Traction Force Screening Enabled by Compliant PDMS Elastomers.

Actomyosin contractility is an essential element of many aspects of cellular biology and manifests as traction forces that cells exert on their surroundings. The central role of these forces makes them a novel principal therapeutic target in diverse diseases. This requires accurate and higher-capacity measurements of traction forces; however, existing methods are largely low throughput, limiting their utility in broader applications. To address this need, we employ Fourier-transform traction force microscopy in a parallelized 96-well format, which we refer to as contractile force screening. Critically, rather than the frequently employed hydrogel polyacrylamide, we fabricate these plates using polydimethylsiloxane rubber. Key to this approach is that the polydimethylsiloxane used is very compliant, with a lower-bound Young's modulus of ∼0.4 kPa. We subdivide these monolithic substrates spatially into biochemically independent wells, creating a uniform multiwell platform for traction force screening. We demonstrate the utility and versatility of this platform by quantifying the compound and dose-dependent contractility responses of human airway smooth muscle cells and retinal pigment epithelial cells. By directly quantifying the endpoint of therapeutic intent, airway-smooth-muscle contractile force, this approach fills an important methodological void in current screening approaches for bronchodilator drug discovery, and, more generally, in measuring contractile response for a broad range of cell types and pathologies.

Changes in growth plate extracellular matrix composition and biomechanics following in vitro static versus dynamic mechanical modulation.

The objective of this study was to investigate the effects of mechanical modulation parameters on structural proteins biocomposition and mechanical properties of the growth plate. Establishing these parameters is a crucial step in the development of fusionless treatment of scoliosis. In this study, ulna explants from 4-weeks-old (pubertal) swines were used. The biocomposition was characterized using biochemical content evaluation and immunohistochemistry. Mechanical properties were characterized by fitting the data of the stress relaxation curves using a fibril reinforced biphasic model. For the mechanical loading, one static modulation condition and three different dynamic modulation conditions, with similar average stress but different amplitude and frequency values, were performed using a bioreactor. Results showed that static loading triggers a decrease in proteoglycan content and type X collagen in specific zones of the growth plate. These changes can be associated with the observed decrement of permeability in the static group. None of the three conditions evaluated for dynamic modulation affected the growth plate biocomposition and biomechanical responses. Results of this study provides an improved understanding of growth plate responses to mechanical environment, which will be useful in finding the optimal and non-damaging parameters for fusionless treatments based on the mechanical modulation of bone growth.

Growth plate cartilage shows different strain patterns in response to static versus dynamic mechanical modulation.

Longitudinal growth of long bones and vertebrae occurs in growth plate cartilage. This process is partly regulated by mechanical forces, which are one of the underlying reasons for progression of growth deformities such as idiopathic adolescent scoliosis and early-onset scoliosis. This concept of mechanical modulation of bone growth is also exploited in the development of fusionless treatments of these deformities. However, the optimal loading condition for the mechanical modulation of growth plate remains to be identified. The objective of this study was to evaluate the effects of in vitro static versus dynamic modulation and of dynamic loading parameters, such as frequency and amplitude, on the mechanical responses and histomorphology of growth plate explants. Growth plate explants from distal ulnae of 4-week-old swines were extracted and randomly distributed among six experimental groups: baseline ([Formula: see text]), control ([Formula: see text]), static ([Formula: see text]) and dynamic ([Formula: see text]). For static and dynamic groups, mechanical modulation was performed in vitro using an Indexed CartiGen bioreactor. A stress relaxation test combined with confocal microscopy and digital image correlation was used to characterize the mechanical responses of each explant in terms of peak stress, equilibrium stress, equilibrium modulus of elasticity and strain pattern. Histomorphometrical measurements were performed on toluidine blue tissue sections using a semi-automatic custom-developed MATLAB toolbox. Results suggest that compared to dynamic modulation, static modulation changes the strain pattern of the tissue and thus is more detrimental for tissue biomechanics, while the histomorphological parameters are not affected by mechanical modulation. Also, under dynamic modulation, changing the frequency or amplitude does not affect the biomechanical response of the tissue. Results of this study will be useful in finding optimal and non-damaging parameters for the mechanical modulation of growth plate in fusionless treatments.

Compressive mechanical modulation alters the viability of growth plate chondrocytes in vitro.

The aim of this study was to investigate the effect of compressive modulation parameters (mode, magnitude, duration, as well as frequency and amplitude for cyclic modulation) on the viability of growth plate chondrocytes. Swine ulnar growth plate explants (n = 60) were randomly distributed among 10 groups: baseline (n = 1 × 6); culture control (n = 1 × 6); static (n = 3 × 6); and dynamic (n = 5 × 6). Static and dynamic samples were modulated in vitro using a bioreactor. Different compression magnitudes (0.1 MPa or 0.2 MPa), durations (12 h or 24 h), frequencies (0.1 Hz or 1.0 Hz), and amplitudes (30% or 100%) were investigated. Viability was assessed by automatic quantification of number of live/dead cells from confocal images of Live/Dead labeled tissues. Chondrocyte viability was found to be dependent on compression magnitude, duration, frequency, and amplitude in a way that increasing each parameter decreased viability in certain zones of growth plate. More specifically, proliferative and hypertrophic chondrocytes were found to be more sensitive to the applied compression. This study provides an in vitro protocol for studying the effects of compressive modulation on biomechanical and biological responses of growth plate explants, which will be useful in finding efficient and non-detrimental parameters for mechanical modulation of bone growth exploited in scoliosis fusionless treatments.