Effects of polyelectrolytic peptides on the quality of mineral crystals grown in vitro
Effects of polyelectrolytic peptides on the quality of mineral crystals grown in vitro
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DOI：
10.1007/s00774-008-0869-x
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发表时间：
2008-11-01
影响因子：
3.3
通讯作者：
Akkus, Ozan
中科院分区：
文献类型：
--
作者：
Dziak, Katherine L.;Akkus, Ozan
带电氨基酸如精氨酸、赖氨酸、谷氨酸和天冬氨酸在调节矿化的非胶原蛋白中含量丰富。这些氨基酸的合成肽形式已被证明会影响溶液中矿物晶体沉淀中的晶体生长。然而，很少有人知道这些肽对骨髓基质细胞（BMSC）的活力和表型或体外矿化过程的影响。从新生大鼠股骨收获骨髓，并在诱导矿化结节形成的条件下培养。矿化骨结节生长，同时补充培养物与五种聚电解质之一：聚苯乙烯磺酸盐（PSS），聚-L-谷氨酸（PLG），聚-L-赖氨酸（PLL），聚-L-天冬氨酸（PLA），柠檬酸钠（SC），以及未处理的对照组。分别通过细胞计数和染料结合试验表征了这些试剂作用下的生存力和胶原合成速率。对矿化骨结节进行拉曼显微光谱分析，以确定聚电解质对矿化、B型碳酸化和矿物相结晶度的影响。用X射线衍射谱线展宽分析（XRD）研究了所得矿物晶体的形态。PSS对细胞具有毒性作用，而其余试剂是生物相容性的，因为细胞活力更大（PLG）或与对照无差异。对于PLL和PSS，第21天的总胶原蛋白产量分别比对照低27%和42%。在存在聚电解质的情况下，培养威尔斯孔的碱性磷酸酶染色呈阳性，表明成骨分化没有受到负面影响。拉曼显微光谱表明，B型碳酸化的晶格增加时，处理PLG，PLL，或PSS。PLL和PSS的结晶度小于对照。结核的矿物质/基质比没有改变，除了PSS治疗组，这是较少的矿化。骨结节的XRD分析表明，PLA处理的样品沿002方向比对照组沿着明显更长。总之，结果表明，由带电氨基酸组成的多肽是生物相容的，并且它们在细胞存在下具有影响体外晶体质量和形态的潜力。然而，这些影响发挥作用的机制仍有待研究。
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.