Role of cortico-cancellous heterologous bone in human periodontal ligament stem cell xeno-free culture studied by Synchrotron radiation phase-contrast microtomography

This study was designed to quantitatively demonstrate via three-dimensional (3D)images, through the Synchrotron Radiation Phase-Contrast Microtomography (SR-PhC-MicroCT),the osteoinductive properties of a cortico-cancellous scaffold (Osteobiol Dual Block—DB) culturedwith human Periodontal Ligament Stem Cells (hPDLSCs) in xeno-free media. In vitro cultures ofhPDLSCs, obtained from alveolar crest and horizontal fibers of the periodontal ligament, were seededonto DB scaffolds and cultured in xeno-free media for three weeks. 3D images were obtained bySR-PhC-microCT after one and three weeks from culture beginning. MicroCT data were successivelyprocessed with a phase-retrieval algorithm based on the Transport of Intensity Equation (TIE).The chosen experimental method, previously demonstratively applied for the 3D characterization ofthe same constructs in not xeno-free media, quantitatively monitored also in this case the early stagesof bone formation in basal and differentiating conditions. Interestingly, it quantitatively showedin the xeno-free environment a significant acceleration of the mineralization process, regardlessof the culture (basal/differentiating) medium. This work showed in 3D that the DB guides theosteogenic differentiation of hPDLSCs in xeno-free cultures, in agreement with 2D observationsand functional studies previously performed by some of the authors. Indeed, here we fully provedin 3D that expanded hPDLSCs, using xeno-free media formulation, not only provide the basis forGood Manufacturing Practice (preserving the stem cells’ morphological features and their abilityto differentiate into mesenchymal lineage) but have to be considered, combined to DB scaffolds,as interesting candidates for potential clinical use in new custom made tissue-engineered constructs.