CN104055795B - A kind of injectable implant and preparation method thereof - Google Patents
A kind of injectable implant and preparation method thereof Download PDFInfo
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- CN104055795B CN104055795B CN201310071109.XA CN201310071109A CN104055795B CN 104055795 B CN104055795 B CN 104055795B CN 201310071109 A CN201310071109 A CN 201310071109A CN 104055795 B CN104055795 B CN 104055795B
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Abstract
The present invention relates to a kind of injectable implant and preparation method thereof.The implant is：Amnion particulate is mixed with pH is 7.0~7.5 phosphoric acid physiological salt solution.Its preparation method is：By disinfecting, de- cell processing, modification, complete the step of prepare amnion particulate, i.e. using amnion particulate as component A and phosphoric acid physiological salt solution as component B, according to mass volume ratio 20:1~100:1 concussion is mixed, and concentration is prepared into for 20~100mg/mL amnion microparticulate suspensions through high-speed micro-jet equipment, and the particle diameter of amnion particulate is 50~200 μm.Present invention preserves the native three dimensional stereochemical structure of people's amnion and substantial amounts of active skull cap components, reduce beauty injection product immunological rejection because of caused by raw material sources after Clinical practice, suitable for being injected to subcutaneous dermal layer depth layer to subcutaneous shallow-layer to repair moderate to severe wrinkle or fold, with good beauty repairing effect.
Description
Technical field
The invention belongs to medical cosmetology shaping technique field, and in particular to a kind of can promote from amnion biomaterial
Enter injectable implant of autologous collagen regeneration and preparation method thereof.
Background technology
Since oneth century, soft tissue augmentation agent is widely used in aesthetic surgery to improve face contour, amendment
Wrinkle, fill and lead up depressed scar and packing volume（Such as lip）Deng the demand for soft tissue filler is increasing now.
Preferable soft tissue filler, it should with cheap and easily-available, good biologically inert, biocompatibility and stably
Property, nontoxicity, non-carcinogenesis, without it is infectious, without acute or chronic inflammatory reaction, be easy to inject and take out, be able to maintain that it is long-term
Even permanent appearance repair effect and recovery time it is short.But so far, being asked still without such preferably injecting type filler
Generation.
All kinds of injectable soft tissue fillers commonly used both at home and abroad at present have following several, including：
（1）Synthesize high polymer material, such as shaping collagen and polymetylmetacrylate（PMMA）Hypodermic implant system, be
Using bovine collagen solution as carrier, the circular smooth microballoons of PMMA are suspended in wherein, simultaneously containing a certain amount of lidocaine anesthesia
Agent；Its major defect is, due to PMMA non-degradables, and longer-term persistence has dislodgment in human body, induces long-term chronic inflammation
Application risk, very high is required to injection site and injecting method.
（2）Bovine collagen albumen, Zyderm uses ox source property NTx albumen for raw material, and non-crosslinked, concentration is 35mg/
ML, action effect is shorter, can typically maintain 4 months；Zyplast, is also to use ox source property NTx albumen for raw material, but warp
Glutaraldehyde cross-linking is crossed, concentration is 35mg/mL, its using effect can be maintained about 6 months；Medical collagen filler (trade name：Skin is beautiful
Up to) be 3.5% bovine collagen albumen and 0.3% lidocaine hydrochloride physiological saline suspension, it is pre-filled glass syringe (containing note
Penetrate pin) in.The shortcoming of above bovine collagen albumen implant is：Skin test need to be carried out to prevent patient from allergy occur within 30 days before the injection
Phenomenon, the beauty repairing effect duration is more of short duration, often needs repeatedly to inject repeatedly, there being possible complication after injection causes
Blood spots, granulation knurl, immediate allergic, retardance allergic reaction and general malaise reaction etc..
（3）Porcine collagen, such as double U.S. collagen implants, its raw material is the type i collagen egg purified by pigskin
It is scattered in phosphoric acid physiological saline cushioning liquid and is filled in aseptic injection syringe through filtration sterilization and sterile working mode in vain
It is made.Its product is without crosslinking Treatment, and the degradation in vivo cycle is about 4~6 months.The shortcoming of the product is pig source property glue
Original, may cause rejection, and general animal derived collagen is both needed to carry out allergy skin test, there is the production of species collagen implants
Product cause bleeding spot, the complication report such as granulation knurl.
（4）Human collagen, is the collagen implantation that the extracellular matrix obtained from human fibroblasts in vitro culture is obtained
Product, such as Dermalogen and Cosmoderm are the allogeneic materials with hide glue fibril and collagenous tissue matrix suspension of injectable；This
A little materials are constituted by collagen and elastic fibers, glucan.Such material advantages is that immunological rejection is low, no from people
Need to carry out skin test test, but manufacturing cycle is longer, and production technology is complex, and equipment requirement is higher, and product cost is higher.
（5）People's acellular dermal matrix particulate, is by removing epidermis, de- cell, freeze-drying, crushing by people's cadaver skin
The material formed afterwards, such as Cymetre are the Allogeneic acellular dermal matrite of micronized injectable, about 123 μm of average grain diameter, without friendship
Connection processing, document report lip repairing effect can be maintained more than 1 year.The major defect of the material is that final form is particulate, not
Be prepared into uniform suspension, needed when doctor uses it is preoperative be prepared work, i.e., using threeway connection valve by particulate and injection
Agent or anesthetic mixing, add operation preparation time, and mix as manual operations, it is difficult to ensure the uniformity coefficient of suspension.
（6）Hyalomitome acids, including the auspicious indigo plants of Restylane etc., its hyaluronic acid raw material sources is produced in bacterial fermentation, warp
The purge processes such as precipitation, filtering, drying are crossed, and it is partial cross-linked through BDDE, then by heating, sterilizing
Obtained hyaluronic acid implant, its main component by the hyaluronic acid through modification (part be chemically crosslinked), sodium ion, chlorine from
Son, phosphate buffer and water for injection composition, hyaluronic acid concentration are 20 ± 3mg/mL, and 100000 particles/mL are average
Particle diameter is about 250 μm, is dissolved in pH7 phosphate buffered saline solution.Hyaluronic acid material also has certain lack as filling implant
There is adverse reaction such as swelling, erythema or sclerosis of the skin in point, such as a small amount of patient；Water imbibition is stronger, has after grand lip oedema not
Good reaction report；Without anesthetic, pain is stronger.
（7）Hyaluronic acid and cellulose family, such product mainly carry out crosslinking Treatment with hyaluronic acid and cellulose and obtained
The filler of the blending constituent arrived, such as escapes beautiful, the nothing being made up of hydroxypropyl methyl cellulose, hyaluronic acid and balanced salt solution
Bacterium gelatinous solution, is encapsulated in pre- filled glass syringe (no injection needle).Such product composition is complex, production technology
Middle control degree of cross linking difficulty is larger, therefore causes the product degradation cycle shorter, is substantially completely degraded after 4~6 months.
There is certain defect in the beauty implantation product of any of the above type, and country produces for beauty implant class at present
Product require very strict, such as：Crosslinking degree, particle size distribution range, molecular size range, degradation cycle etc. are clearly controlled, need to be expired
The technical conditions of foot are very high.
Chinese patent CN200510044005.5 discloses a kind of preparation method of biological filler for injectable soft tissue,
It, which is used, newly slaughters small pigskin for raw material, by going hypodermis, degreasing, alkali process, cuing open layer, ferment treatment, glutaraldehyde cross-linking, drift
White processing and micronized processing, last packaged and Co 60 sterilizing are formed.The patent system is by skin graft for the method for particulate
It is the particulate that the length of side is 300~500 μm by physical method cutting, but mean particle dia is excessive, it is impossible to use the less pin of diameter
Head injection；And final is simply to be packed particulate, it is impossible to which direct injection is used.
Chinese patent CN95106720.6 discloses a kind of method for using Human plactnta to prepare graininess collagen for raw material, its
It is that irradiation inactivated virus is first carried out to placenta, uses organic reagent to extract the collagen in placenta after cleaning, crushing, it is last concentrated
Dialyse and add Injectable Collagen obtained from lidocaine.The patent is close with this patent using raw material, but in technique
Collagen is extracted using a large amount of organic reagents, easily there is organic reagent residual.
