CN107320777A - A kind of dura mater biological sticking patch and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of dura mater biological sticking patch and preparation method thereof.The dura mater biological sticking patch uses intestinal submucosa tissue for raw material, the removal immunogenic substance technique of system, lipid is removed including the use of Ethanol Treatment, take off cell using trypsase and alkaline solution treatment, handled using DNA enzymatic and remove DNA, the steps such as α Gal antigens are removed using α galactosidase treatments, can effectively remove immunogenic substance will not destroy extracellular matrix normal configuration again.When being clinically applied to dura defect repairing, cerebrospinal fluid seepage can be effectively prevented, bootable tissue is grown into, tissue growth speed is matched with sticking patch degradation speed, and immunological rejection is low, good biocompatibility.

Description

A kind of dura mater biological sticking patch and preparation method thereof

Technical field

The invention belongs to biomedical materials field, in particular it relates to a kind of biological sticking patch repaired for dura defect And preparation method thereof.

Background technology

Dura mater is protection brain（Spinal cord）Organize the important barrier together with cerebrospinal fluid seepage is prevented.Dura defect is outside nerve Section is clinically rather common, and wound, tumour erosion, inflammatory destruction, congenital disorders and operations on cranium and brain, operation on spinal cord etc. can be made Into dura defect, so cause cerebrospinal fluid seepage, subcutaneous hematocele/hydrops, otch prolong it is circuitous be not cured, intracranial infection, brain tissue bulging, The complication such as epilepsy.Thus, for a long time, by operative repair dura mater, the complete of dura mater is kept, protect brain（Spinal cord）Tissue, Prevent cerebrospinal fluid seepage, it has also become the common recognition of neurosurgery circle.Traditional dural reconstruciton material is mainly derived from autologous tissue, material Expect limited source, and the pain of making patients, artificial dura mater patching material has been widely accepted in recent years.

At present, existing a variety of artificial dura mater materials enter clinical practice both at home and abroad, are broadly divided into 3 classes：Artificial synthesized material Material, allogenic material and xenogenic biological materials.

Synthetic material, including nonabsorable synthetic material and absorbable synthetic material.Nonabsorable it is artificial synthesized Material is foreign matter forever to the host for receiving transplanting, and chronic inflammatory reaction, scar or encapsulation reaction, machinery pressure can be caused after implantation The problems such as urgent, delayed hemorrhage, art chamber infect., there is brain bad adaptability, contacted with brain tissue in absorbable synthetic material There are chronic fibrosis, the shortcomings of catabolite is easily caused inflammation and adhesion in place.

Allogenic material, including allosome human dura mater, fascia late, acellular human body corium etc..This kind of material is through a variety of Technological means processing inactivation, histocompatbility is good.But exist some insoluble problems, may such as propagate host disease, Source limitation, expensive, ethics problem, and tissue space are big, are easily caused cerebrospinal fluid seepage etc., limit its application.

Xenogenic biological materials, frequent origins have beef tendon, ox/Pigs Hearts bag, the abdomen/pleura and trees-Osima jacoti, Osima excavata of ox/pig After removing cell component and immunogenicity Deng, these animal tissues, resulting extracellular matrix components can be developed Preferable dura mater biological material for repairing, but the product of separate sources all respectively has advantage and disadvantage.

The collagem membrane extracted by beef tendon is repaired available for dura defect, the existing several such launch in the country, but such Material is used for dura mater repairing and had the disadvantage that：Degradation rate is too fast, and material has been degraded before tissue does not grow up to；Toughness is poor, It is easily broken；Adhesion is not strong enough, and cerebrospinal fluid can ooze out from the gap for sticking periphery, forms hydrops；It is applied to defect When infiltrated by tissue fluid after, it is impossible to adjustment position, it is higher to user's skill requirement.

