CN104524634A - Preparation method of tissue repair material - Google Patents
Preparation method of tissue repair material Download PDFInfo
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- CN104524634A CN104524634A CN201410780482.7A CN201410780482A CN104524634A CN 104524634 A CN104524634 A CN 104524634A CN 201410780482 A CN201410780482 A CN 201410780482A CN 104524634 A CN104524634 A CN 104524634A
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Abstract
The invention relates to a preparation method of a tissue repair material. The preparation method comprises the following steps: carrying out pre-treatment; vibrating in an organic solvent to degrease; deactivating a virus with an ethanol solution; removing cells by high and low osmotic solutions; swelling in an acidic or alkaline liquid; and quickly lyophilizing and sterilizing. The thickness of the tissue repair material is increased by 2-4 times, and the material is loose and porous, small in change of mechanical property and degradation property and good in cell removal effect and has relatively high biosafety and biocompatibility. Due to the structural characteristics of proper thickness and looseness and porosity, the material is suitable for damage repair for filling damaged parts required by repair such as cartilage injury, skin and soft tissue defect, gingival recession and the like. Animal experiments verify that the repaired region after eight weeks has no depressed phenomena and is fully replaced by regenerated tissues. Moreover, the regenerated tissue and autologous tissue are healed well and are free of separating phenomenon, which verifies that the material can provide sufficient three-dimensional space for cell proliferation and crawl, so that the repaired region has good integration degree and filling degree, thereby realizing tissue repair and regeneration.
Description
Technical field
The invention belongs to tissue engineering medical biomaterial technical field, be specifically related to a kind of preparation method of tissue renovation material.
Background technology
Tissue, the forfeiture of organ or dysfunction be faced by human health institute the most frequently, one of the most serious problem.According to statistics, the U.S. have every year people up to a million suffer from various tissue, organ lose or dysfunction, need every year to carry out operation 8,000,000 times, year cost exceed 40,000,000,000 dollars.In recent years, along with the development of organizational project and regenerative medicine, the repairing and treating of tissue, organ has had many breakthroughs, and wherein xenogenesis takes off cell biological membrane material because of the natural structure of its uniqueness, the degradation property synchronous with tissue regeneration and obtains more concerns.Lot of domestic and international research institution and company take off cell biological membrane material to xenogenesis and carry out basic research for many years and applied research, and it is effective that its clinical practice for many years also show material safety.
In foreign material source, study and more comprise corium, submucous layer of small intestine, bladder, peritoneum, fascia, pericardium, cardiac valve etc.Because foreign material itself is containing a large amount of cell, hereditary material, fat, directly apply to people and know from experience the strong immunoreation of initiation, so need through defat, de-cell, go the process such as antigen, retain extracellular matrix components to greatest extent, realize the function of its guide tissue regeneration and reparation.Therefore, the processing procedure of material has important function to its result of use.
Method for removing cells comprises physics, chemistry and biology method.Wherein physical method adopts freeze thawing, pressure, can kill cell and but cell component and hereditary material can not be transported out.Chemical method comprises 1) acid-base method, acid or the process of alkali can cause lysis, cause or the hydrolytic degradation of catalytic activity molecule, reach cell free object, but the long duration of action of acid or alkali can produce heavy damage to material; 2) high hypisotonic solution is adopted, osmotic pressure principle smudge cells is utilized to carry out cell free method, be considered to destroy minimum method for removing cells to the molecular structure of substrate, but the effect of its independent role is not thorough, after process, has a small amount of cell debris and DNA to remain; 3) the cell free method of detergent is adopted, comprise ionic detergent (as SDS) and non-ionic detergent (as TritonX-100), and amphoteric ion type detergent (as CHAPS), detergent can dissolved cell film, by DNA eluting out, but detergent destroys the protein in Sum decomposition material simultaneously, simultaneously not easy cleaning, increase cost on the one hand, cause the production cycle long, reagent remains the safety and the compatibility that can affect material on the other hand; 4) adopt the cell free method of organic reagent, comprise the application of alcohols and ketone etc.Biological method mainly comprises the application of enzyme and chelate, and its existence destruction material structure and reagent remain the problem of more difficult cleaning.
