Evaluating cytotoxicity of recycled Ni-Cr dental casting alloys--an in vitro study.
Abstract: This study was conducted to evaluate the cytotoxicity of two beryllium free nickel-chromium dental casting alloys in their reused state. Three groups of specimens per each alloy were casted, first group with 100% new alloy, second by mixing 50 wt% of new alloy with 50 wt% of once used alloy and the third 100% once used alloy. Distilled water conditioned with these alloy specimens for different time intervals was used for testing the cytotoxicity on Chinese hamster V79-HG04 fibroblasts by performing Cellviability and Clonogenic assays. The highest cytotoxicity was noticed at the end of 4 weeks of conditioning and did not alter significantly after that time. 100% reused groups were most cytotoxic. There were no statically significant differences between the two alloys tested. Considering the elevated cytotoxicity of Ni-Cr alloys in their reused state, the practice of reusing the alloy just for economic reasons should be discouraged.
Article Type: Report
Subject: Nickel alloys (Usage)
Authors: Chandra, Tripuraneni Sunil
Kumar, Namburi Suneel
Kumari, Bellam Kranti
Pub Date: 04/01/2011
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: April, 2011 Source Volume: 25 Source Issue: 2
Product: SIC Code: 3339 Primary nonferrous metals, not elsewhere classified; 3356 Nonferrous rolling and drawing, not elsewhere classified
Accession Number: 308434335
Full Text: Introduction

Historically, precious metals and their alloys have been considered as ideal restorative materials due to their excellent properties such as biocompatibility, castability, percentage elongation, burnishability, capacity to take high polish, and resistance to tarnish and corrosion. However, over time for economic reasons, base metal alloys have become widely used, limiting the routine use of precious alloys in dental practice. Base metal alloys are alloys based on >75 weight % (wt%) of base metals (metals that readily oxidize or dissolve to release ions). Initially, base metal alloys were so inexpensive that new ingots were melted and cast, but the demand for them in dental procedures has now resulted in substantial increases in the price, a point of financial concern for commercial dental laboratories.

There have been several reports on the re-cycling of nickel-chromium (Ni-Cr) denture alloys and the evaluation of their physical properties [1]. Since there are relatively few reports available on the cytotoxicity of Ni-Cr denture alloys in their reused state, it is imperative to ascertain whether there will be any changes in the cytotoxicity of Ni-Cr alloys in their reused state when compared to using fresh alloy. Recasting of Ni-Cr alloys could change the corrosion properties of the alloys and; therefore, affect element release and, subsequently, the cytotoxicity of these alloys. The effects of corroded products released in the oral cavity can range from local irritation and systemic toxicity to carcinogenicity [2- 4].

This study was undertaken to evaluate the cytotoxicity of Ni-Cr alloys in their reused state.

Materials and Methods

Preparation for test specimens

Two beryllium and copper free Ni-Cr alloys Wiron 99 (BEGO, Bremer Goldschlagerei Wilh. Herbst GmbH & Co KG, Germany) and Remanium CSe (Dentaurum J P Winkelstroeter KG, Germany) with compositions as indicated by the manufacturers (Table 1) were selected. Disk shaped test alloy specimens 3 mm in height and 5 mm in diameter were cast using Wiron 99 and Remanium Cse, following the conventional lost wax technique using phosphate bonded investment.

To produce disks with reproducible size and shape, an aluminum mold was used for fabricating wax patterns with inlay wax. Three groups, each comprising of 20 test specimens per group were fabricated with each alloy as follows. The first group comprising 20 specimens cast using 100% new Wiron 99 alloy. The second group, 20 specimens cast by combining 50 wt% of new Wiron 99 alloy and 50 wt% recast (once used Wiron 99) alloy. The third group, 20 specimens cast using 100% recast (once used Wiron 99) alloy. Alloy used for recasting was obtained after sandblasting the left over button and sprues of the previous casting using 250 im aluminum oxide.

A similar procedure was repeated with Remanium CSe to obtain three groups of test specimens. After sand blasting, the sprues were cut off using a clean siliconcarbide separating disk, and the specimens were finished with a sintered diamond bur and aluminum oxide polishing stone to remove any gross irregularities on the surface. The specimens were subjected to the standard metallographic polishing technique using 100, 220, 320, 420, 600, 800 and 1,000 grade water proof silicon carbide papers in a sequential manner. Each group of alloy specimens was finished and polished using separate instruments.

All finishing and polishing processes were carried out to simulate the preparation of the castmetal alloys for clinical cases. The disks were soaked in a detergent solution for 5 minutes, scrubbed using a soft-bristled brush, and rinsed under tap water for 5 min and distilled water for another 5 min. The disks were then placed into 95% ethanol and cleaned by sonication for 5 min, then removed and placed in sterile distilled water and cleaned by sonication for another 5 min. Finally, the disks were autoclaved at a temperature of 150[degrees]C for 60 min [5].

