Objectives To explore the feasibility of constructing microvascular-like structure through the co-culture technique and establish the related platform for detection.
Methods Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human endothelial cells (ECs) were co-cultured on 3D porous silk scaffold. Cell proliferation was analysed by Pico-green DNA assay. Their growth profiles were evaluated by SEM and confocal microscopy (CLSM), respectively. The gene expression level of vWF and CD31, two key markers of functional ECs, in the co-cultured ECs was assayed by real time RT-PCR.
Results BothhBM-MSCs and ECs exhibited an ideal proliferative activity when they were cocultured on 3D porous silk scaffold. This is evidenced by the increased DNA content on silk scaffold during the cultivation. SEM image revealed that all the cells grew well on the scaffold, and abundant extracellular matrix (ECM) was also observed in the cocultures, indicating the cell-cell and cell-ECM interaction has been well-established. In addition, quantitative RT-PCR detection showed that the transcript expression level of vWF and CD31, two important markers of functional ECs, was up-regulated significantly in the cocultured ECs in comparison with the 2D monoculture. Specifically, the expression level of vWF was 8.6 folds higher than the control. Most importantly, some premicrovascular structures formed by ECs were observed in CLSM detection, further supporting their more differentiated phenotype and improved functionality on silk scaffold. Taken together, it could be concluded that the 3D co-culture system constructed with the silk scaffold provide an ideal microenvironment for cellular growth and differentiation.
Conclusions Constructing three-dimensional (3D) tissue-like structure in vitro plays a center role in the field of tissue engineering and regenerative medicine. Several advances have been made in the past decades; however, it is still challenging to promote some vascular-like structure formation to address the issue of limited mass transportation caused by 3D cultivation in vitro. In the present study, a co-culture system with hMSCs and ECs incorporation on silk porous scaffold was constructed with an aim to generate vascular-like structure. This is important because it has been demonstrated that regeneration of microvascular structure plays a critical role in improving cellular activity in vitro. An increased proliferation and differentiation of ECs on 3D silk scaffold was observed evidenced by the premicrovascular-like structure formation. This suggests that the unique co-culture system could promote ECs differentiation and self-assembling in vitro. Thus, this culture system provides a robust tool for those studies on microvascular-based tissue engineering.