CN-122025159-A - Working method of mechanical microenvironment in-vitro simulation system for reproducing arteriovenous internal fistula

CN122025159ACN 122025159 ACN122025159 ACN 122025159ACN-122025159-A

Abstract

A working method for reproducing an in-vitro simulation system of a mechanical microenvironment at an arteriovenous fistula belongs to the technical field of cell mechanics biology experimental devices facing health and rehabilitation engineering. Based on the clinical vascular data of arteriovenous fistula stenosis patients and the calculation hydrodynamic simulation, the real wall shear stress waveform is obtained, the quantitative calculation of the shear stress is realized by means of the hydrodynamic simplified formula of the parallel plate flow cavity, the accurate loading of the multi-form shear stress such as the adaptation pulsation, oscillation and the like is utilized, the wall shear stress microenvironment of arteriovenous fistula anastomosis in different physiological and pathological states can be reproduced in the flow cavity, and simultaneously, the mechanical biological response index of endothelial cells can be quantitatively monitored in real time. The method highly restores the real mechanical microenvironment of the vascular endothelial cells at the arteriovenous fistula, and provides a miniaturized, standardized and quantified experimental platform for researching the quantitative relationship between the wall shear stress signals of the region and the mechanical biological effects of the vascular endothelial cells.

Inventors

LIU SHUXIN
WANG HONGHAN
QIN KAIRONG
NA JINGTONG
LI YONGJIANG
Jie Xiaotong
ZHANG SHUANG
WU YULIN

Assignees

大连理工大学附属中心医院(大连市中心医院)

Dates

Publication Date: 20260512
Application Date: 20260413

Claims (8)

1. The working method of the mechanical microenvironment in-vitro simulation system for reproducing the arteriovenous fistula is characterized by comprising the following steps of: s1, selecting vascular endothelial cells of an arteriovenous internal fistula part which are subjected to primary separation culture, adopting a DMEM (medium-electron microscope) culture medium special for the endothelial cells for carrying out passage expansion, and selecting cells with good growth states of 3 rd to 5 th generations for a mechanical stimulation experiment; S2, before an experiment, inoculating endothelial cells on a glass slide coated with fibronectin and sterilized, and placing the glass slide in a cell incubator for culture, and loading the glass slide into a parallel plate flow cavity when the cells are completely adhered, the growth state is stable and the fusion degree is more than 80%; S3, the parallel flat plate flow cavity is connected into an extracorporeal simulated circulation system, typical wall shear stress waveforms of an arteriovenous fistula anastomosis region and an outflow vein in a physiological state and a pathological stenosis state are respectively applied through a control system, air is introduced into a first liquid storage tank after pressure parameters are accurately regulated and controlled by a pressure controller, and circulation liquid is driven to flow out based on air pressure difference; s4, circulating liquid sequentially passes through a first one-way valve and a flow sensor along a pipeline, and bubbles are efficiently removed through a follicular device; the force signal for mechanical stimulation of endothelial cells is obtained by: s41, acquiring an AVF three-dimensional geometric structure by utilizing a computer tomography angiography technology; S42, measuring the axle center flow velocity, the flow and the blood pressure waveform of the arterial and venous blood flow of each branch at the anastomotic site and 4-6cm away from the anastomotic orifice by using a color ultrasonic Doppler instrument; S43, detecting the change trend of the blood vessel diameter in the same area, and synchronously recording heart rate data; S44, segmenting and three-dimensional reconstructing a blood vessel image obtained by a computed tomography angiography technology based on three-dimensional image processing software, importing a reconstructed blood vessel model into a simulation platform, and carrying out CFD simulation analysis research by combining with in-vivo data acquired at an AVF (automatic volume flow) position; s45, finally, different characteristic wall shear stress signals induced by physiological pulsating flow, low-intensity disturbance flow and oscillating flow at the AVF anastomosis position are obtained in a mode of combining in-vivo and simulation data; S5, quantitatively monitoring mechanical biological response indexes of cell morphology, NO concentration, ROS level and related gene/protein expression in the stimulation process, and systematically analyzing quantitative rules between wall shear stress characteristics and endothelial cell functional response; s6, circulating liquid flowing out of the flow cavity flows into the second liquid storage pool through directional diversion of the second one-way valve.
2. The method according to claim 1, wherein the parallel plate flow chamber is a sandwich structure, the middle is a glass slide carrying inner epidermis cells, and the upper and lower cover plates are made of borosilicate glass.
3. The method for operating the in vitro simulation system for reproducing mechanical microenvironment at an arteriovenous fistula according to claim 1, wherein: the parallel plate flow chamber is further internally provided with a flow chamber with the height H=1.25×10 -4 m and the width W=2.5×10 -2 m.
4. The method according to claim 3, wherein in step S5, the wall shear stress is reduced to the following formula: ; Wherein, the Is a viscosity coefficient, Flow, H is the height of the flow chamber, W is the width of the flow chamber; The circulating fluid used was DMEM medium with a viscosity coefficient mu of 4X 10 -3 Pa.s.
5. The method for simulating the mechanical microenvironment in vitro at the arteriovenous fistula according to claim 1, wherein the flow sensor is used for monitoring and collecting the flow Q of the parallel plate flow cavity in real time, and the collected signals are fed back to a matched computer of the programmable pressure controller to adjust the amplitude and frequency of the flow signals so as to generate the required wall shear stress in the parallel plate flow cavity.
6. The method according to claim 1, wherein the check valve controller automatically switches the working sequence of the liquid storage tanks by a self-defined program, the experimental solution flows into the second liquid storage tank from the first liquid storage tank in the initial stage, and when the experimental solution in the first liquid storage tank consumes 2/3 of the experimental solution, the experimental solution automatically switches to the second liquid storage tank, and the experimental solution flows back into the first liquid storage tank from the second liquid storage tank, so that the time for the circulation system to perform the endothelial cell mechanics biology experiment is prolonged by the reciprocating alternating flow mode.
7. The method of claim 6, wherein when the experimental solution flows from the first reservoir to the second reservoir, the first reservoir sequentially passes through the first three-way valve, the first one-way valve, the flow sensor and the follicular device to enter the parallel plate flow chamber, and then sequentially passes through the second one-way valve and the second three-way valve from the outlet of the parallel plate flow chamber to enter the second reservoir; When the experimental solution enters the parallel flat plate flow cavity from the second liquid storage Chi Liuxiang to the first liquid storage pool through the second three-way valve, the first one-way valve, the flow sensor and the foam filter in sequence, and then enters the first liquid storage pool through the second one-way valve and the first three-way valve from the outlet of the parallel flat plate flow cavity in sequence.
8. The method of claim 7, wherein the experimental fluid in the flow chamber always maintains the same flow direction by the mutual matching of the check valve controller, the check valve and the three-way valve, so as to further determine that the experimental conditions of endothelial cells in the cell experimental process are consistent.