Five sterile cast specimens belonging to the first group, Wiron 99 (ie 100% new Wiron 99) were immersed individually in five microcentrifuge vials containing 1 ml of double distilled water for 1 week, to condition the water. Next, five specimens from that first group were similarly immersed for 2 weeks, the next five for 4 weeks, and the last five specimens for 8 weeks. Similar conditioning was done for the rest of the test specimen groups for both the alloys for the same time intervals, as stated above. The surface area of the alloy in contact with the distilled water was in the range recommended by the ISO. The microcentrifuge vials with specimens were placed in an incubator (Sterti-Cult, Formascientific) at 37[degrees]C for the specified time intervals, as above. Conditioned distilled water in which the alloys specimens were immersed was used for evaluating cytotoxicity. Five vials of distilled water without any alloy were used as controls for each time interval.

Cell Culture and Cytotoxicity Testing

The V79-HG04 fibroblast (known as V79 cells), a transformed cell line of a primary culture derived from embryonic lung fibroblasts of a Chinese hamster (National Center for Cell Sciences, Pune, India), were used for cytotoxicity testing in this study. Cytotoxic effect of alloy specimens was evaluated by performing Cell viability assay and Clonogenic assay

Cell viability assay

The cytotoxicity of test alloy specimens at various time intervals was evaluated by Pratt and Will's test [6]. One lack exponential of V79 cells were seeded in 25 cm2 Petri dishes (triplicate for each sample) containing Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum, 1% L-glutamine and 50 mg/mL gentamicine sulfate. They were allowed to grow for 24 hours and there after they were treated with 200^l of distilled water in which various groups of alloy specimens have been kept for 1,2,4 and 8 weeks respectively. The cells were allowed to grow for another 48 hours and harvested by trypsin-EDTA treatment. The harvested cells were counted using a haemocytometer. The viability of cells was determined using Trypan blue dye exclusion test. This method is based on the principle that viable cells do not take up Trypan blue dye, where as dead cells do. Staining facilitates visualization of cell morphology and microscopically it is possible to differentiate dead and viable cells.

Clonogenic Assay

Effect of corrosion products released from test alloy specimens after different time intervals on clonogenicity or reproductive integrity of cells was determined by colony forming assay of Puck and Marcus [7]. One million exponential of V79 cells were grown for 16 hours on MEM supplement medium similarly as above. Then the cells were treated for 24 hours with the distilled water samples in which alloys specimens of different groups were immersed for 1,2,4, and 8 weeks respectively. After 24 hours cells were dislodged by trypsin-EDTA treatment. A fixed number of cells (usually 200) will be inoculated into several individual Petri dishes in triplicate for each specimen (3 Petri dishes for specimen) and were allowed to grow for 8 days for colony formation. The cultures were terminated at the end of 8 days. The resultant colonies were stained with 1%crystal violet in methanol and cultures containing 50 or more cells were scored as a colony. Plating efficiency for all alloy soaking time intervals was determined and the data was expressed as surviving fraction. Statistical analysis was done by one way analysis of variance (one way ANOVA) to determine whether the group means differed from one another significantly. Posthoc analysis was done by Bonferroni's t test (to obtain Bonferroni's corrected p value) to compare whether the mean values of one particular group differed from another significantly.

Results

Results of Cell viability test

This test acts as a primary screening test to know whether corrosion products released from alloys induce any cytotoxic effect or not.

After 1 week of conditioning time Table 2, the viable cell counts in all the three groups of both alloys show that p values (a, b, c) when compared to control, p values (a, a, #) when compared to new metal were coded to each group respectively. Viable cell counts of 100% new metal groups of both the alloys showed highly significant (p < 0.01) decrease when compared to control. Viable cell counts of 50% new + 50% reused groups and 100% reused groups of both the alloys showed very highly significant (p < 0.001) reduction in number of viable cells when compared to control. When compared to 100% new metal groups of both the alloys 50% new + 50% reused and 100% reused groups showed very highly significant(p < 0.001) reduction in viable cell counts.

All the three groups of both alloys after 2 weeks of conditioning time Table 3 showed very highly significant (p < 0.001) reduction in number of viable cells when compared to control. 50% new + 50% reused groups and 100% reused groups showed very highly significant (p < 0.001) reduction in viable cell counts when compared to viable cell counts for 100% new metal groups of both the alloys.

All the three groups of both alloys after 4 weeks of conditioning time Table 4 showed very highly significant (p < 0.001) reduction in number of viable cells when compared to control. 50% new + 50% reused group of Wiron 99 showed non significant (p > 0.05) deference in number of viable cells when compared to 100% new group. 50% new + 50% reused group of Remanium CSe showed significant (p < 0.05) decrease in viable cell counts when compared to 100% new group. 100% reused groups of both the alloys showed very highly significant (p < 0.001) decrease in viable cell counts when compared to 100% new groups.

