CN106822995A - A kind of preparation method for observing the optical table of osteocyte - Google Patents
A kind of preparation method for observing the optical table of osteocyte Download PDFInfo
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- CN106822995A CN106822995A CN201710080265.0A CN201710080265A CN106822995A CN 106822995 A CN106822995 A CN 106822995A CN 201710080265 A CN201710080265 A CN 201710080265A CN 106822995 A CN106822995 A CN 106822995A
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- Prior art keywords
- osteocyte
- hydroxyapatite
- optical table
- calcium
- observe
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/30—Inorganic materials
- A61L27/32—Phosphorus-containing materials, e.g. apatite
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/10—Ceramics or glasses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
Abstract
A kind of preparation method for observing the optical table of osteocyte, belong to composite biological material synthesis technical field, the present invention is in optical substrate by the method for hydro-thermal method film hydroxyapatite films, base material after these films can be used to the situation of easily observation of cell in hydroxyapatite surface Growth and Differentiation, while the interaction of mankind's osteocyte and orthopedic drug can be observed.
Description
Technical field
It is particularly mutual with orthopedic drug for observing osteocyte the invention belongs to composite biological material synthesis technical field
The manufacturing technology of the optical table of effect.
Background technology
Hydroxyapatite（Hydroxyapatite, HA）It is the main part of skeleton.Hydro-thermal method synthesis
Hydroxyapatite not only has nano level crystal formation acicular texture, while there is extraordinary biocompatibility again, in medical science branch
Support Material Field has boundless application value.Artificial synthesized hydroxyapatite powder sample has special superficiality
The advantages of energy, bioactivity, biological degradability, osteoconductive, non-immunogenic, after implantation human body, in the presence of body fluid, calcium
Dissociate material surface with phosphorus, and formed with body bone tissue and be chemically bonded, participate in neoblastic formation, and it is in various phosphorus
It is the most stable of form of thermodynamics in sour calcium, therefore it is had a wide range of applications in biomedical sector, it has also become is domestic
One of major subjects of outer biomedical materials field.
Nowadays most of container of cell culture that scientific research field is used when pursuing cell late growing stage and observing, suddenly
The facilitation of Tissue Culture Plate cell growth is omited.Many people are considering to smear one layer of hydroxyl by cell growth surface
Base apatite improves the growth activity of cell.The technology for coating of hydroxyapatite has many kinds, such as plating spraying technology, colloidal sol
Gel method film and hydro-thermal method film etc..It is the more commonly used painting membrane meanses wherein with hydro-thermal method, by adjusting pH, reaction density
Hydroxyapatite films performance is controlled with hydrothermal temperature.Particularly some optical substrates are in film perhydroxyl radical apatite film
Afterwards, cell can not only be made in substrate surface fast-growth, while being also beneficial to the observation of light microscope.
The Material Field of medical treatment implantation now is mainly based on alloy material, but the biocompatibility of these materials is very
Difference, if after being combined together with hydroxyapatite, the implantation material that biocompatibility and mechanical performance all get both can be obtained.Specially
Sharp document CN 100356991C describe a kind of method that film hydroxyapatite films are carried out on medical material, first match somebody with somebody
Good hydroxyapatite suspension, then by czochralski method, the method such as sol-gal process and spraying process is uniform by hydroxyapatite
Be applied in substrate.But this material cannot use the growing way of light microscope observation of cell and state etc. at the experiment initial stage,
In the testing stage of materials application early stage, generate very big not convenient.
The content of the invention
It is contemplated that a kind of preparation method for observing the optical table of osteocyte is introduced, to facilitate observation of cell to exist
The situation of hydroxyapatite surface Growth and Differentiation.
