Original articleOrthosilicic acid stimulates collagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells in vitro
Introduction
Silicon (Si) is a ubiquitous environmental element found mainly as insoluble silicates, although small amounts of soluble Si are also present in natural waters, chiefly as orthosilicic acid [Si(OH)4] [1]. Around neutral pH, orthosilicic acid polymerises at concentrations much above 2 mM, forming a range of silica species from soluble dimers to colloids and solid phase silica [2]. Some plants and lower animals may promote this reaction, as they use polymeric silica for structure and growth [3], [4], [5]. The normal diet contains (a) orthosilicic acid present in water or following hydrolysis of foods in the gastrointestinal tract, (b) nonhydrolyzed polymeric silica from plants [6], and (c) silicates due mainly to soil and dust contamination or as food additives [7], [8], [9]. Absorption studies have shown that only orthosilicic acid is in a bioavailable form with uptake in humans exceeding 50% of the ingested dose [10], [11]. Fasting concentrations of Si in plasma are 2–10 μM, rising to 20–30 μM after meals, and approximately 700 μmol/day is normally excreted in urine.
In 1972, Carlisle [12] and Schwarz and Milne [13] first reported that silicon deficiency in chicks and rats led to abnormally shaped bones and defective cartilagenous tissue, both of which were restored upon the addition of soluble Si to their diet. This led to the suggestion that Si may play an important role in connective tissue metabolism especially in bone and cartilage. The element’s primary effect in bone and cartilage is thought to be on matrix synthesis rather than mineralization, although its influence on calcification may be an indirect phenomenon through its effects on matrix components [14].
Many of these earlier studies and more recent ones on extracellular matrix formation have been mainly carried out in animals such as chicks [10], rats [15], [16], [13], and calves [17]. The effects of soluble Si on bone matrix synthesis has not been confirmed in humans and species differences may exist. There is also a paucity of studies on the effects and the mechanisms of cellular action of soluble Si on human osteoblasts. Studies of the effects of soluble Si on human osteoblasts in vitro have been done using Zeolite A (ZA) which is a silicon-containing compound [18]. Keeting et al. [18] showed that ZA stimulated the proliferation and differentiation of cultured cells of the osteoblast lineage but they failed to demonstrate any effect of ZA on matrix synthesis. Furthermore, ZA hydrolyzes to release both silicic acid and aluminium salts, and thus the active component of this compound is not established. An important aspect of bone formation is the synthesis and deposition of collagen type 1, which constitutes 90% of the total organic extracellular matrix in mature bone, by preosteoblasts or early undifferentiated osteoblast-like cells [19]. In the present study we examined the effects of soluble Si (orthosilicic acid) on collagen type 1 synthesis in the more early osteoblastic cells. We used (1) the human osteosarcoma cell line MG-63, which represents a homogeneous clonal cell population derived from a specific stage of the osteoblastic lineage [20], (2) primary osteoblast-like cells derived from human bone marrow stromal cells, and (3) a near homogeneous preparation of an osteoblast precursor cell line. This cell line is an immortalized clonal human bone marrow cell line (HCCl) which has been shown to differentiate along the adipogenic and osteogenic lineages with manipulation of culture conditions [21]. As type 1 collagen is also a major constituent of skin, we assessed the effects of soluble Si on human skin fibroblasts too. We also determined the effects of soluble Si on osteoblastic differentiation and sought to ascertain its cellular mechanisms of action in the MG-63 cells.
Section snippets
Study design
The study was divided into two parts. In the first series of experiments we investigated the effects of varying concentrations of soluble Si in the form of orthosilicic acid (Si (OH)4) on collagen synthesis in (a) human osteoblastic cells and (b) skin fibroblasts. In the second set of experiments we examined the influence of soluble Si on (a) osteoblastic differentiation and (b) two of the mechanistic pathways involved in collagen type 1 synthesis (mRNA expression and proline hydroxylation of
Si concentrations in the culture medium
The baseline Si concentration in serum-free DMEM kept in plastic containers was (mean ± SD) 1.60 ± 0.5 μM. Following addition of 10, 20, and 50 μM orthosilicic acid, the final concentrations measured in triplicate were 11.7 ± 1.8, 21.9 ± 2.2, and 50.9 ± 3.1 μM Si, respectively.
Effect of soluble Si on collagen type 1 synthesis
Collagen type 1 synthesis increased in all cell lines tested following treatment with orthosilicic acid at 10 and 20 μM. Treatment of the MG-63 cells with orthosilicic acid at 10 and 20 μM increased the amount of collagen
Discussion
Our results demonstrate that physiological concentrations of Si in the form of orthosilicic acid stimulate collagen type 1 synthesis in human osteoblast-like cells and skin fibroblasts. Treatment with Si also enhanced osteoblastic differentiation. Orthosilicic acid did not alter collagen type 1 gene expression but our results suggest that it may modulate prolyl hydroxylase activity. Further studies are required to confirm this.
The human osteoblastic cell lines were treated with orthosilicic
Acknowledgements
This work was supported by research grants from the Special Trustees of St. Thomas’ Hospital and the Royal Society, London, UK. We thank Dr. B.A. Ashton from the Robert Jones and Agnes Hunt Orthopaedic Hospital, Department of Rheumatology, Oswestry, UK, and Dr Caroline Silve from INSERM U426, Hospital Bichat, Paris, France, for the gift of the HCC1 and MG-63 cells, respectively.
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