CN104224842A - Preparation method of compound amniotic membrane powder and compound amniotic membrane powder prepared thereby - Google Patents
Preparation method of compound amniotic membrane powder and compound amniotic membrane powder prepared thereby Download PDFInfo
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- CN104224842A CN104224842A CN201410445608.5A CN201410445608A CN104224842A CN 104224842 A CN104224842 A CN 104224842A CN 201410445608 A CN201410445608 A CN 201410445608A CN 104224842 A CN104224842 A CN 104224842A
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Abstract
The invention discloses a preparation method of compound amniotic membrane powder. The preparation method disclosed by the invention comprises the following steps of: (1), preparing and cleaning an amniotic membrane material; (2), protecting active factors; (3), crushing at a low temperature; and (4), freeze-drying. The activity protection problem of cell factors in the process of crushing an amniotic membrane is solved; the active factors of the amniotic membrane in the compound amniotic membrane powder material can be retained and stored at normal temperature; furthermore, the compound amniotic membrane powder can be compounded with a plurality of powdery, liquid, aqueous and oily medicines or medicinal materials and the like, and thus, the compound amniotic membrane powder is applied to requirements of interventional therapy or special wound repair.
Description
Technical field
The present invention relates to field of biomedical materials, the preparation method especially relating to a kind of compound amniotic membrane powder and the compound amniotic membrane powder prepared.
Background technology
Amniotic membrane is the innermost layer of Placenta Hominis, thick about 0.02 ~ 0.5mm, under light microscopic and Electronic Speculum, epithelial layer and hypothallus can be divided into, according to fiber alignment structure and contained cell difference, hypothallus can be divided into again basement membrane, compacted zone, fibroblast layer and spongy layer 4 layers, and hypothallus contains a large amount of collagen, be mainly i, iii, iv, v, vii Collagen Type VI and the composition such as fibronectin splicing variants, laminin，LN, can be tissue repair and nutritional support is provided.
Amnioic epithelium and substrate contain some somatomedin as basic fibroblast growth factor (bfgf), hepatocyte growth factor (hgf) and transforming growth factor-β (tgf-β) etc., these factors can promote that epithelization occurs, be conducive to epithelial differentiation, divide a word with a hyphen at the end of a line and strengthen epithelial adhesiveness, cytokine is that a class has bioactive protein molecule.
Research display: amnion stroma layer contains unique interstitial composition, transforming growth factor β signal hypertrophy can be suppressed and suppress the propagation of the myofibroblast of normal human subject cornea, limbus of corneae fibroblast and break up to fibrocyte, thus amniotic membrane also has suppression fibrosis, the function that lessen scar formation is formed.
Hla-a, b, c, dr antigen or β2-microglobulin are not expressed in human amnion membrane surface.Express ib antigen, restriction ia antigen, the feature in these immunologys makes amniotic membrane non-immunogenicity.
Amniotic membrane is degradable natural macromolecular material, its excellent biological character, makes its natural resources as a kind of preciousness have the bright prospects of Application and Development.Membrane film is transplanted and not only be may be used for the reconstruction of 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, gynecological, Urology Surgery etc. rebuild mucosa and skin histology, also achieve gratifying results in the application in stem-cell research and Tissue Engineering Study field.
The clinical application range of domestic and international amniotic membrane is in continuous expansion; apply in some Minimally Invasive Surgerys due to amnion transplantation sheet and be restricted; the present invention adopts people's amniotic membrane in protective agent by machining at low temperature powdering; retain amniotic membrane primary bioactivity; make amniotic membrane powder can be used for Minimally Invasive Surgery and intervention material; play the prevention of inflammation of amniotic membrane at privileged sites, the due function such as acceleration of tissue repair.
Application publication number is the compoistion and method of use that the PCT application for a patent for invention entering National Phase in China of CN101316602A (application number is 200680044389.3) discloses amniotic membrane preparations and purification, that placenta tissue is through homogenate disclosed in this patent is concrete, centrifugal, obtained a kind of containing hyaluronic acid (HA) after lyophilizing, tumor necrosis factor irritates gene 6 (TSG-6), the purified composition of PTX-3 (PTX-3) and thrombospondin (TSP-1) and application thereof, do not mention the protection problem of preparation process activated protein, the defect that this mode exists is:
(1) Placenta Hominis, amniotic membrane are in crushing process, and localized hyperthermia and oxidation can make the active factors deactivation of amniotic membrane itself.