Chinese patent CN201010117490.5 discloses a kind of method for the hyaluronic acid derivatives particulate for preparing injection.
The raw materials used patent is hyaluronic acid dry powder, adds cross-linking agents after it is dissolved with alkaline solution, prepares transparent
Crosslinking agent is removed using dialysis process again after matter acid gel.This method possibly can not thoroughly remove the reagents such as the crosslinking agent in gel
Residual, cleaning efficiency is relatively low, it is necessary to dialyse 4~5 days in addition.
Chinese patent CN200810007484.7 discloses a kind of use human hair keratin and prepares soft tissue filling material
Method, the patent utilization human hair stage casing is as raw material, at over cleaning, bleaching, attrition process, lyophilized, packaging, 60Coradiation
Reason, powder homogenate and liquid human hair keratin particle are prepared into by human hair.The patent prepares particle diameter only with physics method for grinding
60~80 μm of human hair keratin particle homogenate, according to inventor's experiment experience, water insoluble different solid matters are carried out
Physics is milled without the subsequent step such as sieve, homogenize, obtained particle size distribution range generally at 100~1000 μm, it is difficult to
Obtain compared with small particle scope.
Chinese patent CN02156797.2 discloses a kind of preparation method of injectivity collagen and products thereof and application,
The patent prepares injectivity collagen by extracting the collagen fabric in animal tissue, can be complete in preparation process
Destroy animal tissue's original structure and other compositions, have lost have in natural tissues bioactivity or biological function it is natural into
Point, simple collagen filling effect can only be played.
Therefore, beauty implant system disclosed in the various beauty implantation product listed at present and domestic patent family
There is certain shortcoming in Preparation Method, especially similar collagen implants product is in addition to comprising collagen stroma, not comprising biology
Active factors, therefore its effect is only to fill, after these packing materials are decomposed by body to be absorbed, beauty repairing effect can subtract
Weak or disappearance, thus the cosmetic result of such implant product is temporary.
Amnion, is the internal layer of embryo's duplicature from cytotrophoblast derivation, by epithelium layer, basilar memebrane and nothing
Vascular stroma is constituted.A variety of collagen components and active factors are contained in amnion, including：
（1）Collagen：Contain more I, III, IV, V and VII Collagen Type VI in amnion, with epithelium basalis（By IV type glue
The composition such as former, VII Collagen Type VIs and laminin）Composition is close, also contains main component I, the type III glue of skin composition
It is former.The more collagen of content is I, III, IV Collagen Type VI in amnion, accounts for more than 90% of all collagens in amnion.
（2）Laminin（LN）：It is distributed widely in the hyaline layer of basilar memebrane, is close to cell basal part, with IV Collagen Type VI
The skeleton of basilar memebrane is combined to form, adhesion, the motion of cell is influenceed, adjusts the growth and differentiation of cell.
（3）EGF（EGF）:It can be synthesized and promoted on cell propagation and skin by DNA, RNA and protein
Chrotoplast epithelialization.
（4）Keratinocyte growth factor（KGF）：Research shows that the synthesis of application on human skin epithelial cell dna, promotion cell can be promoted
Hyperplasia.
（5）Basic fibroblast growth factor（bFGF）：With promoting the propagation of stromal fibroblast cells, divide a word with a hyphen at the end of a line and divide
Change, promote the effect of wound healing, can effectively stimulating animal and the Epithelial cell of people propagation.
（6）HGF（HGF）：It can stimulate the growth of various target cells, including it is liver cell, epithelial cell, interior
Chrotoplast, keratinocyte and hematopoietic cell.
（7）Expression of TIMP（TIMP）：Fibroblast proliferation and collage synthesis can be promoted, cell is caused
Epimatrix deposits and suppresses its degraded.
（8）Other protease inhibitors：Alpha1-antitrypsin, alpha2-macroglobulin, α 2- ACTs etc., can press down
Make corresponding protease and play antiinflammatory action.
It is more containing more than exactly in amnion to suppress angiogenesis, the active factors of anti-inflammatory（Hao, Y.et,
al.,Identification of antiangiogenic and antiinflammatory proteins in human
amniotic membrane.Cornea,2000.19(3):p.348-52；BK,N.,et al.,Analysis of human
amniotic membrane components as proteinase inhibitors for development of
therapeutic agent of recalcitrant keratitis.Trophoblast Res,1999(13):p.459-
466.）, therefore amnion can play promotion ocular epithelialization, mitigation inflammatory reaction, suppression proliferation of fibrous tissue and new vessels
The effect of formation, so as to clinically be widely used in terms of the reparation of ocular various diseases, skin burnt wound restoration.
The product for using amnion to prepare injectable filler for raw material is there is no at present, also without patent and document report.
The content of the invention
For the defect of above-mentioned various implants, it can suppress inflammation it is an object of the invention to provide one kind, suppress scar
Generation, the permanently effective beauty implant for promoting autologous collagen synthesis and preparation method thereof, this method remains the day of amnion
Right 3-D solid structure and substantial amounts of active skull cap components, reduce beauty injection product after Clinical practice because of raw material sources
Caused by immunological rejection.
Injectable prepared by present invention amnion particulate filler, is molten for 7.0~7.5 phosphoric acid sodium chloride physiology in pH
Amnion particulate is mixed with liquid；The mean particle dia of the injectable implant is 50~200 μm, and concentration is 30~100mg/mL.
The phosphoric acid physiological salt solution, is the sodium chloride containing final concentration of 3~8g/L using water for injection as solvent
Solution, 0.2~0.4g/L sodium dihydrogen phosphate and 1.2~2.0g/L disodium phosphate soln；
The amnion particulate and phosphoric acid physiological salt solution are according to mass volume ratio 20:1~100:1 mixes.
The preparation method of the injectable of present invention amnion particulate filler, is, using amnion as raw material, by de- cell, to hand over
Connection is modified, homogenate and filling, be prepared from, including amnion is disinfected, and takes off cell processing, modification, amnion particulate
Preparation and the preparation of amnion particulate implant, are comprised the following steps that：
Step 1: disinfecting：Aseptically, amnion is immersed in phosphate buffer（PBS）It is middle to clean to nothing
Color, dirt, then cleaned 1~5 time with purified water, the hydrogen peroxide solution for being then 1~3% with volumetric concentration, volumetric concentration are 60
One or several kinds in~90% ethanol water, the peroxide acetate aqueous solution that volumetric concentration is 0.1~2%, successively to sheep
Film carries out disinfection processing 5~60 minutes；
This step passes through the processing that carried out disinfection to amnion, it is possible to decrease the load of microorganisms of amnion, while inactivating a variety of possibility
The virus of presence, it is ensured that security of the amnion in subsequent treatment, it is to avoid the microorganism pollution carried on amnion.
Step 2: de- cell processing：Amnion after step one is disinfected first is cleaned 1~3 time with purified water, then is used
0.1~1mg/mL ethanedioic acid triethylammonium tetrakis（EDTA）The aqueous solution, 0.01~0.1mol/L sodium hydrate aqueous solutions, 1~5mol/L
One or several kinds in sodium-chloride water solution, the 0.1~1mg/mL pancreatin aqueous solution, carry out concussion processing 5 to amnion successively
~120 minutes, frequency of oscillation was 50~80rpm, finally using purified water or PBS 3~10 times；
This step removes the cell component in amnion by gentle chemistry and physical method, reduces the immunogene of amnion
Property, and the collagen and active factors of amnion are at utmost remained simultaneously, the implant for retaining amnion bioactivity for preparation is carried
For raw material.
