Bionic breathing type exposure system for inhalant and application

Abstract

The invention belongs to the field of in-vitro inhalation and exposure, and particularly relates to a bionic breathing type exposure system for an inhalant and application of the bionic breathing type exposure system. The system comprises a breathing power device, a mouth branch and a nose branch, the mouth branch and the nose branch are connected with the breathing power device, the downstream of the mouth branch and the downstream of the nose branch converge to form a throat structure and then are connected with a trachea branch, and the trachea branch comprises a bronchial tree with a plurality of branches. The mouth branch and / or the nose branch are / is connected with a diverter valve used for adjusting the diverter flow of the mouth branch and the nose branch; at least one of the branches is connected with a lung unit exposure device; the lung unit exposure device comprises a microporous membrane for cell growth, a gas chamber and a liquid chamber, wherein the gas chamber and the liquid chamber are located on the two sides of the microporous membrane respectively. According to the invention, the grading and shunting environment of the bronchial tree where the cells are located can be accurately simulated, the respiratory physiological microenvironment where the cells are located can be reduced, and the exposure dose of the cells can be accurately evaluated, so that a more accurate cell exposure experiment result can be obtained.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro inhalation exposure, and in particular relates to a bionic breathing exposure system for inhaled objects and applications thereof. Background Art

[0002] Pulmonary respiratory toxicology and biological effects research in the fields of environmental pollution, occupational hazards, bioaerosols, inhalation preparations, industrial chemicals, pesticides, cosmetics and disinfectants cannot be separated from in vitro inhalation exposure studies. Traditional in vitro inhalation exposure studies mostly use liquid-liquid interface exposure methods. This method cannot objectively and real-time simulate the air-liquid interface environment of lung tissue, the periodic respiratory state and the actual physical state of the inhaled material, resulting in differences between the test results and the actual results.

[0003] In order to more realistically simulate the toxicological or biological effects of inhaled substances through breathing or special inhalation in vitro, full smoke exposure systems represented by Vitrocell and aerosol exposure systems based on bionic breathing developed in recent years have been developed.

[0004] The full smoke exposure system represented by Vitrocell consists of a smoking machine, a smoke transmission system, and an exposure chamber for air-liquid interface exposure. It is mainly used for smoke exposure, that is, for exposure experiments simulating special inhalation. When conducting smoke exposure experiments, synthetic air at a certain flow rate is continuously introduced into the exposure chamber at the air-liquid interface to simulate inhalation during breathing, and synthetic gas at a certain flow rate is continuously pumped out to simulate exhalation during breathing; a smoking machine is used to draw cigarettes to simulate the generation of smoke, and the smoke is diluted by the synthetic air simulated for inhalation and introduced into the exposure chamber to simulate special inhalation. Although the exposure experiment based on this principle can simulate the air-liquid interface environment of lung tissue, it cannot simulate the periodic breathing state and the changes in the concentration of O2 and CO2 in the inhaled and exhaled gases, nor can it reflect the transmission and distribution of the inhaled / exhaled gas by the bronchial tree in the body, as well as the exposure dose and exposure fluid mechanics changes caused by this. The bionics level of the exposure experiment still needs to be improved.

[0005] The Chinese invention patent application with application publication date October 25, 2022 and application publication number CN 115232733 A discloses an aerosol exposure system capable of realizing bionic breathing, including a breathing power device, the breathing power device is connected to a bionic oral cavity (i.e., oral branch) and a bionic nasal cavity (i.e., nasal branch) arranged in parallel, and a bionic throat and a bionic tracheobronchial tree (i.e., tracheal branch) are connected downstream of the bionic oral cavity and the bionic nasal cavity, the bronchial tree includes a first lung unit simulating the left lung and second and third lung units simulating the right lung, an intrapulmonary culture unit is arranged in the second lung unit, and the lower mouths of the first, second and third lung units are connected to a carbon dioxide supply device to simulate the increase in CO2 content during exhaled gas.

[0006] The above-mentioned aerosol exposure system further improves the bionic degree of the respiratory exposure device, simulates the changes in CO2 content during inhalation and exhalation, and can also reflect the expansion and contraction state of the lung unit under a constant exposure pressure. However, the following problems still exist: First, no specific device and method for distributing different flow rates through the mouth and nose are given. Therefore, when studying the oral and nasal inhalation of different volumes, resistances, and different compositions of inhaled substances (such as smoking), it is impossible to simulate the actual oral and nasal distribution of different volumes of inhalation and exhalation; second, although the system gives the construction method of the respiratory tract, it does not give the distribution ratio of inhalation and exhalation at different levels of the bronchial tree, which will be difficult to scientifically determine. Explore the effective exposure doses at different bronchial tree levels and their different branches; although relevant experiments and computer modeling have given certain bronchial tree diversion conditions at different levels, the modeling devices and data used in the experiments and modeling are different from the bronchial trees and exposure units used in the actual human body and system, so the exposure doses under each branch of the bronchial tree under the exposure mode of the system are not of much reference significance; thirdly, there is no specific cell culture, exposure device, and method that adapts to the diversion flow at different levels of the bronchial tree, supports cell culture, exposure, and CO2 aeration, which will not be able to scientifically characterize the real physiological microenvironment of the cells. Therefore, it is impossible to give a reliable and definite specific experimental plan and exposure dose. Summary of the invention

[0007] The purpose of the present invention is to provide a bionic breathing exposure system for inhaled materials to solve the problems that the existing exposure system cannot accurately characterize the respiratory physiological microenvironment of cells, cannot accurately simulate the oral-nasal shunt problem, cannot provide the exposure dose of different branches at levels 0-3 of the bronchial tree, and further cannot accurately evaluate the exposure dose.

[0008] The second object of the present invention is to provide an application of the above-mentioned bionic breathing exposure system for inhalation to solve the problem that the existing exposure system cannot accurately perform exposure evaluation of the respiratory exposure experiment.

[0009] In order to achieve the above first purpose, the technical solution adopted by the present invention is:

[0010] A bionic breathing exposure system for inhaled objects comprises a breathing power device and an oral branch and a nasal branch connected to the breathing power device, wherein the downstream of the oral branch and the nasal branch merge to form a throat structure and then are connected to a tracheal branch, wherein the tracheal branch comprises a bronchial tree having multiple branches, and the oral branch and / or the nasal branch are connected to a shunt valve for adjusting the shunt flow of the oral branch and the nasal branch; at least one of the multiple branches is connected to a lung unit exposure device adapted to the shunt ratio of the corresponding branch or adapted to the target shunt ratio; the lung unit exposure device comprises a microporous membrane for cell growth and a gas chamber and a liquid chamber respectively located on both sides of the microporous membrane, wherein the liquid chamber is provided with a CO2 exhalation device and a refinement baffle for refining the CO2 exhaled by the CO2 exhalation device.