The present invention includes that step is as follows：
1）Under magnetic stirring, by phosphorus source dissolving in deionized water, phosphorus source solution is obtained；
2）Under magnetic stirring, calcium salt is dissolved in deionized water, calcium source solution is obtained；
3）Under the conditions of magnetic agitation, phosphorus source solution and calcium source solution are mixed and is reacted, obtain hydroxyapatite suspension；
4）Optical substrate is put in polytetrafluoroethyllining lining, and to adding the hydroxyapatite suspension in liner；Then
It is put into autoclave, 12~36h of hydro-thermal reaction is carried out under 180 DEG C of temperature environment, is cooled to after the completion of hydro-thermal reaction
Room temperature, obtains the optical table for observing osteocyte.
The present invention is in optical substrate by the method for hydro-thermal method film hydroxyapatite films, the substrate after these films
Material can be used to the situation of easily observation of cell in hydroxyapatite surface Growth and Differentiation, while it is thin to observe mankind's bone
Born of the same parents and the interaction of orthopedic drug.
Further, phosphorus source of the present invention is ammonium dihydrogen phosphate, or sodium ammonium biphosphate.The phosphorus source is water-soluble, convenient
The carrying out of reaction.
The calcium salt is calcium nitrate.Water-soluble as the calcium salt in present invention process with calcium nitrate, that conveniently reacts enters
OK.
When phosphorus source solution and calcium source solution mix, the mol ratio of calcium source and phosphorus source is 1.67: 1.The rate of charge can be saved
About raw material, reduces the pollution and waste for causing as far as possible.
Step 3）During middle hybrid reaction, the pH value with ammoniacal liquor regulation system is 9~11, can make reaction system in alkalescence, can
To form hydroxyapatite.
In addition, being using the pH value advantage of ammoniacal liquor regulation system：Ammoniacal liquor is volatile, soluble, facilitates late-run sample to wash
Work.
The present invention dries also with the hydro-thermal reaction sample of ultrasonic washing cooling to room temperature under the conditions of being subsequently placed in 40~45 DEG C
It is dry.Ultrasound washing can remove the unstable hydroxyapatite of surface attachment, and drying can be with reducing energy consumption, and not under the conditions of 40~45 DEG C
Change the crystal formation of nano-particle.
Brief description of the drawings
Fig. 1 is that the transparency level of the optical table of coating and uncoated hydroxyapatite compares picture.
Fig. 2 is the low power and high magnification microphoto for diluting the film of hydroxyapatite synthesized under 4 times on the glass substrate.
Fig. 3 is that the hydroxyapatite ultrathin membrane on the plate glass that original concentration is 0,1/2,1/4,1 times exists
Transmissivity at 300 600nm wavelength.
Fig. 4 is the infared spectrum of hydroxyapatite structure.
Fig. 5 is the XRD spectrum of hydroxyapatite structure.
Fig. 6 is the Raman collection of illustrative plates of hydroxyapatite structure.
Fig. 7 is the activity figure of the MG-63 cells observed in glass surface culture 3 days and under white light.
Fig. 8 is to cultivate the ALP activity figures of the MG-63 cells of 4 days on glass system surface.
Fig. 9 is the osteoblast mineralization phenomenon figure of the glass strengthening with HA coatings.
Figure 10 is that the enhanced osteocyte dendron shape network of HA coated glassization forms phenomenon figure.
Figure 11 is that HA coated glass promotes osteoclast formation and triggers osteoclast absorbing phenomenon figure.
Specific embodiment
First, preparation technology：
Case study on implementation 1
Weigh 0.5968g four water-calcium nitrate 50mL deionized waters to be completely dissolved, obtain calcium nitrate solution.
Weigh 0.1996g diammonium hydrogen phosphates to be equally completely dissolved with 50mL deionized waters, obtain ammonium dihydrogen phosphate.
Under the conditions of magnetic agitation, ammonium dihydrogen phosphate is slowly added into calcium nitrate solution and is reacted, added
During by ammoniacal liquor adjust reaction system pH=10, the white hydroxyapatite suspension for obtaining.
After the substrate of glass that will be handled well is put in polytetrafluoroethyl-ne alkene reaction liner, hanged to hydroxyapatite is added in liner
Turbid liquid.
