Cell death after cartilage impact occurs around matrix cracks

Journal o f

ELSEGIER

Orthopaedic Research

Journal of Orthopaedic Research 21 (2003) 881-887

www.elsevier.com/locate/orthres

Cell death after cartilage impact occurs around matrix cracks * Jack L. Lewis a, Laurel B. Deloria a, Michelle Oyen-Tiesma Roby C. Thompson Jr. a, Marna Ericson ‘, Theodore R. Oegema Jr. Department of Orthopuedic Surgery, University of Minnesotu, Minneapolis. M N 55455. USA Department of Chemical Engineering and Muteriuls Science, University of Minnesotu, Minneapolis, M N 55455, U S A Department of Dermatology, University of Minnesota, Minneapolis, M N 55455, U S A Depurtment of Binchemi.stry, Moleculur Biology and Biophysic.v, University of Minnesota, Minnrapoh, M N 55455. USA

Accepted 27 January 2003

Abstract The damage from rapid high energy impacts to cartilage may contribute to the development of osteoarthritis (OA). Understanding how and when cells are damaged during and after the impact may provide insight into how these lesions progress. Mature bovine articular cartilage on the intact patella was impacted with a flat impacter to 53 MPa in 250 ms. Cell viability was determined by culturing the cartilage with nitroblue tetrazolium for 18 h or for 4 days in medium containing 5% serum before labeling (5-day sample) and compared to adjacent, non-impacted tissue as viable cells per area. There was a decrease in viable cell density only in specimens with macroscopic cracks and the loss was localized primarily near matrix cracks, which were in the upper 25% of the tissue. This was confirmed using confocal microscopy with a fluorescent livddead assay, using 5’-chloromethylfluorescein diacetate and propidium iodide. Cell viability in the impacted regions distant from visible cracks was no different than the non-impacted control. At 5 days, viable cell density decreased in the surface layer in both the control and impacted tissue, but there was no additional impact-related change. In summary, cell death after the impaction of cartilage on bone occurred around impact induced cracks, but not in impacted areas without cracks. If true in vivo, early stabilization of the damaged area may prevent late sequelae that lead to OA. 0 2003 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Kqword.s: Cartilage; Impact; Crack; Surface; Cell viability

Introduction

Although acute joint injury is related to a subset of osteoarthritic conditions, the pathway that leads from impact to osteoarthritis (OA) is unknown [7,10,11, 13,151. Osteochondral injury with cell death and matrix damage is a suspected factor [4]. Several investigators have studied the fate of cartilage with osteochondral damage using either in vivo animal models of joint trauma [19,27,29], or by following the changes after impaction of cartilage explants [2,9,13,23,24,28]. Thompson et al. used a model of closed impaction of the canine

’

This work was presented in part at the 45th Annual Meeting of the Orthopaedic Research Society, Anaheim, CA. *Corresponding author. Address: Department of Biochemistry, Rush Medical College, 1653 W. Congress Pkwy, Chicago, I L 60612, USA. Tel.: +I-312-942-2711; fax: +1-312-942-271113053. E-mail addre.rs: [email protected] (T.R. Oegema Jr.).

patella in which subchondral bone and calcified cartilage fractures were observed without full-thickness cracks in the cartilage [27]. Osteoarthritic-like degenerative changes were reported at 6 months, but these had stabilized at 12 months. Newberry et al. [19] impacted the mature Flemish Giant rabbit patella-femoral joint and found cartilage softening and subchondral bone thickening of the patella after 12 months. It is not clear if the damage seen in either of these models is sufficient for the joint to progress to full-blown OA. Since it is known that even in the canine anterior cruciate resection model [3] OA takes many years to develop, it may be that insufficient time had passed in either model for end-stage OA to become apparent. This raises the question of what aspect of cartilage damage is sufficient or necessary for progression. Since chondrocytes are required for matrix repair and chondrocyte death would eventually lead to matrix loss [25], chondrocyte death either by apoptosis or necrosis has become a focus of OA research and more recently, cartilage trauma research.

