CN102188746A - Artificial extracellular matrix and preparation method thereof - Google Patents
Artificial extracellular matrix and preparation method thereof Download PDFInfo
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- CN102188746A CN102188746A CN2010101222773A CN201010122277A CN102188746A CN 102188746 A CN102188746 A CN 102188746A CN 2010101222773 A CN2010101222773 A CN 2010101222773A CN 201010122277 A CN201010122277 A CN 201010122277A CN 102188746 A CN102188746 A CN 102188746A
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Abstract
The invention discloses an artificial extracellular matrix and a preparation method thereof. The artificial extracellular matrix is mainly prepared from chitosan, collagen, sodium hyaluronate and lysine, wherein the mass ratio of the chitosan to the collagen to the sodium hyaluronate to the lysine is (0.1-400):(1-400):(0.1-5000):(0.1-5000). The preparation method of the artificial extracellular matrix comprises the following steps: mixing a chitosan colloidal solution, a collagen hydrogel, a sodium hyaluronate aqueous solution and a lysine aqueous solution, and adding a crosslinking agent to react; and carrying out freeze-drying on the crosslinked mixed gel, thus obtaining the artificial extracellular matrix. The artificial extracellular matrix disclosed by the invention has good controllable degradation property, biocompatibility and tissue repair performance, and can be used as a clinically applicable artificial dura mater.
Description
Technical field
The present invention relates to artificial extracellular matrix and preparation method thereof.
Background technology
The neurosurgical doctor often utilizes in operation from the body cranial periosteum clinically, cervical muscle fascia, medicated cap shape chamber film, autologous tissue's cerebral dura mater repairing such as fascia lata are damaged, but not only spended time is long to get these materials, sometimes these are organized self to sustain damage and can not be utilized, the position that has self does not just have complete available tissue, often causes not having enough areas to be utilized.Transplanting its hetero-organization not only needs the art preplanning, prepares second operative site, increases operation and anesthesia duration, also will produce new wound.In addition, even use autologous tissue, the anoxia regular meeting of autologous tissue induces the inflammatory reaction of below cortex, causes the adhesion between meninges and the brain.
The artificial dura mater material of clinical practice at present mainly is divided into allogeneic material, foreign material, synthetic material and four types of natural material.
The allogeneic meninges is to be used for clinical non-repair materials from body the earliest.This material is to obtain from the human body brain of death, processes the back as commodity selling through lyophilization.Advantage is: preparation is simple, can directly use, implant conveniently, can long preservation, and the physiological property that has kept fresh meninges is early stage fibroblastic infiltration and revascularization, eliminates rejection in the normal structure around it can be fused to.But the allosome cerebral dura mater has 3 significant weakness: the one, and material source is restricted, and the donor lacks, but also has the problem of ethics aspect; The 2nd, the existence of infectious protein prime factor (Prion), investigation report according to Japanese health ministry in March, 1997 issue, 766 Creutz Fil spy-Jacob (Creutzfeldt-JakobSyndrome (disease), CJD) among the patient, 28 examples live through the dry duramater reparation art of human body, compare with the millionth spontaneous generation rate of JCD, the people JCD sickness rate of making the cerebral dura mater kposthesis is apparently higher than the natural occurrence rate, for this reason, the ban of the dry meninges of allosome is used in the Health and human services department issue, World Health Organization (WHO) also issues and bans use of the dural urgent notice of people's allosome in the operation simultaneously, so some country has cancelled the freezing human cerebral dura mater of use as patching material at present; The 3rd, costing an arm and a leg has surpassed patient's ability to bear.
Both at home and abroad, xenogenesis cerebral dura mater repair materials is in the more kind of clinical practice, mainly is with bovine pericardium and the preparation of Cor Caprae seu ovis peplos.The advantage of xenogenesis dural repairment material is to be convenient to obtain, and preparation is simple, and leakageresistance is better.But because the safety problem of animal derived material in recent years, the country that has has limited the use of such material.
