CN104524634B - Preparation method of tissue repair material - Google Patents
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- CN104524634B CN104524634B CN201410780482.7A CN201410780482A CN104524634B CN 104524634 B CN104524634 B CN 104524634B CN 201410780482 A CN201410780482 A CN 201410780482A CN 104524634 B CN104524634 B CN 104524634B
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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 organizational engineering medical biomaterial technical field is and in particular to a kind of preparation method of tissue renovation material.
Background technology
Tissue, the forfeiture of organ or dysfunction are one of most frequent, most serious problems faced by human health institute.According to statistics, have in the U.S. that people up to a million suffer from various tissues, organ is lost or dysfunction every year, annual need to carry out performing the operation 8,000,000 times, year cost exceed 40,000,000,000 dollars.In recent years, with the development of organizational project and regenerative medicine, tissue, the repairing and treating of organ have many breakthroughs, and wherein xenogenesis takes off cell biological membrane material and obtains more concerns because of its unique natural structure and the synchronous degradation property of regeneration.Lot of domestic and international research institution and company take off cell biological membrane material to xenogenesis and have carried out basic research for many years and application study, and it is safe and effective that its clinical practice for many years also show material.
In terms of foreign material source, study more inclusion corium, submucous layer of small intestine, bladder, peritonaeum, manadesma, pericardium, cardiac valves etc..Because foreign material itself contains a large amount of cells, inhereditary material, fat, directly apply to people and know from experience the strong immune response of initiation, so needing through degreasing, de- cell, going antigen etc. to process, retain extracellular matrix components to greatest extent, realize its guide tissue regeneration and the function of repairing.Therefore, the processing procedure of material is used for effect and has important function.
Method for removing cells includes physics, chemistry and biology method.Wherein physical method adopts freeze thawing, pressure, can kill cell and but can not transport out by cell component and inhereditary material.Chemical method includes 1) acid-base method, acid or the process of alkali can lead to cell cracking, cause or catalysis activity molecule hydrolytic degradation, reach cell free purpose, but the long duration of action of acid or alkali can produce heavy damage to material；2) adopt high hypotonic solution, carry out cell free method using osmotic pressure principle smudge cells it is considered to be minimum method for removing cells is destroyed to the molecular structure of matrix, but the effect of its independent role is not thorough, after process, have a small amount of cell fragment and dna residual；3) adopt the cell free method of detergent, including ionic detergent (as sds) and non-ionic detergent (as tritonx-100), and amphoteric ion type detergent (as chaps), detergent can dissolve cell membrane, dna is eluted out, but detergent destroys simultaneously and decomposed the protein in material, simultaneously not easy cleaning, on the one hand increase cost, cause the production cycle long, another aspect reagent residual can affect security and the compatibility of material；4) adopt the cell free method of organic reagent, including the application of alcohols and ketone etc..Biological method mainly includes the application of enzyme and chelate, and it there is a problem of destroying material structure and reagent remains more difficult cleaning.
It was verified that being used alone of said method is all unable to reach preferably de- cell effect, therefore generally selects the combination of two or more methods or be used in mixed way.United States Patent (USP) us6206931 carries out de- cell with Peracetic acid-ethanol-water solution collective effect and processes to trees-Osima jacoti, Osima excavata, less to the structure influence of material；And the present inventor presses the document and records experiment it was demonstrated that still it is observed that the presence of intact cell configuration after processing using the method, this will produce potential immunological rejection and inflammatory reaction risk.United States Patent (USP) us5387278 is using acid treatment after first alkali, de- cell effect can be reached, the strong method for removing cells of both is superimposed use for a long time, material structure can be caused with irreversible destruction, lead to that material degradation is too fast, mechanical property is low, the synchronous degradation with regeneration can not be reached it is impossible to be applied to clinic；Additionally, the method is dehydrated after de- cell, there is the risk of organic reagent residual using acetone degreasing, organic reagent；So, the method still has room for improvement from the feasibility that technique is amplified and science.The series of products of tutogen company of the U.S. carry out de- cell using hydrogen peroxide and process, and processing procedure can produce a large amount of foams, be not easy to clean, easily cause the security of reagent remaining influence material.United States Patent (USP) us5413798 is to carry out de- cell with diluted alkaline, dilute sodium chloride, complexing agent, there is also the risk of reagent residual, can cause stronger toxic reaction in vivo.
