CN1274803C - Model of mouth cavity biomembrane and method for forming mouth cavity biomembrane - Google Patents
Model of mouth cavity biomembrane and method for forming mouth cavity biomembrane Download PDFInfo
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Abstract
The present invention relates to a model device for an oral cavity biological film and a method for forming the oral cavity biological film. The model device for an oral cavity biological film is characterized in that the device is composed of an alkali liquid tank (1), a culture medium tank (2), a fermentation tank (3), constant flow culture chambers (4) and a sample adding bottle (7), wherein the alkali liquid tank (1) and the culture medium tank (2) are installed at the anterior pole of the fermentation tank (3); the fermentation tank (3) and the sample adding bottle (7) are installed at the anterior poles of the constant flow culture chambers (4). The devices and apparatuses are connected by a pipeline system and connecters thereof. After the oral cavity biological film is completed in a sterile connection way by the model device, experiment bacterial strain are inoculated in the fermentation tank and are mixed and continuously cultivated. A pH controller keeps the pH of the experiment strains to be 7.0, and manual sputum culture media are added in a constant speed to serve as the nutrition sources of the experiment bacteria. After the bacteria stably grow, a peristaltic pump P6 leads bacteria suspension in the fermentation tank into the constant flow culture chambers connected in parallel, culture media are continuously added, and medium guide forms an oral cavity biological film on a hydroxyapatite sheet. The present invention provides a scientific research method and treatment reference for the cariology and the science of dental materials, and obtains obvious medical effects.
Description
One, technical field
The present invention relates to the method for a kind of oral biological film mould unit and formation oral biological film thereof, belong to microbial film oral medical model apparatus or stomatology research field.
Two, background technology
As single celled life form, bacterium in research for a long time all the time to be free on unicellular state in the liquid nutrient medium as research object, do the influence of having simplified external environment to the full extent like this, also obtained a large amount of achievements in research, for modern microbiology is laid a good foundation.Yet under state of nature, bacterium seldom is free on the form in the pure culture and exists with this.Microbial film is the modal existence form of bacterium, is meant at solid-state organic or inorganic dielectric surface, and a large amount of microorganisms are collected in the matrix that exocellular polysaccharide constitutes and crosslinked mutually and microorganism ecological environment that form.
In medical field, the microbial film disease has occupied the infected by microbes disease more than 80%.Microbial film provides the good ecotope of resisting outside stimulus by the effect of its physics and chemistry barrier to pathogenic bacterium, and the gene of development conducts and engages therein.Help also that thereby the level transmission to drug resistant gene causes very big difficulty to traditional antibiotic therapy between different microorganisms.The principal disease dental caries disease of harm humans oral Health, its main paathogenic factor also are to be the main cariogenicity microbial film (plaque) that constitutes with the streptococcus mutans.
Set up the oral biological film model of simulation mouthful interior environment, help learning research the external oral microorganism that carries out high confidence level.Can obtain than the more accurate and comprehensive result of experiment in vitro.Biological film model can external creation a kind of with body in proximate ecotope, the growth metabolism condition of regulation and control bacterium, make it both to have consistence with natural surroundings, the remarkable advantage that has stdn and Modulatory character again, carrying out the oral biology experiment with biological film model compares with simple experiment in vitro, confidence level is higher, and is repeatable better, is a kind of very potential laboratory facilities.
Biological film model system main purpose is to simulate as much as possible ecotope in the oral cavity, and this artificial environment is maintained at stable status.Relevant biological film model (the Chinese stomatology magazine .1996 of report such as present domestic Liu Zheng; 31 (2): 110-112.) only be easy chemostat microbial culture system, though can satisfy basic microorganism continuous culture requirement, but its structure is numerous and jumbled, core component is cultivated for the fermentor tank continuous static, still can not effectively simulate the continual flowing environment of nutritive medium such as the inner saliva in oral cavity, and not good enough for the control of polluting; And external like product, equal Journal of Dental Research as Sissons, 1991,70 (11): the perseverance of foundation such as the 1409-16 report artificial oral system of multiple-workstation (MAM) and the Kinniment thing membrane modle (Microbiology that improves people's living condition, 1996,142,631-638), although control accuracy height to envrionment conditions, but its complex structure is corresponding also high to the accuracy requirement of peripherals, so it costs an arm and a leg, application cost is high, although to the good but still application that can't put it over of oral cavity environmental simulation.
