Characteristics of lung resistance and elastance associated with tracheal stenosis and intrapulmonary airway narrowing in ex vivo sheep lungs.

BACKGROUND: Understanding the characteristics of pulmonary resistance and elastance in relation to the location of airway narrowing, e.g., tracheal stenosis vs. intrapulmonary airway obstruction, will help us understand lung function characteristics and mechanisms related to different airway diseases. METHODS: In this study, we used ex vivo sheep lungs as a model to measure lung resistance and elastance across a range of transpulmonary pressures (5-30 cmH2O) and ventilation frequencies (0.125-2 Hz). We established two tracheal stenosis models by inserting plastic tubes into the tracheas, representing mild (71.8% lumen area reduction) and severe (92.1%) obstructions. For intrapulmonary airway obstruction, we induced airway narrowing by challenging the lung with acetylcholine (ACh). RESULTS: We found a pattern change in the lung resistance and apparent lung elastance as functions of ventilation frequency that depended on the transpulmonary pressure (or lung volume). At a transpulmonary pressure of 10 cmH2O, lung resistance increased with ventilation frequency in severe tracheal stenosis, whereas in ACh-induced airway narrowing the opposite occurred. Furthermore, apparent lung elastance at 10 cmH2O decreased with increasing ventilation frequency in severe tracheal stenosis whereas in ACh-induced airway narrowing the opposite occurred. Flow-volume analysis revealed that the flow amplitude was much sensitive to ventilation frequency in tracheal stenosis than it was in ACh induced airway constriction. CONCLUSIONS: Results from this study suggest that lung resistance and apparent elastance measured at 10 cmH2O over the frequency range of 0.125-2 Hz can differentiate tracheal stenosis vs. intrapulmonary airway narrowing in ex vivo sheep lungs.

Technical standards for respiratory oscillometry.

Oscillometry (also known as the forced oscillation technique) measures the mechanical properties of the respiratory system (upper and intrathoracic airways, lung tissue and chest wall) during quiet tidal breathing, by the application of an oscillating pressure signal (input or forcing signal), most commonly at the mouth. With increased clinical and research use, it is critical that all technical details of the hardware design, signal processing and analyses, and testing protocols are transparent and clearly reported to allow standardisation, comparison and replication of clinical and research studies. Because of this need, an update of the 2003 European Respiratory Society (ERS) technical standards document was produced by an ERS task force of experts who are active in clinical oscillometry research.The aim of the task force was to provide technical recommendations regarding oscillometry measurement including hardware, software, testing protocols and quality control.The main changes in this update, compared with the 2003 ERS task force document are 1) new quality control procedures which reflect use of "within-breath" analysis, and methods of handling artefacts; 2) recommendation to disclose signal processing, quality control, artefact handling and breathing protocols (e.g. number and duration of acquisitions) in reports and publications to allow comparability and replication between devices and laboratories; 3) a summary review of new data to support threshold values for bronchodilator and bronchial challenge tests; and 4) updated list of predicted impedance values in adults and children.

Defective Fibrillar Collagen Organization by Fibroblasts Contributes to Airway Remodeling in Asthma.

Rationale: Histologic stains have been used as the gold standard to visualize extracellular matrix (ECM) changes associated with airway remodeling in asthma, yet they provide no information on the biochemical and structural characteristics of the ECM, which are vital to understanding alterations in tissue function.Objectives: To demonstrate the use of nonlinear optical microscopy (NLOM) and texture analysis algorithms to image fibrillar collagen (second harmonic generation) and elastin (two-photon excited autofluorescence), to obtain biochemical and structural information on the remodeled ECM environment in asthma.Methods: Nontransplantable donor lungs from donors with asthma (n = 13) and control (n = 12) donors were used for the assessment of airway collagen and elastin fibers by NLOM, and extraction of lung fibroblasts for in vitro experiments.Measurements and Main Results: Fibrillar collagen is not only increased but also highly disorganized and fragmented within large and small asthmatic airways compared with control subjects, using NLOM imaging. Furthermore, such structural alterations are present in pediatric and adult donors with asthma, irrespective of fatal disease. In vitro studies demonstrated that asthmatic airway fibroblasts are deficient in their packaging of fibrillar collagen-I and express less decorin, important for collagen fibril packaging. Packaging of collagen fibrils was found to be more disorganized in asthmatic airways compared with control subjects, using transmission electron microscopy.Conclusions: NLOM imaging enabled the structural assessment of the ECM, and the data suggest that airway remodeling in asthma involves the progressive accumulation of disorganized fibrillar collagen by airway fibroblasts. This study highlights the future potential clinical application of NLOM to assess airway remodeling in vivo.