The easy processing and forming of synthetic material is easy to sterilization, does not have the risk of the virus transmitted.The material that is usually used in the repairing of hard brain (ridge) film has silicone rubber, polypropylene, politef etc.But these materials only have the function of filling-up mechanically, and do not have the ability of short tissue regeneration, especially under the bad situation of material processed, and day long seepage that is easy to generate, even can cause capillary hemorrhage, produce chronic inflammatory disease.
Natural material is the natural biological macromolecular substances of purifying through processing, and the material that can be used for the pachymeninx repairing has collagen, chitosan, chondroitin sulfate etc.They are called as the artificial extracellular matrix, are the favorable tissue engineering scaffold materials.They also can be used as the good substrate of cell growth except having the mechanical barrier effect, the function of transfer sell chemical signal is arranged, and therefore can promote tissue regeneration.In this process, material itself is degraded, and finally the autologous tissue of being healed substitutes.Modern just regenerative medicine requirement of this point and promotion.But these materials also have shortcoming, and are too fast such as degradation rate, so that self new meninges also is not completed into and material is degraded.
Summary of the invention
The purpose of this invention is to provide a kind of artificial extracellular matrix that good controllable degradation property is arranged and preparation method thereof.
Artificial extracellular matrix provided by the present invention mainly is prepared from by chitosan, collagen protein, hyaluronate sodium and lysine.
Artificial extracellular matrix of the present invention, wherein, described chitosan, described collagen protein, described hyaluronate sodium and described lysine are crosslinked by cross-linking agent.
Artificial extracellular matrix of the present invention, wherein, the mass ratio of described chitosan, described collagen protein, described hyaluronate sodium and described lysine is (0.1～400): (1～400): (0.1～5000): (0.1～5000).
Artificial extracellular matrix of the present invention, wherein, described cross-linking agent be selected from following any or appoint several: formaldehyde, glutaraldehyde, carbodiimides, diepoxides, genipin and procyanidin.
Artificial extracellular matrix's provided by the present invention preparation method comprises the steps:
Chitosan gum liquid solution, collagen protein hydrogel, aqueous solution of sodium hyaluronate and lysine solution are mixed, add the cross-linking agent reaction again;
Crosslinked good mixed gel lyophilization obtains described artificial extracellular matrix.
Artificial extracellular matrix's of the present invention preparation method, wherein, the mass ratio of described chitosan, described collagen protein, described hyaluronate sodium and described lysine is (0.1～400): (1～400): (0.1～5000): (0.1～5000).
Artificial extracellular matrix's of the present invention preparation method, wherein, described cross-linking agent be selected from following any or appoint several: formaldehyde, glutaraldehyde, carbodiimides, diepoxides, genipin and procyanidin.
Artificial extracellular matrix's of the present invention preparation method, wherein, described chitosan gum liquid solution is the acetic acid solution of 0.1～5mg/ml chitosan, the concentration of acetic acid is 0.1ml/100ml～0.5ml/100ml in the described acetic acid solution; The concentration of collagen protein is 1～50mg/ml in the described collagen protein hydrogel; The concentration of hyaluronate sodium is 0.1～100mg/ml in the described aqueous solution of sodium hyaluronate; The concentration of lysine is 0.1～100mg/ml in the described lysine solution.
Artificial extracellular matrix's of the present invention preparation method, wherein, described cryodesiccated temperature is-80 ℃～40 ℃.
Artificial extracellular matrix of the present invention has good controllable degradation property, and excellent biological compatibility energy and tissue repair performance can be used as clinical suitable novel artificial cerebral dura mater.
The specific embodiment
Embodiment 1, artificial extracellular matrix
0.01ml/100ml acetic acid solution in add the purified chitosan of deacetylation 60%, stirred 2 hours, be mixed with the chitosan gum liquid solution of 0.1mg/ml.
