CN101948745B - Artificial mouth simulating device - Google Patents
Artificial mouth simulating device Download PDFInfo
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- CN101948745B CN101948745B CN2010102769424A CN201010276942A CN101948745B CN 101948745 B CN101948745 B CN 101948745B CN 2010102769424 A CN2010102769424 A CN 2010102769424A CN 201010276942 A CN201010276942 A CN 201010276942A CN 101948745 B CN101948745 B CN 101948745B
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Abstract
The invention discloses an artificial mouth simulating device, comprising a culture medium tank, multi-channel pumps, flow chambers, drug sample solution bottles and a waste solution collection tank, wherein the culture medium tank is connected with one multi-channel pump by pipelines; the multi-channel pump is connected with the flow chambers by pipelines; Luer connectors are arranged on the pipelines between the multi-channel pump and the flow chambers and are connected with the drug sample solution bottles by pipelines; the flow chambers and the waste solution collection tank are connected by pipelines; and the other multi-channel pump is arranged on the connection pipelines between the flow chambers and the waste solution collection tank. The device has the beneficial effects of simple structure, low cost, short cycle of experiment, good repeatability of the experiment result, high precision control and low possibility of being polluted.
Description
Technical field
The present invention relates to a kind of simulator that carries out experimental study under simulating oral cavity environment, especially relate to a kind of simulating device for artificial oral cavity.
Background technology
For a long time, people carry out bacteriological research in the mode of microorganism pure culture usually, the impact of external environment has been simplified in this research to a great extent, help people to be familiar with the habit of a lot of individual microorganisms, a lot of important achievements in research have been obtained, for huge contribution has been made in medical science and microbiological development.Yet the most of bacteriums of occurring in nature are not with the existence of the unbound state under pure culture, but exist with biomembranous form.Microbial film be a large amount of germs collect on certain dielectric surface and be wrapped in a kind of special cells group structure of forming in the exocellular polysaccharide of himself secretion, it can be planted bacterium and be consisted of by single, but is the body of living in concentrated communities of various bacteria under more susceptible condition.Compare with the bacterium of unbound state; biomembranous Clinicopathological significance is even more important; because it has the protectiveness exocellular polysaccharide of one deck densification; therefore has the ability that stronger resistance and stronger opposing host immune kill and wound; and also exist one to overlap complicated information transport system in microbial film between different bacterium; some drug resistant genes can shift between different bacterial classifications, thereby have strengthened the difficulty of traditional antibiotic therapy.
Plaque is a kind of microbial film that sticks on dental surface, gingival edge, gum inside or other soft tissues of oral cavity.It is comprised of a large amount of oral cavity bacteriums, extracellular matrix, a small amount of white corpuscle, Exfoliative cells and swill; Its adhere firmly is difficult to remove.Plaque is considered to the main pathogenic of most of oral diseases, comprises carious tooth, gingivitis, periodontitis etc., therefore the research of plaque is become a focus of stomatology research.On research method, the method for " original position " can reflect truth, and difficulty is larger but this method actually operating is got up, and has very large uncertainty; So on the research method of oral biological film, what generally adopt is the method for external model.External model can be simplified the internal milieu factor of many complexity on the one hand, can realize on the other hand the autotelic control to the biofilm development condition.
At present, the external model research method of oral biological film has following several:
1, chemostat-flow chamber model.Referring to Herles S, Olsen S, Afflitto J, Garffar A.C hemostat flow cell system:an in vitro model for evaluation of antiplaque age nts.J Dent Res.1994; 73 (11): 1748-1755.Shown in Figure 1, this model comprises perseveranceization chamber 101, additional bacterial solution chamber 102, mixing section 103, multi-channel pump 104, a plurality of flow chamber 105 and waste collection chamber 106; Are connected with mixing section by pipeline to connect and communicate in described perseveranceization chamber 101, are connected with mixing section by pipeline to connect and communicate in described additional bacterial solution chamber 102, described mixing section 103 is connected with the entrance of multi-channel pump 104 by pipeline and communicates, the outlet of described multi-channel pump 104 is connected with a plurality of flow chambers 105 by pipeline and communicates, described flow chamber 105 is connected with waste collection chamber 106 by pipeline and communicates, and described mixing section 103 also is connected with waste collection chamber 106 by pipeline and communicates.The principle of work of this model is: cultivate 5 kinds of oral cavity bacteriums in perseverance chamber 101, in the lower cultivation through certain hour (being generally 2-7 days) of suitable growth conditions (pH, temperature, nutrition and anaerobism have the requirement of regulation), each bacterial classification can reach a kind of state (being the stable of flora ratio, the balance of bacterial growth and decline etc.) of relatively stable balance; After bacterial growth reaches balance, bacterium liquid is extracted in mixing section 103 with pump, with multi-channel pump 104, the bacterium liquid in mixing section 103 is assigned in a plurality of flow chambers 105 again, bacterium liquid is flowed through after flow chamber 105, and bacterium adheres to and breeds growth on can carrier 107 surfaces (hydroxyapatite HAP sheet or germanium wafer) in flow chamber 105 and forms bacterial plaque sample microbial film.This chemostat-flow chamber model has a plurality of flow chamber 105 parallel connections, therefore can carry out simultaneously the test of a plurality of samples, and convenient sampling.But the defective of this model is also apparent in view: 1) flow chamber does not have temperature to control, and therefore can not simulate the real biofilm development temperature of human oral; 2) the whole system pipeline is more complicated, and contaminated possibility is larger; 3) experimental period longer, complete one and take turns test and need about week age.
