The Influence of Physiological Blood Clot on Osteoblastic Cell Response to a Chitosan-Based 3D Scaffold-A Pilot Investigation.

Background The use of ex vivo assays associated with biomaterials may allow the short-term visualization of a specific cell type response inserted in a local microenvironment. Blood is the first component to come into contact with biomaterials, providing blood clot formation, being substantial in new tissue formation. Thus, this research investigated the physiological blood clot (PhC) patterns formed in 3D scaffolds (SCAs), based on chitosan and 20% beta-tricalcium phosphate and its effect on osteogenesis. Initially, SCA were inserted for 16 h in rats calvaria defects, and, after that, osteoblasts cells (OSB; UMR-106 lineage) were seeded on the substrate formed. The groups tested were SCA + OSB and SCA + PhC + OSB. Cell viability was checked by MTT and mineralized matrix formation in OSB using alizarin red (ARS). The alkaline phosphatase (ALP) and bone sialoprotein (BSP) expression in OSB was investigated by indirect immunofluorescence (IF). The OSB and PhC morphology was verified by scanning electron microscopy (SEM).Results The SCA + PhC + OSB group showed greater cell viability (p = 0.0169). After 10 days, there was more mineralized matrix deposition (p = 0.0365) and high ALP immunostaining (p = 0.0021) in the SCA + OSB group. In contrast, BSP was more expressed in OSB seeded on SCA with PhC (p = 0.0033).Conclusions These findings show the feasibility of using PhC in ex vivo assays. Additionally, its inclusion in the experiments resulted in a change in OSB behavior when compared to in vitro assays. This "closer to nature" environment can completely change the scenario of a study.