Clinical research finds, in some surgical operations, when material thickness relatively thin it is impossible to when being sufficient filling with damage location height, can cause to repair position depression or surface irregularity, have a strong impact on its repairing effect and functional rehabilitation.Additionally, during Clinical practice, relatively thin 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 limited by raw material itself, such as the material thickness such as pericardium, peritonaeum, pericystium is only 0.1 ~ 0.4mm；For making up this defect, typically increase thickness using biomaterial superposition or by the way of being combined other materials, so inevitably resulting in cost increases and yield decline, the risk of layers of material, the recovery effects of impact tissue during also bringing use.
Content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of preparation method of tissue renovation material, the tissue renovation material of gained is made to take off cell thoroughly, no the residual of intact cell configuration and cell fragment, dna residual and reagent residual risk are low, thickness increases by 2 ~ 4 times, there is preferable mechanical property and anti-degradability, the requirement of clinical practice can be met.
The preparation method of tissue renovation material proposed by the present invention, the step swollen, be lyophilized and sterilize including pre-treatment, degreasing, inactivation of virus, de- cell, material, specifically include:
Step one, pre-treatment: the membrane tissue of mammal source is laid in flat board, removes attachment fat, connective tissue and edge breakage tissue, be washed to no color afterwards, obtain biomembrane material；Described mammal include pig, ox, sheep any one；Described membrane tissue includes bladder base film, peritonaeum, manadesma, pericardium, any one in cardiac valves；
Step 2, degreasing: gained biomembrane material is placed in organic solvent and after concussion degreasing 2 ~ 10h, changes liquid, repeat degreasing 2 ~ 4 times；It is washed to odorlessness；Described organic solvent is acetone, isopropanol, methyl alcohol and chloroform mixed liquor, n-hexane, ethyl acetate, ethanol or petroleum ether any one；The volume ratio 1 0.25 ~ 4 of wherein methyl alcohol and chloroform mixed liquor.
Wherein, processed using organic solvent degreasing, and be placed on whole technique position earlier above, through subsequent treatment be cleaned multiple times, organic solvent residual can be removed to greatest extent it is ensured that the security of material.
Step 3, sterilization: the biomembrane material after degreasing is placed in the ethanol solution of volumetric concentration 70 ~ 75% and soaks at least 2h, reach disinfection effect, be washed to no alcohol taste；
Step 4, de- cell: the biomembrane material after sterilization is placed in concussion 10 ~ 60min in de- cell hypertonic solution, then proceeds to concussion 10 ~ 60min in de- cell hypotonic solution, so repeatedly 2 ~ 5 times；Wherein, de- cell hypertonic solution is the naoh of the hcl or 0.05 ~ 1m in the nacl solution of 0.5 ~ 5m added with 0.01 ~ 0.5m；De- cell hypotonic solution is water；
The present invention adopts osmotic pressure principle, and in high osmotic treatment, cell shrinks, and can elute dna, and during Hypotonic treatment, cell water suction, can rupture cell.But high hypotonic effect carries out de- cell just with the swelling of physics and shrinkage effect, still there are some inhereditary materials dna, cell fragment to stick to material internal after clasmatosis it is more difficult to wash-out, therefore take off cell effect not good enough (see accompanying drawing 1).And add acid or alkali in hypertonic solution, hydrolysis inhereditary material dna can be destroyed with the degraded of catalyzing factor, accelerate wash-out, improve de- cell efficiency (see accompanying drawing 2).
