CN101785876A - Decalcification method for bone implant materials - Google Patents
Decalcification method for bone implant materials Download PDFInfo
- Publication number
- CN101785876A CN101785876A CN201010019515A CN201010019515A CN101785876A CN 101785876 A CN101785876 A CN 101785876A CN 201010019515 A CN201010019515 A CN 201010019515A CN 201010019515 A CN201010019515 A CN 201010019515A CN 101785876 A CN101785876 A CN 101785876A
- Authority
- CN
- China
- Prior art keywords
- decalcification
- semisolid
- bone implant
- stock solution
- implant material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Materials For Medical Uses (AREA)
Abstract
The invention provides a decalcification method for bone implant materials, comprising the following steps: dissolving a decalcification agent and a semisolid excipient in phosphate buffer, physiological saline or water to obtain a semisolid stock solution; pouring the obtained semisolid stock solution into a container containing the bone implant materials fixed by formaldehyde or paraformaldehyde until the bone implant materials are completely covered by the stock solution, and standing until semisolid is formed; and placing the semisolid covering the bone implant materials into the environment the humidity of which is 100% for decalcifying. The bone implant materials decalcified by utilizing the method of the invention can be directly subjected to common group dyeing, immunity group dyeing, observation under electron microscopes and fluorescence microscopes and the like with no need of post-fixation after curing and frozensection.
Description
Technical field
The present invention relates to the processing method of bone implant material.
Background technology
It is calcareous higher that bone implant material is meant that a class contains, can replace skeleton and the material that is used for bore regenerating, can be " bioceramic " of synthetic, or the similar sub of occurring in nature is as " corallite ", or the bone of deriving of allosome donor (through Deproteinated corpse bone of defat or animal bone), these materials all can be used as the regeneration that bone implant material replaces damaged skeleton and induces skeleton, the final desirable bones that forms should comprise osteocyte and be rich in calcareous extracellular matrix, collagen, blood vessel, nerve, even perimyelis, periosteum, structures such as medullary cavity.
Bone implant material calcium content height, hardness height, light transmission are poor, need handle and cut into slices and just can carry out histological observation it.Present stage is handled the three kinds of approach that mainly contain for the histological observation of this type of material: routine paraffin wax embedded section after (1) decalcification; (2) undecalcified plastic embedding sclerous tissues section; (3) conventional frozen section after the decalcification.Because hard bone implant material belongs to different character with soft tissues such as the cell of wherein growing, blood vessels, observe for the ease of preparation clear in structure, complete slice, conventional treatment method is many with strong acid decalcification (making it whole deliquescing) back routine paraffin wax embedding film-making or conventional frozen section, a few methods select undecalcified and with plastic embedding (making it whole hardening) afterwards with sclerous tissues's microtome film-making.Yet there is the defective that is difficult to avoid in above-mentioned processing method, and comprising: (1) strong acid decalcification meeting causes the Partial Protein degeneration, influences follow-up research; (2) there are some hot injurys in the routine paraffin wax embedding to tissue unavoidably, and general plastic embedding semithin section also can suppress antigen antibody reaction, makes it to be difficult to carry out the observation and the research of some immunohistochemical staining; (3) what is more important originally depended on after the soft tissue decalcification of hard bone implant material growth owing to lose the support of hard tissues, had left original position, had lost original structure, and related Morphological and Mechanism Study are caused difficulty; (4) as carrying out undecalcified section, not only film-making with high costs, waste time and energy, and, be difficult to carry out meticulous observation because section is very thick.
In order to overcome above-mentioned defective, the researcher that has prepares the osseous tissue frozen section of undecalcified, carry out normal dyeing and immune labeled painted observation, though effect is better, but also there are the following problems for this type of frozen section: need special instrument, and expense is very high, and there are indivedual reports in rarely seen Japan, Germany etc.; The more important thing is, this type of frozen section be not suitable for implant before or the bone implant material of the short period that implants, because ossein, osteocyte, blood vessel and the nerve etc. of bone implant material inside are seldom at this moment, material is very crisp, material meeting disintegrate fragmentation can't be finished the observation of film-making and its internal structure when undecalcified was directly cut into slices.
Pu Quan professor's plastic embedding technology is a kind of bone and bone implant material processing method through improvement, the plastics that will have a certain degree of hardness are filled in the space of bone and material and on every side, need not decalcification, it is the unit of plastic film-making of available this method preparation, section can keep proteic immunocompetence preferably, carry out panimmunity groupization (antigen-antibody) reaction, but also there are some problems in this method: (1) only is applicable to cell (diameter 2-3mm is following), this moment, available typical microtome cooperated special wolfram steel cutter (price is more expensive) film-making, as be used for bigger organizing and then need special sclerous tissues's microtome (this quasi-instrument price is very expensive, has only minority unit to have at home); (2) the plastic embedding agent can't fully enter material internal under normal condition, thereby material internal can form bubble, can cause the cell loss on material disintegrate and the material during section; (3) its used fixative can make albuminous degeneration, thereby makes the fluorescence of green fluorescent protein (GFP) or red fluorescent protein (RFP) etc. disappear, so can't be used to observe the fluorecyte of material internal.
Summary of the invention
The technical problem to be solved in the present invention is to make bone implant material when decalcification the cell of institute's load is retained in original position, keep original form and keep tissue and proteic immunocompetence to greatest extent.
