Use of confocal microscopy imaging for in vitro assessment of adipose-derived mesenchymal stromal cells seeding on acellular dermal matrices: 3D reconstruction based on collagen autofluorescence.

BACKGROUND: Both mesenchymal stromal cells (MSCs) and acellular dermal matrices (ADMs) represent fascinating therapeutic tools in the wound healing scenario. Strategies aimed at combining these two treatment modalities are currently under investigation. Moreover, scarcity of quantitative, nondestructive techniques for quality assessment of engineered tissues poses great limitations in regenerative medicine and collagen autofluorescence-based imaging techniques are acquiring great importance in this setting. OBJECTIVE: Our goals were to assess the in vitro interactions between ADSCs and ADMs and to analyze extracellular-matrix production. METHODS: Adipose-derived MSCs (ADSC) were plated on 8-mm punch biopsies of a commercially available ADM (Integra®). Conventional histology with hematoxylin-eosin staining, environmental scanning electron microscopy, and confocal-laser scanning microscopy were used to obtain imaging of ADSC-seeded ADMs. Collagen production by ADSCs was quantified by mean fluorescence intensity (MFI), expressed in terms of positive pixels/field, obtained through ImageJ software processing of three-dimensional projections from confocal scanning images. Control conditions included: fibroblast-seeded ADM, ADSC- and fibroblast-induced scaffolds, and Integra® alone. RESULTS: ADSCs were efficiently seeded on Integra® and were perfectly incorporated in the pores of the scaffold. Collagen production was revealed to be significantly higher when ADSCs were seeded on ADM rather than in all other control conditions. Collagen autofluorescence was efficiently used as a surrogate marker of ECM production. CONCLUSIONS: Combined therapies based on MSCs and collagenic ADMs are promising therapeutic options for chronic wounds. Not only ADSCs can be efficiently seeded on ADMs, but ADMs also seem to potentiate their regenerative properties, as highlightable from fluorescence confocal imaging.

Comparative study of in-vitro autofluorescence of normal versus non-melanoma-skin-cancer cells at different excitation wavelengths.

In this experimental study the autofluorescence of squamous carcinoma cells, stimulated by 6 different excitation wavelengths in the range 280-533 nm, has been compared with the autofluorescence of normal control keratinocytes. Skin cells were cultivated in vitro, to isolate their characteristic autofluorescence form the more complex one that would be originated by the complete skin tissue. Autofluorescence spectra in the visible range were complemented by absorption measurements. It was observed that the control cells showed characteristic emission (and absorption) structures due to typical endogenous chromophores [FAD and NAD(P)H, lipo-pigments, porphyrins], that were severely dumped in pathological cells. The autofluorescence spectra were then elaborated by multivariate analysis: after a first exploratory data analysis by means of Principal Component Analysis, the whole dataset was used to develop classification models using partial least squares-discriminant analysis, to differentiate between normal and pathological cells. This permitted us to identify the most suitable fluorescence spectral interval, in the 550-670 nm range, to discriminate between normal and pathological behavior, independently on the excitation wavelength.