0736-0266/$ - see front matter 0 2003 Orthopaedic Research Society, Published by Elsevier Ltd. All rights reserved doi: 10.1016/S0736-0266(03)00039-1

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Numerous investigators have studied cell death that resulted from a single impaction of cartilage explants [9,13,23,24,28]. In a classic study, Rep0 and Finlay [23] found that cell death (as measured by failure to incorporate radioactive amino acid precursors into proteins as detected by autoradiography) occurred with high rates of loading at stress levels above 25 MPa in old human cartilage that was radially unconstrained, but with an underlying thin layer of bone. They noted cell death around cracks, extensive maceration and fissuring. Jeffrey et al. [ 131 found a linear relationship between loss of cell viability (as measured by uptake of trypan blue after enzymatic release of the cells from the matrix) and impact energy with stress levels up to 200 MPa. Furthermore while the study had no spatial resolution because they released the cells from the matrix, Jeffrey et al. [ 131, stated that chondrocyte viability was unaffected by moderate impacts, even though tissue disruption was severe. Torzilli et al. [28] impacted young mature bovine cranial cartilage with no bone attached at different stress levels and graded the cells as dead (unable to exclude propidium iodide) or live (able to hydrolyze and retain fluorescein diacetate). They found dead cells in the surface layer at a nominal stress level of 15-20 MPa. They also reported extensive cell death in the deep layer at higher levels of nominal stress. Torzilli et al. [28] stated that cell death already occurred when there was evidence of collagen fiber matrix damage as measured by tissue swelling. The goal of the present study was to study cell death after impact, as a function of spatial location and time. The primary hypothesis was that no additional impactrelated loss of viable cell density occurs over time if the impacted cartilage is placed in medium with low serum [12]. After a preliminary study where we noted more cell death around the cracks, the second hypothesis was that with a high rate of loading of cartilage on bone, the loss of viable cells after cartilage impaction occurs only around cracks and if cracks do not occur during the impact there will be no loss of viable cell density.

tissue were removed with a scalpel from the bone and paired with similarly sized, immediately adjacent, non-impacted control specimens. In order to assure there was not damage in the patella before impaction, the cut edges were visually inspected for tissue defects. Specimens were stored during collection in Dulbecco's phosphate-buffered saline (DPBS) with high glucose and 0.1'%1gentamycin for several hours. In a pilot experiment, this collection and testing procedure was sufficient to protect even the surface cells of controls during the time of potting, impaction and collection (see Fig. IA). Pairs of impacted and adjacent cartilage samples were assigned to short- o r long-term culture groups. Short-term specimens ( n = 20 pairs) were incubated in 0.75 mglml nitroblue tetrazolium (NBT) for 18 h in Ham's F12 media with O.I'%i gentamycin and 5'%1 fetal calf serum [21]. Tetrazolium salts, such as NBT, are cell-permeable and are reduced to highly colored formazan products in viable cells stable to fixation, ethanol and xylene dehydration, and paraffin embedding [18,30]. While viable cells rapidly turned blue, this 18 h incubation time was used to assure all viable cells through out the depth of the samples would contain the formazan product above the detection threshold of the imaging system. Since the formazan product is insoluble, cells that accumulate enough t o be detected and then die would register as alive. Since we were interested if cells in damaged tissue might be more

A

Materials and methods Spcitneri prcparation

The surface of whole, fresh, mature (estimated to be I$-3 years of age) bovine patellae with smooth, intact surfaces, continuously moistened with phosphate-buflered saline were potted in poly(methy1 methacrylate) and mounted in a custom fixture. The cartilage s u r f a x was aligned parallel to the flat surface of a 6 mm diameter cylindrical impacter with rounded edges with a radius of curvature of 0.5 mm. Controlled loads were applied at 6000 Nls to a target peak load of 1500 N using an MTS 858 Bionix Test System (MTS Systems Corp.) and the load history recorded. The nominal stress was 53 MPd, defined as the peak load divided by the cross-sectional area of the indenter and created both cracked and non-cracked surfaces at the center of the indenter area. This stress was chosen from a pilot experiment because in this experimental setup it produced a distribution of cracked and noncracked samples. Cartilage squares ( 8 - ~ 9mm) containing the impacted

Fig. 1. A single imaged section with the regions created for the two different analyses. (A) Four zones for analysis I and (B) further division of the first two zones into 12 segments for analysis 11. The categorization for the segments is C = cracked o r disrupted, A =adjacent, and D =distant.

