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Anal. Chem. 1993, 65, 3378-3381
Osteoblast Attachment Monitored with a Quartz Crystal Microbalance Jody Redepenning,' T. K. Schlesinger,+Eric J. Mechalke, David A. Puleo,' and Rena Bizioss Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304
A quartz crystal microbalanceis used in aqueous solutions to monitor the rate of attachment of osteoblasts, bone-formingcells, to the surface of the crystal. Changes in resonant frequency of the crystal are measured for various surface coverages by osteoblasts. Crystal surface coverages are determinedby digital image processingof scanning electron micrographs. A linear relationship is established between the surface coverages and the changes in resonant frequency of the crystal. The osteoblastsare observed to behave viscoelastically. Hence, the Sauerbrey equation can not be used to describe the relationship between the change in mass of osteoblasts on the surface and the change in resonant frequency of the crystal. Apparent viscosities at 5.0 MHz are also determined for osteoblasts. INTRODUCTION The quartz crystal microbalance (QCM) is a sensitive massmeasuring device based on the piezoelectric properties of a single crystal of quartz. QCMs are frequently used to monitor the thickness of metal coatings deposited under vacuum by evaporation or by sputtering. A comprehensive treatise concerning applications of the quartz crystal microbalance has been prepared by Lu and Czanderna.' It has recently been shown that a quartz crystal microbalance can be used to monitor the mass of species attached to the surface of a crystal while that surface is immersed in s ~ l u t i o n .Mass ~~~ changes in solution as small as a monolayer of iodide ions adsorbing on gold have been measured with a QCM.4 Another report has described the use of a QCM to perform a mass immunosorbent assay.5 Since the initial demonstration that the quartz crystal microbalance can be used in solutions, several reviews of the subject matter have appeared.- The QCM has been demonstrated to be useful in relatively simple systems; however, ita application to complicated biological systems is limited. Noteworthy exceptions are the recent work by Nivens et al., which demonstrated that a QCM can
* T o whom correspondence should be addressed. + Present address: University of Colorado Medical School, Denver, co.
f Center for Biomedical Engineering, university of Kentucky, Lexington, KY 40506. f Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180. (1)Applications of Piezoelectric Quartz Crystal Microbalances, Lu, C., Czanderna, A. W., Eds.; Elsevier: New York, 1984. (2)Konash, P. L.;Bastiaans, G. J. Anal. Chem. 1980,52,1929. (3)Nomura, T.Anal. Chem. Acta 1981,124,81. (4) Deakin, M. R.; Tomi, T. L.; Melroy, 0. R. J. Electroanal. Chem. 1988,243,343. ( 5 ) Ebersole, R. C.; Ward, M. D. J.Am. Chern. SOC.1988,110, 8623. ( 6 ) Deakin, M.R.; Buttry, D. A. Anal. Chem. 1989,61,1147A. (7) Suhumacher. R. Angew. Chem. Int. Ed. Engl. 1990,29,329. (8)Buttry, D. A. Applications of the Quartz Crystal Microbalance to Electrochemistry. InElectroanaZytical Chemistry; Bard, A. J.,Ed.; Marcel Dekker, Inc.: New York, 1991;Vol. 17.
be used to monitor bacterial growth: and the work of Ebersole et al., which showed that growth of Escherichia coli can be monitored indirectly by measuring metabolite production.10 In the discussion that follows, we describe use of a quartz crystal microbalance to monitor a complex system in which osteoblasts attach to the surface of a QCM. We believe that further development of the analytical capabilities of the QCM for continuously monitoring relatively complex biological systems may lead to new devices for remote sensing. Additionally, such devices might serve as alternatives to performing animal studies for drug testing or for monitoring the growth of cells on thin films of new biomaterials.
