Dental Traumatology 2014; 30: 265–269; doi: 10.1111/edt.12098
Temporary preservation of avulsed tooth in oral submucosal tissue: an experimental study in cat Mohammad Reza Jamalpour1, Ali Reza Soltanian2, Amir Sasan Tootunchi3, Milad Roshanipaian4 1 Department of Oral and Maxillofacial Surgery, School of Dentistry, Hamadan University of Medical Sciences; 2Department of Biostatistics and Epidemiology, School of Public Health, Hamadan Medical Sciences University; 3 Department of Biochemistry and Biotechnology, Hamadan Medical Sciences University; 4General Dental Practitioner, Hamadan, Iran
Abstract – Objective: The purpose of this study was to determine the ability of oral submucosal tissue to serve as a temporary storage medium for the maintenance of periodontal ligament (PDL) cell viability of avulsed teeth. Materials and methods: Thirty cats were divided into five groups. After extraction of three teeth in each cat, one tooth was put in the depth of cat’s oral submucosal tissue and the other two teeth were put in Hanks’ balanced salt solution (HBSS) and tap water. The teeth were removed after 8, 24, 48, 72, and 168 h from their mediums and sent for laboratory processing and counting of vital periodontal cells. Results: Statistical analysis demonstrated that submucosal tissue kept PDL cells viable as good as HBSS. Conclusion: This animal study showed that the efficacy of oral submucosal tissue in maintaining the viability of human PDL cells is similar to that of HBSS.
Key words: periodontal ligament cells; storage media; submucosal tissue; tooth avulsion Correspondence to: Mohammad Reza Jamalpour, Department of Oral and Maxillofacial Surgery, School of Dentistry, Hamadan University of Medical Sciences, Hamadan, Iran Tel.: +989183134395 Fax: +988118350126 e-mails: [email protected]; [email protected] Accepted 26 January, 2014
Maxillofacial emergencies vary from odontogenic infections to facial fractures. Approximately 15% of all emergency room visits are for maxillofacial trauma and 2% of these cases are pure dentoalveolar trauma (1). On the basis of World Health Organization (WHO) classification, avulsion is the complete displacement of a tooth from its alveolar socket secondary to traumatic injury (1). Avulsion of a permanent tooth is a real dental emergency. It consists of 0.5–3% of all dental injuries in the permanent dentition (2, 3). Although damage to the periodontal ligament (PDL) cells during an avulsion injury is unavoidable, maintaining their viability is critical. One of the most important factors that determine the prognosis for the avulsed tooth is the duration of extra-alveolar time (4, 5). In fact, immediately after avulsion, the number of viable PDL cells on the root surface decreases with increasing drying time and it is impossible to detect viable cells after 2 h (6). Thus, the best treatment at the time of avulsion is immediate replantation and hence re-establishing the natural nutrient supply to the PDL cells (7). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Although immediate replantation has the best prognosis (3, 8–10), certain situations, like the presence of more severe injuries, which requires medical management, can cause the delay in immediate replantation of the avulsed tooth. Generally, where such situations exist, the tooth should be stored in a storage medium until definitive dental treatment can be accomplished. This storage medium should preserve PDL cells’ viability, adherence, and clonogenic capacity (11). Likewise, an ideal storage medium should be readily available or easily accessible at the site of an accident. There are many storage media available for avulsed teeth, including milk, physiologic saline solution, egg white, and Hanks’ balanced salt solution (HBSS) (12). These days, the best known storage medium is HBSS (13), but except for a few areas of the world, it is not easily available at the scene of the injury (14). In this study, oral submucosal tissue was tested for the ability to maintain the viability of PDL cells because, when avulsion occurs in a hospital setting, for example during intubation of endotracheal tube, in comatose 265