The present inventor is by pancreatin, sds, Peracetic acid-ethanol-water solution, soda acid processes several method for removing cells and the inventive method is contrasted, and assessment item includes the de- cell effect of material, material surface structure, mechanical property, degradability etc..Result shows, it is observed that intact cell configuration in material structure section after Peracetic acid PROCESS FOR TREATMENT, observe that dna band is brighter in agarose gel electrophoresis, illustrate that dna residual is higher, the bad (see figure 3) of de- 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 fragment, the no bright dna band (see figure 2) of agarose gel electrophoresis figure display, illustrate that de- cell is thorough；After ESEM result display pancreatin PROCESS FOR TREATMENT, the few fibers of material surface are dissolved, and illustrate that its surface texture is subject to destroy (see figure 4)；Shown by tensile strength, suture tears power and external Collagenase Type degradation results, material mechanical performance after pancreatin, sds and soda acid process is poor, degraded is very fast, and the material mechanical performance that the present invention is processed and degradability are more or less the same with raw material, and the present invention less to the structure composition of material and performance impact (see Fig. 5 and 6) is described.By cell effect is taken off to material, material surface structure, mechanical property, the contrast of degradability can be seen that, the present invention is ensureing the thoroughly cell free impact that simultaneously can reduce to greatest extent to material composition, structure and performance, is one kind gently effective method for removing cells.
Step 5, process of swelling: the biomembrane material sloughing cell is soaked 5 ~ 60min in solution of swelling, is washed to neutrality；Described solution of swelling refers to any one solution of hydrochloric acid, Peracetic acid, phosphoric acid, NaOH, potassium hydroxide, calcium hydroxide or ammoniacal liquor, and concentration is 0.2 ~ 1m；
Described immersion can be realized using two ways: one is all to be immersed in material to swell in solution；Two is that material tiles, and simultaneously contacts solution, another side is exposed to air, if material has loose and fine and close bilateral structure, loose face is contacted solution, dense face is exposed to air.
The present inventor's research confirms, the band electrical properties that tropocollagen molecule in material can be changed are processed using acid or aqueous slkali soaking, make to produce electrostatic repulsion between tropocollagen molecule, cause biomembrane material institutional framework to expand the increase of loose, aperture and porosity and thickness；By comparing result it can be seen that (see figure 7), material thickness increases 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, the attaching beneficial to cell and Proliferation, Differentiation, 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 grown into regeneration and barrier action that material guides cell.
Step 6, lyophilized, sterilizing: the biomembrane material that step 5 is obtained flattens, and is affixed on flat board, places into freeze dryer after being directly placed into freeze dryer or the covering material surface that adds water on flat board；Lyophilized is to be cooled to -80 DEG C ~ -20 DEG C with the speed of 5 DEG C ~ 12 DEG C/min, keeps 10h under the conditions of -10 DEG C, carries out cutting, packs after completing to be lyophilized, and using oxirane or 60Coradiation sterilizing, obtains tissue renovation material.
Compare and dry and vacuum and heating drying, lyophilized open structure and the porous property that can keep material.So that material is fully absorbed water, keep collagenous fibres to be in loose condition (of surface) wherein in the material surface covering that adds water, moisture removal after being lyophilized and material structure is still loose, thickness increases (see accompanying drawing 8)；It is applied to the tissue repair higher to material thickness requirement, be conducive to guiding moving into and regeneration of cell；The quick pre-freeze of material is avoided that the ice crystal of formation is excessive and pulls apart material fiber, destroys internal structure, affects material property.
The inventive method compared with prior art, has the advantage that
(1) experiment proves, using 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, filling is complete and good with organization healing, ensure that restoring area does not form depression, and avoid easy-adhesion operating theater instruments, convenient use after material rehydration；With existing cladding increase thickness approach compared with, will not repair during or postoperative layers of material occurs and affect regeneration and repair situation.
(2) freeze-dried can guarantee that the loose porous of material, rapid freeze-drying is avoided that the fibre structure of larger crystalline fracture material internal；And add water covering in material surface when lyophilized, lyophilized material fiber can be made to be in loose condition (of surface), structure is more loose；The short texture of immersion one side, the compact texture of another side is made not to be destroyed by soaking to the one side of material, thus ensureing that loose face guide tissue regeneration, dense face prevent the function that cell runs off.