EXPERIMENTAL SECTION The experimental apparatus we used consists of a 2.54-cm 5-MHz AT-cut crystal (Valpey-Fisher) and an oscillator which drives the crystal at its resonant frequency. Resonant frequencies were measured with a Philips PM6654C counter and transferred to a personal computer through an IEEE interface. Gold electrodes and chromium underlayers were deposited on the crystals by thermal evaporation techniques. The crystals were mounted between two O-rings in a glass O-ring joint. The attachment of osteoblasts to the microbalance was monitored at ambient temperature. Neonatal rat calvaria osteoblasts were used in all experiments. In previous work, the phenotype and function of the osteoblasts were established by the presence of alkaline phosphatase, production of CAMP in response to parathyroid hormone, synthesis of type I collagen matrix, formation of calcium phosphate mineral deposits, and expression of mRNA for bonerelated proteins including osteocalcin, osteonectin, and osteopontin.",'* Sterile procedures were used to maintain the cell line. The growth medium used was Dulbecco's modified Eagle medium (Gibco) with 10% fetal bovine serum (Gibco). The cultures were maintained in a humidified incubator at 37OC in an atmosphere of 5% COd95% air. The growth medium was replaced every 7 days. Before the QCM crystals were exposed to cell suspensions, the cells were lifted from the surface of the culture flask using an enzymatic (trypsin) solution.'* This solution was also used to split the cells in 2-week intervals. Scanningelectron microscopy was performedon a JEOL JSMMOA. Sampleswere fixed for scanningelectronmicroscopy using a 2.5% glutaraldehyde solution in 0.1 M cacodylate buffer (pH 7.2). This was followed by exposure to a 1% osmium tetraoxide solution in 0.1 M cacodylate buffer (pH 7.2). After dehydration in a graded ethanol series, the samples were immersed in hexamethyldisilazane, dried in air, and then sputter-coated with gold. A Hewlett-PackardScanjet was used to digitizethe scanning electron micrographs at 300 dpi and 256 shades of gray. The digitized micrographs were analyzed using NIH Image 1.41, a public domain digital image processing program. In order to determine the percent of the surface covered by osteoblasts, all areas in the digitized images that were covered by osteoblasts (9)Nivens, D. E.; Chambers, J. Q.; Anderson, T. R.; White, D. C. Anal. Chem. 1993,65, 65. (10)Ebersole, R. C.; Foss, R. P.; Ward, M. D. BiolTechnology 1991, 9,450.
(ll)Puleo,D.A.;Holleran,L.A.;Doremus,R.H.;Bizios,R.J.Biorned. Mater. Res. 1991,25, 711. (12)Puleo, D. A.; Preston, K. E.; Shaffer, J. B.; Bizios, R. Biornaterials 1993,14,111.
0003-2700/93/0365-3378$04.00/0 0 1993 Amerlcan Chemlcal Soclety
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 23,DECEMBER I, 1003
were converted to black; i.e., these areas were assigned a value of 255. All arm that were not covered by cells were assigned a value of 0, thereby converting thew areas to white. This processing of the image was nBc888ary because of artifah in the SEM images: some areas of individual c e h appear to be brighter than the uncoated crystal surface and other areas of the same cell appear to be darker thanthe uncoated cryatalsurface. After the entire gray scale image was converted to a black and white image,the percent surface coveragewas determined by calculating the average ugrayness"for all pixels; i.e., the blackness values for all pixels in the image were summed and the result was divided by the number of pixels. The result obtained by dividing this value by 255 was taken to be the fraction of the surface covered by oeteoblasts.
