CN103800941B - Type-I collagen material, meninx and meninge biological membrane and preparation method and application of thereof - Google Patents
Type-I collagen material, meninx and meninge biological membrane and preparation method and application of thereof Download PDFInfo
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- CN103800941B CN103800941B CN201210445760.4A CN201210445760A CN103800941B CN 103800941 B CN103800941 B CN 103800941B CN 201210445760 A CN201210445760 A CN 201210445760A CN 103800941 B CN103800941 B CN 103800941B
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Abstract
The invention relates to a type-I collagen material with a high sewing concentration and a tri-helical structure, a meninx and meninge biological membrane prepared from the type-I collagen material, and a preparation method and application of the meninx and meninge biological membrane, and the meninx and meninge biological membrane can be used for the preparation of dura meninx and meninge tissue repair materials.
Description
Technical field
The present invention relates to medical science, biomaterial, tissue engineering and regeneration medicine technology field, be specifically related to biological diaphragm of a kind of type i collagen material, meninges or spinal meninges and its preparation method and application, that is: a kind of high-purity keeps the I type medical collagen material of collagen fiber original specific triple helix structure to make high concentration, high density, the meninges of high strength or spinal meninges preparation method and application.
More particularly, the present invention relates to a kind of highly purified type i collagen material, a kind of meninges or the biological diaphragm of spinal meninges and preparation method thereof prepared by described type i collagen material with triple-helix structure, and described meninges or the biological diaphragm of spinal meninges are preparing the application in cerebral dura mater or mater tissue repair materials.
Background technology
Twentieth century end is along with the high speed development of medical science, and cytobiology, molecular biology, gene are learned, and tissue engineering and regenerative medicine be constantly progress and development thereupon also.The biomaterial with premium properties is also being found rapidly and is being utilized simultaneously.In recent years, tissue engineering and the regenerative medicine quick turn up in clinical medicine just demonstrates this point.The type i collagen of natural biology material is used to obtain significant development and apply as support or the organizational project of carrier and the product of regenerative medicine.
An important topic in this field is: how to apply above-mentioned material and be prepared into the support having different physics and chemistry, biological characteristics and also can serve as extracellular matrix material, make the histiocyte of body damaged can have the environment of good reparative regeneration, and impel damaged tissues organ to be able to reparative regeneration close to normal internal and external environment as far as possible for damaged tissue cell provides.This not only needs to consider natural biology material self character, but also will consider to use in technology to have more superperformance with operating aspect.This needs the application tissue engineering in different medical field and the histoorgan reparative regeneration technology of regenerative medicine.
Cerebral dura mater/spinal dura mater is timely at wound and tumors invading, can cause damage and the defect of meninges in various degree or spinal meninges, then form cerebrospinal fluid seepage, secondary infection, the cicatrix even formed because of the abnormal healing of meninges and cause epilepsy.Serious infringement defect, will cause the substantive irreversible damage of cranium mesencephalic centre nervous system organs and degeneration.Current common Therapeutic Method is when damaging defect, selects the cranium brains film at other positions autologous, body muscular fasciae, or the higher biomass product of other antigenicities carries out tight stitching.Because autologous tissue is difficult to obtain, or do not reach expected effect because of insufficient often treatment of chosen material, and other succedaneums often have higher immunoreation, and can form scar tissue, affect therapeutic effect.
Chinese patent application CN101569765A " keeps I type medical collagen material of collagen original specific triple helix structure and products thereof and application " and discloses a kind of the I type medical collagen material and the extracting method thereof that keep collagen original specific triple helix structure, the biological diaphragm of the meninges adopting this I type medical collagen material to prepare/spinal meninges, and the preparation technology of this biological diaphragm and preparing the application in meninges/mater tissue repair materials.The artificial hard brain/spinal meninges invention feature of the type i collagen that this patent application relates to is emphatically to use the meninges of damage defect or the defect of spinal meninges to be had some strength, has the spongy meninges of class of high open three dimensional pore structures or the biological diaphragm of spinal meninges carries out coverings parcel, object is to exempt from stitching.So not only mechanical organizing of protection brain essential site is not disturbed by the intrusion of its hetero-organization of periphery; also establish the regeneration environment of the fibrous tissue at impaired meninges or spinal meninges position simultaneously; prevent the somatomedin of cerebrospinal fluid and Qi Nei from excessively losing; keeping the relatively stable of the environment of damaged tissues, providing suitable environment for repairing meninges or spinal meninges and its regeneration.The time of postoperative stitching defect dura mater can be reduced simultaneously widely, shorten the operation overall time, reduce patient's expense.