2, chemostat-constant current chamber model.Chinese patent application number: CN200610020608.6 discloses a kind of oral biological film dynamic model device and has formed the method for oral biological film.Shown in Figure 2, this model comprises alkali liquid tank 201, culture tank 202, fermentor tank 203, constant flow culture 204 and application of sample bottle 205.The principle of work of this model is similar with " chemostat-flow chamber model ": namely carry out the oral cavity bacterium cultivation in constant flow culture 204, pH can be controlled by alkali liquid tank 201, and nutrition is provided by culture tank 202; The key point of this invention is its constant flow culture 204, and a plurality of sample injectors 206 can vertically be placed by this culturing room 204, and each sample injector 206 can be fixed again a plurality of biofilm carriers 207 surfaces as the hydroxyapatite flag etc.; And this system can a plurality of constant flow culture 204 in parallel.After microbial culture is good, the bacterium liquid pump is entered in constant flow culture 204, bacterium just can adhere to and biofilm growth formation on carrier 206 surfaces on sample injector 205.The relative merits of this model with " chemostat-flow chamber model: compare; the improvements of this model are exactly that the constant flow culture temperature of biofilm development is controlled; but the shortcoming of " chemostat-flow chamber model " does not have much improvement in this model; this model still has following defective: the 1) structure of whole system very complex still, feeding-system, pipeline and pump are all a lot; This convenient loaded down with trivial details degree that increases operation has also increased on the other hand and has polluted the difficulty of controlling; 2) experimental period is long.
3, Zurich model.Referring to Guggenheim B, Giertsen E, Schupbach P, Shapiro S.Validation of an in vitro Biofilm Model of Supragingival Plaque.J Dent Res.2001; 80 (1): 363-370.Guggenheim B, Guggenheim M, Gmur R, Giertsen E, Thurnheer T.Application of the Zurich Biofilm Model to Problems of Cariology.Caries Res.2004; 38:212-222.Shown in Figure 3, this model comprises Tissue Culture Plate 301, and described Tissue Culture Plate 301 is provided with 24 apertures 302, is provided with biofilm development carrier 30 as one kind 3 in this aperture 302.The principle of work of this model is: utilize 24 porocyte culture plates as biofilm growth device, place hydroxyapatite flag or baurodont in the hole as the biofilm development carrier; Concrete step is: 5 kinds of typical oral cavity bacteriums of vitro culture at first, then with its combined inoculation to 24 orifice plates, anaerobism is cultivated, oral cavity bacterium just can deposit also apposition growth formation microbial film on growing carrier like this; Incubation time needs 64 hours altogether, 2 replaced mediums of intermediate demand; When carrying out the pharmaceutical activity test, need to select 6 time points to carry out drug treating; Utilize this model can simulate the growth of people's supragingival plaque.The advantage of the maximum of this model is the device simplicity of design, and is with low cost, operates also more convenient; But this model has following defective: 1) its microbial film training mode is static cultivation, can't simulate the salivary flow in human oral; 2) anaerobism is cultivated, and also there is certain influence in aerobic operation to biomembranous growth; 3) its substratum is prepared more complicated.
