Abstract
Bone defects repair represents a public and urgent problem in clinical practice, in fact, every year, more than two million patients required new treatments for bone injuries. Today a complete vascularization is strategic in bone formation, representing a new frontier for clinical application. Aim of this research has been developed a three-dimensional (3D) coculture platform using a bovine pericardium collagen membrane (BioR) loaded with human periodontal ligament stem cells (hPDLSCs) and endothelial differentiated cells from hPDLSCs (E-hPDLSCs) able to undergo toward osteoangiogenesis differentiation process. First, we have characterized at confocal laser scanning microscopy (CLSM) level the E-hPDLSCs phenotype profile, through CD31 and CD34 markers expression and the ability to tube vessel formation. Real Time-Polimerase Chain Reaction (RT-PCR) and western blotting analyses revealed the upregulation of Runt-related transcription factor 2 (RUNX2), Collagen 1A1 (COL1A1), Vascular Endothelial Growth Factor-A (VEGF-A) genes and proteins in the living construct composed by hPDLSCs + E-hPDSCs/BioR. Human PDLSCs + E-hPDLSCs/BioR construct showed also an enhacement of de novo synthesis of osteocalcin. Given that, the extracellular-signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) transduction signaling was involved in the osteogenesis and angiogenesis process, the ERK1/2 protein level at biochemical level, in our experimental model, has been investigated. Our results evidenced an upregulation of ERK1/2 proteins level born in the living construct. In conclusion, we believe that the use of the hPDLSCs and E-hPDLSCs coculture togheter with BioR as substrate, could represent an efficient model able to activate through ERK1/2 signaling pathway the osteoangiogenesis process, and then representing a new potential engineered platform for surgeons during the repair and the healing of bone defects.
Highlights
Besides supporting the human body, bone tissue regulates blood pH, acts as a mineral source, and generates hematopoietic stem cells and Mesenchymal Stem Cells (MSCs) [1].Materials 2019, 12, 2157; doi:10.3390/ma12132157 www.mdpi.com/journal/materialsToday, tissue engineering represents a novel approach to repair bone-tissue defects in oral, orthopedic, and maxillofacial surgery and it has as principal actors: i) the cells and ii) the scaffold [2].Scaffolds material are three-dimensional (3D) structures used in tissue engineering that mime the role of extracellular matrix and provide a mechanical support for cells
The aim of the present study is focused on the functionalization, in vitro, of a bovine pericardium collagen membrane with human periodontal ligament stem cells (hPDLSCs) and E-hPDLSCs coculture to obtain an improvement of healing process in terms of bone regeneration and neovascularization when the living construct is implanted in vivo
HPDLSCs at passage 2 (P2) were fixed with paraformaldehyde, stained with toluidine blue and observed with an inverted light microscopy Leica DMIL (Leica Microsystem, Milan, Italy)
Summary
Scaffolds material are three-dimensional (3D) structures used in tissue engineering that mime the role of extracellular matrix and provide a mechanical support for cells. An appropriate scaffold should be osteogenic, i.e. able to induce the formation of bone tissue, osteoinductive, i.e. able to enroll MSCs derived from human tissues, and osteoconductive, i.e., able to support the growth of the bone tissue. The latter aspect of the scaffold properties is focused on the chemical composition and on the spatial geometric model qualified to provide to endothelial cells migration and sprouting of new vessels
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