Adipose tissue engineering is investigated for native fat substitutes and wound healing model systems. Research and clinical applications of bioartificial fat require a quantitative and objective method to continuously measure adipogenesis in living cultures as opposed to currently used culture-destructive techniques that stain lipid droplet (LD) accumulation. To allow standardization, automatic quantification of LD size is further needed, but currently LD size is measured mostly manually. We developed an image processing-based method that does not require staining to monitor adipose cell maturation in vitro nondestructively using optical micrographs taken consecutively during culturing. We employed our method to monitor LD accumulation in 3T3-L1 and mesenchymal stem cells over 37 days. For each cell type, percentage of lipid area, number of droplets per cell, and droplet diameter were obtained every 2-3 days. In 3T3-L1 cultures, high insulin concentration (10 microg/mL) yielded a significantly different (p < 0.01) time course of all three outcome measures. In mesenchymal stem cell cultures, high fetal bovine serum concentration (12.5%) produced significantly more lipid area (p < 0.01). Our method was able to successfully characterize time courses and extents of adipogenesis and is useful for a wide range of applications testing the effects of biochemical, mechanical, and thermal stimulations in tissue engineering of bioartificial fat constructs.

Deep tissue injury (DTI) is a serious pressure ulcer, involving a mass of necrotic soft tissue under bony prominences as a consequence of sustained tissue deformations. Though several processes are thought to participate in the onset and development of DTI (e.g., cellular deformation, ischemia, and ischemia-reperfusion), the specific mechanisms responsible for it are currently unknown. Recent work indicated that pathological processes at the cell level, which relate to cell deformation, are involved in the etiology. We hypothesized that sustained tissue deformations can lead to elevated intracellular concentration of cell metabolites, e.g., calcium ion (Ca 2+ ), due to a stretch-induced increase in the local permeability of plasma membranes. This may ultimately lead to cell death due to intracellular cytotoxic concentrations of metabolites. In order to investigate this hypothesis, computational models were developed, for determining compression-induced membrane stretches and trends of times for reaching intracellular cytotoxic Ca 2+ levels due to uncontrolled Ca 2+ influx through stretched membranes. The simulations indicated that elevated compressive cell deformations exceeding 25% induce large tensional strains (>5%, and up to 11.5%) in membranes. These are likely to increase Ca 2+ influx from the extracellular space into the cytosol through the stretched sites. Consistent with this assumption, the Ca 2+ transport model showed high sensitivity of times for cell death to changes in membrane resistance. These results may open a new path in pressure ulcer research, by indicating how global tissue deformations are transformed to plasma membrane deformations, which in turn, affect transport properties and eventually, cell viability.

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