OBJECTIVES: Angiogenesis is closely associated with osteogenesis where reciprocal interactions between endothelial and osteoblast cells play an important role in bone regeneration. For these reasons, the aim of this work was to develop a co-culture system to study in detail any time-dependent interactions between human mesenchymal stem cells (HMSC) and human dermal microvascular endothelial cells (HDMEC), co-cultured in a 2D system, for 35 days. MATERIALS AND METHODS: HMSC and HDMEC were co-cultured at a ratio of 1:4, respectively. Single-cell cultures were used as controls. Cell viability/proliferation was assessed using MTT, DNA quantification and calcein-AM assays. Cell morphology was monitored using confocal microscopy, and real time PCR was performed. Alkaline phosphatase activity and histochemical staining were evaluated. Matrix mineralization assays were also performed. RESULTS: Cells were able to grow in characteristic patterns maintaining their viability and phenotype expression throughout culture time, compared to HMSC and HDMEC monocultures. HMSC differentiation seemed to be enhanced in the co-culture conditions, since it was observed an over expression of osteogenesis-related genes, and of ALP activity. Furthermore, presence of calcium phosphate deposits was also confirmed. CONCLUSIONS: This work reports in detail the interactions between HMSC and HDMEC in a long-term co-culture 2D system. Endothelial and mesenchymal stem cells cultured in the present co-culture conditions ensured proliferation and phenotype differentiation of cell types, osteogenesis stimulation and over-expression of angiogenesis-related genes, in the same culture system. It is believed that the present work can lead to significant developments for bone tissue regeneration and cell biology studies.
OBJECTIVES: Angiogenesis is closely associated with osteogenesis where reciprocal interactions between endothelial and osteoblast cells play an important role in bone regeneration. For these reasons, the aim of this work was to develop a co-culture system to study in detail any time-dependent interactions between human mesenchymal stem cells (HMSC) and human dermal microvascular endothelial cells (HDMEC), co-cultured in a 2D system, for 35 days. MATERIALS AND METHODS: HMSC and HDMEC were co-cultured at a ratio of 1:4, respectively. Single-cell cultures were used as controls. Cell viability/proliferation was assessed using MTT, DNA quantification and calcein-AM assays. Cell morphology was monitored using confocal microscopy, and real time PCR was performed. Alkaline phosphatase activity and histochemical staining were evaluated. Matrix mineralization assays were also performed. RESULTS: Cells were able to grow in characteristic patterns maintaining their viability and phenotype expression throughout culture time, compared to HMSC and HDMEC monocultures. HMSC differentiation seemed to be enhanced in the co-culture conditions, since it was observed an over expression of osteogenesis-related genes, and of ALP activity. Furthermore, presence of calcium phosphate deposits was also confirmed. CONCLUSIONS: This work reports in detail the interactions between HMSC and HDMEC in a long-term co-culture 2D system. Endothelial and mesenchymal stem cells cultured in the present co-culture conditions ensured proliferation and phenotype differentiation of cell types, osteogenesis stimulation and over-expression of angiogenesis-related genes, in the same culture system. It is believed that the present work can lead to significant developments for bone tissue regeneration and cell biology studies.
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