(3), before organic solvent degreasing step being placed in de- cell process, processing through follow-up multiple links and can clean, reduce the risk of organic solvent residual to greatest extent, make the material of preparation have higher biological safety and biocompatibility.
(4) acid or alkali is added biomembrane material to be carried out with de- cell and processes in hypertonic solution, compared with Peracetic acid technique, it is more preferable that the present invention takes off cell effect；Compared with pancreatin, sds and soda acid technique, after process the mechanical property of material and degradability change less it was demonstrated that the present invention is low to the destructiveness of material；The material of present invention preparation takes off cell thoroughly, has relatively low immunogenicity, structural behaviour be not affected by too big impact it is ensured that material safe and reliable.
The tissue renovation material of present invention preparation, there is suitable thickness and loose porous design feature it is adaptable to cartilage damage, skin tissue defects and gingival recession etc. repair needed for injury repair that defect is filled with.Through zoopery checking, confirmations of drawing materials after 8 weeks, restoring area no depressed phenomenon, replaced by cambium completely, and cambium is with autologous tissue healing well, and segregation phenomenon does not occur.Illustrate that the tissue renovation material prepared by the present invention can be cell proliferation and the sufficient three dimensions of offer of creeping, make restoring area reach good degree of integration and compactedness, realize reparation and the regeneration of tissue.
Brief description
Accompanying drawing 1 is to take off materials microstructure section and dna agarose gel photograph after cell is processed using height infiltration method；In figure a be the microphoto of Histological section it can be seen that material internal no intact cell configuration, but still have cell fragment to remain, illustrate to take off cell not thorough；In figure b is dna agarose gel photograph it can be seen that dna band is clearly substantially it was demonstrated that have dna to remain, inhereditary material removes not thorough in material.
Accompanying drawing 2 is the material structure section and dna agarose gel photograph after de- cell process in the present invention；In figure a is Histological section's microphoto it can be seen that no intact cell configuration in material, does not also have cell fragment to remain, and illustrate to take off cell thorough；, for dna agarose gel photograph it can be seen that no obvious dna band, in illustrative material, dna residual is relatively low in figure b.
Accompanying drawing 3 is to take off cell using Peracetic acid technique to process materials microstructure section and dna agarose gel photograph；In figure a is Histological section's microphoto it can be seen that with the presence of complete cell in material, illustrating that de- cell is not thorough；, for dna agarose gel photograph it can be seen that dna band is clearly obvious, in illustrative material, dna residual is higher, and it is poor that the method takes off cell effect in figure b.
Accompanying drawing 4 be surface scan electromicroscopic photograph using pancreatin PROCESS FOR TREATMENT material it can be seen that the few fibers of material surface are dissolved, destructurized.
Accompanying drawing 5 be processed respectively using pancreatin technique, sds technique, soda acid technique and the present invention after material and raw material mechanical property comparison diagram；Wherein a figure is tensile strength comparison diagram, and b figure is suture tears power comparison diagram；It can be seen that, compare raw material, mechanical property using pancreatin technique, sds technique and soda acid PROCESS FOR TREATMENT material has all declined, and the material mechanical performance that the present invention is processed is more or less the same with raw material, illustrate compared with prior art, to process less to the Effect on Mechanical Properties of material using the present invention.
Accompanying drawing 6 be processed respectively using pancreatin technique, sds technique, soda acid technique and the present invention after material and raw material degradability comparison diagram；It can be seen that, compare raw material, the material degradation using pancreatin technique, sds technique and soda acid PROCESS FOR TREATMENT is very fast, and the material degradation speed that the present invention is processed is close with raw material, illustrate compared with prior art, to process the degradation property impact on material using the present invention less.
Accompanying drawing 7 is the thickness change schematic diagram of material after (alkali) before processing of swelling in the present invention；Show that material thickness significantly increases after swelling and processing, illustrate that alkali immersion has obvious effect to material thickness.
