Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure 1/28th the area of the starting gel in 24 hr. The rate of the process can be regulated by varying the protein content of the lattice, the cell number, or the con- centration of an inhibitor such as Colcemid. Fibroblasts of high population doubling level propagated in vitro, which have left the cell cycle, can carry out the contraction at least as efficiently as cycling cells. The potential uses of the system as an immu- nologically tolerated "tissue" for wound hea ing and as a model for studying fibroblast function are discussed.

Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative

Chemical and topographic substrate surface patterning is recognized as a powerful tool for regulating cell functions. We discuss the relative role of scale and pattern of chemically and topographically patterned surfaces in regulating cell behavior. Chemical patterning achieved using spatial cell-adhesive molecular organization regulates different cell functions depending on its scale (micropattern for cell patterning and derived cell functions, nanopattern for collective cell functions such as adhesion, proliferation, and differentiation). In chemical patterning, a direct and specific cell-sensing mechanism such as integrin-ligand binding governs. Alternatively, topographic modification affects different cell functions depending on its pattern (anisotropic ridges and grooves for contact-guided cell alignment, isotropic textures having randomly or evenly distributed topographic features for collective functions). For all topographic patterns, micro- or nanotopographic scale determines whether specific cel...

Cell Sensing and Response to Micro- and Nanostructured Surfaces Produced by Chemical and Topographic Patterning

Cell biology heavily relies on the behavior of fibrillar structures, such as the cytoskeleton, yet the analysis of their behavior in tissues often remains qualitative. Image analysis tools have been developed to quantify this behavior, but they often involve an image pre-processing stage that may bias the output and/or they require specific software. Here we describe FibrilTool, an ImageJ plug-in based on the concept of nematic tensor, which can provide a quantitative description of the anisotropy of fiber arrays and their average orientation in cells, directly from raw images obtained by any form of microscopy. FibrilTool has been validated on microtubules, actin and cellulose microfibrils, but it may also help analyze other fibrillar structures, such as collagen, or the texture of various materials. The tool is ImageJ-based, and it is therefore freely accessible to the scientific community and does not require specific computational setup. The tool provides the average orientation and anisotropy of fiber arrays in a given region of interest (ROI) in a few seconds.

FibrilTool, an ImageJ plug-in to quantify fibrillar structures in raw microscopy images

Organ printing, a novel approach in tissue engineering, applies layered computer-driven deposition of cells and gels to create complex 3-dimensional cell-laden structures. It shows great promise in regenerative medicine, because it may help to solve the problem of limited donor grafts for tissue and organ repair. The technique enables anatomical cell arrangement using incorporation of cells and growth factors at predefined locations in the printed hydrogel scaffolds. This way, 3-dimensional biological structures, such as blood vessels, are already constructed. Organ printing is developing fast, and there are exciting new possibilities in this area. Hydrogels are highly hydrated polymer networks used as scaffolding materials in organ printing. These hydrogel matrices are natural or synthetic polymers that provide a supportive environment for cells to attach to and proliferate and differentiate in. Successful cell embedding requires hydrogels that are complemented with biomimetic and extracellular matrix components, to provide biological cues to elicit specific cellular responses and direct new tissue formation. This review surveys the use of hydrogels in organ printing and provides an evaluation of the recent advances in the development of hydrogels that are promising for use in skeletal regenerative medicine. Special emphasis is put on survival, proliferation and differentiation of skeletal connective tissue cells inside various hydrogel matrices.

Hydrogels as extracellular matrices for skeletal tissue engineering: state-of-the-art and novel application in organ printing.

Apparatus and method for increasing the sensitivity in the detection of optical coherence tomography and low coherence interferometry (“LCI”) signals by detecting a parallel set of spectral bands, each band being a unique combination of optical frequencies. The LCI broad bandwidth source is split into N spectral bands. The N spectral bands are individually detected and processed to provide an increase in the signal-to-noise ratio by a factor of N. Each spectral band is detected by a separate photo detector and amplified. For each spectral band the signal is band pass filtered around the signal band by analog electronics and digitized, or, alternatively, the signal may be digitized and band pass filtered in software. As a consequence, the shot noise contribution to the signal is reduced by a factor equal to the number of spectral bands. The signal remains the same. The reduction of the shot noise increases the dynamic range and sensitivity of the system.

Apparatus and method for ranging and noise reduction of low coherence interferometry LCI and optical coherence tomography OCT signals by parallel detection of spectral bands

Alignment maps of tissues: I. Microscopic elliptical polarimetry.

A method to image fiber alignment during mechanical testing of soft tissues was developed based on quantitative polarized light microscopy. Images were acquired after passing light through a rotating polarizer, a tissue sample, and an effective circular analyzer at multiple polarizer positions during uniaxial mechanical testing. The image set was analyzed offline using harmonic analysis to generate an alignment image, which contains the direction and strength of alignment at each image pixel. Alignment images of the entire tissue sample were generated every 3-5 s during the mechanical test allowing stress-strain behavior to be correlated with fiber alignment. Loading of fresh tissue-equivalent samples in the direction normal to the initial direction of fiber alignment revealed a spatially inhomogeneous realignment into the loading direction, with most realignment occurring near the free edges undergoing maximum lateral contraction and prior to significant load developing. Glutaraldehyde-fixed samples, in contrast, showed little realignment until yielding occurred.

Fiber alignment imaging during mechanical testing of soft tissues.

Alignment Maps of Tissues: II. Fast Harmonic Analysis for Imaging

We present an easily applicable and inexpensive method for patterning cells on arbitrary surfaces including biological gels with little loss of viability or function. Single-cell suspensions of human umbilical vein endothelial cells and NIH 3T3 fibroblasts were sprayed with an off-the-shelf airbrush through a mask to create 100-µm scale patterns on collagen gels. Three-dimensional patterns were created by layering a collagen gel on top of the first pattern and patterning the top gel. Coculture of rat hepatocytes with NIH 3T3 patterns on collagen gels resulted in localized increased activity of cytochrome P-450 along the pattern. These results suggest that cell spraying is a useful tool for the study of heterotypic cellular interactions and tissue-engineering applications on biologically relevant matrices, and for the creation of three-dimensional cell patterns in vitro.

Cell patterning on biological gels via cell spraying through a mask.

A quantitative analysis of data from an in vitro assay presented allows for simultaneous study of cell migration within as well as cell traction on tissue equivalents (collagen gels seeded with tissue cells to serve as model tissues). Using our anisotropic biphasic theory for modeling tissue equivalents, the ability of the model to simulate the data was validated by accurately predicting the alignment of fibroblasts and collagen fibrils in the compacting collagen gels and the effects of gel aspect ratio on gel compaction. The effects of migration and adhesion modulating factors (platelet-derived growth factor-BB and an anti-β1 integrin antibody), as well as mechanical stress in the collagen network (attached vs. floating gels), on cell migration and traction during compaction were investigated. In all cases, cell migration was negatively correlated with cell traction. The relevance of the results to wound contraction is discussed.

Theodore T. Tower

Papers

Alignment maps of tissues: I. Microscopic elliptical polarimetry.

Fiber alignment imaging during mechanical testing of soft tissues.

Alignment Maps of Tissues: II. Fast Harmonic Analysis for Imaging

Cell patterning on biological gels via cell spraying through a mask.

Estimation of cell traction and migration in an isometric cell traction assay