collagem membrane surface, then after pure water cleaning, be placed in mass volume ratio be 2% NaOH solution soak 1h, collagem membrane is formed hydroxyapatite back-up coat (thickness 0.05mm), after pure water cleaning, obtains the biomembrane of bootable tissue regeneration through vacuum drying.
The biofilm thickness that this example obtains is 0.15mm.Its enough thick protective layer adds the stability of material when exposing, and is very suitable for applying in the oral cavity.This material is used for dog teeth socked and fills reparative experiment, take not exclusively to close mode when gingiva is sewed up, fraction membrane material is exposed in oral cavity, to draw materials after January observation, find that the area of new bone height of this material group still can reach the effect consistent with normal bone, and simple collagen membrane material group can observe significantly local bone amount growth not enough (see accompanying drawing 2) at field of operation.
embodiment 3,
With Cor Sus domestica peplos for raw material, prepare guide tissue regeneration biomembrane according to the following steps.
The pretreatment of step one, film: after Cor Sus domestica peplos pure water is cleaned up, put into normal saline oscillation cleaning, then insert in 4 DEG C of environment and be cooled to-80 DEG C with the speed of 0.5 DEG C ∕ min, keep 12h, insert vibration in the solution of potassium carbonate of pH8.5 after taking-up to thaw, concussion speed is 85rpm; Insert after cleaning in the alcoholic solution of 75% volume ratio and soak 2h, then with pure water cleaning, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: being wrapped up and be placed in extraction vessel by the membrane material absorbent cotton of acquisition, take acetone as degreasing agent, heating and control temperature at 90 DEG C; Condensing reflux extracting 2.5h; Take out the membrane material after defat, clean 8 times with pure water;
De-cell process: the membrane material after cleaning is inserted in the PBS buffer containing glucosan (glucosan concentration is 5% in mass ratio), seal with plasticity bag, insert in cold isobaric static pressure machine, control temperature is at 30 DEG C, progressively 500MPa is forced into the speed of 20Mpa/min, keep 5min, then reduce pressure at the same rate, take out membrane material PBS buffer solution for cleaning; Then the mixed solution being placed in the NaCl of NaOH and 1M containing 0.2M 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 obtained is laid on steel plate as electrophoresis negative electrode, is mixed with the mass ratio of deionized water by 1 ︰ 15 by alumina particle, using ultrasonic disperse evenly after as electrophoresis liquid; Carry out electrophoresis (DC voltage 8.0V), in electrophoresis liquid, alumina particle is gradually to movable cathode, is evenly laid in the surface of collagem membrane, forms protective layer of alumina (thickness 0.3 μm);
Step 4, prepare back-up coat: it is negative pole that the one side of collagem membrane matcoveredn step 3 obtained is laid in steel plate, collagem membrane upward, carbon-point connects positive source, the mixed solution of electrolyte to be the sodium dihydrogen phosphate dihydrate of anhydrous calcium chloride containing 0.136mol/L and 0.081mol/L and volume ratio the be ethanol of 20%; Connect pulse current, electric current density is 2mA/cm
2voltage is 30mV, power on/off is a cycle in each 1 minute, 8 times repeatedly, form calcium phosphate coating on the surface of collagem membrane, then after pure water cleaning, be placed in mass volume ratio be 2% NaOH solution soak 2h, collagem membrane is formed hydroxyapatite back-up coat (thickness 0.03mm), after pure water cleaning, obtains the biomembrane of bootable tissue regeneration through vacuum drying.
The biofilm thickness that this example obtains is 0.15mm, and be applicable to shallow-layer osseous tissue defect repair, protective layer and back-up coat add stability and the plasticity of material, are supported on defective region with making material settling out.This material is used for the reparation of rabbit Ulna injuries, 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 caused by (see accompanying drawing 3).
embodiment 4,
With cattle peritoneum for raw material, prepare guide tissue regeneration biomembrane according to the following steps.