Facts have proved, being used alone of said method all cannot reach desirable de-cell effect, therefore usually selects two or more method couplings or used in combination.US Patent No. 6206931 carries out de-cell process with peracetic acid-ethanol-water solution combined effect to trees-Osima jacoti, Osima excavata, less to the structure influence of material; And the present inventor records experiment by the document, still can observe the existence of intact cell configuration after proving to adopt the method process, this will produce potential immunological rejection and inflammatory reaction risk.US Patent No. 5387278 utilizes acid treatment after first alkali, de-cell effect can be reached, these two kinds strong method for removing cells superpose use for a long time, irreversible destruction can be caused to material structure, cause that material degradation is too fast, mechanical property is low, the synchronous degradation with tissue regeneration can not be reached, cannot be applied to clinical; In addition, the method adopts acetone defat, organic reagent dehydrate after de-cell, there is the risk that organic reagent is residual; So the feasibility that the method is amplified from technique and science still have room for improvement.The series of products of TUTOGEN company of the U.S. adopt hydrogen peroxide to carry out de-cell process, and processing procedure can produce a large amount of foam, is not easy to cleaning, easily causes the safety of reagent remaining influence material.US Patent No. 5413798 carries out de-cell with diluted alkaline, dilute sodium chloride, chelating agent, also there is the risk that reagent is residual, can cause stronger toxic reaction in vivo.
Clinical research finds, in some surgical operations, when material thickness is thinner, when fully can not fill damage location height, can cause and repair position depression or surface irregularity, have a strong impact on its repairing effect and functional rehabilitation.In addition, in Clinical practice process, thinner material, because of easily shrinkage after rehydration or absorption tissue fluid, sticks on operating equipment, brings inconvenience to operation technique.And the thickness of biomaterial is by the restriction of raw material itself, as the material thicknesses such as pericardium, peritoneum, pericystium are only 0.1 ~ 0.4mm; For making up this defect, the general mode of biomaterial superposition or compound other materials that adopts increases thickness, so inevitably causes cost to increase and production declining, also brings in use procedure the risk occurring layers of material, the recovery effects of impact tissue.
Summary of the invention
For the deficiency that prior art exists, the object of this invention is to provide a kind of preparation method of tissue renovation material, make the tissue renovation material of gained take off cell thoroughly, to remain without residual, the DNA of intact cell configuration and cell debris and reagent to remain risk low, thickness increases by 2 ~ 4 times, there is good mechanical property and anti-degradability, the requirement of clinical practice can be met.
The preparation method of tissue renovation material that the present invention proposes, comprises pre-treatment, defat, inactivation of virus, de-cell, material is swollen, the step of lyophilizing and sterilizing, specifically comprises:
Step one, pre-treatment: the membrane tissue that mammal is originated is laid in flat board, remove attachment fat, connective tissue and edge breakage tissue, be washed to afterwards without color, obtain biomembrane material; Described mammal comprise pig, cattle, sheep any one; Described membrane tissue comprises any one in bladder base film, peritoneum, fascia, pericardium, cardiac valve;
Step 2, defat: be placed in after organic solvent shakes defat 2 ~ 10h by gained biomembrane material and change liquid, repeat defat 2 ~ 4 times; Be washed to odorlessness; Described organic solvent be acetone, isopropyl alcohol, methanol and chloroform mixed liquor, normal hexane, ethyl acetate, ethanol or petroleum ether any one; Wherein volume ratio 1 ︰ 0.25 ~ 4 of methanol and chloroform mixed liquor.
Wherein, adopt organic solvent degreasing process, and be placed on whole technique comparatively before position, through subsequent treatment with repeatedly clean, organic solvent residual can be removed to greatest extent, the safety of guarantee material.
Step 3, sterilization: the alcoholic solution biomembrane material after defat being placed in volumetric concentration 70 ~ 75% soaks at least 2h, reaches disinfection effect, be washed to without alcohol taste;
Step 4, de-cell: the biomembrane material after sterilization is placed in de-cell hyperosmotic solution concussion 10 ~ 60min, then proceed to concussion 10 ~ 60min in de-cell hypisotonic solution, 2 ~ 5 times so repeatedly; Wherein, de-cell hyperosmotic solution is in the NaCl solution of 0.5 ~ 5M, be added with the HCl of 0.01 ~ 0.5M or the NaOH of 0.05 ~ 1M; De-cell hypisotonic solution is water;
The present invention adopts osmotic pressure principle, and when high osmotic treatment, cell shrinks, energy eluted dna, and cell water suction during Hypotonic treatment, can ruptured cell.But high hypotonic effect just utilizes the swelling of physics and shrinkage effect to carry out de-cell, still has some hereditary material DNA after cell breakage, cell debris sticks to material internal, more difficult eluting, therefore de-cell effect not good enough (see accompanying drawing 1).And in hyperosmotic solution, add acid or alkali, and can the degraded of catalyzing factor, destroy hydrolysis hereditary material DNA, accelerate eluting, improve de-cell efficiency (see accompanying drawing 2).
