Oxidative Stress Alters the Morphological Responses of Myoblasts to Single-Site Membrane Photoporation.

The responses of single cells to plasma membrane damage is critical to cell survival under adverse conditions and to many transfection protocols in genetic engineering. While the post-damage molecular responses have been much studied, the holistic morphological changes of damaged cells have received less attention. Here we document the post-damage morphological changes of the C2C12 myoblast cell bodies and nuclei after femtosecond laser photoporation targeted at the plasma membrane. One adverse environmental condition, namely oxidative stress, was also studied to investigate whether external environmental threats could affect the cellular responses to plasma membrane damage. The 3D characteristics data showed that in normal conditions, the cell bodies underwent significant shrinkage after single-site laser photoporation on the plasma membrane. However for the cells bearing hydrogen peroxide oxidative stress beforehand, the cell bodies showed significant swelling after laser photoporation. The post-damage morphological changes of single cells were more obvious after chronic oxidative exposure than that after acute ones. Interestingly, in both conditions, the 2D projection of nucleus apparently shrank after laser photoporation and distanced itself from the damage site. Our results suggest that the cells may experience significant multi-dimensional biophysical changes after single-site plasma membrane damage. These post-damage responses could be dramatically affected by oxidative stress.

Preventive Effects of Poloxamer 188 on Muscle Cell Damage Mechanics Under Oxidative Stress.

High oxidative stress can occur during ischemic reperfusion and chronic inflammation. It has been hypothesized that such oxidative challenges could contribute to clinical risks such as deep tissue pressure ulcers. Skeletal muscles can be challenged by inflammation-induced or reperfusion-induced oxidative stress. Oxidative stress reportedly can lower the compressive damage threshold of skeletal muscles cells, causing actin filament depolymerization, and reduce membrane sealing ability. Skeletal muscles thus become easier to be damaged by mechanical loading under prolonged oxidative exposure. In this study, we investigated the preventive effect of poloxamer 188 (P188) on skeletal muscle cells against extrinsic oxidative challenges (H2O2). It was found that with 1 mM P188 pre-treatment for 1 h, skeletal muscle cells could maintain their compressive damage threshold. The actin polymerization dynamics largely remained stable in term of the expression of cofilin, thymosin beta 4 and profilin. Laser photoporation demonstrated that membrane sealing ability was preserved even as the cells were challenged by H2O2. These findings suggest that P188 pre-treatment can help skeletal muscle cells retain their normal mechanical integrity in oxidative environments, adding a potential clinical use of P188 against the combined challenge of mechanical-oxidative stresses. Such effect may help to prevent deep tissue ulcer development.

Effects of Biowastes Released by Mechanically Damaged Muscle Cells on the Propagation of Deep Tissue Injury: A Multiphysics Study.

Deep tissue injuries occur in muscle tissues around bony prominences under mechanical loading leading to severe pressure ulcers. Tissue compression can potentially compromise lymphatic transport and cause accumulation of metabolic biowastes, which may cause further cell damage under continuous mechanical loading. In this study, we hypothesized that biowastes released by mechanically damaged muscle cells could be toxic to the surrounding muscle cells and could compromise the capability of the surrounding muscle cells to withstand further mechanical loadings. In vitro, we applied prolonged low compressive stress (PLCS) and short-term high compressive stress to myoblasts to cause cell damage and collected the biowastes released by the damaged cells under the respective loading scenarios. In silico, we used COMSOL to simulate the compressive stress distribution and the diffusion of biowastes in a semi-3D buttock finite element model. In vitro results showed that biowastes collected from cells damaged under PLCS were more toxic and could compromise the capability of normal myoblasts to resist compressive damage. In silico results showed that higher biowastes diffusion coefficient, higher biowastes release rate, lower biowastes tolerance threshold and earlier timeline of releasing biowastes would cause faster propagation of tissue damage. This study highlighted the importance of biowastes in the development of deep tissue injury to clinical pressure ulcers under prolonged skeletal compression.

Change in viability of C2C12 myoblasts under compression, shear and oxidative challenges.