[0011] The present invention belongs to an improved invention creation. The shunt valves on the oral branch and the nasal branch can accurately control the shunt flow of the gas / aerosol inhaled through the mouth and nose. The lung unit exposure device is adapted to the corresponding branch shunt ratio or the target shunt ratio to accurately evaluate the exposure dose of bronchial tree branches of different levels. The refined baffle is used to provide uniform, dense, and fine CO2 to meet the physiological environment of CO2 during exhalation. The system truly restores the real respiratory physiological microenvironment of cells, and combined with the precise shunt of the mouth and nose and the shunt ratio of the lung unit exposure device, it can accurately evaluate the exposure dose of cells, thereby obtaining more accurate cell exposure experimental results.

[0012] Preferably, the diversion ratio of the diversion valve is 100% oral branch, 100% nasal branch or mixed diversion, and the mixed diversion is that the oral branch and the nasal branch are diverted according to a set ratio.

[0013] Preferably, the diverter valve is a rotary diverter valve, a pressure pipe diverter valve, a relative diverter valve or a pneumatic diverter valve. These diverter valves mainly adjust the airflow distribution ratio of the oral branch and the nasal branch by changing the ratio of the inner diameter cross-sectional area of ​​the path through which the oral branch and the nasal branch airflow pass, or the suction resistance of the path through which the oral branch and the nasal branch airflow pass.

[0014] Preferably, the multiple branches of the bronchial tree determine the branch shunt ratio by measuring the flow rate, and the target shunt ratio deviates from the branch shunt ratio to simulate different signs; a lung unit exposure device with a matching gas chamber volume is selected according to the branch shunt ratio and the target shunt ratio, and the gas chamber volume increases or decreases with breathing.

[0015] Preferably, the lung unit exposure device includes a culture unit, which includes the microporous membrane, a gas chamber and a liquid chamber. The top wall of the gas chamber is a soft elastic material, and the soft elastic material expands during inhalation and collapses during exhalation to cause the volume of the gas chamber to change with breathing.

[0016] More preferably, the lung unit exposure device comprises a constant temperature water bath, and the culture unit is arranged in the constant temperature water bath.

[0017] In order to achieve the above second purpose, the technical solution adopted by the present invention is:

[0018] The above-mentioned bionic breathing exposure system is used in the bionic breathing exposure experiment. Cells are inoculated and cultured on the microporous membrane of the lung unit exposure device to construct a cell layer at the air-liquid interface. Then, the exposure experiment is carried out in the breathing mode. The exposure dose is determined according to the shunt ratio of the oral branch and the nasal branch, and the proportion of the airflow entering the lung unit exposure device, and the exposure evaluation is carried out at the exposure dose.

[0019] The application of the bionic breathing exposure system of the present invention in the bionic breathing exposure experiment determines the exposure dose by combining the inhaled air volume and the shunt ratio of the oral branch and the nasal branch, the airflow proportion of the terminal branch of the bronchial tree connected to the lung unit exposure device, and the air intake volume entering the lung unit exposure device. For the first time, the exposure dose of cells in the bionic breathing state is obtained, creating basic conditions for accurate cell exposure evaluation.

[0020] Preferably, the exposure dose is calculated as follows:

[0021] The number of puffs of inhaled material is counted: exposure dose (puffs) = number of puffs of inhaled material N 吸入物口 ×Diverter valve retention coefficient S 保 ×The proportion of airflow entering the exposure chamber V 进装置 / V 吸 ;

[0022] When the inhaled material is measured by flow rate: exposure dose (mL) = inhaled material flow rate V 口 / 鼻特殊吸 ×Diverter valve retention coefficient S 保 ×The proportion of airflow entering the exposure chamber V 进装置 / V 吸 .

[0023] Preferably, the diverter valve retention coefficient S is determined according to the loss of the suction flow through the diverter valve. 保 , the diverter valve retention coefficient S 保 The range is 0 to 100%.

[0024] Preferably, the breathing pattern is selected from natural breathing, special breathing containing the inhalant to be studied, and a combination of natural breathing and the special breathing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A schematic diagram of the structure of a bionic breathing exposure system for inhaled objects according to an embodiment of the present invention;

[0026] Figure 2 for Figure 1 Schematic diagram of the structure of the main components of the mid-lung unit exposure device;

[0027] Figure 3 A schematic structural diagram of a bionic breathing exposure system for inhaled objects according to another embodiment of the present invention;

[0028] Among them, 1-respiratory power device; 2-oral branch; 3-nasal branch; 4-tracheal branch; 5-lung unit exposure device; 6-CO2 exhalation device; 7-computer controller; 8-artificial lip; 9-first solenoid valve; 10-diverter valve; 11-first flow sensor; 12-generator for inhaled material; 13-second solenoid valve; 14-upper pharyngeal tube; 15-lower pharyngeal tube, 150-third solenoid valve; 16-bronchial tree; 17-left branch unit; 18-right branch unit; 19-bionic alveoli, 20-constant temperature water bath; 21-culture Unit; 22-culture tank; 23-sealing cover; 24-cell chamber; 25-microporous membrane; 26-breathing circuit; 27-first connecting pipe; 28-second flow sensor; 29-CO2 exhalation circuit; 30-refining baffle; 31-tank body; 32-tank cover; 33-water inlet pipe; 34-water outlet pipe; 35-water bath; 36-CO2 exhalation power device; 37-gas storage bin; 38-CO2 cylinder; 39-synthetic air cylinder; 40-CO2 outlet pipe; 41-second connecting pipe; 42-fourth solenoid valve; 43-fifth solenoid valve. DETAILED DESCRIPTION

[0029] The technical concept of the present invention is to construct a multi-level, multi-branch terminal bronchial tree, clarify the airflow proportion of different branch terminals, and connect different branch terminals to either alveoli that match the airflow proportion, or to lung unit exposure devices that can simulate the air-liquid interface environment in which cells are located. Combined with oral and nasal shunts, the different exposure environments of cells can be truly restored, and the exposure dose can be accurately measured, thereby conducting exposure evaluation more realistically and accurately.