But this have mushy spongy film in Clinical practice process because its mechanical property is more weak, the poor performance sewed up, cause in neurosurgery, receive certain restriction.In the operation of neurosurgery, there is the operation case of considerable part to need must need tight suture defect when repairing in art as the pathological changes at basis cranii position, invent the inexorable trend that a kind of artificial meninges sewed up improving material mechanical performance becomes development for this reason.
Summary of the invention
The object of the present invention is to provide biological diaphragm of a kind of type i collagen material, meninges or spinal meninges sewed up and its preparation method and application.
The object of the present invention is to provide a kind of highly purified type i collagen material, a kind of meninges or the biological diaphragm of spinal meninges and preparation method thereof prepared by described type i collagen material with triple-helix structure, and described meninges or the biological diaphragm of spinal meninges are preparing the application in cerebral dura mater or mater tissue repair materials.
Biological diaphragm of a kind of type i collagen material, meninges or spinal meninges and preparation method thereof uses a kind of I type medical collagen material of collagen triple-helix structure that keeps under the special freezing conditions of pentanediol, make its collagen solution remain on the state of temperature of-4 ~+4 degree, and with per minute 3,000 ~ 18,000 rotating speed stirs, the temperature making it collagen serosity remains on above-mentioned centigrade state, and the triple-helix structure of its I collage raw material must not be destroyed.Collagen fiber in the biological diaphragm of the meninges for this reason produced or spinal meninges manage its triple-helix structure.
Type i collagen is the one of the multiple collagen of human body, is prevalent in a lot histoorgans; It is made up of 2 α 1 peptide chains and 1 α 2 peptide chain; To spiral composition by right-handed helix structure.As shown in Figure 4.
This kind of structure keeps the attribute of type i collagen, and as aminoacid close-packed arrays in certain sequence, the amino acid residue on adjacent peptide chains can make it crosslinked by distinct methods.But this structure becomes unstable when uniform temperature, the helical form between triple-helix structure opens the loose next stage product-gelatin that coordinates.So a kind of highly purified total protein content is more than 98%, Hydroxyproline content is greater than the type i collagen material that 10% structure keeps self distinctive triple-helix structure, it is characterized in that manufacturing in formation keeping carrying out stirring with certain speed under cryogenic just making its structure do not destroyed.
First aspect of the present invention is to provide a kind of highly purified type i collagen material with triple-helix structure, it is characterized in that, total protein content is more than 98%, and Hydroxyproline content is greater than 10%, and type i collagen fibre structure keeps self distinctive triple-helix structure.
And the biological diaphragm to be made up of the above-mentioned type i collagen material with triple-helix structure.
Second aspect of the present invention is to provide a kind of meninges or the biological diaphragm of spinal meninges and preparation method thereof prepared by type i collagen material described above.
Wherein, the making concentration of above-mentioned meninges or the biological diaphragm of spinal meninges is 1.5% ~ 3%, and diaphragm thickness is 1 ~ 3 millimeter, is densification micro-three dimensional pore structures membrane-like thing with 20 ~ 80 microns of large fine pores.
There being length and width, thick depositing just form three-dimensional, in case when the pore structure in it and size can be referred to as micro-pore structure when micrometer/nanometer rank.
