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Characterization of a decellularized rat larynx: comparison between microscopy techniques
Gentilin Erica1, Simoni Edi1, Albertin Giovanna2
1Bioacoustics Research Laboratory, Department of Neurosciences, University of Padua, via G. Orus, 2b, 35129 Padua, Italy; LIFELAB Program, Consorzio per la Ricerca Sanitaria-CORIS, Veneto Region, 35128 Padua, Italy.
Summary
A new whole larynx decellularization protocol effectively removes cells while preserving the extracellular matrix. Multiphoton laser scanning microscopy (MPM) offers superior collagen quantification and elastin detection compared to traditional histological stains.
Area of Science:
- Biomaterials Science
- Tissue Engineering
- Regenerative Medicine
Background:
- Laryngeal reconstruction remains a significant clinical challenge.
- Decellularization offers a promising approach by utilizing extracellular matrix (ECM) scaffolds.
- Current methods for evaluating ECM components like collagen and elastin are variable.
Purpose of the Study:
- Develop an efficient whole organ decellularization protocol for the larynx.
- Compare microscopy techniques for quantitative collagen and elastin assessment in decellularized tissues.
Main Methods:
- Hematoxylin/eosin, Masson Trichrome, DAPI staining, and DNA quantification for decellularization assessment.
- Van Gieson stain, Picrosirius Red stain (PRS), and multiphoton laser scanning microscopy (MPM) for collagen analysis.
- Weigert stain and MPM for elastin detection and quantification.
Main Results:
- Decellularization successfully removed cells without altering glycosaminoglycan, collagen, or elastin levels.
- MPM provided accurate collagen quantification, outperforming Van Gieson and PRS.
- Polarized microscopy revealed decellularization's impact on the 3D collagen network homogeneity.
- MPM and Weigert stain yielded comparable elastin detection results.
Conclusions:
- An effective whole larynx decellularization protocol was established, preserving ECM integrity.
- MPM is recommended for precise collagen quantification and elastin assessment.
- Specific microscopy techniques are highlighted for visualizing collagen fibers and the 3D network.