4, artificial oral-cavity model (Artificial Mouth), referring to Sissons CH, Cutress TW, Hoffman MP, Wakefield JSJ.A multi-station dental plaque microcosm (artificial mouth) for the study of plaque growth, metabolism, pH, and mineralization.JDent Res.1991; 70 (11): 1409-1416.Shown in Fig. 4,5, this model comprises the seal hatch 401 of a multi-drop multiport, sealing cabin 401 is provided with inoculation mouth 402, substratum entrance 403, detecting electrode entrance 404 and thief hole 405, be provided with carrier supported platform 406 in sealing cabin 401, establish biomembranous growing carrier 407 on this carrier supported platform 406.the principle of work of this model is: the biomembranous growing carrier 407 of this model is selected a kind of tissue culture cover glass, inject salivas to the cover glass carrier 407 of carrier supported platform 406 toward inoculation mouth 402, drip nutrient solution or other impact solution from top substratum entrance 403 subsequently, microbial film will be grown on cover glass carrier 407, culturing process need to pass into anaerobic gas to make anaerobic environment, pH by detecting electrode entrance 404 electrode insertion observable biofilm development processes changes, also can take out the physiological and biochemical analysis that diffraction patterns for biomembrane samples is correlated with from thief hole 405.This pattern layout is very exquisite, can realize biomembranous growing environment and process are carried out some high-precision control and monitorings, and can independently control the growth of a plurality of microbial film samples; But this model has following defective: 1) just because of this model control accuracy have relatively high expectations, so its construction cost is high; 2) experimental period of this model long, once experiment may need time in several weeks.3) this model bacterial plaque need to be grown in anaerobic environment, with biofilm development environment in actual body, difference is arranged.
5, the perseverance thing membrane reactor (Constant Depth Film Fermenter, CDFF) of improving people's living condition; Referring to Pe ters AC, Wimpenny JWT.A constant-depth laboratory model film fermentor.Biotechnol Bioeng.1988; 32:263-270.Pratten J, Wilson M.Antimicrobial sus ceptibility and composition of microcosm dental plaque supplemented with su crose.Antimicrob Agents Chemother.1999; 43 (7): 1595-1599.Shown in Figure 6, this biofilm reactor comprises a cavity 500, and this cavity 500 is provided with sample inlet 501, gas inlet 502 and substratum entrance 503; Be provided with rotating disk 504 in this cavity 500, this rotating disk is provided with the little groove 505 of a plurality of same sizes, is provided with enamel sheet or hydroxylapatite plate 506 in little groove 505; The upper surface of described little groove 505 is provided with the blade 507 of rotation.The principle of work of this biofilm reactor is: biomembranous growth is to carry out in cavity 500 (space of a similar fermentor tank); Be provided with rotating disk 504 in cavity 500, in the little groove 505 of a plurality of even depth is arranged, the surface to provide bacterial plaque to form in groove 505 can be provided for enamel sheet or hydroxylapatite plate 506, in this reactor, the bacterium of inoculation can be cultivated the mixed bacteria liquid in chemostat or come from saliva/bacterial plaque inoculum from anaerobism, start rotating disk 504 during experiment, substratum entrance 503 inoculated bacterias and fed-batch medium by rotating disk 504 tops, can breed gradually after bacterium adheres on the surface of recess and grow form microbial film, and thickening gradually; When in groove 505, microbial film reached certain thickness, the blade 507 of top rotation will be removed unnecessary microbial film, is in constant thickness to guarantee microbial film.This reactor is a relatively ripe biofilm reactor, aspect the characteristic of studying biofilm and antimicrobial substance activity, application is comparatively widely being arranged.But it still has following defective: 1) the temperature of reactor is not controlled; 2) experimental period is long; 3) poor repeatability of experimental result.
In sum, all there is certain shortcoming in the external model of existing oral biological film, can not satisfy the needs of research fully.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of simulating device for artificial oral cavity, and this system and device is simple in structure, and is with low cost, and experimental period is short, the good reproducibility of experimental result, and precision is controlled high, is not vulnerable to pollute.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
Simulating device for artificial oral cavity, it comprises:
Culture tank, this culture tank are used for the cultivation of bacterial classification, and the cultivation of bacterial classification is originated as biomembranous bacterial classification with people's saliva or bacterial plaque, with the state of simulation people's true bacterial plaque at utmost;
Multi-channel pump for delivery of inoculum and drug sample solution, also is responsible for waste liquid in flow chamber is transported to the waste collection tank;
Flow chamber, be used for inoculation and drug treating, the hydroxyapatite flag that is about to saliva coated is put into flow chamber, then fill with the diluent of saliva or bacterial plaque in the flow chamber with pipettor, and with the drug sample solution pump enter flow chamber to adhere to and the certain hour of growing plaque process experiment;
The drug sample solution bottle is used for providing drug sample solution;
With the waste collection tank, be used for the collection of waste liquid.
Described culture tank is connected with multi-channel pump by pipeline, and described multi-channel pump is connected with flow chamber by pipeline; Pipeline between described multi-channel pump and flow chamber is provided with female Luer, and this female Luer is connected with the drug sample solution bottle by pipeline, and this female Luer can disconnect the state that is connected with the drug sample solution bottle when carrying out sample preparation; Described flow chamber is connected with the waste collection tank by pipeline, and the connecting tube between described flow chamber and waste collection tank is provided with multi-channel pump.
