There is increasing interest in designing new biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used a hydrophobic crosslinked polyurethane in a typical tissue-engineering approach, that is, the seeding and in vitro culturing of cells using a porous scaffold. Using an electromagnetic bioreactor (magnetic field intensity, 2 mT; frequency, 75 Hz), we investigated the effect of the electromagnetic stimulation on SAOS-2 human osteoblast proliferation and calcified matrix production. Cell proliferation was twice as high; expression of decorin, osteocalcin, osteopontin, type I collagen, and type III collagen was greater (1.3, 12.2, 12.1, 10.0, and 10.5 times as great, respectively); and calcium deposition was 5 times as great as under static conditions without electromagnetic stimulation. RT-PCR analysis revealed the electromagnetically upregulated transcription specific for decorin, fibronectin, osteocalcin, osteopontin, transforming growth factor-β, type I collagen, and type III collagen. The immunolocalization of the extracellular matrix constituents showed their colocalization in the cell-rich areas. The bioreactor and the polyurethane foam were designed to obtain cell colonization and calcified matrix deposition. This cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair.

Human mesenchymal stem cells (MSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications. Exposure of MSCs to physical stimuli favors early and rapid activation of the tissue repair process. In this study we investigated the in vitro effects of pulsed electromagnetic field (PEMF) treatment on the proliferation and osteogenic differentiation of bone marrow MSCs (BM-MSCs) and adipose-tissue MSCs (ASCs), to assess if both types of MSCs could be indifferently used in combination with PEMF exposure for bone tissue healing. We compared the cell viability, cell matrix distribution, and calcified matrix production in unstimulated and PEMF-stimulated (magnetic field: 2 mT, amplitude: 5 mV) mesenchymal cell lineages. After PEMF exposure, in comparison with ASCs, BM-MSCs showed an increase in cell proliferation (p<0.05) and an enhanced deposition of extracellular matrix components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type-I and -III collagens (p<0.05). Calcium deposition was 1.5-fold greater in BM-MSC–derived osteoblasts (p<0.05). The immunofluorescence related to the deposition of bone matrix proteins and calcium showed their colocalization to the cell-rich areas for both types of MSC-derived osteoblast. Alkaline phosphatase activity increased nearly 2-fold (p<0.001) and its protein content was 1.2-fold higher in osteoblasts derived from BM-MSCs. The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis revealed up-regulated transcription specific for bone sialoprotein, osteopontin, osteonectin, and Runx2, but at a higher level for cells differentiated from BM-MSCs. All together these results suggest that PEMF promotion of bone extracellular matrix deposition is more efficient in osteoblasts differentiated from BM-MSCs.

Bioactive glasses synthesized by the sol-gel technique possess many of the qualities associated with an ideal scaffold material for a bone graft substitute. In view of the potential clinical applications, we performed a detailed in vitro study of the biological reactivity of synthesized 58S bioactive glass containing-zinc, in terms of osteoblast morphology, proliferation, and deposition of a mineralized extracellular matrix (ECM). Human Sarcoma Osteoblast (SAOS-2) cells were used to i) assess cytotoxicity by lactate dehydrogenase (LDH) release and ii) evaluate the deposition of a calcified extracellular matrix by ELISA assay and quantitative RT-PCR (qRT-PCR). In comparison with pure silica and 58S, the 58S-Zn0.4 bioglass showed a significant increase in cellular proliferation and deposition of ECM components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, type-I and -III collagens. Calcium deposition was significantly higher than on pure silica and 58S samples. Also Alkaline phosphatase (ALP) activity and its protein content was higher with respect to pure silica and 58S. qRT-PCR analysis revealed the up-regulation of type-I collagen, bone sialoprotein and osteopontin genes. All together these results demonstrate the cytocompatibility of 58S-Zn0.4 bioglass and its capability to promote osteoblast differentiation.