Accompanying drawing 8 is the material thickness contrast schematic diagram covering with the covering that do not add water that adds water in frozen dried of the present invention；As can be seen that the material thickness of the covering that adds water significantly improves, the covering that illustrates to add water can make material water suction, fiber be in loose condition (of surface), and lyophilized material is more loose, thickness is bigger.
Accompanying drawing 9 is the change curve using pig peritonaeum material material thickness after each step process of the present invention；As can be seen that the thickness of rear material processed by the invention significantly improves, wherein alkali soaking step plays an important role in material thickness increase.
Accompanying drawing 10 is the comparison diagram using pig peritonaeum material through the mechanical strength before and after alkali immersion treatment of the present invention and degradability change；Wherein a figure be the contrast of tensile strength and the change of suture tears power it can be seen that there is not significant change in the suture tears power of material, and it is to be increased by material thickness to cause that tensile strength decreases, and illustrate that alkali soaks little on material mechanical performance impact；B figure be the contrast of material degradation change it can be seen that there is not significant change in degradability, illustrate that alkali soaks little on material degradation impact.
Accompanying drawing 11 is to carry out the pig cartilage injury repair zoopery photo of 8 weeks using without tissue renovation material prepared by alkali immersion treatment；Wherein a figure is that substantially photo, it can be seen that restoring area has depression, is not implemented and repairs the smooth of surface；B figure be Histological section's microphoto it can be seen that restoring area is not concordant with autologous cartilaginous tissue, illustrate that repair materials are too thin it is impossible to realize being filled up completely with of defect area.
Accompanying drawing 12 is to carry out the pig cartilage injury repair zoopery photo of 8 weeks using the cladding tissue renovation material of prior art preparation；Wherein a figure is that substantially photo, it can be seen that restoring area still suffers from being recessed, is not carried out being filled up completely with of defect area；B figure is Histological section's microphoto, can be seen that restoring area does not plan a successor, heal between cambium and between cambium and autologous tissue not good, the regeneration grown into and organize with cell in repair of cartilage is described, layering in plied timber, have impact on reparation and the synergy of cartilaginous tissue.
Accompanying drawing 13 is to carry out the pig cartilage injury repair zoopery photo of 8 weeks using the tissue renovation material of present invention preparation；Wherein a figure be substantially photo it can be seen that repair smooth surface smooth, color and luster is close with autologous tissue, and defect face is completely filled；B figure is Histological section's microphoto it can be seen that integrating good inside cambium and autologous tissue's flush, cambium and between cambium and autologous tissue, does not have fault-layer-phenomenon between tissue.Illustrate that the tissue renovation material of present invention preparation can guide regenerative agent of cartilaginous tissue, realize the reparation of cartilaginous tissue.
Specific embodiment
Specific embodiment below in conjunction with example in detail technical solution of the present invention.Membrane tissue employed in example is bought in the fresh dead meal in Pucheng herding Development Co., Ltd of Shaanxi bharal group slaughterhouse.
Embodiment 1 、Tissue renovation material is prepared for raw material with pig peritonaeum
Step one, pre-treatment: pig peritonaeum is laid on flat board, after removing large stretch of fat, removes remaining fat and connective tissue, cleaned to no color with water, obtain pig peritonaeum material；
Step 2, degreasing: gained membrane material is placed in methyl alcohol and chloroform mixed liquor (volume ratio 1 2), changes liquid after concussion degreasing 10h, repeat degreasing 3 times, cleaned with water to tasteless；
Step 3, sterilization: the membrane material after degreasing is placed in the ethanol solution that volumetric concentration is 75% immersion 3h, is cleaned with water to tasteless；
Step 4, de- cell: in the de- cell hypertonic solution of the naoh membrane material after sterilization being placed in nacl containing 3m and 0.25 m, after concussion 30min, proceed to concussion 30min in water, so repeatedly complete de- cell for 5 times；
Step 5, process of swelling: loose for the membrane material sloughing cell facing down is laid in the koh solution surface of 0.5m, after keeping 5min, is cleaned to neutrality with water；
Step 6, lyophilized, sterilizing: the membrane material flattening that step 5 is obtained is laid on corrosion resistant plate, covering is added water on steel plate to just submergence material surface, afterwards steel plate is put in freeze dryer together with material, be cooled to -20 DEG C with the speed of 5 DEG C/min, keep completing to be lyophilized for 10 hours at -10 DEG C；After cutting membrane material again, packing, through co60Irradiation sterilization.