Modelling resistance and reactance with heterogeneous airway narrowing in mild to severe asthma.

Ventilation heterogeneity is an important marker of small airway dysfunction in asthma. The frequency dependence of respiratory system resistance (Rrs) from oscillometry is used as a measure of this heterogeneity. However, this has not been quantitatively assessed or compared with other outcomes from oscillometry, including respiratory system reactance (Xrs) and the associated elastance (Ers). Here, we used a multibranch model of the human lung, including an upper airway shunt, to match previously reported respiratory mechanics in mild to severe asthma. We imposed heterogeneity by narrowing a proportion of the peripheral airways to account for patient Ers at 5 Hz, and then narrowed central airways to account for the remaining Rrs at 18 Hz. The model required >75% of the small airways to be occluded to reproduce severe asthma. While the model produced frequency dependence in Rrs, it was upward-shifted below 5 Hz compared with in-vivo results, indicating that other factors, including more distributed airway narrowing or central airway wall compliance, are required. However, Ers quantitatively reflected the imposed heterogeneity better than the frequency dependence of Rrs, independent of the frequency range for the estimation, and thus was a more robust measure of small-airway function. Thus, Ers appears to have greater potential as a clinical measure of early small-airway disease in asthma.

Development and characterization of a 3D multicell microtissue culture model of airway smooth muscle.

Airway smooth muscle (ASM) cellular and molecular biology is typically studied with single-cell cultures grown on flat 2D substrates. However, cells in vivo exist as part of complex 3D structures, and it is well established in other cell types that altering substrate geometry exerts potent effects on phenotype and function. These factors may be especially relevant to asthma, a disease characterized by structural remodeling of the airway wall, and highlights a need for more physiologically relevant models of ASM function. We utilized a tissue engineering platform known as microfabricated tissue gauges to develop a 3D culture model of ASM featuring arrays of ∼0.4 mm long, ∼350 cell "microtissues" capable of simultaneous contractile force measurement and cell-level microscopy. ASM-only microtissues generated baseline tension, exhibited strong cellular organization, and developed actin stress fibers, but lost structural integrity and dissociated from the cantilevers within 3 days. Addition of 3T3-fibroblasts dramatically improved survival times without affecting tension development or morphology. ASM-3T3 microtissues contracted similarly to ex vivo ASM, exhibiting reproducible responses to a range of contractile and relaxant agents. Compared with 2D cultures, microtissues demonstrated identical responses to acetylcholine and KCl, but not histamine, forskolin, or cytochalasin D, suggesting that contractility is regulated by substrate geometry. Microtissues represent a novel model for studying ASM, incorporating a physiological 3D structure, realistic mechanical environment, coculture of multiple cells types, and comparable contractile properties to existing models. This new model allows for rapid screening of biochemical and mechanical factors to provide insight into ASM dysfunction in asthma.

Hyperpolarized (3) He and (129) Xe MRI: differences in asthma before bronchodilation.

PURPOSE: To compare hyperpolarized helium-3 ((3) He) and xenon-129 ((129) Xe) MRI in asthmatics before and after salbutamol inhalation. MATERIALS AND METHODS: Seven asthmatics provided written informed consent and underwent spirometry, plethysmography, and MRI before and after salbutamol inhalation. (3) He and (129) Xe ventilation defect percent (VDP) and ventilation coefficient of variation (COV) were measured. To characterize the airways spatially related to ventilation defects, wall area percent (WA%) and lumen area (LA) were evaluated for two subjects who had thoracic x-ray computed tomography (CT) acquired 1 year before MRI. RESULTS: Before salbutamol inhalation, (129) Xe VDP (8 ± 5%) was significantly greater than (3) He VDP (6 ± 5%, P = 0.003). Post-salbutamol, there was a significant improvement in both (129) Xe (5 ± 4%, P < 0.0001) and (3) He (4 ± 3%, P = 0.001) VDP, and the improvement in (129) Xe VDP was significantly greater (P = 0.008). (129) Xe MRI COV (Pre: 0.309 ± 0.028, Post: 0.296 ± 0.036) was significantly greater than (3) He MRI COV (Pre: 0.282 ± 0.018, Post: 0.269 ± 0.024), pre- (P < 0.0001) and post-salbutamol (P < 0.0001) and the decrease in COV post-salbutamol was significant ((129) Xe, P = 0.002; (3) He, P < 0.0001). For a single asthmatic, a sub-segmental (129) Xe MRI ventilation defect that was visible only before salbutamol inhalation but not visible using (3) He MRI was spatially related to a remodeled fourth generation sub-segmental airway (WA% = 78%, LA = 2.9 mm(2) ). CONCLUSION: In asthma, hyperpolarized (129) Xe MRI may help reveal ventilation abnormalities before bronchodilation that are not observed using hyperpolarized (3) He MRI.