Wherein, the preparation method of meninges described above or the biological diaphragm of spinal meninges, comprises the steps:
Step one, selects 1 ~ 4%(weight/volume) type i collagen be dissolved in the hydrochloric acid of 0.001 ~ 1N or the glacial acetic acid of 0.01 ~ 0.5M;
Step 2, be carry out homogenizing under the environment of pH1 ~ 5 to stir 0.5 ~ 3 hour in acid-base value, mixing speed is 3,000 ~ 6,000 rev/min;
Step 3, the sodium hydroxide solution adding 0.001 ~ 4N in whipping process regulates acid-base value to make it progressively be promoted to the alkaline environment of pH9 ~ 14;
Step 4, stir 0.5 ~ 1 hour, mixing speed is 10,000 ~ 18,000 rev/min.Thereafter the hydrochloric acid adding 0.001 ~ 1N in whipping process again regulates acid-base value again, makes its acid-base value be adjusted to down the environment of pH4 ~ 8 again;
Step 5, then keep whipping temp lower than 15 degrees Celsius, rapid stirring 1 ~ 3 hour, mixing speed is 18,000 ~ 30,000 rev/min;
Step 6, filters homogenizing fluid;
Step 7, then the ethanol adding 100%;
Step 8, be 0 degree Celsius ~+15 degrees Celsius at whipping temp thereafter, mixing speed is 3, under the condition of 000 ~ 10,000 rev/min, continue stirring 0.5 ~ 1 hour, poured into by the suspension obtained in medical stainless steel refrigerator tray, application Freeze Drying Technique makes meninges or the biological diaphragm of spinal meninges.
Wherein, in preparation method described above, above-mentioned in steps in through at a slow speed or high-speed low temperature carry out homogenizing stirring, whipping temp is 0 degree Celsius ~+15 degrees Celsius, and mixing time is 1 ~ 8 hour, and mixing speed is 3,000 ~ 30,000 rev/min.Described " homogenizing " is exactly the meaning of " stirring ", and homogenizing stirs and refers to that the stirring of 0.5% concentration collagen requires the stirring into quality homogeneity.The spongy biomembranous stirring of low concentration 0.5% of prior art to completing all constant under fixing same sour environment high-speed stirred, and the condition of collagen under its sour environment is constant; But difference of the present invention is, mixing time change is different, rotation speed change is different; The acid-base value of collagen in agitating solution is different simultaneously.Wherein key is that the environment that stirs is different, its acid-base value be changed to acidity-neutral-alkaline-weakly acidic pH.
Wherein, the preparation method of meninges described above or the biological diaphragm of spinal meninges, also comprises special lyophilization step, is specially:
1) cryogenic temperature be set to (1)+4 degree Celsius/30 minutes/in persistent state; (2)-75 ~-50 degrees Celsius/60-120 minute/in quick hold mode; (3)-70 ~-50 degrees Celsius/120 ~ 300 minutes/linearly hold mode; In process, the temperature of freezer dryer refrigerator keeps-90 ~-80 degrees Celsius;
2) temperature of drying stage is set to (1) 0 degree Celsius/maintenance 15 ~ 24 hours; (2) temperature of secondary temperature elevation is set to 20 degrees Celsius, and the time is 30 ~ 120 minutes;
3) above-mentioned all programs carry out that its vacuum in the middle of process arranges should at 200 ~ 350 millitorrs.
More specifically,
1) cryogenic temperature be set to (1)+4 degree Celsius/30 minutes/in persistent state; (2)-60 ~-40 degrees Celsius/120 ~ 300 minutes/curved state; (3)-60 ~-40 degrees Celsius/60 ~ 180 minutes/in persistent state; In process, the temperature of freezer dryer refrigerator should keep-80 degrees Celsius, and freezer temperature is-45 degrees Celsius;
2) temperature of drying stage is set to (1) 0 degree Celsius/maintenance 15 ~ 24 hours; (2) temperature of secondary temperature elevation is set to 20 degrees Celsius, and the time is 30 ~ 120 minutes;
3) above-mentioned all programs are carried out its vacuum in the middle of process and are set to 200 ~ 350 millitorrs.
Wherein, the preparation method of meninges described above or the biological diaphragm of spinal meninges, also comprise vacuum high-temperature dehydration (DHT) step, to strengthen the hinge of tropocollagen molecule peptide interchain, be specially: the meninges obtained or the biological diaphragm of spinal meninges are put into vacuum oven, its negative pressure arranges and should be negative 1 atmospheric pressure, and temperature is 100 ~ 120 degrees Celsius of process 48 ~ 96 hours.
Wherein, the preparation method of the biological diaphragm of meninges described above or spinal meninges, also comprises oxirane disinfection sterilization steps, make hole 20 ~ 80 microns there is micro-three dimensional pore structures meninges or the biological diaphragm of spinal meninges.
