Innovations in Food Technology | February 2009 | www.innovfoodtech.com62

Evaluation of beneficial health effects ofSikapur® Kieselsäure-Gel on culturedconnective tissue fibroblastsPeter C. Dartsch, Dartsch Scientific GmbH

esides oxygen, silicon is the most abundant element in the earth’s crust. In humans, it belongs to the most abundant trace elements and is

especially associated with bone, skin andblood vessels1-3. Compelling data suggest that silicon is essential for bone formation by affectingcartilage composition and ultimately cartilage calcification4. Silicon has been shown to stimulatecollagen type 1 synthesis and osteoblastic differentiation in human osteoblast-like cells invitro5, improve hair tensile strength6 and posi-tively influences skin surface and its mechanicalproperties as well as hair and nails7. Dietary siliconis predominately derived from solid plant foodsand is readily broken down and absorbed in thegastrointestinal tract in the form of orthosilicicacid2, 8. Its bioavailability seems to be stronglyinfluenced by its speciation such as chemical formor matrix9.

Aim of this studyThis study was undertaken to evaluate some

health effects such as cellular regeneration orvitality of a formulation consisting of silicic acidwith disperse-colloidal silicon dioxide. The questions which should be answered were as follows: 1 Is this specific silicon formulation able to

stimulate basal energy metabolism of culturedconnective tissue fibroblasts in short-termexperiments?

2 Is the formulation able to stimulate vitality andmitotic activity/proliferation in long-termexperiments?

3 Is the formulation able to induce cellular and/orcytoskeletal alterations in cultured connectivetissue fibroblasts as a sign of cellular uptake andresponse?

Silicon formulationSikapur® Kieselsäure-Gel (Silicium Gel F):

Silicic acid gel with disperse-colloidal silicon dioxide (MEDOPHARM Arzneimittel GmbH & Co.KG, D-79238 Ehrenkirchen, Germany). Charge642003; expiry date: 09/2010.

Test concentrationsAccording to the data sheet, the manu-

facturer recommends a daily dosage of 15 ml(approximately 15 g). This dosage results in a concentration of 0.25 mg/ml for a distributionwithin the body fluid (50 to 60 liters) and approx-imately 5 mg/ml for a distribution within theblood fluid (3.5 liters). Depending on the testassay, the test concentrations varied between 0.1 mg/ml and 20 mg/ml.

Cell cultureThis study was carried out with connective

tissue fibroblasts (cell line L-929; established fromnormal subcutaneous and adipose tissue of amale C3H/An mouse; ACC2; Deutsche Sammlungfür Mikroorganismen und Zellkulturen,Braunschweig, Germany). The cells were routinelycultured as mass cultures (cultivated up to 80% to 90% confluency before subculturing) in ahumidified atmosphere of 95% air and 5% CO2 at37°C in an incubator. Cells were taken for theexperiments at passage 28 to 34. Routine culturemedium was RPMI 1640 supplemented with 5%foetal bovine serum and 100 U/ml of penicillinand 100 μg/ml of streptomycin (all cell culturereagents were from PAA Laboratories, Cölbe,Germany).

Direct metabolic stimulation of connective tissue fibroblasts

Experimental design. Cells were grown in 96-well plates for three days to reach approxi-mately 90% confluency. After washing of the cellswith phosphate-buffered saline without calciumand magnesium (PBS-), cells were stimulated withphosphate-buffered saline with calcium and magnesium (PBS+) and containing 5 mM glucose± different Sikapur® concentrations. Energymetabolism was measured by the total cellularredox activity resulting in the cleavage and colour

change of an externally added orange-red tetrazolium dye (WST-1; Roche Diagnostics,Mannheim, Germany) to yield a yellowish water-soluble formazan. The colour change was continuously monitored (one reading per minute;four sec shaking interval before each reading) by atwo-wavelength difference measurement ΔOD450 – 690 nm at 37°C ± 0.1°C using a BioTekElx808 ELISA reader. The double-wavelengthmeasurement proved necessary to eliminateunspecific readings by dye or solution turbidity.For data analysis, ΔOD 450 – 690 nm was corrected by the corresponding blank values. Thelinear increase in ΔOD 450 – 690 nm during theobservation period was taken for the evaluation of the stimulating potential of Sikapur®. All experiments were done in triplicate. Results. As