[0030] By further refining the baffles and constant temperature water bath settings in the lung unit exposure device, the constant temperature environment of cells in the body and the process of gas smoothly and evenly passing through the gas-blood barrier (i.e., the gas-liquid interface for cell growth) were completely restored, further improving the device's degree of bionics of the breathing process.

[0031] The implementation process of the present invention is described in detail below in conjunction with specific embodiments.

[0032] 1. Specific embodiments of the bionic breathing exposure system for inhaled objects of the present invention

[0033] Example 1

[0034] The bionic breathing exposure system for inhaled objects of this embodiment includes a breathing power device 1, an oral branch 2, a nasal branch 3, a tracheal branch 4, a lung unit exposure device 5, a CO2 exhalation device 6 and a computer controller 7.

[0035] The respiratory power device 1 provides respiratory power for the oral branch 2, the nasal branch 3, and the tracheal branch 4, and is specifically in the form of an electric syringe. The computer controller 7 controls the movement of the syringe of the respiratory power device 1. The movement of the syringe includes pulling the piston outward to allow the gas / aerosol to enter the syringe, and pushing the piston inward to allow the gas / aerosol to be discharged from the syringe, that is, providing power for the inhalation and exhalation process of breathing. The computer controller 7 can set the change of the gas / aerosol flow waveform (respiratory wave, bell wave, square wave, sine wave, triangle wave, etc.), push-pull time, push-pull time interval, etc. Specifically, the driving syringe range is 0-1000mL, accuracy 0.05-5mL, frequency 0-100 times / min, push / pull time 0-60s, accuracy 0.01s, push-pull interval 0-999min, accuracy 0.01s.

[0036] Oral branch 2 mainly simulates the respiratory path of oral inhalation. Figure 1 The device comprises an oral inhalation pipeline and an artificial lip 8 connected to the inlet of the oral inhalation pipeline, and the oral inhalation pipeline is provided with a first electromagnetic valve 9, a diverter valve 10 and a first flow sensor 11. The artificial lip 8 can be connected to a generator 12 for inhaled objects, such as cigarettes.

[0037] Nasal branch 3 mainly simulates the breathing path of nasal inhalation. Figure 1 The nasal inhalation pipeline is included, and a second solenoid valve 13 is arranged on the nasal inhalation pipeline.

[0038] The downstream of the oral branch 2 and the nasal branch 3 merge to form a pharyngeal structure and then connect to the tracheal branch 4. The pharyngeal structure is composed of an upper pharyngeal tube 14 and a lower pharyngeal tube 15. The oral branch 2 and the nasal branch 3 are both connected to one end of the upper pharyngeal tube 14, and the other end of the upper pharyngeal tube 14 is connected to the respiratory power device 1 and the lower pharyngeal tube 15. The tracheal branch 4 simulates the situation where the lower respiratory tract of the human body is connected to the lungs, that is, the gas or aerosol enters the tracheal branch 4 after being inhaled from the mouth and nose and completes gas exchange in the lungs. The above oral branch 2, nasal branch 3, tracheal branch 4, upper pharyngeal tube 14, and lower pharyngeal tube 15 completely simulate the upper and lower respiratory tract conditions of the human body, creating good conditions for achieving accurate simulation of in vivo exposure. A third solenoid valve 150 is provided on the lower pharyngeal tube 15.

[0039] The bronchial tree 16 of the tracheal branch 4 includes a left branch unit 17 and a right branch unit 18. Figure 1In the figure, the left branch unit 17 and the right branch unit 18 both include two-level branches, so that after adding the main branches that form the left branch unit 17 and the right branch unit 18, the bronchial tree 16 forms a three-level branch structure, which is closer to the hierarchical structure of the bronchial tree 16 in the body. The bronchial tree 16 can be constructed and generated by computer tomography imaging technology and 3D printing technology. There are 10 terminal branches of the left branch unit 17 and 8 terminal branches of the right branch unit 18. Among all the terminal branches, one is connected to the lung unit exposure device 5, and the remaining 17 are connected to the bionic alveoli 19, wherein the bionic alveoli 19 are spherical and can expand and contract (i.e., swell when inhaling and concave when exhaling), and the expandable volume ratio is matched with the airflow distribution ratio of each terminal branch of the bronchial tree 16, so that the air pressure of each lung unit exposure device is consistent when expanding, and the airflow distribution ratio is ensured not to change with the same proportion expansion of each lung unit.

[0040] The airflow distribution ratio of each final branch of the left branch unit 17 is Ln, and the airflow distribution ratio of each final branch of the right branch unit 18 is Rn. The specific airflow distribution ratio of Ln and Rn can be determined by the flow measurement method, that is, a flow sensor is connected to the end of each branch, and the flow ratio measured by the flow sensor at the end of each branch of different grades under the same total air intake is obtained by examining the flow ratio measured by the flow sensor at the end of each branch of different grades under the same total air intake. The specific distribution ratio is shown in Table 1 below. The distribution ratio of level 2, level 1, and level 0 is the sum of the branches of the three-level bronchial tree corresponding to each branch.

[0041] Table 1 Airflow distribution ratio of each terminal branch of the bronchial tree

[0042]

[0043]

[0044] Combined with the above-mentioned setting of the bionic alveoli 19 adapting to the shunt ratio of the terminal branches of the bronchial tree 16, the increase of the bionic alveoli 19 does not cause the change of the shunt ratio of the terminal branches of the bronchial tree 16, so as to better simulate the transmission, distribution and shunt of the inhaled matter by the healthy human body, and further improve the quantification and bionic simulation degree of the airflow through the bronchial tree 16. When studying different pathological signs (such as right lung atelectasis), the shunt ratio of the terminal branches of the bronchial tree 16 can also be adjusted by the bionic alveoli 19, so as to achieve the change of the shunt ratio of the terminal branches of the bronchial tree 16 caused by the increase of the bionic alveoli 19, so as to better simulate the transmission, distribution and shunt of the inhaled matter by the human body with specific pathological signs, and further improve the quantification and bionic simulation degree of the airflow through the bronchial tree 16.