The three groups of both alloys at 8 weeks time interval Table-5 showed very highly significant (p < 0.001) reduction in number of viable cells when compared to control. 50% new + 50% reused group of Wiron 99 showed non significant (p > 0.05) deference in number of viable cells when compared to 100% new group. 50% new + 50% reused group of Remanium CSe showed significant (p < 0.05) decrease in viable cell counts when compared to 100% new group. 100% reused groups of both the alloys showed very highly significant (p < 0.001) decrease in viable cell counts when compared to 100% new groups.

The correlation between viable cell counts and conditioning time of all the three groups of Wiron 99 was determined by plotting mean and standard error of mean of viable cell counts on Y-axis and time interval on X-axis (Graph 1). A gradual but significant decline in viable cells was observed with increase in conditioning time of Wiron 99 alloy and a greatest cytotoxic effect was describable after 4 weeks. The cytotoxic effect did not altered significantly when the length of conditioning time of alloy was increased to 8 weeks. The difference in cytotoxicity between 4 and 8 weeks of conditioning time was statistically non significant. Although the viable cell counts showed a conditioning time dependent decline for all the three groups of Wiron 99 alloy, the greatest viable cell counts were observed for 100% new metal, intermediate viable cell counts were observed for 50% new + 50% reused alloy group and the lowest viable cell counts were observed for 100% reused group. Similar results were obtained for Remanium CSe in all the three groups Graph 2.

[GRAPHIC 1 OMITTED]

[GRAPHIC 2 OMITTED]

Results of clonogenic assay

This test was used to evaluate the effect of corrosion products released from alloys on reproductive integrity or clonogenicity of the cells. The plating efficiency for all groups of both the alloys was determined and the data was expressed as mean and standard error of mean of surviving fraction.

After one week of conditioning time Table 6 all the three groups of both the alloys showed very highly significant (p < 0.001) decrease in surviving fractions when compared to control. Surviving fractions of 50% new + 50% reused groups and 100% reused groups of both the alloys showed very highly significant (p < 0.001) decrease when compared to surviving fraction of 100% new metal groups. 100% reused groups of both the alloys showed highly significant (p < 0.01) decrease in surviving fractions when compared to 50% new + 50% reused groups.

After two weeks conditioning time Table 7 all the three groups of both the alloys showed very highly significant (p < 0.001) reduction in surviving fractions when compared to control. Surviving fractions of 50% new+50% reused groups and 100% reused groups of both the alloys showed very highly significant (p < 0.001) reduction when compared to surviving fraction of 100% new metal groups. 100% reused groups of both the alloys showed significant (p < 0.05) reduction in surviving fractions when compared to 50% new + 50% reused groups.

After four weeks of conditioning time Table-8 all the three groups of both the alloys showed very highly significant (p < 0.001) decrease in surviving fractions when compared to control. Surviving fractions of 50% new + 50% reused groups and 100% reused groups of both the alloys showed very highly significant (p < 0.001) decrease when compared to surviving fraction of 100% new metal groups. 100% reused groups of both the alloys showed highly significant (p < 0.01) decrease in surviving fractions when compared to 50% new + 50% reused groups.

After eight weeks of conditioning time Table 9 all the three groups of both the alloys showed very highly significant (p < 0.001) decrease in surviving fractions when compared to control. Surviving fractions of 50% new+50% reused groups and 100% reused groups of both the alloys showed very highly significant (p < 0.001) decrease when compared to surviving fraction of 100% new metal groups. 100% reused groups of both the alloys showed highly significant (p < 0.01) decrease in surviving fractions when compared to 50% new + 50% reused groups. The difference between surviving fractions of Remanium CSe and Wiron99 was not significant (p>0.05).

[GRAPHIC 3 OMITTED]

[GRAPHIC 4 OMITTED]

The correlation between surviving fraction and time interval of all the three groups of Wiron 99 was determined by plotting mean and standard error of mean of surviving fraction on Y-axis and time interval on Xaxis Graph 3. Surviving fraction decreased with increase in time interval. A gradual but significant decline in surviving fraction was observed with increase in conditioning time of Wiron 99 alloy and a greatest reduction in surviving fraction was describable after 4 weeks. The surviving fraction did not altered significantly when the length of conditioning time of alloy was increased to 8 weeks. Although the cell survival showed a conditioning time dependent decline for all the three groups of Wiron 99 alloy, the greatest surviving fraction was observed for 100% new metal; intermediate results were observed for 50% new + 50% reused alloy group and lowest surviving fraction was observed for 100% reused alloy group. Similar results were obtained for all the three groups of Remanium CSe when plotted Graph 4.

