CN105284673A - Method for renewing old pearl - Google Patents
Method for renewing old pearl Download PDFInfo
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- CN105284673A CN105284673A CN201510304841.6A CN201510304841A CN105284673A CN 105284673 A CN105284673 A CN 105284673A CN 201510304841 A CN201510304841 A CN 201510304841A CN 105284673 A CN105284673 A CN 105284673A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a method for renewing an old pearl. The method comprises following steps: firstly removing stains on the surface of the old pearl; secondly removing the pearl layer with the modified surface and bleaching a surface layer; covering the surface with a layer of a biodegradable high molecular polymer tissue engineering scaffold suitable for development of tissue; cultivating a mantle free cell taken from a pearl mussel and the old pearl adhered to the tissue engineering scaffold in vitro such that the tissue is adhered to the tissue engineering scaffold of the surface of the old pearl, splits and develops; inserting the old pearl to a nucleus insertion position for the pearl mussel; engraving a clamshell with a related mark; sleeving the clamshell with a net bag having mesh apertures less than diameter of the pearl; and adopting a conventional pearl mussel culture method for cultivation. The tissue engineering scaffold is three-dimensional net space allowing mitosis and growth of tissue. The method for renewing the old pearl has following beneficial effects: clams are picked and pearls are taken according to the appointed time by adoption of the conventional pearl mussel culture method for cultivation; the luster of the surface pearl can be kept for a long time; an internal structure of the pearl is preserved; and therefore precious pearls can be stored for a long time so that pearls with commemorative significance can be stored for a long time.
Description
Technical field
The present invention relates to one makes old pearl renovate, thus makes pearl be able to permanent method of preserving.Belong to pearl culture field.
Background technology
Pearl is the natural gemstone that people like, the development propagated artificially along with pearl, and the output of pearl is very high, but the pearl of jewelry level is also few.What pearl circle had " seven pearl eight treasures (choice ingredients of certain special dishes) " says, the pearl meaning more than diameter 8mm is very difficult, large and pearl that is circle is very rare.Rare pearl is usually by as rare treasure, and the crown pearl, Quan Zhu, the necklace worn by official, imperial robe pearl, armor pearl etc. of China and foreign countries emperor are all the symbols of state supremacy power.The crown of British empire queen studs with 227 pearls, and wherein a pearl diameter originated from white butterfly shell reaches 1.8 centimetres, and the imperial crown of Russian queen Ye Kajielinna bis-studs with priceless large pearl 80.It is documented, kind auspiciousness time head on have on one top gold hat, inlay the dragon and phoenix pearl of the egg size that is paid tribute from Nan Yang.The necklace worn by official of Qianrong is also made with 108 pearls.Regrettably, as time goes by, thus the gloss that these famous and precious pearls all lose its beauty lose its jewel value.
The beauty of pearl, depends on its gloss to a great extent.Although the color and luster of pearl is very attractive in appearance, be unable to undergo long test.General through decades, pearl will become common yellow, loses beautiful pearly luster simultaneously, Here it is proverb said " in the sear and yellow ".Its reason is that nacre is staggered by aragonite crystals and organic substrate to form, and the time one is long, and it will become common calcite.Aragonite and calcite, although chemical composition is the same, and their crystal habit and gloss are made a world of difference.The reason of pearl variable color that Here it is.Pearl is once turn yellow, and its value will be had a greatly reduced quality.Time is excessively of a specified duration, and the nacre of pearl inside also gradually becomes calcite, that just only remaining pearl shape and without the essence of pearl.Pearl general life-span is greatly about about 100 years.The yellowing gradually of general use time surface about tens to decades, As time goes on, pearl interior also starts change gradually, finally becomes one piece of broken at a touch calcium carbonate powder.
The pearl of treasure level how is made to maintain the gloss of its beauty, really can become the treasure of passing on from generation to generation, particularly there is souvenir or the pearl energy long-term inheritance of historical relic meaning goes down, be one highly significant with the thing be worth, regrettably have not yet to see someone and study.The present invention has invented one and has cultivated by again being implanted in pearl shell by old pearl; the way of new nacre is covered again on the surface of former pearl; pearl can be made to keep its lustrous surface for a long time; also the interior of pearl can be protected; thus the pearl of some preciousnesses can be made to be able to persistence go down, be particularly suitable for the long-term preservation of memorable pearl.