Coming from the dura mater sticking patch product of ox/Pigs Hearts bag, ox/pig peritonaeum or pleura has mechanical strength high, and compactness is good The advantages of.But needing, through the Chemical Treatments such as epoxides or glutaraldehyde, to have the following disadvantages its process more：Have potential thin Cellular toxicity；Need to clean repeatedly before use to remove chemical residue；Degradation rate is slower, mismatches, can cause with regeneration Fibrosis, chronic inflammatory reaction and the complication such as skull or brain tissue adhesion；Quality is hard, it is impossible to pasted with the perfection of brain tissue surface Close, it is necessary to tight suture.

In xenogenic biological materials, submucous layer of small intestine (small intestinal submucosa, SIS) source Acellular matrix is one of generally acknowledged optimal tissue renovation material of academic circles at present.SIS host materials have advantages below： Good toughness, overcomes beef tendon source and extracts the inadequate shortcoming of obtained collagen film toughness；Without epoxides or glutaraldehyde cross-linking, Avoid the potential cytotoxicity of crosslinking agent；Material is soft, can be fitted with the perfection of brain tissue surface；It can induce tissue growth, group Knit growth synchronous with material degradation, absorb complete, regeneration plasticity is perfect.In addition, also contain a variety of growth factors in SIS, Suitable microenvironment can be created for regeneration, still contain these growth factors even across the SIS of de- cell processing, such as FGF-2 and TGF-β.These growth factors have important facilitation in the reparation and reconstruct of tissue, and this is other thin Extracellular matrix materials and artificial polymer's (such as collagen, chitosan, PLA and polyglycolic acid) can not compare.

SIS host materials are as a kind of preferable dura mater alternative materials, and the current domestic dura mater sticking patch with this kind of material is only There is an imported product --- the biological duramater reparation piece of U.S. Cook Biotech Incorporated productions（Commodity Name：Biodesign Surgisis）.The product has occupied 10~30% biological dacron patch market in American-European countries, enters China market nearly 10 years.But Surgisis production technology（CN 102176929 B）Only carry out SDS cleaning agents, peroxide The process steps such as acetic acid, isopropanol and aqueous slkali, although consider the steps such as sterilization, degreasing, but pointedly use does not go to exempt from Epidemic disease immunogenic substance technique, result in that cell residue amount, DNA content are too high, such as the Oasis in Surgisis series of products^TM's DNA content is 0.42ng/mg（Gilbert TW, etc, Quantification of DNA in biologic scaffoldmaterials. J Surg Res 2009, 152 (1): 135–139.）, and to being present in extracellular matrix In native antigen composition --- α-gal antigens also do not consider.

In recent years, preparation of some the domestic producers to SIS products has made intensive studies.Patent of invention CN 103272275 B uses inbred strais animal intestinal submucosa tissue to ensure that the stability and uniformity of different batches of product for raw material, with machine Tool vibrates removes residual DNA with supersonic oscillations technique, overcomes Biodesign Surgisis product dna residual quantities more The problem of, the DNA residual quantities of the patented product are 120 ± 15pg/g, hence it is evident that less than Biodesign Surgisis DNA residuals 250 ± 45pg/g of amount, but the de- cell of the patent and skimming processes only with low concentration aqueous slkali within a short period of time Processing, takes off cell and degreasing effect is not good enough, and the technique for being also not directed to removal α-Gal antigens.Patent of invention CN The 101366975 B and B of CN 101366979 SIS materials are employed peracetic acid disinfectant, aqueous slkali take off cell, DNA enzymatic and α- The technique of galactosidase treatments, although consider removal immunogenic substance DNA and α-Gal antigens, but it takes off cell and de- Fat technique is only the alkaline solution treatment of short time, less effective.Though processing of the B of patent of invention CN 103405811 to SIS materials So include the steps such as sterilization, trypsase, DNA enzymatic and alpha-galactoside ferment treatment, but be not directed to degreasing process.Invention is special Although the sharp B of CN 101433735 employ " Peracetic acid+ethanol " sterilization, organic solvent degreasing, " high osmotic treatment to SIS materials + trypsin treatment+acid treatment+alkali process " takes off the technique such as cell and surfactant de-sludging, but poison is used in the technique Property stronger organic solvent degreasing, if organic solvent removes not thorough, can there is the potential risk for causing tissue toxicity to be reacted；The work Trypsinase concentration is too high in skill and processing time is long, may destroy extra-cellular matrix structure；And this invented technology is simultaneously The step of being not directed to remove DNA and α-Gal antigens.