Discussion

Currently, the majority of commercial dental laboratories are reusing scrap alloy (used once or more times) either alone or in combination with the new alloy for fabricating the dental prosthesis, considering only the cost factor of the alloy, without any regard to manufacturers' instructions or alterations in its properties on reusage. Hence, it becomes imperative to ascertain if there is any elevation in cytotoxicity of Ni-Cr alloys in the reused state when compared to its first time use.

Two copper and beryllium free, commercially available Ni-Cr (Wiron 99 and Remanium CSe), base metal dental alloys were tested in this study, because base metal alloys containing copper and beryllium are more prone to corrosion [8, 9]. Beryllium also increases the release of nickel from Ni-Cr alloys into the corrosive conditioning media (eg, saliva) when compared to the beryllium free Ni-Cr alloys. It was observed in previous studies that test alloys containing copper and beryllium exhibited elevated levels of cytotoxicity compared to alloys without these elements [10, 11]. The presence of these elements in the test specimens could exaggerate the cytotoxic effects of the remaining constituents. In addition to the above mentioned reasons, the alloys in this study varied in their composition of minor elements, ie carbon, silicon, niobium, cerium, aluminum and cobalt (Table 1). These elements can be affected by recasting. Most previous studies evaluating the biological affects of base metal alloys in vitro, analyzed the elements released from these alloys by atomic absorption spectrometry and correlated the amount of element released with cytotoxic effects [5, 10].

Different types of conditioning media such as artificial saliva, saline, dilute acids and cell culture media were used in previous studies [12]. These media contain various minerals or organic constituents that could have an effect on the corrosion susceptibility of the alloys, and they also interfere with cell metabolism of the cell lines used for testing cytotoxicity. In this study, distilled water was used as a conditioning medium to avoid such effects. Most of the previous studies used the conditioning time of 24168 hr. In some of the previous studies evaluating the long-term cytotoxic effects of casting alloys, conditioning media (cell culture media) were changed at regular time intervals to prevent microbial colonization [11]; this could result in the loss of corrosion products previously released into the conditioning media. In this study, sterile double distilled water was used for conditioning the alloy specimens. Distilled water did not support any microbial colonization, and the same distilled water was maintained throughout the entire test period for conditioning the alloy specimens; this simulates an intra oral situation where the restorations were in continuous contact with the oral environment.

For evaluation of cell death due to corrosion products released from test alloy specimens Cell viability assay (Pratt and Wills test) was used [6]. Treatment of V79 cells with the corrosion products released into distill water from test alloy specimens during 1to 8 weeks period showed a time dependent decline in viable cell counts (Graphs 1and 2) in all the three groups with both the alloys. However the difference in cytotoxicity between 4 and 8 weeks of conditioning time was not significant. The decline in the release of corrosion products from the test alloy specimens into the conditioning media after certain periods of conditioning time is probably the reason for the non significant increase in toxicity beyond 4 weeks of conditioning time as was observed in this study. All the three groups of both the alloys showed statistically significant decline in viable cell counts when compared to control, that is distilled water without any alloy specimen. The greatest decline in viable cell counts was observed for 100% reused alloy groups with both the alloys. The differences between Wiron 99 and Remanium CSe were also statistically non significant.

For evaluation of effects of corrosion products released from alloys on reproductive integrity or clonogenicity of the cells and to further confirm the results obtained with Pratt and Wills test, Clonogenic assay or colony forming assay of Puck and Marcus was used [7]. There is a time dependent decline in the surviving fraction or ability of a cell to divide and form colonies when compared to control. This decline was gradual and greatest decline was observed after 4 weeks of conditioning time. The difference between surviving fraction after 4 and 8 weeks of conditioning time was statistically non significant (Graphs 3and 4). Although cell survival showed a conditioning time dependent decline for all the three groups of both the alloys the greatest decline was observed for 100% reused groups, intermediate results for 50% new + 50% reused groups and the lowest decline for 100% new alloy groups. These results further confirm the findings of cell viability assay.

100% reused groups of both the alloys showed a statistically significant decline in surviving fractions when compared to 50%new +50% reused groups. Where as in cell viability assay the difference between viable cell counts of same groups was non significant. In intra oral environment decline in surviving fraction of cells due to corrosion products released from restorations fabricated from reused Ni-Cr base metal alloy may affect the tissue regeneration and wound healing of oral tissues surrounding these restorations. The Clonogenic assay was gold standard for evaluation of cytotoxicity, where as other assays give an approximate estimation.

In intraoral environment, elevated cell death due to corrosion products released from restorations fabricated from reused Ni-Cr base metal alloy may result in damage and necrosis of the oral tissues surrounding these restorations. Several mechanisms were proposed in the literature to explain these cytotoxic and genotoxic effects of Ni-Cr alloys [13- 16]. Changes in the chemical composition of alloys, the reaction of alloy constituents with atmospheric gases like sulfur, oxygen and carbon during the repeated melting of alloys in atmospheric air could result in the formation of toxic byproducts (i.e. NiS, [Ni.sub.2[S.sub.3]], Ni [(CO).sub.4], [Cr.sub.6+] [Cr.sub.4+]), which were known as cytotoxic and genotoxic. Contamination of alloys from crucibles used for melting and investment molds can be a contributing factor.