Step 3: modification：The amnion that step 2 is obtained is with processing is either physically or chemically modified, according to most
The requirement of finished product degradation property, may be selected the method for modifying of different disposal intensity.Modified chemical reagent used or physics side
Method includes but is not limited to：0.1~0.5mol/L 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride
(EDC.HCL) pH is handled 6~48 hours for 5~6 aqueous solution at 2~40 DEG C, the 0.1~1mg/mL riboflavin aqueous solution
Handle 4~24 hours at room temperature, 3000~10000 μ W/cm at room temperature2Ultraviolet irradiates 4~16 hours, and volumetric concentration is
0.1~1% glutaraldehyde water solution is handled 6~12 hours at room temperature, and the 0.5~1.5mg/mL ribose aqueous solution is at 2~40 DEG C
Processing 3~14 days；
Different modification processing method in this step, can be that the amnion particulate implant for finally preparing different degradation times is carried
Selective, it is adaptable to the reparation of facial different parts, the present inventor passes through experimental verification, the short amnion particulate implantation of degradation time
Agent can be used for skin corium shallow region to repair superficial microgroove, the region such as canthus surrounding, the amnion particulate of degradation time length
Implant can be used for skin corium deep layer position to repair compared with deep wrinkle, the region such as nasolabial groove.In addition, can by modification
The especially various factors of the active component retained in amnion are fixed in amnion, reduced because of follow-up cleaning, dry, disintegrating process
The loss caused.
Step 4: the preparation of amnion particulate：By the amnion purified water after step 3 modification or PBS concussions cleaning 3
~10 times, then carry out room temperature air-dry or vacuum freeze drying processing, most after at 2~8 DEG C by amnion through ultrahigh speed low temperature from
Heart pulverizer is ground into the particle of 100~1000 μm of sizes.
Processing is first dried to amnion and is prepared into particle again for this step, is conducive to amnion to crush, in addition, step 3 and step
Rapid four methods being homogenized afterwards using being first crosslinked, facilitate modified cleaning, can thoroughly remove reagent residual.
Step 5: the preparation of amnion particulate implant：PH, as component A, is 7.0 by amnion particulate prepared by step 4
~7.5 phosphoric acid physiological salt solution is as component B, by component A and component B according to mass volume ratio 20:1~100:1（g/
L）Concussion is mixed, and is circulated 3~8 times through high-speed micro-jet equipment.Then, it is first that amnion microparticulate suspensions are filling through Sterile vacuum, then
Sterilize or amnion microparticulate suspensions sterilize through 10~30kGy radiated by gamma-ray through 10~30kGy radiated by gamma-ray first, and
Carry out Sterile vacuum it is filling, that is, be made can Clinical practice amnion particulate implant：Concentration is 20~100mg/mL amnion particulates
Suspension, the particle diameter of amnion particulate is 50~200 μm.
The composition of the phosphoric acid physiological salt solution of the component B is：Using water for injection as solvent, contain final concentration of 3
~8g/L sodium chloride solution, 0.2~0.4g/L sodium dihydrogen phosphate and 1.2~2.0g/L disodium phosphate soln.
Can also be added in component B phosphoric acid physiological salt solution final concentration of 0.001~0.01mg/mL Porcine HGF,
Any of 0.3~2mg/mL lidocaine, 1~10mg/mL hyaluronic acid etc. are several.
The high-speed micro-jet equipment that the step 5 is used, the operating pressure of high-speed micro-jet equipment is 5000~15000Pa,
A diameter of 0.1~the 0.25mm. of outlet nozzle.The shearing force produced by the fluid motion of high-speed micro-jet equipment is come further powder
The amnion particulate that broken step 4 is prepared, obtains the amnion particulate that particle diameter is 50~200 μm.The present inventor passes through macrophage
Swallow experimental verification, the small amnion particulate of diameter（10~40 μm）After implanting, easily swallowed by macrophage, it is impossible to reach
Repairing effect；It is generally clinical that in order to which precise control is injected to the position that skin corium needs to repair, the syringe needle used is 27~30g,
Excessive easy block of particle diameter injects beauty syringe needle.
The preparation facilities of the implant of the present invention, fixed amnion is put using hollow semielliptical shape dress, i.e., the device is hollow
Some apertures are evenly distributed with semielliptical shape housing, housing.Because natural amnion in elliposoidal wraps up fetus, from puerperal
The isolated amnion of placenta can not often be sprawled in plane completely, and the present invention puts fixed, place using porose semielliptical shape dress
Amnion, can entirely be covered in hemispheric surface apparatus by reason, cleaning amnion, apparatus surface have multiple apertures in favor of
Penetration by liquid（Referring to accompanying drawing 1）.During using the device, amnion is put into outer surface top from semielliptical shape dress and is spread to bottom, and
Amnion and device are fixed together with fixture or suture, device can be totally submerged in liquid or using spray,
The modes such as flowing water are handled amnion, compared to the non-processing method for sprawling formula, it is to avoid caused by amnion is curled, folded
Cleaning efficiency declines, and device can also be put in dry environment directly amnion is carried out to air-dry or vacuum drying treatment, the device
Material is the material that stainless steel etc. is difficult to be corroded by reagents such as water, ethanol, soda acid, salt.It is laid in compared to amnion non-porous stainless
Steel operating desk, the device, which can be effectively improved, to be air-dried or lyophilized efficiency, is prevented effectively from processing, cleaning process that amnion is damaged, twine
Around greatly improving cleaning, treatment effeciency.The present inventor is by experimental verification, if without using the device, being wound between amnion causes
After cleaning each time, need to untie or even occur damaged, the efficiency of influence scale processing, and winding at amnion winding by hand
Position is unfavorable for Liquid Penetrant and cleaning, can cause between batch and testing result is unstable in batch.It is demonstrated experimentally that not using this
Invention device fixes cleaning amnion, because vibration can caused by the case of 70% amnion wind, untie and twine by hand after cleaning terminates every time
Around amnion need to take 5~10 minutes, repeatedly processing amnion can be damaged, integrity degree is only 30~40%；And use amnion
Fixing device, then 100% can avoid amnion from winding, amnion integrity degree is more than 90%.
Amnion particulate implant prepared by the present invention, to remove the amnion of variant cell composition as primary raw material, with amnion
Distinctive natural collagen and all kinds of factors, such as IV Collagen Type VIs, bFGF, EGF, HGF, KGF etc.；Implant is in homogenate state；It is micro-
A diameter of 50~200 μm of grain, concentration is 30~100mg/mL.The implant is applied to be injected to subcutaneous dermal layer depth layer to subcutaneous
Shallow-layer, to repair moderate to severe wrinkle or fold, with fabulous beauty repairing effect.The present invention is implanted into by subcutaneous rat
Experiment, it was confirmed that amnion particulate implant can at least maintain the effect of 52 weeks in subcutaneous rat.
Compared with prior art, the invention has the advantages that being：
（1）Using technology of preparing of the present invention, type i collagen, type III collagen, the IV type glue in most people's amnion can be retained
The natural activity factor such as the collagen such as original natural component and HGF, EGF, bFGF, TIMP, KGF, is effectively retained amnion and suppresses in itself
Inflammation, the ability for suppressing scar generation（Fig. 2, Fig. 3）.
（2）Compared with pure collagen implants, hyaluronic acid implant, synthesis microballoon PMMA, amnion prepared by the present invention is micro-
Grain implant, the extracellular matrix native three dimensional network structure of amnion can be maintained to a certain extent（Fig. 4）, before material degradation
The space of implant site can be effectively maintained, and the three dimensions of autologous tissue's Hemapoiesis is provided, can plant injection amnion particulate
The position for entering agent maintains preferably three-dimensional repairing effect.
（3）The present invention eliminates the immunological rejections such as the cell and soluble protein of amnion by gentle physico-chemical process
Composition, is effectively retained a variety of natural components and extracellular matrix natural structure, and the method being homogenized afterwards using being first crosslinked, conveniently changes
Property after clean, can thoroughly remove reagent residual, make amnion particulate implant that there is good cell compatibility and tissue compatible
Property, cytotoxicity is not shown when amnion particulate implant prepared by the present invention is with human fibroblasts co-cultivation, and it is completeer
Whole characteristics of amniotic extracellular matrix can promote fibroblast to be attached at the progress propagation growth of amnion particulate（Fig. 5, Fig. 6）；Rat skin
Lower implantation test result indicates that, implant site does not occur any redness, festers, hemotoncus, histological stain section result shows 1,
4th, 8,24, the tissue inflammation reaction of 52 weeks is relatively light, and the inflammatory cell quantity of implantation surrounding materials and internal infiltration is less（Figure
9）.
