Response of Monocultured and Co-Cultured Human Microvascular Endothelial Cells and Mesenchymal Stem Cells to Macroporous Granules of Nanostructured-Hydroxyapatite Agglomerates

Recent studies have shown that hydroxyapatite (HA) nanocrystalline have better functional properties that are important to create suitable local conditions for bone formation, when implanted in an osseous environment. Bone formation depends on several complex processes, including a tight communication between endothelial cells and osteoblasts and mesenchymal stem cells. This study examined the interaction between human dermal microvascular endothelial cells (HDMEC) and human mesenchymal stem cells (HMSC), in monoculture and co-culture on,macroporous granules of nanostructured-hydroxyapatite agglomerates. Cell viability/proliferation was assessed through MTT and DNA quantification assays. CLSM and SEM observations allow the study of cell morphology and growth pattern of cells. The angiogenic and osteogenic genes expression were studied using real time PCR and cell differentiation was assessed by ALP activity and matrix mineralization assays. Matrigel tube-like formation assay was also used. Increased expression levels of genes related with osteogenesis and angiogenesis was evident. The osteoblastic phenotype was clearly promoted, as evidenced by the over-expression of osteoblastic genes, increased ALP activity and matrix mineralization. The work clearly demonstrated that the nanostructured-HA granules were able to support cell type's survival, proliferation and individual functionality in a monoculture and co-culture system, for 21 days. HMSC seeded on the granules were able to differentiate into osteoblastic phenotype. The results achieved suggest that nano-structured HA granules may be considered promising implants for bone regeneration and tissue engineering application, in which the granules can be pre-seeded with these two types of autologous cells, before bone graft implant.