, with pig peritonaeum as raw material, fat content is higher for this example, adopts the ungrease treatment of long period and more number of times, to reach more preferable effect therefore in technique；In addition, pig peritonaeum has the design feature of loose face and dense face in itself, swollen process using one side, tissue renovation material thickness can be made to increase to 3 times about (see figure 9)s of material stock thickness；Simultaneously as being lyophilized using covering water layer, the loose face of material can be made more loose porous, there is provided sufficient three dimensions for cell proliferation and differentiation, and dense face structure is unaffected, can play barrier action, avoid cell to run off, be therefore more suitable for repairing cartilage damage.Pig cartilage injury repair 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, promote the functional rehabilitation (see Figure 13) in joint.
Embodiment 2 、Tissue renovation material is prepared for raw material with sheep bladder base film
Step one, pre-treatment: sheep bladder is laid on flat board, un-mixing bases plasma membrane, is cleaned to no color with water, obtain sheep bladder base membrane material；
Step 2, degreasing: gained membrane material is placed in acetone, after concussion degreasing 2h, changes liquid, after continuing concussion degreasing 4h, cleaned with water to tasteless；
Step 3, sterilization: the membrane material after degreasing is placed in the ethanol solution that volumetric concentration is 75% immersion 2h, is cleaned with water to tasteless；
Step 4, de- cell: in the de- cell hypertonic solution of the hcl that the membrane material after sterilization is placed in nacl and 0.2m containing 1m, after concussion 15min, proceed to concussion 15min in water, so repeatedly complete de- cell for 3 times；
Step 5, process of swelling: the membrane material sloughing cell is soaked after 1h with the naoh solution of 1m, is cleaned to neutrality with water；
Step 6, lyophilized and sterilizing: the membrane material flattening that step 5 is obtained is laid on glass plate, puts in freeze dryer, is cooled to -40 DEG C with the speed of 7 DEG C/min, keeps 10h to complete to be lyophilized at -10 DEG C；Carry out cutting, pack, using ethylene oxide sterilizing.
Sheep periplast film thinner thickness compared with peritonaeum, therefore adopts gentle de- cellular processes.Because the tissue of sheep source property has the risk of prion, in process of swelling, 1m is adopted to whole membrane material
Naoh soaks, and can reduce the Viral risks of material, and material structure is more homogeneous while increasing material thickness.Prepared tissue renovation material is de-, and cell is thorough, immunogenicity is low, thickness is uniform it is adaptable to the reparation of skin tissue defects and damage, can make the smooth surface no concave-convex of regenerated bark skin, realize also farthest recovering attractive in appearance while functional rehabilitation.
Embodiment 3 、Tissue renovation material is prepared for raw material with bovine pericardium
Step one, pre-treatment: bovine pericardium is laid on flat board, removes large stretch of fat thereon, remove residual fat and connective tissue, cleaned to no color with water, obtain bovine pericardium material；
Step 2, degreasing: gained membrane material is placed in isopropanol, after concussion degreasing 4h, changes liquid, so more repeatedly concussion degreasing twice, the time is respectively 10h and 4h, cleaned with water to tasteless；
Step 3, sterilization: the membrane material after degreasing is placed in the ethanol solution that volumetric concentration is 70% immersion 4h, is cleaned with water to tasteless；
Step 4, de- cell: in the de- cell hypertonic solution of the naoh that the membrane material after sterilization is placed in nacl and 1m containing 5m, after concussion 1h, proceed to concussion 20min in water, so repeatedly complete de- cell for 2 times；
Step 5, process of swelling: loose for the membrane material sloughing cell facing down is laid in the hcl solution surface of 0.2m, after keeping 15min, is cleaned to neutrality with water；
Step 6, lyophilized and sterilizing: the membrane material flattening that step 5 is obtained is laid on polyfluortetraethylene plate, puts in freeze dryer, is cooled to -80 DEG C with the speed of 12 DEG C/min, keeps completing to be lyophilized for 10 hours at -10 DEG C；Again membrane material cut, pack, using co60Irradiation sterilization.