The pretreatment of step one, film: by cattle peritoneum with after pure water cleaning, put into PBS buffer oscillation cleaning; Insert in 4 DEG C of environment again and be cooled to-80 DEG C with the speed of 0.5 DEG C of ∕ min, keep 12h, insert vibration in the potassium bicarbonate solution of pH8 after taking-up and thaw, concussion speed is 100rpm; Insert after cleaning in the alcoholic solution of 75% volume ratio and soak 2h, then with pure water cleaning, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: by the membrane material absorbent cotton of acquisition parcel be placed in extraction vessel, take acetone as degreasing agent, heating and control temperature at 95 DEG C; Condensing reflux extracting 2h; Take out the membrane material after defat, clean 5 times with pure water;
De-cell process: the membrane material after cleaning is soaked 1h in the NaOH aqueous solution of 1M, then insert (glucosan concentration is 4% in mass ratio) in the PBS buffer containing glucosan, seal with plasticity bag, insert in cold isobaric static pressure machine, control temperature, at 15 DEG C, is progressively forced into 500MPa with the speed of 20Mpa/min, keeps 5min, reduce pressure at the same rate again, take out membrane material PBS buffer solution for cleaning; Then the mixed solution being placed in the NaCl of NaOH and 1M containing 0.15M 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 obtained being laid on steel plate is electrophoresis negative electrode, is mixed by alumina particle with the mass ratio of deionized water by 1 ︰ 8, using ultrasonic disperse evenly after as electrophoresis liquid; Carry out electrophoresis (DC voltage 3.0V), in electrophoresis liquid, alumina particle is gradually to movable cathode, is evenly laid in the surface of collagem membrane, forms protective layer of alumina (thickness 0.25 μm);
Step 4, prepare back-up coat: it is negative pole that the one side of collagem membrane matcoveredn step 3 obtained is laid in steel plate, collagem membrane upward, carbon-point connects positive source, the mixed solution of electrolyte to be the sodium dihydrogen phosphate dihydrate of anhydrous calcium chloride containing 0.21mol/L and 0.125mol/L and volume ratio the be ethanol of 20%; Connect pulse direct current, electric current density 2mA/cm
2voltage 50mV, power on/off is a cycle in each 1 minute, 11 times repeatedly, form calcium phosphate coating on collagem membrane surface, then after pure water cleaning, be placed in mass volume ratio be 3% NaOH solution soak 2.5h, collagem membrane is formed hydroxyapatite back-up coat (thickness 0.08mm), after pure water cleaning, obtains the biomembrane of bootable tissue regeneration through vacuum drying.
The biofilm thickness 0.2mm that this example is obtained, is applicable to the reparation of osseous tissue defect on a large scale, forms sufficient chemical bond, can be close to above defective region, ensure the growing space of new bone between its back-up coat and osseous tissue surface.This material is used for image processing reparation, draw materials after January 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 subsided owing to fixing insufficient causing, bone amount growth not enough (see accompanying drawing 4).
embodiment 5,
Take trees-Osima jacoti, Osima excavata as raw material, prepare guide tissue regeneration biomembrane according to the following steps.
The pretreatment of step one, film: by trees-Osima jacoti, Osima excavata with after pure water cleaning, put into PBS buffer oscillation cleaning; Insert in 4 DEG C of environment again and be cooled to-80 DEG C with the speed of 0.5 DEG C of ∕ min, keep 12h, insert vibration in the sodium radio-phosphate,P-32 solution of pH8.5 after taking-up and thaw, concussion speed is 85rpm; Insert after cleaning in the alcoholic solution of 75% volume ratio and soak 2h, then with pure water cleaning, obtain membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: by the membrane material absorbent cotton of acquisition parcel be placed in extraction vessel, take acetone as degreasing agent, heating and control temperature at 90 DEG C; Condensing reflux extracting 3h; Take out the membrane material after defat, clean 8 times with pure water;
De-cell process: the membrane material after cleaning is inserted in the PBS buffer containing glucosan (glucosan concentration is 5% in mass ratio), seal with plasticity bag, insert in cold isobaric static pressure machine, control temperature is at 30 DEG C, progressively 500MPa is forced into the speed of 20Mpa/min, keep 5min, then reduce pressure at the same rate, take out membrane material PBS buffer solution for cleaning; Then the mixed solution being placed in the NaCl of NaOH and 1M containing 0.1M 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 obtained is laid on steel plate as electrophoresis negative electrode, is mixed with the mass ratio of deionized water by 1 ︰ 3 by alumina particle, using ultrasonic disperse evenly after as electrophoresis liquid; Carry out electrophoresis (DC voltage 10V), in electrophoresis liquid, alumina particle is gradually to movable cathode, is evenly laid in the surface of collagem membrane, forms protective layer of alumina (thickness 0.5 μm);
Step 4, prepare back-up coat: it is negative pole that the one side of collagem membrane matcoveredn step 3 obtained is laid in steel plate, collagem membrane upward, carbon-point connects positive source, the mixed solution of electrolyte to be the sodium dihydrogen phosphate dihydrate of anhydrous calcium chloride containing 0.42mol/L and 0.25mol/L and volume ratio the be ethanol of 20%; Connect pulse direct current, electric current density is 2mA/cm
2voltage is 100mV, power on/off is a cycle in each 1 minute, 7 times repeatedly, form calcium phosphate coating on collagem membrane surface, then after pure water cleaning, be placed in mass volume ratio be 4% NaOH solution soak 3h, collagem membrane is formed hydroxyapatite back-up coat (thickness 0.1mm), after pure water cleaning, obtains the biomembrane of bootable tissue regeneration through vacuum drying.
The biofilm thickness that this example obtains is 0.15mm, is applicable to deep layer osseous tissue defect repair, and the calcium that back-up coat degraded produces, phosphonium ion are distributed in defect area, for guiding function is played in the regeneration of freshman bone tissue, can accelerate the repair process of osseous tissue defect.This material is used for bag reparative experiment under Os Canitis, discovery of drawing materials after January, area of new bone and normal bone basically identical, and simple collagen membrane material has the intrusion of a large amount of connective tissue in Cranial defect district, affect the growth (see accompanying drawing 5) of new bone.