Airway contractility and remodeling: links to asthma symptoms.

Respiratory symptoms are largely caused by obstruction of the airways. In asthma, airway narrowing mediated by airway smooth muscle (ASM) contraction contributes significantly to obstruction. The spasmogens produced following exposure to environmental triggers, such as viruses or allergens, are initially responsible for ASM activation. However, the extent of narrowing of the airway lumen due to ASM shortening can be influenced by many factors and it remains a real challenge to decipher the exact role of ASM in causing asthmatic symptoms. Innovative tools, such as the forced oscillation technique, continue to develop and have been proven useful to assess some features of ASM function in vivo. Despite these technologic advances, it is still not clear whether excessive narrowing in asthma is driven by ASM abnormalities, by other alterations in non-muscle factors or simply because of the overexpression of spasmogens. This is because a multitude of forces are acting on the airway wall, and because not only are these forces constantly changing but they are also intricately interconnected. To counteract these limitations, investigators have utilized in vitro and ex vivo systems to assess and compare asthmatic and non-asthmatic ASM contractility. This review describes: 1- some muscle and non-muscle factors that are altered in asthma that may lead to airway narrowing and asthma symptoms; 2- some technologies such as the forced oscillation technique that have the potential to unveil the role of ASM in airway narrowing in vivo; and 3- some data from ex vivo and in vitro methods that probe the possibility that airway hyperresponsiveness is due to the altered environment surrounding the ASM or, alternatively, to a hypercontractile ASM phenotype that can be either innate or acquired.

Cell-contact-mediated assembly of contractile airway smooth muscle rings.

Microtissues in the shape of toroidal rings provide an ideal geometry to better represent the structure and function of the airway smooth muscle present in the small airways, and to better understand diseases such as asthma. Here, polydimethylsiloxane devices consisting of a series of circular channels surrounding central mandrels are used to form microtissues in the shape of toroidal rings by way of the self-aggregation and -assembly of airway smooth muscle cell (ASMC) suspensions. Over time, the ASMCs present in the rings become spindle-shaped and axially align along the ring circumference. Ring strength and elastic modulus increase over 14 d in culture, without significant changes in ring size. Gene expression analysis indicates stable expression of mRNA for extracellular matrix-associated proteins, including collagen I and lamininsα1 andα4 over 21 d in culture. Cells within the rings respond to TGF-ß1 treatment, leading to dramatic decreases in ring circumference, with increases in mRNA and protein levels for extracellular matrix and contraction-associated markers. These data demonstrate the utility of ASMC rings as a platform for modeling diseases of the small airways such as asthma.

Regional pulmonary response to a methacholine challenge using hyperpolarized (3)He magnetic resonance imaging.