Skeletal and epidermal loadings can damage muscle cells and contribute to the development of deep tissue injury (DTI) - a severe kind of pressure ulcers affecting many people with disability. Important predisposing factors include the multiaxial stress and strain fields in the internal tissues, particularly the vulnerable muscles around bony prominences. A careful study of the mechanical damage thresholds for muscle cell death is critical not only to the understanding of the formation of DTI, but also to the design of various body support surfaces for prevention. In this paper, we measured the mechanical damage thresholds of C2C12 myoblasts under prescribed compressive strains (15% and 30%) and shear strains (from 0% to 100%), and studied how oxidative stress, as caused potentially by reperfusion or inflammation, may affect such damage thresholds. A flat plate was used to apply a uniform compressive strain and a radially increasing shear strain on disks of Gelatin-methacrylate (GelMA) hydrogel with myoblasts encapsulated within. The percentages of cell death were estimated with propidium iodide (PI) and calcein AM staining. Results suggested that cell death depended on both the level and duration of the applied strain. There seemed to be a non-linear coupling between compression and shear. Muscle cells often need to function biomechanically in challenging oxidative environments. To study how oxidative stress may affect the mechanical damage thresholds of myoblasts, cell viability under compressive and shear strains was also studied after the cells were pre-treated for different durations (1h and 20h) with different concentrations (0.1mM and 0.5mM) of hydrogen peroxide (H2O2). Oxidative stress can either compromise or enhance the cellular resistance to shear damage, depending on the level and duration of the oxidative exposure.

Effects of oxidative stress-induced changes in the actin cytoskeletal structure on myoblast damage under compressive stress: confocal-based cell-specific finite element analysis.

Muscle cells are frequently subjected to both mechanical and oxidative stresses in various physiological and pathological situations. To explore the mechanical mechanism of muscle cell damage under loading and oxidative stresses, we experimentally studied the effects of extrinsic hydrogen peroxides on the actin cytoskeletal structure in C2C12 myoblasts and presented a finite element (FE) analysis of how such changes in the actin cytoskeletal structure affected a myoblast's capability to resist damage under compression. A confocal-based cell-specific FE model was built to parametrically study the effects of stress fiber density, fiber cross-sectional area, fiber tensile prestrain, as well as the elastic moduli of the stress fibers, actin cortex, nucleus and cytoplasm. The results showed that a decrease in the elastic moduli of both the stress fibers and actin cortex could increase the average tensile strain on the actin cortex-membrane structure and reduce the apparent cell elastic modulus. Assuming the cell would die when a certain percentage of membrane elements were strained beyond a threshold, a lower elastic modulus of actin cytoskeleton would compromise the compressive resistance of a myoblast and lead to cell death more readily. This model was used with a Weibull distribution function to successfully describe the extent of myoblasts damaged in a monolayer under compression.

Rheological behavior of actin stress fibers in myoblasts after nanodissection: Effects of oxidative stress.

BACKGROUND: Cytoskeletal stress fibers (SFs) play important roles in cell rheology. Oxidative stress, as caused by excessive hydrogen peroxide (H2O2) or other reactive oxygen species, can cause cell damages via multiple pathways. Stress fiber mechanics in an oxidative environment is important for the understanding of such pathological challenges. OBJECTIVE: This investigation aimed to assess the effects of oxidative stress on the mechanical conditions of single stress fibers in living cells. METHODS: Utilizing a femtosecond (fs) laser to sever single SFs inside living C2C12 myoblasts, we investigated the retraction rheology of the severed single SFs to probe the mechanical conditions of the cells and the effect of H2O2 on them. RESULTS: The equilibration time of the retraction of the severed SFs became longer in the H2O2-treated myoblasts compared to the control. The initial gap between the two severed ends of the SF immediately after fs laser severing was larger in the H2O2-treated groups. This suggested that H2O2 exposure could promote the pre-stress in individual SFs in-situ. CONCLUSION: Oxidative stress could significantly affect the mechanical conditions of cytoskeletal SFs in myoblasts. The results were consistent with cell stiffness measured on single myoblasts under oxidative stress.

Oxidative Stress and Plasma Membrane Repair in Single Myoblasts After Femtosecond Laser Photoporation.