In summary, current SIS preparation process not yet has one kind intactly to consider to remove residual cell, lipid, DNA With the technique of the immunogenic substance such as α-Gal antigens, product immunogenic substance too high levels can be caused, be easily caused and implant Immunotoxicity reaction occurs afterwards.

The content of the invention

It is an object of the invention to overcome the shortcomings of existing dura mater biological sticking patch technology of preparing, there is provided one kind use SIS systems Dura mater biological sticking patch that standby, immunogenic substances content is low, can retain natural extracellular matrix three-dimensional structure and preparation method thereof.

In order to realize the purpose of the present invention, the present invention is prepared the preparation method of dura mater biological sticking patch using SIS, employed pre- Processing, sterilization, degreasing, de- cell, remove DNA, go the PROCESS FOR TREATMENTs such as α-Gal antigens, freeze-drying and sterilizing.

The preparation method of above-mentioned dura mater biological sticking patch, is comprised the following steps that：

In below step, in addition to special instruction, the ratio of SIS materials and solution is 1:5~1:20（v/v）Between.

（1）Pretreatment

Take the chitterlings tissue wash of fresh slaughtered animals clean, be placed in 0.01% ~ 0.5% acetum and soak 10 ~ 120min, go Raw meat, scrapes mucous layer, muscle layer, placenta percreta, lymph node that division removes chitterlings jejunum using physics, isolates submucosa, cut Into fragment, rinsed at least 3 times using purified water.

（2）Sterilization

The mixed aqueous solution for the ethanol that the Peracetic acid and concentration that concentration is 0.5~1.5% are 15~25%, ultrasound condition Under, 30 ~ 120min of soaking at room temperature carries out disinfection.It is cleaned by ultrasonic at least 3 times using purified water afterwards.

（3）Degreasing

Concentration is 90-100% ethanol, under ultrasound condition, soak at room temperature 0.5-12h.It is cleaned by ultrasonic afterwards using purified water At least 3 times.

（4）Cell is taken off, DNA is removed and removes α-Gal antigens

Using the mixed aqueous solution containing 0.01~0.10% trypsase and containing 0.01~0.05%EDTA, under ultrasound condition, in 36 15-40min is soaked under the conditions of ± 2 DEG C.It is cleaned by ultrasonic at least 3 times using PBS afterwards；

Using the aqueous solution of the DNA enzymes containing 0.05~10U/ml, under ultrasound condition, 15-40min is soaked under the conditions of 36 ± 2 DEG C. It is cleaned by ultrasonic at least 3 times using PBS drifts afterwards；

Using the aqueous solution of the alpha-galactosidase containing 0.05~10U/ml, under ultrasound condition, 15- is soaked under the conditions of 20-37 DEG C 40min.It is cleaned by ultrasonic at least 3 times using PBS afterwards；

Concentration is the 5- 40mM NaOH aqueous solution, under ultrasound condition, soak at room temperature 20-60min.Surpassed afterwards using PBS Sound cleaning is until neutral.

（5）Lyophilized, sterilizing

The stacking of SIS sheet materials 3~12 is added, is fixed on mould, after freeze-drying, is cut into specification needed for Clinical practice, is used PET packaging bags are packed, and finally carry out irradiation sterilization.

Compared with prior art, the present invention has following remarkable advantage and beneficial effect：

Employ the removal immunogenic substance technique of system, remove lipid including the use of Ethanol Treatment, using trypsase and Alkaline solution treatment takes off cell, is handled using DNA enzymatic and removes DNA, and the step such as α-Gal antigens is removed using alpha-galactoside ferment treatment Suddenly.Compared with existing similar products, immunogenic substances content is lower, and such as residual cell amount is not more than 1/mirror downward view（400 ×）, DNA residual quantities be not more than 120pg/g, α-Gal antigens for feminine gender（Immunofluorescence assay）, lipid content is not more than 0.1%, so biocompatibility is more preferable after implanting, immunological rejection is lower, and security is more preferable.