BACKGROUND AND OBJECTIVE: Spirometry is insensitive to small airway abnormalities in asthma. Our objective was to evaluate regional lung structure and function using hyperpolarized (3)He magnetic resonance imaging (MRI) before, during and after a methacholine challenge (MCh). METHODS: Twenty-five asthmatics (mean age = 34 ± 11 years) and eight healthy volunteers (HV) (mean age = 33 ± 11 years) underwent spirometry, plethysmography and hyperpolarized (3)He MRI prior to a MCh. MRI was repeated following the MCh and again 25 min after salbutamol administration. (3)He MRI gas distribution was quantified using semiautomated segmentation of the ventilation defect percent (VDP). Tissue microstructure was measured using the (3)He apparent diffusion coefficient (ADC). Analysis of variance with repeated measures was used to evaluate changes at each time point as well as to determine interactions between regions of interest (ROI) and subject group. Pearson's correlations were performed to evaluate associations between (3)He MRI measurements and established clinical measures. RESULTS: In asthmatics, but not HV, whole-lung ADC was increased post-MCh (P < 0.01). In asthmatics only, ADC was increased post-MCh in posterior ROI (P < 0.01) and all ROI in the superior-inferior direction (P < 0.01). VDP was increased in posterior and inferior ROI (P < 0.001). There was a correlation between VDP and specific airway resistance (r = 0.74, P < 0.0001), dyspnoea score (r = 0.66, P < 0.01) and fractional exhaled nitric oxide (r = 0.45, P < 0.05). CONCLUSIONS: We evaluated the regional pulmonary response to methacholine and salbutamol using (3)He MRI and showed heterogeneous VDP and ADC consistent with bronchoconstriction and gas trapping, respectively, post-MCh. These regional alterations resolved post-salbutamol.

Clinical significance and applications of oscillometry.

Recently, "Technical standards for respiratory oscillometry" was published, which reviewed the physiological basis of oscillometric measures and detailed the technical factors related to equipment and test performance, quality assurance and reporting of results. Here we present a review of the clinical significance and applications of oscillometry. We briefly review the physiological principles of oscillometry and the basics of oscillometry interpretation, and then describe what is currently known about oscillometry in its role as a sensitive measure of airway resistance, bronchodilator responsiveness and bronchial challenge testing, and response to medical therapy, particularly in asthma and COPD. The technique may have unique advantages in situations where spirometry and other lung function tests are not suitable, such as in infants, neuromuscular disease, sleep apnoea and critical care. Other potential applications include detection of bronchiolitis obliterans, vocal cord dysfunction and the effects of environmental exposures. However, despite great promise as a useful clinical tool, we identify a number of areas in which more evidence of clinical utility is needed before oscillometry becomes routinely used for diagnosing or monitoring respiratory disease.

Mechanical determinants of airways hyperresponsiveness.

Asthmatic individuals typically experience exaggerated decrements in their ability to breathe after receiving standardized doses of smooth muscle agonist, a phenomenon known as airways hyperresponsiveness (AHR). Breathing difficulties are caused by excessive narrowing of the pulmonary airways, which is instigated by shortening of the airway smooth muscle (ASM). Exactly why many asthmatic individuals are hyperresponsive, however, remains controversial because of the many varied mechanisms that could possibly be involved. Nevertheless, much of the understanding of AHR comes down to a matter of considering the spatial configuration of the components that make up the airway, and the static and dynamic physical forces these components experience. In this review, we consider these mechanical factors, which are conveniently subdivided into three groups involving (i) the active forces construing to narrow the airways, (ii) the mechanical loads against which these forces must work, and (iii) the geometric transformation of a given degree of ASM shortening into airway narrowing. Each of these groups of factors has potent potential to influence AHR. It is likely, however, that they operate together to produce the AHR characteristic of severe asthma.

Epithelial-interleukin-1 inhibits collagen formation by airway fibroblasts: Implications for asthma.

In asthma, the airway epithelium has an impaired capacity to differentiate and plays a key role in the development of airway inflammation and remodeling through mediator release. The study objective was to investigate the release of (IL)-1 family members from primary airway epithelial-cells during differentiation, and how they affect primary airway fibroblast (PAF)-induced inflammation, extracellular matrix (ECM) production, and collagen I remodeling. The release of IL-1α/ß and IL-33 during airway epithelial differentiation was assessed over 20-days using air-liquid interface cultures. The effect of IL-1 family cytokines on airway fibroblasts grown on collagen-coated well-plates and 3-dimensional collagen gels was assessed by measurement of inflammatory mediators and ECM proteins by ELISA and western blot, as well as collagen fiber formation using non-linear optical microscopy after 24-hours. The production of IL-1α is elevated in undifferentiated asthmatic-PAECs compared to controls. IL-1α/ß induced fibroblast pro-inflammatory responses (CXCL8/IL-8, IL-6, TSLP, GM-CSF) and suppressed ECM-production (collagen, fibronectin, periostin) and the cell's ability to repair and remodel fibrillar collagen I via LOX, LOXL1 and LOXL2 activity, as confirmed by inhibition with ß-aminopropionitrile. These data support a role for epithelial-derived-IL-1 in the dysregulated repair of the asthmatic-EMTU and provides new insights into the contribution of airway fibroblasts in inflammation and airway remodeling in asthma.