The technical scheme that the present invention addresses the above problem is:
A kind of decalcification method of bone implant material, this method may further comprise the steps:
(1) get bone implant material, fixing with the formaldehyde immersion of 4% paraformaldehyde or 10%;
(2) will fix the back bone implant material and be soaked in the semisolid stock solution, leave standstill to forming semi-solid;
(3) will being embedded in bone implant material in the semisolid, to place humidity be that decalcification to bone implant material structure disappears or do not have hard composition under 100% the environment;
Semisolid stock solution described in the step (2) is prepared by following method: get decalcifying agent and semisolid excipient, join phosphate buffer, normal saline or water, dissolving forms uniform semisolid stock solution;
Described decalcifying agent is a decalcifying agent commonly used during clinical pathology is checked, as EDTA, formic acid, acetic acid, hydrochloric acid etc.; When selecting different decalcifying agent for use, its concentration in the decalcification system is also different, and this is that those of ordinary skills should grasp routine techniques, and as when decalcifying agent is EDTA, the concentration of EDTA in described semisolid stock solution is 0.05g/mL～0.15g/mL; When decalcifying agent was formic acid, the concentration of formic acid in described semisolid stock solution was 4%～15% (v/v).The inventor finds that when the mixture of 0.05g/mL～0.15g/mL EDTA and 2%～8% (v/v) formic acid was the decalcifying agent of decalcification system of the present invention, decalcification effect was best, and required time is also less;
Described semisolid excipient can be agar, and its concentration in described semisolid stock solution is 0.004～0.005g/mL;
Described semisolid excipient also can be sodium alginate, and its concentration in described semisolid stock solution is 0.05g/mL～0.3g/mL, and wherein the concentration ratio of 0.18g/mL～0.23g/mL is fit to form semi-solid decalcification system.
In order to accelerate the speed of decalcification, during decalcification, also available buffer soaked described semisolid, changed a buffer every 2～3 days; Described buffer is the mixed solution of decalcifying agent and phosphate buffer (PBS), and wherein the concentration of decalcifying agent in described mixed solution is identical with the concentration of decalcifying agent in semisolid stock solution.
Bone implant material of the present invention be meant a class contain calcareous higher, can replace skeleton and be used for the material of bore regenerating, specifically can be " bioceramic " of synthetic, or the similar sub of occurring in nature is as " corallite ", or the bone of deriving of allosome donor (as through Deproteinated corpse bone of defat or animal bone).
When implementing the inventive method, should note following item (following points for attention are the general knowledge of this area, also are laboratory routine operation skills):
1, when adopting agar, needs the heating hydrotropy as semisolid excipient; When adopting sodium alginate as excipient, can need not to heat hydrotropy, directly superfines and the decalcifying agent with sodium alginate is dissolved in phosphate buffer, normal saline or the water.
2, when described decalcifying agent contains EDTA, EDTA is insoluble in water at normal temperatures and can separates out under less than 4 situation at PH, therefore need heating for dissolving and guarantee pH 〉=4 of place solution environmental, can mix up the pH value of PBS during concrete operations earlier, after dissolving with an amount of PBS Hybrid Heating separately then, mix with semisolid excipient again; Perhaps join heating for dissolving among the PBS with semisolid excipient.When described decalcifying agent contained formic acid, formic acid was volatile liquid, therefore should be cooled to add formic acid about 50 ℃ again after the indissoluble material is dissolved in PBS.
3, the set time of step (1) is generally 10min to 24h, and the decalcification time of step (3) was generally for 1～4 week, specifically should decide according to the bone implant material size, can visit thorn by perusal or fine needle in fixing and decalcification process and determine the concrete time.
The method of the invention adopts agar or sodium alginate to make the decalcification system form semi-solid state as excipient, decalcifying agent wherein can be with semi-solid sluggish flow, promote the calcareous calcium ion that is dissolved as in the bone implant material, the progressively calcareous composition performance supporting role in the replacement bone embedded material of the excipient in the semisolid simultaneously makes the cell that grows in originally on the calcareous composition remain on original position.Bone implant material after the decalcification of use the inventive method just can directly carry out common group of change dyeing, immunohistochemical staining, electron microscopic observation and Fluirescence observation etc. after quick-freezing curing, frozen section.
Description of drawings
The fluorescence inverted microscope observed result of the bone biologic material of Fig. 1 is load fluorecyte.
Fig. 2 is result's (green fluorescence protein gene labeled cell) of the frozen section of the semi-solid decalcification bone of fluorescence microscope example 1 method gained.
Fig. 3 is result's (red fluorescent protein genetic marker cell) that laser confocal microscope is observed the frozen section of example 2 method gained decalcification bones.
The specific embodiment
Example 1 adopts the semi-solid decalcification system of the agar of formic acid+EDTA decalcifying agent
1, waits to observe the preparation of bone implant material
1) extract rabbit bone marrow, prepare former generation rabbit mesenchymal cell, In vitro culture is to the third generation.
2) preparation of LV-GFP recombinant slow virus and titer determination
Utilize calcium phosphate method, with 3 plasmids of LV carrier system in (5 μ g: 4 μ g: the ratio of 1 μ g) import the PhoenixA cell simultaneously, collect the culture supernatant that 36h～84h is rich in LV-GFP virus, the membrane filtration degerming of 0.45 μ m ,-80 ℃ of preservations.