Wherein, oxirane disinfection sterilising conditions as above is as follows:
A, 50% pure ethylene oxide in addition 50% carbon dioxide in disinfector, carry out sterilizing;
B, sterilising temp are 50 degrees Celsius ± 5 degrees Celsius;
C, sterilizing humidity are 30% ~ 70%RH;
D, forevacuum are-30 ~-100Kpa;
E, ventilation vacuum are-30 ~-50Kpa;
F, rate of ventilation are 6 ~ 10 times;
G, sterilization time are 3 ~ 5 hours.
In said method, hydrochloric acid is N is equivalent.2) to be M be glacial acetic acid mole.
The ice crystal that the inner pore formation basic theory of sponge-like collagenous biological diaphragm is formed in refrigerating process by the liquid in its homogeneous slurry is determined.Because collagen molecules has very strong toughness.The chemistry of high density, high-quality collagenous biological film makes the homogeneous slurry that thinking and operating difficulties part are how to be formed by the material of thickness flowable.How long collagen fiber can be separated out from homogeneous slurry the fiber bodies forming high concentration is key of the present invention for this reason.Add the technology that ethanol is uniqueness of the present invention.
In lyophilization program, the method to set up of low temperature and cold degree temperature form liquid in serosity to become the key of ice crystal.On its of collagen fiber, attachment defines its hole.Collagen solution can control the viscosity of its highdensity collagen serosity according to the height of its acid-base value.This reconciles limit can be that Resolution Center increases acid or aqueous slkali and makes it slurry condition and change according to the acid-base value of collagen isoelectric point, IP.Short fiber in it can be aggregated into longer fiber by the collagen fiber simultaneously stirred at suitable homogenizing after its serosity raises pH value.
3rd aspect of the present invention is to provide meninges described above or the biological diaphragm of spinal meninges preparing the application in cerebral dura mater or mater tissue repair materials.
Wherein, the biological diaphragm of meninges described above or spinal meninges for the preparation of repair impaired cerebral dura mater/spinal dura mater nervous tissue material, be used for the treatment of or prevent because of wound and tumor cause cerebral dura mater or spinal meninges to damage or defect material, be used for the treatment of or prevent the purposes in the material of cerebrospinal leak because dura defect causes, infection, epilepsy.
Wherein, the biological diaphragm of above-mentioned meninges or spinal meninges can in 6 ~ 12 months degraded and absorbed.
Wherein, above-mentioned meninges or the biological diaphragm of spinal meninges are the epidural surfaces at any position covering damage or defect.
Wherein, above-mentioned meninges or the biological diaphragm of spinal meninges can be applicable to any position of craniocerebral injury and can sew up.
Wherein, above-mentioned meninges or the biological diaphragm of spinal meninges are preventing or are reducing the purposes in the material of cerebrospinal fluid seepage.
Wherein, above-mentioned meninges or the biological diaphragm of spinal meninges are preventing or are reducing the purposes in the material of tissue adhesion.
Wherein, for the performance making the I type medical collagen diaphragm with triple-helix structure have good anti-degradation property and anti alpha-smooth muscle contraction albumen, just must carry out hinge and reinforcement to loose tropocollagen molecule, the structure making it collagen molecules peptide interchain is changed.
Wherein, application said method is in preparation process, the mixing suspension of variable concentrations, different cryogenic temperatures and the different strong methods that adds match, and just can reach 20 ~ 80 microns of substantially imporous products with higher-strength and different degraded and absorbed rates.Preferred pore size is 20 ~ 80 microns, more preferably 20 ~ 60 microns, and best preferred value is 25 microns.
Wherein, the concentration of above-mentioned I-type collagen is preferably 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%.Preferred concentration is 1% ~ 4%, and more preferably concentration is 1.5 ~ 3%, and best preferred value is 1.75 ~ 2%.All percentage value units of the present invention are quality/volume.
In a word, the invention provides the I type medical collagen with triple-helix structure that a kind of use 98% high protein purity, the auxiliary histidine content of hydroxyl be greater than 10% be material make high concentration, high density, micro-/without the preparation method of the collagen the sewed up diaphragm of open space.Be used in the repair materials as meninges or mater tissue after meninges or spinal meninges damage and defect, for suture repair, repair impaired cerebral dura mater/dura mater tissue and protection central nervous system tissue.