Cell membranes are susceptible to biophysical damages. These biophysical damages often present themselves in challenging oxidative environments, such as in chronic inflammation. Here we report the damage evolution after single myoblasts were individually subjected to femtosecond (fs) laser photoporation on their plasma membranes under normal and oxidative conditions. A well-characterized tunable fs laser was coupled with a laser scanning confocal microscope. The post-damage wound evolution was documented by real-time imaging. The fs laser could generate a highly focused hole at a targeted site of the myoblast plasma membrane. The initial hole size depended on the laser dosage in terms of power and exposure duration. With the same laser power and irradiation duration, photoporation invoked bigger holes in the oxidative groups than in the control. Myoblasts showed difficulty in repairing holes with initial size beyond certain threshold. Within the threshold, holes could apparently be resealed within 100 s under the normal condition; while in oxidative condition, the resealing process could take 100-300 s. The hole-resealing capacity of myoblasts was compromised under oxidative stress particularly when the oxidative exposure was chronic. It is interesting to note that brief exposure to oxidative stress apparently could promote resealing in myoblasts after photoporation.

The effects of oxidative stress on the compressive damage thresholds of C2C12 mouse myoblasts: implications for deep tissue injury.

Deep tissue injury (DTI) is a severe kind of pressure ulcers formed by sustained deformation of muscle tissues over bony prominences. As a major clinical issue, DTI affects people with physical disabilities, and is obviously related to the load-bearing capacity of muscle cells in various in vivo conditions. It has been hypothesized that oxidative stress, either induced by reperfusion immediately following tissue unloading or in chronic inflammatory conditions, may affect the cellular capacity against subsequent mechanical damages. In this study, we measured the compressive damage threshold of C2C12 mouse myoblasts with or without pre-treatment of hydrogen peroxide as an oxidative agent to understand how changes in the oxidative environment may contribute to the development of DTI. Spherical indentation was applied onto a layer of agarose gel (3 mm thick) covering a monolayer of C2C12 myoblasts. Cell damage was recognized by using a cell membrane damage assay, propidium iodide. The spatial profile of the measured percentage cell damage was correlated with the radially varying stress field as determined by finite element analysis to estimate the compressive stress threshold for cell damage. Results supported the hypothesis that chronic exposure to high-dosage oxidative stress could compromise the capability of muscle cells to withstand compressive damages, while short exposure to low-dosage oxidative stress could enhance such capability.

H2O2 Exposure Affects Myotube Stiffness and Actin Filament Polymerization.

Skeletal muscles often experience oxidative stress in anaerobic metabolism and ischemia-reperfusion. This paper reports how oxidative stress affects the stiffness of cultured murine myotubes and their actin filaments polymerization dynamics. H2O2 was applied as an extrinsic oxidant to C2C12 myotubes. Atomic force microscopy results showed that short exposures to H2O2 apparently increased the stiffness of myotubes, but that long exposures made the cells softer. The turning point seemed to take place somewhere between 1 and 2 h of H2O2 exposure. We found that the stiffness change was probably due to actin filaments being favored for depolymerization after prolong H2O2 treatments, especially when the exposure duration exceeded 1 h and the exposure concentration reached 1.0 mM. Such depolymerization effect was associated with the down-regulation of thymosin beta 4, as well as the up-regulation of both cofilin2 and profilin1 after prolong H2O2 treatments.

Accumulation of loading damage and unloading reperfusion injury--modeling of the propagation of deep tissue ulcers.

Deep tissue injury (DTI) occurs in deep muscles around bony prominences due to excessive and prolonged mechanical loading acting on the skin surface. The condition is clinically challenging because it can escape being noticed till the damage propagates all the way to the skin. In this study, a semi-3D finite element model of a human buttock was used to simulate the process of ulcer evolution based on our recent damage accumulation and repair theory for DTI. The theory included not only the loading damage, but also further reperfusion and inflammatory injuries upon unloading. The results showed that depending on the model parameters and loading conditions, a deep tissue ulcer may initiate around a bony prominence and expand to affect the entire tissue thickness. The damage evolution can be affected by the tissue healing rate, the loading-unloading pattern and the cushion stiffness. The results may help clinical workers appreciate the importance of proper patient turning and the appropriate choice of cushion.

Deformation and reperfusion damages and their accumulation in subcutaneous tissues during loading and unloading: a theoretical modeling of deep tissue injuries.