[0045] The lung unit exposure device 5 simulates the process of gas exchange of alveolar cells under breathing state. It generally carries epithelial cells and can simulate the air-liquid interface environment of epithelial cells and the expansion or contraction of alveoli themselves with breathing. Specifically, Figure 2 As shown, the lung unit exposure device 5 includes a constant temperature water bath 20 and a culture unit 21 installed in the constant temperature water bath 20. The culture unit 21 includes a culture tank 22 and a sealing cover 23 that seals the culture tank 22. A cell chamber 24 is arranged in the culture tank 22. The cell chamber 24 can be a commercialized Transwell chamber, and the bottom wall of the cell chamber 24 is a microporous membrane 25 for cell growth. The culture tank 22 and the sealing cover 23 enclose a closed culture unit cavity. The lower side of the microporous membrane 25 is in contact with the culture medium, and the upper side is in contact with the air. The cells grown on the microporous membrane 25 are in an air-liquid interface environment. The sealing cover 23 is a hemispherical shell and is made of elastic material, so that the culture unit cavity can expand and contract. A breathing tube 26 is arranged on the sealing cover 23, and the breathing tube 26 is connected to the terminal branch of the bronchial tree 16 through a first connecting tube 27. A second flow sensor 28 is arranged on the first connecting tube 27.

[0046] A CO2 exhalation line 29 is connected to the liquid side of the microporous membrane 25 of the culture tank 22, and the CO2 exhalation line 29 is connected to the CO2 exhalation device 6 to exhale a certain concentration of CO2 from the liquid side to the cell layer on the microporous membrane 25 to simulate the high concentration of CO2 in the exhaled air. A thinning baffle 30 is also provided on the liquid side of the microporous membrane 25 of the culture tank 22. The thinning baffle 30 and the microporous membrane 25 are arranged along the horizontal direction, and the microporous membrane 25 is located within the plate surface range of the thinning baffle 30. The thinning baffle 30 can thin the CO2 exhaled from the CO2 exhalation line 29 to generate uniform, dense, and fine CO2 bubbles to simulate the gas exchange process of the gas-blood barrier, and prevent the larger CO2 gas mass directly exhaled from the CO2 exhalation line 29 from causing damage to the cells or destroying the gas-liquid interface environment where the cells are located.

[0047] The constant temperature water bath 20 is used to simulate the constant temperature environment of the human body, and includes an open tank body 31 and a tank cover 32 sealed with the tank body 31. The tank body 31 is connected with a water inlet pipe 33 and a water outlet pipe 34. The water inlet pipe 33 and the water outlet pipe 34 are connected to a water bath 35 to provide 37°C constant temperature water. In addition, the sealing cover 23 of the culture unit 21 is arranged on the tank cover 32, and the sealing cover 23 of the culture unit 21 and the culture tank 22 are provided with an adaptive sealing structure (such as a sealing groove and a sealing ring). When the tank body 31 and the tank cover 32 are correspondingly covered and sealed, the sealing cover 23 of the culture unit 21 and the culture tank 22 are also sealed synchronously.

[0048] The CO2 exhalation device 6 generates a certain concentration of CO2 and exhales it through the CO2 exhalation pipeline 29. The CO2 exhalation device 6 includes a CO2 exhalation power device 36, a gas storage bin 37, a CO2 cylinder 38, and a synthetic air cylinder 39. The CO2 cylinder 38 and the synthetic air cylinder 39 are connected to the gas storage bin 37. The gas storage bin 37 is connected to a CO2 outlet pipe 40. The CO2 outlet pipe 40 is connected to the CO2 exhalation power device 36. The CO2 exhalation power device 36 is connected to the CO2 exhalation pipeline 29 through a second connecting pipe 41. The CO2 exhalation power device 36 uses an electric syringe consistent with the breathing power device 1. The CO2 that meets the requirements in the gas storage bin 37 enters the electric syringe through the CO2 outlet pipe 40, and then pushes the piston of the electric syringe to push the CO2 that meets the requirements into the CO2 exhalation pipeline 29.

[0049] The CO2 exhalation device 6 is connected to the computer controller 7, and the concentration of generated CO2 can be set on the computer controller 7. The CO2 cylinder 38 and the synthetic air cylinder 39 can pass the corresponding gas into the gas storage bin 37 according to the computer command. The gas sensor on the gas storage bin 37 feedbacks the CO2 concentration. When the CO2 concentration in the gas storage bin 37 reaches the set concentration, the intake is stopped. The fourth solenoid valve 42 and the fifth solenoid valve 43 are respectively arranged on the CO2 outlet pipe 40 and the second connecting pipe 41, which can be used in conjunction with the CO2 exhalation power device 36 to meet the breathing rhythm requirements.

[0050] In other implementation situations, the CO2 cylinder 38 and the synthetic air cylinder 39 can both be standard gas cylinders with certain CO2 and O2 concentrations, and the two standard gas cylinders are connected to the gas storage bin 37. The gas storage bin is in a vacuum state, and by setting the CO2 concentration (consistent with the CO2 concentration of the standard gas cylinder) and the pressure, the gas in the two standard gas cylinders enters the gas storage bin 37. When the CO2 concentration and pressure reach the expected level, the gas storage bin 37 contains CO2 and O2 that are consistent with the standard gas concentration. Then, a certain concentration of CO2 and O2 is exhaled into the exposure unit according to the set exhalation rhythm.

[0051] The first, second, third, fourth and fifth solenoid valves can be compression valves, and the opening and closing of the compression valves are controlled by the computer controller 7. The first and second flow sensors can be pipeline flow sensors, both ends of which are connected to the pipeline to be tested to obtain the flow through the pipeline to be tested, so as to calculate the exposure dose. The first flow sensor 11 is used to obtain the gas flow V entering through the mouth. 口 The second flow sensor 28 is used to obtain the inhaled gas flow rate V entering the lung unit exposure device 5 肺 The sensing signals of the first and second flow sensors are received by the computer controller 7, and the flow waveform curve can be displayed on the computer and the flow value can be obtained.

[0052] Example 2

[0053] The bionic breathing exposure system for inhalation of this embodiment is as follows: Figure 3 As shown, it includes an oral branch 2, a nasal branch 3, a tracheal branch 4, a respiratory power device 1, a lung unit exposure device 5 and a CO2 exhalation device 6. Among them, the implementation of the tracheal branch 4, the respiratory power device 1, the lung unit exposure device 5 and the CO2 exhalation device 6 is the same as that of Example 1, and the implementation of the oral branch 2 and the nasal branch 3 is introduced below.