Summary of the invention
The object of this invention is to provide a kind of method that pearl can be made to preserve for a long time; the way of new nacre is covered again by the surface former pearl; pearl can be made to keep its lustrous surface for a long time; also the interior of pearl can be protected; thus the pearl of some preciousnesses can be made to be able to persistence go down, be particularly suitable for the long-term preservation of memorable pearl.
For achieving the above object, present invention employs following technical scheme: the method for a kind of old pearl renovation of the present invention, it is characterized in that, the spot of old pearl surface is first washed away with neutral detergent solution, then the nacre of its surface modification is dissolved with dilute acid soln, and/or bleach its top layer with hydrogen peroxide solution, the degradable high polymer tissue engineering bracket that grows of cell is applicable to wherein uniformly afterwards in its surface coverage a layer thickness, described tissue engineering bracket is a cubic network space that can hold cell merisis wherein, outer embrane free cell culture in vitro together with the old pearl having adhered to tissue engineering bracket of pearl culturing clam will be taken from, cell is attached in the tissue engineering bracket of old pearl surface, division, growth, again old pearl is inserted the slotting nuclear location of pearl culturing clam, clam shell engraves mark of correlation, put the mesh bag that mesh aperture is less than pearl diameter, adopt conventional pearl culturing clam cultural method cultivation, vertical hanging culture supports alternately rotation with flat, adopt freshwater mussel by the time of agreement and get pearl, to get pearl winter as well.
The method of a kind of old pearl renovation of the present invention, its concrete steps are:
1) remove surface blot: by old pearl neutral detergent solution soaked overnight, wiped clean, then in distilled water, cleaning solution is removed in rinsing;
2) dilute acid soln dissolves old pearl surface denatured layer;
3) bleach: use hydrogen peroxide solution bleaching 10 ~ 60 minutes;
4) old pearl shelf layer mould used is prepared: described mould is made up of mould I and mould II, mould I is made up of the hollow hemisphere that two structures are the same, the medial surface of each hemisphere has the rib of 3 projections, wherein 2 ribs lay respectively at hemisphere both sides of the edge, another rib is positioned at the centre of hemisphere, the length of rib is 1/2 of spheroid girth, article 3, the corresponding north and south at ball of the end of rib is extremely crossing, the rib cut-out of end intersection, the South Pole forms recess as hole for injecting glue, height 0.02 ~ the 2mm of rib, edge rib width is the half of middle edge; The effect of rib is to support old pearl, and the height after making that old pearl is put into mould between old pearl with hemisphere inner face is consistent, and the height of rib is exactly the thickness of the tissue engineering bracket that will fill; During use, first old pearl is put into a hemisphere, then another hemisphere is closed up, edge rib its side after two hemispheres closes up of two hemisphere is stitched together and is combined into the width rib the same with middle edge width, two hemisphere close up into a spheroid, the sphere being positioned at middle edge one end is closed, and the sphere place being positioned at the other end reserves an aperture as hole for injecting glue for perfused tissue engineering rack liquid, the rib cut-out of this end joint, reserves duct and makes tissue engineering bracket liquid from then on be filled in 4 spaces separated by rib in mould in duct simultaneously; The first step that mould I is prepared for completing old pearl tissue engineering bracket, obtains the old pearl of reeded tissue engineering bracket; Mould II is made up of the hemisphere of two hollows, its inner surface is without raised structures, hemisphere internal diameter is 2 times of sums of old pearl diameter and mould I rib height, a hole for injecting glue is left for perfused tissue engineering rack liquid after two hemispheres closes up, utilize mould II to complete filling up of groove, obtain the old pearl of bulb tissue's engineering rack that tissue engineering bracket layer thickness is identical;
5) tissue engineering bracket liquid is prepared under aseptic condition: adopt compound that collagen or itself and other materials form as timbering material, under aseptic condition, timbering material is dissolved in pure water, concentration is 1.0wt% ~ 3.0wt% solution, the glutaraldehyde solution of 2.5vol% is added wherein as crosslinking agent after timbering material dissolves completely, make glutaraldehyde final concentration be 0.01vol% ~ 0.05vol%, stir;