J.L. Lcwi.s et (11. I Journal of Orihopaedic Reseurcli 21 (2003) 881-887 vulnerable, we placed the tissue (18 pairs) in long-term culture (5 days) in 5‘%1fetal calf serum [8] with NBT added for the last 18 h. The fetal calf serum level of 5‘Yn was chosen because it will support chondrocyte metabolism, yet is low enough to put metabolic tension on the chondrocytes [I 21. Following culture, samples were washed with serum-free Ham’s F12 medium and fixed in 10%) formalin, cut in half at the middle of the impact, processed for routine paraffin histology and a series of 3-12 serial sections (20 pm thick) was obtained. The thicker sections provided full cell profiles with the included formazan product. Sections were lightly connter-stained with Safranin-0 for visualization of the tissue borders [21]. Three images of the full-depth sections that were as close to center as possible and not folded were collected with a Spot digital camera (Diagnostic Instruments, Inc.) at 40x magnification. To avoid discontinuity effects near the impact edges, the center 113 was imaged, converted to grayscale and filtered (find edges filter) using Adobe Photoshop (Adobe Systems, Inc.). Anu1j.si.y I: Effect of tinie. surfircr damage, und depth

The cartilage was divided into four zones of equal depth with lines running parallel to the cartilage surface (Fig. IA) from the articulating surface (zone I ) to nearest the bone (zone 4). Images were thresholded to distinguish NBT-stained (viable) cells from the background and the Metamorph Imaging System (Universal Imaging Corp.) was then used to compute the viable cell density. which was defined as the viable cells per cartilage area and was not corrected for cell diameter or section thickness [5]. The values were averaged and also normalized to adjacent non-impacted controls. Three observers graded the surface damage on a scale of 0-3 with 0 indicating no surface damage, 1 indicating minimal surface damage such as a single fissure, 2 indicating increased fissuring interspersed with regions of intact surface, and 3 indicating complete surface disruption including extensive cracking (Fig. 2). The kappa statistic was used to assess the level of agreement between observers. The impacted and adjacent control specimens at each time were compared for each zone using a paired t-test [6]. The normalized (impactedhon-impacted adjacent controls) density of viable cells for the two times were compared using unpaired t-tests for each zone. In addition, the zone 1 data was analyzed using a two factor ANOVA with factors of time (18 h, 5 days) and damage score (0, I , 2, 3). Multiple comparisons were performed using Bonferroni’s test. Finally, for the pooled 18 h and 5 day data, the relationship between density of viable cells and damage score was assessed using the non-parametric Spearman correlation coefficient.

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Analysis 11: Efect of pro.witnity to trucks, ond tirnc In order to determine if the cell viability decreased in the region around cracks, the upper half of the cartilage (zones I and 2) was divided into six segments per zone (Fig. IB). In zone I (surface), the segments were assigned to “cracked” if there was any matrix disruption in that segment, “adjacent” if the segment was not cracked or disrupted but was adjacent to a region that was cracked, and “distant“ if the segment was neither “cracked” nor “adjacent”. Specimens with no visible damage were all assigned “distant” and sections that were a 3 were typically assigned to “cracked” for all 6 segments. Occasionally a surface crack did extend slightly down into the zone 2. and these segments were assigned “cracked”. Zone 2 segments that were adjacent to or beneath a cracked segment were called “adjacent”, and others were assigned to “distant”. The viable cell density for each area was normalized by dividing by the corresponding areas of the nonimpacted controls. The data was analyzed by a repeated measure (ANOVA), with time (18 h, 5 days) included as the grouping factor and location (cracked, adjacent, distant) used as the repeated factor. Multiple comparisons between locations were performed using the Bonferroni procedure. The effect of time on just the “cracked” segments was reanalyzed, and confidence intervals calculated. Live

LT.

deadjluoresceni u s s a ~

As a confirming check on the conclusions from the analyses using the NBT, a live vs. dead assay was also used to determine if dead cells were clustered near the crack. Cartilage samples were cut in half and one-half was incubated with NBT as above. The other half was incubated in DPBS with glucose containing 6.7 pg/ml of propidium iodide and 2 pM, 5’-chloromethylfluorescein diacetate (CMFDA), Cell Tracker Green, (Molecular Probes, Eugene. O R ) at 37 “C for I h. The C M F D A was dissolved in dimethylsulfoxide at 100 pM and diluted just before use. The six impacted and six adjacent control samples were washed 2 x 10 min in DPBS at 37 “C. Labeled samples were immediately cut and laser scanning confocal microscopy (LSCM) epifluorescence data sets were acquired using an MRC-1000 LSCM (Bio-Rad Laboratories. Inc., Hercules, CA). LSCM image data sets were collected as sequential I-, 2- or 5-pm serial optical sections (10-20 sections in a z series) in the z plane of focus. The optical z sections were projected, in register, into single in-focus images. Merged color images of red (dead cells) and green (live cells) were generated using Confocal Assistant@and Photoshop-.