[0054] Figure 3 In the embodiment, the oral branch 2 includes an oral inhalation pipeline and an artificial lip 8, and a first electromagnetic valve 9 and a first flow sensor 11 are arranged on the oral inhalation pipeline. The artificial lip 8 can be connected to a generator 12 for inhaled materials, such as cigarettes. The nasal branch 3 includes a nasal inhalation pipeline, and a second electromagnetic valve 13 and a diverter valve 10 are arranged on the nasal inhalation pipeline. The diverter valve 10 adopts a fixed flow diverter valve.

[0055] The above-mentioned diverter valve 10 is mainly set to adjust the diversion ratio of the oral branch 2 and the nasal branch 3. When the inhalation volume is constant (for example, the single inhalation volume of the respiratory power device 1 is controlled to be 500mL), the mouth or nose can be adjusted as the main inhalation, thereby simulating different inhalation states.

[0056] The diverter valve 10 can be a non-fixed flow diverter valve or a fixed flow diverter valve. In different implementation situations, it can be set on the oral branch 2 and the nasal branch 3 to achieve inhalation and exhalation control in different diversion situations through the mouth, nose, and mouth + nose. The diverter valve 10 controls the ratio of the airflow flowing through the oral and nasal tubes by controlling the cross-sectional area ratio of the oral and nasal tubes and / or using the complexity of the internal path to cause a change in the suction resistance.

[0057] When the gas or aerosol shunted through the mouth and nose is not easy to block the shunt pipeline, the shunt valve 10 can be installed on the branch pipeline of the mouth and nose. When the gas or aerosol shunted through the mouth and nose is easy to block the shunt pipeline, the shunt valve 10 needs to be installed on the branch pipeline of the non-inhaled gas or aerosol that is easy to block. When the oral and nasal branch pipelines both shunt the gas or aerosol that is easy to block the shunt pipeline, fixed flow shunt valves are used on both the oral and nasal branch pipelines.

[0058] Furthermore, when the cigarette smoke is diverted through the mouth and nose, the cigarette is connected to the mouth branch 2 pipeline, and the nose branch 3 pipeline is connected to the atmosphere. When the mouth branch 2 pipeline is connected to the non-fixed flow diverter valve, the retention coefficient S of different types of non-fixed flow diverter valves is 保 They are shown in Table 2 below. A fixed flow diverter valve can also be used. Since the internal structure of the fixed diverter valve is a straight-through pipeline or is not connected to the gas / aerosol that is easy to block the diverter pipeline, the loss can be ignored and the retention coefficient S 保 Counted as 100%.

[0059] Table 2 Retention coefficient S of different non-fixed flow diverter valves 保

[0060]

[0061]

[0062] The diverter valve can be selected according to the oral and nasal diversion conditions and aerosol blockage conditions required by the experiment. The lung unit exposure device 5 and the bronchial tree 16 are selected according to the number of exposure unit connections and the exposure level of the bronchial tree 16 to be studied. The selection and matching of the lung unit exposure device and the bronchial tree level branch are generally carried out according to the following principles: when the distribution ratio at the end of the bronchial tree 16 is not changed, the end of each branch should be connected to an exposure device and / or a bionic lung unit whose gas chamber has the same distribution ratio as the bronchial tree; when the distribution ratio at the end of the bronchial tree 16 is changed, the end of each branch should be connected to an exposure device and / or a bionic lung unit whose gas chamber has the target distribution ratio; when the airflow entering the exposure device does not cause cell damage, the volume of the air chamber of the exposure device should be less than 1.6 times the airflow entering the exposure device; when investigating the cell damage caused by the airflow entering the exposure device, the volume of the air chamber of the exposure device is selected according to the cell survival rate caused by the preliminary experiment.

[0063] The bionic breathing exposure system of the above embodiment can be used to implement exposure experiments with different breathing modes and different signs. The cells and culture medium in the exposure device can be collected according to the purpose, or the cells in the chamber can be further incubated or processed, so as to make an exposure evaluation. The following is an explanation of the typical breathing mode based on the above bionic breathing exposure system:

[0064] (1) One-breath mode

[0065] a. Gas / aerosol enters the respiratory dynamics device through the oral branch 2 tube and / or the nasal branch 3 tube 1: Set the respiratory waveform, inhalation flow V 吸 mL, inhalation time S 吸 s, select the airflow distribution coefficient of the oral cavity and nasal cavity (such as oral V 口吸 mL, nasal V 鼻吸 mL) corresponding to the diverter valve, when the oral and nasal flow rates are greater than 0, the oral and nasal communication solenoid valves (the first and second solenoid valves) are set to open at this stage, and the respiratory power device 1 is in the intake mode and enters the next stage waiting time S a等 s.

[0066] b1 and b2 are synchronized.

[0067] b1. Gas / aerosol enters the bronchial tree 16 through the respiratory power device 1 and enters the lung unit exposure device 5: set the respiratory waveform and push the flow rate V 推 mL, push time S推 s, the solenoid valve (third solenoid valve) of the pipeline connected to the bronchial tree 16 lung unit exposure device 5 is opened at this stage, the syringe is in the push mode, and the flow rate entering the lung unit exposure device 5 is monitored by the flow sensor and the computer controller 7, and the flow rate is V 进装置 mL, wait time S to enter the next stage b1等 s.

[0068] b2. CO2 enters the CO2 exhalation power device 36: Set the CO2 concentration of the gas storage bin 37, set the breathing waveform, and the flow rate V of the CO2 entering CO2进 mL, entry time S CO2进 s, the passage connecting the gas storage chamber 37 to the CO2 exhalation power device 36 is opened at this stage, the syringe is in the air intake mode, and the next stage waits for a time S b2等 s.

[0069] c1 and c2 are synchronized.

[0070] c1. CO2 enters the lung unit exposure device 5: Set the breathing waveform and the exhaled CO2 flow rate V CO2呼 mL, exhalation time S CO2呼 s, the passage connecting the CO2 exhalation power device 36 to the CO2 exhalation pipeline 29 is opened at this stage, the syringe is in the air pushing mode, and the next stage waits for a time S c1等 s.

