An Approach for Mineralizing Perennial Ryegrass Using Supersaturated Calcium-Phosphate Solution

Objectives: For developing bio-based tissue engineering scaffolds an attempt was made to utilize phytoliths of plants. The objective of this study was to achieve the mineralization of perennial ryegrass using simulated body fluid (SBF) solutions at various concentrations, enhanced by the addition of S53P4 bioactive glass (BG).
Methods: A pilot study was conducted to grow perennial ryegrass using various concentrations of SBF and Tris-buffered saline (TBS). Among twelve recipes, two promising formulations were selected for further investigation, as determined by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) analysis. The grass seeds were grown on kitchen paper and watered with ultra-pure water for 9 days. Then the experimental grasses were alternately watered with CaCl₂ and NaHPO₄ solutions followed by the two formulas for an additional 7 days. Three experimental groups were used: 1) Ultra-pure water (control group), 2) 1-fold-SBF, and 3) 1.5-SBF. In addition to SBF, BG S53P4 was added to the group 2 and 3 twice to enhance mineralization. At the end of the experiment, the grass was harvested by cutting it 2cm from the seeds, dried at 60°C for 70h, and then burned in the oven for 1h at 500°C. Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) was used elemental analysis of the ash.
Results: SEM-EDS analysis revealed higher calcium (Ca) and phosphorus (P) concentrations in the grass watered with SBF compared to the control group. Quantitative analysis indicated that groups 2 and 3 exhibited higher dry matter and ash weight percentages than the control group. Additionally, ICP-OES analysis of the ash showed Ca concentrations of 18mg/L, 65mg/L, and 140mg/L, and P concentrations of 17mg/L, 21mg/L, and 43mg/L for groups 1, 2, and 3, respectively.
Conclusions: Irrigating perennial ryegrass with SBF increased mineral deposition within the grass. These findings pave the way for future research opportunities for bio-based tissue engineering scaffolds.