Further, described flow chamber comprises at least one flow chamber unit; Once doing simultaneously the identical test of many group experiment conditions in experiment, increase the sample test capacity.
Further, be provided with the breeches joint of cascade between described culture tank and multi-channel pump, the nutrient solution in culture tank is assigned to each mobile chamber unit.
Further, described a plurality of flow chambers unit is arranged on a flow chamber anchor; The flow chamber anchor can one-time fix a plurality of flow chambers, conveniently a plurality of flow chambers is carried out parallel running, guarantees the consistence of each flow chamber operational condition.
Further, described flow chamber anchor is outside equipped with constant water bath box; Biomembranous growth temperature can be accurately controlled and be regulated in water bath with thermostatic control.
Further, described flow chamber comprises flow chamber chassis and flow chamber upper cover; Described flow chamber chassis upper surface is provided with buckled recess, and the size of this buckled recess and flow chamber upper cover outline are complementary, and described flow chamber upper cover is fastened in buckled recess, and both mutually fasten and seal; The middle part of described buckled recess is provided with the liquid-flow groove, is equipped with at least one slide glass identical or close with the tooth material in this liquid-flow groove; Cover on described flow chamber and offer a pair of through hole, be screwed with the thread connection head in this each through hole, described thread connection head is the gangway as liquid in the liquid-flow groove on the chassis, and can be used as the upper cover handle in when dismounting; Described flow chamber chassis and flow chamber upper cover are dismountable fixing by stationary installation.
Described liquid-flow groove middle part is equipped with the slide glass stopper protrusion, is respectively equipped with the position limiting structure for the location slide glass on the liquid-flow groove inner side-wall of slide glass stopper protrusion sidewall and correspondence; The setting of this position limiting structure makes slide glass can be positioned at fixing locus in the liquid-flow groove, is convenient to the growth of microbial film on slide glass.
Offer a ring groove on the buckled recess of described liquid-flow groove outer rim, be embedded with elastic seal ring in this ring groove; Preferably, this elastic seal ring is quality of rubber materials; The setting of elastic seal ring can prevent that the nutrient solution in the liquid-flow groove from oozing out outside groove, pollutes.
The lower surface of described flow chamber upper cover middle part upwards be concaved with the flow chamber chassis on the upper cover groove of liquid-flow groove size coupling, its effect is to increase the volume that microbial film is cultivated nutrient solution in flow chamber.
Described upper cover groove middle part is equipped with upper cover slide glass stopper protrusion downwards, slide glass stopper protrusion position in this upper cover slide glass stopper protrusion and described liquid-flow groove is corresponding, its effect is to prevent that slide glass from arbitrarily moving in the accommodation space of flow chamber chassis and flow chamber upper cover formation, avoids the effect of slide glass moving influence experiment.
Described flow chamber chassis and flow chamber upper cover are dismountable fixing by bolt, in culturing process, and sampling analysis at any time.
The described slide glass identical or close with the tooth material is hydroxyapatite flag or germanium wafer.
The material of described flow chamber chassis and flow chamber upper cover is polycarbonate (PC) or polymethylmethacrylate (PMMC) or other transparent material, thermotolerance and resistance to chemical corrosion polycarbonate (PC) preferably preferably, one of purpose of selecting above-mentioned materials is make with low cost, two of purpose is to be convenient to observe the upgrowth situation of microbial film on slide glass, and nutrient solution or the mobile changing conditions of sample solution in the groove that flows.
Further, described flow chamber anchor comprises upper holder and lower fixed frame; Fixed frame lower surface is provided with groove; The upper surface of described lower fixed frame is provided with and the corresponding groove of the groove location of upper holder lower surface, and the groove of described upper and lower anchor and the size of flow chamber are complementary, thereby flow chamber can be fixed in the groove of described upper and lower anchor; Described upper and lower anchor is fixed together by stationary installation.
Further, described constant water bath box comprises casing and stay-warm case, and described stay-warm case and casing coupling fasten, and stay-warm case can guarantee the even distribution of each several part temperature in water bath, and reduces heat loss; The lower surface of described stay-warm case is provided with breach, and this breach is as the access way of pipeline; The upper surface of described stay-warm case is provided with handle, to facilitate picking and placeing of stay-warm case; Be provided with in described casing and hold the thing plate, be used for laying the flow chamber anchor; Described casing is provided with temperature digital display and adjusting knob, to be adjusted to the temperature condition that needs.