[0071] c2. Lung unit exposure device 5 gas / aerosol is exhaled through the bronchial tree 16 to the respiratory power device 1: set the respiratory waveform, the total exhaled flow rate V of the bronchial tree 16 支呼 mL, the exhaled flow rate from the exposure device is V 装置呼 mL, exhalation time S 装置呼 s, the lung unit exposure device 5 is connected to the exhalation line of the bronchial tree 16, the line between the bronchial tree 16 and the respiratory power device 1 is opened at this stage, the syringe is in the intake mode, and the next stage is entered. The waiting time S c2等 s.

[0072] d. The gas / aerosol in the breathing power device 1 is exhaled through the mouth and / or nose: set the breathing waveform, exhaled flow rate V 呼出 mL, exhalation time S 呼 s, select the airflow distribution coefficient of the oral cavity and nasal cavity (such as oral V 口吸 mL, nasal V 鼻吸 mL), when the oral and nasal flow rates are greater than 0, the oral and nasal communication solenoid valves are set to open at this stage, the syringe is in the air push mode, and the next set of actions is entered with a waiting time of S 组1等循 s.

[0073] Among them, V 吸 mL=V口特殊吸 mL+V 口吸 mL+V 鼻吸 mL≥V 推 mL≥V 进装置 mL; S 推 s≥S CO2进 s;S b1等 s+S 推 s=S CO2进 s+S b2等 s; V CO2进 mL≥V CO2呼 mL; S CO2呼 =S c1等 ; S c1等 =S c2等 ; V 装置呼 mL=V CO2呼 mL+V 进装置 mL; V 支呼 mL≥V 呼出 mL≥V 吸 mL+V CO2呼 mL.

[0074] (2) A special oral inhalation breathing pattern (special inhalation such as cigarette smoke inhalation)

[0075] a. Gas / aerosol enters the respiratory dynamic device 1 through the oral branch 2 tube and / or the nasal branch 3 tube: Set the respiratory waveform, inhalation flow V 吸 mL, inhalation time S 吸 s, select the airflow distribution coefficient of the oral cavity and nasal cavity (such as oral V 口特殊吸 mL、V 口吸 mL, nasal V 鼻吸 mL) corresponding to the diverter valve, when the oral and nasal flow rates are greater than 0, the oral and nasal communication solenoid valves are set to open at this stage, the respiratory power device 1 is in the intake mode, and enters the next stage waiting time S a等 s.

[0076] b1 and b2 are synchronized.

[0077] The rest are the same as (1) a group of breathing patterns b1, b2, c1, c2, d.

[0078] Among them, V 吸 mL=V 口特殊吸 mL+V 口吸 mL+V 鼻吸 mL≥V 推 mL≥V 进装置 mL; S 推 s≥S CO2进 s; S b1等 s+S 推 s=S CO2进 s+Sb2等 s; V CO2进 mL≥V CO2呼 mL; S CO2呼 s=S c1等 s; S c1等 s=S c2等 s; V 装置呼 mL=V CO2呼 mL+V 进装置 mL; V 支呼 mL≥V 呼出 mL≥V 吸 mL+V CO2呼 mL.

[0079] (3) A special nasal inhalation breathing pattern (special inhalation such as drug inhalation)

[0080] a. Gas / aerosol enters the respiratory dynamic device 1 through the oral branch 2 tube and / or the nasal branch 3 tube: Set the respiratory waveform, inhalation flow V 吸 mL, inhalation time S 吸 s, select the airflow distribution coefficient of the oral cavity and nasal cavity (such as oral V 口吸 mL, nasal V 鼻特殊吸 mL、V 鼻吸 mL) corresponding to the diverter valve, when the oral and nasal flow rates are greater than 0, the oral and nasal communication solenoid valves are set to open at this stage, the respiratory power device 1 is in the intake mode, and enters the next stage waiting time S a等 s.

[0081] b1 and b2 are synchronized.

[0082] The rest are the same as (1) a group of breathing patterns b1, b2, c1, c2, d.

[0083] Among them, V 吸 mL=V 鼻特殊吸 mL+V 口吸 mL+V 鼻吸 mL≥V 推 mL≥V 进装置 mL; S 推 s≥S CO2进 s; S b1等 s+S 推 s=S CO2进 s+S b2等 s; V CO2进 mL≥V CO2呼 mL; S CO2呼 s=S c1等 s; S c1等 s=S c2等 s; V 装置呼 mL=V CO2呼 mL+V 进装置 mL; V支呼 mL≥V 呼出 mL≥V 吸 mL+V CO2呼 mL.

[0084] (4) One-time special inhalation breathing mode through the mouth or nose (special inhalation such as drug inhalation)

[0085] a. Gas / aerosol enters the respiratory dynamic device 1 through the oral branch 2 tube and / or the nasal branch 3 tube: Set the respiratory waveform, inhalation flow V 吸 mL, inhalation time S 吸 s, select the airflow distribution coefficient of the oral cavity and nasal cavity (such as oral V 口特殊吸 mL、V 口吸 mL, nasal V 鼻特殊吸 mL、V 鼻吸 mL) corresponding to the diverter valve, when the oral and nasal flow rates are greater than 0, the oral and nasal communication solenoid valves are set to open at this stage, the respiratory power device 1 is in the intake mode, and enters the next stage waiting time S a等 s.

[0086] b1 and b2 are synchronized.

[0087] The rest are the same as (1) a group of breathing patterns b1, b2, c1, c2, d.

[0088] Among them, V 吸 mL=V 口特殊吸 mL+V 鼻特殊吸 mL+V 口吸 mL+V 鼻吸 mL≥V 推 mL≥V 进装置 mL; S 推 s≥S CO2进 s; S b1等 s+S 推 s=S CO2进 s+S b2等 s; V CO2进 mL≥V CO2呼 mL; S CO2呼 s=S c1等 s; S c1等 s=S c2等 s; V 装置呼 mL=V CO2呼 mL+V 进装置 mL; V 支呼 mL≥V 呼出 mL≥V 吸 mL+V CO2呼 mL.

[0089] (5) Multiple sets of breathing or special inhalation actions in series (special inhalation such as cigarette smoke inhalation)

[0090] After the specific parameters of the breathing or special inhalation actions involved are configured, the special inhalation time interval and the breathing time interval are matched, and multiple groups are executed according to the order of the special inhalation times and the breathing times, and the multiple groups are set in series. For example, one smoking action + multiple natural breathing actions can be combined into one group, and multiple groups can be repeated continuously to simulate the overall exposure process of the human body during smoking.