This example is with bovine pericardium as raw material, thick compared with the membrane material in example 2, therefore adopts higher reagent concentration and longer process time, thoroughly takes off cell effect to reach；Additionally, property tissue in ox source equally exists the risk of prion, this example adds the naoh solution of 1m in de- cell processes, has played the effect of inactivation of virus while improving de- cell efficiency.The PROCESS FOR TREATMENT that this example is swollen using one side can meet the requirement to material thickness for the gingival recession reparation, material dense face grows into cell for organization internal and newborn tissue provides barrier function, protect it from erosion and the mechanical damage of oral environment, animal experiments prove that, gum reparation area filling degree and coverage are good, and gum highly has and is obviously improved.
Claims (3)
1. a kind of preparation method of tissue renovation material, including pre-treatment, degreasing, inactivation of virus, de- cell, process of swelling, the step being lyophilized and sterilizing, specifically includes:
Step one, pre-treatment: the membrane tissue of mammal is laid in flat board, removes attachment fat, connective tissue and edge breakage tissue, be washed to no color afterwards, obtain biomembrane material；Described mammal includes any one of pig, ox or sheep；Described membrane tissue include bladder base film, peritonaeum, manadesma, pericardium or valvular any one；
Step 2, degreasing: gained biomembrane material is placed in organic solvent and after concussion degreasing 2 ~ 10h, changes liquid, repeat degreasing 2 ~ 4 times；It is washed to odorlessness；
Step 3, sterilization: the biomembrane material after degreasing is placed in the ethanol solution of volumetric concentration 70 ~ 75% and soaks at least 2h, then be washed to no alcohol taste；
Step 4, de- cell: the biomembrane material after sterilization is placed in concussion 10 ~ 60min in de- cell hypertonic solution, then proceeds to concussion 10 ~ 60min in de- cell hypotonic solution, so repeatedly 2 ~ 5 times；Wherein, de- cell hypertonic solution is the naoh of the hcl or 0.05 ~ 1m that add 0.01 ~ 0.5m in the nacl solution of 0.5 ~ 5m；De- cell hypotonic solution is water；
Step 5, process of swelling: the biomembrane material sloughing cell is soaked 5 ~ 60min in solution of swelling, is washed to neutrality；Described solution of swelling refers to any one solution of hydrochloric acid, Peracetic acid, phosphoric acid, NaOH, potassium hydroxide, calcium hydroxide or ammoniacal liquor, and concentration is 0.2 ~ 1m；
Step 6, lyophilized, sterilizing: the biomembrane material that step 5 is obtained flattens, and is affixed on flat board, places into freeze dryer after being directly placed into freeze dryer or the covering material surface that adds water on flat board；Lyophilized is to be cooled to -80 DEG C ~ -20 DEG C with the speed of 5 DEG C ~ 12 DEG C/min, keeps 10h at -10 DEG C；Carry out cutting after completing to be lyophilized, pack, using oxirane or 60Coradiation sterilizing, obtain tissue renovation material.
2. preparation method according to claim 1 it is characterised in that the organic solvent described in step 2 be acetone, isopropanol, methyl alcohol and chloroform mixed liquor, n-hexane, ethyl acetate, ethanol or petroleum ether any one；Volume ratio 1:0.25 ~ 4 of wherein methyl alcohol and chloroform mixed liquor.
3. preparation method according to claim 1 is it is characterised in that the immersion described in step 5 is all to be immersed in material to swell in solution, or material is tiled, and loose face contacts solution, and dense face is exposed to air.
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