With 0.1mg/ml chitosan gum liquid solution, 1mg/ml collagen protein hydrogel, 0.1mg/ml putting into crosslinked jar, aqueous solution of sodium hyaluronate and 0.1mg/ml lysine solution mix, 0.1mg/ml chitosan gum liquid solution, 1mg/ml collagen protein hydrogel, 0.1mg/ml the volume ratio of aqueous solution of sodium hyaluronate and 0.1mg/ml lysine solution is 1: 1: 1: 1, adding carbodiimides again is mixed, the mass ratio of carbodiimides and chitosan is 0.001: 1,50 ℃ were reacted 2 hours, be cooled to room temperature, the lyophilizing dish vacuum lyophilization under-80 ℃～40 ℃ conditions of packing into of crosslinked good mixed gel was obtained the artificial extracellular matrix in 48 hours.
Artificial extracellular matrix's outward appearance is the lamellar rectangle of white, and there is crystal gloss on the surface, and visible material is random short fiber network shape structure under naked eyes or the magnifier, knows that with feel certain toughness is arranged, the clean inviolateness of appearance.
Artificial extracellular matrix's length is 4.32～4.56cm, and wide is 4.24～4.54cm, and thick is 0.5～0.9cm.
Artificial extracellular matrix's chemical property: artificial extracellular matrix's residue on ignition 0.3%, pH value 7.1, artificial extracellular matrix's content of beary metal (in lead) is less than 10ug/g.
Artificial extracellular matrix's biology performance: steriling test (GB/T 19973.2-2005) artificial extracellular matrix meets sterility specifications.The cytotoxicity that cell toxicity test (GB/T 16886.5-2003) detects the artificial extracellular matrix is 0～1 grade.Bacterial endotoxin test (EN455-3-2000) detects artificial extracellular matrix's bacterial endotoxin less than 0.5EU/ml.Salmonella reversion test (GB/T 16886.3-2008) detects negative.Chromosomal aberration test (GB/T 16886.3-2008) detects negative.Micronucleus test (GB/T 16886.3-2008) detects negative.Sensitization test (STT) (GB/T 16886.10-2005) detects no sensitivity response.Acute for the no acute living body toxic reaction of poison test (ISO 10993-11:2006) detection.Implant back local response test (ISO 10993-6:2007), after the artificial extracellular matrix implanted for 1 week, more lymphocyte, neutrophil cell and a small amount of multinucleated giant cell are arranged around sample material, have granulation tissue to hold around the material.After implanting for 4 weeks, more lymphocyte, neutrophil cell are arranged around sample material.After implanting for 12 weeks, implant site has few lymphocyte, neutrophil cell, and sample material is degraded and absorbed, and no naked eyes can be distinguished foreign body.
Artificial extracellular matrix in the The above results explanation present embodiment has good controllable degradation property, excellent biological compatibility energy and tissue repair performance.
Embodiment 2, artificial extracellular matrix
0.5ml/100ml acetic acid solution in add the purified chitosan of deacetylation 70%, stirred 10 hours, be mixed with the chitosan gum liquid solution of 5mg/ml.
With 5mg/ml chitosan gum liquid solution, 50mg/ml collagen protein hydrogel, 100mg/ml aqueous solution of sodium hyaluronate and 100mg/ml lysine solution are put into crosslinked jar and are mixed, 5mg/ml chitosan gum liquid solution, 50mg/ml collagen protein hydrogel, the volume ratio of 100mg/ml aqueous solution of sodium hyaluronate and 100mg/ml lysine solution is 80: 80: 50: 50, add glutaraldehyde again, the volume ratio of glutaraldehyde and chitosan gum liquid solution is to be mixed at 1: 400,50 ℃ were reacted 2 hours, be cooled to room temperature, the lyophilizing dish vacuum lyophilization under-60 ℃～30 ℃ conditions of packing into of crosslinked good mixed gel was obtained the artificial extracellular matrix in 48 hours.