（4）Amnion particulate implant prepared by the present invention, can effectively facilitate fibroblast rubber polymer after implanting
Original, carries out amnion particulate implant by simulated in vivo environment in vitro and is co-cultured with human fibroblasts, to fibroblast
The ability of synthesis collagen is verified, as a result shows that amnion particulate implant can the raising collagen conjunction of effective stimulus human fibroblasts
Into ability（Fig. 7）, illustrate that preparation method of the present invention can retain the fibroblast growth factor of amnion, can effectively facilitate
Fibroblastic propagation.
（5）Preparation method of the present invention has higher quality controllability, can be by controlling product in product preparation process
The parameters such as modification degree, concentration, average grain diameter and particle size distribution range are so as to control dynamic viscosity, degradation rate of product etc. to join
Number, realizes the stability of product quality.According to the every physical and chemical parameter of the batches of products prepared of the invention such as particle concentration, move
Power viscosity, degradation time in vitro are relatively stable（Table 1）, and 90% amnion diameter of particle can be controlled between 50~200 μm,
Avoid the occurrence of that particle diameter distribution is excessive to be caused to block syringe needle during injection or particle diameter distribution is too small causes the problems such as validity is unstable（Figure
8）.
（6）During cleaning, processing amnion, amnion is fixed on according to the natural elliposoidal feature of amnion by the present invention
Semielliptical shape dress is put, and can be prevented effectively from amnion breakage in processing procedure, and winding greatly improves production efficiency.
（7）Preparation method of the present invention can effectively control the parameters such as product design, viscosity, degradation rate, ensure that preparation
Amnion particulate implant implant after can rapid shaping, with long-term good filling effect, its persistence is at least 12
More than individual month, though amnion particulate implant is progressively being degraded, the phenomenon such as it is not shifted over, spreads（Fig. 9）.
（8）Amnion particulate implant prepared by the present invention, biological safety is good, with using animal origin collagen or passing through
Collagen implants prepared by the recombinant collagen that genetic recombination fermentation is obtained are compared, and homology is greatly improved, and can avoid injecting animal
The skin test experiment that collagen or recombinant collagen must be carried out, saves patient assessment's time and expense.
Brief description of the drawings
Fig. 1 is that the hollow semielliptical shape of the present invention fixes the schematic device of amnion
Fig. 2 is the picture of the immunohistochemical staining result of non-collagen components in amnion particulate implant prepared by embodiment
Fig. 3 is the figure of the quantitative testing result of a variety of collagens and active factors of amnion particulate implant manufactured in the present embodiment
Piece
Fig. 4 is the lyophilized rear ESEM result of amnion particulate implant manufactured in the present embodiment
Fig. 5 is that human fibroblasts are carried out after co-culturing 5 days under ordinary optical microscope with amnion particulate implant
Observe picture
Fig. 6 is that human fibroblasts carry out Histological section's picture after co-culturing 5 days with amnion particulate implant
Fig. 7 be amnion particulate implant manufactured in the present embodiment co-culture 3 with human fibroblasts in vitro, 5, after 7 days,
All human fibroblasts in co-culture system are taken, are extracted after mRNA therein using RT-PCR method detection type i collagen
The picture of the testing result of mrna expression amount
Fig. 8 is the picture of the particle diameter distribution testing result of amnion particulate implant manufactured in the present embodiment
Fig. 9 is progress histology and sheep after amnion particulate implant implantation subcutaneous rat tissue prepared by the present invention
The Study on degradation result picture of film particles implant
Figure 10 is different time points progress outward appearance after amnion particulate implant implantation subcutaneous rat tissue prepared by the present invention
The picture of effect observation
Figure 11 is the histology of different time points after amnion particulate implant implantation subcutaneous rat tissue prepared by the present invention
The picture of dicing effect observation
Embodiment
The present invention will be further described with reference to the accompanying drawings and examples.
The embodiment of the present invention is that puerpera is signed after informed consent form using people's amnion, is obtained through caesarean birth from discarded separation of placenta
, and the preservation of PBS ice baths is soaked in, transported within 24 hours to laboratory；High-speed micro-jet equipment is U.S.'s BEE manufacturer productions
Nano DeBEE.45 models；High-speed low temperature cintrifugal disintegrator is Shanghai good fortune Ritz experimental instruments and equipment limited production customization.
Fig. 1 shows the shape and structure of the fixation amnion device of the present invention
The device is to be evenly distributed many apertures 2 on hollow semielliptical shape housing 1, housing, and hole diameter is
0.5cm, adjacent apertures spacing is 2cm.In use, during cleaning, processing amnion, amnion is fixed on into the semielliptical shape shell
On body, amnion breakage in processing procedure can be prevented effectively from, winding greatly improves production efficiency.
It is set forth below nine and prepares the embodiment that injectable is implanted into agent method：
Embodiment 1,
Step 1: disinfecting：Aseptically, amnion is immersed in PBS and cleaned extremely without color, dirt, then used
Purified water is cleaned 1 time, and then carried out disinfection processing 30 minutes with the ethanol water that volumetric concentration is 75% to amnion；
Step 2: de- cell processing：Amnion after step one is disinfected first is cleaned 3 times with purified water, then is used
0.05mol/L sodium hydrate aqueous solutions carry out concussion processing 30 minutes to amnion, and frequency of oscillation is 50rpm, finally using purifying
Water is cleaned 3 times；
Step 3: modification：The amnion that step 2 is obtained 0.1mol/L 1- ethyls-(3- dimethylaminos third
Base) pH of carbodiimide hydrochloride handles 6 hours for 5 aqueous solution at 2 DEG C；
Step 4: the preparation of amnion particulate：Amnion after step 3 modification purified water is shaken into cleaning 3 times, so
Vacuum freeze drying afterwards, most after amnion is ground into the micro- of about 200 μm of particle diameter through ultrahigh speed low-temperature centrifugation pulverizer at 2 DEG C
Grain；
Step 5: the preparation of amnion particulate implant：Amnion particulate prepared by 3.5g step 4 is used as component A
100mL waters for injection prepare pH and are used as component B for 7.2 phosphoric acid physiological salt solution（The phosphoric acid sodium chloride life of the component B
Reason solution composition be：Using water for injection as solvent, the sodium chloride solution containing final concentration of 5.5g/L, 0.312g/L phosphoric acid
The disodium phosphate soln of dihydro sodium solution and 1.6g/L）, by component A and component B according to mass volume ratio 35:1（g/L）Quickly
Concussion is mixed, and is circulated 6 times through high-speed micro-jet equipment, operating pressure is in 15000Pa, and a diameter of 0.1mm of outlet nozzle is made
It is standby into the amnion microparticulate suspensions that concentration is 35mg/mL, about 60~100 μm of the particle diameter of amnion particulate.Finally by amnion microparticulate suspensions
It is filling through Sterile vacuum, then through 25kGy radiated by gamma-ray sterilize, that is, prepare can Clinical practice amnion particulate implantation
Agent.
The amnion particulate implant cell compatibility that this example is prepared is good, is co-cultured 5 days with human fibroblasts
The result observed and detected using light microscope and Histological section afterwards shows that amnion is micro- as shown in Figure 5, Figure 6, as a result
Grain implant can support fibroblast docile, grow.In addition, this example prepare amnion particulate implantation agent concentration and
Modification degree is relatively low, therefore degradation rate is very fast, carries out external degradation using 0.2mg/mL Proteinase K, degradation time is about
24 hours, subcutaneous rat implanting result showed that amnion particulate implant is degraded substantially after 4~6 months.Amnion prepared by this method
Particulate implant can be used for the reparation of microgroove around canthus.