3) GFP labelling
Selecting the former rabbit BMSC and the LV-GFP virus of supporting the third generation of being commissioned to train is 1: 10 mixed by infestation index (MOI), and the adding final concentration is the protamine of 4ug/mL in cultivating system, changing fresh complete culture solution behind the 12h continues to cultivate, obtain rabbit BMSC-GFP cell behind the 48h, with fluorescence inverted microscope observation of cell fluorescence, and detect the ratio of fluorecyte with flow cytometer.
As seen most BMSC-GFP cells send green fluorescence under fluorescence microscope, detect GFP+%＞95% with flow cytometer.With load the biomaterial of cell place the fluorescence inverted microscope to observe, visible cell enters material internal, along circular-hole internal-wall growth (Fig. 1).
4) RT-PCR detects GFP expression of gene and ratio.
Reference reagent box description.Extract cell total rna, and carry out reverse transcription and PCR reaction, GFP upstream region of gene primer is: 5 '-GGC AAG CTG ACC CTG AAG TTC ATC-3 '; Downstream primer is: 5 '-AAC TCC AGC AGG ACCATG TGA TCG-3 '.Product is identified through 1.2% sepharose electrophoresis.The distinctive band of RT-PCR product electrophoresis showed GFP gene.
5) with rabbit BMSC-GFP cell loading in bone implant material.
2, with the inventive method to load the bone implant material of rabbit BMSC-GFP cell carry out decalcification and handle
(1) configuration of semisolid stock solution
Get EDTA and agar powder, join among the neutral PBS (Ph=7.4), it is that 0.1g/mL, agar concentration are the solution of 0.004g/mL that the alcohol burner heating for dissolving is made into EDTA concentration, be cooled to about 50 ℃, the formic acid that adds 5% (v/v) (quickens decalcification, the final pH value of system should be more than or equal to 4), use immediately behind the mixing.
(2) semi-solid decalcification of bone implant material and preparation frozen section
In order to guarantee the form and the position of bone implant material internal blood vessel, cell etc., can soak with the formaldehyde of 4% paraformaldehyde or 10% before the material decalcification and fix 10 minutes.
The water white polyethylene preservative film of place mat in advance in 6 well culture plates bottoms has the bone implant material 5mm * 5mm of cell to put into 6 orifice plates load, pours semisolid stock solution immediately into, and material is wrapped up fully; After cooling forms semisolid, add the PBS solution that contains 0.1g/mL EDTA, PH 〉=4 above it again, changed once to quicken decalcification in per 2 days; Can be interrupted with fine needle and spy understanding decalcification situation, and place wet box to keep humidity 6 orifice plates, 1 all backs are visited to sting with fine needle and are found no hard composition, the decalcification end.In the decalcification whole process, can observe with the fluorescence inverted microscope at any time.
Material is after semi-solid decalcification, and-70 ℃ of freezing 0.5h make its curing, and under the low temperature, gently carry the preservative film edge piece of tissue is taken out, after repairing with blade, the continuous frozen section of preparation OCT parcel.With the fluorescence microscope frozen section, the results are shown in Figure 2, showed cell can be sent green fluorescence (highlighted position among the figure) among the figure, well-grown in osseous tissue, and along circular-hole internal-wall growth, to be inverted the microexamination result consistent with the fluorescence before the decalcification.
It is the semi-solid decalcification system of agar of decalcifying agent that example 2 adopts EDTA
1, waits to observe the preparation of bone implant material
Identical with example 1.
2, with the inventive method to load the bone implant material of rabbit BMSC-GFP cell carry out decalcification and handle
1) the semi-solid decalcification stock solution of preparation
Contain among the PBS 20mL of EDTA of 0.1g/mL and add agar, the agar final concentration is 0.004g/mL, be heated to boiling after, be cooled to about 50 degrees centigrade, make the semi-solid decalcification stock solution of agar.
3) semi-solid decalcification of bone implant material and preparation frozen section
In order to guarantee the form and the position of bone implant material internal blood vessel, cell etc., can soak with the formaldehyde of 4% paraformaldehyde or 10% before the material decalcification and fix 10 minutes.
The water white polyethylene preservative film of place mat in advance in 6 well culture plates bottoms, there is the bone implant material 5mm * 5mm of cell to put into 6 orifice plates load, pour semi-solid decalcification stock solution immediately into, material is wrapped up fully, the cooling back forms semi-solid decalcification system, is interrupted available fine needle and spies understanding decalcification situation, and place wet box to keep humidity 6 orifice plates, visit thorn with fine needle after 1 week and find no hard composition, can prepare frozen section.In the decalcification whole process, can observe with the fluorescence inverted microscope at any time.
Material is after semi-solid decalcification, and-70 ℃ of freezing 0.5h make its curing, and under the low temperature, gently carry the preservative film edge piece of tissue is taken out, after repairing with blade, the continuous frozen section of preparation OCT parcel.With the fluorescence microscope frozen section, the results are shown in Figure 3, showed cell can be sent green fluorescence (highlighted position among the figure) among the figure, well-grown in osseous tissue, and along circular-hole internal-wall growth, to be inverted the microexamination result consistent with the fluorescence before the decalcification.