Autoclave is put in baking oven the hydro-thermal reaction 24h under 180 DEG C of temperature conditionss, question response terminates to be cooled to room
Wen Hou, sample ultrasonic washing 5min washes away the unstable hydroxyapatite of surface attachment, finally washes, and dried under the conditions of 45 DEG C
It is dry.
Case study on implementation 2
Weigh 0.2984g four water-calcium nitrates and spend 50mL deionized waters and be completely dissolved, obtain calcium nitrate solution.
Weigh 0.0998g diammonium hydrogen phosphates to be equally completely dissolved with 50mL deionized waters, obtain ammonium dihydrogen phosphate.
Under the conditions of magnetic agitation, ammonium dihydrogen phosphate is slowly added into calcium nitrate solution and is reacted, added
During by ammoniacal liquor adjust reaction system pH=10, the white hydroxyapatite suspension for obtaining.
After the substrate of glass that will be handled well is put in polytetrafluoroethyl-ne alkene reaction liner, hanged to hydroxyapatite is added in liner
Turbid liquid.
Autoclave is put in baking oven the hydro-thermal reaction 24h under 180 DEG C of temperature conditionss, question response terminates to be cooled to room
Wen Hou, sample ultrasonic washing 5min washes away the unstable hydroxyapatite of surface attachment, finally washes, and dried under the conditions of 45 DEG C
It is dry.
Case study on implementation 3
Weigh 0.1492g four water-calcium nitrates and spend 50mL deionized waters and be completely dissolved, obtain calcium nitrate solution.
Weigh 0.0449g diammonium hydrogen phosphates to be equally completely dissolved with 50mL deionized waters, obtain ammonium dihydrogen phosphate.
Under the conditions of magnetic agitation, ammonium dihydrogen phosphate is slowly added into calcium nitrate solution and is reacted, added
During by ammoniacal liquor adjust reaction system pH=10, the white hydroxyapatite suspension for obtaining.
After the substrate of glass that will be handled well is put in polytetrafluoroethyl-ne alkene reaction liner, hanged to hydroxyapatite is added in liner
Turbid liquid.
Autoclave is put in baking oven the hydro-thermal reaction 24h under 180 DEG C of temperature conditionss, question response terminates to be cooled to room
Wen Hou, sample ultrasonic washing 5min washes away the unstable hydroxyapatite of surface attachment, finally washes, and dried under the conditions of 45 DEG C
It is dry.
Case study on implementation 4
Weigh 0.1492g four water-calcium nitrates and spend 50mL deionized waters and be completely dissolved, obtain calcium nitrate solution.
Weigh 0.0449g sodium ammonium biphosphates to be equally completely dissolved with 50mL deionized waters, obtain ammonium hydrogen phosphate sodium solution.
Under the conditions of magnetic agitation, ammonium hydrogen phosphate sodium solution is slowly added into calcium nitrate solution and is reacted, added
During by ammoniacal liquor adjust reaction system pH=10, the white hydroxyapatite suspension for obtaining.
After the substrate of glass that will be handled well is put in polytetrafluoroethyl-ne alkene reaction liner, hanged to hydroxyapatite is added in liner
Turbid liquid.
Autoclave is put in baking oven the hydro-thermal reaction 24h under 180 DEG C of temperature conditionss, question response terminates to be cooled to room
Wen Hou, sample ultrasonic washing 5min washes away the unstable hydroxyapatite of surface attachment, finally washes, and dried under the conditions of 50 DEG C
It is dry.
Note：The preprocess method of substrate of glass is the flushing repeatedly by soft detergent and pure water in each example above, with
Obtain the substrate of glass of cleaning.
2nd, the base material effect analysis after the film that the method for the present invention is obtained：
Compare picture from the transparency level of the optical table of the coating and uncoated hydroxyapatite of Fig. 1 can be seen that：Coating
The substrate of glass translucency of hydroxyapatite is good.