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patients or patients with low Glasgow Coma Scale (GCS), the easiest and fastest way for a physician is creating a stab incision in the oral mucosa, placing the avulsed tooth in the depth of incision, and finally suturing the incision with one or two stitches. Then, after improving the condition, for example recovery from general anesthesia (GA) or increasing the level of patient consciousness, the hidden tooth can be removed and replanted in its socket by that physician or a dentist. The purpose of this study was to evaluate the capability of oral submucosal tissue in comparison with HBSS (as the positive control) and water (as the negative control) in maintaining the viability of PDL cells. Materials and methods
This study was approved by the Ethics Committee of the Hamadan University of Medical Sciences (P/16/35/ 1/120151) prior to the initiation of the study. Thirty healthy cats were examined in this experiment. The animals were housed in individual cages and maintained on a commercial animal diet. After each surgical procedure, a soft diet was administered for 10 days. Each cat was operated twice. All surgical procedures were performed under general anesthesia, achieved by 2–3 mg kg 1 Xylazine (Eurovet Animal Health B.V., Bladel, The Netherlands) intramuscularly, followed by an intramuscular injection of ketamine at 15 mg kg 1 (Rotexmedica, Trittau, Germany). For reduction in pain, all incised sites were infiltrated with lidocaine 1% (Pasteur Institute of Iran, Tehran, Iran) before incision. Before the first surgery, the mouth was rinsed with chlorhexidine 0.2% (Behsa Pharmaceutical, Arak, Iran). Then, three lower incisors of each cat were extracted as atraumatically as possible. Teeth with moderate-to-severe periodontal disease or with caries were not included in this study. Following extractions, the teeth were held with forceps by the coronal region, and coronal 2 mm of PDL was scraped with a curette to remove cells that may have been damaged. One tooth was placed in tap water as a negative control, and the other one was put in HBSS as a positive control. Before extraction of third incisor, a stab incision was performed in the lower vestibule, and after creation of adequate space in the depth of submucosa, this tooth was extracted and immediately placed in this submucosal space. The incision was sutured with 4-0 silk suture (SUPA Medical Devices, Tehran, Iran). Totally, 90 permanent periodontal disease-free lower incisor teeth were extracted. Thirty teeth were placed in the water, 30 were placed in the HBSS, and 30 were inserted in the depth of oral submucosal tissue. All cats received intramuscular injection of penicillin G 800 000 U (20 000 U kg 1, BID) (Jaber Ebne Hayyan Pharmaceutical Co, Tehran, Iran) postsurgically for 5 days and were fed on a soft diet for 10 days. At the end of the first surgery, the cats were randomly divided into five groups and the teeth in groups 1–5 were removed from submucosa of each cat and its controls (HBSS and water) after 8, 24, 48, 72, and
168 h in order. Therefore, for each tooth that was maintained in submucosal tissue for the determined time, there was one tooth that was preserved in HBSS for the same time (as positive control) and there was one tooth that was preserved in water for the same time (as negative control). The teeth that were removed from submucosa were washed in sterile saline solution to wash out the residual blood. Then, the specimens were transferred to the laboratory in 15-ml Falcon tubes with 10 ml of HBSS. The method used in this study for preparation and counting of the viable PDL cells was the same as the method used by Doyle et al. (15). To obtain the PDL cells, the teeth were trypsinized using a 2.5% trypsin solution (Sigma-Aldrich, St. Louis, MO, USA) in normal saline at 37°C for 10 min with occasional shaking, and then, the supernatant was removed. Each specimen tooth, after washing, was incubated for 40 min in 10 ml of 0.1% collagenase (SigmaAldrich) in normal saline at 37°C with occasional shaking. After incubation, the enzyme activity was stopped by the addition of 5 ml of RPMI 1640 culture medium (Sigma-Aldrich) with 10% fetal calf serum. Then, the tooth was drawn out and the suspension was centrifuged at 172 g for 5 min at 4°C. Finally, after removing the supernatant , the precipitate was re-suspended in 10 ml of HBSS at 4–6°C and filtered through a 100lm-size-pore filter. Cell viability was determined by adding trypan blue (1 volume per 2.5 volumes of medium; Sigma-Aldrich) to the suspension (16). Then, cells were examined for staining under light microscope with 400% magnification. An average of 100 cells per field was counted in four different fields per culture. Data were statistically analyzed using a two-way repeated measurement complemented by the Bonferroni test. Cell viability was transformed by Arcsin. The level of statistical significance was lower than 5% (P < 0.05). Results