(2) amnion tissue is mainly based on collagen protein, is water-insoluble structural protein; Abundant active factors is contained in amnioic epithelium layer and substrate; i.e. functional protein; conventional activated protein protective agent kind is a lot; comprise sugar, aminoacid, macromolecular material, surfactant etc., the characteristics such as the acid and alkali resistance of most protective agent in the course of processing, salt, heat and its ability controlling fluid stream character all can not reach the protection requirement to amniotic membrane powder.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of compound amniotic membrane powder; solve the problem of membrane film crushing process cytokine activity protection; this compound amniotic membrane powder material amniotic membrane active factors is retained; can preserve at normal temperatures; and can carry out composite with multiple powdery, aqueous, aqueous, oiliness medicine or medicinal materials etc., be applicable to Wicresoft and get involved or the demand of special wound repair.
Another object of the present invention is the compound amniotic membrane powder providing the method to prepare.
The present invention adopts following technical scheme to realize: a kind of preparation method of compound amniotic membrane powder, comprises the following steps:
1) amniotic membrane is drawn materials and cleaning: carry out amniotic membrane by standard (YZB/ state 1340-2013) and draw materials, and under cleaning sterile environment, fresh amnion physiological saline solution is rinsed removing bloodstain, controls dry saline;
2) the prolection factor: xanthan gum being dissolved in configuration quality concentration in the buffer solution of 0.1M ~ 1M, PH=6.8 ~ 7.5 is the xanthan gum solution of 0.01% ~ 0.5%, the amniotic membrane processed through step 1 is inserted in this xanthan gum solution, the mass ratio of amniotic membrane and xanthan gum solution is 1:1 ~ 100, rotate mixing 20min ~ 30min, the rotating speed rotating mixing is 10r/min ~ 100r/min;
3) pulverize at low temperature: under the low temperature environment of-190 DEG C ~-4 DEG C, the mixture of the amniotic membrane processed through step 2 and xanthan gum solution is carried out grinding process, 100 order ~ 200 mesh sieves are crossed again at 4 DEG C ~ 10 DEG C, filtered solution is centrifuge dehydration 5min ~ 10min at 4 DEG C ~ 10 DEG C, the rotating speed of centrifuge dehydration is 8000r/min ~ 15000r/min, obtains centrifugal sediment;
4) lyophilization: be 3% ~ 10% by centrifugal sediment lyophilization to water content obtained in step 3, i.e. obtained compound amniotic membrane powder.
Further, buffer solution described in step 2 is the one in sodium dihydrogen phosphate/sodium hydrogen phosphate, citric acid/sodium citrate.
Further, low temperature environment described in step 3 is by liquid nitrogen or dry ice manufacture.
Further, grinding process described in step 3 is carry out milled processed by ball mill or refiner.
The compound amniotic membrane powder that the preparation method of compound amniotic membrane powder of the present invention prepares.
Compared with prior art, the present invention has following beneficial effect:
(1) the present invention adopts xanthan gum and amniotic membrane to combine, make amniotic membrane in pulverize at low temperature process, the sulfydryl of activated protein is subject to the protection of the physical barriers of xanthan gum, decrease the oxidation of active group, active factors remains 30% ~ 60% compared with fresh amnion, and amniotic membrane powder prepared by conventional method active factors compared with fresh amnion only remains less than 10%, obviously, the active factors that compound amniotic membrane powder prepared by the inventive method retains is far away higher than amniotic membrane powder prepared by conventional method, the compound amniotic membrane powder made still has prevention for clinical, reduce inflammation, the functions such as acceleration of tissue repair,
(2) compound amniotic membrane powder pseudoplastic behavior also given by xanthan gum, this characteristic is because xanthan gum shows high rheological variation in the crushing process of low temperature, high shear force, the rheological property of xanthan gum makes insoluble collagen membrane composition suspend for long periods, the small amniotic membrane granule pulverizing stylish generation is wrapped up by xanthan gum rapidly, makes protective value reach best; After pulverizing completes, mixture becomes rapidly high glutinous, stable gel, and this is the speciality that other protective agents do not possess, and is conducive to amniotic membrane pulverize at low temperature, room temperature is preserved;
(3) due to xanthan gum, amniotic membrane combination, compound amniotic membrane powder is applied convenient, can need to carry out composite with other drug or medicinal materials according to difference, as long as product of the present invention is being revolved high speed rotating on misfortune mixed instrument together with the material added, xanthan gum viscosity degradation, the material added can be uniform and stable be distributed in compound amniotic membrane powder, this characteristic is very convenient in clinical practice, can at any time compound amniotic membrane powder and multiple powdery, aqueous, aqueous, oiliness medicine or medicinal materials etc. be carried out composite, get involved for Wicresoft or the wound repair of specific demand.