Artificial extracellular matrix's outward appearance is the lamellar rectangle of white, and there is crystal gloss on the surface, and visible material is random short fiber network shape structure under naked eyes or the magnifier, knows that with feel certain toughness is arranged, the clean inviolateness of appearance.
Artificial extracellular matrix's length is 4.32～4.56cm, and wide is 4.24～4.54cm, and thick is 0.5～0.9cm.
Artificial extracellular matrix's chemical property: artificial extracellular matrix's residue on ignition 0.5%, pH value 7.1, artificial extracellular matrix's content of beary metal (in lead) is less than 10ug/g.
Artificial extracellular matrix's biology performance: steriling test (GB/T 19973.2-2005) artificial extracellular matrix meets sterility specifications.The cytotoxicity that cell toxicity test (GB/T 16886.5-2003) detects the artificial extracellular matrix is 0～1 grade.Bacterial endotoxin test (EN455-3-2000) detects artificial extracellular matrix's bacterial endotoxin less than 0.5EU/ml.Salmonella reversion test (GB/T 16886.3-2008) detects negative.Chromosomal aberration test (GB/T 16886.3-2008) detects negative.Micronucleus test (GB/T 16886.3-2008) detects negative.Sensitization test (STT) (GB/T 16886.10-2005) detects no sensitivity response.Acute for the no acute living body toxic reaction of poison test (ISO 10993-11:2006) detection.Implant back local response test (ISO 10993-6:2007), after the artificial extracellular matrix implanted for 1 week, more lymphocyte, neutrophil cell and a small amount of multinucleated giant cell are arranged around sample material, have granulation tissue to hold around the material.After implanting for 4 weeks, more lymphocyte, neutrophil cell are arranged around sample material.After implanting for 12 weeks, implant site has few lymphocyte, neutrophil cell, and sample material is degraded and absorbed, and no naked eyes can be distinguished foreign body.
Artificial extracellular matrix in the The above results explanation present embodiment has good controllable degradation property, excellent biological compatibility energy and tissue repair performance.
Embodiment 3, artificial extracellular matrix
0.3ml/100ml acetic acid solution in add the purified chitosan of deacetylation 85%, stirred 30 hours, be mixed with the chitosan gum liquid solution of 2mg/ml.
With 2mg/ml chitosan gum liquid solution, 10mg/ml collagen protein hydrogel, 10mg/ml aqueous solution of sodium hyaluronate and 20mg/ml lysine solution are put into crosslinked jar and are mixed, 2mg/ml chitosan gum liquid solution, 10mg/ml collagen protein hydrogel, the volume ratio of 10mg/ml aqueous solution of sodium hyaluronate and 20mg/ml lysine solution is 80: 20: 30: 30, adding diepoxides again is mixed, the volume ratio of diepoxides and chitosan gum liquid solution is 1: 500,50 ℃ were reacted 2 hours, be cooled to room temperature, the lyophilizing dish vacuum lyophilization under-40 ℃～40 ℃ conditions of packing into of crosslinked good mixed gel was obtained the artificial extracellular matrix in 48 hours.
Artificial extracellular matrix's outward appearance is the lamellar rectangle of white, and there is crystal gloss on the surface, and visible material is random short fiber network shape structure under naked eyes or the magnifier, knows that with feel certain toughness is arranged, the clean inviolateness of appearance.
Artificial extracellular matrix's length is 4.32～4.56cm, and wide is 4.24～4.54cm, and thick is 0.5～0.9cm.
Artificial extracellular matrix's chemical property: artificial extracellular matrix's residue on ignition 0.4%, pH value 7.2, artificial extracellular matrix's content of beary metal (in lead) is less than 10ug/g.