Stress and strain in the contractile and cytoskeletal filaments of airway smooth muscle.

Stress and strain are omnipresent in the lung due to constant lung volume fluctuation associated with respiration, and they modulate the phenotype and function of all cells residing in the airways including the airway smooth muscle (ASM) cell. There is ample evidence that the ASM cell is very sensitive to its physical environment, and can alter its structure and/or function accordingly, resulting in either desired or undesired consequences. The forces that are either conferred to the ASM cell due to external stretching or generated inside the cell must be borne and transmitted inside the cytoskeleton (CSK). Thus, maintaining appropriate levels of stress and strain within the CSK is essential for maintaining normal function. Despite the importance, the mechanisms regulating/dysregulating ASM cytoskeletal filaments in response to stress and strain remained poorly understood until only recently. For example, it is now understood that ASM length and force are dynamically regulated, and both can adapt over a wide range of length, rendering ASM one of the most malleable living tissues. The malleability reflects the CSK's dynamic mechanical properties and plasticity, both of which strongly interact with the loading on the CSK, and all together ultimately determines airway narrowing in pathology. Here we review the latest advances in our understanding of stress and strain in ASM cells, including the organization of contractile and cytoskeletal filaments, range and adaptation of functional length, structural and functional changes of the cell in response to mechanical perturbation, ASM tone as a mediator of strain-induced responses, and the novel glassy dynamic behaviors of the CSK in relation to asthma pathophysiology.

Time-Varying Respiratory Mechanics as a Novel Mechanism Behind Frequency Dependence of Impedance: A Modeling Approach.

Frequency dependence of respiratory mechanics is a well-established behavior of the respiratory system and is known to be an indicator of severity of obstructive disease, attributed to both tissue viscoelasticity and heterogeneity of airflow in the lung. Despite the fact that respiratory parameters are known to vary in time, often amplified in disease, all analysis methods assume stationarity or short-time stationarity in the parameters used to describe the respiratory system, and the effects of this assumption have not yet been examined in any detail. Here, using a generalized approach, we developed a theory for time-varying respiratory mechanics in time-frequency domain for analysis of linear time-varying systems, then, we analyzed the same respiratory system model with time-varying parameters in the time domain. Both time-frequency domain and time-domain derivations revealed a striking correlation between time-varying behavior of the respiratory system and frequency dependence of resistance. Remarkably, this phenomenon arose from the amplitude of time variations of the elastance. This links two mechanisms that are known to increase in obstructive disease: apparent low frequency increases in resistance and the time variations of reactance.

Oscillometry and pulmonary magnetic resonance imaging in asthma and COPD.

Developed over six decades ago, pulmonary oscillometry has re-emerged as a noninvasive and effort-independent method for evaluating respiratory-system impedance in patients with obstructive lung disease. Here, we evaluated the relationships between hyperpolarized 3 He ventilation-defect-percent (VDP) and respiratory-system resistance, reactance and reactance area (AX ) measurements in 175 participants including 42 never-smokers without respiratory disease, 56 ex-smokers with chronic-obstructive-pulmonary-disease (COPD), 28 ex-smokers without COPD and 49 asthmatic never-smokers. COPD participants were dichotomized based on x-ray computed-tomography (CT) evidence of emphysema (relative-area CT-density-histogram ≤ 950HU (RA950 ) ≥ 6.8%). In asthma and COPD subgroups, MRI VDP was significantly related to the frequency-dependence of resistance (R5-19 ; asthma: ρ = 0.48, P = 0.0005; COPD: ρ = 0.45, P = 0.0004), reactance at 5 Hz (X5 : asthma, ρ = -0.41, P = 0.004; COPD: ρ = -0.38, P = 0.004) and AX (asthma: ρ = 0.47, P = 0.0007; COPD: ρ = 0.43, P = 0.0009). MRI VDP was also significantly related to R5-19 in COPD participants without emphysema (ρ = 0.54, P = 0.008), and to X5 in COPD participants with emphysema (ρ = -0.36, P = 0.04). AX was weakly related to VDP in asthma (ρ = 0.47, P = 0.0007) and COPD participants with (ρ = 0.39, P = 0.02) and without (ρ = 0.43, P = 0.04) emphysema. AX is sensitive to obstruction but not specific to the type of obstruction, whereas the different relationships for MRI VDP with R5-19 and X5 may reflect the different airway and parenchymal disease-specific biomechanical abnormalities that lead to ventilation defects.

