The verification of the MTT assay on the viability of periodontal ligamental cells in rat molars through the histologic examination

Hyun-Ki Kim; Eui-Seoung Kim; In-Bok Choi; Jin Kim; Seung-Jong Lee

doi:10.5395/JKACD.2003.28.5.385

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Original Article The verification of the MTT assay on the viability of periodontal ligamental cells in rat molars through the histologic examination: Hyun-Ki Kim, Eui-Seoung Kim, In-Bok Choi, Jin Kim, Seung-Jong Lee; 2003;28(5):-391.
DOI: https://doi.org/10.5395/JKACD.2003.28.5.385
Published online: September 30, 2003

Department of Conservative Dentistry, College of dentistry, Yonsei University, Korea.

Corresponding author (sjlee@yumc.yonsei.ac.kr)

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Abstract
REFERENCES

Abstract

The purpose of this study is to examine the viability of PDL cells in rat molars by using MTT assay and to verify the MTT assay through the histologic observation. Thirty of Sprague-Dawley white female rats of 4-weeks old with a body weight of about 100 grams were used. Groupings are as follows:

Immediate Group : Positive control group(n=10)-after extraction immediately.

Dried Group : Negative control group(n=10)-after drying for an hour under warm dry.

ViaSpan® Group : 1hour ViaSpan® group(n=10)-after storing in ViaSpan® at 4℃ for 1hour.

Ten teeth of each group were treated as same as above and replanted to the original socket of experimental animals. After two weeks of replantation, all the experimental animals were sacrificed. And after fixation, extracted maxillary jaw was dimineralized. After it was embedded in paraffin, serial section by 5µm was carried out and for construction of specimen, hematoxylin-eosin dye was used.

The mean MTT measurement of immediate group(positive control) is 2.81 and the mean measurement of dried group(negative control) is 0.98 which is significant differnt(P<0.05). The mean measurement of ViaSpan® group is 2.65 and there is significant difference between dried group and ViaSpan® group(P<0.05). However, there is no difference between immediate group and ViaSpan® group. The average resorption points of immediate group is 3.03 points. In the dried group, average 6.44 points resorption and 2.68 points showed resorption in the ViaSpan® group. Unlike with MTT assay, there was no significant difference between the immediate group and ViaSpan® group.

The usage of MTT assay as a viable cell marker may give us a better indication of the maintenance of periodontal ligament cell vitality.
Keywords: MTT assay; Root resorption; ViaSpan®

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Table 1

Mean and standard deviation of the optical density(OD)