Table 1 shows the mean percentage and standard deviation values of cell viability in different examined times. The percentage of viable PDL cells in submucosal tissue were significantly more than in tap water (negative control) in all tested times (P < 0.001). According to these results, there is no statistically significant difference between oral submucosal tissue and HBSS (Table 1, Fig. 1). Discussion
The prognosis of an avulsed tooth is largely dependent on the number of viable PDL cells at the moment of replantation (17). On the other hand, the viability of these cells is highly dependent on the extra-alveolar time (3, 4). Hence, the ideal treatment of choice at the time of avulsion is immediate replantation (9). But some conditions may occur that delay immediate replantation. Usually, there are concomitant injuries with tooth avulsion, as facial lacerations or fractures (3), or even more serious injuries as intracranial or abdominal traumas. Obviously, these injuries should be © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Avulsed tooth preservation in oral submucosa Table 1. Mean percentage and standard CRUD values of cell viability in different times Time (h) Material 8
HBSS Submucosal tissue HBSS Submucosal tissue HBSS Submucosal tissue HBSS Submucosal tissue
24 48 72
Mean SD
P (95% CI)
85.56 9.27 86.67 8.34
0.731 ( 7.42, 5.20)3
30.87 4.71 35.54 4.99
0.134 ( 6.14, 1.19)3
18.81 4.7 16.44 3.92
0.146 ( 0.73, 0.93)3
9.34 1.87 8.42 2.79
0.304 ( 0.78, 2.62)3
1
P2 (95% CI) 0.126
1 The P values to compare cell viability mean between HBSS and submucosal clefts at 8, 24, 48, and 48 h separately based on the Bonferroni test. 2 The P value of repeated measurement analysis to compare cell viability mean between HBSS and submucosal clefts at 8, 24, 48, and 48 h. 3 95% confidence interval for mean difference in cell viability between HBSS and submucosal clefts at 8, 24, 48, and 48 h.
Materials: HBSS Submucosal tissue Water
100.00
Means
80.00
60.00
40.00
20.00
0.00 8.00
24.00
48.00 Hours
72.00
Fig. 1. Time-dependent results according to the experimental solutions and cell viability.
managed before tooth replantation. Also, avulsion may be seen in patients with decreased or lost consciousness. For example, some avulsions may be occurred during the management of the comatose patients (1) or nasotracheal intubation procedure (1, 18–20). Its importance will be more obvious if we know that perioperative tooth damages are the most common medicolegal claimsrelated anesthesia (21). And also the knowledge of the emergency management of tooth avulsion by emergency medical staffs and physicians is not adequate (22). It is clear that replantation of avulsed tooth in patients with decreased consciousness could be resulted in aspiration of tooth and it is better to delay replantation until increasing the level of patient consciousness. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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In these situations, the avulsed tooth should be preserved in a storage medium that can maintain the PDL cells’ viability for a few days until definite dental treatment is fulfilled. Although HBSS is the best known storage medium, its ability to maintain tooth viability is proven for only 4 days. Likewise, HBSS may not be found in some operating rooms or medical emergency departments. On the other hand, surgeons usually use subcutaneous or submucosal tissue for temporary preserving autografts. For example, during a maxillary osteotomy in cleft patients, the excised septal cartilage is easily banked in the vestibule under mucosa and used for subsequent rhinoplasty some months later (23). Are these tissues, which can maintain the viability of cartilage cells for so long time, capable of preserving PDL cells’ viability, too? If it is possible, when avulsion occurs in hospital setting and there is not suitable storage medium or the tooth have to be replanted after 4 days, the clinician can place the avulsed tooth in submucosal tissue and use it in appropriate time for replantation. Therefore, this study was conducted to compare the effectiveness of submucosal tissue in maintaining the viability of PDL cells in comparison with HBSS. This study was carried out considering storage