Build a simulation device to apply Bacillus subtilis and verify the ability of Bacillus subtilis to reduce simulated blood uric acid concentration in the device.
Based on the theory of hemodialysis, we built a dialysis-like device utilizing B. subtilis to reduce uric acid in simulated human vein and completed two schemes aiming at human body veins that is probably available. The working condition, the penetrability of bacteria and uric acid, and biosafety of our device was fully evaluated.
In our subject, the main idea of the second part is to directly remove blood uric acid. Although B. subtilis is a gram-negative bacterium and human infection of B. subtilis is rarely reported, it is still infeasible to let the bacteria work inside human body considering the risk of causing body's blood coagulation and immune response, which may also impair its function. As a result, we turned to seek inspiration from current medical therapeutics and learnt a lot from hemodialysis. On the one hand, the semipermeable membrane of the dialyzer can efficiently separate bacteria and blood. On the other hand, the larger contact area and high throughput allow the device to ensure the effective exchange of uric acid. Unlike the purpose of conventional dialysis treating uremia, our ultimate goal is to eliminate the uratoma, and we think it is feasible as long as the blood uric acid concentration remains a low level for some time. So we can choose an available vein as a target and avoid arteriovenous fistula which is requisite to support long term hemodialysis in a normal therapy. In consideration of the difficulty of human or animal experiments with hemodialysis, our dialysis-like device included the simulation of human blood vessel to help us test the ability of bacteria under the real situation.
Our dialysis-like device is composed of four parts, the human body blood vessel simulation part, the simulated blood extracorporeal circulation part, the bacterial circulation part, and the dialyzer part. The blood vessel simulation part includes a peristaltic pump, a blood storage conical flask, and silicone tubes. As its name suggests, it works to simulate the function of human veins. The conical flask is used to store the simulated circulation blood. The peristaltic pump provides power. The silicone tube has the same inner diameter and blood flow volume with the vein we chose. The simulated blood extracorporeal circulation part includes a peristaltic pump, connecting pipes and needles, and this part’s function is to pump simulated blood into the dialyzer at a certain rate and then make it return to the simulated blood vessel. The bacteria circulation part includes a peristaltic pump, a bacteria storage conical flask, and connecting pipes. Its function is to make the bacteria be recycled into the hollow fiber of the dialyzer. The HD-150 dialyzer is provided by Chengdu OCI Medical Devices Co., Ltd. Its internal dialysis fibrous membrane area is 1.5m2, and its surface dialysis microporous aperture is average of 7 ~ 9nm. Although this is a high throughput product, we think it is still suitable for our work. We let the bacteria go through the hollow fiber while blood flow through outside of the hollow fiber in order to achieve the purpose of material exchange. The assembling of the device is shown below in figure 1 and figure 2.
The working principle of our mechanism is as the follows. We use medium to simulate human blood, adding uric acid to let the final concentration equals that of the hyperuricemia. Silicone tubes with smooth internal wall are used to mimic blood vessels. We can modulate the flow velocity and flux of our mimetic blood to be as same as the blood flow rate by mediating the peristaltic pump, and our simulated blood extracorporeal circulation part will lead the liquid flow into the dialyzer. In terms of the bacterial circulation part with bacteria solution, we let it have the same flow rate but adverse direction, gaining the effect of convection to accelerate the interchange of material. Meanwhile, the same flow rate of those two parts can avoid ultrafiltration furthest and guarantee the least change of blood elements. What’s more, as the small substances are in free diffusion and under the convection effect, they will mainly diffuse rely on their concentration gradient. In our device the main exchange refers to the uric acid in blood and the small proteins in the bacteria solution. Circumstance will be more complicated in vivo, like more protein will need to be taken into consideration, but as Bacillus Subtilis is a safe strain, we regard this difference to be acceptive.
When uric acid flows into the bacteria solution, bacteria can utilize it. The circulation of bacteria solution can let the concentration of uric acid in the dialysis device keep low. The stable and relatively high concentration gradient in those two parts guarantee the reaction happen at express speed.
security is always what we concern first, and we are aware that the foundation of achieving function is guaranteeing safety.
1)bacteria lekage experiment
Though the theoretical parameters of our device already showed no possibility of bacteria revealing into mimetic blood and the whole device is enclosed that no bacterium can invade, we still designed bacteria leakage experiment and protein leaking experiment to test the safety of our device. bacteria leakage experiment is used to test whether Bacillus Subtilis can penetrate the fibrous wall.
As shown in figure 3, the sample from the opening for the bacteria circulation grew bacteria, but the sample from the opening for the blood circulation was with no bacteria growth, which indicated that the bacteria cannot pass through the hollow fiber.
2)Protein leakage experiment
After ensuring that the bacteria cannot pass through the dialyzer, we considered that bacteria might secret protein when grown or after death, which might pass through the hollow fiber and get into the blood, leading to immune response. So we did protein leakage experiment to find out how much protein can be leaked.
The result showed that after the running of the device, protein concentration was the same or even decreased (Table 1). We suspected that it’s due to that the dialyzer can absorb some protein in it (the surface of the fiber tube, eg.), which means that, at least in our experiment, the biosafety of our device is highly promising.