^*Statistically significant at the 95% level of confidence

Table 2

The mean number of resorption points

^*Statistically significant at the 95% level of confidence

REFERENCES

1. Lee SJ, Jung IY, Lee CY, Choi SY, Kum KY. Clinical application of computer-aided rapid prototyping for tooth transplantation. Dent Traumatol. 2001;17: 114-119.Article PubMed PDF
2. Andreasen JO, Kristerson L. The effect of limited drying or removal of the periodontal ligament. Periodontal healing after replantation of mature permanent incisors in monkeys. Acta Odontol Scand. 1981;39: 1-13.PubMed
3. Söder PO, Otteskog P, Andreasen JO, Modeer T. Effect of drying on viability of the periodontal membrane. Scand J Dent Res. 1977;85: 164-168.PubMed
4. Andreasen JO, Hjorting-Hansen E. Replantation of teeth. I. Radiographic and clinical study of 110 human teeth replanted after accidental loss. Acta Odontol Scand. 1966a;24: 263-286.Article PubMed
5. Andreasen JO, Hjorting-Hansen E. Replantation of teeth. II. Histological study of 22 replanted anterior teeth in humans. Acta Odontol Scand. 1966b;24: 287-306.Article PubMed
6. Blomlöf L. Milk and saliva as possible storage media for traumatically exarticulated teeth prior to replantation. Swed Dent J Suppl. 1981;8: 1-26.PubMed
7. Krasner P, Rankow HJ. New philosophy for the treatment of avulsed teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1995;79: 616-623.Article PubMed
8. Layug ML, Barrett EJ, Kenny DJ. Interim storage of avulsed permanent teeth. J Can Dent Assoc. 1998;64: 357-363. 365-369.PubMed
9. Harkacz OM Sr, Carnes DL Jr, Walker WA 3rd. Determination of periodontal ligament cell viability in the oral rehydration fluid Gatorade and milks of varying fat content. J Endod. 1997;23: 687-690.Article PubMed
10. Ashkenazi M, Sarnat H, Keila S. In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in six different media. Endod Dent Traumatol. 1999;15: 149-156.Article PubMed
11. Patel S, Dumsha TC, Sydiskis RJ. Determining periodontal ligament (PDL) cell vitality from exarticulated teeth stored in saline or milk using fluorescein diacetate. Int Endod J. 1994;27: 1-5.Article PubMed
12. Hupp JG, Trope M, Mesaros SV, Aukhil I. Tritiated thymidine uptake in periodontal ligament cells of dogs' teeth stored in various media for extended time periods. Endod Dent Traumatol. 1997;13: 223-227.Article PubMed
13. Lekic PC, Kenny DJ, Barrett EJ. The influence of storage conditions on the clonogenic capacity of periodontal ligament cells: implications for tooth replantation. Int Endod J. 1998;31: 137-140.Article PubMed
14. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65: 55-63.Article PubMed
15. Andreasen JO. Analysis of pathogenesis and topography of replacement root resorption (ankylosis) after replantation of mature permanent incisors in monkeys. Swed Dent J. 1980a;4: 231-240.PubMed
16. Andreasen JO. Analysis of topography of surface- and inflammatory root resorption after replantation of mature permanent incisors in monkeys. Swed Dent J. 1980b;4: 135-144.PubMed
17. Birkedal-Hansen H. External root resorption caused by luxation of rat molars. Scand J Dent Res. 1973;81: 47-61.Article PubMed
18. Bornstein P. The cross-linking of collagen and elastin and its inhibition in osteolathyrism. Is there a relation to the aging process? Am J Med. 1970;49: 429-435.PubMed
19. Barrington EP, Meyer J. Recovery of the rat dental organ from experimental lathyrism. J Periodontol. 1966;37: 453-467.Article PubMed
20. Cho MI, Garant PR. The effect of beta-aminoproprionitrile on the periodontal ligament. I. Ultrastructure of fibroblasts and matrix. J Periodontal Res. 1984a;19: 247-260.PubMed
21. Cho MI, Garant PR. The effect of beta-aminoproprionitrile on the periodontal ligament: II. Radioautographic study of collagen secretion from fibroblasts. Anat Rec. 1984b;209: 41-52.Article PubMed
22. Ashkenazi M, Marouni M, Sarnat H. In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in four media at room temperature. Endod Dent Traumatol. 2000;16: 63-70.Article PubMed PDF
23. Hiltz J, Trope M. Vitality of human lip fibroblasts in milk, Hanks balanced salt solution and Viaspan storage media. Endod Dent Traumatol. 1991;7: 69-72.PubMed
24. Fischer S, Maclean AA, Liu M, Cardella JA, Slutsky AS, Suga M, Moreira JF, Keshavjee S. Dynamic changes in apoptotic and necrotic cell death correlate with severity of ischemia-reperfusion injury in lung transplantation. Am J Respir Crit Care Med. 2000;162: 1932-1939.Article PubMed
25. Hyon SH, Kim DH. Long-term preservation of rat pancreatic islets under physiological conditions. J Biotechnol. 2001;85: 241-246.Article PubMed
26. Sitasawad SL, Patole MS. Effect of cryopreservation on lipid peroxidation in chick cornea. Indian J Exp Biol. 1998;36: 1025-1027.PubMed
27. Santanam N, Ramachandran S, Parthasarathy S. Oxygen radicals, antioxidants, and lipid peroxidation. Semin Reprod Endocrinol. 1998;16: 275-280.PubMed
28. Buttke TM, Trope M. Effect of catalase supplementation in storage media for avulsed teeth. Dent Traumatol. 2003;19: 103-108.Article PubMed PDF
29. Chung WG. In vitro assessment on viability of human periodontal ligament cells after storage in chlorophyllin-added medium. 2002;Seoul: Yonsei Universtiy; Doctorial thesis.

Tables & Figures

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Table 1

Mean and standard deviation of the optical density(OD)