Three, summary of the invention
The objective of the invention is to provide the method for a kind of oral biological film mould unit and formation oral biological film thereof at the deficiencies in the prior art, be characterized in adopting hydroxylapatite plate to place and contain the continuous culture system of quantitative experiment bacterial strain, by regularly giving sucrose solution to replenish the required substrate of each bacterium anaerobic glycolysis.
Purpose of the present invention is realized by following technical measures:
The oral biological film mould unit is made up of alkali liquid tank, culture tank, fermentor tank, constant flow culture and application of sample bottle.Alkali liquid tank and culture tank are installed in the preceding utmost point of fermentor tank, and fermentor tank and application of sample bottle are installed in the preceding utmost point of constant flow culture, will install with each device by piping system and web member thereof and connect and compose integral body.
Alkali liquid tank is connected with fermentor tank by peristaltic pump, and culture tank is connected with fermentor tank by peristaltic pump, and fermentor tank is introduced rare gas element N by filter and pump
2And CO
2Fermentor tank discharges waste liquid, waste gas by peristaltic pump and strainer, fermentor tank is connected with a plurality of constant flow culture by peristaltic pump, each constant flow culture discharges waste liquid, waste gas by peristaltic pump and strainer, and culture tank is connected with a plurality of constant flow culture in parallel by peristaltic pump respectively with the application of sample bottle.
Fermentor tank is an interlayer, and interlayer is provided with circulating water intake and outlet, and fermentor tank places on the magnetic stirrer.
Fermentor tank is that glass, plastics or metal are made.
The built-in hydroxylapatite plate of each constant flow culture, the hydroxylapatite plate vertical fixing is on platform, also be provided with hyperchannel pH registering instrument electrode and waste liquid outlet port in the constant flow culture, constant flow culture places the interior constant temperature of constant incubator to hatch, by the flow circuit of circulation tube saliva in peristaltic pump P8 keeps the simulation oral environment.
Constant flow culture and platform material are glass, plastics or metal.
Hydroxylapatite plate is provided by oral cavity biomedical engineering key lab of West China College of Stomatology Sichuan University investigation of materials chamber.
The formation method of oral biological film
1, after the aseptic connection of mould unit is finished, fermentor tank is placed on the magnetic stirrer, obtain the stirring velocity of 30-100rpm, temperature is maintained about 37 ℃ by the recirculated water bath cabinet, alkali liquid tank provides basic solution to make about the interior pH=7.0 of fermentor tank, is regulated by pH controller and peristaltic pump.
2, the interior Sheng of culture tank artificial saliva substratum is added by peristaltic pump constant speed in fermentor tank and constant flow culture as the main source of nutrition of experimental bacteria.
3, contain medicine and sucrose in the application of sample bottle, and the experiment microbial film provides the substrate sucrose that causes dental caries and the anticariogenic agent of required research in constant flow culture, can adopt different medicines and concentration according to the difference of research purpose.
4, constant flow culture places in the constant incubator of sealing, keeps the circulation velocity of 50～100ml/min to simulate the dynamic environment of salivary flow in the oral environment by circulation tube through peristaltic pump.
The present invention has following advantage:
1, can effectively simulate ecotope and the microbial film of formation experiment stably in the outlet, verify its application usefulness by big quantity research at cariology and science of dental materials;
2, adopt hyperchannel culture technique in parallel, difference that can more different simultaneously microbial film treatment measures has also been simplified workflow greatly, is more convenient for using;
3, the major equipment of selecting for use is product commonly used on the market, and cost control is good, compares with international like product, has goodish price advantage;
Thereby 4, the pollution of adopting airtight training method effectively to control environment.