periods from 8 h to 7 days to be clinically relevant. Even though extra-alveolar time and storage medium mainly determine the prognosis of an avulsed tooth (24), some studies have shown that storage medium is more determinant (6, 25). The storage medium must supply the optimal osmolality, cell nutrients, and pH (26). It has been indicated that the growth of cells mainly happens at an osmolality of 230–400 mosmol kg 1 and a pH of 6.6–7.8, but its optimal growth occurs at an osmolality 290– 300 mosmol kg 1 and a pH of 7.2–7.4(27, 28). Hanks’ balanced salt solution is the most extensively tested solution that was recommended by International Association of Dental Traumatology as a storage medium for avulsed tooth (5). The osmolality and pH of HBSS are 270–290 mosmol kg 1 and 7.2, respectively. Also, it contains all of the nutrients that are necessary to maintain regular cell metabolism (26). Therefore, in this study, HBSS was used as a positive control. On the other hand, tap water is a hypotonic solution whose use leads to rapid cellular lysis (26); hence, we used it as a negative control. It sounds that the PDL cells in the depth of oral submucosa are in close contact with interstitial fluid. This fluid is a favorable storage medium for these cells, because the interstitial fluid osmolality and pH are 281 mosmol l 1 and 7.35; also, it has many necessary nutrients (29). Based on the results of the present study, there was no significant difference between HBSS and oral submucosal tissue at storage times of 8, 24, 48, and 72 h. Both could not maintain viability of PDL cells at 7 days after extraction. Our results demonstrated that the oral submucosal tissue can maintain the PDL cells’ viability just similar to HBSS and it cannot preserve PDL cells’ viability longer than HBSS.
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As our study, Andreasen used submucosal tissue for storing the extracted teeth (30). The purpose of that study was to determine whether root resorption after replantation could be inhibited by delayed replantation of teeth after submucosal storage to regenerate injured PDL cells. The extracted monkeys’ teeth were preserved in submucosal sites for 2 or 6 weeks. Then, they were recovered and replanted. The results of Andreasen’s study indicated that storing the teeth in submucosa cannot prevent root resorption, or in the other words, submucosal tissue is not able to regenerate damaged PDL cells. The explanation for the difference between our results and Andreasen’s findings is that, on the basis of our study, there are not any vital PDL cells on the extracted root surface after storing tooth in submucosa for 1 week or more. Therefore, if the extracted monkeys’ teeth were replanted before 1 week, the results might be different. In the dental literature, various techniques have been used to quantitate the number of viable PDL cells. We chose the method used by Doyle et al. (15). In this technique, the extracted tooth is placed directly in the storage medium. After a predetermined time, the PDL cells are isolated using enzymes and the tooth is taken out of the medium to evaluate cell viability. This method replicates more closely what actually occurs in clinical practice. It should be emphasized that this study is a preliminary animal study, and we never suggest its application in clinical practice. It is clear that there is need to other studies that confirm these results. Our intention was to present and evaluate a new concept for storing avulsed teeth especially when immediate replantation is not allowed. Conclusion
Within the parameters of this study, it appears that the ability of oral submucosal tissue to maintain Cat PDL cells viability is statistically similar to HBSS. Acknowledgements
The authors would like to extend their gratitude to the Deputy of Research at Hamadan University of Medical Sciences and the Dental Research Center for the support provided. Conflict of interest statement
The authors declare no conflict of interests. References 1. Reynolds JS, Reynolds MT, Powers MP. Diagnosis and management of dentoalveolar injuries. In: Fonseca RJ, Walker RW, Barber HD, Powers MP, Frost DE, editors. Oral and maxillofacial trauma, 4th edn. China: Elsevier Saunders, St. Louis; 2013. p. 248–92. 2. Glendor U, Halling A, Andersson L, Eilert-Peterson E. Incidence of traumatic tooth injuries in children and adolescents in the county of V€astmanland, Sweden. Swed Dent J 1996;20:15–28.
