The duramater reparation support of degradable people's amnion and ox back tendon compound and preparation method
Technical field
The present invention relates to a kind of duramater reparation support, specifically the duramater reparation support of a kind of degradable people's amnion and ox back tendon compound and preparation method.
Background technology
Endocranium is the important barrier together of protection cerebral tissue, is a thick and tough and tensile duplicature. The skin of endocranium is the periosteum in face in skull, loosely attached in braincap, particularly more loose at occipitalia and temples attachment, is called periosteum layer; The relatively outer thickness of internal layer and tough and tensile, with endorchis at foramen magnum place continuous even, be called meninx layer. The factors such as the erosion of wound, tumour and operation all can cause dura defect, it is necessary to repairs, to prevent cerebrospinal leak, intracranial infection and cortex adhesion cause the complication such as epilepsy.
Current duramater reparation Clinical Processing mode has two kinds: 1. suture type, with artificial dural repairment material (such as " meninx bodyguard ", process by bovine pericardium) directly sew up defect, it is stitched together with self meninx, this method is applicable to little leak, acute high cranium is pressed to the patient of the rear Naoning tablet of hernia cerebri decompression, sew application difficulty, it is easy to injure cerebral tissue when sewing up. 2. pasting formula, be directly covered in defect place with artificial dural repairment material (such as collagen protein sponge), clinical existence pastes not tight, cerebrospinal fluid seepage, easily forms meninx apposition liquid.
Research shows, the endocranium of people each square millimeter can bear the tension stress intensity of about 37kg, can bear the stitching tension force of various model suture. Desirable duramater reparation support should comprise following quality factor: (1) has promotion regeneration and restoration function; (2) after sewing up, it is possible to accomplish to sew up densification waterproof, prevent cerebrospinal fluid seepage, protection cerebral tissue; (3) can stop to transmit potential known or unknown infection; (4) use safety, without rejection, non-toxic reaction, without carcinogenic, teratogenesis; (5) can be completely absorbed after cambium generates; (6) easily preserving, pre-operative preparation is simple and convenient;(7) material source is convenient; (8) price rather moderate.
The factors such as the erosion of wound, tumour and operation all can cause dura defect, and cerebrospinal fluid seepage, intracranial infection and cortex adhesion can be occurred after art to cause the complication such as epilepsy. About meninx repair document report can trace back to 1890 the earliest, experienced by from goldleaf to manadesma, by people's corpse meninx to the development course of xenogenesis fascia tissue and even the macromolecule polymer material of synthetic.
At present for repairing the material of endocranium and mainly contain four classes such as autologous manadesma (autologous film material draw materials limited cause secondary insult intensity relatively endocranium is poor), allogenic material (corpse endocranium), foreign material (common bovine pericardium, Pigs Hearts bag), natural and synthetic material, after front two class equivalent material are transplanted, effect is relatively good, limited owing to originating, limit its clinical middle widespread use. Rear two class surrogates are drawn materials and conveniently can effectively be alleviated the shortage of donor graft materials of the same race to a certain extent.
Domestic in recent years apply more product clinically and be: biological artificial dura mater's collagen protein sponge, primarily of NTx protein gelatin after lyophilize obtained, xenogenic biological materials " meninx bodyguard " (the bovine pericardium product through glutaraldehyde process) etc. after going antigen to process, has the trend becoming main product. but owing to animal tendon is normally become collagen gel through acid, alkali or enzymic hydrolysis by collagen protein sponge, spongy collagen microbial film is become again through lyophilize, its sewing properties, mechanical property are poor, and degradation speed is too fast, can not being commonly used to dural repair as conventional material, especially relatively big at dura defect, cerebral can not use higher. " meninx bodyguard " is in preparation process simultaneously, adopt glutaraldehyde organized processing technology remove foreign protein antigenic while, the a small amount of aldehyde radical constantly occurred in material, introduce residual toxicity, not easily thoroughly remove in additional subsequent disposal, although this kind of toxicity intensity is lower, but the time length is very long, almost suitable with the implantation life-span of material, although faint residual toxicity is not obvious after fibrillar connective tissue forms Nang Bi affects histocompatibility, it but block cell to the migration of implanting tissue, it is difficult to metabolism and assimilates implanting tissue further, it is unfavorable for tissue repair.
Reproducibility reparation is the trend of biomedical advancement, not only requires that material has good histocompatibility and desirable mechanical characteristics, also to be allowed autogenous cell to grow into wherein, gradually metabolism patching material and be formed with the cambium of function. Current substituted type dural repairment material does not all reach this requirement.