Further, the stay-warm case of described flow chamber, flow chamber anchor and constant water bath box all can select polymethylmethacrylate (PMMC) or polycarbonate (PC) materials processing to make, also can select other organic materials to make, preferred resistance to chemical attack, resistant to elevated temperatures, transparent material.
The present invention has following beneficial effect: simple in structure, with low cost, experimental period is short, the good reproducibility of experimental result, and precision is controlled high, is not vulnerable to pollute.
Description of drawings
Fig. 1 is existing chemostat-flow chamber model structure schematic diagram;
Fig. 2 is existing chemostat-constant current chamber model structure schematic diagram;
Fig. 3 is existing Zurich model structure schematic diagram;
Fig. 4 is existing artificial oral-cavity model Facad structure schematic diagram;
Fig. 5 is the side-looking structural representation of Fig. 4;
Fig. 6 is the existing perseverance thing membrane reactor structural representation of improving people's living condition;
Fig. 7 is a kind of technical scheme structural representation of the present invention;
Fig. 8 is the concrete structure schematic diagram of flow chamber in Fig. 7;
Fig. 9 is Fig. 8 flow chamber chassis vertical view;
Figure 10 is Fig. 8 flow chamber upper cover top view;
Figure 11 is that the A-A of Fig. 9 is to sectional view;
Figure 12 is that the B-B of Figure 10 is to sectional view;
Figure 13 is the vertical view of flow chamber anchor in Fig. 7;
The side-looking exploded view of Figure 14 Figure 13;
Figure 15 is the concrete structure schematic diagram of constant water bath box in Fig. 7;
External bacterial plaque 24h bacterial plaque accumulation and viable bacteria growth curve that Figure 16 obtains for the simulated experiment of using apparatus of the present invention to carry out;
External bacterial plaque 24h bacterial plaque accumulation and pH curve that Figure 17 obtains for the simulated experiment of using apparatus of the present invention to carry out;
The vitro culture bacterial plaque gramstaining figure that Figure 18 obtains for the simulated experiment of using apparatus of the present invention to carry out;
The bacterium colony figure of vitro culture bacterial plaque after cultivating on the plumbic acetate flat board that Figure 19 obtains for the simulated experiment of using apparatus of the present invention to carry out;
Bacterial plaque after the natural bacterial plaque that Figure 20 obtains for the simulated experiment of using apparatus of the present invention to carry out, natural saliva, 6h cultivate, bacterial plaque PCR-DGGE comparison diagram after 24h cultivates;
Scanning electron microscope structure iron when the 24h bacterial plaque that Figure 21 obtains for the simulated experiment of using apparatus of the present invention to carry out is amplified 300 times;
Scanning electron microscope structure iron when the 24h bacterial plaque that Figure 22 obtains for the simulated experiment of using apparatus of the present invention to carry out is amplified 5000 times.
Embodiment
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Shown in Figure 7, a kind of simulating device for artificial oral cavity 600, it comprises:
Multi-channel pump 602,603;
Drug sample solution bottle 605;
With waste collection tank 606;
Described culture tank 601 is connected with multi-channel pump 602 by pipeline, and described multi-channel pump 602 is connected with flow chamber 604 by pipeline; Pipeline between described multi-channel pump 602 and flow chamber 604 is provided with female Luer 607, this female Luer 607 is connected with drug sample solution bottle 605 by pipeline, and this female Luer can disconnect the state that is connected with the drug sample solution bottle when carrying out sample preparation; Each unit of described flow chamber 604 is connected with waste collection tank 605 by pipeline respectively, and the connecting tube between described flow chamber 604 and waste collection tank 605 is provided with multi-channel pump 603;
Above-mentioned multi-channel pump 602,603 does not use simultaneously, use the multi-channel pump 602 between culture tank 601 and flow chamber 604 in the microbial film culturing process, and the draw-in groove of the multi-channel pump 603 between flow chamber 604 and waste collection groove 605 is in the state of getting loose; When carrying out the drug solution processing, disconnect female Luer 607, an end that will be connected with flow chamber 604 is put into drug sample solution bottle 605, utilizes multi-channel pump 603 to complete injection and the removing of sample solution; The pipeline of whole system uses silica gel hose;
Be provided with the breeches joint 608 of cascade between described culture tank 601 and multi-channel pump 602, the nutrient solutions in culture tank 601 are assigned to each flow chamber Unit 604;
Shown in Fig. 8～12, described flow chamber unit 604 comprises flow chamber chassis 609 and flow chamber upper cover 610; Described flow chamber chassis 609 upper surfaces are provided with buckled recess 611, and the size of this buckled recess 611 and flow chamber upper cover 610 outlines are complementary, and described flow chamber upper cover 610 is fastened in buckled recess 611; The middle part of described buckled recess 611 is provided with liquid-flow groove 612, is equipped with four hydroxyapatite flag slide glasses 613 in this liquid-flow groove 612; Offer a pair of through hole 614 on described flow chamber upper cover 610, be screwed with thread connection 615 in this each through hole 614; Described flow chamber chassis 609 and flow chamber upper cover 610 are dismountable fixing by bolt 616;