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RESULTS A large decrease in the resonant frequency is immediately observed upon addition of a suspension of osteoblasts to a dry crystal. This stepwise change in resonant frequency is associated with viscous damping of the oscillations by the medium. The magnitude of the viscous damping, which has been studied carefully by other workers, depends on the viecoeity and density of the solution13J4as well as the pressure exerted by the solution on the crystal.0 In all of our experiments the frequency change associated with viscous damping of the crystal was much larger than the frequency change produced by the subsequentattachment of osteoblaete. Because viscous damping occurs on a time scale much faster than that associated with cell attachment, it was possible to use the viscous damping as a marker to identify the time at which the cell suspension was added to the surface of the crystal and subsequent cell attachment began. Figure l a shows a typical example of the response of the microbalance when a suspension of osteoblasts in the growth medium ie added to a dry crystal. In this experiment data points were collected using a 60-8 measuring time. The line shown connecta all of the data points. The f i t data point collected corresponds to the average resonant frequency of the dry crystal over the f i t 60-8 interval of data collection. During the second minute of data collection, an osteoblast suspension was added to the surface of the crystal. Consequently, the third minute of data collection was the f i t interval over which the crystal was viscously damped for the entire period. Following the stepwise decrease in resonant frequency associatedwith viscous damping of the crystal, an additional gradual change in resonant frequency, which is associated with osteoblast attachment to the surface, was observed over the next 6 h. This gradual change in resonant frequency was not observed in the absence of osteoblasts. In Figure lb, the f i t two data points are not shown and the time for the third data point is set to zero. Additionally, the change in resonant frequency is measured from the resonant frequency of the viscously damped crystal (Le., from the resonant frequencyof the third data point) to give the change in resonant frequency caused by osteoblast attachment. In thisparticular experiment, excess cells were used to produce a crystal surface completelycovered with attached osteoblasts. Multiple layers of cells resulted. Note that the resonant frequency changes by approximately 500 Hz over the 6 h required to complete the attachment process. The dashed line in Figure l b is a kinetic fit to the data which assumes that the rate of osteoblast attachment is f i t order in the area of the crystal that remains uncoated. It was originallyhoped that the Sauerbrey equationl6could be used to describe the relationship between the mass of osteoblasts on the surface of the crystal and the change in (13) Kanazawa, K.K.;Gordon, J. G. Anal. Chim. Acta 1986,175,99. (14) Kanazawa, K. K.;Gordon, J. G. A d . Chem. 1886,57,1770. (16) Sauerbrey, G. 2.Phys. 1959,155,206.
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FIguro 1. Change In resonant frequency of crystal upon addltkn of osteoblasts suspendedIn growth mediun: (a,top)raw daw,(b,bottom) same expdmemt with tlme set to zero and change h freqwncy set to zero after crystal Is vkousty damped. -dashed llne b a klnotlc fit WMCh assumes that the rate of the reectkn LB first order h the percentage of surface which remains uncoated ( f I 1 2 = 57.5 mln).
resonant frequency. Having a relationship between the maee of cells on the surface and the change in resonant frequency, it was hoped that it would be possible to monitor the maee of cells on the surface in real time by measuring the change in resonant frequency in real time. Unfortunately, the Sauerbrey equation has been of no use in quantifying osteoblast attachment in the present studies. The Sauerbrey equation applies when thin and rigid layers of material are attached to the surface, but osteoblasts are neither thin nor rigid. In fact, there is no correlation between the change in resonant frequency of the crystal and the change in the mass of cells on the surface of the crystal. However, as will be described below, a good correlation between the change in resonant frequency and the change in crystal surface coverage is observed. In order to establish a relationship between osteoblast attachment and change in resonant frequency, it was necBBBary to resort to procedures currently used to monitor cell attachment; Le., it was necessary to expoae the crystals to osteoblast suspensions for varying periods of time to obtain different surface coverages. Each sample was then prepared for later analysis. This process is typically labor intensive. Our measurement of surface coverages for osteoblast-coated crystals was no exception. We used scanning electron microscopy to provide a correlation between the change in surface coverage by osteoblasts and the change in resonant frequency of the QCM. Figure 2a shows one region of a digitized grayscale image of a crystal partially covered with oeteoblasts. Figure 2b is the same region after the image was converted to the black and white format used to determine
ANALMICM CHEMISTRY. VOL. 85. NO. 23. DECEMBER 1. 1093
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BO 80 100 120 z mm a . c a h t m ar~eshowlnochangel n r e s a ~ n t ~ ~ o t crystal plottedwmwguw permmsvfam owerage by osteoblasts: &pe 5.00 Hr/%;plntercept-25 Hr. Error bars htheprcent surfam coverage are one standard deviaHon determined from thee dMereni miu~aphsfaeachcr/stai.The+30-Hrenorbanon~frequency axis are associated wkh +3oHr random fiuctuaiions of the resonant frequency occwing on tm lima scala of attachment pmceas. The nw drawn mrough the data is me best fn to the average sulace 0
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coverages fa each of the eight crystals.
