Ex vivo study – a more suitable way for 3D scaffold analysis

4QABH ePOSTER BASIC RESEARCH

Background: Biomaterials have been widely used in regenerative process. Blood is the first component to come into contact with biomaterials and regeneration depends substantially on the cell type recruited after coagulum installation. This will serve as a substrate for cells recruitment. In this context, three-dimensionally (3D) printed scaffolds have been researched for providing homogeneous cell distribution and, consequently, improving osteoconductivity.

Aim/Hypothesis: The purpose of this research was to investigate how previous formation of physiological coagulation (PhC) on 3D scaffolds (SCA) of hydrogel and 20% beta-tricalcium phosphate will ultimately change the patterns of osteoblastic cells behavior in vivo.

Materials and Methods: For PhC formation, SCA were placed in defects created in male Hannover rats calvaria, aged 12 weeks. After 16 hours, the SCA was removed from calvaria defect and transported to lab. Osteoblastic cells (OSB) derived from rat (UMR-106 lineage) were then seeded either on SCA with PhC or without. The groups tested were: SCA + OSB and SCA + PhC + OSB. After 72 hours of OSB cultivation, cell viability and OSB morphology were verified by MTT colorimetric assay and scanning electron microscopy (SEM), respectively. To observe mineralized matrix formation, Alizarin red stain (ARS) assay was performed at 7 and 10 days. Alkaline phosphatase (ALP) and bone sialoprotein (BSP) expression in OSB was investigated after 10 days using indirect immunofluorescence assay. The proteins immunodetection was verified in multiphoton microscopy and quantified by ImageJ software. All results were analyzed by GraphPadPrism 5.0 software using Student's t and Mann-Whitney tests, based on data distribution.

Results: MTT analysis demonstrated more cellular viability in PhC group (P = 0.0169). After 72 hours of cell culture, it was possible to verify in detail PhC phenotyping, enriched with a fibrin network composed with white and red blood cells, and OSB morphology. After 10 days, in ARS assay there was greater mineralized matrix formation (P = 0.0365) and high ALP expression (P = 0.0021) in SCA + OSB group, while BSP expression was more prevalent in the SCA + PhC + OSB group (P = 0.0033).

Conclusions and Clinical Implications: These results show that there is a different effect when an in vitro and ex vivo study is conducted in the same cell type. PhC formation model can be useful when analyzing 3D scaffold that goes to osseous regeneration and this “closer to natural” environment may change completely the scenario of a study.

Acknowledgements: This research was supported by São Paulo Research Foundation – FAPESP (process number #18/12036-3).

Keywords: Blood clot, Hydrogel, Biomaterial, Bone regeneration