Shown in Figure 9, described liquid-flow groove 612 middle parts are equipped with slide glass stopper protrusion 617, are respectively equipped with the position limiting structure 618 for the location slide glass on slide glass stopper protrusion 617 sidewalls and corresponding liquid-flow groove 612 inner side-walls;
Shown in Fig. 8,9,11, offer a ring groove 619 on the buckled recess 611 of described liquid-flow groove 612 outer rims, be embedded with rubber seal 620 in this ring groove 619;
Shown in Fig. 8,12, the lower surface of described flow chamber upper cover 610 middle part upwards be concaved with flow chamber chassis 609 on the upper cover groove 621 of liquid-flow groove 612 size couplings;
Shown in Fig. 8,12, described upper cover groove 621 middle parts are equipped with upper cover slide glass stopper protrusion 622 downwards, and slide glass stopper protrusion 617 positions in this upper cover slide glass stopper protrusion 622 and described liquid-flow groove 612 are corresponding;
The material of described flow chamber chassis 609 and flow chamber upper cover 610 is thermotolerance and resistance to chemical corrosion polycarbonate (PC) preferably;
Shown in Figure 13,14, described 24 flow chamber unit 604 are placed on flow chamber anchor 623 fixing; Described flow chamber anchor 623 comprises upper holder 624 and lower fixed frame 625; Fixed frame 624 lower surfaces are provided with groove 626; The upper surface of described lower fixed frame 625 is provided with the groove 626 corresponding grooves 627 in position with upper holder 624 lower surfaces, described upper and lower anchor 624,625 groove 626,627 and the size of flow chamber unit 604 be complementary, thereby flow chamber unit 604 can be fixed in described upper and lower anchor 624,625 groove 626,627 spaces that form, described upper and lower anchor 624,625 is fixed together by bolt 628;
Shown in Fig. 7,15, described flow chamber anchor 623 is outside equipped with constant water bath box 629; Water bath with thermostatic control can make the variation of temperature condition in whole experimentation very little, is not enough to affect the accuracy of whole experiment;
Shown in Figure 15, described constant water bath box 629 comprises casing 630 and stay-warm case 631, and described stay-warm case 631 and casing 30 couplings fasten; The lower surface of described stay-warm case 631 is provided with breach 632, and this breach 632 is as the access way of pipeline; The upper surface of described stay-warm case 631 is provided with handle 633, to facilitate picking and placeing of stay-warm case 631; Be provided with in described casing 630 and hold thing plate 634, be used for laying flow chamber anchor 623; Described casing 630 is provided with temperature digital display 635 and adjusting knob 636, to be adjusted to the temperature condition that needs;
The stay-warm case 631 of described flow chamber anchor 623 and constant water bath box 629 is all selected the polymethyl methacrylate materials processing and fabricating.
Utilize simulating device for artificial oral cavity of the present invention to carry out the method for simulated experiment, comprise the steps:
1) selection of substratum
Substratum is selected basic Saliva Orthana substratum (BMM, Sissons, et al.J Dent Res.1991; 70 (11): 1409-16), fill a prescription as follows:
Hog gastric mucin 2.5g/L
Proteose peptone 10.0g/L
Trypticase peptone 5.0g/L
Yeast extract 5.0g/L
KCl 2.5g/L
Haemin 5mg/L
Arginine 1mmol/L
L-cys 0.1g/L
Urea 1mmol/L
Vitamin K1 1mg/L
2) the hydroxyapatite flag is coated
Collector's saliva, the centrifuging and taking supernatant with the uv irradiating sterilization, then is immersed the hydroxyapatite flag, and soak time at least 2 hours makes hydroxyapatite flag surface coverage skim sialoprotein;
3) bacterial classification source
Saliva or bacterial plaque with the people are originated as biomembranous bacterial classification, the state of simulation people's true bacterial plaque at utmost, and the saliva of collecting or bacterial plaque can dilute to ensure enough inoculum sizes with the BMM substratum;
4) inoculation
The hydroxyapatite flag 613 of saliva coated is put into flow chamber 604, then fill with the diluent of saliva or bacterial plaque with pipettor in the flow chamber 604
5) culture condition
Water bath with thermostatic control temperature: 37 ℃
The flow velocity of multi-channel pump 602 is configured such that the clearance rate of each flow chamber is 0.7h
-1
6) the antibiont film activity of medicine test
A compounding pharmaceutical solution;
B disconnects female Luer 607 after the microbial film incubation time reaches 8h, use multi-channel pump 603 first with the emptying rate of liquid in flow chamber 604;
Joint one end that c will be connected with flow chamber 604 is put into drug sample solution, then with multi-channel pump 603, drug solution is pumped in flow chamber 604, determines action time by the experimental plan requirement;
After finishing action time, d use again multi-channel pump 603 that liquid is emptying;
E is same with pumping into-emptying method sterile water wash flow chamber, removes as far as possible liquor residue;
F reconnects female Luer 607, and the multi-channel pump 603 that gets loose, with the multi-channel pump 602 past interior injection fresh culture of flow chamber 604 again;
G continues to cultivate 16h, can complete one and take turns test.