[0091] The exposure dose in the above exposure experiment process can be calculated as follows:

[0092] (1) Counting the number of puffs of inhaled material: Exposure dose (puffs) = Number of puffs of inhaled material (N 吸入物口 )× diverter valve retention coefficient (S 保 )×the proportion of airflow entering the exposure chamber (V 进装置 / V 吸 )=Number of inhalation ports (N 吸入物口 )× diverter valve retention coefficient (S 保 )×the proportion of airflow entering the exposure chamber (V 吸 ×R n or V 吸 ×L n ).

[0093] (2) Measure the flow rate of the inhaled material: Exposure dose (mL) = inhaled material flow rate (V 口 / 鼻特殊吸 )× diverter valve retention coefficient (S 保 )×the proportion of airflow entering the exposure chamber (V 进装置 / V 吸 )=Number of inhalation ports (V 口 / 鼻特殊吸 )× diverter valve retention coefficient (S 保 )×the proportion of airflow entering the exposure chamber (V 吸 ×R n or V 吸 ×L n ).

[0094] (3) The mass of the inhaled substance is measured as follows: Exposure dose (mg) = mass per puff (M 每口 )×exposure dose (mouth) = mass per milliliter (M 每毫升 )×exposure dose (mL).

[0095] 2. Specific Examples of Application of the Bionic Breathing Exposure System for Inhaled Objects of the Present Invention in Bionic Breathing Exposure Experiments Example 3

[0096] The application of the bionic breathing exposure system for inhaled objects of this embodiment in the bionic breathing exposure experiment uses the bionic breathing exposure system of the above-mentioned embodiment 2, and is specifically described as follows:

[0097] 1) Mode setting

[0098] Select the special oral inhalation mode. According to the special inhalation of 35mL of smoke inhaled through the mouth and the situation of smoke blocking the pipeline, select the nasal branch 3 pipeline to connect the diverter valve 10 to adjust the oral and nasal diversion. According to the breathing parameters (breathing 500mL, exhaling 520mL, breathing waiting 1s, inhalation and exhalation time are both 2s) and smoking parameters (puffing 0, 84, and 168 puffs of smoke respectively, the puffing volume is 35mL, the puffing time is 2s, and the puffing interval is 55s), set the following mode parameters:

[0099] (1) One-breath mode

[0100] a. Gas / aerosol enters the respiratory system through the oral cavity and / or nasal cavity 1: Set the respiratory waveform and inhalation flow rate V 吸 mL (500mL), inhalation time S 吸 s(2s), select the airflow distribution coefficient of the oral cavity and nasal cavity (such as oral V 口吸 0mL, nasal V 鼻吸 500mL), when the oral and nasal flow rates are greater than 0, the oral and nasal communication solenoid valves are set to open at this stage, the syringe is in the air intake mode, and the next stage waits for a time S a等 s(1s).

[0101] b1 and b2 are synchronized.

[0102] b1. Gas / aerosol enters the bronchial tree 16 through the respiratory power device 1 and enters the lung unit exposure device 5: set the respiratory waveform and push the flow rate V 推 mL(500mL), pushing time S 推 s(2s), the solenoid valve of the pipeline connecting the bronchial tree 16 to the lung unit exposure device 5 is opened at this stage, the syringe is in the push mode, and the flow rate entering the lung unit exposure device 5 is V 进装置 mL (20mL), enter the next stage waiting time S b1等 s(1s).

[0103] b2. CO2 enters the CO2 exhalation power device 36: The CO2 concentration (4%) in the gas storage bin 37 is set in advance, and the CO2 of the set concentration is mixed in the gas storage bin 37 at this stage. Set the breathing waveform, the flow rate V of the CO2 entering CO2进 mL (20 mL), entry time S CO2进 s(2s), the passage connecting the gas storage chamber 37 to the CO2 exhalation power device 36 is opened at this stage, the syringe is in the air intake mode, and the next stage waits for a time S b2等 s(1s).

[0104] c1 and c2 are synchronized.

[0105] c1. CO2 enters the lung unit exposure device 5: Set the breathing waveform and the exhaled CO2 flow rate V CO2呼 mL (20 mL), exhalation time S CO2呼 s(2s), the passage connecting the CO2 exhalation power device 36 to the CO2 exhalation pipeline 29 is opened at this stage, the syringe is in the air pushing mode, and the next stage waits for a time S c1等 s(1s).

[0106] c2. Lung unit exposure device 5 gas / aerosol is exhaled into the syringe through the bronchial tree 16: set the breathing waveform, bronchial tree exhaled flow V 支呼 mL (520 mL), the exhaled flow rate from the exposure device is V 装置呼 mL (40 mL), exhalation time S 装置呼 s(2s), the exhalation line of the lung unit exposure device 5 is connected to the bronchial tree 16. The line connecting the bronchial tree 16 and the lung unit exposure device 5 is opened at this stage, the syringe is in the air intake mode, and the next stage is entered. The waiting time S c2等 s(1s).

[0107] d. The gas / aerosol in the breathing power device 1 is exhaled through the mouth and / or nose: set the breathing waveform, exhaled flow rate V 呼出 mL (520 mL), exhalation time S 呼 s(2s), select the airflow distribution coefficient of the oral cavity and nasal cavity (such as oral V 口吸 mL, nasal V 鼻吸 mL), when the oral and nasal flow rates are greater than 0, the oral and nasal communication solenoid valves are set to open at this stage, the syringe is in the air push mode, and the next set of actions is entered with a waiting time of S 组1等循 s(1s).

[0108] (2) Breathing pattern of a single cigarette

[0109] a. Smoke enters the respiratory power device 1 through the oral branch 2 pipe and air enters the nasal branch 3 pipe: Set the respiratory waveform and the inhalation flow V 吸 mL (500mL), inhalation time S 吸 s(2s), select the airflow distribution coefficient of the oral cavity and nasal cavity (such as oral V 吸烟 mL(35mL), V 口吸 0mL, nasal V 鼻吸 465mL), when the oral and nasal flow rates are greater than 0, the oral and nasal communication solenoid valves are set to open at this stage, the syringe is in the air intake mode, and the next stage waits for a time S a等 s(1s).

[0110] b1 and b2 are synchronized.

[0111] The rest are the same as (1) a group of breathing patterns b1, b2, c1, c2, d.

