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The capacity of bone grafts to repair critical size defects can be greatly enhanced by the delivery of mesenchymal stem cells (MSCs). Adipose tissue is considered the most effective source of MSCs (ADSCs); however, the efficiency of bone regeneration using undifferentiated ADSCs is low. Therefore, this study proposes scaffolds based on polycaprolactone (PCL), which is widely considered a suitable MSC delivery system, were used as a three-dimensional (3D) culture environment promoting osteogenic differentiation of ADSCs. PCL scaffolds enriched with 5% tricalcium phosphate (TCP) were used. Human ADSCs were cultured in osteogenic medium both on the scaffolds and in 2D culture. Cell viability and osteogenic differentiation were tested at various time points for 42 days. The expression of RUNX2, collagen I, alkaline phosphatase, osteonectin and osteocalcin, measured by real-time polymerase chain reaction was significantly upregulated in 3D culture. Production of osteocalcin, a specific marker of terminally differentiated osteoblasts, was significantly higher in 3D cultures than in 2D cultures, as confirmed by western blot and immunostaining, and accompanied by earlier and enhanced mineralization. Subcutaneous implantation into immunodeficient mice was used for in vivo observations. Immunohistological and micro-computed tomography analysis revealed ADSC survival and activity toward extracellular production after 4 and 12 weeks, although heterotopic osteogenesis was not confirmed -probably resulting from insufficient availability of Ca/P ions. Additionally, TCP did not contribute to the upregulation of differentiation on the scaffolds in culture, and we postulate that the 3D architecture is a critical factor and provides a useful environment for prior-to-implantation osteogenic differentiation of ADSCs.

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Creating a functional vascularized bone tissue remains one of the main goals of bone tissue engineering. Recently, a growing interest in the crosstalk between endothelial cells (EC) and osteoblasts (OB), the two main players in a new bone formation, has been observed. However, only a few reports have addressed a mutual influence of OB and EC on cell proliferation. Our study focuses on this issue by investigating cocultures of human bone-derived cells (HBDC) and human umbilical vein endothelial cells (HUVEC). Three various proportions of cells have been used that is, HBDC:HUVEC 1:1, 1:4, and 4:1 and the cocultures were investigated on day 1, 4, and 7, while HUVEC and HBDC monocultures served as reference. We have detected enhanced alkaline phosphatase (ALP) activity in a direct HBDC-HUVEC coculture. This effect was not observed when cells were separated by an insert, which is consistent with other reports on various OB-EC lineages. The appearance of gap-junctions in coculture was confirmed by a positive staining for connexin 43. The number of cells of both phenotypes has been determined by flow cytometry: CD-31-positive cells have been considered EC, while CD-31-negative have been counted as OB. We have observed an over 14-fold increase in OB number after a week in the 1:4 HBDC:HUVEC coculture as compared with less than fourfold in monoculture. The increase in HBDC number in 1:1 coculture has been less pronounced and has reached the value of about sevenfold. These results correspond well with the cell proliferation rate, which has been measured by 5-bromo-2¢-deoxyuridine incorporation. Moreover, at day 7 EC have been still present in the coculture, which is inconsistent with some other reports. Real-time polymerase chain reaction analysis has revealed the upregulation of ALP and collagen type I genes, but not osteocalcin gene, in all the cocultures grown without pro-osteogenic additives. Our study indicates that HUVEC significantly promote HBDC expansion and upregulate collagen I gene expression in these cells. We believe that these findings have application potency in bone tissue engineering.