shown in Figure 1, addition of Sikapur®Kieselsäure-Gel resulted in a marked stimulationof basal energy metabolism of cultured connec-tive tissue fibroblasts for all tested concentrationsin the range from 0.1 to 10 mg/ml. However, the stimulation pattern of the test substanceshowed a Gaussian distribution with a maximumstimulation by approximately 17% for a Sikapur®concentrations of 1 mg/ml. Lower and higher testconcentrations caused stimulation to a lowerextent.

Figure 1: (A) Typical reading of the changes in optical density (ΔOD 450 – 690 nm) with increasing incubation timedemonstrating the linear part of the sigmoidal curve between 40 and 80 min (arrows) as taken in the present setof experiments for quantification and comparison of the results. The curve from 0 to about 30 min demonstratesthe uptake of the test substance until the first effects are achieved and the slowering in the course of the curvebeyond 100 min is due to the increase of tetrazolium dye concentration in the reaction mixture. (B) Graphical presentation of the stimulatory effect of Sikapur® Kieselsäure-Gel on total energy metabolism of cultured connec-tive tissue fibroblasts between 40 to 80 min after addition of the test substance (linear part of the measurementcurves). Data represent mean value ± S.E.M. (standard error of the mean). BoFl = calculated body fluid concen-tration = 0.25 mg/ml; BlFl = calculated blood fluid concentration = 5 mg/ml.

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Long-term effect on vitality and mitoticactivity/cell proliferation

L-929 cells from 80 to 90% confluent masscultures were seeded into 12-well plates (cell density: 25,000 cells/well; 1.8 ml of culture medium) and 96-well plates (cell density: 5,000 cells/well; 180 μl of cul-ture medium) and allowed to attachand spread for 24 hours. Thereafter, cellswere exposed to Sikapur® Kieselsäure-Gel for 1 and 3 days at various concen-trations. Two different examinationswere done for the evaluation of cellvitality and mitotic activity/cell proliferation (all experiments weredone in triplicate):

Enzymatic activity of mitochon-drial dehydrogenases. After exposureof the cells in 96-well plates, culturemedium was discarded and cells werewashed with PBS-. Then, 180 μl of fresh culture medium and 20 μl of XTT wereadded to each well and the cleavage ofthe dye was measured as an end-pointdetermination using a Biotek ELx808ELISA reader with a two-wavelengthdifference measurement ΔOD 450 –690 nm. The optical density was deter-mined at t = 0 min and t = 120 min.The difference was taken to calculateabsolute and relative measurement values in comparison to untreated controls. XTT is a tetrazolium dye whichis cleaved by the activity of mito-chondrial dehydrogenases and yields awater-soluble formazan. Direct cellcounting. After exposure of the cells in12-well-plates, culture medium wasdiscarded and cells were washed withPBS-. Adherent cells were detached bytrypsin/EDTA treatment for 10 min at37°C to yield a homogenous cell suspension and the cell numbers wereexamined by using a Neubauer chamber. At least the cells within 4 different squares of the chamber werecounted. Results. Long-term exposureof L-929 fibroblasts to Sikapur®Kieselsäure-Gel resulted in a stimula-

tion of cell vitality in terms of an increased mito-chondrial dehydrogenase activity (Figure 2). Theincrease in cell vitality/metabolic state wasobserved for both exposure times (1 and 3 days)and was in the range of 5 to 9% at body and blood

fluid concentrations. At concentrations higher than10 mg/ml, cell vitality decreased and became evennegative (= inhibitory effect) at overdosage concentrations of 20 mg/ml. This activated metabolic state was not related to an increasedmitotic or proliferative activity of the cells. A significant difference between the cell numbers ofthe exposed cells in comparison to the untreatedcontrols was not observed (not depicted).