Claims (5)
1. a biomembrane for bootable tissue regeneration, it is characterized in that taking collagem membrane as support, described collagem membrane is derived from animal membrane tissue, has natural three-D space structure; At the protective layer that the one side of collagem membrane has aluminium oxide or zirconia particles to be formed, thickness is 0.01 ~ 0.5 μm; Another side has hydroxyapatite back-up coat, and thickness is 0.01 ~ 0.1mm.
2. prepare the biomembranous method of bootable tissue regeneration according to claim 1, it is characterized in that, animal membrane tissue is obtained the collagem membrane with natural extracellular matrix 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, hydroxyapatite back-up coat is formed, finally by the biomembrane obtaining bootable tissue regeneration after dry, packaging, sterilizing in another surface of collagem membrane.
3. preparation method according to claim 2, is characterized in that, concrete steps comprise:
The pretreatment of step one, film: after adopting pure water to clean up in animal membrane tissue, put into normal saline or PBS buffer oscillation cleaning, insert again in 4 DEG C of environment and be cooled to-80 DEG C with the speed of 0.5 DEG C ∕ min, keep 12 hours, insert vibration in the aqueous slkali of pH 7.5 ~ 9.0 after taking-up to thaw, concussion speed 80 ~ 100rpm; Inserting volumetric concentration after cleaning is soak 2 hours in the alcoholic solution of 75%, then with pure water cleaning, obtains membrane material;
The preparation of step 2, collagem membrane
Ungrease treatment: by the membrane material absorbent cotton of acquisition parcel be placed in extraction vessel, take acetone as degreasing agent, heating and control temperature at 80 ~ 100 DEG C, condensing reflux extracting 2 ~ 3 hours; By the pure water cleaning 4 ~ 8 times of the membrane material after defat;
De-cell process: the membrane material after cleaning is inserted in the PBS buffer containing glucosan, seal with plasticity bag, insert in cold isobaric static pressure machine, control temperature is 10 ~ 30 DEG C, progressively 500MPa is forced into the speed of 20Mpa/min, keep 5min, then reduce pressure at the same rate, take out membrane material PBS buffer solution for cleaning; The mixed solution being placed in the NaCl of NaOH and 1M containing 0.1 ~ 0.25M again 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: collagem membrane step 2 obtained is laid on steel plate as electrophoresis negative electrode, zirconium oxide or alumina particle is mixed with the mass ratio of deionized water by 1 ︰ 3 ~ 15, using ultrasonic disperse evenly after as electrophoresis liquid; Carry out electrophoresis, form zirconium oxide or protective layer of alumina on collagem membrane surface;
Step 4, prepare back-up coat: it is negative pole that the one side of collagem membrane matcoveredn step 3 obtained is laid in steel plate, carbon-point connects positive source, and electrolyte is contain the mixed solution that the anhydrous calcium chloride of 0.004 ~ 0.42mol/L and the sodium dihydrogen phosphate dihydrate of 0.003 ~ 0.25mol/L and volume ratio are the ethanol of 20%; Connect pulse direct current, electric current density is 2mA/cm
2, voltage is 1 ~ 100mV, and be energized after 1 minute power-off again 1 minute, 5 ~ 50 times so repeatedly, forms calcium phosphate coating on collagem membrane surface; Again after pure water cleaning, be placed in NaOH solution immersion 1 ~ 3h that mass volume ratio is 0.4% ~ 4%, collagem membrane is formed hydroxyapatite back-up coat; After pure water cleaning, obtain the biomembrane of bootable tissue regeneration through vacuum drying.
4. preparation method according to claim 3, is characterized in that, in the de-cell process of step 2, 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 process of step 2, needs in the NaOH solution of 1M, first soak 1h and removes virus, then proceeds in the PBS buffer containing glucosan and soak.
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| CN108525009A (en) * | 2018-05-22 | 2018-09-14 | 北京清源伟业生物组织工程科技有限公司 | A kind of preparation method of Acellular trachea matrix material |
| CN108853586A (en) * | 2018-06-29 | 2018-11-23 | 四川大学 | A modified tea polyphenol collagen film and its application |
| CN111001040A (en) * | 2019-12-24 | 2020-04-14 | 北京晞思迈特生物科技有限公司 | Preparation method of extracellular matrix material of biological tissue |
| CN114425104B (en) * | 2021-12-21 | 2023-03-03 | 中国人民解放军空军军医大学 | A drug-loaded bone conduction/induction composite structure and its preparation method and application |
| CN116306034B (en) * | 2023-05-18 | 2023-07-21 | 成都康盛科泰生物技术有限公司 | Construction method and application of universal biomembrane thickness distribution model |
| 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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