Effects of polyelectrolytic peptides on the quality of mineral crystals grown in vitro

Abstract

Charged amino acids such as arginine, lysine, glutamic acid, and aspartic acid are abundant in noncollagenous proteins that regulate mineralization. Synthetic peptide forms of these amino acids have been shown to affect crystal growth in precipitation of mineral crystals in solution. However, little is known about the effects of these peptides on the viability and phenotype of bone marrow stromal cells (BMSCs) or on the in vitro mineralization process. Bone marrow was harvested from neonatal rat femora and cultured under conditions to induce mineralized nodule formation. Mineralized bone nodules were grown while supplementing the cultures with one of five polyelectrolytes: polystyrene sulfonate (PSS), poly-l-glutamic acid (PLG), poly-l-lysine (PLL), poly-l-aspartic acid (PLA), and sodium citrate (SC), as well as a nontreated control group. The viability and the rate of collagen synthesis under the effect of these agents were characterized by cell-counting and dye-binding assays, respectively. Raman microspectroscopy was conducted on mineralized bone nodules to determine the effect of the polyelectrolytes on the mineralization, type-B carbonation, and crystallinity of the mineral phase. Morphology of resulting mineral crystals was investigated using X-ray diffraction line-broadening analysis (XRD). PSS had toxic effects on cells whereas the remaining agents were biocompatible, as the cell viability was either greater (PLG) or not different from controls. The total collagen production by day 21 was 27% and 42% lower than controls for PLL and PSS, respectively. Culture wells stained positively for alkaline phosphatase in the presence of polyelectrolytes, indicating that osteogenic differentiation was not impacted negatively. Raman microspectroscopy revealed that the type-B carbonation of the crystal lattice increased when treated with PLG, PLL, or PSS. Crystallinity of PLL and PSS was smaller than that of control. The mineral/matrix ratios of nodules did not change with polyelectrolyte treatment, with the exception of the PSS-treated group, which was less mineralized. XRD analysis of bone nodules indicated that PLA-treated samples were significantly longer than controls along the 002 direction. Overall, the results suggest that the polypeptides consisting of charged amino acids are biocompatible and that they have the potential to affect crystal quality and morphology in vitro in the presence of cells. However, the mechanisms by which these effects come into play remain to be investigated.