Pancreatin, SDS, peracetic acid-ethanol-water solution, several method for removing cells of acid-alkali treatment and the inventive method contrast by the present inventor, and assessment item comprises the de-cell effect of material, material surface structure, mechanical property, degradability etc.Result shows, intact cell configuration can be observed in material structure section after peracetic acid PROCESS FOR TREATMENT, DNA band is observed brighter in agarose gel electrophoresis, illustrate that DNA is residual higher, the de-bad (see figure 3) of cell effect, and the biomembrane material after the inventive method process is examined under a microscope and be there is not intact cell configuration and cell debris, agarose gel electrophoresis figure shows without bright DNA band (see figure 2), illustrates that to take off cell thorough; After scanning electron microscope result display pancreatin PROCESS FOR TREATMENT, the few fibers of material surface is dissolved, and illustrates that its surface texture is damaged (see figure 4); Shown by hot strength, suture tears power and external NTx enzymatic degradation result, material mechanical performance after pancreatin, SDS and acid-alkali treatment is poor, degraded is very fast, and the material mechanical performance of process of the present invention and degradability and raw material are more or less the same, illustrate that the present invention is to the structure composition of material and performance impact less (see Fig. 5 and 6).By taking off cell effect to material, material surface structure, mechanical property, the contrast of degradability can be found out, the present invention ensure thorough cell free while can reduce impact on material composition, structure and performance to greatest extent, be a kind of gentle effective method for removing cells.
Step 5, process of swelling: by sloughing the biomembrane material of cell at the immersion 5 ~ 60min in solution that swells, be washed to neutrality; Described solution of swelling refers to any one solution of hydrochloric acid, peracetic acid, phosphoric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide or ammonia, and concentration is 0.2 ~ 1M;
Described immersion can be adopted and be realized in two ways: one is all be immersed in by material to swell in solution; Two is tiled by material, and one side contacts solution, and another side is exposed to air, if material has loose and fine and close bilateral structure, then will loosen face contact solution, and dense face is exposed to air.
The present inventor studies confirmation, adopt acid or aqueous slkali soaking process can change the band electrical properties of tropocollagen molecule in material, make to produce electrostatic repulsion between tropocollagen molecule, cause biomembrane material organizational structure to expand the increase of loose, aperture and porosity and thickness; Can find out (see figure 7) by comparing result, material thickness is increased to 0.4 ~ 1mm by 0.1 original ~ 0.4mm.And the method adopting one side to soak, especially to the biomembrane material with bilateral structure, the one side structure of soaking solution can be made more loose, be beneficial to attaching and the proliferation and differentiation of cell, another side then keeps original compact texture; The architectural difference of loose face and dense face can be increased by one side immersion treatment, improve the effect that material guides growing into of cell and tissue regeneration and barrier action.
Step 6, lyophilizing, sterilizing: biomembrane material step 5 obtained flattens, and is affixed on flat board, directly puts into freeze dryer or put into freeze dryer again behind the cladding material surface that adds water on flat board; Lyophilizing is cooled to-80 DEG C ~-20 DEG C with the speed of 5 DEG C ~ 12 DEG C/min, under-10 DEG C of conditions, keep 10h, carries out cutting, packing after completing lyophilizing, adopts oxirane or 60Coradiation sterilizing, obtain tissue renovation material.
Compare and dry and vacuum and heating drying, lyophilizing can keep open structure and the porous character of material.Wherein material is fully absorbed water, keep collagen fiber to be in loose condition (of surface) in the material surface covering that adds water, moisture removal after lyophilizing and material structure still loosens, thickness increases (see accompanying drawing 8); Be applicable to the tissue repair higher to material thickness requirement, be conducive to guiding moving into and tissue regeneration of cell; The ice crystal that the quick pre-freeze of material can avoid the formation of is excessive and pull apart material fiber, destruction internal structure, affects material property.