Significant elevation in cytotoxicity of reused Ni-Cr dental casting alloys when compared to new alloys has been proven beyond doubt through this in vitro study.

Considering the elevated cytotoxicity and genotoxicity of Ni-Cr base metal alloys in their reused state, the practice of reusing these alloys in dental laboratories for economic reasons should be discouraged. Even though various intra oral protective mechanisms play a major role in combating these harmful stimuli, we are not sure of their threshold limits. However, further studies can be directed to evaluate the tissue response to corrosion products released from Ni-Cr alloys in vivo in order to consolidate the results of this study.

References

(1.) Hesby DA, Kobes P, Garver DG Pelleu GB, "Physical properties of repeatedly used non-precious metal alloys", J Prosthet Dent., 44, 291-293 (1980).

(2.) Wataha JC, Hanks CT, Malcolm CT, "Correlation between cytotoxicity and elements released by dental casting alloys", Int J Prosthodont., 8, 9-14 (1995).

(3.) Wataha JC, Malcolm CT, "Effect of alloy surface composition on release of elements from dental casting alloys", J Oral Rehabit. 23, 583-589 (1996).

(4.) Ozdemir S, Arikan A, " Effect of recasting on the amount of corrosion products released from two Ni-Cr base metal alloys", Eur J Prosthodont Restor Dent., 6, 149-153 (1998).

(5.) Al-Hiyasta AS, Bashabsheh OM, Darmani H, "Elements released from dental casting alloys and their cytotoxic effects", Int J Prosthodont., 15, 473-478 (2002).

(6.) Pratt RM, Wills WD, "In vitro screening assay for teratogens using growth inhibition of human embryonic cells", Proc Natl ACAD Sci., USA, 82, 5791-5794 (1985).

(7.) Puck TT, Marcus PI, "A rapid method for viable cell titration and clone production with Hela cells in tissue culture-The use of X irradiated cells to supply conditioning factors", Pros Nat Acad. Sci., USA, 41, 432-437 (1995).

(8.) Al-Hiyasta AS, Bashabsheh OM, Darmani H, "An investigation of cytotoxic effects of dental casting alloys", Int J Prosthodont., 16, 8-12 (2003).

(9.) Covington JS, Bride MC, Slagle WE, Disney AL, " Quantization of nickel and beryllium leakage from base metal casting alloys", J Prosthet Dent., 54, 127-35 (1984).

(10.) Bumgardner JD, Lucas LC, "Cellular response to metallic ions released from nickel-chromium dental casting alloys", J Dent Res., 74, 1521 1527 (1995).

(11.) Wataha JC, Lockwood PE, "Release of elements from dental casting alloys into cell culture medium over 10 months", Dent Mater., 14, 158 163 (1998).

(12.) Wataha JC, Lockwood PE, Nelson SK, Bouillaguet S, "Long term cytotoxicity of dental casting alloys", Int J Prosthodont., 12, 242-248 (1999).

(13.) Sjogren G Odont LDS, Sletten G, Scient C, et al., "Cytotoxicity of dental alloys, metals and ceramics assessed by Millipore filter, agar overlay and MTT tests", J Prosthet Dent., 84, 229-236 (2000).

(14.) Pierce LH, Goodkind RJ, "A status of possible risks of base metal alloys and their components", J Prosthet Dent., 62, 234-237 (1989).

(15.) Freeman MAR, Miller AT, Roberts BV, "Metal sensitivity as a cause of bone necrosis and loosening of the prosthesis in total joint replacement", J Bone Joint Surg Br., 56B, 426-442 (1974).

(16.) Roe FJC, Carter RL, "Chromium carcinogenesis, calcium chromate as a potent carcinogen for the subcutaneous tissue of the rat", Br J Cancer 23, 172-176 (1969)

Tripuraneni Sunil Chandra, Namburi Suneel Kumar *, Bellam Kranti Kumari (1)

Department of Prosthodontics, (1) Department of Conservative Dendistry and Endodontics, St. Joseph Dental College and Hospital, Duggirala, Eluru, Andhrapradesh

Corresponding author: Dr. Namburi Suneel Kumar, e-mail:dearsuneelin@gmail.com

Received 31 July 2010; Accepted 27 April 2011; Available online 4 May 2011
Table 1: Composition according to weight
percentage by manufacturer
                 Wiron 99   RemanuimCSe

Nickel(Ni)         65           61
Chromium(Cr)      22.5          26
Molybdenum(Mn)    9.5           11
Silicone(Si)       1           1.5
Niobium(Nb)        1            0
Ferrum(Fe)        0.5          >0.5
Cerium(Ce)        0.5          >0.5
Carbon(c)         0.02          0
Aluminum(Al)       0           >0.5
Cobalt(Co)         0           0.5