3. Andreasen JO, Andreasen FM. Avulsions. In: Andreasen JO, Andreasen FM, Andersson L, editors. Textbook and color atlas of traumatic injuries to the teeth, 4th edn. Oxford, UK: Wiley-Blackwell; 2007. p. 444–88. 4. 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. 5. Andersson L, Andreasen JO, Day P, Heithersay G, Trope M, DiAngelis AJ et al. International association of dental traumatology guidelines for the management of traumatic dental injuries: 2. Avulsion of permanent teeth. Dent Traumatol 2012;28:88–96. 6. Soder P-O, Otteskog P, Andreasen JO, Modeer T. Effect of drying on viability of periodontal membrane. Scand J Dent Res 1977;85:164–8. 7. Andersson L, Bodin I. Avulsed human teeth replanted within 15 minutes a long-term clinical follow-up study. Endod Dent Traumatol 1990;6:37–42. 8. Andreasen JO, HjØrting-Hansen E. Replantation of teeth. Part I. Radiographic and clinical study of 110 human teeth replanted after accidental loss. Acta Odontol Scand 1966;24:263–86. 9. Andreasen JO, HjØrting-Hansen E. Replantation of teeth. Part II. Histological study of 22 replanted anterior teeth in humans. Acta Odontol Scand 1966;24:287–306. 10. Andreasen JO, Borum MK, Jacobsen HL. Replantation of 400 avulsed permanent incisors. Part 4. Factors related to periodontal ligament healing. Endod Dent Traumatol 1995;11:76–89. 11. 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. 12. Udoye CI, Jafarzadeh H, Abbott PV. Transport media for avulsed teeth: a review. Aust Endod J 2012;38:129–36. 13. Trope M, Friedman S. Periodontal healing of replanted dog teeth stored in Viaspan, milk and Hanks Balanced Salt Solution. Endod Dent Traumatol 1992;8:183–8. 14. Pohl Y, Filippi A, Kirschner H. Results after replantation of avulsed permanent teeth. II. Periodontal healing and the role of physiologic storage and antiresorptive-regenerative therapy. Dent Traumatol 2005;21:93–101. 15. Doyle DL, Dumsha TC, Sydiskis RJ. Effect of soaking in Hank’s Balanced Salt Solution or milk on PDL cell viability of dry stored human teeth. Endod Dent Traumatol 1998;14:221–4. 16. Altman SA, Randers L, Rago G. Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations. Biotechnol Prog 1993;9:671–4. 17. Hammer H. Replantation and implantation of teeth. Int Dent J 1955;5:439–57. 18. Guadio RM, Barbieri S, Feltracco P, Tiano L, Galligioni H, Uberti M et al. Traumatic dental injuries during anesthesia. Part II: medico-legal evaluation and liability. Dent Traumatol 2011;27:40–5. 19. Newland MC, Ellis SJ, Peters KR, Simonson JA, Durham TM, Ullrich FA et al. Dental injury associated with anesthesia: a report of 161,687 anesthetics given over 14 years. J Clin Anesth 2007;19:339–45. 20. Givol N, Gershtansky Y, Halamish-Shani T, Perel A, Segal E. Perianesthetic dental injuries: analysis of incident reports. J Clin Anesth 2004;16:173–6. 21. Cass NM. Medicolegal Claims against anaesthetists: a 20 year study. Anaesth Intensive Care 2004;32:47–58. 22. Qazi SR, Nasir KS. First-aid knowledge about tooth avulsion among dentists, doctors and lay people. Dent Traumatol 2009;25:295–9. 23. Tiwana PS, Turvey TA, Ruiz RL, Costello BJ. Cleft orthognathic surgery. In: Fonseca RJ, Marciani RD, Turvey TA, © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Avulsed tooth preservation in oral submucosa editors. Oral and maxillofacial surgery, volume 3, 2nd edn. China: Elsevier Saunders, St. Louis; 2009. p. 813–27. 24. Andreasen JO. The effect of extra-alveolar period and storage media upon periodontal and pulpal healing after replantation of mature permanent incisors in monkeys. Int J Oral Surg 1981;10:43–5. 25. Blomlof L, Lindskog S, Hammarstorm L. Periodontal healing of the exarticulated monkey teeth stored in milk or saliva. Scand J Dent Res 1989;88:251–9. 26. Blomlof L. Milk and saliva as possible storage media for traumatically exarticulated teeth prior to replantation. Swed Dent J Suppl 1981;8:1–26.