The inventive method compared with prior art, has the following advantages:
(1) experiment proves, adopt acid or aqueous slkali soaking, material thickness is made to increase by 2 ~ 4 times, prepared material gives full play to the effect of filling defect in operation, fill complete and good with organization healing, ensure that restoring area does not form depression, and avoid easily adhering to operating theater instruments, easy to use after material rehydration; Compared with increasing thickness approach with existing cladding, can not or postoperatively there is layers of material and affect the situation of tissue regeneration and reparation in repair process.
(2) can ensure the loose porous of material through lyophilizing, rapid freeze-drying can avoid the fibre structure of larger crystalline fracture material internal; And to add water covering at material surface during lyophilizing, the material fiber of lyophilizing can be made to be in loose condition (of surface), and structure is more loose; Make the short texture of immersion one side by soaking the one side of material, the compact texture of another side be not destroyed, thus ensure loose face guide tissue regeneration, function that dense face prevents cell to run off.
(3), before organic solvent degreasing step is placed in de-cell process, through follow-up multiple link process and cleaning, the risk of organic solvent residual can be reduced to greatest extent, makes the material of preparation have higher biological safety and biocompatibility.
(4) in hyperosmotic solution, add acid or alkali carries out de-cell process to biomembrane material, compared with peracetic acid technique, it is better that the present invention takes off cell effect; Compared with pancreatin, SDS and soda acid technique, mechanical property and the degradability change of the rear material of process are less, prove that the destructiveness of the present invention to material is low; It is thorough that material prepared by the present invention takes off cell, and have lower immunogenicity, structural behaviour is not subject to too large impact, ensure that the safe and reliable of material.
Tissue renovation material prepared by the present invention, has suitable thickness and loose porous construction features, is applicable to the injury repairing of filling defect needed for the reparations such as cartilage injury, skin tissue defects and gingival recession.Through zoopery checking, confirmation of drawing materials after 8 weeks, restoring area, without depressed phenomenon, is replaced by cambium completely, and cambium and autologous tissue heal well, and segregation phenomenon does not occur.Illustrating that the tissue renovation material prepared by the present invention can provide sufficient three dimensions for cell proliferation with creeping, making restoring area reach good degree of integration and compactedness, realizing reparation and the regeneration of tissue.
Accompanying drawing explanation
Accompanying drawing 1 is that after adopting height infiltration method to take off cell process, materials microstructure is cut into slices and DNA agarose gel photograph; In figure, A is the microphotograph of Histological section, can find out that material internal is without intact cell configuration, but still has cell debris to remain, and illustrates that de-cell is not thorough; In figure, B is DNA agarose gel photograph, can find out that DNA band is obviously clear, and have DNA to remain in testimonial material, hereditary material is removed not thorough.
Accompanying drawing 2 is that the material structure in the present invention after de-cell process is cut into slices and DNA agarose gel photograph; In figure, A is Histological section's microphotograph, can find out without intact cell configuration in material, does not also have cell debris to remain, and illustrates that de-cell is thorough; In figure, B is DNA agarose gel photograph, can find out without obvious DNA band, and in illustrative material, DNA is residual lower.
Accompanying drawing 3 takes off the section of cell process materials microstructure and DNA agarose gel photograph for adopting peracetic acid technique; In figure, A is Histological section's microphotograph, can find out in material and have complete cell to exist, and illustrates that de-cell is not thorough; In figure, B is DNA agarose gel photograph, can find out that DNA band is obviously clear, and in illustrative material, DNA is residual higher, and the method takes off cell effect difference.
Accompanying drawing 4 is the surface scan electromicroscopic photograph adopting pancreatin PROCESS FOR TREATMENT material, can find out that the few fibers of material surface is dissolved, destructurized.
Accompanying drawing 5 is the comparison diagram adopting pancreatin technique, SDS technique, soda acid technique and the present invention to process rear material and raw material mechanical property respectively; Wherein A figure is hot strength comparison diagram, and B figure is suture tears power comparison diagram; Can find out, compare raw material, the mechanical property of pancreatin technique, SDS technique and soda acid PROCESS FOR TREATMENT material is adopted to decline all to some extent, and the material mechanical performance of process of the present invention and raw material are more or less the same, illustrate compared with prior art, adopt the Effect on Mechanical Properties of process of the present invention to material less.