It is the semi-solid decalcification system of agar of decalcifying agent that example 3 adopts formic acid
1, waits to observe the preparation of bone implant material
Identical with example 1.
2, with the inventive method to load the bone implant material of rabbit BMSC-GFP cell carry out decalcification and handle
1) the semi-solid decalcification stock solution of preparation
Weigh agar powder 0.4g, agar powder is mixed with 100mlPBS, the ebuillition of heated dissolving is cooled to about 50 degrees centigrade, adds the formic acid of volume ratio 8%, forms semi-solid decalcification stock solution.
2) semi-solid decalcification of bone implant material and preparation frozen section
In order to guarantee the form and the position of bone implant material internal blood vessel, cell etc., can soak with the formaldehyde of 4% paraformaldehyde or 10% before the material decalcification and fix 10 minutes.
The water white polyethylene preservative film of place mat in advance in 6 well culture plates bottoms, there is the bone implant material 5mm * 5mm of cell to put into 6 orifice plates load, pour semi-solid decalcification stock solution immediately into, material is wrapped up fully, cooling forms semi-solid decalcification system, is interrupted available fine needle and spies understanding decalcification situation, and place wet box to keep humidity 6 orifice plates, observe the bone implant material structure after 1 week and disappear, can prepare frozen section.In the decalcification whole process, can observe with the fluorescence inverted microscope at any time.
Material is after semi-solid decalcification, and-70 ℃ of freezing 0.5h make its curing, and under the low temperature, gently carry the preservative film edge piece of tissue is taken out, after repairing with blade, the continuous frozen section of preparation OCT parcel.With the fluorescence microscope frozen section, see that cell can send green fluorescence, well-grown in osseous tissue, and along circular-hole internal-wall growth, to be inverted the microexamination result consistent with the fluorescence before the decalcification.
It is the semi-solid decalcification system of sodium alginate of decalcifying agent that example 4 adopts EDTA and formic acid
1, waits to observe the preparation of bone implant material
Identical with example 1.
2, with the inventive method to load the bone implant material of rabbit BMSC-GFP cell carry out decalcification and handle
1) the semi-solid decalcification stock solution of the sodium alginate of preparation EDTA and formic acid
Weigh an amount of sodium alginate, configuration EDTA concentration is 0.1g/mL earlier, and the formic acid volume ratio is 5% PBS mixed liquor 10mL, dissolves in the 2g sodium alginate again, forms the semi-solid decalcification system of the EDTA, 5% volume ratio formic acid and the 0.2g/mL sodium alginate that contain 0.1g/mL.
3) semi-solid decalcification of bone implant material and preparation frozen section
In order to guarantee the form and the position of bone implant material internal blood vessel, cell etc., can soak with the formaldehyde of 4% paraformaldehyde or 10% before the material decalcification and fix 10 minutes.
Be poured on the material of required decalcification with the semi-solid decalcification stock solution of the sodium alginate that step 1) disposed, concrete operations are as follows: the water white polyethylene preservative film of place mat in advance in 6 well culture plates bottoms, there is the bone implant material 5mm * 5mm of cell to put into 6 orifice plates load, pour semi-solid decalcification stock solution immediately into, material is wrapped up fully; Should semisolid decalcification system, placing humidity is decalcification under 100% the environment, be interrupted available fine needle and spy and understand the decalcification situation, and place wet box to keep humidity 6 orifice plates, 1 week the back visit thorn with fine needle and find no hard composition, can prepare frozen section.In the decalcification whole process, can observe with the fluorescence inverted microscope at any time.
Material is after semi-solid decalcification, and-70 ℃ of freezing 0.5h make its curing, and under the low temperature, gently carry the preservative film edge piece of tissue is taken out, after repairing with blade, the continuous frozen section of preparation OCT parcel.With the fluorescence microscope frozen section, see that cell can send green fluorescence, well-grown in osseous tissue, and along circular-hole internal-wall growth, to be inverted the microexamination result consistent with the fluorescence before the decalcification.
It is the semi-solid decalcification system of sodium alginate of decalcifying agent that example 5 adopts EDTA
1, waits to observe the preparation of bone implant material
Identical with example 1.
2, with the inventive method to load the bone implant material of rabbit BMSC-GFP cell carry out decalcification and handle
1) the semi-solid decalcification stock solution of preparation sodium alginate
Weigh an amount of sodium alginate, as the 2g sodium alginate being dissolved in the 10mLPBS solution that EDTA concentration is 0.1g/mL, forming concentration is the semi-solid decalcification system of 0.2g/mL sodium alginate.
3) semi-solid decalcification of bone implant material and preparation frozen section
In order to guarantee the form and the position of bone implant material internal blood vessel, cell etc., can soak with the formaldehyde of 4% paraformaldehyde or 10% before the material decalcification and fix 10 minutes.
Be poured on the material of required decalcification with the semi-solid decalcification stock solution of the sodium alginate that step 1) disposed, concrete operations are as follows: the water white polyethylene preservative film of place mat in advance in 6 well culture plates bottoms, there is the bone implant material 5mm * 5mm of cell to put into 6 orifice plates load, pour semi-solid decalcification stock solution immediately into, material is wrapped up fully; Should semisolid decalcification system, placing humidity is decalcification under 100% the environment, is interrupted available fine needle and spies and understand the decalcification situation, and place wet box to keep humidity 6 orifice plates, observes the bone implant material structure after 1 week and disappears, and can prepare frozen section.In the decalcification whole process, can observe with the fluorescence inverted microscope at any time.