Amnion, as the natural resources of a kind of preciousness, has excellent biological character, and development prospect is wide. Research shows, human amnion membrane has obvious plasticity-and Multidirectional Differentiation potential, under the adjustment of different somatomedin, the multiple histocytes such as hepatic lineage, cardiac-like muscle cell, neurogliocyte, neuronal cell and insulin-like cell can be divided into, medically have broad application prospects at cell replacement therapy and tissue regeneration. Containing multiple adjustment cytoactive, the biologically active substance promoting damaged tissue healing in amnion fibre substrate, comprise multiple enzyme, activated protein, cytokine etc. The isolation effect of amnion stroma layer and the various kinds of cell factor, biological activity protein jointly play the protection surface of a wound, prevent adhesion, suppress local inflammation reaction, promote that damage field histocyte repairs the biological actions such as hyperplasia, are a kind of desirable biological restoration timbering materials.
The clinical application range of domestic and international amnion is in continuous expansion, it is possible not only to rebuild for eye table, can also be used for promoting tendon injury reparation, promote peripheral nerve regeneration reparation, promote periosteum growth and knitting, burns unit, plastic surgery, gynaecology, Urology Surgery etc. rebuild mucous membrane and skin histology, and the application in stem-cell research and Tissue Engineering Study field also achieves gratifying results. But there is, as endocranium repair materials, the problem that mechanical property does not reach clinical requirement in independent people's amnion.
In sum, the defect that prior art exists is:
1. adopting the combination of mucous membrane of small intestine lower floor (about 0.08mm) with people's amnion (about 0.05mm), thickness differs relatively big with endocranium, the poor requirement not reaching clinical application of sewing properties, mechanical property;
2. in bovine pericardium or Pigs Hearts coating, attach autologous vein endotheliocyte or the umbilical cord venous endothelial cell that one layer of cultivation survives, the cultivation of these viable cell, preservation condition and pre-operative preparation require higher, in-convenience in use, the production technique of product, production cost and costs of commodity circulation also all corresponding raising;
3. collagen sponge is taking animal tendon collagen as raw material, but collagen protein generating portion hydrolysis after peracid or alkaline purification in its making, the structure of collegen filament has a very large change, and its mechanical property is good not as native state;
4. the mechanical property of synthesized polymer material can meet the clinical requirement of duramater reparation, but its biology performance comprises regeneration and restoration, biocompatibility etc. is all not so good as natural macromolecular material.
Summary of the invention
It is an object of the invention to solve in prior art the defect existing for dural repairment material, it is provided that duramater reparation support and the preparation method of a kind of degradable people's amnion and ox back tendon compound solve the problem.
In order to realize above-mentioned purpose, the technical scheme of the present invention is as follows:
The preparation method of the duramater reparation support of degradable people's amnion and ox back tendon compound, it is characterised in that, comprise the following steps:
(1) amnion is prepared: carry out amnion by national standard YZB/ state 0593-2005 and draw materials and prepare;
(2) ox back tendon tendon sheet is prepared: fresh ox back tendon production technique water cleans, by concentration is the Peracetic Acid soaking disinfection 1-2hr of 0.1%-0.5%, then by washed with de-ionized water; After the saline soak 1-2hr of 1%-5%, the temperature being placed in-20 DEG C-40 DEG C carries out freezing, moves towards longitudinally to be cut into tendon sheet along ox back tendinous fibres; Tendon sheet is put-20 DEG C-40 DEG C freezing rear clear water putting into 20 DEG C-40 DEG C that take out to be melted rinse-added, after multigelation like this 2-5 time, comprise with nuclease: concentration is the DNase of 100-200U/mL and concentration is the RNase process of 50-150U/mL, temperature is 20-45 DEG C, shaking table concussion is after 8-24 hour, cleaning 3-5 time to PH6.5-7.5 through PBS, obtained ox back tendon tendon sheet, refrigerates for subsequent use under putting the environment of 4-8 DEG C;
(3) amnion and ox back tendon tendon sheet is nontoxic crosslinked: by above-mentioned amnion and ox back tendon collagem membrane after PBS or physiological saline infiltrate 5-10 minute, the epithelial lining of amnion is tiled down, again the above-mentioned ox back tendon tendon sheet handled well is layered on amnion, amnion and ox back tendon tendon sheet are placed in non-toxic crosslinker, linking agent comprises: molecular weight 1,500 5000 glycoprotein, the hydroxy fatty acid of carbonatoms less than 30, through 20-60 minute, 25 DEG C-37 DEG C, after the crosslinked bath of PH4.5-7.5, abandon solution, and with deionized water rinsing 2-5 time repeatedly, drain water postlyophilization, packaging, sterilizing, the duramater reparation support of obtained people's amnion and ox back tendon tendon sheet compound.