Embodiment 2,
Step 1: disinfecting：Aseptically, amnion is immersed in PBS and cleaned extremely without color, dirt, then used
Purified water cleans 5 times, is then that 75% ethanol water carries out disinfection processing 15 minutes to amnion with volumetric concentration, then with 0.1%
Peracetic acid carries out disinfection to amnion and handled 15 minutes；
Step 2: de- cell processing：Amnion after step one is disinfected first is cleaned 1 time with purified water, then is used
0.01mol/L sodium hydrate aqueous solutions carry out concussion processing 10 minutes to amnion, and frequency of oscillation is 80rpm, then uses 3mol/L
Sodium-chloride water solution carries out concussion processing 100 minutes, is finally cleaned 10 times using purified water；
Step 3: modification：The amnion that step 2 is obtained is handled 3 days with the 0.5mg/mL ribose aqueous solution at 2 DEG C；
Step 4: the preparation of amnion particulate：Amnion after step 3 modification purified water is shaken into cleaning 10 times, so
Rear chamber warm air is done, most after that amnion is ground into about 500 μm of sizes of particle diameter at 8 DEG C through ultrahigh speed low-temperature centrifugation pulverizer
Grain；
Step 5: the preparation of amnion particulate implant：Amnion particulate prepared by 5g step 4 uses 100mL as component A
Water for injection prepares pH and is 7.0 phosphoric acid physiological salt solution, then adds 50mg lidocaines and 200mg hyaluronic acids, with
As component B after above-mentioned phosphoric acid physiological salt solution mixing, by component A and component B mass volume ratios 50:1（g/L）Quick shake
Mixing is swung, is circulated 5 times through high-speed micro-jet equipment, operating pressure is in 8000Pa, prepared by a diameter of 0.25mm of outlet nozzle
Into the amnion microparticulate suspensions that concentration is 50mg/mL, about 150~200 μm of the particle diameter of amnion particulate.Finally amnion microparticulate suspensions are passed through
Sterile vacuum is filling, then through 15kGy radiated by gamma-ray sterilize i.e. prepare can Clinical practice amnion particulate implant.Institute
The composition for stating phosphoric acid physiological salt solution is：It is respectively 4.0g/L sodium chloride containing final concentration using water for injection as solvent
Solution, 0.2g/L sodium dihydrogen phosphate and 1.0g/L disodium phosphate soln.
The amnion particulate implant particle diameter that the present embodiment is prepared is larger, while with the addition of lidocaine as local fiber crops
Liquor-saturated dose, it with the addition of hyaluronic acid to improve amnion particulate implant viscosity.The amnion particulate implantation that this other example is prepared
Agent concentration and modification degree are higher, therefore degradation rate is slower, and external degradation, degraded are carried out using 0.2mg/mL Proteinase K
Time is about 72 hours, Study on degradation result such as Fig. 9 institutes of the detection of subcutaneous rat implanting tissue and amnion particulate implant
Show, amnion particulate implant still has lot of materials not to be degraded and absorbed after showing 52 weeks, it is contemplated that sheep after 18~24 months after injection
Film particles implant can degrade substantially.Amnion particulate implant prepared by this method can be used for the repairing compared with deep wrinkle such as nasolabial groove
It is multiple.
Embodiment 3,
Step 1: disinfecting：Aseptically, amnion is immersed in PBS and cleaned extremely without color, dirt, then used
Purified water is cleaned 3 times, and then amnion is carried out disinfection for 75% ethanol water with volumetric concentration and handled 10 minutes；Volume is used again
Concentration is sterilized 10 minutes for 1% hydrogen peroxide solution to amnion；
Step 2: de- cell processing：First the amnion after sterilization is cleaned 2 times with purified water, then 1mg/mL EDTA water-soluble
Liquid carries out concussion processing 60 minutes, 0.1mg/mL pancreatin aqueous solution oscillation treatment 10 minutes to amnion, and frequency of oscillation is
60rpm, is finally cleaned 6 times using purified water；
Step 3: modification：The glutaraldehyde water solution that the amnion volumetric concentration that step 2 is obtained is 1% is in room temperature
Lower processing 6 hours；
Step 4: the preparation of amnion particulate：Amnion after step 3 modification purified water is shaken into cleaning 6 times, so
Rear chamber warm air is done, most after that amnion is ground into about 280 μm of sizes of particle diameter at 4 DEG C through ultrahigh speed low-temperature centrifugation pulverizer
Grain；
Step 5: the preparation of amnion particulate implant：Amnion particulate prepared by 3.5g step 4 is used as component A
100mL waters for injection prepare pH and are used as component B for 7.5 phosphoric acid physiological salt solution（The phosphoric acid sodium chloride life of the component B
Reason solution composition be：It is respectively 8g/L sodium chloride solution, 0.4g/L phosphoric acid containing final concentration using water for injection as solvent
The disodium phosphate soln of dihydro sodium solution and 2.0g/L）, by component A and component B according to mass volume ratio 35:1（g/L）Quickly
Concussion is mixed, and is circulated 6 times through high-speed micro-jet equipment, operating pressure is in 12000Pa, and a diameter of 0.2mm of outlet nozzle is made
It is standby into the amnion microparticulate suspensions that concentration is 35mg/mL, about 80~120 μm of the particle diameter of amnion particulate.Finally by amnion microparticulate suspensions
First through 25kGy radiated by gamma-ray sterilize, then through Sterile vacuum it is filling i.e. prepare can Clinical practice amnion particulate be implanted into
Agent.
Three batches of amnion particulate implants that this example is prepared carry out degradation time in vitro, amnion particle concentration, put down
Equal particle diameter and dynamic viscosity detection, as a result as shown in table 1, testing result difference less, shows that the inventive method can between each batch
To be effectively ensured and control the stability of the amnion particulate implant quality prepared.
Embodiment 4,
Step 1: disinfecting：Aseptically, amnion is immersed in PBS and cleaned extremely without color, dirt, then used
Purified water is cleaned 3 times, then amnion is carried out disinfection 60 minutes for 3% hydrogen peroxide solution with volumetric concentration, then use volumetric concentration
Amnion is carried out disinfection 60 minutes for 90% ethanol water, then with the peroxide acetate aqueous solution that volumetric concentration is 2% to amnion
Carry out disinfection processing 60 minutes；
Step 2: de- cell processing：Amnion after step one is disinfected first is cleaned 3 times with purified water, then uses 1mg/
ML EDTA aqueous solution concussion processing 120 minutes, then handled 120 minutes with the concussion of 0.1mol/L sodium hydrate aqueous solutions, then use
5mol/L sodium-chloride water solutions oscillation treatment 120 minutes, is finally carried out at concussion with the 1mg/mL pancreatin aqueous solution to amnion again
Reason 120 minutes, frequency of oscillation is 80rpm.Finally use PBS 10 times；
Step 3: modification：The amnion that step 2 is obtained is located at room temperature with the 0.1mg/mL riboflavin aqueous solution
Reason 4 hours；
Step 4: the preparation of amnion particulate：By the PBS concussions cleaning 3 times of the amnion after step 3 modification, Ran Houzhen
Vacuum freecing-dry, most after that amnion is ground into about 300 μm of sizes of particle diameter at 6 DEG C through ultrahigh speed low-temperature centrifugation pulverizer
Grain；
Step 5: the preparation of amnion particulate implant：Amnion particulate prepared by 2g step 4 uses 100mL as component A
Water for injection prepares pH and is 7.5 phosphoric acid physiological salt solution, then adds 30mg lidocaine and above-mentioned phosphoric acid sodium chloride
As component B after physiological solution mixing, by component A and component B mass volume ratios 20:1（g/L）Quick concussion is mixed, through at a high speed
Microfluidizer is circulated 8 times, and operating pressure is 15000Pa, and a diameter of 0.1mm of outlet nozzle is prepared into concentration for 20mg/mL sheep
Film particles suspension, the particle diameter of amnion particulate is about 50~200 μm.Finally by amnion microparticulate suspensions through 15kGy radiated by gamma-ray
Sterilizing, then through Sterile vacuum it is filling i.e. prepare can Clinical practice amnion particulate implant.The phosphoric acid sodium chloride physiology
The composition of solution is：It is respectively 8g/L sodium chloride solution, 0.4g/L di(2-ethylhexyl)phosphate containing final concentration using water for injection as solvent
The disodium phosphate soln of hydrogen sodium solution and 2.0g/L.
The amnion particulate implant action effect and embodiment 1 that this example is prepared are basically identical, available for canthus week
Enclose the reparation of microgroove.