6) support preparation: first the old pearl of step 3) process is positioned in mould I, tissue engineering bracket perfusion step 5) prepared enters mould I, vibration shakes up 10 minutes, and left at room temperature makes tissue engineering bracket material be cross-linked for 2 ~ 12 hours, the demoulding after being cooled to 4 DEG C, 10 minutes; Then open mould I and take out old pearl, be placed in mould II again, the indent of the groove of tissue engineering bracket is made to aim at hole for injecting glue, tissue engineering bracket solution prepared by step 3) of reinjecting, during to make first time injecting glue, the groove that stays of old pearl surface fills up tissue engineering bracket liquid, and left at room temperature makes tissue engineering bracket material be cross-linked for 2 ~ 12 hours; Then take out, by the surface rubbing at hole for injecting glue place, under-20 DEG C of conditions, place 1 ~ 6 hour freeze forming, then freeze drier vacuum drying is put into 6 ~ 24 hours, vacuum is 12 ~ 20Pa, and freeze temperature is-20 ~-70 DEG C, is soaked in distilled water by old pearl and washes away residual glutaraldehyde;
7) outer embrane free cell obtains: cleaning clam shell, tears and gets nacreous mantle tissue, margins of excision colo(u)r streak part, remove endepidermis, be cut into about 1mm after cleaning and disinfecting
2fritter, adopt trypsin digestion digestion tissue block, usual employing is containing the 0.25wt% trypsin solution of EDTA, digested 0.5 ~ 4 hour of 25 ~ 37 DEG C of scopes, filter with 200 order tulles and remove residual tissue block, centrifugation in 1000r/ minute goes out cell, adds culture fluid and dispels cell precipitation, obtains the mantle tissue free cell of pearl shell;
8) the free cell culture in vitro in the constant incubator together old pearl after step 6) process is separated with outer embrane, culture fluid is complete culture solution, adopt RPM1640 or M199 or DMEM, add the serum that final volume mark is 10vol% ~ 30vol%, serum adopts freshwater mussel serum or cow's serum, cultivation temperature 26 ~ 37 DEG C, incubation time 1 ~ 6 day, repeatedly can change culture fluid therebetween to ensure the attaching of cell, division, growth, cell attachment is on the tissue engineering bracket layer fiber of old pearl surface or after dividing 2 ~ 6 times further, remove culture fluid, with the old pearl of PBS wash buffer, remove the cell do not sticked on tissue engineering bracket,
9) old pearl step 8) obtained is implanted in pearl culturing clam body and cultivates, and for preventing the excessive compressing visceral mass of old pearl, adopts vertical hanging culture and horizontal hanging culture to replace rotation; Overlap at pearl culturing clam the mesh bag that a grid aperture is less than pearl diameter outward during cultivation, the cultivation time is generally 6 ~ 24 months.
Wherein said diluted acid refers to the nitric acid of the hydrochloric acid of 0.1 ~ 10wt% or the acetic acid of 0.1 ~ 20wt% or 0.1 ~ 5wt% or the sulfuric acid of 0.1 ~ 3wt%.
Wherein said hydrogen peroxide solution concentration is 5 ~ 50vol%.
Specifically, first old pearl neutral detergent is washed away surface blot, clean rear diluted acid and soak a period of time, diluted acid uses hydrochloric acid solution usually, concentration is between 1.0wt% ~ 3.0wt%, soak time length is relevant to the thickness of pearl surface yellowing layer, and object is to dissolve surperficial yellowing layer, soaks pickling is clean after completing with clear water; Old pearl can also be soaked further, by its superficial bleaching with hydrogen peroxide.Then using process after pearl as pearl core, be applicable at its surface adhesion one deck the degradable high polymer tissue engineering bracket material that cell grows wherein, cause a three-dimensional porous network space that can hold cell merisis wherein at old pearl surface.By separation from nacreous outer embrane free cell culture in vitro together with the old pearl being stained with tissue engineering bracket, make cell attachment growing multiplication on the tissue engineering bracket fiber of old pearl surface, after cell attaches on tissue engineering bracket fiber or multiple fission grows multi-layer cellular, again old pearl is inserted the slotting nuclear location of pearl culturing clam, clam shell engraves mark of correlation, put the mesh bag that mesh is less than pearl, adopt conventional pearl culturing clam cultural method cultivation, vertical hanging culture and horizontal hanging culture replace rotation, adopt freshwater mussel by the time of arranging with trustee and get pearl, to get pearl winter as well.