Above-mentioned degradable people's amnion and the preparation method of the duramater reparation support of ox back tendon compound, it is characterized in that, in described step (3), ox back tendon tendon sheet comprises two layers, two layers of ox back tendon tendon sheet intersect according to the transverse and longitudinal that tendon collagen fabric moves towards and stack, and then are layered on amnion by fold two layers of ox back tendon tendon sheet.
Above-mentioned degradable people's amnion and the preparation method of the duramater reparation support of ox back tendon compound, it is characterised in that, described step (3) is covered lid layer amnioic epithelium layer amnion upward on ox back tendon tendon sheet again.
Above-mentioned degradable people's amnion and the preparation method of the duramater reparation support of ox back tendon compound, it is characterised in that, the concentration of the Peracetic Acid in described step (2) is 0.2%, and concentration be 0.2% Peracetic Acid in include 5% dehydrated alcohol; The temperature of nuclease is 37 DEG C; By obtained ox back tendon tendon sheet, refrigerate for subsequent use under putting the environment of 4 DEG C.
Above-mentioned degradable people's amnion and the preparation method of the duramater reparation support of ox back tendon compound, it is characterised in that, in described step (2), the thickness of tendon sheet is 0.3-0.5mm, and length is: 10-100mm, and width is: 10-50mm.
Above-mentioned degradable people's amnion and the preparation method of the duramater reparation support of ox back tendon compound, it is characterised in that, in described step (3) when tiling amnion, the nitrocellulose filter paper of place mat 0.45 μm of micropore below amnion.
The duramater reparation support of degradable people's amnion and ox back tendon compound, it is characterised in that, comprise the amnion and ox back tendon tendon sheet that are obtained by above-mentioned preparation method, by the duramater reparation support of nontoxic crosslinked people's amnion of obtaining and ox back tendon tendon sheet compound.
Above-mentioned degradable people's amnion and the duramater reparation support of ox back tendon compound, it is characterized in that, ox back tendon tendon sheet comprises two layers, and two layers of ox back tendon tendon sheet intersect according to the transverse and longitudinal that tendon collagen fabric moves towards and stack, and then are layered on amnion by fold two layers of ox back tendon tendon sheet.
Above-mentioned degradable people's amnion and the duramater reparation support of ox back tendon compound, it is characterised in that, lid layer amnioic epithelium layer amnion upward is covered in the upper of ox back tendon tendon sheet again.
The useful effect of the present invention is: adopting people's amnion and ox back tendon tendon sheet through the duramater reparation support of nontoxic crosslinked compound, amnion treatment process meets national standard, can retain the bioactive functions of amnion; The preparation of ox back tendon tendon sheet is different from the preparation method of other collagem membranes, had both eliminated the immunogenic substance such as cell, nucleic acid, fat, and had not destroyed again the structure of natural ox back tendon collagen so that it is mechanical property is retained, and biological safety is improved. Two kinds of natural collagem membranes through nontoxic crosslinked and after lyophilize, its every performance meets the clinical requirement of duramater reparation. Specifically comprise: 1, the preparation method of ox back tendon tendon sheet, have employed Hypertonic Solutions to soak and add multigelation and make cell rupture, desmoenzyme method destroys nucleic acid material again, both can have been removed the immunogenicity of ox back tendon by present treatment, the natural ox back original structure of tendon collagen and mechanical property thereof can have been retained again as far as possible; 2, the compound of two kinds of collagem membranes have employed the method that nontoxic crosslinked bath adds lyophilize, and the biological safety of product is increased, and the preparation method of ox back tendon tendon sheet and permutation and combination method, make the mechanical property of product of the present invention better; 3, the combination of people's amnion and ox back key collagem membrane, had both remained the biology performance that people's amnion is excellent, particularly Regeneration and Repair performance, turn increased the mechanical property of ox back tendon tendon sheet so that it is can meet the clinical demand of dural repairment material.
Accompanying drawing explanation
Fig. 1 is the structural representation of the embodiment of the present invention 1;
Fig. 2 is the structural representation of the embodiment of the present invention 2;
Fig. 3 is the structural representation of the embodiment of the present invention 3.