Material is after semi-solid decalcification, and-70 ℃ of freezing 0.5h make its curing, and under the low temperature, gently carry the preservative film edge piece of tissue is taken out, after repairing with blade, the continuous frozen section of preparation OCT parcel.With the fluorescence microscope frozen section, see that cell can send green fluorescence, well-grown in osseous tissue, and along circular-hole internal-wall growth, to be inverted the microexamination result consistent with the fluorescence before the decalcification.
It is the semi-solid decalcification system of sodium alginate of decalcifying agent that example 6 adopts formic acid
1, waits to observe the preparation of bone implant material
Identical with example 1.
2, with the inventive method to load the bone implant material of rabbit BMSC-GFP cell carry out decalcification and handle
1) the semi-solid decalcification stock solution of preparation
Add formic acid among the 40mlPBS, making its volume ratio is 5%, adds sodium alginate behind the mixing, and the concentration of sodium alginate is 0.2g/mL, makes the semi-solid decalcification stock solution of formic acid and sodium alginate.
3) semi-solid decalcification of bone implant material and preparation frozen section
In order to guarantee the form and the position of bone implant material internal blood vessel, cell etc., can soak with the formaldehyde of 4% paraformaldehyde or 10% before the material decalcification and fix 10 minutes.
With step 1) in 6 well culture plates bottoms the water white polyethylene preservative film of place mat in advance, there is the bone implant material 5mm * 5mm of cell to put into 6 orifice plates load, pour semi-solid decalcification stock solution immediately into, material is wrapped up fully, form semi-solid decalcification system, be interrupted available fine needle and spy understanding decalcification situation, and place wet box to keep humidity 6 orifice plates, visit thorn with fine needle after 1 week and find no hard composition, can prepare frozen section.In the decalcification whole process, can observe with the fluorescence inverted microscope at any time.
Material is after semi-solid decalcification, and-70 ℃ of freezing 0.5h make its curing, and under the low temperature, gently carry the preservative film edge piece of tissue is taken out, after repairing with blade, the continuous frozen section of preparation OCT parcel.With the fluorescence microscope frozen section, see that cell can send green fluorescence, well-grown in osseous tissue, and along circular-hole internal-wall growth, to be inverted the microexamination result consistent with the fluorescence before the decalcification.
Other gets section and adopts Rui Shi-Jim Sa (R-S) dyeing, and step is as follows:
(1) gets pathological material of disease smear, natural drying;
(2) drip Wright's stain and dyed 1 minute, specimen is fixed by formaldehyde wherein;
(3) phosphate buffer (or equivalent ultra-pure water) that adds equivalent PH6.4 rocks slide gently, evenly leaves standstill 5 minutes;
(4) clear water rinsing, blot, microscopy.
Observed result: visible more spindle cell keeps the cell growthform, and along the circular-hole internal-wall growth, the cell microstructure is clear, the karyon navy blue, and endochylema is deep mixed.
It is the semi-solid decalcification system of sodium alginate of decalcifying agent that example 7 adopts hydrochloric acid
1, waits to observe the preparation of bone implant material
Identical with example 1.
2, with the inventive method to load the bone implant material of rabbit BMSC-GFP cell carry out decalcification and handle
1) the semi-solid decalcification stock solution of preparation
Add hydrochloric acid among the 40mlPBS, making its volume ratio is 0.5% (PH＞2), adds sodium alginate behind the mixing, and the concentration of sodium alginate is 0.2g/mL, makes the semi-solid decalcification stock solution of hydrochloric acid and sodium alginate.
3) semi-solid decalcification of bone implant material and preparation frozen section
In order to guarantee the form and the position of bone implant material internal blood vessel, cell etc., can soak with the formaldehyde of 4% paraformaldehyde or 10% before the material decalcification and fix 10 minutes.
With step 1) in 6 well culture plates bottoms the water white polyethylene preservative film of place mat in advance, there is the bone implant material 5mm * 5mm of cell to put into 6 orifice plates load, pour semi-solid decalcification stock solution immediately into, material is wrapped up fully, form semi-solid decalcification system, be interrupted available fine needle and spy understanding decalcification situation, and place wet box to keep humidity 6 orifice plates, visit thorn with fine needle after 1 week and find no hard composition, can prepare frozen section.In the decalcification whole process, can observe with the fluorescence inverted microscope at any time.
Material is after semi-solid decalcification, and-70 ℃ of freezing 0.5h make its curing, and under the low temperature, gently carry the preservative film edge piece of tissue is taken out, after repairing with blade, the continuous frozen section of preparation OCT parcel.Haematoxylin-Yihong method (HE) is to section statining in employing, and step is as follows:
(1) section was soaked 3～5 minutes with 1% aqueous sodium persulfate solution, clear water rinsing 5 minutes, and distilled water embathed 0.5～1 minute;
(2) with haematoxylin dye liquor 7～15 minutes, regular hydrochloric acid color separation 5～7 seconds, clear water rinsing oil blackeite;
(3) soaked 1 minute with 0.5% Yihong liquid;
(4) with dehydration, color separation in 3: 2 the n-butyl alcohol dehydrated alcohol mixed liquor, transparent, sealing.