Concrete technical scheme is as follows:
1. remove surface blot: by old pearl neutral detergent solution soaked overnight, wiped clean, then rinsing in distilled water;
2. dissolve old pearl surface denatured layer with dilute acid soln, preferably use the dilute hydrochloric acid solution between mass concentration 1.0 ~ 3.0wt%, also can adopt other dilute acid soln, such as dilute sulfuric acid, dust technology, spirit of vinegar etc.
3. old pearl bleaching: adopt the hydrogen peroxide solution of about 30vol% concentration to soak 10 ~ 60 minutes, according to the pearl actual yellowing degree adjustment processing time.Then use the old pearl of distilled water rinsing, wash away the hydrogen peroxide on surface, adhere to one deck poly-D-lysine film at old pearl surface.
4. prepare old pearl shelf layer mould: mould I is made up of the hollow hemisphere that two structures are the same, during use, 2 hemisphere are closed up and become a complete spheroid.The medial surface of each hemisphere has 3 south poles (with hole for injecting glue present position for the South Pole, its offside is the arctic) direction arrangement strip projection (hereinafter referred to as rib), 2 ribs wherein lay respectively at the both sides of the edge of hemisphere, another rib is positioned in the middle of hemisphere, preferably be positioned at the middle of hemisphere, the length of rib is 1/2 of spheroid girth, and therefore the end of 3 ribs intersects at the south poles place of ball, and the rib cut-out of end intersection, the South Pole forms recess as hole for injecting glue; The height H of rib is 0.02 ~ 2mm, preferably about 1mm, middle edge width 2 ~ 4mm, and edge rib width is the half of middle edge width; The effect of rib is to support old pearl, and the spatial altitude after making old pearl put into mould outside old pearl is consistent, and the height H of rib is exactly the thickness of tissue engineering bracket.During use, first old pearl is put into a hemisphere, then another hemisphere is closed up, edge rib its side after two hemispheres closes up of two hemisphere is stitched together, be combined into the rib that a width is the same with middle edge width, two hemisphere close up into a spheroid, the sphere of rib one end is closed, other end sphere place reserves an aperture as hole for injecting glue for perfused tissue engineering scaffold material, the rib of this end is slightly short, reserves duct and timbering material can be filled in 4 spaces separated by rib in mould simultaneously.The first step that mould I is prepared for completing old pearl tissue engineering bracket, obtains the old pearl of surperficial reeded tissue engineering bracket.Mould II is made up of the hemisphere of two hollows, its inner surface is without raised structures, hemisphere internal diameter is 2 times of sums of old pearl diameter and mould I rib height, a hole for injecting glue is left for perfused tissue engineering scaffold material after two hemispheres closes up, utilize mould II to complete filling up of groove, finally obtain the old pearl of bulb tissue's engineering rack that shelf layer thickness is identical.
5. prepare tissue engineering bracket liquid under aseptic condition: tissue engineering bracket material can adopt natural macromolecular material, comprises collagenous fibres, the compound that fibrin, agarose, shitosan, hyaluronic acid etc. or they and other materials form; Also can adopt degradable synthesized polymer material, comprise the compound that PLA, the poly-liquor-saturated acid of second (PGA) and PLGA (PLGA) etc. or they and other materials form.Preferred material is the compound that collagen or itself and other materials form.Collagen concentration is 1.0wt% ~ 3.0wt%, and the glutaraldehyde solution adding 2.5vol% after collagen dissolves completely wherein, as crosslinking agent, makes glutaraldehyde final concentration be 0.01 ~ 0.2vol%, stirs.
6. support preparation: the old pearl first step 3 processed is positioned in mould I, tissue engineering bracket perfusion step 5 prepared enters mould I, vibration shakes up 10 ~ 30 minutes, left at room temperature makes tissue engineering bracket material be cross-linked for 1 ~ 24 hour, form a reeded gel layer at old pearl surface, be cooled to 4 ~ 10 DEG C of process and within about 10 minutes, make the demoulding of pearl core; Then open mould and take out pearl core, again the timbering material at its hole for injecting glue place is cut out on a small quantity, form the recess that is communicated with 4 road grooves, be placed in mould II again, the indent of tissue engineering bracket is made to aim at hole for injecting glue, the tissue engineering bracket solution of step 5 of reinjecting preparation, the groove that when making first time injecting glue, pearl core surface stays fills up tissue engineering bracket liquid, and left at room temperature makes tissue engineering bracket material be cross-linked for 1 ~ 12 hour; Take out, by the tissue engineering bracket surface rubbing at hole for injecting glue place, freeze forming in freezing temperatures, then puts into freeze drier vacuum drying and removes solvent.