Detailed description of the invention
Below in conjunction with specific embodiment, the preparation method of a kind of compound amniotic membrane of the present invention powder and the compound amniotic membrane powder for preparing are described in further detail.
Embodiment 1
Carry out people's amniotic membrane by standard (YZB/ state 1340-2013) to draw materials, under cleaning sterile environment, Freshman amniotic membrane physiological saline solution is rinsed removing bloodstain, controls dry saline; Xanthan gum is dissolved in the citric acid/sodium citrate buffer solution of 0.1M, PH=6.8, configuration quality concentration is the xanthan gum solution of 0.01%, above-mentioned amniotic membrane is inserted in this xanthan gum solution, the mass ratio of amniotic membrane and xanthan gum solution is 1:1, rotate mixing 20min, the rotating speed rotating mixing is 100r/min; Then under the low temperature environment of-190 DEG C that utilize liquid nitrogen to manufacture, undertaken grinding process by ball mill, then cross 200 mesh sieves at 4 DEG C, filtered solution is centrifuge dehydration 5min at 4 DEG C, and the rotating speed of centrifuge dehydration is 15000r/min, obtains centrifugal sediment; Be 3% by centrifugal sediment lyophilization to water content again, i.e. obtained compound amniotic membrane powder.People's amniotic membrane used in the present embodiment can be replaced with the amniotic membrane of other animal origins.
Cytokine content analysis design mothod in compound amniotic membrane powder:
One, titer configuration and standard curve making
Precision takes cytokine standards product (equal purchased from American Merck company), and described cytokine standards product comprise: a, recombinant human epidermal growth factor; B, recombinant human fibroblast growth factor; C, recombinant human hepatocyte growth factor; D, rhTGF-BETA β; E, recombinant human insulin-like growth factor; F, recombinant human platelet-derived growth factor; G, Recombinant human vascular endothelial growth factor; H, recombinant human nerve growth factor; I, recombination human ciliary neurotrophy factor; J, recombined human Brain Derived Neurotrophic Factor; Not commensurability cytokine standards product are joined in the PBS buffer of PH=7.4, the concentration of preparation cytokine are respectively 1,5,10,15,20,25,30,35,40,45, the titer of 50ng/mL; Radio immunoassay is adopted to measure each titer activity, production standard curve.
Two, sample treatment
Add the PBS buffer of PH=7.4 in compound amniotic membrane powder and fresh amnion, make corresponding sample solution, deposit for subsequent use for 4 DEG C.
Three, immunoradiometric assay experiment
1) insolubilized antibody preparation
Liquid (carbonate buffer solution of 0.1mol/L, PH=9.5) is buffered by cytokine monoclonal antibody (mouse-anti hEGF monoclonal antibody with bag, equal purchased from American Immulomedics company) be diluted to 500mg/L, add in polystyrene hexagonal test tube, dosage 200 μ L/ manages, and puts 4 DEG C of refrigerator overnight.Next day, incline coating buffer, adds confining liquid (phosphate buffer containing 0.05mol/L, PH=7.4 of 1%BSA) 37 DEG C of closed 1h with 500 μ L/ pipes; Discard confining liquid, naturally dry rear for subsequent use.