Amnion particulate implant that this example is prepared carries out the result of droplet measurement as shown in figure 8, from droplet measurement
As a result it can be seen that particle size there are about more than 90% in 50~200 μ ms, show that amnion particulate implant can be controlled effectively
Product cut size processed.
Embodiment 5,
Step 1: disinfecting：Aseptically, amnion is immersed in PBS and cleaned extremely without color, dirt, then used
Purified water is cleaned 3 times, and then carried out disinfection processing 5 minutes with the hydrogen peroxide solution that volumetric concentration is 1% to amnion, then uses volume
The ethanol water that concentration is 60% carries out disinfection to amnion to be handled 5 minutes, then water-soluble for 0.1% Peracetic acid with volumetric concentration
Liquid carries out disinfection to amnion and handled 5 minutes；
Step 2: de- cell processing：Amnion after step one is disinfected first is cleaned 3 times with purified water, then is used
0.1mg/mL EDTA aqueous solution concussion processing 5 minutes, then handled 5 minutes with the concussion of 0.01mol/L sodium hydrate aqueous solutions, then
With 1mol/L sodium-chloride water solutions oscillation treatment 5 minutes, finally amnion is carried out at concussion with the 0.1mg/mL pancreatin aqueous solution
Reason 5 minutes, frequency of oscillation is 50rpm.Finally using purified water or PBS 3 times；
Step 3: modification：The amnion that step 2 is obtained uses 3000 μ W/cm at room temperature2Ultraviolet irradiation 4 is small
When；
Step 4: the preparation of amnion particulate：Amnion after step 3 modification PBS is shaken into cleaning 10 times, then
Room temperature is air-dried, most after the particle that amnion is ground into about 600 μm of sizes of particle diameter at 5 DEG C through ultrahigh speed low-temperature centrifugation pulverizer；
Step 5: the preparation of amnion particulate implant：Amnion particulate prepared by 10g step 4 is used as component A
100mL waters for injection prepare pH and are 7.2 phosphoric acid physiological salt solution, then add 1mg bFGF and above-mentioned phosphoric acid sodium chloride
As component B after physiological solution mixing, by component A and component B mass volume ratios 100:1（g/L）Quick concussion is mixed, through at a high speed
Microfluidizer is circulated 3 times, and operating pressure is 5000Pa, a diameter of 0.25mm of outlet nozzle, is prepared into concentration for 100mg/mL
Amnion microparticulate suspensions, the particle diameter of amnion particulate is 150~180 μm.It is finally that amnion microparticulate suspensions are filling through Sterile vacuum, then pass through
10kGy radiated by gamma-ray sterilizing i.e. prepare can Clinical practice amnion particulate implant.The phosphoric acid sodium chloride physiology
The composition of solution is：Using water for injection as solvent, the sodium chloride solution containing final concentration of 5.5g/L, 0.312g/L di(2-ethylhexyl)phosphate
The disodium phosphate soln of hydrogen sodium solution and 1.6g/L.
The amnion particulate implant action effect and embodiment 2 that this example is prepared are basically identical, available for nasolabial groove
Deng the reparation compared with deep wrinkle.
Amnion particulate implant that this example is prepared, which carries out the detection of collagen immunization group and collagen, factor content, to be determined
Amount detection, as a result as shown in Figures 2 and 3, from testing result as can be seen that this example prepares the reservation of amnion particulate implant
Natural collagen and all kinds of factor components in amnion.
Embodiment 6,
Step 1: disinfecting：Aseptically, amnion is immersed in PBS and cleaned extremely without color, dirt, then used
Purified water is cleaned 2 times, and then carried out disinfection processing 30 minutes with the hydrogen peroxide solution that volumetric concentration is 2% to amnion, then uses volume
The ethanol water that concentration is 75% carries out disinfection to amnion to be handled 30 minutes, finally with the Peracetic acid water that volumetric concentration is 1%
Solution carries out disinfection to amnion and handled 30 minutes；
Step 2: de- cell processing：Amnion after step one is disinfected first is cleaned 2 times with purified water, then is used
The 0.5mg/mL EDTA aqueous solution is handled 60 minutes, then is handled 60 minutes with 0.05mol/L sodium hydrate aqueous solutions, then is used
3mol/L sodium-chloride water solutions are handled 60 minutes, are finally handled 60 minutes with the 0.5mg/mL pancreatin aqueous solution, frequency of oscillation is
65rpm.Finally use PBS 7 times；
Step 3: modification：The aqueous solution that the amnion that step 2 is obtained is 6 with 0.5mol/L EDC.HCL pH
In, handled 48 hours at 40 DEG C；
Step 4: the preparation of amnion particulate：By the PBS concussions cleaning 7 times of the amnion after step 3 modification, Ran Houzhen
Vacuum freecing-dry, most after the particle that amnion is ground into 500 μm of sizes at 3 DEG C through ultrahigh speed low-temperature centrifugation pulverizer.
Step 5: the preparation of amnion particulate implant：Amnion particulate prepared by 5g step 4 uses 100mL as component A
Water for injection prepares pH and is 7.3 phosphoric acid physiological salt solution, then adds 1g hyaluronic acids and above-mentioned phosphoric acid sodium chloride physiology
As component B after solution mixing, by component A and component B mass volume ratios 50:1（g/L）Quick concussion is mixed, and is penetrated through high speed is micro-
Flow device is circulated 5 times, and operating pressure is 10000Pa, a diameter of 0.15mm of outlet nozzle, is prepared into concentration for 50mg/mL amnions
Microparticulate suspensions, the particle diameter of amnion particulate is 120~170 μm.Finally amnion microparticulate suspensions are first gone out through 10kGy radiated by gamma-ray
Bacterium, then filling through Sterile vacuum, that is, prepare can Clinical practice amnion particulate implant.The phosphoric acid sodium chloride physiology is molten
The composition of liquid is：Using water for injection as solvent, the sodium chloride solution containing final concentration of 5.8g/L, 0.356g/L biphosphate
The disodium phosphate soln of sodium solution and 1.4g/L.
The amnion particulate implant action effect and embodiment 2 that this example is prepared are basically identical, available for nasolabial groove
Deng the reparation compared with deep wrinkle.
The amnion particulate implant that this example is prepared, carries out ESEM detection, testing result is such as after being lyophilized
Shown in Fig. 4, from testing result as can be seen that amnion particulate still remains the good three-dimensional net structure of characteristics of amniotic extracellular matrix.
Embodiment 7,
The present embodiment is substantially the same manner as Example 1, is different only in that：" 1- ethyls-(3- dimethyl in the step 3
Aminopropyl) carbodiimide hydrochloride " it is changed to that " the 1mg/mL riboflavin aqueous solution handles 24 hours at room temperature, at room temperature
10000μW/cm2Ultraviolet irradiates 16 hours ".In the step 5：100mg lidocaines, 100mg are added in component B saturating
Bright matter acid.
The amnion particulate implant that this example is prepared and human fibroblasts co culture system in vitro, to co-culturing one section
Human fibroblasts after time extract mRNA therein, and type i collagen mrna expression amount, detection knot are detected using RT-PCR method
Fruit is as shown in fig. 7, the amnion particulate implant that as can be seen from the figure prepared by this example, can effectively facilitate fibroblastic growth
Propagation.
Embodiment 8,
The present embodiment is substantially the same manner as Example 1, is different only in that：" 1- ethyls-(3- dimethyl in the step 3
Aminopropyl) carbodiimide hydrochloride " it is changed to " volumetric concentration is that 0.1% glutaraldehyde water solution is handled 12 hours at room temperature ".Institute
State in step 5：0.1mg bFGF, 500mg hyaluronic acid, 200mg lidocaines are added in component B.
Embodiment 9,
The present embodiment is substantially the same manner as Example 1, is different only in that：" 1- ethyls-(3- dimethyl in the step 3
Aminopropyl) carbodiimide hydrochloride " it is changed to " the 1.5mg/mL ribose aqueous solution is handled 14 days at 40 DEG C ".