7. outer embrane free cell obtains: cleaning clam shell, tear and get nacreous mantle tissue, margins of excision colo(u)r streak part, is cut into about 1mm after cleaning and disinfecting
2fritter, adopt trypsin digestion digestion tissue block, usual employing is containing the 0.25wt% pancreatin of EDTA, digested 0.5 ~ 4 hour of 25 ~ 37 DEG C of scopes, filter with 200 order tulles and remove residual tissue block, centrifugation in 1000r/ minute goes out cell, adds culture fluid and dispels cell precipitation, obtain the mantle tissue free cell of pearl shell, wherein build the various cells needed for pearl sac containing outer embrane epidermal cell and phoirocyte etc.
8. the free cell that the old pearl after step 6 being processed is separated with outer embrane culture in vitro in constant incubator together, culture fluid is that complete culture solution is (as RPM1640, M199, DMEM etc.) add the serum that final volume mark is 10vol% ~ 30vol%, serum can adopt freshwater mussel serum or cow's serum, cultivation temperature 26 ~ 37 DEG C, incubation time 1 ~ 5 day, repeatedly can change culture fluid therebetween to ensure the attaching of cell, division, growth, cell attachment is on the tissue engineering bracket layer fiber of old pearl surface or after dividing 2 ~ 6 times further, remove culture fluid, with the old pearl of PBS wash buffer, remove the cell do not sticked on tissue engineering bracket.
9. old pearl step 8 obtained is implanted in pearl culturing clam body and cultivates.For preventing the excessive extruding visceral mass of old pearl, vertical hanging culture and horizontal hanging culture is adopted to replace rotation.For anti-antiemetic core causes old pearl to be lost, put the mesh mesh bag less than old pearl to pearl culturing clam, making provision against emergencies there is telling core situation and can bring back and reform.The cultivation time is generally 6 ~ 24 months.To get pearl winter as well.
Beneficial effect of the present invention:
After adopting technical finesse of the present invention, can make again to cover upper new nacre because Long-Time Service causes surperficial yellowing to lose nacreous pearl, thus recover the gloss of pearl and prevent pearl aging further, can the indefinite prolongation pearl life-span by Reusability method of the present invention, make historical value, memorable pearl, valuable pearl etc. be preserved use for a long time.The pearl culturing method that traditional external carbon dosage small pieces and pearl core are implanted altogether, secrete nacreous outer embrane exocuticle skewness around pearl core, easily cause pearl surface color and luster uneven, the problems such as flaw pearl or tail pearl, be not suitable for the renovation of old pearl, old pearl of the present invention is adopted to cover the method for tissue engineering bracket material and outer embrane free cell Dual culture outward, a uniform pearl sac of thickness can be formed at old pearl surface, thus make the new nacre formed be evenly distributed on the surface of old pearl, make old pearl surface cover the new nacre of last layer when not changing the shape of old pearl, recover its pearly luster, reach the object of old pearl renovation.
Accompanying drawing explanation
Fig. 1 is the front elevation of mould I 1 hemisphere described in the embodiment of the present invention 1.
Fig. 2 is the cross-sectional view (face vertical with hole for injecting glue position) of mould I hemisphere, and middle circle represents the old pearl of putting in the future.
Fig. 3 is the vertical view after mould I two hemispheres closes up.
Fig. 4 is the profile being enclosed with the old pearl of tissue engineering bracket.
Embodiment
The present invention will be further described by reference to the accompanying drawings for following specific embodiment, but scope not thereby limiting the invention.
Embodiment 1
1. by old pearl neutral detergent soaked overnight, clean gently, wash away surface blot, then wash away cleaning solution with clear water.
2. old pearl to be immersed in the hydrochloric acid solution of concentration 1.0wt% 30 minutes, to take out, be soaked in rinsing in distilled water, change clothes 3 times, often all over 10 minutes.