Beneficial effect of the present invention comprises: the invention provides I type medical collagen material that a kind of use has a triple-helix structure and prepare high density and can sew up the meningeal use and preparation method that degradation time extends.Can promote that the cerebral dura mater/spinal dural cell damaging defect is divided a word with a hyphen at the end of a line, breaks up, regenerated on the collagen scaffold provided, form self identical tissue, disturb fibroblastic hypertrophy of non-sequence to form cicatrix simultaneously.Invention increases the mechanical property of mechanical brains membrane material, can in neurosurgery while repairing (pathological changes as basis cranii position need in art) tight suture defect, overcome the defect that prior art makes neurosurgery limited because mechanical property is weak and sewing properties is poor, effective tissue repairing damage defect, reduces the misery of patient.
Accompanying drawing explanation
Figure 1A is entirety solid (transparent effect) schematic diagram of the biological diaphragm of meninges or spinal meninges.
Figure 1B is the front view of the biological diaphragm of meninges or spinal meninges.
Fig. 2 A has the I type medical collagen material meninges of triple-helix structure or the biological diaphragm riding position of spinal meninges, stitching and and the relation schematic diagram of cranium cerebral tissue.
Fig. 2 B is the profile along the A-B section in Fig. 2 A.
Fig. 3 A is the schematic diagram of one of biomembrane using method clinically.
Fig. 3 B is the schematic diagram of biomembrane using method two clinically.
Fig. 4 is the schematic diagram of the triple-helix structure that type i collagen material has.
Detailed description of the invention
Below in conjunction with accompanying drawing and instantiation, the invention will be further described.
Figure 1A is entirety solid (transparent effect) schematic diagram of the biological diaphragm of meninges or spinal meninges; Figure 1B is the front view of the biological diaphragm of meninges or spinal meninges.Wherein, Reference numeral implication is: 1---hole (aperture is 20 ~ 80 microns).Wherein, meninges or the biological diaphragm of spinal meninges are the I type medical collagen material meninges with triple-helix structure or the spinal meninges biomembrane of 20 ~ 80 micro-void/1.5 ~ 3 millimeters thick 98% purity, namely, pore size is the high density diaphragm of the micro three-dimensional structure hole of 20 ~ 80 microns.
Fig. 2 A be have the I type medical collagen material meninges of triple-helix structure or the biological diaphragm riding position of spinal meninges, stitching and and the relation schematic diagram of cranium cerebral tissue; Fig. 2 B is the profile along the A-B section in Fig. 2 A.Wherein, Reference numeral implication is: 2---skull hone lamella; 3---collagenous biological film; 4---normal meninges; 5---brain entity; 6---suture.
Fig. 3 A is the schematic diagram of one of biomembrane using method clinically, and wherein, biomembrane size is slightly larger than the meninges of defect; Fig. 3 B is the schematic diagram of biomembrane using method two clinically, and wherein biomembrane size meets the meninges of defect.Wherein, Reference numeral implication is: 2---skull hone lamella; 3---collagenous biological film; 4---normal meninges; 5---brain entity; 6---suture.
Fig. 4 is the schematic diagram of the triple-helix structure that type i collagen material has.Wherein, Reference numeral implication is: 7---Article 1 α 1-peptide chain right-handed helix structure; 8---Article 2 α 1-peptide chain right-handed helix structure; 9---α 2-peptide chain right-handed helix structure.
Preparation embodiment
Prepare meninges or the biological diaphragm example of spinal meninges:
Embodiment 1: prepare the biological diaphragm of 1.0% meninges/spinal meninges
Apply 7.2 grams of highly purified I type medical collagen materials with triple-helix structure uses 10,000 and 18,000 rotating speed stir 30 minutes and within 1.5 hours, make the collagen composite suspension of 1.% concentration under the environment of hydrochloric acid.Thereafter use sodium hydroxide to regulate its acid-base value, make it to reach the satisfactory state of viscosity.Getting 500 milliliters pours in the freezing pallet of 50X50 centimetre, under the cryogenic conditions of-60 degrees Celsius, freezing 3 ~ 4 hours, dry 15 hours, makes 60 microns, hole, the biological diaphragm of meninges/spinal meninges of 3 millimeters thick.
