The radioactive chromium (51Cr) release method is one of the commonly used screening tests recom­ mended by FDI and ADA. This method has been developed by Spangberg1 for the evaluation of the cytotoxicity of endodontic filling materials using established cell lines in in vitro model. In that method Spangberg used a cell monolayer pre-labeled for 20 hours with 51Cr in sodium chromate. The test material is prepared in culture chambers and a suspension of labelled cells is added. After various incubation periods,51Cr released from the cells into the media and values obtained from cells exposed to the material are measured and com­ pared with those of the controls.2,31Cr has been used widely in vitro and in vivo by many investigators to measure the volume of cir­ culating red cells,4 to tag and measure red blood cells survival,5,6,7 to study the life span of leuko­ cytes,8 and to study hemolysis.9 In addition, this method has been used for quantitative and kinetic studies of immunologically induced lysis of nuc­ leated cells,10,11 and for material toxicity evalua-tion2,3
5lCr, as an isotonic solution of Na2 Cr 04, non-covalently binds to proteins and other cell constituents.5 This method has the following advantages:
(a)      the labeling of the cells is very simple and rapid;
(b)      the half-life of51Cr is short (about 27 days);
(c)       in contrast to many other isotopes such as 1H-thymidine and 32P, the released 59Cr can not be reutilized by the cells6,8,12,13,14 as the chromate is reduced during binding;
(d)   non-specific leakage of the isotope is very low;
(e)   quantitative cytotoxicity assays are accurate because of minimum handling;
(f)     only small amount of 59Cr is required, and
(g)    it is not expensive.
In addition to the radiochromium release method, several othertechniques such as quantita­ tive enzyme cytochemistry methods,15 vital stain exclusion,16 filter technique using Millipore fil­ ters,17 and hanging drop cultures18 have been used to study cellular changes caused by endodontic fil­ling materials. The question has been raised whether the chromium labeling procedure could affect the cell and alter the ultrastructure of the cell during the incubation time or not. In addition, elec­ tron microscopic evaluation of the chromium labeled cell has not yet been reported.
Thus, the purpose of the present investigation is to evaluate on electron microscopic level, the mor­ phological effects of the 51Cr labeling on the cul­ tured cells. Two different cultured cells will be used, established cell lines and primary cells.

  1.    Cells
A.      L 929 cell line
Three to five day old culture of L 929 cells were used (Flow laboratories, McLean, VA, USA). The cells were suspended in culture medium at a density of 5 X 105 cells per milliliter. The culture medium was changed every other day and the day before an experiment. Cells were harvested with 0.02% trypsin in phosphate buffered saline (PBS).
B.      Human periodontal ligament fibroblasts
Periodontal tissue was obtained from a maxillary premolar tooth extracted during orthodontic therapy from a 14 year old male patient. After extraction, the tooth was stored in PBS. The periodontal ligament tissue was scraped off, washed several times with PBS and then cut into small pieces with scissors. The tissue fragments were washed again with PBS, suspended in culture medium and centrifuged at lOOXg for 5 minutes. The tissue fragments were transferred into culture flasks and allowed to settle on the bottom of the flasks using a small amount of medium. The flasks were placed in a humidified incubator at 37°C for 30 minutes to allow the tissue fragments to attach to the plastic substrate. More medium was added and incubation continued for one week. The fourth subculture was used. The cells were suspended in culture medium at a density of 5 X 105 cells per mil­ liliter. The cells were grown and harvested in the same manner as the L 929 cells.
2.      Culture Medium
Eagle's MEM with Earl's BSS (Flow laboratories, McLean,VA,USA), supplemented with 10% (v/v) fetal calf serum, 2mM L-glutamine and 2.2mg sodium bicarbonate per ml was used. In addition, 100 I.U/ml penicillin and 50 ug/ml streptomycin were added to the culture medium.
3.      Cell Labeling
1Cr was supplied as sodium chromate in a sterile isotonic solution. The activity of the sodium chro­ mate was 350 to 450mC per mg. The cells were labeled with approximately 2uC per 105 cells 12 to 20 hours before the experiment. The radioc­ hromium labeling procedure described by Spangberg1 was followed.
4.      Experimental Procedures
The labeled cells were harvested with 0.02% trypsin and washed in PBS solution before being suspended in culture medium. The cell suspension was washed and centrifuged in culture medium four times at 500Xg. The experiments were per­ formed in plastic tissue culture clusters (Costar Cambridge, Mass. USA) containing 24 wells each with an inner diameter of 16mm. One ml of the cell suspension and one ml of the culture medium were added to culture wells and incubated at 37CC and 100% humidity for two and four hours. At the end of the incubation period, the cell were prepared for electron microscope examination in the following ways:
A.    Scanning electron microscopy(SEM)
The cell cultures (L 929 and PDL fibroblasts) were prefixed in 0.1 % glutaraldehyde in tissue cul­ ture medium for 5 minutes. The medium was then decanted and replaced with 2.0% glutaraldehyde in 100mM Na cacodylate buffer (pH 7.2) at room temperature and fixed for half an hour in the same solution. Specimens weredehydrated (5 minutes each) through 50%, 70%, 90% and 100% ethanol, then criticalpoint dried with C02. The tissue culture cluster wells were trimmed, mounted using silver con­ ducting paint and gold sputter coated to a thick­ ness of 5-7mm. Specimens were then examined with a Hitachi H300 microscope equipped with a H3010 scanning attachment, operated at an accelerating voltage of 20Kv.
B.    Transmission electron microscopy (TEM)
The cell cultures were prefixed in 0.1% gfutaraldehyde in tissue culture medium for 5 minutes. The medium was then decanted and replaced with 2.0% glutaraldehyde in 100mM Na cacodylate buffer (pH 7.2) at room temperature and fixed for 5 minutes in the same solution. An equal volume of 2.0% osmium tetraoxide in 100mM cacodylate buffer (pH 7.2) was added and the culture fixed for one hour followed by three washes of 5 minutes each with distilled water. The cells were stained with 1% uranyl acetate for 30 minutes, and the washed three times (10 minutes each) with dis­ tilled water. The specimens were dehydrated (10 minutes each) in a graded series of 50%, 70%, 90%, 95% and two 100% ethanol washes.
Cells were infiltrated in 1:1 poly Bed:ethanol for one hour then replaced with fresh poly Bed (Polysciences Inc. Warrington, PA,USA). The resin was changed again after one hour and left overnight. Finally, the specimens were embedded in fresh poly Bed and cured in a 60°C oven for 24 hrs. Semi-thin sections were cut with a glass knife, stained and viewed with the light microscope to check the general condition and orientation of the specimen and to select areas of interest. Thin sections were cut with a dia­mond knife, mounted on 300 mesh copper grids, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM10 at 80Kv accelerating voltage.
Unlabeled cells grown under control culture conditions were prepared for electron microscopy in the same manner.