Morphological and cytoskeletal alterations after Sikapur® treatment

The cytoskeleton of eukaryotic cells is adynamic three-dimensional structure that fills thecytoplasm and reacts very fast to changes of theinternal or external conditions. Basically, the primary types of fibres comprising the cyto-skeleton are microfilaments (actin, myosin), micro-tubules (�- and �-tubulin), and intermediate filaments (vimentin, desmin, cytokeratin). Whilemicrofilaments and microtubules are highlydynamic structures and are responsible for movement, cell shape and spindle fibres duringmitosis, the intermediate filaments provide tensile strength to the cell and are the cytoskeletalcomponents which are most stable and lessdynamic10. Experimental design. Cells from 80-90% confluent mass cultures were seeded onsterile round glass coverslips and allowed to attachand spread for 48 hours. Thereafter, cells wereexposed to Sikapur® Kieselsäure-Gel at concentra-tions ranging from 1 mg/ml to 20 mg/ml for 16hours. Cells were then fixed by treatment withmethanol p.a. for 6 min at -20°C. Fixed cells wereexamined for morphological alterations. For stain-ing of non-muscle myosin and microtubules,

methanol-fixed cells were incubated for45 min at 37°C in a moist chambereither with anti-pan myosin antibodiesor anti-�-tubulin antibodies (both anti-bodies were from Amersham Buchler,Braunschweig, Germany). After a PBS-wash, the second TRITC-labelled goatanti-mouse IgG + IgM antibody (SigmaChemie, Deisenhofen, Germany) wasapplied for 60 min at 37°C in a moistchamber. Then, cover slips were washedin PBS- and embedded in Mowiol 4-8811. Results. As depicted in Figure 3,exposure to Sikapur® Kieselsäure-Gelfor 16 hours did not result in markedalterations of cell morphology such asrounding, retraction or flattening of thecells. However, a dose-dependent cyto-plasmic vacuolization was observed attest concentrations ≥ 5 mg/ml. Thecause for this phenomenon remainsunclear, but demonstrates that the cellsobviously responded to the exposure toSikapur® Kieselsäure-Gel. Exposure ofcells to Sikapur® Kieselsäure-Gel didnot result in marked alterations of thecytoskeleton (Figure 4). Althoughmicrotubules seemed to become moreabundant with higher Sikapur® concen-trations, a marked difference was notobserved between all concentrationstested. The intracellular distribution of non-muscle myosin was also unaffected for concentrations up to 5mg/ml in comparison to untreated controls. Thereafter, non-muscle myosinstaining became less prominent withseveral distinct cytoplasmic vacuoles asalready observed at phase contrast (notdepicted).

Summary and conclusionsThe investigations presented here

demonstrate that Sikapur® Kieselsäure-

Figure 2: Graphical presentation of the effect of Sikapur® Kieselsäure-Gel on cell vitality of cultured L-929 fibro-blasts after an exposure time of 1 day (A) and 3 days (B) as calculated from the mitochondrial dehydrogenase activity. Positive changes represent a stimulatory effect, whereas negative changes represent an inhibitory effect.Data represent mean value ± S.E.M. (standard error of the mean). BoFl = calculated body fluid concentration =0.25 mg/ml; BlFl = calculated blood fluid concentration = 5 mg/ml.

Figure 3: Cell morphology of L-929 cells after treatment with Sikapur®Kieselsäure-Gel for 16 hours and methanol fixation. (A) Untreated control cells, (B)1 mg/ml Sikapur®, (C) 5 mg/ml Sikapur®, and (D) 20 mg/ml Sikapur®. Note thedose-dependent vacuolization of the cells at both highest concentrations.Micrographs were taken at phase contrast with an Olympus IX50 inverted micro-scope equipped with an Olympus 20x LC achromate lens. Micrographs weretaken with an Olympus E-10 digital camera at 4 megapixels and optimised usingAdobe Photoshop 7.0.