As shown in Fig. 2 at 180 DEG C, hydro-thermal reaction is clearly illustrated after 24 hours by SEM image under 4 times of diluted concentrations
The configuration of surface of the film of hydroxyapatite formed on base material.In fig. 2, in the view with hydroapatite particles in glass table
Hydroxyapatite coating layer is equably prepared on face, and it was found that the hydroxyapatite coating layer of synthesis by diameter in 80nm extremely
Hydroapatite particles composition between 100nm.
Solution after synthesis using various concentrations forms hydroxyapatite coating layer, by ultraviolet specrophotometer study sample
Transmissivity of the wavelength between 300nm and 600nm；Result is as shown in Figure 3.In figure 3, it is displayed without hydroxyapatite coating layer
Foliated glass there is highest transmissivity, and with the increase of solution concentration, wavelength between 300nm and 600nm it is saturating
Penetrating rate gradually reduces, and in initial concentration decrease in transmission to 70%.
By IR Characterization molecular structure, characterization result is as shown in Figure 4.The FTIR spectrum of hydroxyapatite coating layer such as Fig. 4
It is shown.In fig. 4,963cm-1The absworption peak at place is the symmetrical stretching vibration peak of phosphate radical, 1030cm-1And 1088cm-1The peak at place
Belong to the asymmetric stretching vibration peak of phosphate radical, and 563cm-1And 603cm-1It is the antisymmetric stretching vibration peak of phosphate radical.
In Fig. 5, in 1500-1400cm-1The peak in region is attributed to C-O（υ3）Antisymmetric stretching vibration.Generally, these peaks are substantially
Meet the standard IR spectrum of hydroxyapatite.Further research on hydroxyapatite films passes through Raman（Fig. 5）, phosphoric acid
The characteristic peak of root appears in 959.4cm-1, and appear in 3568.3cm-1Peak belong to OH-.
The XRD case of the ultra-thin hydroxyapatite coating layer of synthesis is shown in Figure 6.By the hydroxyapatite coat of Hydrothermal Synthesiss
The diffraction maximum of layer includes（200）,（111）,（002）,（102）,（210）,（211）,（300）,（202）,（310）,（213）,
（004）.And all these diffraction maximums of hydroxyapatite coating layer can find in the standard diagram of pure HAp, it shows to apply
Layer is hydroxyapatite, and the HA of synthesis also has good crystal structure.
3rd, it is used for observing osteocyte in hydroxyapatite table using the base material after the film of method of the present invention acquisition
The situation of face Growth and Differentiation：
It is to study hydroxyapatite microthin coating to observe the MG-63 cultivated on sheet material by inverted microscope in Fig. 7 to 8
The influence of the transparency of sheet material to being made from a different material.The shape of cell is can clearly be observed that using inverted microscope
State, wherein cell growth are good and keep good on sheet material in the case where preparing or not preparing hydroxyapatite microthin coating
Form.This shows that hydroxyapatite microthin coating does not reduce the transparency of glass plate.
As shown in figure 9, examining under a microscope more ALP positive nodules.Statistical analysis display HA coatings are enhanced
Osteoblast mineralization 4%.The further qPCR of Gegenbaur's cell related gene expression confirms Gegenbaur's cell base in the coated glass groups of HA
Because the multiple expressed increases.
As shown in Figure 10, the osteocyte on the glass of HA coatings forms more more than osteocyte on uncoated glass and more
Dendron process long.
As shown in figure 11, the TRAP positive osteoclasts observed in the glass of HA coatings are more than uncoated glass.
There are about 100 TRAP positive osteoclasts in group is uncoated, but there are 150 TRAP positive osteoclasts in HA coating groups.
Additionally, the expression of osteoclast specific gene also strengthens in HA coating groups.It was noted that many needle-like HA particles are inhaled
In receiving osteoclast.Other Confocal Images also demonstrate that these HA particles are absorbed by mature osteoclast.