Artificial extracellular matrix's biology performance: steriling test (GB/T 19973.2-2005) artificial extracellular matrix meets sterility specifications.The cytotoxicity that cell toxicity test (GB/T 16886.5-2003) detects the artificial extracellular matrix is 0～1 grade.Bacterial endotoxin test (EN455-3-2000) detects artificial extracellular matrix's bacterial endotoxin less than 0.5EU/ml.Salmonella reversion test (GB/T 16886.3-2008) detects negative.Chromosomal aberration test (GB/T 16886.3-2008) detects negative.Micronucleus test (GB/T 16886.3-2008) detects negative.Sensitization test (STT) (GB/T 16886.10-2005) detects no sensitivity response.Acute for the no acute living body toxic reaction of poison test (ISO 10993-11:2006) detection.Implant back local response test (ISO 10993-6:2007), after the artificial extracellular matrix implanted for 1 week, more lymphocyte, neutrophil cell and a small amount of multinucleated giant cell are arranged around sample material, have granulation tissue to hold around the material.After implanting for 4 weeks, more lymphocyte, neutrophil cell are arranged around sample material.After implanting for 12 weeks, implant site has few lymphocyte, neutrophil cell, and sample material is degraded and absorbed, and no naked eyes can be distinguished foreign body.
Artificial extracellular matrix in the The above results explanation present embodiment has good controllable degradation property, excellent biological compatibility energy and tissue repair performance.
Embodiment 4, artificial extracellular matrix
0.1ml/100ml acetic acid solution in add the purified chitosan of deacetylation 99%, stirred 60 hours, be mixed with the chitosan gum liquid solution of 3mg/ml.
With 3mg/ml chitosan gum liquid solution, 30mg/ml collagen protein hydrogel, 30mg/ml aqueous solution of sodium hyaluronate and 30mg/ml lysine solution are put into crosslinked jar and are mixed, 3mg/ml chitosan gum liquid solution, 30mg/ml collagen protein hydrogel, the volume ratio of 30mg/ml aqueous solution of sodium hyaluronate and 30mg/ml lysine solution is 50: 30: 10: 10, adding formaldehyde again is mixed, the volume ratio of formaldehyde and chitosan gum liquid solution is 1: 600,50 ℃ were reacted 2 hours, be cooled to room temperature, the lyophilizing dish vacuum lyophilization under-80 ℃～30 ℃ conditions of packing into of crosslinked good mixed gel was obtained the artificial extracellular matrix in 48 hours.
Artificial extracellular matrix's outward appearance is the lamellar rectangle of white, and there is crystal gloss on the surface, and visible material is random short fiber network shape structure under naked eyes or the magnifier, knows that with feel certain toughness is arranged, the clean inviolateness of appearance.
Artificial extracellular matrix's length is 4.32～4.56cm, and wide is 4.24～4.54cm, and thick is 0.5～0.9cm.
Artificial extracellular matrix's chemical property: artificial extracellular matrix's residue on ignition 0.4%, pH value 7.1, artificial extracellular matrix's content of beary metal (in lead) is less than 10ug/g.
Artificial extracellular matrix's biology performance: steriling test (GB/T 19973.2-2005) artificial extracellular matrix meets sterility specifications.The cytotoxicity that cell toxicity test (GB/T 16886.5-2003) detects the artificial extracellular matrix is 0～1 grade.Bacterial endotoxin test (EN455-3-2000) detects artificial extracellular matrix's bacterial endotoxin less than 0.5EU/ml.Salmonella reversion test (GB/T 16886.3-2008) detects negative.Chromosomal aberration test (GB/T 16886.3-2008) detects negative.Micronucleus test (GB/T 16886.3-2008) detects negative.Sensitization test (STT) (GB/T 16886.10-2005) detects no sensitivity response.Acute for the no acute living body toxic reaction of poison test (ISO 10993-11:2006) detection.Implant back local response test (ISO 10993-6:2007), after the artificial extracellular matrix implanted for 1 week, more lymphocyte, neutrophil cell and a small amount of multinucleated giant cell are arranged around sample material, have granulation tissue to hold around the material.After implanting for 4 weeks, more lymphocyte, neutrophil cell are arranged around sample material.After implanting for 12 weeks, implant site has few lymphocyte, neutrophil cell, and sample material is degraded and absorbed, and no naked eyes can be distinguished foreign body.
