Autofluorescence-aided assessment of integration and μ-structuring in chitosan/gelatin bilayer membranes with rapidly mineralized interface in relevance to guided tissue regeneration.

Beyond providing barrier function, the advanced materials in guided tissue regeneration (GTR) concept are further prompt to foster regeneration of distinct interfacing tissues. Herein we develop chitosan (CHT)/gelatin (GEL) bilayer membranes via successive solvent- and freeze-casting procedures and genipin (GEN) cross-linking chemistry. By utilizing the autofluorescence signal from GEN cross-linking products (i.e. the secondary CHT (GEL) amines and GEN esters), the Confocal Fluorescent Microscopy (CFM) identifies the chemical inter-linking as well as physical integration between interface layers. The presence of non- and highly μ-porous and pore-interconnecting regions is demonstrated within cross-sections of membranes with (by weight) prevalent GEL contribution in contrast to the sheet-like organization in membrane with equal presence of components. The constant processing conditions on variable compositions did not significantly affect the pore size distributions (in 1-230 μm range), while pore wall thickness increase up to 220 μm with GEL increase, which also improves the yield stress at compression (from 10 kPa to 19 kPa) and elastic modulus (from 26 kPa to 34 kPa). The rapid mineralization procedure resulted in deposition of non-regular to spherical minerals, containing nonstoichiometric carbonated apatite with Ca/P ration in 1.7-2 range, which demonstrates formation of osseointegrative interface. The fast and high (up to 580%), composition-dependent swelling, as well as 67% to 100% weight loss in 4 weeks in vitro degradation experiment point on membranes' relevance in GTR.