Below for above-described embodiment, Binding experiment data, picture information etc. are analyzed：
Fig. 2 is the picture of the immunohistochemical staining result of non-collagen components in amnion particulate implant prepared by embodiment
In figure, 1 is Ι Collagen Type VIs, and 2 be type III collagen, and 3 be IV Collagen Type VIs, and 4 be negative control.
Picture is shown：Ι Collagen Type VIs, type III collagen content in amnion particulate implant is more, and IV Collagen Type VI contents are slightly few
But also there is obvious positive expression.
Fig. 3 is the figure of the quantitative testing result of a variety of collagens and active factors of amnion particulate implant manufactured in the present embodiment
Piece
In figure, Ι Collagen Type VIs, type III collagen content are more, and IV Collagen Type VI contents are slightly few, with non-collagen components qualitative results phase
Symbol.In addition, HGF in each active factors（HGF）, keratinocyte growth factor（KGF）And（Fibroblast
Growth factor）BFGF contents are more.As a result show that amnion particulate implant manufactured in the present embodiment can be effectively retained various active
The factor.
Fig. 4 is the lyophilized rear ESEM result of amnion particulate implant manufactured in the present embodiment
It is visible in figure：Amnion particulate after lyophilized is in the sheep in more loose fibre morphology, display amnion particulate implant
Film particles still remain the good three-dimensional net structure of characteristics of amniotic extracellular matrix.
Fig. 5 is that human fibroblasts are carried out after co-culturing 5 days under ordinary optical microscope with amnion particulate implant
Observe picture
In figure, fibroblast attaches well with amnion particulate implant, and fibroblast energy normal proliferative grows, knot
Fruit shows that amnion particulate implant prepared by the present invention has good cell compatibility.
Fig. 6 is that human fibroblasts carry out Histological section's picture after co-culturing 5 days with amnion particulate implant
As a result show：Amnion particulate implant top layer and part amnion interparticle have attached continuous fibroblast
Layer, illustrates that amnion particulate implant prepared by the present invention has fabulous cell compatibility, and also illustrate that amnion particulate is planted
Enter agent and remain preferable characteristics of amniotic extracellular matrix three-dimensional net structure, cell can be promoted to grow into.
Fig. 7 be amnion particulate implant prepared by embodiment co-culture 3 with human fibroblasts in vitro, 5, after 7 days, take
All human fibroblasts in co-culture system, are extracted after mRNA therein using RT-PCR method detection type i collagen mRNA
The picture of the testing result of expression quantity
As can be seen from the figure：After 7 days, the type i collagen mrna expression amount ratio of the human fibroblasts under the conditions of co-cultivation
Individually the expression quantity of culture human fibroblasts will have more 60% or so, illustrate amnion particulate implant prepared by the present invention, can have
Effect retains the fibroblast growth factor isoreactivity composition in amnion, can play the work for promoting proliferation of fibroblasts
With.
Table 1 is the external degradation of many batches of amnion particulate implants prepared according to the present invention, averagely amnion particle concentration, grain
Footpath and the testing result of dynamic viscosity
| Product batches code name | Degradation time in vitro（min） | Amnion particle concentration（mg/mL） | Average grain diameter (μm) | Dynamic viscosity (mPa ﹒ s) |
| 1 | 4358 | 35.04 | 98.23 | 9341 |
| 2 | 4403 | 35.19 | 99.84 | 9580 |
| 3 | 4278 | 35.74 | 98.91 | 9803 |
Table 1
Illustrate that amnion particulate implant prepared by the present invention can effectively control the stability of product above mass parameter.
Fig. 8 is the picture of the particle diameter distribution testing result of amnion particulate implant prepared by embodiment
Illustrate that amnion particulate implant prepared by the present invention can effectively control product cut size distribution.
Fig. 9 is progress histology and sheep after amnion particulate implant implantation subcutaneous rat tissue prepared by the present invention
The Study on degradation result picture of film particles implant
In figure, A is 1 week materials outward appearance picture, and B is 1 week materials histological stain section picture, and C is 4 weeks materials outside drawings
Piece, D is 4 weeks materials histological stain section pictures, and E is 8 weeks materials outward appearance pictures, and F is 8 weeks materials histological stain slice maps
Piece, G is 24 weeks materials outward appearance pictures, and H is 24 weeks materials histological stain section pictures, and I is 52 weeks materials outward appearance pictures, and J is
52 weeks materials histological stain section pictures.As a result show：Amnion particulate implant has good histocompatbility, good
Long-term filling effect, the phenomenon such as will not be shifted over, spread.
Laboratory report（Take passages）
1st, amnion particulate implant is introduced
Amnion（Amniotic membrane AM）For it is smooth, without blood vessel, impassivity, the transparent membrane without lymph, it is thick about
0.02~0.50mm, is a kind of preferable organizational project repair materials, has been used for the treatment and reparation of Various Tissues defect.
The amnion particulate implant that this experiment is manufactured, is, by this patent technique, to be prepared as injection type particulate by amnion
Implant.The variant cell composition in amnion can be effectively removed in preparation process, and at utmost retains amnion natural collagen
And all kinds of factors, good biocompatibility, without obvious immune induction activity, with the good collagenogenic ability of induction,
It is a kind of preferable beauty injection fillers material.
2nd, wrinkle of skin Plastic renovation is introduced
2.1 wrinkle formation mechanisms
The reason for skin wrinkle, there is a variety of, is broadly divided into endogenous and exogenous reason, endogenous mainly with
The growth at age, the aging of skin, the balance of synthesis and the degraded of collagen and elastin laminin is broken, and causes collagen
With the reduction of elastin laminin content, skin corium thickness reduces, and collagenous fibres stereochemical structure is destroyed, fibroblast in skin corium
Aging and disappearance and cause wrinkle to produce；The various radiation such as the ultraviolet that exogenous reason is mainly in environment cause base in skin
Matter metalloproteinases increases, so that causing the content of collagen in skin reduces, in addition skin health state with nicotine, again
Renaturation muscular movement, diet, sleeping posture and holistic health degree have compared with Important Relations.
The 2.2 amnion particulate implant mechanisms of action
The injection site of amnion particulate implant is corium deep layer, and main function is that corium supplement has humanized after injection
Collagen, polysaccharide extracellular matrix, including I, the type III collagen being rich in amnion etc., and amnion particulate implant is rich in
A variety of factors such as bFGF can effectively facilitate the fibroblast proliferation in the skin corium of injection site, so as to greatly promote true
Collage synthesis in cortex, a variety of anti -inflammatory cytokines such as TIMP contained etc., it can effectively reduce the Metal Substrate in extracellular matrix
Matter proteinase activity, suppresses its degradation rate to the extracellular matrix in skin corium；Meeting syringe needle is caused during additionally, due to injection
Slight damage, can also stimulate body to carry out remodeling effect to the dermal tissue of injection site during wound healing simultaneously.
3rd, animal experimental model, operation method and evaluation criterion
3.1 animal experimental model
This Report on Animal uses the efficacy for be subcutaneously injected filling effect to 30 SD rats to verify, moves
Thing male or female, animal age is adult.
3.2 operation method
1 foundation
Chinese name republic standard GB/T/T16886.10-2005《GBT16886.2-2000 medicine equipment biology
Evaluate part 2：Animal protection requirement》
2 animal subjects
（1）Animal strains：SD rats
（2）Rearing conditions：Full nutrition pellet feeding, free choice feeding, free water, 18-26 DEG C of room temperature, per the daylight
According to 12 hours, pre-operative anxiety 8 hours.
3 operation methods
（1）The sodium intravenous carry out deep anaesthesia of 1% amobarbital is used according to 35mg/kg dosage.
（2）Treat that animal enters stage of surgery, animal prone position is fixed on operating table.
（3）Conventional preserved skin, iodophor disinfection operative site.
（4）Patient opens amnion particulate implant product, injection needle is installed, using point-like injecting method, by material
It is injected to the subcutaneous shallow-layer position of rat thoracic vertebrae both sides skin.Every injection volume 0.5ml.
（5）Injection carries out iodophor disinfection again after finishing to injection site.
（6）Postoperative animal warm is recovered, and cage tool is put into after reviving.
（7）Cage tool label is finished writing, test data sheet is carried out.
