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

Abstract

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.

Optical coherence tomography for assessment of epithelialization in a human ex vivo wound model

“…Although the VivoSight Dx is already in clinical use, the lack of HD of its OCT images does not provide adequate resolution to differentiate between individual cells or all five strata of epidermis as traditional H&E histology. However, newer improved HD‐OCT devices can provide real‐time three‐dimensional images with higher resolution that can visualize individual cells and have been successfully utilized to assess skin in vitro and in vivo . The depth at which an OCT device can reach as well as the resolution that an OCT device can provide depends on wavelength .…”

“…It is able to distinguish epidermis, cornified epidermis, papillary dermis, and reticular dermis as well as skin appendages such as hair follicles, blood vessels, and sebaceous glands . Newer high‐definition (HD) OCT devices can produce real‐time three‐dimensional images with adequate resolution to visualize individual cells and have been used to assess fibroblasts in culture, recellularization of acellular dermal matrices, and age‐related morphological skin changes in vivo …”

Monitoring calcium-induced epidermal differentiation in vitro using multiphoton microscopy

“…For example, these techniques have already been used for noninvasive in vivo studies of tissue regeneration. 46,47 Our rationale for choosing MPM was primarily based on its ability to visualize intrinsic tissue fluorescence, and future work will explore the possibility of combining the morphological data with spectral signatures and FLIM [27][28][29][30] to shed further light on the metabolic aspects of Ca 2þ -induced in vitro epidermal differentiation in the context of in vitro tissue culturing.…”

Swim bladder-derived biomaterials: structures, compositions, properties, modifications, and biomedical applications

“…Thus, allogeneic or xenogeneic tissues or organs can be reused for repair and reconstruction after tissue injury in vivo and/or in vitro. To improve the efficiency of decellularization, physical, chemical, and biological methods are often combined [ 11 , 94 – 107 ]. Generally, the decellularization step involves destroying the cell membrane using physical or chemical solvents, separating the cell components from the ECM using enzymes, and finally separating the cell fragments from the ECM [ 11 ].…”