3. old pearl to be soaked in 30vol% hydrogen peroxide solution 30 minutes, to take out, be soaked in rinsing in distilled water, change clothes 3 times, often all over 10 minutes; Put into 75vol% alcohol sterilization treatment, the Poly-L-Lysine Solution then putting into 0.01wt% soaks 30 minutes, takes out, dries under aseptic condition.
4. prepare old pearl shelf layer mould: mould I is made up of the hollow hemisphere that two structures are the same, during use, 2 hemisphere are closed up and become a complete spheroid.The medial surface of each hemisphere has 3 south poles (with hole for injecting glue present position for the South Pole, its offside is the arctic) direction arrangement strip projection (hereinafter referred to as rib), wherein 2 ribs lay respectively at hemisphere both sides of the edge (hereinafter referred to as edge rib), another rib is positioned at middle (hereinafter referred to as middle edge) (Fig. 1 of hemisphere, Fig. 2), the length of rib is 1/2 of spheroid girth, and therefore the end of 3 ribs intersects at the south poles place of ball, and the rib cut-out of end intersection, the South Pole forms recess as hole for injecting glue; Width about the 3mm of middle edge 2, height about 1mm, edge rib 3 width is the half of middle edge.Mould II is made up of the hemisphere of two hollows, and its inner surface is without raised structures, and hemisphere internal diameter is 2 times of sums of old pearl diameter and mould I rib height, and after two hemispheres closes up, (Fig. 3) leaves a hole for injecting glue 1 for perfused tissue engineering scaffold material.
5. timbering material process: under aseptic condition, recombinant human collagen albumen I is mixed with the solution that concentration is 1.5wt%, the glutaraldehyde solution of 2.5vol% is added wherein as crosslinking agent after collagen dissolves completely, make glutaraldehyde final concentration be 0.02vol%, stir.
6. support preparation: the old pearl 6 first step 3 processed is positioned in mould I, tissue engineering bracket perfusion step 5 prepared enters mould I, vibration shakes up 10 minutes, left at room temperature makes tissue engineering bracket material be cross-linked for 2 ~ 12 hours, form a reeded gel layer at old pearl surface, be cooled to 4 DEG C and make the demoulding in 10 minutes; Then open mould and take out old pearl, again the timbering material at its hole for injecting glue place is cut out on a small quantity, form the recess that is communicated with 4 road grooves, be placed in mould II again, the indent of tissue engineering bracket is made to aim at hole for injecting glue, the tissue engineering bracket solution of step 5 of reinjecting preparation, when making first time injecting glue, the groove that stays of old pearl surface fills up tissue engineering bracket liquid, and left at room temperature makes tissue engineering bracket material be cross-linked for 2 ~ 12 hours; Take out, by the surface rubbing at hole for injecting glue place, under-20 DEG C of conditions, place 2 hours freeze formings, then put into freeze drier vacuum drying 6 hours, vacuum is 12Pa, and freeze temperature is-20 DEG C.Obtain the old pearl 5(Fig. 4 being coated with tissue engineering bracket 4), old for tissue engineering bracket pearl is soaked in distilled water and washes away residual glutaraldehyde, change clothes 3 times fully to wash most glutaraldehyde.
7. outer embrane free cell obtains: cleaning clam shell, cut off closed shell flesh, get mantle tissue, first clean 3 times with PBS buffer solution, the PBS buffer solution be placed in by tissue containing 4000 ~ 5000U/mL antibiotic (penicillin+streptomycin) soaks 10 minutes, be pulled through rapidly → PBS buffer solution (10 minutes) in PBS buffer solution (5 minutes) → 70vol% alcoholic solution again, put into culture dish, tissue shear is broken into about 1mm
2small pieces, add the 0.25wt% Trypsin Induced 30 minutes containing EDTA, then the RPM1640 culture fluid containing 10vol% freshwater mussel serum is added, termination enzymic digestion is reacted, and filter with 200 order tulles and remove residual tissue block, 1000r/ minute centrifugal 5 minutes, abandon supernatant, the RPM1640 culture fluid added containing 10vol% freshwater mussel serum dispels precipitation, obtains the mantle tissue free cell of pearl shell, wherein builds the various cells needed for pearl sac containing outer embrane epidermal cell and phoirocyte etc.