Wherein, monoclonal antibody comprises: a, mouse-anti hEGF monoclonal antibody, 125I-mouse-anti hEGF monoclonal antibody; B, mouse-anti human fibroblastic growth factor monoclonal antibody, 125I-mouse-anti human fibroblastic growth factor monoclonal antibody; C, mouse-anti human hepatocyte growth factor monoclonal antibody, 125I-mouse-anti human hepatocyte growth factor monoclonal antibody; D, mouse-anti transforming growth factor-beta monoclonal antibody, 125I-mouse-anti transforming growth factor-beta monoclonal antibody; E, mouse-anti human insulin-like growth factor monoclonal antibody, 125I-mouse-anti human insulin-like growth factor monoclonal antibody; F, mouse-anti human platelet-derived growth factor monoclonal antibody, 125I-mouse-anti human platelet-derived growth factor monoclonal antibody; G, mouse-anti human vascular endothelial growth factor monoclonal antibody, 125I-mouse-anti human vascular endothelial growth factor monoclonal antibody; H, mouse-anti growth factor of human nerve monoclonal antibody, 125I-mouse-anti growth factor of human nerve monoclonal antibody; I, mouse-anti human ciliary nerve nutrition factor monoclonal antibody, 125I-mouse-anti human ciliary nerve nutrition factor monoclonal antibody; J, mouse-anti Neurotrophic Factor monoclonal antibody, 125I-mouse-anti Neurotrophic Factor monoclonal antibody.
2) immunoradiometric assay program
Wrap by the bag of corresponding antibodies by pipe in add 100 μ L respective fine intracellular cytokine titers or testing sample and 100 μ L 125I-mouse-anti hEGF monoclonal antibodies, mixing, 37 DEG C of isothermal reaction 3h; Abandon reactant liquor, wash 3 times (each 500 μ L) with cleaning mixture (phosphate buffer of 0.02mol/L, PH=7.4), mouth of pipe residual liquid is blotted in absorbent paper, measures bag by the radiocounting of pipe with radioimmunoassay system; With cytokine concentrations (ng/mL) for abscissa, the combination rate (B/T) of each standard point is vertical coordinate, drawing standard curve on log-log paper; B/T per sample, finds corresponding cytokine concentrations from standard curve, or uses the calculation procedure of the subsidiary analysis software of instrument directly to obtain corresponding cytokine concentrations in testing sample.
3) result of the test
Shown in the various cytokine content following tables of compound amniotic membrane powder and fresh amnion:
According to above experimental result, the amniotic membrane prepared by said method still maintains the basis of fresh amnion, and the cytokine content retained still reaches therapeutic dose level.
Therefore the compound amniotic membrane powder prepared by the method still maintains the original cytokine of amniotic membrane and biological activity thereof, comprises the effect of inflammation-inhibiting reaction and the characteristic etc. of Promote cell's growth.
Embodiment 2
Carry out people's amniotic membrane by standard (YZB/ state 1340-2013) to draw materials, under cleaning sterile environment, Freshman amniotic membrane physiological saline solution is rinsed removing bloodstain, controls dry saline; Xanthan gum is dissolved in the sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution of 0.2M, PH=6.9, configuration quality concentration is the xanthan gum solution of 0.05%, above-mentioned amniotic membrane is inserted in this xanthan gum solution, the mass ratio of amniotic membrane and xanthan gum solution is 1:25, rotate mixing 25min, the rotating speed rotating mixing is 60r/min; Then under the low temperature environment of-80 DEG C that utilize dry ice to manufacture, undertaken grinding process by ball mill, then cross 150 mesh sieves at 5 DEG C, filtered solution is centrifuge dehydration 6min at 5 DEG C, and the rotating speed of centrifuge dehydration is 12000r/min, obtains centrifugal sediment; Be 5% by centrifugal sediment lyophilization to water content again, i.e. obtained compound amniotic membrane powder.People's amniotic membrane used in the present embodiment can be replaced with the amniotic membrane of other animal origins.
Embodiment 3
Carry out people's amniotic membrane by standard (YZB/ state 1340-2013) to draw materials, under cleaning sterile environment, Freshman amniotic membrane physiological saline solution is rinsed removing bloodstain, controls dry saline; Xanthan gum is dissolved in the sodium dihydrogen phosphate/disodium hydrogen phosphate buffer solution of 0.5M, PH=7.2, configuration quality concentration is the xanthan gum solution of 0.2%, above-mentioned amniotic membrane is inserted in this xanthan gum solution, the mass ratio of amniotic membrane and xanthan gum solution is 1:60, rotate mixing 28min, the rotating speed rotating mixing is 25r/min; Then under the low temperature environment of-30 DEG C that utilize liquid nitrogen to manufacture, undertaken grinding process by ball mill, then cross 120 mesh sieves at 8 DEG C, filtered solution is centrifuge dehydration 8min at 8 DEG C, and the rotating speed of centrifuge dehydration is 10000r/min, obtains centrifugal sediment; Be 8% by centrifugal sediment lyophilization to water content again, i.e. obtained compound amniotic membrane powder.People's amniotic membrane used in the present embodiment can be replaced with the amniotic membrane of other animal origins.