Method of enzymatically treating used in the present invention, its concentration and action time can effectively remove immunogenic substance again not It can destroy under extracellular matrix normal configuration, product ESEM and show collagenous fiber bundle structural integrity；Water preventing ability detection display Tool prevents liquid from passing through effect；Cerebrospinal fluid seepage, and implantation material can be effectively prevented when being repaired for beasle dog dura defect It is good with normal surrounding tissue compatibility.

The dura mater biological sticking patch of the present invention is made up of 3~12 layers of individual layer SIS, when being repaired for dura defect, can be carried in short term For a good compactness barrier to prevent cerebrospinal fluid seepage；It can be grown into for a long time as organization bracket induction dura mater tissue, by Gradually with one entirety of formation of dura mater tissue around, repair deficiency prevents cerebrospinal fluid seepage, while sticking patch is progressively degraded, tissue is again Life is synchronous with sticking patch degradation speed.

Brief description of the drawings

Shown in Fig. 1 is the sample drawing of the embodiment of the present invention 1.

Shown in Fig. 2 is the section HE colored graphs of the embodiment of the present invention 1.

Shown in Fig. 3 is the electron-microscope scanning figure of the embodiment of the present invention 1.

Shown in Fig. 4 is the histotomy figure for beasle dog dural repair of the embodiment of the present invention 1.

Embodiment

The present invention is further described in detail with reference to embodiments, but is not limited to this.

Embodiment 1

（1）Pretreatment

Take the chitterlings tissue wash of fresh slaughtered animals clean, be placed in 0.5% acetum immersion 30min, chitterlings and acetic acid The ratio of solution is 1:5, mucous layer, muscle layer, placenta percreta, lymph node that division removes chitterlings jejunum are scraped using physics, separation Go out submucosa, be cut into fragment, rinsed 3 times using purified water.

（2）Sterilization

Using the mixed aqueous solution containing 1.0% Peracetic acid and 15% ethanol, the ratio of SIS materials and mixed aqueous solution is 1: 10, under ultrasound condition, soaking at room temperature 100min carries out disinfection.It is cleaned by ultrasonic 3 times using purified water afterwards.

（3）Degreasing

Concentration is 95% ethanol, and the ratio of SIS materials and ethanol is 1:10, under ultrasound condition, soak at room temperature 2h.Afterwards It is cleaned by ultrasonic 3 times using water for injection.

（4）Cell is taken off, DNA is removed and removes α-Gal antigens

Using the mixed aqueous solution containing 0.02% trypsase and containing 0.02%EDTA, SIS materials and trypsase/EDTA solution Ratio be 1:5, under ultrasound condition, 30min is soaked under the conditions of 37 DEG C.It is cleaned by ultrasonic 3 times using PBS afterwards；

Using the aqueous solution of the enzymes of DNA containing 5U/ml, the ratio of SIS materials and DNA enzymatic solution is 1:5, under ultrasound condition, in 37 20min is soaked under the conditions of DEG C.It is cleaned by ultrasonic 3 times using PBS drifts afterwards；

Using the aqueous solution of the alpha-galactosidase containing 5U/ml, the ratio of SIS materials and alpha-galactoside enzyme solutions is 1:5, surpass Under the conditions of sound, 20min is soaked under the conditions of 30 DEG C.It is cleaned by ultrasonic 3 times using PBS afterwards；

Concentration is the 25mM NaOH aqueous solution, and the ratio of SIS materials and NaOH solution is 1:20, under ultrasound condition, normal temperature Soak 50min.It is cleaned by ultrasonic afterwards using PBS until neutral.

（5）Lyophilized, sterilizing

SIS materials 4 are laminated and added, are fixed on mould, is freeze-dried, is cut into 4cm × 7cm specifications, is entered using PET packaging bags Row packaging, finally carries out irradiation sterilization.Product is as shown in Figure 1.