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Gel is able to stimulate total energy metabolismof cultured connective tissue fibroblasts at con-centrations adequate to a calculated distributionwithin the body or blood fluid in vivo. This meta-bolic stimulation might result in an increased cellular regeneration in the course of wound healing processes. Moreover, the investigationsdemonstrate that long-term exposure of fibro-blasts to Sikapur® Kieselsäure-Gel results in anactivated metabolic state without a coincidentalincrease in mitotic activity or cell proliferation.Thus, this activated metabolic state might result inan increase in cellular regeneration, not by anenhanced proliferative activity of the cells, but bya shorter lag-phase for regenerative activitybecause of the higher basal metabolic state of cellspresent in the subcutaneous tissue.

It should be mentioned here, that Calommeet al.12 have shown in a double-blind clinical studywith 14 healthy subjects that the absorption of silicon is largely related to its speciation and isapproximately 2% for colloidal silicic acid such asSikapur® Kieselsäure-Gel. When calculating the silicon concentrations in the blood fluid at thisabsorption rate, the effects of the lowest test concentration of 0.1 mg/ml have to be taken intoaccount. However, even when considering thisminor absorption rate as stated by Calomme et al.,Sikapur® Kieselsäure-Gel has proved its effective-ness in the investigation presented here.

Author’s address:Prof. Dr. rer. nat. habil.

Peter C. DartschDartsch Scientific GmbHInstitut für zellbiologische

TestsystemeOskar-von-Miller-Straße 10

D-86956 Schongau/Oberbayern,Germany

Tel: +49 (0) 8861 256-5250Fax: +49 (0) 8861 256-7162

Email: pc.dartsch@dartsch-scientific.com

Figure 4: Microtubule network (A, B) and distribution of non-muscle myosin in microfilaments (C, D) in L-929 cellsafter exposure to 5 mg/ml of Sikapur® Kieselsäure-Gel for 16 hours (B, D) in comparison to untreated controls (A, C). For further explanations, see text. Indirect immunofluorescence micrography with a Zeiss IM35 invertedmicroscope equipped for epifluorescence microscopy and a Canon D350 digital camera at 8 megapixels and 400ASA rating. Micrographs were optimised using Adobe Photoshop 7.0.

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3 Martin KR (2007): The chemistry of silica and itspotential health benefits. J Nutr Health Aging 11:94–97.

4 Nielsen FH (1991): Nutritional requirements for boron,silicon, vanadium, nickel, and arsenic: current knowl-edge and speculation. FASEB J 5: 2661-1667.

5 Reffitt DM, Ogston N, Jugdaohsingh R et al (2003):Orthosilicic acid stimulates collagen type 1 synthesisand osteoblastic differentiation in human osteoblast-like cells in vitro. Bone 32: 127–135.

6 Wickett RR, Kossmann E, Barel A et al (2007): Effectof oral intake of choline-stabilized orthosilicic acid onhair tensile strength and morphology in women withfine hair. Arch Dermatol Res 299: 499–505.

7 Barel A, Calomme M, Timchenko A et al (2005): Effectof oral intake of choline-stabilized orthosilicic acid onskin, nails and hair in women with photodamaged skin.Arch Dermatol Res 297: 147–153.

8 Jugdaohsingh R, Anderson SHC, Tucker KL et al.(2002): Dietary silicon intake and absorption. Am JClin Nutr 75: 887-893.

9 Van Dyck K, Van Cauwenbergh R, Robberecht H et al.(1999): Bioavailability of silicon from food and foodsupplements. Analyt Bioanalyt Chem 363: 541-544.

10 Brinkley Br (1982): The cytoskeleton: a perspective.Meth Cell Biol 24: 1-8.

11 Dartsch PC (1990): Mounting media for epifluores-cence microscopy. A comparative study on thecytoskeleton of cultivated eye lens epithelial cells. GITLabormedizin 13: 450-456.

12 Calomme MR, Cos P, D’Haese PC et al. (1998):Absorption of silicon in healthy subjects. In: Collery P,Brätter P, Negretti de Brätter V, Khassanova L, EtienneJ-C, eds. Metal Ions in Biology and Medicine. Vol 5.Paris: John Libbey Eurotext, pp. 228-232.

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