Artificial extracellular matrix in the The above results explanation present embodiment has good controllable degradation property, excellent biological compatibility energy and tissue repair performance.
Above embodiment is described preferred implementation of the present invention; be not that scope of the present invention is limited; design under the prerequisite of spirit not breaking away from the present invention; various distortion and improvement that the common engineers and technicians in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (10)
1. an artificial extracellular matrix mainly is prepared from by chitosan, collagen protein, hyaluronate sodium and lysine.
2. artificial extracellular matrix according to claim 1 is characterized in that: described chitosan, described collagen protein, described hyaluronate sodium and described lysine are crosslinked by cross-linking agent.
3. artificial extracellular matrix according to claim 1 and 2 is characterized in that: the mass ratio of described chitosan, described collagen protein, described hyaluronate sodium and described lysine is (0.1～400): (1～400): (0.1～5000): (0.1～5000).
4. artificial extracellular matrix according to claim 3 is characterized in that: described cross-linking agent be selected from following any or appoint several; Formaldehyde, glutaraldehyde, carbodiimides, diepoxides, genipin and procyanidin.
5. artificial extracellular matrix's preparation method comprises the steps:
Chitosan gum liquid solution, collagen protein hydrogel, aqueous solution of sodium hyaluronate and lysine solution are mixed, add the cross-linking agent reaction again;
Crosslinked good mixed gel lyophilization obtains described artificial extracellular matrix.
6. method according to claim 5 is characterized in that: the mass ratio of described chitosan, described collagen protein, described hyaluronate sodium and described lysine is (0.1～400): (1～400): (0.1～5000): (0.1～5000).
7. according to claim 5 or 6 described methods, it is characterized in that: described cross-linking agent be selected from following any or appoint several; Formaldehyde, glutaraldehyde, carbodiimides, diepoxides, genipin and procyanidin.
8. method according to claim 7 is characterized in that: described cryodesiccated temperature is-80 ℃～40 ℃.
9. method according to claim 7 is characterized in that: described chitosan gum liquid solution is the acetic acid solution of 0.1～5mg/ml chitosan, and the concentration of acetic acid is 0.1ml/100ml～0.5ml/100ml in the described acetic acid solution; The concentration of collagen protein is 1～50mg/ml in the described collagen protein hydrogel; The concentration of hyaluronate sodium is 0.1～100mg/ml in the described aqueous solution of sodium hyaluronate; The concentration of lysine is 0.1～100mg/ml in the described lysine solution.
10. the application of arbitrary described artificial extracellular matrix in preparation artificial dura mater material in the claim 1 to 4.
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| CN103435837A (en) * | 2013-09-06 | 2013-12-11 | 杨树林 | Preparation method of recombinant human-like collagen biological sponge |
| CN105903081A (en) * | 2015-12-14 | 2016-08-31 | 上海其胜生物制剂有限公司 | Preparation method of novel double layer proteoglycan-based restoration material |
| WO2020171723A3 (en) * | 2019-02-21 | 2020-10-22 | Uniwersytet Jagielloński | Hydrogel hybrid material, method of its preparation and application |
| WO2021134082A1 (en) * | 2019-12-26 | 2021-07-01 | Allergan, Inc. | Crosslinked ha-collagen hydrogels as dermal fillers |
| WO2021261355A1 (en) * | 2020-06-22 | 2021-12-30 | 凸版印刷株式会社 | Gel composition and production method therefor, and three-dimensional tissue body and production method therefor |
| US11833269B2 (en) | 2011-09-06 | 2023-12-05 | Allergan, Inc. | Hyaluronic acid-collagen matrices for dermal filling and volumizing applications |
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