Effects of Daidzein, Tempeh, and a Probiotic Digested in an Artificial Gastrointestinal Tract on Calcium Deposition in Human Osteoblast-like Saos-2 Cells

Abstract

Adequate calcium intake is crucial for the prevention and treatment of bone-related issues. Developing a nutritional source of readily bioavailable calcium is particularly significant for individuals deficient in this essential element and at risk of developing osteoporosis. This research aimed to evaluate the impact of tempeh (T), daidzein (D), and Lactobacillus acidophilus (LA) within a simulated intestinal environment consisting of Caco-2 epithelial and Saos-2 cells, focusing on their implications for bone mineralization mechanisms. In the initial phase, calcium bioaccessibility from calcium citrate (CaCt), LA, D, the daidzein combination D–CaCt–LA (D1:1:1), and the tempeh combination T–CaCt–LA (T1:1:1) was assessed through digestion simulation. The calcium content of both untreated and digested samples was determined using atomic absorption spectrometry (AAS). In the subsequent stage, the digested samples were used to induce intestinal absorption in differentiated enterocyte-like Caco-2 cells. The permeable fractions were then evaluated in a culture of osteoblast-like Saos-2 cells. Preliminary cellular experiments employed the MTT assay to assess cytotoxicity. The results indicated that the analyzed products did not influence the deposition of extracellular calcium in Saos-2 cells cultured without mineralization stimulators. The combined formulations of permeable fractions of digested CaCt, LA, D, and T demonstrated the capacity to enhance the proliferation of Saos-2 cells. In Saos-2 cells, D, D1:1:1, and LA showed no discernible impact on intracellular calcium accumulation, whereas T and T1:1:1 reduced the calcium deposits. Additionally, mRNA transcripts and alkaline phosphatase (ALP) activity levels in Saos-2 cells cultured without mineralization induction were unaffected by the analyzed products. An examination of the products revealed no discernible effect on ALP activity or mRNA expression during Saos-2 cell differentiation. Our findings suggest that tempeh, daidzein, and L. acidophilus did not positively impact cellular calcium deposition in Saos-2 cells. However, tempeh, daidzein and its combination, and L. acidophilus might enhance the process of osteogenic differentiation in Saos-2 cells. Nevertheless, this study did not identify any synergistic impact on calcium deposition and the process of osteogenic differentiation in Saos-2 cells of isoflavones and probiotics.

Effects of daily probiotic supplementation with Lactobacillus acidophilus on calcium status, bone metabolism biomarkers, and bone mineral density in postmenopausal women: a controlled and randomized clinical study

“…It revealed promising trends suggesting a potential impact of L. acidophilus on calcium metabolism and bone health. Additionally, our in vitro cell study provided valuable insights by demonstrating that L. acidophilus may enhance osteogenic differentiation in Saos-2 cells ( 25 ), indicating its potential mechanisms of action on bone health. These findings underscored the need for further exploration in a human study.…”

“…This specific strain has garnered significant attention due to its proven effectiveness in promoting bone health and preventing the progression of osteoporosis ( 24 ). L. acidophilus may aid in the process of osteogenic differentiation during bone mineralization, as evidenced by studies conducted on human osteosarcoma Saos-2 cells ( 25 ). In our previous research, we extensively investigated the effects of L. acidophilus supplementation using in vitro digestion models and healthy female rats ( 26 – 30 ), shedding light on its potential implications for calcium status and bone health.…”