Embodiment 2：The sample prepared to embodiment 1 carries out physical property detection, chemical property detection, histology Detection, growth factor detection, biology performance detection and animal experiment.

1. physical property is detected

1）Water preventing ability

Method：Reference《YY/T 0471.3-2004 contact Wound dressing test method third portions：Water preventing ability》Method enter Row detection.

As a result：With water preventing ability.

2）Suture strength

Method：With 3-0 non-absorbing suture in sticking patch both sides center away from being sutured at edge 2mm, by the suture other end and sample The other end be separately fixed on tensiometer two ends, stretched with 20mm/min speed, until stitch points are torn, record Maximal force.3 batches of samples are detected as stated above.

As a result：Suture strength is in 3N ~ 5N scopes.

3）Tensile strength

Method：Sample is cut to width for 10mm shapes respectively along both direction；In relative humidity 40% ~ 60%, temperature after cutting Tested after being placed 2 hours in 22 ± 2 DEG C of environment of degree.Fixture spacing is 25mm, and sample two ends are fixed on into cupping machine Chuck on, with 100mm/min speed tensile, maximal force during record fracture.3 batches of samples are carried out as stated above Detection.

As a result：Tensile strength is in 30N ~ 35N scopes.

4）Bursting strength

Method：According to《YY 0500-2004 cardiovascular implant artificial blood vessels》8.3.3.2 the measurement side for rupture strength of popping one's head in Method is chosen 9.5mm diameters probe and detected.3 batches of samples are detected as stated above.

As a result：Bursting strength is in 85N ~ 90N scopes.

2. chemical property is detected

1）Acid-base value

Method：According to《GB/T14233.1 medical infusions, blood transfusion, the part of the instrument used for injection method of inspection the 1st：Chemical analysis method》 Specified in acid-base value method carry out.

As a result：The difference of the PH values of test liquid and blank control liquid is no more than 1.5.

2）Bacterial endotoxin

Method：According to GB/T 14233.2-2005《Medical infusion, blood transfusion, the part of the instrument used for injection method of inspection the 2nd:Biology Test method》Specified in bacterial endotoxin test method carry out.

As a result：Bacteria endotoxin content is less than 2.15EU/ pieces, meets and contacts medicine equipment to bacterial endotoxin with cerebrospinal fluid The requirement of limitation.

3）DNA residual quantities

Method：According to YY/T 0606.25-2014《Animal derived biomaterial DNA determination of residual amount methods：Fluorescence colour》Enter Row detection.

As a result：DNA residual quantities are 80 ± 12 pg/g.

4）α-Gal antigenic contents

Method：After sample is fixed with paraformaldehyde, routine paraffin wax embedded section, piece is thick 3 microns.Utilize biotin labeling BSI- B4 and α-Gal antigens special affinity characteristic carry out immunohistochemical reaction.Coloration result judges：Dark brown yellow particle is strong sun Property（+++）, brown yellow granule is the positive（++）, yellow particle is weakly positive（+）, have no and be colored as feminine gender（-）.

As a result：For feminine gender（-）.

5）Lipid content

Method：Reference《Fatty measure in the food of GB/T 5009.6》Middle soxhlet extraction methods are measured.

As a result：Lipid content is 0.08%.

3. histology

1）Observation by light microscope

Method：Fixed with 10% neutral formalin, FFPE, be cut into 0.4 micron of thin slice, dewaxed through dimethylbenzene, series wine Essence dehydration, haematine-eosin stains, micro- Microscopic observation cell residue situation and matrix fiber structure.

As a result：Acellular and cell fragment residual；Collagenous fibres are continuous, no fracture, as shown in Fig. 2.

2）Ultrastructural observation

Method：It is scanned using electron scanning mirror.

As a result：Material porous structure, collagenous fibres are without fracture, as shown in Fig. 3.

4. growth factor is detected

Method：The content that fibroblast growth factor is detected using ELLISA methods, concrete operations side will be used after tissue mashing Method refers to kit operation instructions.

As a result：Content is 9540-9937pg/g.

5. biology performance is detected

Method：Tested with reference to GB/T16886 series methods.