Embodiment 2: prepare the biological diaphragm of 2% meninges/spinal meninges
Apply 14.4 grams of highly purified I type medical collagen materials with triple-helix structure and use 3,000,10,000 and 18 under the environment of hydrochloric acid, 000 rotating speed stirs 30 minutes, 30 minutes and within 1.0 hours, makes the collagen composite suspension of 2% concentration.Thereafter use sodium hydroxide to regulate its acid-base value, make it to reach the satisfactory state of viscosity.Thereafter add 100% ethanol and continue stirring 30 minutes.Getting 500 milliliters pours in the freezing pallet of 50X50 centimetre, under the cryogenic conditions of-70 degrees Celsius, freezing 3 ~ 4 hours, dry 20 hours, makes 20 microns, hole, the biological diaphragm of meninges/spinal meninges of 2.5 millimeters thick.
Embodiment 3: prepare the biological diaphragm of 2.5% meninges/spinal meninges
Apply 18 grams of highly purified I type medical collagen materials with triple-helix structure and adopt 3 under the environment of hydrochloric acid, 000,10,00,18,000 and 28,000 rotating speed stirs 15 minutes, 30 minutes, 30 minutes and within 1 hour, makes the collagen composite suspension of 2.5% concentration.Thereafter use sodium hydroxide to regulate its acid-base value and 100% ethanol, make it to reach the satisfactory state of viscosity.Continue stirring 15 minutes.Getting 500 milliliters pours in the freezing pallet of 50X50 centimetre, under the cryogenic conditions of-75 degrees Celsius, freezing 3 ~ 6 hours, dry 25 hours, makes hole and is less than 20 microns, the biological diaphragm of meninges/spinal meninges of 2 millimeters thick.
Effect example
Test method and result: the cerebral dura mater covering damaged or defect/spinal dura mater position that meninges or the biological diaphragm of spinal meninges expand, platelet in blood around its tissue is adsorbed in a large number, simultaneously prothrombing will permeate the blood coagulation activity that to be activated in the three-dimensional pore space of collagem membrane, and blood coagulation fiber makes collagem membrane contact with normal brain activity film close.Due to its diaphragm three-dimensional pore space is very little or imporosity so the adhesion time of biological diaphragm may be longer, the raw material of high concentration and the biomembrane of freezing have high-strength tensile performance, prevent oozing out of cerebrospinal fluid so can carry out sewing up with meninges, make the environment of intracranial corresponding stable.The fibroblast of dura mater itself is divided a word with a hyphen at the end of a line by the three dimensional structure of collagem membrane and profile, differentiation, regeneration thus promote the regeneration of dura mater newborn in damaged tissue.
Embodiment 4: relevant meninges or the biomembranous zoopery of spinal meninges
The zoopery of meninges or the biological diaphragm of spinal meninges: 6 male hunting dogs carry out outside craniectomy before right side.By the trepan every trade right front outside parietal bone otomy of diameter 3 centimetres.Do not damage any skull undertissue.After careful hemostasis, the cerebral dura mater under it is mentioned, the cerebral dura mater of careful shearing diameter 3 centimetres.Protect the arachnoidea under it and pia mater encephali without any damage.
By the position of implantation dura defect careful for the collagen diaphragm of diameter 3.5 centimetres, cover the surface of dural defect and brain essence and sew up.Thereafter after careful hemostasis, the Cranial-bone-flap of excision is put back to the position of defect of skull.
The whole health of postoperative animal survives, and without any nerve, psychological problem symptom, anencephaly cerebrospinal fluid leakage, without infecting.Put to death animal after week, the center of the primordial skull bone after resetting with first time operation bone lobe with the trepan of diameter 6 centimetres is new center, and expanded scope excision skull, exposes the cerebral dura mater under it and brain parenchymal tissue.
Perusal: the right front outside parietal bone healing of 6 laboratory animals is bright good, without any infection.After raising bone lobe, without obvious adhesion between skull and cerebral dura mater, newborn cerebral dura mater tissue-smooth, cerebral dura mater, without obvious adhesion and cicatrix between arachnoidea and brain essence.Brain material surface is normal, changes without any degenerative.