4 evaluation indexes
30 experimental rats are carried out in postoperative 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, 24 weeks, 32 weeks, 40 weeks, 52 weeks
Put to death dissection materials, each time point draws materials 3 rats, gross examination of skeletal muscle amnion particulate implant in subcutaneous rat residual condition,
And carry out histocompatbility, inflammatory reaction and the degraded situation of Histological section's detection material implant site etc..
4th, results of animal
4.1 substantially materials appearance results are as shown in Figure 10
As shown in Figure 10, each time point observes subcutaneous rat implant site to injection site general condition during each time point materials
Do not occur the red and swollen, adverse reaction such as fester, skin appearance is normal, and Biocompatibility is good.Amnion particulate implant exists
Implantation subcutaneous rat remains to go out from the appearance the protuberance effect at injection material position after 52 weeks.
4.2 histology results are as shown in figure 11
Material shape after each time point materials, as shown in figure 11, each time point material is kept Histological section's result
Good filling effect, the bad phenomenon such as is not shifted over, spreads.Amnion particulate implant is after implantation subcutaneous rat 52 weeks
Remaining to see from Histological section has more material remaining, and material internal inflammatory cell infiltration is less, and fibroblast gradually moves
Enter material internal, amnion particulate implant biocompatibility is good.
The above results illustrate that amnion particulate implant has good histocompatbility, good long-term filling effect, no
It the phenomenon such as can be shifted over, spread.
Claims (5)
1. a kind of injectable implant, it is characterised in that：It is to be mixed in pH is 7.0~7.5 phosphoric acid physiological salt solution
There is amnion particulate；The mean particle dia of the injectable implant is 50~200 μm, and concentration is 30~100mg/mL：
Amnion particulate and phosphoric acid physiological salt solution, are mixed according to mass volume ratio 20: 1~100: 1；The phosphoric acid sodium chloride
Physiological solution, is the sodium chloride solution containing final concentration of 3~8g/L using water for injection as solvent, 0.2~0.4g/L phosphoric acid
The disodium phosphate soln of dihydro sodium solution and 1.2~2.0g/L.
2. a kind of preparation method of injectable implant, it is characterised in that：Using amnion as raw material, by de- cell, it is cross-linking modified,
Homogenate and filling, is prepared from, it includes disinfecting for amnion, takes off cell and handle, modification, the preparation of amnion particulate with
And the preparation of amnion particulate implant, comprise the following steps that：
Step 1: disinfecting：Aseptically, amnion is immersed in phosphate buffer and cleaned extremely without color, dirt,
Cleaned again with purified water 1~5 time, the hydrogen peroxide solution for being then 1~3% with volumetric concentration, volumetric concentration are 60~90%
Ethanol water, volumetric concentration are the one or several kinds in 0.1~2% peroxide acetate aqueous solution, and amnion is carried out successively
Disinfect 5~60 minutes；
Step 2: de- cell processing：Amnion after step one is disinfected first is cleaned 1~3 time with purified water, then with 0.1~
1mg/mL ethanedioic acid tetrem amine aqueous solution, 0.01~0.1mol/L sodium hydrate aqueous solutions, 1~5mol/L aqueous sodium chlorides
One or several kinds in liquid, the 0.1~1mg/mL pancreatin aqueous solution, carry out concussion processing 5~120 minutes to amnion successively,
Frequency of oscillation is 50~80rpm, finally using purified water or PBS 3~10 times；
Step 3: modification：The amnion that step 2 is obtained is with processing is either physically or chemically modified, according to final production
The requirement of product degradation property, may be selected the method for modifying of different disposal intensity；Modified chemical reagent or physical method bag used
Include but be not limited to：The pH of 0.1~0.5mol/L 1- ethyls-(3- dimethylaminopropyls) carbodiimide hydrochloride is 5~6
The aqueous solution is handled 6~48 hours at 2~40 DEG C, and it is small that the 0.1~1mg/mL riboflavin aqueous solution handles 4~24 at room temperature
When, 3000~10000 μ W/cm at room temperature2Ultraviolet irradiates 4~16 hours, and volumetric concentration is 0.1~1% glutaraldehyde water solution
Handle 6~12 hours at room temperature, the 0.5~1.5mg/mL ribose aqueous solution is handled 3~14 days at 2~40 DEG C；
Step 4: the preparation of amnion particulate：By the amnion purified water after step 3 modification or PBS concussions cleaning 3~10
It is secondary, then carry out room temperature air-dry or vacuum freeze drying processing, most after at 2~8 DEG C by amnion through ultrahigh speed low-temperature centrifugation powder
Broken machine is ground into the particle of 100~1000 μm of sizes；
Step 5: the preparation of amnion particulate implant：Amnion particulate prepared by step 4 as component A, by pH be 7.0~
7.5 phosphoric acid physiological salt solution is as component B, by component A and component B according to mass volume ratio 20: 1~100: 1 (g/L)
Concussion is mixed, and is circulated 3~8 times through high-speed micro-jet equipment；Then, it is first that amnion microparticulate suspensions are filling through Sterile vacuum, then pass through
10~30kGy radiated by gamma-ray sterilizes or first amnion microparticulate suspensions sterilizes through 10~30kGy radiated by gamma-ray, goes forward side by side
Row Sterile vacuum is filling, that is, be made can Clinical practice amnion particulate implant；The phosphoric acid physiological salt solution of the component B
Composition be：Using water for injection as solvent, the sodium chloride solution containing final concentration of 3~8g/L, 0.2~0.4g/L di(2-ethylhexyl)phosphate
The disodium phosphate soln of hydrogen sodium solution and 1.2~2.0g/L.
3. the preparation method of injectable implant according to claim 2, it is characterised in that：In component B, also include thin
Any of the intracellular growth factor, lidocaine, hyaluronic acid are several, final concentration of 0.001~0.01mg/mL cell
Any of growth factor, 0.3~2mg/mL lidocaine, 1~10mg/mL hyaluronic acid are several.
4. injectable implant preparation method according to claim 2, it is characterised in that：In step 5, the high speed is micro- to penetrate
The operating pressure of flow device is 5000~15000Pa, a diameter of 0.1~0.25mm of outlet nozzle.
5. the fixed shell for injectable implant preparation method described in claim 2, it is characterised in that：For fixing sheep
Film, the fixed shell is to be evenly distributed with some apertures on hollow semielliptical shape housing, the housing.
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| CN106492285A (en) * | 2016-10-31 | 2017-03-15 | 广州昕生医学材料有限公司 | Injectable Acellular cartilaginous matrix particulate and its application in implant |
| CN106492288A (en) * | 2016-10-31 | 2017-03-15 | 广州昕生医学材料有限公司 | Injectable de- cellular fat matrix particles and its application in implant |
| CN107213028A (en) * | 2017-05-26 | 2017-09-29 | 陕西慧康生物科技有限责任公司 | A kind of collagen implant and preparation method thereof |
| CN107308494A (en) * | 2017-07-27 | 2017-11-03 | 北京华信佳音医疗科技发展有限责任公司 | A kind of injection collagen, preparation method and filler |
| AU2019269210B2 (en) * | 2018-05-18 | 2025-02-27 | Mimedx Group, Inc. | Placental tissue component compositions for treatment of skin defects and methods using same |
| CN109568663B (en) * | 2018-11-27 | 2021-01-15 | 中山大学中山眼科中心 | Preparation method of corneal stent material and corneal stent material |
| CN111359020B (en) * | 2018-12-26 | 2022-05-13 | 深圳兰度生物材料有限公司 | Soft tissue repair material and preparation method and application thereof |
| CN110585456B (en) * | 2019-09-25 | 2021-06-01 | 赛克赛斯生物科技股份有限公司 | Virus inactivation method, treatment method and application of biological material |
| CN113769168A (en) * | 2021-08-16 | 2021-12-10 | 江苏优创生物医学科技有限公司 | Acellular matrix particle product for soft tissue filling repair and preparation method thereof |
| CN114949359B (en) * | 2022-06-27 | 2024-01-23 | 西安臻研生物科技有限公司 | Acellular matrix particle filler and preparation method thereof |
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