Table 2: Cell vitality after one week of conditioning time
Test Group

                                Viable Cells

                             Mean [+ or -] SEM

Control                     448000 [+ or -] 5375.87
Wiron99 100% new          399600 [+ or -] 4142.46 (b)
Wiron99 50% + 50%        351200 [+ or -] 4789.57 (c), (#)
Wiron99 100% reused       250200 [+ or -] 5499.09 (c),
Remanium CSe 100% New     399400 [+ or -] 4843.55 (b)
Remanium CSe 50% + 50%   311200 [+ or -] 5489.99 (c), (#)
Remanium CSe 100%        235200 [+ or -] 5112.72 (c), (#)
Reused

Table 3: Cell vitality after two weeks of conditioning time

Test Group                         Cell Viability

                                  Mean [+ or -] SEM

Control                       447940 [+ or -] 3068.159
Wiron99 100% new             372920 [+ or -] 5955.37 (c)
Wiron99 50% + 50%          334960 [+ or -] 6175.88 (c), (#)
Wiron99 100% reused        224400 [+ or -] 5626.72 (c), (#)
Remanium CSe 100% New        362200 [+ or -] 9467.84 (c)
Remanium CSe 50% + 50%     308200 [+ or -] 7213.87 (c), (#)
Remanium CSe 100% Reused   210000 [+ or -] 7609.20 (c), (#)

Table 4: Cell vitality after four weeks of conditioning time

                                    Viable Cells

Test Group                        Mean [+ or -] SEM

Control                        445600 [+ or -] 5065.57
Wiron99 100%New              314340 [+ or -] 3356.42 (c)
Wiron 99 50% + 50%           305400 [+ or -] 5617.82 (c)
Wiron 99 100% Reused        195000 [+ or -] 6252.99 (c), (#)
Remanium CSe 100% New       304800 [+ or -] 10542.29 (c)
Remanium CSe 50% + 50%     287880 [+ or -] 6656.90 (c), (?)
Remanium CSe 100% Reused   184600 [+ or -] 5230.67 (c), (#)

Table 5: Cell vitality after eight weeks of conditioning time

                               Viable Cells

Test Group                   Mean [+ or -] SEM

Control                     448520 [+ or -] 877.72
Wiron99 New               304860 [+ or -] 4871.71 (c)
Wiron 99 50% + 50%        288760 [+ or -] 5737.73 (c)
Wiron 99 100% Reused     190000 [+ or -] 4909.17 (c), (#)
Remanium CSe 100% New     300840 [+ or -] 5010.54 (c)
Remanium CSe 50% + 50%   268000 [+ or -] 10540.39 (c), (?)
Remanium CSe 100%        180800 [+ or -] 1529.70 (c), (#)
Reused

Table 6: Clonogenic assay after one week of conditioning time

Test group           Number      Number of      Plating
                    of cells      Colonies     Efficiency
                     in each    formed from      (PE)
                    petridish   seeded cells

Conrol                 200         173.33       86.665
                       200         172.33       86.165
                       200          175          87.5
                       200         168.33       84.165
                       200         173.33       86.665
Wiron 99 New           200          160           80
                       200          162           81
                       200          160           80
                       200         162.66       81 .33
                       200         160.33       80.165
Wiron 50% + 50 %       200         151.33       75.665
                       200         148.33       74.165
                       200          152           76
                       200         149.33       74.665
                       200         151.33       75.665
Wiron 100% Reused      200          143          71.5
                       200         140.66        70.33
                       200         146.66        73.33
                       200         142.33       71.165
                       200          144           72
Remanium CSe New       200         157.66        78.83
                       200          160           80
                       200          159          79.5
                       200         158.33       79.165
                       200         156.33       78.165
Remanium CSe           200         148.66        74.33
  50% + 50%            200          146           73
                       200          150           75
                       200          148           74
                       200         150.66        75.33
Remanium CSe 100%      200         146.66        73.33
  Reused               200          138           69
                       200          144           72
                       200         144.33       72.165
                       200         140.33       70.165

Test group          Average PE     Surviving          Average
                                   Fraction         SF [+ or -]
                                     (SF)               SEM