Since we are aiming at using our device on human body, we have to select veins which could be utilized and make simulation of them in our hardware. There are some conditions that we should consider in our selection of veins: blood flow, convenience, clinical usage, safety. At last, we chose cephalic vein and femoral vein.
Cephalic vein(figure 4) is a superficial vein in the arm. It has a diameter ranging from 2.2mm to 5mm1,2,3 and the blood flow rate of it is around 28ml/min2. Cephalic vein is a common site for venipuncture, and it’s also wildly used to construct radio cephalic fistula or brachiocephalic fistula in dialysis2. The extensive clinical usage of it ensures that it could also be used together with our device. But the blood flow rate of it is not so high which means the process of our device’s lowering uric acid concentration in the whole blood would need a long time.
Femoral vein(figure 5) is a deep vein accompanies the femoral artery in the femoral sheath. It has a average diameter ranging from 4.4mm to 7.9mm4,5,6, and the blood flow rate of it is around 138ml/min4. Venous catheterization can be conducted in femoral vein and femoral vein is also potential for dialysis7. Although puncturing into femoral vein is a little more difficult because of the complex distribution of vessels and nerves around it, the flow rate and dialysis use potential ensure its’ work proficiency with our device.
The two veins we choose have a same feature that they are not veins with great diameters or flow rates. This is because we want to reduce the harm of immunogen leakage from our device. Most immunogen can be obstructed by the dialyzer, but there is risk that some micromolecular immunogen getting through the hollow fiber membrane and cause immunoreaction. In large vein with high flow rate, the immunoreaction could be much more acute and even do harm to heart.
Experiments are taken to measure the device’s ability of uric acid filtration with the simulation of the two veins. The diameters of silicone tube we settled are 3mm for simulating cephalic vein and 6.4mm for femoral vein. The flow rates inside them are 28ml/min for simulating cephalic vein and 138ml/min for femoral vein. And LB medium with uric acid concentration of 420μg/L, which is the critical concentration of hyperuricemia, is used to simulate the blood and blank LB medium is used to replace the bacteria solution.
Before the experiment, we thought about building a model to predict the variation tendency of the uric acid concentration in two mediums referring to a classic model8. We set up assumption that uric concentration is uniform in the medium. According to Fick’s law, uric acid crossing a section of membrane of unit area in unit time δt is approximately
Where k is the proportionality constant, and u(t) is the uric acid concentration of blood simulating medium, v(t) is the uric acid concentration in bacteria solution replacing medium.
The quantity of uric acid getting into blank medium is equal to the quantity reduction of the uric acid in the blood simulating medium in δt, so that we can get the equation
Where VB is the volume of the blood simulating medium and S is the membrane area of the dialyzer. And we get
Let δt → 0 gives
(4)Considering situations in blank medium, we can get the equation,
Where VD is the volume of bacteria solution replacing medium. Add (4) and (5)
Set z=u-v. Thus
Where α=Sk/VB +Sk/VD. Thus,
A being an arbitrary constant. Take (8) into (4) and we get
And integrating gives
Where B is an arbitrary constant. With the same method, we can get
Since we have the starting conditions that u=u0=420μg/L at t=0, and v=0 at t=0, we can get
Because in our experiment, VB=VD=V=500ml, thus
Take (14) into (13) and (12)
For every simulated vein, we took out one experiment. For simulated femoral vein, we set the flow rate in the simulated blood extracorporeal circulation part and the bacterial circulation part to be 60ml/min; for simulated cephalic vein, we set the flow rate in the simulated blood extracorporeal circulation part and the bacterial circulation part to be 14ml/min. The experiment is carried out as protocol and the data we get is shown below in table 2:
We transferred the data into the concentration of uric acid and plotted them. Data-fitting is done to verify that the data conforms to the model, and the result is shown below:
According to figure 6 and figure 7 and parameters (in table 3 and table 4) got from data-fitting, we can find that for femoral vein simulation, the fitting curve matches the plotted data very well. But the match between experiment data and fitting curve of cephalic vein simulation is not so good. While the values of k in the two results are not the same. The reason for those conditions could be that when the flow rate is low, the assumption of uniform uric acid concentration is not valid. Which can be justified in the future work. The results show that efficiency of uric permeating in femoral vein simulation is much better than that in cephalic vein simulation, because for the former one approximately 30 minutes are needed to reach concentration equilibrium but for the later one the time consumed is about 1 hour. It suggests that femoral vein could be more feasible for our device to work with. But there is also an application for cephalic vein. The traditional dialysis demands abundant dialysate fluid to keep the concentration gradient but our bacteria can metabolize uric acid permeated into the bacterial circulation which means we do not need vast dialysate fluid. This working characteristic together with the microminiaturization of sensors make it possible for our hardware to become a wearable device.
In fact, our device is not usable for cleaning of uric acid. With the development of synthetic biology, it is not hard to speculate that more engineered bacteria that have different metabolizing ability can be produced and they can be applied in our device and deal with some other metabolic diseases. It is promising that our device to be a clinical stage for a lot of metabolic diseases in the futher.
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