Four, description of drawings
Fig. 1 is an oral biological film mould unit synoptic diagram
1. alkali liquid tank 2. culture tanks 3. fermentor tanks 4. constant flow culture 5. hydroxylapatite plates 6. platforms 7. application of sample bottles 8. magnetic stirrers 9. constant water bath box 10. hyperchannel pH survey meters
P1～P8 is a peristaltic pump
F1～F3 is a strainer
Fig. 2 is the fermentor tank structural representation
Fig. 3 is the constant flow culture structural representation
Fig. 4 is the pH response curve figure in the cultured continuously
Fig. 5 is microbial film live bacterial count figure on the hydroxylapatite plate
Fig. 6 is the constituent ratio of each experimental bacteria in experimental group and the control group microbial film
The negative control group microbial film of Fig. 7 scanning electron microscope is seen SEM X5000
The positive control group microbial film of Fig. 8 scanning electron microscope is seen SEM X5000
Fig. 9 sees SEM X5000 for the experimental group scanning electron microscope
Five, embodiment
Below by embodiment the present invention is carried out concrete description; be necessary to be pointed out that at this present embodiment only is used for the present invention is further specified; but can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of the invention described above.
Embodiment:
The oral biological film mould unit is shown in Fig. 1～3, form by alkali lye bottle 1, culture tank 2, fermentor tank 3, constant flow culture 4 and application of sample bottle 7, alkali liquid tank 1 and culture tank 2 are installed in the preceding utmost point of fermentor tank 3, fermentor tank 3 and application of sample bottle 7 are installed in 4 the preceding utmost point of constant flow culture, by piping system and web member thereof each device and device are connected and composed integral body.Alkali liquid tank 1 is connected with fermentor tank 3 by peristaltic pump P1; Culture tank 2 is connected with fermentor tank 3 by peristaltic pump P2; Fermentor tank 3 is introduced rare gas element N2 and CO2 by filter F2 and pump P4; Fermentor tank 3 is by peristaltic pump P5 and filter F1 effluent discharge, waste gas, fermentor tank 3 is connected with a plurality of constant flow culture 4 by peristaltic pump P6, each constant flow culture 4 discharges waste liquid, waste gas by peristaltic pump P7 and filter F 3, by peristaltic pump P2, P3 is connected with the constant flow culture 4 of a plurality of parallel connections respectively for culture tank 2 and application of sample bottle 7.Fermentor tank 3 is an interlayer, and interlayer is established circulating water intake and outlet, is about 37 ℃ by temperature in the circulator bath maintenance fermentor tank, and fermentor tank places on the magnetic stirrer 8.Fermentor tank useable glass, plastics or metal are made.Each constant flow culture 4 built-in hydroxylapatite plate 5, hydroxylapatite plate 5 vertical fixing are on platform 6, establish hyperchannel pH survey record instrument electrode and waste liquid, outlet port in the constant flow culture 4, constant flow culture is hatched as for constant temperature in the constant incubator 9, by the flow circuit of circulation tube saliva in peristaltic pump P8 keeps the simulation oral environment, keeping temperature is about 37 ℃.Constant flow culture and platform useable glass, plastics or metal are made.Hydroxylapatite plate is made by oral cavity biomedical engineering key lab of West China College of Stomatology Sichuan University investigation of materials chamber and is provided.
The formation method of oral biological film:
1, after the aseptic connection of mould unit is finished, fermentor tank 3 is placed on the magnetic stirrer 8, obtain the stirring velocity of 30～100rpm, temperature is maintained about 37 ℃ by the recirculated water bath cabinet, alkali liquid tank 1 provides 0.1N NaOH basic solution to make about the interior pH=7.0 of fermentor tank, regulate by pH controller and peristaltic pump P1, treat that each stable back of experimental bacteria growth is standby.
2, culture tank 2 interior Sheng artificial saliva substratum are added by peristaltic pump P2 constant speed in fermentor tank 3 and constant flow culture 4 as the main source of nutrition of experimental bacteria.
3, contain medicine and sucrose in the application of sample bottle 7, and to constant flow culture 4 in, test microbial film the substrate sucrose that causes dental caries and the anticariogenic agent of required research are provided, can adopt different medicines and concentration according to the difference of research purpose.