does not contribute to the overall frequency change observed in the presence of osteoblasts.
DISCUSSION Flgun 2. (a, top) Oignized gayscale image of a scanning elsclmn mlcro@aphf a a surfacepartially coated wim osteoblasts; (b. boilom) same image converted to black and whne fwmat.
the surface coverage. The dimensions of the region shown in these images are approximately 560 pm hy 820 pm. The percent surface coverage for the entire image, of which the images in Figures 2 are only a part, was found to be 47% when the procedure described in the Experimental Section was used. By measuring the change in resonant frequency and the surface coverage by osteoblasts for different crystals, it was possible to construct the calibration curve shown in Figure 3. Eight crystals with different cell coverages were used to establish this calibration curve. Three micrographs were obtained in different arean of each crystal to obtain more aenurancethat the SEMmicrographs, whichshow only asmall fraction of the entire surface, accurately represent the entire surface. After the surface coverage for each of the three micrographs wan determined, the coverages were averaged to estimate the true coverage. The exception to this method of estimating the true coverage is the m e when the surface coveragewaslOO%:asthesurfacecoverageapproacheslOO%, the error in determining the surface coverage approaches zero because there is little error associated with establishing that the crystal surface is completely covered by cells. No calculation of the percent surface coverage from the digitized image was necessary. Visual inspection of the SEM micrographswassufficienttoestablishthatnoportionofthecrystal surface remained uncovered. To demonstrate that adsorption of species from solution were not contributing to the long-term frequency change, blanks were performed in which aliquots of the growth medium that did not contain suspended cells were added to clean crystals. As long as care was taken to establish that the crystaland themedium wereinitiallyatthesametemperature, no change in resonant frequency was observed following the initial viscous damping. We take this an an indication that adsorptionofsolutionconstituentstothesurfaceofthecrystal
ThefittothedatainFigure lbisstrictlyanempiricalone. Upon initial inspection of the frequency response when a large excess of cells wan used, it appeared that the rate of attachment wan first order in surface area. A more rigorous examination of the frequency response produced the fit that is shown in Figure lb. The time required for hall-maximal attachment of osteoblasts is 55-60 min. While we are not eware of a model for cellular attachment that accounts for the firstorder kinetica, our results are consistent with recent work by Malik et al., which describes the morphological changes undergone by osteoblasts during the firstfew hours of attachment to surfaces.16 After settling to the surface in a matter of a few minutes, the cells (which are essentially spherical and 1C-15 pm in diameter when suspended in solution) attach to the surface and begin to spread radially. Although the surface coverage by the cells increases during thespreadingproceas, themassof cellsattachedto the crystal is essentially constant because the rate of cell growth is relativelyslow. In short, even though the maea of cells on the surfaceisfixed,theresonantfrequencychangesasthesurface coverage increases. The cells gradually spread over 2 h to form disk shapea with outer diameters of 40-50 pm and average thicknesses of about 1-2 pm; the thicker regions near the nuclei are approximately 5-10 pm in diameter. Once the osteoblasts have spread to their maximum diameter, their dimensions in the plane of the crystal are much greater than their thickness. but this thickness appears to he large compared to the viscoelastic decay length. Consequently, it is not possible to distinguish between a single layer of cells thatcompletelycovers thesurface and multiple layersof cells. At the 5.0-MHz frequency used in our experiments the cells behave viscoelastically. The Sauerbrey equation does not apply. If one estimates that a single layer of cells on the surface is 1pm thick and that the density of cells is 1ped, then the mass of cells per unit area is lo5n.cm-2. This mass would produce a -5600-Hz change in the resonant frequency (16) MaliL,M.11;Puleo,D.A.;Bizios,R.;Doremlls,R.H.Bio~teMb 1992,13. 123.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 23, DECEMBER 1, 1993