Results

Damage Scoring Scale

Fig. 2. The damage scoring scale utilized in analysis I. Each image was graded independently by three observers using this scale, where 0 = n o disruption of the cartilage surface, I =minimal damage such as one crack. 2 =additional damage interspersed with regions of intact matrix, and 3 =complete disruption of the cartilage surface. Kappa score for interobserver variability between the three graders was 0.72.

An average of six paired impact/adjacent non-impacted control sets was obtained for each patella. Unless the impacter was incorrectly aligned and skipped, the peak force reached was within 5%) of the target 1500 N and occurred in less than 250 ms. Thirty-eight of the impact-control sets from eight different patellae from mature bovine were available for analysis (20 pairs at 18 h and 18 pairs at 5 days). For both methods of determining cell viability, we had a very low backgrounds, so if the samples were freeze-thawed before incubation, there were no NBT- or CMFDA-labeled cells. Using the 18 h and 5 day impacted samples with our sample size and variability, we had a power of 0.79 at an a of 0.05 for these experiments. Analysis I: Efect of time, surface damage, and depth

Viable cell density depended on the depth from the surface. There was a statistically significant difference in

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of’ Orilloptiedic Reseurch 21 (2003) 881-887

Table I Average cell density values and corresponding average viability scores (with standard deviation)

Zone Zone Zone Zone

1 (surface) 2 3 4 (bone)

18 h impact (cells/mm’)

18 h controls (cellslmm’ )

18 h viability score (ratio)

5 day impact (ceils/mm’)

5 day controls (cells/mm’)

5 day viability score (ratio)

575i201,‘ 532 i 152 489 i 154 451 i 127

696 f 164’ 546 f 127b 498 i 116 466 100

0.83 f0.22 1.OO f0.29 0 . 9 9 f 0.26 0.99 0.34

394 f207” 441 f 134 4 4 3 f I28 416+ 100

540 f 139‘ 457 k 104h 427 3 120 456f130

0.72 i0.29 0.98 i0.23 1.05i0.21 0.95 f0.23

*

*

‘’ Impacted and control are statistically different by paired t-test, p < 0.05. 18 h and 5 day controls are statistically different by unpaired t-test, p < 0.05.

viable cell density between impacted and control samples in the surface zone (zone 1) of both the 18 h and 5 day groups (Table I). In contrast to the semi-quantitative data from high stress levels reported by Torzilli et al. [28] with samples impacted off the bone, there was no difference between impacted and control specimens in any other zone. Time in culture after impact affected the density of viable cells, In medium containing 5% serum, there was a generalized loss of viable cell density in all four zones in the controls after 5 days (Table 1). As expected, the greatest loss was in the surface zone, but the difference was statistically significant in both zones 1 and 2. When comparing the impacted samples, which had no difference in average damage score between the groups, there was no statistical difference between the 18 h and 5 day values in any zone due to impact (p = 0.71), even when compared as normalized values. Damage at the surface (zone 1) was a significant factor in determining normalized viable cell density ( p < 0.001). Viable cell density, in specimens with a damage score of 0, was significantly higher than those with a damage score of 1, 2 or 3 (p < 0.01 for each). Since time after impact was not significant, this could be shown another way where the data for both times were pooled and the viability score or the ratio of viable cell density in the impacted tissue to the nonimpacted control analyzed as a function of damage score by Spearman correlation. There was a statistically significant negative Spearman correlation coefficient of -0.60 (p = < O . O O l ) (Fig. 3), so in the most damaged tissue in the surface layer, viable cell density was only 63%)of the controls.