4, constant flow culture 4 places in the constant incubator of sealing, keeps the circulation velocity of 50～100ml/min to simulate the dynamic environment of salivary flow in the oral environment by circulation tube through peristaltic pump P8.
The artificial saliva substratum is a BM series microbiological culture media.
Below be the application example in cariology research of this mould unit:
1. experimental strain
Streptococcus mutans Streptococcus mutans ATCC25175
Streptococcus sanguis Streptococcus sanguis SB 179
Streptococcus-salivarius Streptococcus salivarius SS 196
Actinomyces naeslundii Actinomyces naeslundii WVU 627
Above bacterial strain provides by School of Stomatology oral cavity, West China microbial room of biomedical engineering key lab.
2. culture condition
2.1 fermentor cultivation condition
Temperature: keep by the recirculated water bath cabinet for 37 ℃;
Stir: low speed, by magnetic stirring apparatus 8 controls, so that bacteria suspension mixing in the fermentor tank;
Anaerobic environment: 95%N2,5%CO2, flow velocity 10ml/min is controlled by peristaltic pump P4;
PH: maintain about 7.0 with 0.1N NaOH solution, regulate by pH controller and peristaltic pump P4;
Clearance rate: D=0.1/hr is controlled by peristaltic pump P5.
2.2 constant flow culture culture condition
Temperature: keep by constant incubator for 37 ℃;
Circulation: 60ml/min is controlled by peristaltic pump P8;
Clearance rate: D=0.75/hr is controlled by peristaltic pump P7;
Sucrose (comparing with distilled water): gave 1 time in per 12 hours, each 10ml makes it reach certain final concentration, by peristaltic pump P3 and divider control.
3. experiment flow
After the aseptic connection of mould unit was finished, the inoculation experiments bacterial strain mixed cultured continuously in fermentor tank wherein, kept the balance of its pH by the pH controller, and constant speed adds artificial saliva substratum BM-5, as the main source of nutrition of experimental bacteria.Treat the stable back of each bacteria growing (nutrient solution was than turbid and live bacterial count there was no significant difference in continuous two days); By peristaltic pump the bacteria suspension in the fermentor tank is introduced in each constant flow culture in parallel, and continued to add the BM-5 substratum, mediation forms the experiment microbial film on hydroxylapatite plate.Can be by to wherein regularly adding different drug solutions, to observe according to the difference of experiment purpose to testing biomembranous influence.
4. artificial saliva
The artificial saliva prescription that this experiment is adopted is microbial film improved culture medium BM-5:
III type hog gastric mucin (Hog gastric mucin, Type III, Sigma) 2.50g/l
Peptide peptone (Proteose peptone, Fluka) 2.00g/l
Casein peptone (Peptone from casein, tryptic digest, Fluka) 1.00g/l that pancreatin decomposes
Yeast extract (Yeast extract, Oxoid) 1.00g/l
Repone K 2.50g/l
Glucose (Sucrose) 0.50g/l
Cystine hydrochloride (Cystiene-Hydrochloride, Sigma) 0.10g/l
Protohemine (Heamin, Shanghai east wind Biochem Technology, INC.) 1.00mg/l
Dipotassium hydrogen phosphate 0.114g/l
Potassium primary phosphate 0.20g/l
Adjusting its pH value with 1N NaOH solution is 7.0.121 ℃, 15psi, sterilization 70min.
5. experimental result example
This example is for studying the natural drug honeycomb to testing biomembranous influence
A tests grouping
After bacterial growth in the constant flow culture was stable, application of sample was 1 time in per 12 hours, and each 10ml makes it reach certain final concentration, by peristaltic pump and divider control.According to difference, be divided into following each group to application of sample in the constant flow culture:
Positive controls: the 250mM sucrose solution, the final concentration in the culturing room is about 25mM.
Negative control group: the distilled water that does not contain any carbohydrate is as blank.
Experimental group: the 250mM sucrose solution that contains the Nidus Vespae extract of 4mg/ml.