Description

Working method of mechanical microenvironment in-vitro simulation system for reproducing arteriovenous internal fistula Technical Field The invention belongs to the technical field of cell mechanics biology experimental devices facing health and rehabilitation engineering, and relates to a high-precision in-vitro simulation circulating system which is designed based on a wall shear stress generation principle and a microfluidic technology and consists of a shear stress generation unit, a peripheral fluid loading device, a signal acquisition processing and feedback control system and is used for researching quantitative relations between wall shear stress signals caused under different physiological and pathological state stimulation conditions and vascular endothelial cell mechanics biological effects at arteriovenous fistulae. Background Vascular access is the core support for Hemodialysis (HD) treatment of End Stage Renal Disease (ESRD) patients, whose patency directly determines dialysis adequacy and long-term survival of the patient. Autologous arteriovenous fistula (AVF) is recommended by international guidelines such as KDIGO as a preferred vascular access for HD patients due to the advantages of high long-term patency rate, fewer complications, no implantation of foreign matters, low cost and the like. However, AVF has high clinical power loss, and its core cause is neointimal hyperplasia (NIH) and vascular stenosis in the peripheral venous segment of the stoma, and this pathological process is closely related to the disturbance of the microenvironment of Wall Shear Stress (WSS) at the stoma. The vascular endothelial cells are used as barriers of blood flow and blood vessel walls, and can sense and respond to hemodynamic signals such as WSS, blood pressure, circumferential stress and the like through a mechanical biological mechanism, wherein physiological laminar WSS can promote endothelial cells to secrete protective factors such as Nitric Oxide (NO) and the like, maintain vascular homeostasis, abnormal WSS breaks the balance of NO and Reactive Oxygen Species (ROS), causes oxidative stress and inflammation, induces NIH to form, leads to vascular stenosis and insufficient dialysis, even needs intervention operation intervention, and increases pain and death risk of patients. The existing AVF dysfunctional prevention and treatment uses surgery as a main body, and mechanical therapies such as external counterpulsation, exercise intervention and the like also become important directions. However, how to accurately regulate endothelial cell function by WSS signal change induced by the therapy is not yet studied systematically, which hinders clinical application. In-vivo experiments (animals/human bodies) are main means for exploring the regulation mechanism, but the defects of complicated WSS microenvironment, multi-factor interference, limited detection means, low precision, long period, high cost, ethical disputes and the like exist, and the accurate research requirement is difficult to meet. The extracorporeal simulated circulation system becomes a key scheme for breaking through the limitation, and can be used for exploring the quantitative relation between WSS and endothelial cell mechanical biological effect. The existing system has obvious defects of low integration level, large size, more consumption materials and high cost of the system built based on a parallel flat plate flow cavity, a silicone tube and the like, system theory and method for realizing WSS simulation by relying on a micro-fluidic chip technology, but lacking in accurate simulation of the WSS under physiological/pathological states and mechanical intervention conditions, insufficient integration level and consumption material optimizing space of a hydrodynamic circuit formed by large-size centralized parameter components outside the chip, open-loop control of dynamic loading of the WSS, lack of closed-loop accurate regulation and control, and incapability of realizing online and real-time quantitative monitoring due to off-line sampling of endothelial cell function detection. In conclusion, the existing in-vitro simulation circulation system cannot meet the requirements of accurate simulation and on-line monitoring of WSS microenvironment at an AVF anastomosis site and quantitative research on WSS signals and endothelial cell mechanics biological effects. Disclosure of Invention Based on ultrasonic and angiography data of an AVF (automatic flow chart) of a patient, combining computational fluid dynamics (ComputationalFluidDynamics, CFD) simulation analysis, acquiring the space-time distribution characteristics of the ECs endothelial surface of the AVF anastomosis relative to the real wall surface shear stress, combining a parallel plate flow cavity with a programmable pressure controller on the basis, and constructing an AVF endothelial cell in-vitro micro-fluidic culture model. And developing wall shear stress and ECs mechanic