Accompanying drawing 6 is the comparison diagram adopting pancreatin technique, SDS technique, soda acid technique and the present invention to process rear material and raw material degradability respectively; Can find out, compare raw material, adopt the material degradation of pancreatin technique, SDS technique and soda acid PROCESS FOR TREATMENT very fast, and the material degradation speed of process of the present invention is close with raw material, illustrate compared with prior art, adopt the degradation property impact of process of the present invention on material less.
Accompanying drawing 7 is the varied in thickness schematic diagram of material before and after (alkali) process of swelling in the present invention; Demonstrate material thickness after process of swelling obviously to increase, illustrate that alkali immersion has obvious effect to material thickness.
Accompanying drawing 8 is add water in frozen dried of the present invention to cover to contrast schematic diagram with the material thickness covered that do not add water; Can find out, the material thickness covered that adds water significantly improves, and illustrates that the covering that adds water can make material water suction, fiber is in loose condition (of surface), and the material of lyophilizing is more loose, thickness is larger.
Accompanying drawing 9 is the change curve adopting pig peritoneum material material thickness after each step process of the present invention; Can find out, after process of the present invention, the thickness of material significantly improves, and wherein alkali soaking step plays an important role in material thickness increases.
Accompanying drawing 10 is the comparison diagram of mechanical strength and the degradability change adopting pig peritoneum material before and after alkali immersion treatment of the present invention; Wherein A figure is the contrast of hot strength and the change of suture tears power, and can find out, significant change does not occur the suture tears power of material, and hot strength to decrease be increased by material thickness to cause, illustrates that alkali soaks on material mechanical performance impact not quite; B figure is the contrast of material degradation change, and can find out, significant change does not occur degradability, illustrates that alkali soaks material degradation impact little.
Accompanying drawing 11 is adopt the tissue renovation material prepared without alkali immersion treatment to carry out the pig cartilage injury repairing zoopery photo of 8 weeks; Wherein A figure is substantially photo, can find out that restoring area exists depression, unrealizedly repairs the smooth of surface; B figure is Histological section's microphotograph, can find out that restoring area is not concordant with autologous cartilaginous tissue, illustrate that repair materials is too thin, can not realize the filling completely of defect area.
Accompanying drawing 12 is that the cladding tissue renovation material adopting prior art to prepare carries out the pig cartilage injury repairing zoopery photo of 8 weeks; Wherein A figure is cardinal principle photo, can find out that restoring area still exists depression, not realize the filling completely of defect area; B figure is Histological section's microphotograph, can find out that restoring area does not plan a successor, heal between cambium and between cambium and autologous tissue not good, the regeneration of growing into and organizing along with cell in repair of cartilage is described, there is layering in plied timber, have impact on reparation and the synergy of cartilaginous tissue.
Accompanying drawing 13 is that the tissue renovation material adopting the present invention to prepare carries out the pig cartilage injury repairing zoopery photo of 8 weeks; Wherein A figure is cardinal principle photo, and can find out that reparation smooth surface is smooth, color and luster is close with autologous tissue, and defect face is completely filled; B figure is Histological section's microphotograph, can find out that cambium is concordant with autologous tissue surface, and cambium is inner and integrate good between cambium and autologous tissue, does not have fault-layer-phenomenon between tissue.Illustrate that tissue renovation material prepared by the present invention can guide cartilage tissue regeneration, realize the reparation of cartilaginous tissue.
Detailed description of the invention
Below in conjunction with the detailed description of the invention of example in detail technical solution of the present invention.The membrane tissue adopted in example buys the fresh dead meal in Pucheng herding Development Co., Ltd of Shaanxi bharal group slaughterhouse.