The dynamic change of pH in the B cultured continuously
As shown in Figure 2, in the process of 6 timing dosings, negative control group is a distilled water owing to what add, so its pH does not see tangible fluctuation.In positive controls, owing to added the sucrose solution of the about 25mM of final concentration, from administration for the third time, its pH value just continues to be suppressed in the low scope.And in experimental group, although the adding of the sucrose of same concentration is arranged, but owing to added Nidus Vespae extract simultaneously, thereby only at the dosing initial stage, its pH is visible obviously to descend, and after 4-5 hour, along with the progressively removing of meta-bolites, its pH value all can progressively return to normal range at every turn, and curve is the waviness of rule.
C tests biomembranous live bacterial count
As seen from Figure 6, along with regularly addings in per 12 hours of 6 sucrose solutions, streptococcus mutans on the microbial film, the viable bacteria numeration of Streptococcus sanguis and actinomyces naeslundii is compared with negative control group (distilled water adding group) has tangible growth (P＜0.05) more.Honeycomb has been compared significant inhibition performance (P＜0.05) to the growth of streptococcus mutans on microbial film with the positive controls that adds same concentration sucrose.Shown in Fig. 4,5, adhere at microbial film certain difference is quantitatively arranged although can find for the different experiments bacterium it, on constituting, it does not see to have significant change.
D tests biomembranous scanning electron microscopic observation
To testing in the biomembranous scanning electron microscopic observation as shown in Figure 7 in the positive controls, the existence of a large amount of exocellular polysaccharides makes it to be the performance of fine hair sample, and in negative control group, as shown in Figure 8, boundary is clear between each mycetocyte, and extracellular matrix is less.The sucrose adding image that group becomes that contains honeycomb solution then falls between as shown in Figure 9.
Experimental result shows
Experiment as seen thus; along with the timing of sucrose solution is additional; microbial film extracellular matrix showed increased on the hydroxylapatite plate; combination is more tight between bacterium; and each bacterium quantitatively also has and significantly increases; this has verified that from another aspect bacterial plaque causes the dental caries hypothesis; promptly along with a large amount of absorptions of carbohydrate; acidic metabolite is accumulated in a large number in the ripe bacterial plaque; be in for a long time under the low pH environment; the amount of each cariogenic bacteria has remarkable increase, produces acid and further increases, thereby normal bacterial plaque is changed to the cariogenicity bacterial plaque.But the adding of honeycomb crude extract solution; then can significantly reduce streptococcus mutans adherent amount on hydroxylapatite plate; also show the minimizing of extracellular matrix in the observation of scanning electron microscope, the adding of prompting honeycomb solution may have certain restraining effect to the dental caries transformation that causes of bacterial plaque.
Claims (5)
1, oral biological film mould unit, it is characterized in that this device comprises alkali liquid tank (1), culture tank (2), fermentor tank (3), constant flow culture (4) and application of sample bottle (7), alkali liquid tank (1) and culture tank (2) are installed in the preceding utmost point of fermentor tank (3), fermentor tank (3) and application of sample bottle (7) are installed in the preceding utmost point of constant flow culture (4), alkali liquid tank (1) is connected with fermentor tank (3) by peristaltic pump P1, culture tank (2) is connected with fermentor tank (3) by peristaltic pump P2, and fermentor tank (3) is introduced rare gas element N by filter F2 and pump P4
2And CO
2Fermentor tank (3) discharges waste liquid, waste gas by peristaltic pump P5 and filter F 1, fermentor tank (3) is connected with a plurality of constant flow culture (4) by peristaltic pump P6, each constant flow culture (4) discharges waste liquid, waste gas by peristaltic pump P7 and filter F 3, by peristaltic pump P2, P3 is connected with a plurality of constant flow culture (4) respectively for culture tank (2) and application of sample bottle (7);
Wherein, fermentor tank (3) is an interlayer, and interlayer is established circulator bath import and outlet, jar built-in pH controller electrode, and fermentor tank (3) places on the magnetic stirrer (8); The built-in hydroxylapatite plate of each constant flow culture (4) (5), the hydroxylapatite plate vertical fixing is on platform (6), also be provided with hyperchannel pH survey record instrument (10) electrode and waste liquid, outlet port in the constant flow culture, constant flow culture (4) places the interior constant temperature of constant incubator (9) to hatch, by the flow circuit of circulation tube saliva in peristaltic pump P8 keeps the simulation oral environment.