Deep tissue injuries (DTI) involve damages in the subcutaneous tissues under intact skin incurred by prolonged excessive epidermal loadings. This paper presents a new theoretical model for the development of DTI, broadly based on the experimental evidence in the literatures. The model covers the loading damages implicitly inclusive of both the direct mechanical and ischemic injuries, and the additional reperfusion damages and the competing healing processes during the unloading phase. Given the damage accumulated at the end of the loading period, the relative strength of the reperfusion and the healing capacity of the involved tissues system, the model provides a description of the subsequent damage evolution during unloading. The model is used to study parametrically the scenario when reperfusion damage dominates over healing upon unloading and the opposite scenario when the loading and subsequent reperfusion damages remain small relative to the healing capacity of the tissues system. The theoretical model provides an integrated understanding of how tissue damage may further build-up paradoxically even with unloading, how long it would take for the loading and reperfusion damages in the tissues to become fully recovered, and how such loading and reperfusion damages, if not given sufficient time for recovery, may accumulate over multiple loading and unloading cycles, leading to clinical deep tissues ulceration.

Biomechanics of pressure ulcer in body tissues interacting with external forces during locomotion.

Forces acting on the body via various external surfaces during locomotion are needed to support the body under gravity, control posture, and overcome inertia. Examples include the forces acting on the body via the seating surfaces during wheelchair propulsion, the forces acting on the plantar foot tissues via the insole during gait, and the forces acting on the residual-limb tissues via the prosthetic socket during various movement activities. Excessive exposure to unwarranted stresses at the body-support interfaces could lead to tissue breakdowns commonly known as pressure ulcers, often presented as deep-tissue injuries around bony prominences or as surface damage on the skin. In this article, we review the literature that describes how the involved tissues respond to epidermal loading, taking into account both experimental and computational findings from in vivo and in vitro studies. In particular, we discuss related literature about internal tissue deformation and stresses, microcirculatory responses, and histological, cellular, and molecular observations.

A one-step method to fabricate PLLA scaffolds with deposition of bioactive hydroxyapatite and collagen using ice-based microporogens.

Porous poly(l-lactic acid) (PLLA) scaffolds with bioactive coatings were prepared by a novel one-step method. In this process, ice-based microporogens containing bioactive molecules, such as hydroxyapatite (HA) and collagen, served as both porogens to form the porous structure and vehicles to transfer the bioactive molecules to the inside of PLLA scaffolds in a single step. Based on scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction and Fourier transform infrared spectroscopy analysis, the bioactive components were found to be transferred successfully from the porogens to PLLA scaffolds evenly. Osteoblast cells were used to evaluate the cellular behaviors of the composite scaffolds. After culturing for 8days, MTT assay and alkaline phosphatase activity results suggested that HA/collagen could improve the interactions between osteoblast cells and the polymeric scaffold.

Post pressure response of skin blood flowmotions in anesthetized rats with spinal cord injury.

Pressure ulcer is a common complication developed in persons with spinal cord injury (SCI) when prolonged unrelieved pressure was applied to the body/skin and underlying tissues. The objective of this study is to assess the hyperemic response of the skin blood flowmotions in anesthetized rats with spinal cord injury subjected to prolonged pressure using spectral analysis based on wavelets transform of the periodic oscillations of the cutaneous laser Doppler flowmetry (LDF) signal. A total of twenty-eight Sprague-Dawley rats were used in this study, of which 14 were normal rats and the other 14 were spinal cord injured rats with transection of the T1 spinal nerves. External pressure of 13.3 kPa (100 mmHg) was applied to the trochanter area of rats via a specifically designed indentors. The loading duration was 6 h. LDF measurement was monitored for 20 min prior to and after the prescribed compression period. Five frequency intervals were identified (0.01-0.05 Hz, 0.05-0.15 Hz, 0.15-0.4 Hz, 0.4-2 Hz and 2-5 Hz) corresponding to endothelial related metabolic, neurogenic, myogenic, respiratory and cardiac origins. The absolute amplitude of oscillations of each particular frequency interval and the normalized amplitude were calculated for quantitative assessments. Comparisons of hyperemic response were performed between SCI rats and normal ones. The results showed that the normalized amplitude in the frequency interval II (0.05-0.15 Hz) was significantly lower on SCI rats than that in normal ones (p<0.01). Also, decreased reactive hyperemic response was observed in rats suffered from spinal cord injury.

Hybrid coating of hydroxyapatite and collagen within poly(D,L-lactic-co-glycolic acid) scaffold.