Histological examination: by skull/cerebral dura mater/brain essence entirety excision.Through decalcification, after cured piece of embedding, make 5 ~ 6 microns of slabs tissues observed form after HE dyeing.
Under microscope, all no abnormal histiocyte of conventional cut sections for microscopic examination occurs.Entirety is shown as fibroblast and the fiber knot tellurium tissue of sequence.Compare except having activeness regenerative cell to divide in newborn cerebral dura mater substrate outside the pale of civilization with normal cerebral dura mater, in structure, form and normal cerebral dura mater are as good as.Without adhesion, without cicatrization.Newborn fibrous tissue is shown under polarizing microscope
Present the distinctive structure of type i collagen-interim spectral composition, and the collagem membrane degraded and absorbed all implanted, without any residual collagen-based materials.
The invention provides I type medical collagen material that a kind of use has a triple-helix structure to prepare high density and can sew up the meningeal use and preparation method that degradation time extends, can promote that the cerebral dura mater/spinal dural cell damaging defect is divided a word with a hyphen at the end of a line, breaks up, regenerated on the collagen scaffold provided, form self identical tissue, disturb fibroblastic hypertrophy of non-sequence to form cicatrix simultaneously, effective tissue repairing damage defect, reduce the misery of patient, for larger welfare is sought by society.
Claims (7)
1. a preparation method for the biological diaphragm of type i collagen material, meninges or spinal meninges, it is characterized in that, this preparation method comprises the steps:
Step one, selects the type i collagen of 1 ~ 4% weight/volume percent to be dissolved in the hydrochloric acid of 0.001 ~ 1N or the glacial acetic acid of 0.01 ~ 0.5M;
Step 2, be carry out homogenizing under the environment of pH1 ~ 5 to stir 0.5 ~ 3 hour in acid-base value, mixing speed is 3,000 ~ 6,000 rev/min;
Step 3, the sodium hydroxide solution adding 0.001 ~ 4N in whipping process regulates acid-base value to make it progressively be promoted to the alkaline environment of pH9 ~ 14;
Step 4, stir 0.5 ~ 1 hour, mixing speed is 10,000 ~ 18,000 rev/min, and the hydrochloric acid adding thereafter 0.001 ~ 1N in whipping process again regulates acid-base value again, makes its acid-base value be adjusted to down the environment of pH4 ~ 8 again;
Step 5, then keep whipping temp lower than 15 degrees Celsius, rapid stirring 1 ~ 3 hour, mixing speed is 18,000 ~ 20,000 rev/min;
Step 6, filters homogenizing fluid;
Step 7, then the ethanol adding 100%;
Step 8, be 0 degree Celsius ~+15 degrees Celsius at whipping temp thereafter, mixing speed is 3, under the condition of 000 ~ 10,000 rev/min, continue stirring 0.5 ~ 1 hour, poured into by the suspension obtained in medical stainless steel refrigerator tray, application Freeze Drying Technique makes meninges or the biological diaphragm of spinal meninges;
Above-mentioned type i collagen, comprise a kind of highly purified type i collagen material with triple-helix structure, its total protein content is more than 98%, and Hydroxyproline content is greater than 10%, and type i collagen fibre structure keeps self distinctive triple-helix structure;
Above-mentioned biological diaphragm, comprises the biological diaphragm that the above-mentioned type i collagen material with triple-helix structure is made;
Above-mentioned meninges or the biological diaphragm of spinal meninges, comprise the meninges or the biological diaphragm of spinal meninges be made up of above-mentioned type i collagen, its diaphragm thickness is 1 ~ 3 millimeter, is densification micro-three dimensional pore structures membrane-like thing with 20 ~ 80 microns of large fine pores.
2. preparation method as claimed in claim 1, is characterized in that, above-mentioned in steps in through at a slow speed or high-speed low temperature carry out homogenizing stirring, whipping temp is 0 degree Celsius ~+15 degrees Celsius, and mixing time is 1 ~ 8 hour, and mixing speed is 3,000 ~ 20,000 rev/min.
