Enzymatically crosslinked carboxymethyl–chitosan/gelatin/nano-hydroxyapatite injectable gels for in situ bone tissue engineering application

Present study reports synthesis and characterization of an enzymatically crosslinked injectable gel (iGel) suitable for cell based bone tissue engineering application. The gel comprises of carboxymethyl–chitosan (CMC)/gelatin/nano-hydroxyapatite (nHAp) susceptible to tyrosinase/p-cresol mediated in situ gelling at physiological temperature. Study revealed that a combination of tyrosinase (60U) and p-cresol (2 mM) as crosslinking agents yield rigid gels at physiological temperature when applied to CMC/gelatin within 35 min in presence or absence of nHAp. Rheological study in conjugation with FT-IR analysis showed that an increase in CMC concentration in the gel leads to higher degree of crosslinking and higher strength. Scanning electron microscopy showed that pore sizes of iGels increased with higher gelatin concentration. In vitro study of osteoblast cell proliferation and differentiation showed that, although all iGels are supportive towards the growth of primary osteoblast cells, GC1:1 supported cellular differentiation to the maximum. Application of iGels in mice revealed that stability of the in situ formed gels depends on the degree of crosslinking and CMC concentration. In conclusion, the iGels may be used in treating irregular small bone defects with minimal clinical invasion as well as for bone cell delivery.

Research Highlights
► Enzymatically crosslinked injectable gel made up of CM-chitosan (C)/gelatin (G)/nHAp.
► Tyrosinase/p-cresol used for crosslinking and in situ gelling of polymers at 37°C.
► 60U tyrosinase and 2mM p-cresol is needed for gelation in 35 min.
► Higher GC ratio manifests lower crosslinking and gel strength but higher porosity.
► GC1:1 shows maximum in vivo gel stability and in vitro osteoblast differentiation.