Embodiment 4
Carry out people's amniotic membrane by standard (YZB/ state 1340-2013) to draw materials, under cleaning sterile environment, Freshman amniotic membrane physiological saline solution is rinsed removing bloodstain, controls dry saline; Xanthan gum is dissolved in the citric acid/sodium citrate buffer solution of 1M, PH=7.5, configuration quality concentration is the xanthan gum solution of 0.5%, above-mentioned amniotic membrane is inserted in this xanthan gum solution, the mass ratio of amniotic membrane and xanthan gum solution is 1:100, rotate mixing 30min, the rotating speed rotating mixing is 10r/min; Then under the low temperature environment of-4 DEG C that utilize dry ice to manufacture, undertaken grinding process by ball mill, then cross 100 mesh sieves at 10 DEG C, filtered solution is centrifuge dehydration 10min at 10 DEG C, and the rotating speed of centrifuge dehydration is 8000r/min, obtains centrifugal sediment; Be 10% by centrifugal sediment lyophilization to water content again, i.e. obtained compound amniotic membrane powder.People's amniotic membrane used in the present embodiment can be replaced with the amniotic membrane of other animal origins.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. a preparation method for compound amniotic membrane powder, is characterized in that, comprises the following steps:
1) amniotic membrane is drawn materials and cleaning: carry out amniotic membrane by standard and draw materials, and under cleaning sterile environment, fresh amnion physiological saline solution is rinsed removing bloodstain, controls dry saline;
2) the prolection factor: xanthan gum being dissolved in configuration quality concentration in the buffer solution of 0.1M ~ 1M, PH=6.8 ~ 7.5 is the xanthan gum solution of 0.01% ~ 0.5%, the amniotic membrane processed through step 1 is inserted in this xanthan gum solution, the mass ratio of amniotic membrane and xanthan gum solution is 1:1 ~ 100, rotate mixing 20min ~ 30min, the rotating speed rotating mixing is 10r/min ~ 100r/min;
3) pulverize at low temperature: under the low temperature environment of-190 DEG C ~-4 DEG C, the mixture of the amniotic membrane processed through step 2 and xanthan gum solution is carried out grinding process, 100 order ~ 200 mesh sieves are crossed again at 4 DEG C ~ 10 DEG C, filtered solution is centrifuge dehydration 5min ~ 10min at 4 DEG C ~ 10 DEG C, the rotating speed of centrifuge dehydration is 8000r/min ~ 15000r/min, obtains centrifugal sediment;
4) lyophilization: be 3% ~ 10% by centrifugal sediment lyophilization to water content obtained in step 3, i.e. obtained compound amniotic membrane powder.
2. the preparation method of compound amniotic membrane powder according to claim 1, is characterized in that, buffer solution described in step 2 is the one in sodium dihydrogen phosphate/sodium hydrogen phosphate, citric acid/sodium citrate.
3. the preparation method of compound amniotic membrane powder according to claim 1, is characterized in that, low temperature environment described in step 3 is by liquid nitrogen or dry ice manufacture.
4. the preparation method of compound amniotic membrane powder according to claim 1, is characterized in that, grinding process described in step 3 is carry out milled processed by ball mill or refiner.
5. according to the compound amniotic membrane powder that the preparation method of the arbitrary described compound amniotic membrane powder of Claims 1-4 prepares.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106978389A (en) * | 2017-05-05 | 2017-07-25 | 广州市天河诺亚生物工程有限公司 | A kind of combination culture medium for cultivating amniotic epithelial cells |
| CN109394398A (en) * | 2018-09-14 | 2019-03-01 | 江西瑞济生物工程技术股份有限公司 | A kind of degradable foldable bioamnion complex repairation bracket |
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| CN1943796A (en) * | 2006-10-20 | 2007-04-11 | 关志广 | The preparation method of the radiant crosslink porous reticular cells amnionic hydro gel dressing and its preparation method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106978389A (en) * | 2017-05-05 | 2017-07-25 | 广州市天河诺亚生物工程有限公司 | A kind of combination culture medium for cultivating amniotic epithelial cells |
| CN109394398A (en) * | 2018-09-14 | 2019-03-01 | 江西瑞济生物工程技术股份有限公司 | A kind of degradable foldable bioamnion complex repairation bracket |
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