Observed result: visible more spindle cell keeps the cell growthform, and along the circular-hole internal-wall growth, visible karyon, endochylema are painted deep mixed, and microstructures such as cell membrane, karyon, endochylema and cell endoparticle are clear and legible.
It is the semi-solid decalcification system of agar of decalcifying agent that example 8 adopts hydrochloric acid
1, waits to observe the preparation of bone implant material
Identical with example 1.
2, with the inventive method to load the bone implant material of rabbit BMSC-GFP cell carry out decalcification and handle
1) the semi-solid decalcification stock solution of preparation
Weigh agar powder 0.4g, agar powder is mixed with 100mlPBS, the ebuillition of heated dissolving is cooled to about 50 degrees centigrade, adds the hydrochloric acid of volume ratio 0.5%, forms semi-solid decalcification stock solution.
2) semi-solid decalcification of bone implant material and preparation frozen section
In order to guarantee the form and the position of bone implant material internal blood vessel, cell etc., can soak with the formaldehyde of 4% paraformaldehyde or 10% before the material decalcification and fix 10 minutes.
The water white polyethylene preservative film of place mat in advance in 6 well culture plates bottoms, there is the bone implant material 5mm * 5mm of cell to put into 6 orifice plates load, pour semi-solid decalcification stock solution immediately into, material is wrapped up fully, cooling forms semi-solid decalcification system, is interrupted available fine needle and spies understanding decalcification situation, and place wet box to keep humidity 6 orifice plates, visit thorn with fine needle after 1 week and find no hard composition, can prepare frozen section.In the decalcification whole process, can observe with the fluorescence inverted microscope at any time.
Material is after semi-solid decalcification, and-70 ℃ of freezing 0.5h make its curing, and under the low temperature, gently carry the preservative film edge piece of tissue is taken out, after repairing with blade, the continuous frozen section of preparation OCT parcel.Adopt the Gomori silver staining that reticulum fiber dyeing is carried out in section, step is as follows:
(1) section is with 1% potassium permanganate oxidation 1～2 minute, clear water rinsing;
(2) 2.5% oxalic acid decolouring 1 minute, the clear water rinsing;
(3) 2% iron alum liquid were handled 1 minute, clear water rinsing 2～3 minutes;
(4) ammargenum soaked 3 minutes, and distilled water speed is washed;
(5) be dipped in 10% formalin solution interior 3 minutes, distillation clear water rinsing 2～3 minutes;
(6) 5% sodium thiosulfate fixations 1 minute, the clear water rinsing;
(7) dehydration, transparent, sealing.
Observed result: visible black color or brownish black fiber-like species distribution are between cell, and collagen fiber are yellow or orange colour, and karyon is the grey black navy blue.
It is the semi-solid decalcification system of sodium alginate of decalcifying agent that example 9 adopts acetic acid
1, waits to observe the preparation of bone implant material
Identical with example 1.
2, with the inventive method to load the bone implant material of rabbit BMSC-GFP cell carry out decalcification and handle
1) the semi-solid decalcification stock solution of preparation
Add acetic acid among the 40mlPBS, making its volume ratio is 1% (PH＞2), adds sodium alginate behind the mixing, and the concentration of sodium alginate is 0.2g/mL, makes the semi-solid decalcification stock solution of acetic acid and sodium alginate.
3) semi-solid decalcification of bone implant material and preparation frozen section
In order to guarantee the form and the position of bone implant material internal blood vessel, cell etc., can soak with the formaldehyde of 4% paraformaldehyde or 10% before the material decalcification and fix 10 minutes.
With step 1) in 6 well culture plates bottoms the water white polyethylene preservative film of place mat in advance, there is the bone implant material 5mm * 5mm of cell to put into 6 orifice plates load, pour semi-solid decalcification stock solution immediately into, material is wrapped up fully, form semi-solid decalcification system, be interrupted available fine needle and spy understanding decalcification situation, and place wet box to keep humidity 6 orifice plates, visit thorn with fine needle after 1 week and find no hard composition, can prepare frozen section.In the decalcification whole process, can observe with the fluorescence inverted microscope at any time.
Material is after semi-solid decalcification, and-70 ℃ of freezing 0.5h make its curing, and under the low temperature, gently carry the preservative film edge piece of tissue is taken out, after repairing with blade, the continuous frozen section of preparation OCT parcel.To gained section carrying out MASSON dyeing, step is as follows:
(1) section clear water rinsing;
(2) haematoxylin was contaminated 7～15 minutes, flowing water flushing, the differentiation of 1% hydrochloric acid, flowing water oil blackeite;
(3) Ponceaux-fuchsin solution was contaminated 5～10 minutes; 1% acetum was washed 30 seconds～1 minute;
(4) 1% phosphomolybdic acid differentiation 3～5 minutes; 1% acetum was washed 30 seconds;
(5) 2% viride nitens dyed 3 minutes, clear water rinsing 1 minute;
(6) conventional dehydration, transparent, sealing.
Observed result: visible chondrigen is green, and collagen is red green alternate in bone and the calcified cartilage, and sclerotin is ripe more, red dye many more.Collagen in the ripe osseous tissue is cerise, and the collagen in the inmature osseous tissue mainly is green.In maturation process, red collagen is more and more by new life for experimental union of fracture specimen, callus.