1.4 0

18 hours

0

5 days

1.2

0

0.2 -

€4 y = -0.1365~+ 0.9824

R2 = 0.345

2

-

.-%

075

05

.-m> 0 25

n

Aitulysis II: Effect cf proxinzity to cracks

Fig. 4. Analysis I1 results: Data for 18 11, 5 day, and combined data, for both zones 1 and 2. The abscissa is separated by crack condition (Fig. 2). *: Significantly different from both adjacent and distant in the same zone, p < 0.01,

Since the presence of visible damage had a significant effect on viable cell density, we next determined if the decreased viable cell density was localized to the disrupted areas or was diffusely distributed throughout zones 1 and 2 (Fig. 1B). In zone 1, by ANOVA, areas that were cracked had a significant decrease in viable cell density relative to controls (p < 0.001). By the Bonferroni test, viable cell densities in the crack segments were

significantly lower than those found in either the adjacent or distant segments. The adjacent segments were not different from the distant segments (Fig. 4). Analysis of the data by two-way ANOVA revealed that there was no effect of time after impact on viable cell density near the cracks. The confidence interval for the difference in mean values of the ratios of impacted to non-impacted tissue of 0.70 and 0.63 was calculated as

J. L. Leu7i.yrt ul. I Jouriiul of Ortlzopuedic RiJseurcli21 (2003) 881487

(-0.11, 0.24) and included zero, which is consistent with the hypothesis that time was not an influencing factor on cell viability in the cracked segments. If the viable cell density decreases near cracks, there should be corresponding increases in dead cells near cracks. This was shown directly with a live vs. dead assay (Fig. 5). As before, in the adjacent controls stained with NBT, there was an even distribution of viable cells with formazan precipitates (Fig. 5a). In the impacted tissue (Fig. 5b), there are numerous cells near the cracks with no formazan precipitate that appear as clear ovals (compare with Fig. 5a). In the corresponding PI/ CMFDA stained control section, only occasional dead (red) cells are seen. This is in contrast to impacted tissue where there are many dead (red) and few living (green) cells near the cracks (Fig. 5c and e). There is a clear steep gradient of dead cells near the cracks. In the low power

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photomicrograph (Fig. 50, a specimen is shown across the area of impaction. The effect at the large stress discontinuity edge of the impaction can be appreciated on the right and left sides of the sample. In these regions there was always cell death even if there were no cracks (compare left side with no crack to the right side with a large crack) along with localized cell death, which is around the cracks in the central region of the indenter. The edge effect as also seen as a white ring in the nitrotetrazolium blue stained samples. There was very little cell death in the deep region.

Discussion In this study with cartilage on an intact patella, cell death due to cartilage impact occurred around impact

Fig. 5. Montage of impacted and adjacent cartilage: (a) histological section adjacent to impact area that had been incubated with NBT and processed for conventional histology. There is a high percentage of cells with included black precipitates indicative of live cells. (l00x magnification). (b) Conventional histological section of impact area near the crack where there are many cells without any black precipitates, only near the crack (100x magnification). (c) LSCM image of non-impacted tissue immediately adjacent to impact area (scale bar = 200 pm). (d) LSCM image of impact area (scale bar =200 pm) ( e ) LSCM image of impact area (scale bar=200 pn).(0 LSCM image of impact area. Lower magnification of Fig. 5d (scale bar = 200 pm).