3. preparation method as claimed in claim 1, is characterized in that, step 8 is dry through special lyophilization Slow-rate freezing, is specially:
1) cryogenic temperature be set to (1)+4 degree Celsius/30 minutes/in persistent state; (2)-75 ~-50 degrees Celsius/60 ~ 120 minutes/in quick hold mode; (3)-70 ~-50 degrees Celsius/120 ~ 300 minutes/linearly hold mode; In process, the temperature of freezer dryer refrigerator keeps-90 ~-80 degrees Celsius;
2) temperature of drying stage is set to (1) 0 degree Celsius/maintenance 15 ~ 24 hours; (2) temperature of secondary temperature elevation is set to 20 degrees Celsius, and the time is 30 ~ 120 minutes;
3) above-mentioned all programs carry out that its vacuum in the middle of process arranges should at 200 ~ 350 millitorrs.
4. preparation method as claimed in claim 1, it is characterized in that, also comprise vacuum high-temperature dehydration DHT step, to strengthen the hinge of tropocollagen molecule peptide interchain, be specially: the meninges obtained or the biological diaphragm of spinal meninges are put into vacuum oven, its negative pressure arranges and should be negative 1 atmospheric pressure, and temperature is 100 ~ 120 degrees Celsius of process 48 ~ 96 hours.
5. preparation method as claimed in claim 1, is characterized in that, also comprise oxirane disinfection sterilization steps, makes hole at densification micro-three dimensional pore structures membrane-like thing of 20 ~ 80 microns.
6. the preparation method as described in claim 1 or 3, is characterized in that, in described Freeze Drying Technique
1) cryogenic temperature be set to (1)+4 degree Celsius/30 minutes/in persistent state; (2)-60 ~-40 degrees Celsius/120 ~ 300 minutes/curved state; (3)-60 ~-40 degrees Celsius/60 ~ 180 minutes/in persistent state; In process, the temperature of freezer dryer refrigerator should keep-80 degrees Celsius, and freezer temperature is-45 degrees Celsius;
2) temperature of drying stage is set to (1) 0 degree Celsius/maintenance 15 ~ 24 hours; (2) temperature of secondary temperature elevation is set to 20 degrees Celsius, and the time is 30 ~ 120 minutes;
3) above-mentioned all programs are carried out its vacuum in the middle of process and are set to 200 ~ 350 millitorrs.
7. preparation method as claimed in claim 5, is characterized in that, use oxirane disinfection sterilization steps is:
A, 50% pure ethylene oxide in addition 50% carbon dioxide in disinfector, carry out sterilizing;
B, sterilising temp are 50 degrees Celsius ± 5 degrees Celsius;
C, sterilizing humidity are 30% ~ 70%RH;
D, forevacuum are-30 ~-100Kpa;
E, ventilation vacuum are-30 ~-50Kpa;
F, rate of ventilation are 6 ~ 10 times;
G, sterilization time are 3 ~ 5 hours.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210445760.4A CN103800941B (en) | 2012-11-09 | 2012-11-09 | Type-I collagen material, meninx and meninge biological membrane and preparation method and application of thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210445760.4A CN103800941B (en) | 2012-11-09 | 2012-11-09 | Type-I collagen material, meninx and meninge biological membrane and preparation method and application of thereof |
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| Publication Number | Publication Date |
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| CN103800941A CN103800941A (en) | 2014-05-21 |
| CN103800941B true CN103800941B (en) | 2015-04-22 |
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| US6440167B2 (en) * | 1996-11-20 | 2002-08-27 | Yasuhiko Shimizu | Collagen material and its production process |
| CN101564552A (en) * | 2009-03-20 | 2009-10-28 | 华南理工大学 | Method for preparing high-strength collagen tissue repair material product |
| CN101569765A (en) * | 2009-06-23 | 2009-11-04 | 许和平 | I-type medical collagen material keeping original specific triple helix structure of collagen, product and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6440167B2 (en) * | 1996-11-20 | 2002-08-27 | Yasuhiko Shimizu | Collagen material and its production process |
| CN101564552A (en) * | 2009-03-20 | 2009-10-28 | 华南理工大学 | Method for preparing high-strength collagen tissue repair material product |
| CN101569765A (en) * | 2009-06-23 | 2009-11-04 | 许和平 | I-type medical collagen material keeping original specific triple helix structure of collagen, product and application thereof |
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