It is the semi-solid decalcification system of agar of decalcifying agent that example 10 adopts acetic acid
1, waits to observe the preparation of bone implant material
Identical with example 1.
2, with the inventive method to load the bone implant material of rabbit BMSC-GFP cell carry out decalcification and handle
1) the semi-solid decalcification stock solution of preparation
Weigh agar powder 0.4g, agar powder is mixed with 100mlPBS, the ebuillition of heated dissolving is cooled to about 50 degrees centigrade, adds the acetic acid of volume ratio 1%, forms semi-solid decalcification stock solution.
2) semi-solid decalcification of bone implant material and preparation frozen section
In order to guarantee the form and the position of bone implant material internal blood vessel, cell etc., can soak with the formaldehyde of 4% paraformaldehyde or 10% before the material decalcification and fix 10 minutes.
The water white polyethylene preservative film of place mat in advance in 6 well culture plates bottoms, there is the bone implant material 5mm * 5mm of cell to put into 6 orifice plates load, pour semi-solid decalcification stock solution immediately into, material is wrapped up fully, cooling forms semi-solid decalcification system, is interrupted available fine needle and spies understanding decalcification situation, and place wet box to keep humidity 6 orifice plates, visit thorn with fine needle after 1 week and find no hard composition, can prepare frozen section.In the decalcification whole process, can observe with the fluorescence inverted microscope at any time.
Material is after semi-solid decalcification, and-70 ℃ of freezing 0.5h make its curing, and under the low temperature, gently carry the preservative film edge piece of tissue is taken out, after repairing with blade, the continuous frozen section of preparation OCT parcel.With the fluorescence microscope frozen section, see that cell can send green fluorescence, well-grown in osseous tissue, and along circular-hole internal-wall growth, to be inverted the microexamination result consistent with the fluorescence before the decalcification.Immunohistochemical staining (the SP test kit is an example) is carried out in section to gained, and step is as follows:
(1) frozen section, room temperature was placed after 30 minutes, went into 4 ℃ of acetone fixed 10 minutes, and PBS washes, and 5 minutes * 3.Hatched 5～10 minutes with 3% hydrogen peroxide, eliminate the activity of endogenous peroxydase.PBS washes, and 5 minutes * 2.
(2) 5～10% normal goats serum (PBS dilution) sealing, incubated at room 10 minutes.The serum deprivation that inclines is not washed, and drips an anti-or anti-working solution of proper proportion dilution, hatches for 37 ℃ to spend the night in 1～2 hour or 4 ℃.
(3) PBS flushing, 5 minutes * 3 times.
(4) drip the biotin labeling two anti-(1%BSA-PBS dilution) that proper proportion is diluted, hatched 10～30 minutes for 37 ℃; Or drip second filial generation biotin labeling two anti-working solutions, 37 ℃ or incubated at room 10～30 minutes.The PBS flushing, 5 minutes * 3 times.
(5) drip the Radix Cochleariae officinalis enzyme labelling strepto-avidin (PBS dilution) that proper proportion is diluted, hatched 10～30 minutes for 37 ℃; Or second filial generation Radix Cochleariae officinalis enzyme labelling strepto-avidin working solution, 37 ℃ or incubated at room 10～30 minutes.
(6) PBS flushing, 5 minutes * 3 times.
(7) chromogenic reagent (DAB or AEC).
(8) tap water fully washes, and redyes mounting.
Observed result: the areal area colour developing of visible local organization related antigen, be brown granular shape, wire or some lamellar, background does not have pale brown color or homogeneous is light dyes, and nucleus is redyed and is blue.
Claims (3)
1. the decalcification method of a bone implant material, this method may further comprise the steps:
(1) get bone implant material, fixing with the formaldehyde immersion of 4% paraformaldehyde or 10%;
(2) will fix the back bone implant material and be soaked in the semisolid stock solution, leave standstill to forming semi-solid;
(3) will being embedded in bone implant material in the semisolid, to place humidity be that decalcification to bone implant material structure disappears or do not have hard composition under 100% the environment;
Semisolid stock solution described in the step (2) is prepared by following method:
Get decalcifying agent and semisolid excipient, join phosphate buffer, normal saline or water, dissolving forms uniform semisolid stock solution; Wherein,
Described decalcifying agent is a decalcifying agent commonly used during clinical pathology is checked;
Described semisolid excipient is: agar, and its concentration in described semisolid stock solution is 0.004g/mL～0.005g/mL; Perhaps sodium alginate, its concentration in described semisolid stock solution is 0.05g/mL～0.3g/mL.
2. the method for claim 1 is characterized in that, will be embedded in bone implant material in the semisolid place humidity be under 100% the environment decalcification during, soak described semisolid with buffer, changed a buffer every 2～3 days; Described buffer is the mixed solution of decalcifying agent and PBS, and wherein the concentration of decalcifying agent in described mixed solution is identical with the concentration of decalcifying agent in semisolid stock solution.