8. cell concentration is diluted to about 5 × 10
6cell/mL, drop on old pearl surface, make cell be evenly distributed on whole old pearl surface as far as possible, then old pearl is positioned over a kind of with in the culture vessel of hemispherical groove, slowly add a small amount of RPMI-1640 containing 20vol% freshwater mussel serum, try not to stir cell, make whole old pearl be immersed in culture fluid, cultivate 2 hours in 26 DEG C of constant incubators, make cell attachment on timbering material, and then add in culture dish into culture fluid, make liquid level cover old pearl more than 2mm.Culture fluid is the RPM1640 culture fluid containing 10vol% freshwater mussel serum, cultivate about 2 ~ 3 days at 26 DEG C, check under the microscope, see that cell is bred in a large number, when being flooded with a large amount of cell in tissue engineering bracket, stopping cultivating, remove culture fluid, with the old pearl of PBS wash buffer, remove the cell do not sticked on tissue engineering bracket.
9. the old pearl handled well by previous step implants the visceral mass position of pearl culturing clam.
Clam shell engraves mark of correlation, and putting mesh bag to pearl culturing clam in case tell core causes old pearl to be lost, and adopts vertical hanging culture to cultivate with flat pearl culturing clam cultural method of supporting alternately rotation, adopts freshwater mussel get pearl by the time of arranging with trustee.
Above-described embodiment is section Example of the present invention, not any pro forma restriction is done to the present invention, any above embodiment is done according to technical spirit of the present invention any simple modification, equivalent variations and modification, all belong to the scope of technical solution of the present invention.
Claims (4)
1. the method for an old pearl renovation, it is characterized in that, the spot of old pearl surface is first washed away with neutral detergent solution, then the nacre of its surface modification is dissolved with dilute acid soln, and/or bleach its top layer with hydrogen peroxide solution, the degradable high polymer tissue engineering bracket that grows of cell is applicable to wherein uniformly afterwards in its surface coverage a layer thickness, described tissue engineering bracket is a cubic network space that can hold cell merisis wherein, outer embrane free cell culture in vitro together with the old pearl having adhered to tissue engineering bracket of pearl culturing clam will be taken from, cell is attached in the tissue engineering bracket of old pearl surface, division, growth, again old pearl is inserted the slotting nuclear location of pearl culturing clam, clam shell engraves mark of correlation, put the mesh bag that mesh aperture is less than pearl diameter, adopt conventional pearl culturing clam cultural method cultivation, vertical hanging culture supports alternately rotation with flat, adopt freshwater mussel by the time of agreement and get pearl, to get pearl winter as well.
2. the method for a kind of old pearl renovation according to claim 1, is characterized in that, comprise the following steps:
1) remove surface blot: by old pearl neutral detergent solution soaked overnight, wiped clean, then in distilled water, cleaning solution is removed in rinsing;
2) dilute acid soln dissolves old pearl surface denatured layer;
3) bleach: use hydrogen peroxide solution bleaching 10 ~ 60 minutes;
4) old pearl shelf layer mould used is prepared: described mould is made up of mould I and mould II, mould I is made up of the hollow hemisphere that two structures are the same, the medial surface of each hemisphere has the rib of 3 projections, wherein 2 ribs lay respectively at hemisphere both sides of the edge, another rib is positioned at the centre of hemisphere, the length of rib is 1/2 of spheroid girth, article 3, the corresponding north and south at ball of the end of rib is extremely crossing, the rib cut-out of end intersection, the South Pole forms recess as hole for injecting glue, height 0.02 ~ the 2mm of rib, edge rib width is the half of middle edge; The effect of rib is to support old pearl, and the height after making that old pearl is put into mould between old pearl with hemisphere inner face is consistent, and the height of rib is exactly the thickness of the tissue engineering bracket that will fill; During use, first old pearl is put into a hemisphere, then another hemisphere is closed up, edge rib its side after two hemispheres closes up of two hemisphere is stitched together and is combined into the width rib the same with middle edge width, two hemisphere close up into a spheroid, the sphere being positioned at middle edge one end is closed, and the sphere place being positioned