embodiment 1,with pig peritoneum for tissue renovation material prepared by raw material
Step one, pre-treatment: be laid on flat board by pig peritoneum, after removing large stretch of fat, remove remaining fat and connective tissue, with water cleaning to without color, obtains pig peritoneum material;
Step 2, defat: gained membrane material is placed in methanol and chloroform mixed liquor (volume ratio 1 ︰ 2), change liquid after concussion defat 10h, repeat defat 3 times, with water cleaning to tasteless;
Step 3, sterilization: the membrane material after defat is placed in volumetric concentration be 75% alcoholic solution soak 3h, with water cleaning to tasteless;
Step 4, de-cell: the membrane material after sterilization is placed in the de-cell hyperosmotic solution containing the NaCl of 3M and the NaOH of 0.25 M, after concussion 30min, proceed in water and shake 30min, so repeatedly complete de-cell 5 times;
Step 5, process of swelling: the KOH solution surface that being loosened by the membrane material sloughing cell faces down is laid in 0.5M, after keeping 5min, with water cleaning to neutral;
Step 6, lyophilizing, sterilizing: membrane material step 5 obtained flattens and is laid on corrosion resistant plate, steel plate adds water and is covered to just submergence material surface, afterwards steel plate is put into freeze dryer together with material, be cooled to-20 DEG C with the speed of 5 DEG C/min, keep completing lyophilizing in 10 hours at-10 DEG C; After again membrane material being cut, packing, through Co
60irradiation sterilization.
This example is with pig peritoneum for raw material, and fat content is higher, therefore adopts the ungrease treatment of long period and more number of times in technique, to reach better effect; In addition, pig peritoneum itself has the construction features of loose face and dense face, adopts one side to swell process, tissue renovation material thickness can be made to be increased to about 3 times (see figure 9)s of material stock thickness; Meanwhile, cover water layer lyophilizing owing to adopting, material face of loosening can be made more loose porous, for cell proliferation and differentiation provides sufficient three dimensions, and dense face structure is unaffected, can play barrier action, avoid cell to run off, be therefore more suitable for and repair cartilage injury.Pig cartilage injury repairing results of animal shows, the cartilage surface that this tissue renovation material is repaired is smooth, and neocartilage organizes the phenomenon that do not plan a successor, and heals well with autologous tissue, achieve the regeneration of defective tissue, facilitate the functional rehabilitation (see Figure 13) in joint.
embodiment 2,with sheep bladder base film for tissue renovation material prepared by raw material
Step one, pre-treatment: sheep bladder is laid on flat board, un-mixing bases plasma membrane, with water cleaning to without color, obtains sheep bladder base membrane material;
Step 2, defat: gained membrane material is placed in acetone, change liquid after concussion defat 2h, after continuing concussion defat 4h, with water cleaning to tasteless;
Step 3, sterilization: the membrane material after defat is placed in volumetric concentration be 75% alcoholic solution soak 2h, with water cleaning to tasteless;
Step 4, de-cell: the de-cell hyperosmotic solution membrane material after sterilization being placed in the HCl of NaCl and 0.2M containing 1M, after concussion 15min, proceed in water and shake 15min, so repeatedly complete de-cell 3 times;
Step 5, process of swelling: after the NaOH solution of sloughing the membrane material 1M of cell is soaked 1h, with water cleaning to neutral;
Step 6, lyophilizing and sterilizing: membrane material step 5 obtained flattens and is laid on glass plate, puts into freeze dryer, is cooled to-40 DEG C with the speed of 7 DEG C/min, keep 10h to complete lyophilizing at-10 DEG C; Carry out cutting, packing, adopt ethylene oxide sterilizing.
Sheep periplast film thinner thickness compared with peritoneum, therefore adopts gentle de-cellular processes.There is the risk of Protein virus in the tissue due to sheep source property, adopts 1M NaOH to soak, can reduce the Viral risks of material, and material structure is more homogeneous while increasing material thickness in process of swelling to whole membrane material.Prepared tissue renovation material takes off that cell is thorough, immunogenicity is low, thickness is even, is applicable to the reparation of skin tissue defects and damage, can makes the smooth surface no concave-convex of regeneration skin, also farthest recover attractive in appearance while practical function recovery.
