In vitro osteoblastic differentiation of human mesenchymal stem cells and human dental pulp stem cells on poly-L-lysine-treated titanium-6-aluminium-4-vanadium.

Three-dimensional (3D) titanium-6-aluminium-4-vanadium (Ti6Al4V) is a widely used biomaterial for orthopedic prosthesis and dental implants; thanks to its very high-mechanical strength and resistance to corrosion. Human mesenchymal stem cells (hMSCs) and dental pulp stem cells (hDPSCs) are responsible for bone regeneration following colonization of prosthesis or dental implants. Both hMSCs and hDPSCs have lower ability to colonize this biomaterial in comparison with tissue culture-treated plastic. Both hMSCs and hDPSCs show lack of focal adhesion kinase (FAK) activation when grown on Ti6Al4V. This signal is restored in the presence of poly-L-lysine (poly-L-lys). Poly-L-lys has been used as part of organoapatite or together with zinc and calcium ions. Our results suggest that poly-L-lys alone induces FAK activation through ß1-INTEGRIN, because the presence of ß1-INTEGRIN blocking antibody avoided FAK autophosphorylation. Presence of poly-L-lys also increases expression of osteoblastic differentiation marker genes in hMSCs and hDPSCs grown on Ti6Al4V.

In vitro calcified matrix deposition by human osteoblasts onto a zinc-containing bioactive glass.

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.

In vitro enhancement of SAOS-2 cell calcified matrix deposition onto radio frequency magnetron sputtered bioglass-coated titanium scaffolds.

In bone tissue engineering, bioglass coating of titanium (Ti) scaffolds has drawn attention as a method to improve osteointegration and implant fixation. In this in vitro study, bioactive glass layers with an approximate thickness of 1 microm were deposited at 200 degrees C onto a three-dimensional Ti-6Al-4V scaffold using a radio frequency (r.f.) magnetron sputtering system. After incubation with SAOS-2 human osteoblasts, in comparison with the uncoated scaffolds, the bioglass-coated scaffolds showed a twofold increase in cell proliferation (p < 0.05) up to 68.4 x 10(6), and enhanced the deposition of extracellular matrix components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type-I and -III collagens (p < 0.05). Calcium deposition was twofold greater on the bioglass-coated scaffolds (p < 0.05). The immunofluorescence related to the preceding bone matrix proteins and calcium showed their colocalization to the cell-rich areas. Alkaline phosphatase activity increased twofold (p < 0.001) and its protein content was threefold higher with respect to the uncoated sample. Quantitative reverse transcriptase-polymerase chain reaction analysis revealed upregulated transcription specific for type-I collagen and osteopontin (p < 0.001). All together, these results demonstrate that the bioglass coating of the three-dimensional Ti scaffolds by the r.f. magnetron sputtering technique determines an in vitro increase of the bone matrix elaboration and may potentially have a clinical benefit.

Effects of electromagnetic stimulation on calcified matrix production by SAOS-2 cells over a polyurethane porous scaffold.

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-beta, 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.