Sheet-based tissue engineering is an innovative field that has provided the scientific community with new tissue-engineered products such as skin, cornea, heart valves, and vascular grafts. As this area of tissue engineering progresses towards clinical implementation, quality control becomes more and more important. Imaging methods advertise themselves because of their high resolution and good tissue-fluid contrast. We present and compare two methods, one based on a custom-designed automatized large-area confocal scanner that uses backscattered light for image formation, and one based on optical coherence tomography (OCT). In both modalities, additional image processing is used to extract sheet thickness and density information and to create a quantitative tissue thickness map in a fully automated fashion. In test objects (glass of known thickness and scattering samples) and engineered tissue sheets with artificially introduced defects we found high agreement between the two methods in the measurement of thickness and the visual representation of the defects. Both the OCT and the confocal scanner were able to provide high-detail images visually consistent to those obtained with brightfield microscopy. Both OCT and large-area confocal scanning in combination with specialized image processing algorithms promise to provide information on tissue homogeneity, density, and the presence of potential defects in tissue sheets in an unsupervised fashion and thus help establish new quality control methods in sheet-based tissue engineering.
BackgroundConfocal microscopy has become an important option for examining tissues in vivo as a diagnostic tool and a quality control tool for tissue-engineered constructs. Collagen is one of the primary determinants of biomechanical stability. Since collagen is also the primary scattering element in skin and other soft tissues, we hypothesized that laser-optical imaging methods, particularly confocal scattered-light scanning, would allow us to quantify scattering intensity and determine collagen content in biological layers.MethodsWe built a fully automated confocal scattered-light scanner to examine how light scatters in Intralipid, a common tissue phantom, and three-dimensional collagen gels. Intralipid with 0.5%, 1.0%, 1.5%, and 2.0% concentration was filled between precisely spaced glass coverslips. Collagen gels at collagen concentrations from 0.30 mg/mL to 3.30 mg/mL were prepared, and all samples underwent A-mode scanning with multiple averaged scans. In Intralipid samples, light reflected from the upper fluid-glass interface was measured. In collagen gels, average scattering intensity inside the actual gel was measured. In both cases, intensity was correlated with concentration.ResultsBy measuring light attenuation at interface reflections of various thicknesses using our device, we were able to determine that the scattering coefficient at 660 nm of Intralipid at increasing concentrations in water to be 39 cm-1 for each percent increase of Intralipid. We were also able to measure the amount of scattering of various concentrations of collagen in gels directly using backscattered light. The results show a highly linear relationship with an increase of 8.2 arbitrary units in backscattering intensity for every 1 mg increase of collagen within a 1 mL gel volume.ConclusionThe confocal scattered-light scanner allows to accurately quantify scattering in Intralipid and collagen gels. Furthermore, a linear relationship between collagen concentration and intensity was found. Confocal scattered-light scanning therefore promises to allow imaging of collagen content in soft tissue layers.
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