Development of a Custom Fluid Flow Chamber for Investigating the Effects of Shear Stress on Periodontal Ligament Cells

Abstract

The periodontal ligament (PDL) is crucial for maintaining the integrity and functionality of tooth-supporting structures. Mechanical forces applied to the tooth during orthodontic tooth movement generate pore pressure gradients, leading to interstitial fluid movement within the PDL. The generated fluid shear stress (FSS) stimulates the remodeling of PDL and alveolar bone. Herein, we present the construction of a parallel fluid-flow apparatus to determine the effect of FSS on PDL cells. The chamber was designed and optimized using computer-aided and computational fluid dynamics software. The chamber was formed by PDMS using a negative molding technique. hPDLCs from two donors were seeded on microscopic slides and exposed to FSS of 6 dyn/cm² for 1 h. The effect of FSS on gene and protein expression was determined using RT-qPCR and Western blot. FSS upregulated genes responsible for mechanosensing (FOS), tissue formation (RUNX2, VEGFA), and inflammation (PTGS2/COX2, CXCL8/IL8, IL6) in both donors, with donor 2 showing higher gene upregulation. Protein expression of PTGS2/COX2 was higher in donor 2 but not in donor 1. RUNX2 protein was not expressed in either donor after FSS. In summary, FSS is crucial in regulating gene expression linked to PDL remodeling and inflammation, with donor variability potentially affecting outcomes.

Keywords: PDMS; cells; fluid flow; laminar; mechanical stimulation; orthodontics; periodontal ligament; shear stress; tissue formation; tooth movement.

Figure 1

Chamber design and chamber production. (A) Inner chamber dimension. Dimensions of the master chamber model (identical to the inner part of the chamber). (B–D) Chamber production using the negative molding technique. (B) The master model of the inner chamber (1) was glued to the 3D-printed flask consisting of a base and frame (2) using modeling wax. Threaded nozzles (3) were placed onto the chamber’s inlet and outlet and degassed PDMS was poured into the flask. (C) The final chamber is made from PDMS with threaded fittings. (D) Exploded 3D image of the final chamber with all parts including chamber closing frame (4), chamber closing lid (5), and polyurethane nano tape (6). The parts not made from PDMS were 3D printed using an SLA printer.

Figure 2

Computational fluid flow shear stress simulation of the parallel flow chamber using Autodesk CFD (Autodesk, San Rafael, CA, USA). (A) The shear stress magnitude was determined to confirm the mathematical calculations and distribution of wall shear stress at 6 dyn/cm² across the desired chamber cell seeding area. Arrows represent the flow direction. (B) The velocity was simulated to determine undesirable phenomena such as turbulence. Streamline visualization of the flow field shows no flow turbulence at the seeding area of the flow chamber. Turbulence lines near the inlet and outlet are shown. (C) The region of consistent FSS was identified by fluid flow simulation (Autodesk CFD; Autodesk, San Rafael, CA, USA). The graphs depict FSS along the length (left) and across the middle of the chamber (right). A custom-made gasket was designed using Autodesk Inventor (Autodesk, San Rafael, CA, USA) (see Figure 3 for details).

Figure 3

Workflow of the experimental setup. (A) Custom-made culture well gaskets. The gasket was constructed by blocking the desired area of the glass slide using modeling wax and molded with 1:10 PDMS in cell culture dishes. (B) First, the gaskets were placed onto microscopic slides and coated with collagen. Second, cells were seeded in the gasket well at a density of 3 × 10⁵ cells/cm² and incubated overnight. Third, the slides were loaded into a parallel flow chamber and secured using clamps, after which the two chambers were stimulated in parallel. The complete setup consists of (1) a water bath used to keep the culturing medium temperature at ~37 °C; (2) culturing medium reservoir; (3) a peristaltic pump; (4) a pulse damper; (5) a bubble trap composed of a T-connector and a valve; (6) parallel flow chamber; (7) clamps; (8) silicon tubing (black: chamber 1; red: chamber 2).

Figure 4

Calibration of the temperature in the parallel flow chamber. The temperature within the chamber was calibrated with the water heating bath using a digital thermometer implanted within the parallel flow chamber using a fluid flow rate of 166.67 mL/min.

Figure 5

Cell attachment of human periodontal ligament cells (hPDLCs) and human osteosarcoma cell line (SaOS-2) was assessed by microscopy before and after applying FSS. Microscopic images of cells growing in the corners and center of the seeding area of the microscopic slide are shown. (Scale bar: 1000 μm).

Figure 6

Cell viability of human periodontal ligament cells (hPDLCs) and human osteosarcoma cell line (SaOS-2) was assessed by live/dead cell staining. Microscopic images of cells growing in the center of the seeding area of the microscopic slide are shown. Live cells are indicated by calcein AM staining (green), and dead cells are indicated by ethidium homodimer-1 (EthD-1) staining (red arrows). (Scale bar: 400 μm).

Figure 7

Reference gene primer stability was assessed using RefFinder [25]. (a) Descriptive statistics of the Cq values of the reference gene panel (FSS: 1 h FSS) (n = 4); Control: negative control (n = 4); All: FSS and control groups combined (n = 8). (b) The result from the comprehensive analysis of gene stability for the reference gene panel from RefFinder. Lower values in this analysis correspond to higher gene stability.

Figure 8

Gene expression of the early mechanosensitive responder FOS after 1 h fluid shear stress. Each test group is represented by the mean (━), with error bars that indicate the standard deviation (SD). The 2^−ΔΔCq technique was used, with RPL0 and RPL22 as reference genes. The differences between the test and control groups were evaluated using the Mann-Whitney U Test. Groups with significant differences are highlighted as follows: * p < 0.05; ** p < 0.01.

Figure 9

Gene expression of inflammation-related genes after 1 h fluid shear stress: (A) PTGS2, (B) CXCL8 (IL8), and (C) IL6. Each test group is represented by the mean (━), with error bars that indicate the standard deviation (SD). The 2^−ΔΔCq technique was used, with RPL0 and RPL22 as reference genes. The differences between the test and control groups were evaluated using the Mann-Whitney U Test. Groups with significant differences are highlighted as follows: * p < 0.05; *** p < 0.001.

Figure 10

Gene expression of osteogenic differentiation-related genes after 1 h fluid shear stress: (A) RUNX2, (B) VEGFA, (C) TNFRSF11B, and (D) SP7. Each test group is represented by the mean (━), with error bars that indicate the standard deviation (SD). The 2^−ΔΔCq technique was used, with RPL0 and RPL22 as reference genes. The differences between the test and control groups were evaluated using the Mann-Whitney U Test. Groups with significant differences are highlighted as follows: * p < 0.05; ** p < 0.01.

Figure 11

Western blot analysis of PTGS2/COX2 and RUNX2 proteins after 1 h fluid shear stress. FSS induced the expression of PTGS2 but not RUNX2. Lysates from donor 1 (A) and donor 2 (B) from 1 h FSS, the corresponding control (Ctrl), and the positive controls for GAPDH (HeLa), COX2, and RUNX2 (both expressed in baculovirus-insect cells) were separated by PAGE on a 14% SDS gel and transferred onto a PVDF membrane.

Figure 12

Force-related tooth displacement alters PDL dynamics (pore size, pore pressure, flow rate, and FSS). (A) The amount of displacement was presented in a table. As illustrated, the greatest displacement happens during the first hour using a force of 1 N [modified from [18]. (B,C) Conceptual representation of FSS magnitude during low and high orthodontic force. By applying low orthodontic force, the duration of fluid flow will be longer with a lower FSS magnitude over time and vice versa.