The Use of Optical Clearing and Multiphoton Microscopy for Investigation of Three-Dimensional Tissue-Engineered Constructs

Calle, Elizabeth A.; Vesuna, Sam; Dimitrievska, Sashka; Zhou, Kevin C.; Huang, Angela; Zhao, Liping; Niklason, Laura E.; Levene, Michael J.

doi:10.1089/ten.tec.2013.0538

Cited by 24 publications

(21 citation statements)

References 29 publications

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“…For example, Calle et al recently investigated the use of the clearing technique BABB in conjunction with two-photon-induced tissue autofluorescence and secondharmonic generation to visualize extracellular matrix molecules in thick samples. 35 Chen et al have applied the commercially available clearing reagent FocusClear to 3D intestinal crypt organoids and biopsied tissues to supplement conventional 2D slice histopathology. 36 Combining acquisition techniques with clearing protocols will allow researchers to maximize the information obtained from tissue-engineered samples and increase the throughput of imaging and analysis of intact 3D tissues.…”

Section: Discussionmentioning

confidence: 99%

Application and Assessment of Optical Clearing Methods for Imaging of Tissue-Engineered Neural Stem Cell Spheres

Boutin

Hoffman–Kim

2015

Tissue Engineering Part C: Methods

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Section: Discussionmentioning

confidence: 99%

Application and Assessment of Optical Clearing Methods for Imaging of Tissue-Engineered Neural Stem Cell Spheres

Boutin

Hoffman–Kim

2015

Tissue Engineering Part C: Methods

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“…Although providing a superior 2-D en face imaging of elastic and collagen fibres, the 3-D assessment by this technique is not real-time in/ex vivo. 32 Real-time in/ex vivo 3-D HD-OCT imaging seems to be relevant to visualize the impact on basic dermal microarchitecture and vascular hole retention during HADM processing.…”

Section: Accepted Articlementioning

confidence: 99%

Recellularizing of human acellular dermal matrices imaged by high‐definition optical coherence tomography

Boone

Draye

Verween

et al. 2015

Experimental Dermatology

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High-definition optical coherence tomography (HD-OCT) permits real-time 3D imaging of the impact of selected agents on human skin allografts. The real-time 3D HD-OCT assessment of (i) the impact on morphological and cellular characteristics of the processing of human acellular dermal matrices (HADMs) and (ii) repopulation of HADMs in vitro by human fibroblasts and remodelling of the extracellular matrix by these cells. Four different skin decellularization methods, Dispase II/Triton X-100, Dispase II/SDS (sodium dodecyl sulphate), NaCl/Triton X-100 and NaCl/SDS, were analysed by HD-OCT. HD-OCT features of epidermal removal, dermo-epidermal junction (DEJ) integrity, cellularity and dermal architecture were correlated with reflectance confocal microscopy (RCM), histopathology and immunohistochemistry. Human adult dermal fibroblasts were in vitro seeded on the NaCl/Triton X-100 processed HADMs, cultured up to 19 days and evaluated by HD-OCT in comparison with MTT proliferation test and histology. Epidermis was effectively removed by all treatments. DEJ was best preserved after NaCl/Triton X-100 treatment. Dispase II/SDS treatment seemed to remove all cellular debris in comparison with NaCl/Triton X-100 but disturbed the DEJ severely. The dermal micro-architectural structure and vascular spaces of (sub)papillary dermis were best preserved with the NaCl/Triton X-100. The impact on the 3D structure and vascular holes was detrimental with Dispase II/SDS. Elastic fibre fragmentation was only observed after Dispase II incubation. HD-OCT showed that NaCl/Triton X-100 processed matrices permitted in vitro repopulation by human dermal fibroblasts (confirmed by MTT test and histology) and underwent remodelling upon increasing incubation time. Care must be taken in choosing the appropriate processing steps to maintain selected properties of the extracellular matrix in HADMs. Processing HADMs with NaCl/Triton X-100 permits in vitro the proliferation and remodelling activity of human dermal fibroblasts. HD-OCT provides unique real-time and non-invasive 3D imaging of tissue-engineered skin constructs and complementary morphological and cytological information.

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“…While other techniques such as spectral ultrasound imaging are applicable to soft tissue, some features of cells and tissue smaller than micrometric resolution are missed [3]. Two-photon and multiphoton microscopy, as well as visualizing the second harmonic generation signals, can potentially provide information at sub-micrometric level and detect ECM fibers in tissue, but these techniques suffer from light scattering and are not recommended for complex structures [4,7,8]. Among available strategies, scanning electron microscopy (SEM), allows for high resolution analysis of various features, from the visualization of the nano-sized architecture of extracellular matrix (ECM) fibers to the morphology of cells [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Bimodal morphological analyses of native and engineered tissues

Baharvand

Martin

Scherberich

2017

Materials Science and Engineering: C

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Assessing the morphological features of native and engineered tissues is pivotal to evaluate their degree of development and to identify possible structure-function relationships. Conventional histological or immunohistochemical imaging of stained sections provides limited information about their architecture. Scanning electron microscopy (SEM) yields sub-micrometric resolution images of tissues, but typically cannot be associated with the morphological structures identified by histology. The aim of this study was to establish a technique based on SEM analysis of sections of paraffin-embedded tissues prepared for histological processing (Histo-SEM) for the assessment of morpho-architectural properties of native and engineered tissues. Histo-SEM was performed on sections of cartilaginous, bone and fibrous tissues, native or engineered, in parallel with histological/immunohistochemical staining. Histo-SEM technique allowed evaluating morpho-architectural features typically unreachable by conventional histological staining, like (i) the extent of cartilage maturation based on collagen fibers' diameter and orientation, (ii) the formation of bone tissue/osteoid based on the presence of nanoscale-dense matrix structures, and (iii) tissue integration and vascularization based on collagen fibers' density and average vessel walls thickness of fibrous tissue growing within ectopically implanted porous materials. In conclusion, Histo-SEM allows integrating bimodal morphological assessments of native or engineered tissues and deriving complementary qualitative and quantitative parameters related to their structural organization and level of maturation.

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The Use of Optical Clearing and Multiphoton Microscopy for Investigation of Three-Dimensional Tissue-Engineered Constructs

Cited by 24 publications

References 29 publications

Application and Assessment of Optical Clearing Methods for Imaging of Tissue-Engineered Neural Stem Cell Spheres

Application and Assessment of Optical Clearing Methods for Imaging of Tissue-Engineered Neural Stem Cell Spheres

Recellularizing of human acellular dermal matrices imaged by high‐definition optical coherence tomography

Bimodal morphological analyses of native and engineered tissues

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