at the other end reserves an aperture as hole for injecting glue for perfused tissue engineering rack liquid, the rib cut-out of this end joint, reserves duct and makes tissue engineering bracket liquid from then on be filled in 4 spaces separated by rib in mould in duct simultaneously; The first step that mould I is prepared for completing old pearl tissue engineering bracket, obtains the old pearl of reeded tissue engineering bracket; Mould II is made up of the hemisphere of two hollows, its inner surface is without raised structures, hemisphere internal diameter is 2 times of sums of old pearl diameter and mould I rib height, a hole for injecting glue is left for perfused tissue engineering rack liquid after two hemispheres closes up, utilize mould II to complete filling up of groove, obtain the old pearl of bulb tissue's engineering rack that tissue engineering bracket layer thickness is identical;
5) tissue engineering bracket liquid is prepared under aseptic condition: adopt compound that collagen or itself and other materials form as timbering material, under aseptic condition, timbering material is dissolved in pure water, concentration is 1.0wt% ~ 3.0wt% solution, the glutaraldehyde solution of 2.5vol% is added wherein as crosslinking agent after timbering material dissolves completely, make glutaraldehyde final concentration be 0.01vol% ~ 0.05vol%, stir;
6) support preparation: first the old pearl of step 3) process is positioned in mould I, tissue engineering bracket perfusion step 5) prepared enters mould I, vibration shakes up 10 minutes, and left at room temperature makes tissue engineering bracket material be cross-linked for 2 ~ 12 hours, the demoulding after being cooled to 4 DEG C, 10 minutes; Then open mould I and take out old pearl, be placed in mould II again, the indent of the groove of tissue engineering bracket is made to aim at hole for injecting glue, tissue engineering bracket solution prepared by step 3) of reinjecting, during to make first time injecting glue, the groove that stays of old pearl surface fills up tissue engineering bracket liquid, and left at room temperature makes tissue engineering bracket material be cross-linked for 2 ~ 12 hours; Then take out, by the surface rubbing at hole for injecting glue place, under-20 DEG C of conditions, place 1 ~ 6 hour freeze forming, then freeze drier vacuum drying is put into 6 ~ 24 hours, vacuum is 12 ~ 20Pa, and freeze temperature is-20 ~-70 DEG C, is soaked in distilled water by old pearl and washes away residual glutaraldehyde;
7) outer embrane free cell obtains: cleaning clam shell, tears and gets nacreous mantle tissue, margins of excision colo(u)r streak part, remove endepidermis, be cut into about 1mm after cleaning and disinfecting
2fritter, adopt trypsin digestion digestion tissue block, usual employing is containing the 0.25wt% trypsin solution of EDTA, digested 0.5 ~ 4 hour of 25 ~ 37 DEG C of scopes, filter with 200 order tulles and remove residual tissue block, centrifugation in 1000r/ minute goes out cell, adds culture fluid and dispels cell precipitation, obtains the mantle tissue free cell of pearl shell;
8) the free cell culture in vitro in the constant incubator together old pearl after step 6) process is separated with outer embrane, culture fluid is complete culture solution, adopt RPM1640 or M199 or DMEM, add the serum that final volume mark is 10vol% ~ 30vol%, serum adopts freshwater mussel serum or cow's serum, cultivation temperature 26 ~ 37 DEG C, incubation time 1 ~ 6 day, repeatedly can change culture fluid therebetween to ensure the attaching of cell, division, growth, cell attachment is on the tissue engineering bracket layer fiber of old pearl surface or after dividing 2 ~ 6 times further, remove culture fluid, with the old pearl of PBS wash buffer, remove the cell do not sticked on tissue engineering bracket,
9) old pearl step 8) obtained is implanted in pearl culturing clam body and cultivates, and for preventing the excessive compressing visceral mass of old pearl, adopts vertical hanging culture and horizontal hanging culture to replace rotation; Overlap at pearl culturing clam the mesh bag that a grid aperture is less than pearl diameter outward during cultivation, the cultivation time is generally 6 ~ 24 months.
3. the method for a kind of old pearl renovation according to claim 2, it is characterized in that, wherein said diluted acid refers to the nitric acid of the hydrochloric acid of 0.1 ~ 10wt% or the acetic acid of 0.1 ~ 20wt% or 0.1 ~ 5wt% or the sulfuric acid of 0.1 ~ 3wt%.
4. the method for a kind of old pearl renovation according to Claims 2 or 3, it is characterized in that, wherein said hydrogen peroxide solution concentration is 5 ~ 50vol%.
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