Relatively the antibiont film activity of each medicine is strong and weak by detecting bacterial plaque amount on the hydroxyapatite flag and the viable bacteria amount in bacterial plaque.
Shown in Figure 16, the figure illustrates the variation tendency of viable bacteria amount in 24 hours culturing process bacterial plaque amounts and bacterial plaque, the bacterial plaque amount keeps rising tendency in culturing process, and the viable bacteria amount in bacterial plaque just reaches balance substantially after 9h, the growth of this explanation bacterial plaque amount comprises that not only the viable count purpose increases, and has also comprised the increase of the peculiar extracellular products of microbial film and the accumulation of other meta-bolites to a great extent.Participate in shown in Figure 17ly, the bacterial plaque amount constantly increases at 24h, but pH approaches neutrally in culturing process, need not to add alkaline matter and regulates the pH value, and this also conforms to the growth conditions of bacterial plaque in body.This is an advantage place of the present invention.
Referring to Figure 18,19, bacterial plaque is carried out gramstaining and dilute the diversity that the dull and stereotyped cultivation of rear plumbic acetate can be observed the plaque bacteria kind.
Utilize 16s rDNA method for determining bacteria to carry out isolation identification to the bacterium in the 24h bacterial plaque, find that the bacterium kind in bacterial plaque has abundant diversity, comprise streptococcus, actinomyces, Wei Rong Pseudomonas, Fusobacterium, general Bordetella and part fusobacterium and enterobacter bacterium, in these bacteriums, in these bacteriums, Wei Rong Pseudomonas, Fusobacterium and general Bordetella all belong to absolute anaerobic bacterium, under the condition of anaerobic gas, anerobe also can obtain good growth in this model not needing to replenish, and this is also another advantage of the present invention place.Analyze relatively by PCR-DGGE, 6h, 24h cultivate bacterial plaque and form highly similar, shown in Figure 20 with natural bacterial plaque, natural salivary bacteria.And utilize scanning electron microscope that the structure of 24h bacterial plaque is observed, can observe the distinctive microcolony form of microbial film when amplifying 300 times, referring to shown in Figure 21; Can see the diversity of flora form in microbial film when amplifying 5000 times, referring to shown in Figure 22; Above evidence has proved absolutely that all this model can be good at simulating people's natural bacterial plaque.
Obviously, the above embodiment of the present invention is only for example of the present invention clearly is described, and is not to be restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here can't give all embodiments exhaustive.Everyly belong to the row that apparent variation that technical scheme of the present invention extends out or change still are in protection scope of the present invention.
Claims (17)
1. simulating device for artificial oral cavity (600), is characterized in that, it comprises:
Culture tank (601);
Multi-channel pump (602,603);
Flow chamber (604);
Drug sample solution bottle (605); With
Waste collection tank (606);
Described culture tank (601) is connected with multi-channel pump (602) by pipeline, and described multi-channel pump (602) is connected with flow chamber (604) by pipeline; Pipeline between described multi-channel pump (602) and flow chamber (604) is provided with female Luer (607), and this female Luer (607) is connected with drug sample solution bottle (605) by pipeline; Described flow chamber (604) is connected with waste collection tank (606) by pipeline, and the connecting tube between described flow chamber (604) and waste collection tank (606) is provided with multi-channel pump (603).
2. simulating device for artificial oral cavity according to claim 1, it is characterized in that: described flow chamber (604) comprises at least one flow chamber unit (604).