To improve the cell-affinity of biodegradable polymer scaffold, coating hydroxyapatite (HA) or collagen on the surface of polymer materials seemed to be a strategy to combine both advantages of them. The objective of this study was to develop a novel method to introduce HA and collagen inside polymer scaffold uniformly. HA and collagen suspension was mixed with paraffin microspheres, and molded to form a composite sample. After the sample was dried, HA/collagen composite was left among and on the surface of paraffin microspheres. Poly(D,L-lactic-co-glycolic acid) (PLGA) (50/50) solution was cast into the inter-space of the paraffin microspheres and dried. Afterwards, the paraffin was dissolved and removed, HA/collagen was transferred to the surface and even inside of the pore wall of PLGA scaffolds. Collagen fibers and HA particles which were inlaid inside the PLGA pore wall could help to enhance the coating strength between HA/Col coating and the pore wall surface of the PLGA scaffold. The scaffolds with HA/Col coating were expected to exhibit desirable properties in bone tissue engineering.

A knowledge-based computer-aided system for closed diaphyseal fracture reduction.

BACKGROUND: We recently developed an algorithm to perform closed fracture reduction using unilateral external fixator. Although its validity has been verified experimentally, the whole reduction process was not evaluated owing to the lack of a device that could facilitate its implementation in clinical practice. The objective of this study is to develop a prototype of such a system, and quantify its reduction accuracy. METHODS: The system consists of a custom-made unilateral external device and a self-contained software package. The device features 7 one degree of freedom joints, each allows for continuous adjustments and is equipped with measurement components to facilitate accurate positioning. A CT-based method was developed, which facilitates virtual reduction and calculates the adjustment requirements that reduce a fracture deformity. The device was adjusted off-the-site and reattached back in place to guide the reduction of the fracture fragments. Reduction accuracy was evaluated using eight phantoms of different types, sides and fracture patterns by calculating the rotation about a screw axis and the displacement between the origins of the distal and proximal local coordinate systems after the reduction. FINDINGS: The mean (SD) of the translational and rotational reduction errors were 1.73 (0.97)mm and 2.57 degrees (1.36 degrees), respectively, which demonstrated the accuracy and reliability of the system. INTERPRETATION: The system allows surgeons to perform fracture reduction in an objective, efficient, and accurate manner yet minimize the radiation exposure and lessens the extent of tissue disruption around the fracture site during the reduction process.

Wavelet analysis of the effects of static magnetic field on skin blood flowmotion: investigation using an in vivo rat model.

BACKGROUND: In the literature, various in vivo studies on animals have demonstrated that a static magnetic field (SMF) might maintain microvascular tone in the cutaneous microcirculatory system by its biphasic effects on vasomotion. Here, the effects of locally applied SMF on skin blood flowmotion within the stressed or unstressed skin in the trochanter area were evaluated using wavelet analysis of skin blood perfusion as measured by laser Doppler flowmetry (LDF) in anesthetized rats. MATERIALS AND METHODS: Forty-eight experimental trials were carried out on twelve Sprague-Dawley rats. Four experimental groups were formed at random: i) Group CNL (no loading or SMF exposure; n = 12 trials); ii) Group SMF (SMF exposure only; n = 12 trials); iii) Group L (stressed skin without SMF exposure; n = 12 trials); iv) Group L + SMF (stressed skin with SMF exposure; n = 12 trials). RESULTS: SMF significantly enhanced endothelial related metabolic activity (0.01-0.05 Hz) in the stressed skin (p = 0.03). However, SMF did not induce significant change in the flowmotion amplitude in the unstressed skin (p = 0.22). CONCLUSION: The modulating effect of SMF on skin blood flowmotion might be related to the vascular tone modified by prolonged loading.

Transfer of apatite coating from porogens to scaffolds: uniform apatite coating within porous poly(DL-lactic-co-glycolic acid) scaffold in vitro.

Strategies to bone tissue engineering have focused on the use of synthetic or natural degradable materials as scaffolds for cell transplantation to guide bone regeneration. Biocompatibility, biodegradability, biomechanical integrity, and osteoconductivity are important requirements for the scaffold materials. This study explored a new approach of apatite coating to enhance the osteoconductivity of a synthetic degradable poly(DL-lactic-co-glycolic acid) (PLGA) scaffold. The new approach was developed to ensure a relatively uniform apatite coating on the interior pore surfaces deep inside a scaffold, even for a relatively thick scaffold with small pores. Apatite was first coated on the surface of paraffin spheres of the desirable sizes. The paraffin spheres were then molded to form a foam. PLGA/pyridine solution was cast into the interspaces among the paraffin spheres. After the paraffin spheres were dissolved and removed by cyclohexane, PLGA scaffold with controlled pore size, good interconnectivity and high porosity was obtained with apatite left on the pore surface uniformly throughout the whole scaffold. The scaffold and apatite coating were characterized using thermogravimetry analysis, scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffractometry.