Differential effects of uniaxial and biaxial strain on U937 macrophage-like cell morphology: influence of extracellular matrix type proteins.

Tissue engineering concepts have expanded in the last decade to consider the importance of biochemical signaling from extracellular matrix (ECM) proteins adhered to substrates such as polymeric and ceramic scaffolds. This study investigated combined ECM/mechanical factors on the key signaling cells (macrophages) for wound healing, since previously, mechanical strain and ECM proteins have only been considered separately for their effects on macrophage morphology. Human U937 macrophage-like cells were cultured on a model elastomeric membrane, coated with either collagen type I or poly-RGD peptide (ProNectin). The cells were subjected to cyclic uniform uniaxial or nonuniform biaxial strain with the Flexercell Tension Plus system to simulate strains that various soft tissue implants may undergo during the critical tissue-implant integration period. The surface coatings affected total cellular protein, which was significantly higher in cells on collagen than ProNectin coated surfaces after biaxial, but not uniaxial strain, whereas ProNectin coated surfaces caused a decrease in DNA following uniaxial, but not biaxial strain. Adding the protein coatings that relate to the wound healing process during tissue regeneration, elicited effects specific to the strain type imposed. The combination of these parameters caused changes in U937 macrophage-like cells that should be considered in the outcome of the desired performance in the tissue-material constructs.

Characterization of the Flexcell Uniflex cyclic strain culture system with U937 macrophage-like cells.

Mechanical forces alter many cell functions in a variety of cell types. It has been recognized that stimulation of cells in culture may be more representative of some physiologic conditions. Although there are commercially available systems for the study of cells cultured in a mechanical environment, very little has been documented on the validation techniques for these devices. In this study, Flexcell's recently introduced Uniflex cyclic strain system was programmed to apply 10% longitudinal sinusoidal strain (0.25 Hz) for 48 h to U937 cells cultured on Uniflex plates. Image analysis was employed to characterize the actual strain field. For a chosen amplitude of 10% the applied strain was highly reproducible and relatively uniform (10.6+/-0.2%) in a central rectangular region of the membrane (dimensions of 9.2+/-2 x 13.6+/-0.8 mm2). The strain increased the release of IL-6, esterase and acid phosphatase activity (p<0.05) from adherent U937 cells. Cells also displayed altered morphology, aligning and lengthening with the direction of strain, whereas static cells maintained a round appearance showing no preferred orientation. These data indicate that cyclic mechanical strain applied by the Uniflex strain system modulates U937 cell function leading to selective increases in enzymatic activities as well as orientation in a favored direction.

Cyclic biaxial strain affects U937 macrophage-like morphology and enzymatic activities.

As monocytes migrate to the site of a foreign body and differentiate into mature monocyte-derived macrophages (MDMs), the cells undergo a morphological transformation that involves mechanical stimulation via membrane stretch. Because the site of many cardiovascular implant devices includes substrates that are also undergoing mechanical change, it is of interest to assess the effect of such dynamic conditions on cellular-biomaterial responses. This study investigated the influence of cyclic (0.25 Hz) biaxial strain (maximum 10% amplitude) on human U937 macrophage-like cells cultured on a flexible siloxane membrane. Cell attachment was unaffected by the strain but total protein levels were significantly higher in stimulated cells. Intracellular esterase and released acid phosphatase activities were elevated by dynamic loading in addition to a strain-induced increase of monocyte-specific esterase protein as demonstrated by immunoblotting analysis. The morphology of static cells changed with cyclic strain from a round cell shape to an irregular, spread phenotype with a progressive reorganization of filamentous actin. The focal adhesion protein vinculin showed distinct reorganization in structure going from a well-defined arrangement in static cells to a diffuse staining pattern in mechano-stimulated cells. This study has demonstrated that U937 cells respond to cyclic deformation with an augmentation of select enzymatic activities that have been identified as being important in polymer biodegradation processes, as well as morphological changes, which may be characteristic of mechanical stress-induced cell activation.