if the Sauerbrey equation were applicable. Thus, the Sauerbrey equation predicts a response 1 order of magnitude larger than that actually seen at 100% ' coverage (Figure 1). Because the cells behave a~ a viscoelastic deposit, there is not a direct correlation between the mass of the cells on the surface and the change in resonant frequency. Additionally, because multiple layers of cells do not produce a greater change in resonant frequency than a single layer, it seems that the shear wave is nearly completely damped by a single layer of cells. This result agrees with calculations of Kanazawa and Gordon which show that in water the shear wave is nearly completely damped over distances of 1 pm.13J4 For comparative purposes it is also valuable to consider changes in frequency due to osteoblasts using a model which assumes that the osteoblasts behave like a viscous medium. In this limit, Kanazawa13.14 has shown that the change in the resonant frequency upon transferring a crystal from vacuum to a solution is given by
= +2(2%)1'2 XPPh
where f o is the resonant frequency of fundamental mode (nominally 5.0 MHz), pl is the density of medium, m is the viscosity of medium, pq is the density of quartz (2.648g.cm3), and is the shear modulus of quartz (2.947x 10" g c m l . ~ - ~ ) . Since m = 1.002 x lo2g c m l d and p1 = 0.9982 g c m 3 for water at 20 OC,.the above equation predicts that a frequency decrease of about 700Hz should be observed upon immersing a dry crystal in water. Given that the decrease in resonant frequency for a single layer of osteoblasts is about 500 Hz greater than that observed for the crystal in the aqueous growth medium, the ratio of the change in frequency in the (17)Dintenfaes, L.Biorheology 1975,12, 253.
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presence of osteoblasts (500 Hz 700 Hz) to the change in frequencyexpected in pure water (700Hz) can be determined. Assuming that the density of the cells is 1 ~ . c m -one ~ , can use this ratio to calculate the ratio of the apparent viscosity of osteoblasts to the viscosity of pure water. Such a calculation gives qmdqwater= 3. So the apparent viscosity of the cells at this frequency is approximately 0.03 g.cmW. Although this apparent viscosity should be used cautiously because osteoblasts are not truly viscous fluids, the value is consistent with those expected for thin lipid bilayers enveloping aqueous intracellular components. For comparative purposes, we point out that at 20 "C the viscosities of ethylene glycol and aniline are 19.9 X lo2and 4.40 X lo2 g.cm1.S1, respectively, and that Dintenfass has estimated the internal viscosity of red blood cells to be between 0.01 and 0.05 g.cm1*s-'.17 Finally,now that a calibrationcurve for the surface coverage of osteoblasts is available, it should be possible to monitor cell growth over a period of severalweeks. It should be possible to monitor cell growth in media with different compositions and on different substrates and to monitor production of mineral deposits by osteoblasts. Although our preliminary experiments over extended periods of time have met with some difficulty because of oscillator drift, we are encouraged by the work of Nivens et al. in which they succeeded in minimizing problems associated with long-term drift of the oscillator during their studies involving bacterial films.9
ACKNOWLEDGMENT The authors acknowledge the Whitaker Foundation for financial support of this work and Mark Deakin for providing a diagram for the oscillator circuit.
RECEIVED May 22,1993. Accepted September 3,1993.' * Abstract published in Advance ACS Abstracts, October 15,1993.