Conrol               0.866654      1.007733          1.002698
                     0.86165       1.001919           [+ or -]
                      0.875        1.017442           0.00651
                     0.84165       0.978663
                     0.86665       1.007733
Wiron 99 New           0.8         0.930233        0.936035 (c)
                       0.81         0.94186           [+ or -]
                       0.8         0.930233           0.00324
                      0.8133       0.945698
                     0.80165       0.932151
Wiron 50% + 50 %     0.75665       0.879826      0.874791 (c), (#)
                     0.74165       0.862384           [+ or -]
                       0.76        0.883721           0.00405
                     0.74665       0.868198
                     0.75665       0.879826
Wiron 100% Reused     0.715        0.831395
                      0.7033       0.817791      0.833314 (c), (#)
                      0.7333       0.852674           [+ or -]
                     0.71165        0.8275            0.00578
                       0.72        0.837209
Remanium CSe New      0.7883       0.916628         0.92014 (c)
                       0.8         0.930233           [+ or -]
                      0.795        0.924419           0.0036
                     0.79165       0.920523
                     0.78165       0.908895
Remanium CSe          0.7433       0.864302      0.86432 (c), (#)
  50% + 50%            0.73        0.848837           [+ or -]
                       0.75        0.872093           0.00474
                       0.74        0.860465
                      0.7533        0.87593
Remanium CSe 100%     0.7333       0.842874      0.819908 (c), (#)
  Reused               0.69        0.793103           [+ or -]
                       0.72        0.827586           0.00888
                     0.72165       0.829483
                     0.70165       0.806494

Table 7: Clonogenic assay after two weeks of conditioning time

Test group          Number of    Number of      Plating
                    cells in      Colonies     Efficiency
                      each      formed from       (PE)
                    petridish   seeded cells

Conrol                 200         178.33        89.165
                       200         174.33        87.165
                       200         170.33        85.165
                       200         177.33        88.665
                       200         176.33        88.165
Wiron 99 New           200          153           76.5
                       200         154.66        77.33
                       200          150            75
                       200          155           77.5
Wiron 50% + 50%        200          149           74.5
                       200         141.33        70.665
                       200         141.66        70.83
                       200         139.66        69.83
                       200         141.66        70.83
                       200         136.33        68.165
Wiron 100% Reused      200          130            65
                       200         133.66        66.83
                       200          138            69
                       200         128.66        64.33
                       200         132.66        66.33
                       200         149.33        74.665
Remanium CSe New       200         147.33        73.665
                       200          158            79
                       200         153.33        76.665
                       200          149           74.5
                       200         134.66        67.33
                       200         143.33        71.665
                       200          134            67
                       200          137           68.5
Remanium CSe           200         135.66        67.83
  50% + 50%            200         127.33        63.665
                       200         127.66        63.83
                       200         133.66        66.83
                       200         123.66        61.83
                       200         130.66        65.33

Test group          Average   Surviving      Average SF
                      PE      Fraction        [+ or -]
                                (SF)             SEM

Conrol              0.89165   1 .036802      1.01936 (c)
                    0.87165   1 .013547       [+ or -]
                    0.85165   0.990291         0.00822
                    0.88665   1 .030988
                    0.88165   1 .025174
Wiron 99 New         0.765     0.87931      0.875471 (c)
                    0.7733    0.888851        [+ or -]
                     0.75     0.862069         0.00698
                     0.775    0.890805
Wiron 50% + 50%      0.745    0.856322    0.805333(c), (#)
                    0.70665   0.812241        [+ or -]
                    0.7083    0.814138         0.00586
                    0.6983    0.802644
                    0.7083    0.814138
                    0.68165   0.783506
Wiron 100% Reused    0.65     0.747126    0.762046 (c), (#)
                    0.6683    0.768161        [+ or -]
                     0.69     0.793103         0.00932
                    0.6433    0.739425
                    0.6633    0.762414
                    0.74665   0.858218
Remanium CSe New    0.73665   0.846724       0.870103(c)
                     0.79     0.908046        [+ or -]
                    0.76665   0.881207         0.01105
                     0.745    0.856322
                    0.6733    0.773908
                    0.71665   0.823736
                     0.67     0.770115
                     0.685    0.787356
Remanium CSe        0.6783    0.779655    0.739046 (c), (#)
  50% + 50%         0.63665   0.731782        [+ or -]
                    0.6383    0.733678         0.00968
                    0.6683    0.768161
                    0.6183     0.71069
                    0.6533     0.75092

Table 8: Clonogenic assay after four weeks of conditioning time

Test group           Number     Number of    Plating
                    of cells    Colonies    Efficiency
                     in each     formed        (PE)
                    petridish     from
                                 seeded
                                  cells

Control                200        177.3       88.65
                       200         176          88
                       200       171.66       85.83
                       200         175         87.5
                       200       170.66       85.33
Wiron 99 New           200         146          73
                       200       145.66       72.83
                       200       149.33       74.665
                       200         144          72
                       200       144.66       72.33
Wiron 50% + 50%        200       126.66       63.33
                       200       134.33       67.165
                       200       126.33       63.165
                       200         122          61
Wiron 100% Reused      200       124.66       62.33
                       200       116.33       58.165
                       200       112.33       56.165
                       200       109.66       54.83
                       200       115.33       57.665
                       200         108          54
                       200       145.66       72.83
Remanium CSe New       200       146.33       73.165
                       200       138.33       69.165
                       200       152.66       76.33
                       200       142.33       71.165
Remanium CSe           200       120.66       60.33
  50% + 50%            200         129         64.5
                       200       126.33       63.165
                       200       118.33       59.165
                       200       119.66       59.83
Remanium CSe 100%      200         110          55
  Reused               200       111.33       55.665
                       200         104          52
                       200       105.33       52.665
                       200         108          54