Effects of prolonged compression on the variations of haemoglobin oxygenation - assessment by spectral analysis of reflectance spectrophotometry signals.

The consequences of rhythmical flow motion for nutrition and the oxygen supply to tissue are largely unknown. In this study, the periodic variations of haemoglobin oxygenation in compressed and uncompressed skin were evaluated with a reflection spectrometer using an in vivo Sprague-Dawley rat model. Skin compression was induced over the trochanter area by a locally applied external pressure of 13.3 kPa (100 mmHg) via a specifically designed pneumatic indentor. A total of 19 rats were used in this study. The loading duration is 6 h per day for four consecutive days. Haemoglobin oxygenation variations were quantified using spectral analysis based on wavelets' transformation. The results found that in both compressed and uncompressed skin, periodic variations of the haemoglobin oxygenation were characterized by two frequencies in the range of 0.01-0.05 Hz and 0.15-0.4 Hz. These frequency ranges coincide with those of the frequency range of the endothelial-related metabolic and myogenic activities found in the flow motion respectively. Tissue compression following the above loading schedule induced a significant decrease in the spectral amplitudes of frequency interval 0.01-0.05 Hz during the pre-occlusion period on day 3 and day 4 as compared to that on day 1 (p < 0.05). In contrast, at a frequency range of 0.15-0.4 Hz, prolonged compression caused a significant increase in spectral amplitude during the pre-occlusion period in the compressed tissue on day 3 (p = 0.041) and day 4 (p = 0.024) compared to that in the uncompressed tissue on day 1. These suggested that the variations of the haemoglobin oxygenation were closely related to the endothelial-related metabolic and myogenic activities. Increased amplitude in the frequency interval 0.15-0.4 Hz indicated an increased workload of the vascular smooth muscle and could be attributed to the increase of O(2) consumption rates of arteriolar walls. The modification of vessel wall oxygen consumption might substantially affect the available oxygen supply to the compressed tissue. This mechanism might be involved in the process leading to pressure ulcer formation.

Effects of prolonged surface pressure on the skin blood flowmotions in anaesthetized rats--an assessment by spectral analysis of laser Doppler flowmetry signals.

The objective of this study is to assess the effect of prolonged surface compression on the skin blood flowmotion in rats using spectral analysis based on wavelets transform of the periodic oscillations of the cutaneous laser Doppler flowmetry (LDF) signal. An external pressure of 13.3 kPa (100 mmHg) was applied to the trochanter area and the distal lateral tibia of Sprague-Dawley rats via two specifically designed pneumatic indentors. The loading duration was 6 hours/day for 4 consecutive days. Five frequency intervals were identified (0.01-0.04 Hz, 0.04-0.15 Hz, 0.15-0.4 Hz, 0.4-2 Hz and 2-5 Hz) corresponding to endothelial related metabolic, neurogenic, myogenic, respiratory and cardiac origins. The absolute amplitude of oscillations of each particular frequency interval and the normalized amplitude were calculated for quantitative assessments. The results showed that (1) tissue compression following the above schedule induced significant decrease in the normalized amplitude in the frequency interval of 0.01-0.04 Hz both in the trochanter area (p < 0.001) and tibialis area (p = 0.023), (2) prolonged compression induced significant increase in the absolute amplitude (p = 0.004 for the trochanter area and p = 0.017 for the tibialis area) but significant decrease in the normalized amplitude (p = 0.023 for the trochanter area and p = 0.026 for the tibialis area) in the frequency interval of 0.15-0.4 Hz, and (3) at the tibialis area, the flowmotion amplitude (frequency interval 0.15-0.4 Hz) measured prior to the daily tissue compression schedule was found to be significantly higher on day 4 than the measurements obtained on day 1. However, this finding was not observed at the trochanter area. Our results suggested that prolonged compression might induce endothelial damage and affect the endothelial related metabolic activities.