[0112] (3) Multiple sets of natural breathing or smoking breathing movements in series

[0113] One special smoking action connected to 10 natural breathing actions is a set of cycle actions. When exposing 0 puffs of cigarettes, insert a non-lit cigarette at the end of the mouth, and perform 168 sets of cycle actions; when exposing 84 puffs of cigarettes, insert a lit cigarette at the end of the mouth, and perform 84 sets of cycle actions; when exposing 168 puffs of cigarettes, insert a lit cigarette at the end of the mouth, and perform 168 sets of cycle actions.

[0114] (2) Device connection: According to Figure 2 Connect the device and prepare the cell chamber 24 to be exposed. Connect the inhalation generator 12 (cigarette) to the oral branch 2, and connect the fixed flow proportional valve to the nasal branch 3. The total inhalation volume of the oral branch 2 and the nasal branch 3 is 500mL, and the oral shunt flow is 35mL. Select the 3rd level bronchial tree 16, connect one lung unit exposure device 5, and the rest are connected to the bionic alveoli 19. The cell chamber 24 in the lung unit exposure device 5 is a 6-hole Transwell chamber.

[0115] (3) Exposure experiment and sample collection: The exposure experiment was carried out according to the above settings. After the exposure experiment, the cells in the lung unit exposure device 5 were collected and incubated for 24 hours before the CCK-8 experiment was carried out.

[0116] (4) Exposure assessment

[0117] Exposure dose is calculated by puffs:

[0118] Exposure dose (mouth) = number of inhaled mouthfuls (N 吸入物口 )× diverter valve retention coefficient (S 保 )×the proportion of airflow entering the exposure chamber (V 进装置 / V 吸 )=Number of inhalation ports (N 吸入物口 )×diverter valve retention coefficient (100)×the proportion of airflow entering the exposure chamber (20mL / 500mL)=0.04×the number of inhaled inhalation ports (N 吸入物口 ), the exposure doses of the exposure device exposing 0, 84, and 168 puffs of cigarettes are 0, 3.36, and 6.72 puffs, respectively.

[0119] The results of Beas-2b cell survival rates at different exposure doses are shown in Table 3 below.

[0120] Table 3 Beas-2b cell survival rate at different exposure doses

[0121] Number of smoke exposures (mouth) Cell survival rate (%) 0 100 3.36 92±3.1％ 6.72 77±6.8％

[0122] As shown in Table 3, with the continuous increase in the number of cigarettes (puffs), the survival rate of Beas-2b cells continues to decrease, which is consistent with the previous research results.

[0123] It can be seen from the above description that the bionic breathing exposure system for inhalation of the present invention well simulates the respiratory physiological microenvironment of cells, and scientifically characterizes the diversion of oral and nasal shunts and the 3rd level bronchial tree 16, thereby ensuring the scientificity and accuracy of the exposure research based on inhalation.

Claims

1. A bionic breathing exposure system for inhaled materials, comprising a breathing power device and an oral branch and a nasal branch connected to the breathing power device, wherein the downstream of the oral branch and the nasal branch merge to form a throat structure and then connect to the tracheal branch, wherein the tracheal branch comprises a bronchial tree with multiple branches, characterized in that: The oral branch and / or nasal branch are connected to a shunt valve for adjusting the shunt flow of the oral branch and the nasal branch; at least one of the multiple branches is connected to a lung unit exposure device that is adapted to the corresponding branch shunt ratio or the target shunt ratio; the lung unit exposure device includes a microporous membrane for cell growth and a gas chamber and a liquid chamber located on both sides of the microporous membrane respectively, and the liquid chamber is provided with a CO2 exhalation device and a refinement baffle for refining the CO2 exhaled by the CO2 exhalation device.

2. The bionic breathing exposure system for inhalation as claimed in claim 1, characterized in that The diversion ratio of the diversion valve is 100% oral branch, 100% nasal branch or mixed diversion, and the mixed diversion is diversion of the oral branch and the nasal branch according to a set ratio.

3. The bionic breathing exposure system for inhalation as claimed in claim 2, characterized in that: The diverter valve is a rotary diverter valve, a pressure pipe diverter valve, a relative diverter valve or a pneumatic diverter valve.

4. The bionic breathing exposure system for inhalation as claimed in claim 1, characterized in that: The multiple branches of the bronchial tree determine the branch shunt ratio by measuring the flow rate, and the target shunt ratio deviates from the branch shunt ratio to simulate different signs; a lung unit exposure device with a matching gas chamber volume is selected according to the branch shunt ratio and the target shunt ratio, and the gas chamber volume increases or decreases with breathing.

5. The bionic breathing exposure system for inhalation as claimed in claim 1, characterized in that: The lung unit exposure device includes a culture unit, which includes the microporous membrane, a gas chamber and a liquid chamber. The top wall of the gas chamber is a soft elastic material. The soft elastic material expands during inhalation and collapses during exhalation to cause the volume of the gas chamber to change with breathing.

6. The bionic breathing exposure system for inhalation as claimed in claim 5, characterized in that: The lung unit exposure device includes a constant temperature water bath, and the culture unit is arranged in the constant temperature water bath.

7. Use of a bionic breathing exposure system for inhalation according to any one of claims 1 to 6 in a bionic breathing exposure experiment, characterized in that: Cells are inoculated and cultured on the microporous membrane of the lung unit exposure device to construct a cell layer at the air-liquid interface, and then an exposure experiment is carried out in a breathing mode. The exposure dose is determined based on the shunt ratio of the oral branch and the nasal branch, and the proportion of the airflow entering the lung unit exposure device, and an exposure evaluation is performed at the exposure dose.

8. The use according to claim 7, characterized in that The exposure dose is calculated as follows: The number of puffs of inhaled material is counted: exposure dose (puffs) = number of puffs of inhaled material N 吸入物口 ×Diverter valve retention coefficient S 保 ×The proportion of airflow entering the exposure chamber V 进装置 / V 吸 ; When the inhaled material is measured by flow rate: exposure dose (mL) = inhaled material flow rate V 口 / 鼻特殊吸 ×Diverter valve retention coefficient S 保 ×The proportion of airflow entering the exposure chamber V 进装置 / V 吸 .

9. The use according to claim 8, characterized in that Determine the diverter valve retention coefficient S according to the loss of the suction flow through the diverter valve 保 , the diverter valve retention coefficient S 保 The range is 0 to 100%.

10. The use according to claim 7, characterized in that The breathing pattern is selected from natural breathing, special breathing containing the inhalant to be studied, and a combination of natural breathing and the special breathing.

Images