Test group          Average    Surviving    Average
                       PE      Fraction       SF
                                 (SF)      [+ or -]
                                              SEM

Control              0.8865    1.030814    1.01 2349
                      0.88     1.023256    [+ or -]
                     0.8583    0.998023     0.00741
                     0.875     1.017442
                     0.8533    0.992209
Wiron 99 New          0.73      0.83908    0.838678
                     0.7283    0.837126       (c)
                    0.74665    0.858218    [+ or -]
                      0.72     0.827586     0.00529
                     0.7233    0.831379
Wiron 50% + 50%      0.6333    0.727931    0.728713
                    0.67165    0.772011    (c), (#)
                    0.63165    0.726034    [+ or -]
                      0.61     0.701149     0.01182
Wiron 100% Reused    0.6233    0.716437
                    0.58165    0.668563    0.645575
                    0.56165    0.645575    (c), (#)
                     0.5483     0.63023    [+ or -]
                    0.57665    0.662816     0.00917
                      0.54      0.62069
                     0.7283    0.837126
Remanium CSe New    0.73165    0.840977     0.83369
                    0.69165      0.795        (c)
                     0.7633    0.877356    [+ or -]
                    0.71 165   0.817989     0.01363
Remanium CSe         0.6033    0.693448
  50% + 50%          0.645     0.741379
                    0.63165    0.726034
                    0.59165    0.680057
                     0.5983    0.687701
Remanium CSe 100%     0.55     0.632184    0.619149
  Reused            0.55665    0.639828    (c), (#)
                      0.52     0.597701    [+ or -]
                    0.52665    0.605345     0.00791
                      0.54      0.62069

Table 9: Clonogenic assay after eight weeks of conditioning time

Test group               Number         Number of      Plating
                    of cells in each     Colonies     Efficiency
                       petridish       formed from       (PE)
                                       seeded cells

Control                   200              173           86.5
                          200             177.6          88.8
                          200              172            86
                          200             172.33        86.165
                          200             179.66        89.83
                          200             145.66        72.83
Wiron 99 New              200             145.33        72.665
                          200              142            71
                          200             146.66        73.33
                          200             145.66        72.83
Wiron 50% + 50%           200             133.33        66.665
                          200             121.33        60.665
                          200              119           59.5
                          200              121           60.5
                          200              118            59
Wiron 100% Reused         200              107           53.5
                          200              115           57.5
                          200              110            55
                          200              111           55.5
                          200              107           53.5
                          200              143           71.5
                          200              145           72.5
Remanium CSe New          200             143.33        71.665
                          200              142            71
                          200              147           73.5
Remanium CSe              200             126.33        63.165
  50% + 50%               200              128            64
                          200             119.66        59.83
                          200             118.66        59.33
                          200              120            60
Remanium CSe 100%         200             108.33        54.165
  Reused                  200              113           56.5
                          200              103           51.5
                          200             109.66        54.83
                          200              104            52

Test group          Average PE     Surviving        Average SF
                                 Fraction (SF)      [+ or -]SEM

Control              0.866654      1.005814          1.01 6965
                      0.888        1.032558           [+ or -]
                       0.86            1              0.00906
                     0.861 65      1.001919
                      0.8983       1.044535
                      0.7283       0.837126
Wiron 99 New         0.72665        0.83523         0.83369 (c)
                       0.71        0.816092           [+ or -]
                      0.7333       0.842874           0.00458
                      0.7283       0.837126
Wiron 50% + 50%      0.66665       0.766264      0.704207 (c), (#)
                     0.60665       0.697299           [+ or -]
                      0.595        0.683908           0.01592
                      0.605        0.695402           0.01592
                       0.59        0.678161
Wiron 100% Reused     0.535        0.614943      0.632184 (c), (#)
                      0.575         0.66092           [+ or -]
                       0.55        0.632184           0.00852
                      0.555        0.637931
                      0.535        0.614943
                      0.71 5       0.821839
                      0.725        0.833333
Remanium CSe New     0.71665       0.823736        0.827966 (c)
                       0.71        0.816092           [+ or -]
                      0.735        0.844828           0.00505
Remanium CSe         0.63165       0.726034
  50% + 50%            0.64        0.735632
                      0.5983       0.687701
                      0.5933       0.681954
                       0.6         0.689655
Remanium CSe 100%    0.541 65      0.622586      0.618379 (c), (#)
  Reused              0.565        0.649425           [+ or -]
                      0.51 5       0.591954           0.0106
                      0.5483        0.63023
                       0.52        0.597701
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