3. simulating device for artificial oral cavity according to claim 2, it is characterized in that: described flow chamber unit (604) comprises flow chamber chassis (609) and flow chamber upper cover (610); Described flow chamber chassis (609) upper surface is provided with buckled recess (611), the size of this buckled recess (611) and flow chamber upper cover (610) outline are complementary, described flow chamber upper cover (610) is fastened in buckled recess (611), and both mutually fasten and seal; The middle part of described buckled recess (611) is provided with liquid-flow groove (612), is equipped with at least one slide glass (613) identical or close with the tooth material in this liquid-flow groove (612); Offer a pair of through hole (614) on described flow chamber upper cover (610), be screwed with thread connection head (615) in this each through hole (614); Described flow chamber chassis (609) and flow chamber upper cover (610) are dismountable fixing by stationary installation (616).
4. simulating device for artificial oral cavity according to claim 3, it is characterized in that: be equipped with slide glass stopper protrusion (617) in the middle part of described liquid-flow groove (612), be respectively equipped with the position limiting structure (618) for the location slide glass on liquid-flow groove (612) inner side-wall of slide glass stopper protrusion (617) sidewall and correspondence.
5. simulating device for artificial oral cavity according to claim 3, it is characterized in that: offer a ring groove (619) on the buckled recess (611) of described liquid-flow groove (612) outer rim, be embedded with elastic seal ring (620) in this ring groove (619).
6. simulating device for artificial oral cavity according to claim 5, it is characterized in that: described elastic seal ring (620) is rubber seal.
7. simulating device for artificial oral cavity according to claim 3 is characterized in that: the lower surface middle part of described flow chamber upper cover (610) upwards be concaved with flow chamber chassis (609) on the upper cover groove (621) of liquid-flow groove (612) size coupling.
8. simulating device for artificial oral cavity according to claim 7, it is characterized in that: described upper cover groove (621) middle part is equipped with upper cover slide glass stopper protrusion (622) downwards, and interior slide glass stopper protrusion (617) position of this upper cover slide glass stopper protrusion (622) and described liquid-flow groove (612) is corresponding.
9. simulating device for artificial oral cavity according to claim 3 is characterized in that: described flow chamber chassis (609) and flow chamber upper cover (610) are dismountable fixing by bolt (616).
10. simulating device for artificial oral cavity according to claim 3, it is characterized in that: the described slide glass (613) identical or close with the tooth material is hydroxyapatite flag or germanium wafer.
11. simulating device for artificial oral cavity according to claim 3 is characterized in that: the material of described flow chamber chassis (609) and flow chamber upper cover (610) is polycarbonate or polymethylmethacrylate.
12. simulating device for artificial oral cavity according to claim 1 is characterized in that: the breeches joint (608) that is provided with cascade between described culture tank (601) and multi-channel pump (602).
13. simulating device for artificial oral cavity according to claim 2 is characterized in that: described flow chamber unit (604) is arranged on flow chamber anchor (623).
14. simulating device for artificial oral cavity according to claim 13 is characterized in that: described flow chamber anchor (623) comprises upper holder (624) and lower fixed frame (625); Fixed frame (624) lower surface is provided with groove (626); The upper surface of described lower fixed frame (625) is provided with the corresponding groove in groove (626) position (627) with upper holder (624) lower surface, and the groove (626,627) of described upper and lower anchor (624,625) is complementary with the size of flow chamber (604); Described upper and lower anchor (624,625) is fixed together by stationary installation (628).
15. simulating device for artificial oral cavity according to claim 13 is characterized in that: described flow chamber anchor (623) is outside equipped with constant water bath box (629).
16. simulating device for artificial oral cavity according to claim 15 is characterized in that: described constant water bath box (629) comprises casing (630) and stay-warm case (631), and described stay-warm case (631) and casing (630) coupling fasten; The lower surface of described stay-warm case (631) is provided with breach (632); The upper surface of described stay-warm case (631) is provided with handle (633); Be provided with in described casing (630) and hold thing plate (634); Described casing (630) is provided with temperature digital display (635) and adjusting knob (636).
17. according to claim 13 or 15 described simulating device for artificial oral cavities is characterized in that: the stay-warm case (631) of described flow chamber anchor (623), constant water bath box (629) is selected polymethylmethacrylate or polycarbonate material.
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| CN2010102769424A CN101948745B (en) | 2010-09-09 | 2010-09-09 | Artificial mouth simulating device |
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| CN106932229B (en) * | 2015-12-31 | 2023-06-06 | 上海烟草集团有限责任公司 | Simulated oral device for collecting buccal tobacco dissolution and application method thereof |
| CN105954476B (en) * | 2016-07-14 | 2019-02-19 | 云南中烟工业有限责任公司 | A kind of Mouthsimulator |
| CN109298146B (en) * | 2018-09-11 | 2021-09-28 | 郑州大学第一附属医院 | Experimental device for simulating oral tooth enamel demineralization and research method for tooth enamel demineralization |
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