As a result：Cell-cytotoxic reaction is less than or equal to 1 grade；Without delayed allergy；Intradermal reaction shows test specimen And the difference of solvent control mean score is less than 1.0；Without pyrogenicity；Without hemolytic reaction；Genetic toxicity test result shows that mouse hinders Cold salmonella back mutation（Ames）The negative, mouse lymphoma assay of experiment is negative, the distortion of dye-free body Property；Without acute generalised toxic reaction；Without sub- chronic generalized toxicity；Muscular grafting 30 days, 60 days, 90 days with negative controls Tissue reaction without significant difference.

6. animal experiment

Method：Using Cook（COOK）The biological duramater reparation piece of biotech company's production（Registration certificate is numbered：State's food medicine prison Tool（Enter）Word 2014 the 3462661st）As control, carry out Beagal dogs dura defect using dura mater biological sticking patch and repair Art.

As a result：After implantation 3 months, implantation material and normal endocranium obscure boundary have been merged；It is implanted into material fine Dimension is substituted by cambium；Material position is implanted into normal endocranium histology and morphology structure without significant difference, it is seen that fiber is female The cells such as cell, macrophage, lymphocyte, thick liquid cell are present, it is seen that angeogenesis；Arachnoid and brain tissue are normal. The dura mater biological sticking patch cerebral dura mater repairing defect histotomy of 3 months is as shown in Figure 4.

The above embodiment of the present invention is the description of the invention and cannot be used for the limitation present invention, the right with the present invention Any change in claim suitable implication and scope, is all considered as being included within the scope of the claims.

Claims (2)

1. the invention discloses a kind of preparation method of dura mater biological sticking patch, it is characterised in that including following operating procedure：

（1）Pretreatment

Take the chitterlings tissue wash of fresh slaughtered animals clean, be placed in 0.01% ~ 0.5% acetum and soak 10 ~ 120min, go Raw meat, scrapes mucous layer, muscle layer, placenta percreta, lymph node that division removes chitterlings jejunum using physics, isolates submucosa, cut Into fragment, rinsed at least 3 times using purified water；

（2）Sterilization

The mixed aqueous solution for the ethanol that the Peracetic acid and concentration that concentration is 0.5~1.5% are 15~25%, ultrasound condition Under, 30 ~ 120min of soaking at room temperature carries out disinfection, and is cleaned by ultrasonic at least 3 times using purified water afterwards；

（3）Degreasing

Concentration is 90-100% ethanol, under ultrasound condition, and soak at room temperature 0.5-12h is cleaned by ultrasonic using purified water afterwards At least 3 times；

（4）Cell is taken off, DNA is removed and removes α-Gal antigens

Using the mixed aqueous solution containing 0.01~0.10% trypsase and containing 0.01~0.05%EDTA, under ultrasound condition, in 36 15-40min is soaked under the conditions of ± 2 DEG C, is cleaned by ultrasonic at least 3 times using PBS afterwards；

Using the aqueous solution of the DNA enzymes containing 0.05~10U/ml, under ultrasound condition, 15-40min is soaked under the conditions of 36 ± 2 DEG C, It is cleaned by ultrasonic at least 3 times using PBS drifts afterwards；

Using the aqueous solution of the alpha-galactosidase containing 0.05~10U/ml, under ultrasound condition, 15- is soaked under the conditions of 20-37 DEG C 40min, is cleaned by ultrasonic at least 3 times using PBS afterwards；

Concentration is the 5- 40mM NaOH aqueous solution, under ultrasound condition, soak at room temperature 20-60min, is surpassed afterwards using PBS Sound cleaning is until neutral；

（5）Lyophilized, sterilizing

The stacking of SIS sheet materials 3~12 is added, is fixed on mould, after freeze-drying, is cut into specification needed for Clinical practice, is used PET packaging bags are packed, and finally carry out irradiation sterilization.

2. a kind of preparation method of dura mater biological sticking patch according to claim 1, it is characterised in that：The step （1）、（2）、（3）、（4）The ratio of middle material and solution is 1:5~1:20（v/v）Between.