The above, is only presently preferred embodiments of the present invention, and any formal limitation is not made to the present invention, any ripe
Professional and technical personnel is known, it is without departing from the scope of the present invention, real to more than according to technical spirit of the invention
Apply any simple modification, equivalent that example made and improve etc., still fall within technical solution of the present invention protection domain it
It is interior.
Claims (6)
1. a kind of preparation method for observing the optical table of osteocyte, it is characterised in that as follows including step：
1）Under magnetic stirring, by phosphorus source dissolving in deionized water, phosphorus source solution is obtained；
2）Under magnetic stirring, calcium salt is dissolved in deionized water, calcium source solution is obtained；
3）Under the conditions of magnetic agitation, phosphorus source solution and calcium source solution are mixed and is reacted, obtain hydroxyapatite suspension；
4）Optical substrate is put in polytetrafluoroethyllining lining, and to adding the hydroxyapatite suspension in liner；Then
It is put into autoclave, 12~36h of hydro-thermal reaction is carried out under 180 DEG C of temperature environment, is cooled to after the completion of hydro-thermal reaction
Room temperature, obtains the optical table for observing osteocyte.
2. according to claim 1 be used for observe osteocyte optical table preparation method, it is characterised in that：Phosphorus source
It is ammonium dihydrogen phosphate, or sodium ammonium biphosphate.
3. according to claim 1 be used for observe osteocyte optical table preparation method, it is characterised in that：The calcium salt
It is calcium nitrate.
4. according to claim 1 be used for observe osteocyte optical table preparation method, it is characterised in that：Phosphorus source
When solution and calcium source solution mix, the mol ratio of calcium source and phosphorus source is 1.67: 1.
5. according to claim 1 or 4 be used for observe osteocyte optical table preparation method, it is characterised in that：Step
3）During middle hybrid reaction, the pH value with ammoniacal liquor regulation system is 9~11.
6. according to claim 1 be used for observe osteocyte optical table preparation method, it is characterised in that：With ultrasonic water
The hydro-thermal reaction sample for being cooled to room temperature is washed, is dried under the conditions of being subsequently placed in 40~45 DEG C.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5018847A (en) * | 1988-09-13 | 1991-05-28 | Asahi Kogaku Kogyo Kabushiki Kaisha | Observation and/or cultivation instrument for cells |
| US5213801A (en) * | 1990-07-19 | 1993-05-25 | Kabushiki Kaisha Sangi | Contact lens material |
| CN104203295A (en) * | 2011-12-23 | 2014-12-10 | 丹格·广·斯文·勒 | A process for modifying the surface morphology of a medical device |
| CN105137509A (en) * | 2015-08-20 | 2015-12-09 | 扬州大学 | Method for smearing biological activity hydroxy apatite transparent film on different optical element substrates |
| CN105194732A (en) * | 2015-09-14 | 2015-12-30 | 扬州大学 | Method for coating fluorine-doped hydroxylapatite on nickel-titanium alloy plate |
-
2017
- 2017-02-15 CN CN201710080265.0A patent/CN106822995A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5018847A (en) * | 1988-09-13 | 1991-05-28 | Asahi Kogaku Kogyo Kabushiki Kaisha | Observation and/or cultivation instrument for cells |
| US5213801A (en) * | 1990-07-19 | 1993-05-25 | Kabushiki Kaisha Sangi | Contact lens material |
| CN104203295A (en) * | 2011-12-23 | 2014-12-10 | 丹格·广·斯文·勒 | A process for modifying the surface morphology of a medical device |
| CN105137509A (en) * | 2015-08-20 | 2015-12-09 | 扬州大学 | Method for smearing biological activity hydroxy apatite transparent film on different optical element substrates |
| CN105194732A (en) * | 2015-09-14 | 2015-12-30 | 扬州大学 | Method for coating fluorine-doped hydroxylapatite on nickel-titanium alloy plate |
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Application publication date: 20170613 |