embodiment 3,be that tissue renovation material prepared by raw material with bovine pericardium
Step one, pre-treatment: be laid in by bovine pericardium on flat board, remove large stretch of fat on it, remove residual fat and connective tissue, with water cleaning to without color, obtain bovine pericardium material;
Step 2, defat: gained membrane material is placed in isopropyl alcohol, change liquid after concussion defat 4h, repeatedly shake defat twice so again, the time is respectively 10h and 4h, with water cleaning to tasteless;
Step 3, sterilization: the membrane material after defat is placed in volumetric concentration be 70% alcoholic solution soak 4h, with water cleaning to tasteless;
Step 4, de-cell: the de-cell hyperosmotic solution membrane material after sterilization being placed in the NaOH of NaCl and 1M containing 5M, after concussion 1h, proceed in water and shake 20min, so repeatedly complete de-cell 2 times;
Step 5, process of swelling: being loosened by the membrane material sloughing cell faces down is laid in the HCl solution surface of 0.2M, after keeping 15min, with water cleaning to neutral;
Step 6, lyophilizing and sterilizing: membrane material step 5 obtained flattens and is laid on polyfluortetraethylene plate, puts into freeze dryer, is cooled to-80 DEG C with the speed of 12 DEG C/min, keep completing lyophilizing in 10 hours at-10 DEG C; Again membrane material cut, pack, adopt Co
60irradiation sterilization.
This example take bovine pericardium as raw material, thick compared with the membrane material in example 2, therefore adopts higher reagent concentration and comparatively long process time, to reach de-cell effect thoroughly; In addition, cattle source property organizes the risk that there is Protein virus equally, and this example adds the NaOH solution of 1M in de-cell processes, while the de-cell efficiency of raising, played the effect of inactivation of virus.The PROCESS FOR TREATMENT that this example adopts one side to swell can meet the requirement of gingival recession reparation to material thickness, material dense face provides barrier function for grow into cell and newborn tissue of organization internal, make its erosion from oral environment and mechanical damage, prove through zoopery, well, gingiva height has obvious lifting for gingiva restoring area compactedness and coverage.
Claims (3)
1. a preparation method for tissue renovation material, comprises the step of pre-treatment, defat, inactivation of virus, de-cell, process of swelling, lyophilizing and sterilizing, specifically comprises:
Step one, pre-treatment: mammiferous membrane tissue is laid in flat board, remove attachment fat, connective tissue and edge breakage tissue, be washed to afterwards without color, obtain biomembrane material; Described mammal comprise pig, cattle or sheep any one; Described membrane tissue comprise bladder base film, peritoneum, fascia, pericardium or valvular any one;
Step 2, defat: be placed in after organic solvent shakes defat 2 ~ 10h by gained biomembrane material and change liquid, repeat defat 2 ~ 4 times; Be washed to odorlessness;
Step 3, sterilization: the alcoholic solution biomembrane material after defat being placed in volumetric concentration 70 ~ 75% soaks at least 2h, then be washed to without alcohol taste;
Step 4, de-cell: the biomembrane material after sterilization is placed in de-cell hyperosmotic solution concussion 10 ~ 60min, then proceed to concussion 10 ~ 60min in de-cell hypisotonic solution, 2 ~ 5 times so repeatedly; Wherein, de-cell hyperosmotic solution is in the NaCl solution of 0.5 ~ 5M, add the HCl of 0.01 ~ 0.5M or the NaOH of 0.05 ~ 1M; De-cell hypisotonic solution is water;
Step 5, process of swelling: by sloughing the biomembrane material of cell at the immersion 5 ~ 60min in solution that swells, be washed to neutrality; Described solution of swelling refers to any one solution of hydrochloric acid, peracetic acid, phosphoric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide or ammonia, and concentration is 0.2 ~ 1M;
Step 6, lyophilizing, sterilizing: biomembrane material step 5 obtained flattens, and is affixed on flat board, directly puts into freeze dryer or put into freeze dryer again behind the cladding material surface that adds water on flat board; Lyophilizing is cooled to-80 DEG C ~-20 DEG C with the speed of 5 DEG C ~ 12 DEG C/min, at-10 DEG C, keep 10h; Carry out after completing lyophilizing cutting, packing, adopt oxirane or 60Coradiation sterilizing, obtain tissue renovation material.
2. preparation method according to claim 1, is characterized in that, the organic solvent described in step 2 be acetone, isopropyl alcohol, methanol and chloroform mixed liquor, normal hexane, ethyl acetate, ethanol or petroleum ether any one; Wherein volume ratio 1 ︰ 0.25 ~ 4 of methanol and chloroform mixed liquor.
3. preparation method according to claim 1, is characterized in that, the immersion described in step 5 is all immersed in by material to swell in solution, or is tiled by material, and loose face contact solution, dense face is exposed to air.
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