Embodiment
For making the constitutional features to the present invention and effect of reaching have a better understanding and awareness, coordinate detailed description in order to preferred embodiment and accompanying drawing, it be described as follows:
The preparation method of the duramater reparation support of degradable people's amnion and ox back tendon compound, comprises the following steps:
(1) amnion is prepared: carry out amnion by national standard YZB/ state 0593-2005 and draw materials and prepare; In the making of people's amnion, amniotic epithelial cells can be retained or remove epithelial cell with enzymolysis process;
(2) preparing ox back tendon tendon sheet: utilize repeatedly freezing with thawing, make the cell in material form ice crystal, the salt concn of residue cytosol increases and causes the swelling fragmentation of cell, changes material and cell-surface antigens thus significantly reduces the antigenicity of biomaterial. Research shows, the tendon thin slice of multigelation desmoenzyme process 0.3mm, de-cell effect is good, disintegration can not be there is in collagen, native three dimensional structure and original mechanical property of tendon collegen filament are retained largely, tensile strength is 17-29MPa (during normal adult's clinostatism, cerebrospinal fluid pressure is 0.78~1.76kpa), about can retain the 90% of green strength.
Concrete mode is: fresh ox back tendon production technique water cleans, by concentration is the Peracetic Acid soaking disinfection 1-2hr of 0.1%-0.5%, then by washed with de-ionized water; After the saline soak 1-2hr of 1%-5%, the temperature being placed in-20 DEG C-40 DEG C carries out freezing, moves towards longitudinally to be cut into tendon sheet along ox back tendinous fibres; Then just tendon sheet put-20 DEG C-40 DEG C freezing after take out the clear water putting into 20 DEG C-40 DEG C and melt rinse-added, after multigelation like this 2-5 time, comprise with nuclease: concentration is the DNase of 100-200U/mL and concentration is the RNase process of 50-150U/mL, temperature is 25-40 DEG C, shaking table concussion is after 12-24 hour, cleaning 3-5 time to PH6.5-7.5 through PBS, obtained ox back tendon tendon sheet, refrigerates for subsequent use under putting the environment of 5-8 DEG C;
(3) amnion and ox back tendon tendon sheet is nontoxic crosslinked: by above-mentioned amnion and ox back tendon collagem membrane after infiltrating 5-10 minute, the epithelial lining of amnion is tiled down, again the above-mentioned ox back tendon tendon sheet handled well is layered on amnion, amnion and ox back tendon tendon sheet are placed in non-toxic crosslinker, linking agent comprises: molecular weight 1,500 5000 glycoprotein, the hydroxy fatty acid of carbonatoms less than 30, through 20-60 minute, 25 DEG C-37 DEG C, after the crosslinked bath of PH4.5-7.5, abandon solution, and with deionized water rinsing 2-5 time repeatedly, drain water postlyophilization, packaging, sterilizing, the duramater reparation support of obtained people's amnion and ox back tendon tendon sheet compound.
Two kinds of natural collagen proteins are all extracellular matrix, its surface has glycosaminoglycan, occur with polysaccharide chain form, sugar chain has many free hydroxyls, carboxyl or sulfate, electronegative in electrolyte solution, it is possible to attract positive charge, such as sodium ion etc., these positively charged ions are again in conjunction with large quantity of moisture, and glycosaminoglycan water-swelling, has bigger toughness; The hairbrush shape structure of glycosaminoglycan, oozes condition process through (slightly) height, and part discharges in molecule Bound moisture glycosaminoglycan and shrinks, and the formation that is hinged of hairbrush shape structure is sticked, and makes amnion and dorsal muscles tendon formation mechanical connection; O-O is formed or/and the glycosidic links such as O-N are covalently bound again through bi-functional cross-linking agent (amino acid, hydroxy fatty acid) effect, polysaccharide and polysaccharide, polysaccharide and collagen protein.Mechanical bond and covalent attachment, again through lyophilize, make amnion and tendon produce highly stable cohering.
Consider that tendon collagen fabric is arranged in parallel, cross directional tensile level is lower than longitudinal anti-tensile level, and endocranium fiber orientation varied (based on arciform, radial, stringer and diagonal fiber), there is multiple directions tension force in clinical stitching place, in wherein another preferred implementation of the present invention, in step (3), ox back tendon tendon sheet comprises two layers, two layers of ox back tendon tendon sheet intersect according to the transverse and longitudinal that tendon collagen fabric moves towards and stack, and then are layered on amnion by fold two layers of ox back tendon tendon sheet; In wherein another preferred implementation of the present invention, described step (3) covers lid layer amnioic epithelium layer amnion upward again in the upper of ox back tendon tendon sheet.