2,, it is characterized in that fermentor tank is that glass, plastics or metal are made according to the described oral biological film mould unit of claim 1.
3,, it is characterized in that constant flow culture and platform are that glass, plastics or metal are made according to the described oral biological film mould unit of claim 1.
4, form the method for oral biological film according to the described oral biological film mould unit of one of claim 1-3, it is characterized in that:
1) after the aseptic connection of mould unit is finished fermentor tank (3) is placed on the magnetic stirrer (8), obtain the mixing speed of 30-100rpm, temperature keeps about 37 ℃ by recirculated water, alkali liquid tank (1) provides alkali lye to make about the interior pH=7.0 of fermentor tank, regulate by pH controller and peristaltic pump P1, treat that the stable back of each bacteria growing is standby;
2) contain the main source of nutrition of artificial saliva substratum in the culture tank (2), add by peristaltic pump P2 constant speed in fermentor tank (3) and constant flow culture (4) as experimental bacteria;
3) contain medicine and sucrose in the application of sample bottle (7), to constant flow culture (4) in, test microbial film the substrate sucrose that causes dental caries and the anticariogenic agent of required research are provided, can adopt different pharmaceutical and concentration according to the difference of research purpose;
4) constant flow culture (4) places in the constant current incubator of sealing, by the flowing environment of circulation tube saliva in peristaltic pump P8 keeps 50～100ml/min simulation oral environment.
5, form the method for oral biological film according to the described oral biological film mould unit of claim 4, it is characterized in that the artificial saliva substratum is a BM series of biologic film substratum.
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| CN 03135320 CN1274803C (en) | 2003-07-01 | 2003-07-01 | Model of mouth cavity biomembrane and method for forming mouth cavity biomembrane |
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Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100408669C (en) * | 2006-03-30 | 2008-08-06 | 四川大学 | Oral biological film dynamic model device and its oral biological film forming method |
| CN101935612B (en) * | 2010-08-24 | 2013-10-16 | 好来化工(中山)有限公司 | Flow cell of manual oral cavity model used for culturing biological film |
| CN101935611B (en) * | 2010-08-24 | 2015-04-22 | 好来化工(中山)有限公司 | Biomembrane culture and flow chamber in artificial mouth simulation device |
| CN101948745B (en) * | 2010-09-09 | 2013-06-26 | 好来化工(中山)有限公司 | Artificial mouth simulating device |
| CN102965272B (en) * | 2012-11-09 | 2015-11-25 | 华东理工大学 | The implementation method of Fermentation linkage device and fermentation linkage |
| CN106148178B (en) * | 2015-04-02 | 2019-10-29 | 复旦大学附属华山医院 | A kind of culture systems and its construction method of the static killing curve for anti-anaerobic agent |
| CN106281998B (en) * | 2015-05-29 | 2020-04-24 | 复旦大学附属华山医院 | Anaerobic in-vitro dynamic pharmacokinetics/pharmacodynamics model system and construction method |
| CN107418891A (en) * | 2017-09-20 | 2017-12-01 | 四川省人民医院 | A kind of perfusion type bladder bacterial biof iotalm dynamic is incubated simulation system |
| CN108641901B (en) * | 2018-06-07 | 2023-09-15 | 云南中烟工业有限责任公司 | Oral cavity biomembrane external anaerobic model device and application thereof |
| CN112816678A (en) * | 2020-12-29 | 2021-05-18 | 广州市华代生物科技有限公司 | Method for detecting efficacy of oral product by using oral micro-ecological biomembrane model |
| CN113956965B (en) * | 2021-09-13 | 2023-11-21 | 北京大学口腔医学院 | Device for simulating waterway of oral cavity comprehensive treatment table and biofilm forming method |
| CN117264818B (en) * | 2023-09-15 | 2024-05-17 | 中山大学附属口腔医院 | Oral bacteria biological film culture medium and application thereof |
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