An in-vitro traumatic model to evaluate the response of myelinated cultures to sustained hydrostatic compression injury

Abstract

While a variety of in-vitro models have been employed to investigate the response of load-bearing tissues to hydrostatic pressure, long-term studies are limited by the need to provide for adequate gas exchange during pressurization. Applying compression in vitro may alter the equilibrium of the system and thereby disrupt the gas exchange kinetics. To address this, several sophisticated compression chamber designs have been developed. However, these systems are limited in the magnitude of pressure that can be applied and may require frequent media changes, thereby eliminating critical autocrine and paracrine signaling factors. To better isolate the cellular response to long-term compression, we created a model that features continuous gas flow through the chamber during pressurization, and a negative feedback control system to rigorously control dissolved oxygen levels. Monitoring dissolved oxygen continuously during pressurization, we find that the ensuing response exhibits characteristics of a second- or higher-order system which can be mathematically modeled using a second-order differential equation. Finally, we use the system to model chronic nerve compression injuries, such as carpal tunnel syndrome and spinal nerve root stenosis, with myelinated neuron-Schwann cell co-cultures. Cell membrane integrity assay results show that co-cultures respond differently to hydrostatic pressure, depending on the magnitude and duration of stimulation. In addition, we find that myelinated Schwann cells proliferate in response to applied hydrostatic compression.

FIG. 1.

The hydrostatic compression chamber system is designed to apply a defined magnitude of pressure to cell cultures while maintaining dissolved oxygen and pH homeostasis.

FIG. 2.

A feedback control system maintains dissolved oxygen homeostasis during pressurization. Dissolved oxygen is measured continuously using a fiberoptic dissolved oxygen sensor and recorded with LabView software. A PID controller compares the actual and baseline dissolved oxygen values to regulated oxygen and nitrogen flow into the incubator.

FIG. 3.

(A) The steady-state dissolved oxygen is directly proportional to the applied hydrostatic pressure. (B) Flow rate is not correlated with steady-state dissolved oxygen standard cubic feet per hour (SCFH).

FIG. 4.

(A) The transient-state dissolved oxygen is directly proportional to the applied hydrostatic pressure. (B) Flow rate is not correlated with transient-state dissolved oxygen standard cubic feet per hour (SCFH).

FIG. 5.

Using the solution calculated from the second-order differential equation [Eqn. (4)], the dissolved oxygen is graphed as a function of time for different applied pressures.

FIG. 6.

The chamber was pressurized to 21 mm Hg with continuous gas flow through the chamber during pressurization of 0.5 standard cubic feet per hour. Using the feedback control system, we maintained the dissolved oxygen response within 2% of the baseline value before pressurization.

FIG. 7.

Myelinated neuron-Schwann cell co-cultures were pressurized at 18 mm Hg for 1–7 days (A), or 18–62 mm Hg for 24 h (B). Cell membrane integrity, as measured by LDH release into the culture medium at 0 and 24 h post-compression, depends on the magnitude and duration of applied pressure (p ≤ 0.05).

FIG. 8.

Schwann cells were subjected to 24 h of hydrostatic compression at 21 mm Hg. The percentage of Schwann cells undergoing proliferation at 24 h post-compression increased significantly (*p ≤ 0.05) when compared with uncompressed control cultures.