  1.    Morphology of Unlabeled Cells
PDL fibroblasts
Fibroblasts were elongated, spindle shaped with tapering cytoplasmic extensions. The outer surface of the cell was generally smooth but sometimes covered with microvilli [Fig. 1]. The nuclei appeared ovoid with a double membrane. Mitochondria were elongated, round or rod-like in shape with double membranes and well preserved cristae. The rough endoplasmic reticulum comprised a network of narrow cisternae distributed around the nucleus and in the cytoplasm. Free ribosomes were widely distributed and some attached to the surface of the endoplasmic reticulum. Polyribosomes and vacules within the cytoplasm were occasionally observed. Several well developed Golgi complexes were seen composed of arrays of packed cisternae and numerous small vesicles.
L929 cells
The cells were elongated, spindle or fusiform-shaped. Dividing cells appeared round in shape and were observed scattered throughout the cul­ ture. The outer surface of the cells were covered with large numbers of hemisphere shaped projec­ tions (blebs).
Few microvilli were observed [Fig. 2]. The nuclei occupied most of the cell with a double mem- brane. The chromatin was evenly distributed in the nucleolus.
Mitochondria  with  double membranes appeared circular or rod-like in shape. Cristae were well preserved. Lysosomes, polysomes and vac- ules were occasionally observed.
A network of rough-surfaced endoplasmic reticulum was distributed in the cytoplasm. Ribo-somes were seen on the surface of the endoplasmic reticulum and in the cytoplasm. Golgi complexes were occasionally seen.
2.  Effect of Chromium Labelling on Cell Morphology
PDL fibroblasts
Figures 3-4 illustrate the 51Cr effect on the PDL cell morphology. After two hours of incubation, the labelled cells were either spherical or irregularly elongated spindle- shaped and the cell body covered with microvilli. After an additional two hours of incubation, the cells were spread and exhibited a spindle to elongated shape with numer­ ous microvilli on the cell body [Fig. 3]. Some cells had smooth surfaces. Lamellar cytoplasm was seen around the cell body at both 2 and 4 hours of incu­ bation and the intracellular organelles were normal and well developed [Fig. 4].
L929 cells
Figures 5-6 illustrate the 51Cr effect on the L 929 cell morphology. After two hours of incubation, the labeled cells exhibited a fusiform, stellate or spindle-like profiles. A number of rounded cells were seen. Most of the cells had microvilli, and some cells had blebs on the surface. The internal structure of the cells were normal. Occasionally, mitotic figures, Golgi complexes and vacules were observed. After an additional two hours of incuba­ tion, the cells were spindle- shape and covered with microvilli [Fig. 5]. Mitotic figures were seen more often and the intracellular structure was well developed [Fig. 6].

It was noticed in this experiment that the outer surface of both cell types were covered with micro­ villi. This was believed to be important for their attachment to the substrate to provide good spreading. The presence of the microvilli was a characteristic feature of the spreading of L 929 cells.19
As the cells begin to spread, the microvilli shor­ ten and decrease in number.20 The degree of spreading varies with time and, therefore, the shape of the spreading cells varied from one cell to another. The labeled L 929 cells spread faster than the labeled PDL cells after two hours or four hours incubation.
Spangberg1 reported that an adequate label would be achieved after 6 hours and the increase of the time after 24 hours would be negligible. In this experiment, the labelling procedure was done 12-20 hours before the experiment. This time was cho­ sen for practical reasons.
Of the 75 uC of 51Cr used to label a 75 cm2 cul­ ture flask, about 5 percent of the isotopes were usu­ ally taken up by the cells during an overnight label­ ing.21 Consequently, when the culture medium was poured off at the end of the labelling period, 95 percent of the radioactivity was discarded. This resulted in less than 0.05 uC per ml of radiation in the cell suspension used for the experiments. Thus, the amount of sodium chromate remaining in the culture after completed labelling was approxi­ mately 4 X 10-7 mg/ml. This was very low and non­ toxic to the cells as observed by Eadie & Brown22, Gibson & Scheitlin23 and Spangberg.1 It has been established that approximately 1 mg/ml of sodium chromate can be tolerated in culture before toxic responses are observed.21 The electron micro­ scope examination of the chromium labelled cells confirmed the above observation. The chromium labelling did not alter the outer ultrastructure or the internal morphology of the cells. Both cell types looked normal and the intra-structural mor­ phologies were similar to the control cells. There­ fore, it was concluded that the use of 51CR to label cells for in vitro testing of the cytotoxicity of dental materials does not alter the cells or influence the experimental results.

  Based on observations from this study, it would appear that: