Effects of dynamic matrix remodelling on en masse migration of fibroblasts on collagen matrices.

Fibroblast migration plays a key role during various physiological and pathological processes. Although migration of individual fibroblasts has been well studied, migration in vivo often involves simultaneous locomotion of fibroblasts sited in close proximity, so-called 'en masse migration', during which intensive cell-cell interactions occur. This study aims to understand the effects of matrix mechanical environments on the cell-matrix and cell-cell interactions during en masse migration of fibroblasts on collagen matrices. Specifically, we hypothesized that a group of migrating cells can significantly deform the matrix, whose mechanical microenvironment dramatically changes compared with the undeformed state, and the alteration of the matrix microenvironment reciprocally affects cell migration. This hypothesis was tested by time-resolved measurements of cell and extracellular matrix movement during en masse migration on collagen hydrogels with varying concentrations. The results illustrated that a group of cells generates significant spatio-temporal deformation of the matrix before and during the migration. Cells on soft collagen hydrogels migrate along tortuous paths, but, as the matrix stiffness increases, cell migration patterns become aligned with each other and show coordinated migration paths. As cells migrate, the matrix is locally compressed, resulting in a locally stiffened and dense matrix across the collagen concentration range studied.

Role of intracellular poroelasticity on freezing-induced deformation of cells in engineered tissues.

Freezing of biomaterials is important in a wide variety of biomedical applications, including cryopreservation and cryosurgeries. For the success of these applications to various biomaterials, biophysical mechanisms, which determine freezing-induced changes in cells and tissues, need to be well understood. Specifically, the significance of the intracellular mechanics during freezing is not well understood. Thus, we hypothesize that cells interact during freezing with the surroundings such as suspension media and the extracellular matrix (ECM) via two distinct but related mechanisms-water transport and cytoskeletal mechanics. The underlying rationale is that the cytoplasm of the cells has poroelastic nature, which can regulate both cellular water transport and cytoskeletal mechanics. A poroelasticity-based cell dehydration model is developed and confirmed to provide insight into the effects of the hydraulic conductivity and stiffness of the cytoplasm on the dehydration of cells in suspension during freezing. We further investigated the effect of the cytoskeletal structures on the cryoresponse of cells embedded in the ECM by measuring the spatio-temporal intracellular deformation with dermal equivalent as a model tissue. The freezing-induced change in cell, nucleus and cytoplasm volume was quantified, and the possible mechanism of the volumetric change was proposed. The results are discussed considering the hierarchical poroelasticity of biological tissues.

Thermomechanical analysis of freezing-induced cell-fluid-matrix interactions in engineered tissues.

Successful cryopreservation of functional engineered tissues (ETs) is significant to tissue engineering and regenerative medicine, but it is extremely challenging to develop a successful protocol because the effects of cryopreservation parameters on the post-thaw functionality of ETs are not well understood. Particularly, the effects on the microstructure of their extracellular matrix (ECM) have not been well studied, which determines many functional properties of the ETs. In this study, we investigated the effects of two key cryopreservation parameters--(i) freezing temperature and corresponding cooling rate; and (ii) the concentration of cryoprotective agent (CPA) on the ECM microstructure as well as the cellular viability. Using dermal equivalent as a model ET and DMSO as a model CPA, freezing-induced spatiotemporal deformation and post-thaw ECM microstructure of ETs was characterized while varying the freezing temperature and DMSO concentrations. The spatial distribution of cellular viability and the cellular actin cytoskeleton was also examined. The results showed that the tissue dilatation increased significantly with reduced freezing temperature (i.e., rapid freezing). A maximum limit of tissue deformation was observed for preservation of ECM microstructure, cell viability and cell-matrix adhesion. The dilatation decreased with the use of DMSO, and a freezing temperature dependent threshold concentration of DMSO was observed. The threshold DMSO concentration increased with lowering freezing temperature. In addition, an analysis was performed to delineate thermodynamic and mechanical components of freezing-induced tissue deformation. The results are discussed to establish a mechanistic understanding of freezing-induced cell-fluid-matrix interaction and phase change behavior within ETs in order to improve cryopreservation of ETs.

Effects of freezing-induced cell-fluid-matrix interactions on the cells and extracellular matrix of engineered tissues.

The two most significant challenges for successful cryopreservation of engineered tissues (ETs) are preserving tissue functionality and controlling highly tissue-type dependent preservation outcomes. In order to address these challenges, freezing-induced cell-fluid-matrix interactions should be understood, which determine the post-thaw cell viability and extracellular matrix (ECM) microstructure. However, the current understanding of this tissue-level biophysical interaction is still limited. In this study, freezing-induced cell-fluid-matrix interactions and their impact on the cells and ECM microstructure of ETs were investigated using dermal equivalents as a model ET. The dermal equivalents were constructed by seeding human dermal fibroblasts in type I collagen matrices with varying cell seeding density and collagen concentration. While these dermal equivalents underwent an identical freeze/thaw condition, their spatiotemporal deformation during freezing, post-thaw ECM microstructure, and cellular level cryoresponse were characterized. The results showed that the extent and characteristics of freezing-induced deformation were significantly different among the experimental groups, and the ETs with denser ECM microstructure experienced a larger deformation. The magnitude of the deformation was well correlated to the post-thaw ECM structure, suggesting that the freezing-induced deformation is a good indicator of post-thaw ECM structure. A significant difference in the extent of cellular injury was also noted among the experimental groups, and it depended on the extent of freezing-induced deformation of the ETs and the initial cytoskeleton organization. These results suggest that the cells have been subjected to mechanical insult due to the freezing-induced deformation as well as thermal insult. These findings provide insight on tissue-type dependent cryopreservation outcomes, and can help to design and modify cryopreservation protocols for new types of tissues from a pre-developed cryopreservation protocol.

Spatiotemporal measurement of freezing-induced deformation of engineered tissues.

In order to cryopreserve functional engineered tissues (ETs), the microstructure of the extracellular matrix (ECM) should be maintained, as well as the cellular viability since the functionality is closely related to the ECM microstructure. Since the post-thaw ECM microstructure is determined by the deformation of ETs during cryopreservation, freezing-induced deformation of ETs was measured with a newly developed quantum dot (QD)-mediated cell image deformetry system using dermal equivalents as a model tissue. The dermal equivalents were constructed by seeding QD-labeled fibroblasts in type I collagen matrices. After 24 h incubation, the ETs were directionally frozen by exposing them to a spatial temperature gradient (from 4 degrees C to -20 degrees C over a distance of 6 mm). While being frozen, the ETs were consecutively imaged, and consecutive pairs of these images were two-dimensionally cross-correlated to determine the local deformation during freezing. The results showed that freezing induced the deformation of ET, and its magnitude varied with both time and location. The maximum local dilatation was 0.006 s(-1) and was always observed at the phase change interface. Due to this local expansion, the unfrozen region in front of the freezing interface experienced compression. This expansion-compression pattern was observed throughout the freezing process. In the unfrozen region, the deformation rate gradually decreased away from the freezing interface. After freezing/thawing, the ET experienced an approximately 28% decrease in thickness and 8% loss in weight. These results indicate that freezing-induced deformation caused the transport of interstitial fluid, and the interstitial fluid was extruded. In summary, the results suggest that complex cell-fluid-matrix interactions occur within ETs during freezing, and these interactions determine the post-thaw ECM microstructure and eventual post-thaw tissue functionality.

High-resolution broadband N2 coherent anti-Stokes Raman spectroscopy: comparison of measurements for conventional and modeless broadband dye lasers.

We have performed high-resolution N2 coherent anti-Stokes Raman spectroscopy (CARS) measurements using a modeless dye laser (MDL) as the Stokes beam source to determine the effects of a reduction in mode noise on the accuracy and precision of the method. These results are compared with previous research that employed a conventional broadband dye laser (CBDL) as the Stokes beam source. A new spectral-fitting procedure was developed to avoid starting-point bias in the least-squares fitting results, which possibly had altered the previous measurements. Single-shot measurements of pressure were performed in a static-pressure vessel over the range of 0.1-4.0 atm to examine the pressure sensitivity of the technique. The precision of these measurements is a measure of the baseline noise level of the system, which sets the detection limit for flow-field pressure fluctuations. Centerline measurements of pressure and temperature in an underexpanded jet (Mj = 1.85) were also used to determine the performance of the technique in a compressible flow field. Our study represents the first known application, to our knowledge, of a MDL CARS system in a low-temperature, low-pressure supersonic environment. Improvements in accuracy for mean single-shot measurements and increased precision were found for pressure vessel conditions above 1.0 atm. For subatmospheric pressure vessel conditions (0.1-1.0 atm) and the underexpanded jet measurements, there was a decrease in accuracy and precision compared with the CBDL results. A comparison with the CBDL study is included, along with a discussion of the MDL system behavior.