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induced cracks. Macroscopic cracking was restricted primarily to the top 25%)of the distance from the surface which is consistent with observations by other investigators [9,13,24] for constrained cartilage attached to bone with rapid loading, Torzilii et al. [28] found cell death throughout the thickness of the impacted cartilage explant at a level comparable to the stress level we used, whereas we found cell death only near the surface. This would be similar to the area of damage they found at 15-20 MPa [28]. We believe this can be explained by the difference in boundary conditions at the base of the explants. Torzilli et al. [28] freed their explants from the bone prior to impact; our specimens were attached to bone. Our specimens would be constrained from lateral expansion in the deep zone compared to Torzilli et al. [28], resulting in less strain. We believe this protected the cells in the deeper zones from damage. Ewers et al. [9] reported in constrained compression with 18-24 month old bovine metatarsal cartilage with no bone attached, the rate of loading determined the site and extent of cell death. At a fast rate of loading of -900 MPa/s, cell death was predominantly around cracks whereas at a 40 MPa/s loading rate for the same load of 40 MPa, the number of dead cells was higher and they had a more diffuse distribution. Another point of disagreement is that other investigators have found cell death after impact increases with time and this death is due to apoptosis. We did not find decreased viable cell density with time related to impact. Small changes could have been missed because of generally lower viable cell density, specifically near the surface at 5 days and the fact that cell viability with a higher background of living cells was measured, not cell death. Ewers et al. [9], Quinn et al. [22] and D'Lima et al. [S] found diffuse cell death due to apoptosis after loading cartilage explants. However, they loaded implants at a much slower rate than we did. It has been suggested that at slow rates, there can be water flow in the tissue and out of the cell, leading to cell death [8]. This type of diffuse cell death and matrix damage is similar to that reported by Loening et al. [16] when very young bovine cartilage was repeatedly slowly compressed to 50% of their original volume and held for 5 min. Similar to the latter study, the predominant mechanism of cell death in bovine cartilage impacted to 23 MPa over 500 ms was found to be apoptosis and could be partially inhibited by caspase inhibitors [S]. In constrained cartilage attached to bone, we hypothesize that loading rate is an important determinant in trauma-induced cell death due to apoptosis and that apoptosis does not occur at the higher loading rates that we used. We also suspect that the higher loading rates are more relevant to in vivo impact trauma, where loading times are in the order of a few milliseconds ~71.

The quantitative localization of cell death to cracks has not been reported previously, although this has been suggested by previous studies [9,23]. This is in contrast to the lack of cell death around cracks produced by cyclic loading at levels below that which causes cracks in a single load [6]. We found cell death at the edge of the impacter where there is a large difference in the stress but not at the center of the impact if no crack occurred. This suggests high stress and elastic deformation alone is not enough to cause cell death. However when a crack forms there are large differences in stress around the elongating crack tip, and the collagen network is damaged and locally disorganized and displaced, and this could be what leads to cell death. The role of cracks in the progression from trauma to OA is not clearly defined. Cracking ofGartilage seen in animal models of OA [17,19,20] have been shown to be co-localized with acellularity in the cracked tissue [ 191, and regions of chondrocyte apoptosis [l]. Since simple cuts in cartilage do not lead to progression [26] it is likely cell death and tissue disruption is needed for progression. Post-traumatic cracking of cartilage has been seen in human joints in vivo, and increased cracking was associated with higher histological damage scores indicating cell death or injury [14]. So the spatial relationship between matrix cracks and non-viable cells may be of clinical importance [20] and it is easy to imagine progression to full-depth cracks, even with normal joint loading [25,28]. Understanding the mechanisms of how trauma contributes to the pathogenesis of OA can lead to rational design to prevent injury or to partially ameliorate the damage. The in vitro studies point out how complicated the process may be. Even with simple uni-axial compressive impacts that vary only the rate and levels of impaction and how the cartilage is constrained, very different responses are found. Our study would suggest normal cartilage on bone would tolerate a fairly high level impact if the impact was rapid, as might be expected in most crashes, and that the cell injury would be around cracks and would be minimal if cracks were not present. Further, in the absence of additional mechanical impact or inflammatory signals, death would be limited to the initial damage and not readily progress. However, if the rates of impact are slower, the magnitude of impact that will cause damage would be lower, there would be cell death without cracking, but with microscopic matrix disruption, and cell death would be by apoptosis. Since apoptosis occurs over a longer timeframe it might be preventable [8]. If the mechanism of cell necrosis caused by rapid loading is typical of most traumatic injuries, standards for crash design aimed at preventing cartilage injury would be considerably different than if the cell death caused by apoptosis caused by slower rates of loading is the typical case. Also, in the first case, post-treatment

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options could include stabilizing the cracked matrix, while in the slower impact rate injuries, acute treatments that prevent apoptosis might be more efficacious.

Acknowledgements

This work was supported by grants from the National Institutes of Arthritis, Musculoskeletal and Skin Diseases (AR41975), the Cathryn Mills-Davis Endowment and a National Science Foundation graduate fellowship to M. Oyen-Tiesma. The authors would like to thank Jerry Sedgewick and the staff of the Biomedical Image Processing Laboratory for their help with the computerbased image analysis, and Bruce Lindgren of the Biometrics Consulting service for advice on statistical analyses. The authors would also like to thank Toni Meglitsch, Sandra Johnson, Mary Slagle, Fred Wentorf, and Dr. Doug Adams for their time and assistance.

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