^*Statistically significant at the 95% level of confidence

Table 2

The mean number of resorption points

^*Statistically significant at the 95% level of confidence

REFERENCES

1. Lee SJ, Jung IY, Lee CY, Choi SY, Kum KY. Clinical application of computer-aided rapid prototyping for tooth transplantation. Dent Traumatol. 2001;17: 114-119.Article PubMed PDF
2. Andreasen JO, Kristerson L. The effect of limited drying or removal of the periodontal ligament. Periodontal healing after replantation of mature permanent incisors in monkeys. Acta Odontol Scand. 1981;39: 1-13.PubMed
3. Söder PO, Otteskog P, Andreasen JO, Modeer T. Effect of drying on viability of the periodontal membrane. Scand J Dent Res. 1977;85: 164-168.PubMed
4. Andreasen JO, Hjorting-Hansen E. Replantation of teeth. I. Radiographic and clinical study of 110 human teeth replanted after accidental loss. Acta Odontol Scand. 1966a;24: 263-286.Article PubMed
5. Andreasen JO, Hjorting-Hansen E. Replantation of teeth. II. Histological study of 22 replanted anterior teeth in humans. Acta Odontol Scand. 1966b;24: 287-306.Article PubMed
6. Blomlöf L. Milk and saliva as possible storage media for traumatically exarticulated teeth prior to replantation. Swed Dent J Suppl. 1981;8: 1-26.PubMed
7. Krasner P, Rankow HJ. New philosophy for the treatment of avulsed teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1995;79: 616-623.Article PubMed
8. Layug ML, Barrett EJ, Kenny DJ. Interim storage of avulsed permanent teeth. J Can Dent Assoc. 1998;64: 357-363. 365-369.PubMed
9. Harkacz OM Sr, Carnes DL Jr, Walker WA 3rd. Determination of periodontal ligament cell viability in the oral rehydration fluid Gatorade and milks of varying fat content. J Endod. 1997;23: 687-690.Article PubMed
10. Ashkenazi M, Sarnat H, Keila S. In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in six different media. Endod Dent Traumatol. 1999;15: 149-156.Article PubMed
11. Patel S, Dumsha TC, Sydiskis RJ. Determining periodontal ligament (PDL) cell vitality from exarticulated teeth stored in saline or milk using fluorescein diacetate. Int Endod J. 1994;27: 1-5.Article PubMed
12. Hupp JG, Trope M, Mesaros SV, Aukhil I. Tritiated thymidine uptake in periodontal ligament cells of dogs' teeth stored in various media for extended time periods. Endod Dent Traumatol. 1997;13: 223-227.Article PubMed
13. Lekic PC, Kenny DJ, Barrett EJ. The influence of storage conditions on the clonogenic capacity of periodontal ligament cells: implications for tooth replantation. Int Endod J. 1998;31: 137-140.Article PubMed
14. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65: 55-63.Article PubMed
15. Andreasen JO. Analysis of pathogenesis and topography of replacement root resorption (ankylosis) after replantation of mature permanent incisors in monkeys. Swed Dent J. 1980a;4: 231-240.PubMed
16. Andreasen JO. Analysis of topography of surface- and inflammatory root resorption after replantation of mature permanent incisors in monkeys. Swed Dent J. 1980b;4: 135-144.PubMed
17. Birkedal-Hansen H. External root resorption caused by luxation of rat molars. Scand J Dent Res. 1973;81: 47-61.Article PubMed
18. Bornstein P. The cross-linking of collagen and elastin and its inhibition in osteolathyrism. Is there a relation to the aging process? Am J Med. 1970;49: 429-435.PubMed
19. Barrington EP, Meyer J. Recovery of the rat dental organ from experimental lathyrism. J Periodontol. 1966;37: 453-467.Article PubMed
20. Cho MI, Garant PR. The effect of beta-aminoproprionitrile on the periodontal ligament. I. Ultrastructure of fibroblasts and matrix. J Periodontal Res. 1984a;19: 247-260.PubMed
21. Cho MI, Garant PR. The effect of beta-aminoproprionitrile on the periodontal ligament: II. Radioautographic study of collagen secretion from fibroblasts. Anat Rec. 1984b;209: 41-52.Article PubMed
22. Ashkenazi M, Marouni M, Sarnat H. In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in four media at room temperature. Endod Dent Traumatol. 2000;16: 63-70.Article PubMed PDF
23. Hiltz J, Trope M. Vitality of human lip fibroblasts in milk, Hanks balanced salt solution and Viaspan storage media. Endod Dent Traumatol. 1991;7: 69-72.PubMed
24. Fischer S, Maclean AA, Liu M, Cardella JA, Slutsky AS, Suga M, Moreira JF, Keshavjee S. Dynamic changes in apoptotic and necrotic cell death correlate with severity of ischemia-reperfusion injury in lung transplantation. Am J Respir Crit Care Med. 2000;162: 1932-1939.Article PubMed
25. Hyon SH, Kim DH. Long-term preservation of rat pancreatic islets under physiological conditions. J Biotechnol. 2001;85: 241-246.Article PubMed
26. Sitasawad SL, Patole MS. Effect of cryopreservation on lipid peroxidation in chick cornea. Indian J Exp Biol. 1998;36: 1025-1027.PubMed
27. Santanam N, Ramachandran S, Parthasarathy S. Oxygen radicals, antioxidants, and lipid peroxidation. Semin Reprod Endocrinol. 1998;16: 275-280.PubMed
28. Buttke TM, Trope M. Effect of catalase supplementation in storage media for avulsed teeth. Dent Traumatol. 2003;19: 103-108.Article PubMed PDF
29. Chung WG. In vitro assessment on viability of human periodontal ligament cells after storage in chlorophyllin-added medium. 2002;Seoul: Yonsei Universtiy; Doctorial thesis.

Citations

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