The concentration of the Peracetic Acid in step (2) is 0.2%, and concentration be 0.2% Peracetic Acid in include 5% dehydrated alcohol; The hydrolysis temperature of nuclease is 37 DEG C; By obtained ox back tendon tendon sheet, refrigerate for subsequent use under putting the environment of 4 DEG C.
In step (2), the thickness of tendon sheet is 0.3-0.5mm, and length is: 10-100mm, and width is: 10-50mm.
In step (3) when tiling amnion, the nitrocellulose filter paper of place mat 0.45 μm of micropore below amnion.
Degradable people's amnion in the present invention and the preparation method of the duramater reparation support of ox back tendon compound are relative to prior art, owing to employing the basic unit of ox back tendon tendon sheet as amnion, for amnion provides enough tension force, ensure that it can be applied in duramater reparation.
Embodiment 1:
Referring to Fig. 1, the duramater reparation support of degradable people's amnion and ox back tendon compound, comprise by the above-mentioned amnion 100 for preparing and ox back tendon tendon sheet 200, the epithelial lining of amnion 100 is tiled down, again the above-mentioned ox back tendon tendon sheet 200 handled well is layered on amnion 100, by the nontoxic duramater reparation support being cross-linked people's amnion and the ox back tendon tendon sheet compound obtained.
Embodiment 2:
Referring to Fig. 2, the duramater reparation support of degradable people's amnion and ox back tendon compound, comprise by the above-mentioned amnion 100 for preparing and ox back tendon tendon sheet 200, ox back tendon tendon sheet 200 comprises two layers, two layers of ox back tendon tendon sheet 200 intersect according to the transverse and longitudinal that tendon collagen fabric moves towards and stack, and then fold two layers of ox back tendon tendon sheet 200 are layered on amnion 100, by the nontoxic duramater reparation support being cross-linked people's amnion and the ox back tendon tendon sheet compound obtained. (Fig. 2 also have a kind of situation be above layer amnion that add again)
Embodiment 3:
Referring to Fig. 3, the duramater reparation support of degradable people's amnion and ox back tendon compound, comprise by the above-mentioned amnion 100 for preparing and ox back tendon tendon sheet 200, lid layer amnioic epithelium layer amnion 100 upward is covered in the upper of ox back tendon tendon sheet 200 again, by the nontoxic duramater reparation support being cross-linked people's amnion and the ox back tendon tendon sheet compound obtained.
The useful effect of the present invention is: adopting people's amnion and ox back tendon tendon sheet through the duramater reparation support of nontoxic crosslinked compound, amnion treatment process meets national standard, can retain the bioactive functions of amnion; The preparation of ox back tendon tendon sheet is different from the preparation method of other collagem membranes, had both eliminated the immunogenic substance such as cell, nucleic acid, and had not destroyed again the structure of natural ox back tendon collagen so that it is mechanical property is retained, and biological safety is improved.Two kinds of natural collagem membranes through nontoxic crosslinked and after lyophilize, its every performance meets the clinical requirement of duramater reparation. Specifically comprise: 1, the preparation method of ox back tendon tendon sheet, have employed Hypertonic Solutions to soak and add multigelation and make cell rupture, desmoenzyme method destroys nucleic acid material again, both can have been removed the immunogenicity of ox back tendon by present treatment, the natural ox back original structure of tendon collagen and mechanical property thereof can have been retained again as far as possible; 2, the compound of two kinds of collagem membranes have employed the method that nontoxic crosslinked bath adds lyophilize, and the biological safety of product is increased, and the preparation method of ox back tendon tendon sheet and permutation and combination method, make the mechanical property of product of the present invention better; 3, the combination of people's amnion and ox back key collagem membrane, had both remained the biology performance that people's amnion is excellent, particularly Regeneration and Repair performance, turn increased the mechanical property of ox back tendon tendon sheet so that it is can meet the clinical demand of dural repairment material.
More than show and describe the ultimate principle of the present invention, the advantage of main characteristic sum the present invention. The technician of the industry should understand; the present invention is not restricted to the described embodiments; the principle of the just the present invention described in above-described embodiment and specification sheets; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the scope of claimed the present invention. The present invention require protection domain by appending claims and etc. jljl define.