3. method as claimed in claim 1 or 2, it is characterized in that described decalcifying agent is the mixture of EDTA and formic acid, wherein the concentration of EDTA in semisolid stock solution is 0.05g/mL～0.15g/mL, and the concentration of volume percent of formic acid in semisolid stock solution is 2%～8%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010019515A CN101785876A (en) | 2010-01-20 | 2010-01-20 | Decalcification method for bone implant materials |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010019515A CN101785876A (en) | 2010-01-20 | 2010-01-20 | Decalcification method for bone implant materials |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101785876A true CN101785876A (en) | 2010-07-28 |
Family
ID=42529344
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010019515A Pending CN101785876A (en) | 2010-01-20 | 2010-01-20 | Decalcification method for bone implant materials |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101785876A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103877615A (en) * | 2014-03-18 | 2014-06-25 | 北京大学第三医院 | Cartilage tissue engineering bracket and preparation method thereof |
| CN103926108A (en) * | 2013-01-11 | 2014-07-16 | 陈军 | Bone slice slicing machine for hard tissue |
| CN106018050A (en) * | 2016-05-20 | 2016-10-12 | 北京九州柏林生物科技有限公司 | Frozen section embedding agent |
| CN109568664A (en) * | 2018-11-29 | 2019-04-05 | 薛志强 | A kind of costal cartilage material preparation method for nose filling shaping |
| CN114755407A (en) * | 2022-04-02 | 2022-07-15 | 北京大学深圳医院 | Preparation and detection method of human bone tissue multi-marker immunohistochemical sample |
| CN115931517A (en) * | 2022-12-05 | 2023-04-07 | 中山大学附属第五医院 | Dyeing method of unwinding collagen in bone and cartilage |
-
2010
- 2010-01-20 CN CN201010019515A patent/CN101785876A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103926108A (en) * | 2013-01-11 | 2014-07-16 | 陈军 | Bone slice slicing machine for hard tissue |
| CN103877615A (en) * | 2014-03-18 | 2014-06-25 | 北京大学第三医院 | Cartilage tissue engineering bracket and preparation method thereof |
| CN103877615B (en) * | 2014-03-18 | 2015-08-19 | 北京大学第三医院 | A kind of cartilage tissue engineering rack and preparation method thereof |
| CN106018050A (en) * | 2016-05-20 | 2016-10-12 | 北京九州柏林生物科技有限公司 | Frozen section embedding agent |
| CN109568664A (en) * | 2018-11-29 | 2019-04-05 | 薛志强 | A kind of costal cartilage material preparation method for nose filling shaping |
| CN114755407A (en) * | 2022-04-02 | 2022-07-15 | 北京大学深圳医院 | Preparation and detection method of human bone tissue multi-marker immunohistochemical sample |
| CN115931517A (en) * | 2022-12-05 | 2023-04-07 | 中山大学附属第五医院 | Dyeing method of unwinding collagen in bone and cartilage |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101785876A (en) | Decalcification method for bone implant materials | |
| CN107300496B (en) | Transparentizing agent for biological material and use thereof | |
| CN102085390B (en) | Cartilage cell removal matrix and preparation method and application thereof | |
| Sano et al. | Acellular adipose matrix as a natural scaffold for tissue engineering | |
| CN105973673A (en) | Paraffin sectioning method for eucalyptus tissue | |
| CN106501053B (en) | The paraffin section method of meadowrueleaf corydalis root blade | |
| CN106075598A (en) | A kind of photo-crosslinking sericin hydrogel and its preparation method and application | |
| CN106644656A (en) | Hematoxylin-eosin one-step dyeing method | |
| Odabas et al. | Auricular cartilage repair using cryogel scaffolds loaded with BMP‐7‐expressing primary chondrocytes | |
| CN106370491A (en) | Environment-friendly transparent dewaxing liquid, preparation method thereof and application of environment-friendly transparent dewaxing liquid in preparing pathological section | |
| CN102147417B (en) | Method for positioning immune tissues of growth hormone for malus plants and application thereof | |
| CN104225667B (en) | Angiogenesis-facilitating temperature-sensitive hydrogel powder and temperature-sensitive hydrogel prepared from same | |
| CN102168065A (en) | Method for inducing human umbilical cord mesenchymal stem cells in vitro into liver cells and application thereof | |
| CN103705542A (en) | Decellularized blood vessel matrix gel, preparation method therefor and applications thereof | |
| CN105331579A (en) | Separation and culture method and application of human testis mesenchymal stem cells | |
| CN108853145A (en) | Application of the excretion body of mescenchymal stem cell secretion in biological agent | |
| CN103852368B (en) | The two dyeing of Cucumber Pollen semithin section MTG-DAPI observation line mitochondrial DNA | |
| CN100564518C (en) | Placenta amnion cell extract and induce application in the differentiation at mescenchymal stem cell | |
| Exbrayat | Classical methods of visualization | |
| Brown et al. | Preparation of decellularized biological scaffolds for 3D cell culture | |
| CN102822677B (en) | HLA-G hypotype is as the purposes becoming bone seeker | |
| Broese et al. | Seeding a human tendon matrix with bone marrow aspirates compared to previously isolated hBMSCs–an in vitro study | |
| Minuth et al. | Advanced fixation for transmission electron microscopy unveils special extracellular matrix within the renal stem/progenitor cell niche | |
| JP2004037215A (en) | Manufacturing method of tissue specimen | |
| CN106706389B (en) | The paraffin section method of meadowrueleaf corydalis root root |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20100728 |