Fluorescent labeling of chitosan for use in non-invasive monitoring of degradation in tissue engineering

Abstract

The establishment of non-invasive analytical tools for assessing the in-situ use of biomaterials for surgical implants or scaffolds in tissue engineering and polymer-based therapies is fundamental. This study established a method for fluorescent tracking of the degradation of a chitosan membrane scaffold for use in vitro in bioreactors and ultimately in vivo. The basis of this tracking system is a fluorescence emitting biomaterial obtained by covalent binding of the fluorophore tetramethylrhodamine isothiocyanate (TRITC) onto the backbone of chitosan. Using confocal microscopy, this study quantitated the reductions in fluorescence intensity of the membrane and correlated these decreases with weight loss during polymer breakdown, thereby providing a technique for non-destructively assessing the extent of degradation of chitosan materials over time in vitro. Using multispectral imaging in a mouse model, the study assessed the degradation profile of the fluorophore-labeled biomaterial in vivo in real time and identified the dispersing pathway of the chitosan membrane degradation products in vivo. The results revealed that TRITC conjugated chitosan was biocompatible and supported bone cell growth. The changes in fluorescence intensity correlated well with weight loss up to 16 weeks of in vitro culture and could be monitored over two weeks in vivo.

Pre-clinical Characterization of Tissue Engineering Constructs for Bone and Cartilage Regeneration

“…The degradation of tetramethylrhodamine isothiocyanate (TRITC)-labeled chitosan membranes were successfully monitored in a subcutaneous mouse model for 2 weeks, and the measured fluorescence intensities correlated well with the weight loss of the implants. 31 Membrane fragments were also observed in the neighboring tissue areas, allowing for monitoring of scaffold clearance. Comparisons have been made between in vitro and in vivo degradation kinetics of hydrolytically degradable fluorescein-tagged poly(ethylene glycol):dextran hydrogels and enzymatically degradable Texas Red-tagged collagen scaffolds.…”

Fluorescent imino and secondary amino chitosans as potential sensing biomaterials

“…So far, fluorescent chitosan amino complexes and derivatives have been prepared for drug releasing (Cui et al, 2011), tissue engineering applications (Cunha-Reis, El Haj, Yang, & Yang, 2013), and live cell imaging (Gonil et al, 2014), among other applications.…”

Zinc Phthalocyanine Labelled Polyethylene Glycol: Preparation, Characterization, Interaction with Bovine Serum Albumin and Near Infrared Fluorescence Imaging in Vivo

“…Near infrared imaging methods have been applied in several medical fields including the diagnosis of cancer, vascular mapping, tissue perfusion, inflammation, atherosclerosis and protease activity [13][14][15][16]. Nowadays, fluorescence imaging has been exploited to track or monitor the fate of biomaterials [17][18][19][20]. Artzi et al investigated in vivo and in vitro tracking of erosion in biodegradable hydrogels using fluorescein and Texas red.…”

“…Lovell et al demonstrated porphyrin cross linked hydrogels for monitoring and surgical resection [18]. Cunha-Reis et al used tetramethylrhodamine isothiocyanate labelling of chitosan to monitor the degradation of chitosan for tissue engineering and identified the dispersion pathway of the chitosan membrane degradation products in vivo [19]. Moller et al synthesized Lucifer yellow tagged hydrogels and monitored the in vivo process by fluorescence imaging [20].…”