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27. Waymouth C. Osmolality of mammalian blood and media for culture of mammalian cells. In Vitro 1970;6:109–27. 28. Lindskog S, Blomlof L. Influence of osmolality and composition of storage media on human periodontal ligaments cells. Acta Odontol Scand 1982;40:435–41. 29. Hall JE. Guyton and Hall textbook of medical physiology, 12th edn. Philadelphia: Saunders Elsevier; 2011. 379–96 pp. 30. Andreasen JO. Delayed replantation after submucosal storage in order to prevent root resorption after replantation. An experimental study in monkeys. Int J Oral Surg 1980;9:394– 403.
Temporary preservation of avulsed tooth in oral submucosal tissue: an experimental study in cat Mohammad Reza Jamalpour1, Ali Reza Soltanian2, Amir Sasan Tootunchi3, Milad Roshanipaian4 1 Department of Oral and Maxillofacial Surgery, School of Dentistry, Hamadan University of Medical Sciences; 2Department of Biostatistics and Epidemiology, School of Public Health, Hamadan Medical Sciences University; 3 Department of Biochemistry and Biotechnology, Hamadan Medical Sciences University; 4General Dental Practitioner, Hamadan, Iran
Abstract – Objective: The purpose of this study was to determine the ability of oral submucosal tissue to serve as a temporary storage medium for the maintenance of periodontal ligament (PDL) cell viability of avulsed teeth. Materials and methods: Thirty cats were divided into five groups. After extraction of three teeth in each cat, one tooth was put in the depth of cat’s oral submucosal tissue and the other two teeth were put in Hanks’ balanced salt solution (HBSS) and tap water. The teeth were removed after 8, 24, 48, 72, and 168 h from their mediums and sent for laboratory processing and counting of vital periodontal cells. Results: Statistical analysis demonstrated that submucosal tissue kept PDL cells viable as good as HBSS. Conclusion: This animal study showed that the efficacy of oral submucosal tissue in maintaining the viability of human PDL cells is similar to that of HBSS.
Key words: periodontal ligament cells; storage media; submucosal tissue; tooth avulsion Correspondence to: Mohammad Reza Jamalpour, Department of Oral and Maxillofacial Surgery, School of Dentistry, Hamadan University of Medical Sciences, Hamadan, Iran Tel.: +989183134395 Fax: +988118350126 e-mails: [email protected]; [email protected] Accepted 26 January, 2014
Maxillofacial emergencies vary from odontogenic infections to facial fractures. Approximately 15% of all emergency room visits are for maxillofacial trauma and 2% of these cases are pure dentoalveolar trauma (1). On the basis of World Health Organization (WHO) classification, avulsion is the complete displacement of a tooth from its alveolar socket secondary to traumatic injury (1). Avulsion of a permanent tooth is a real dental emergency. It consists of 0.5–3% of all dental injuries in the permanent dentition (2, 3). Although damage to the periodontal ligament (PDL) cells during an avulsion injury is unavoidable, maintaining their viability is critical. One of the most important factors that determine the prognosis for the avulsed tooth is the duration of extra-alveolar time (4, 5). In fact, immediately after avulsion, the number of viable PDL cells on the root surface decreases with increasing drying time and it is impossible to detect viable cells after 2 h (6). Thus, the best treatment at the time of avulsion is immediate replantation and hence re-establishing the natural nutrient supply to the PDL cells (7). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Although immediate replantation has the best prognosis (3, 8–10), certain situations, like the presence of more severe injuries, which requires medical management, can cause the delay in immediate replantation of the avulsed tooth. Generally, where such situations exist, the tooth should be stored in a storage medium until definitive dental treatment can be accomplished. This storage medium should preserve PDL cells’ viability, adherence, and clonogenic capacity (11). Likewise, an ideal storage medium should be readily available or easily accessible at the site of an accident. There are many storage media available for avulsed teeth, including milk, physiologic saline solution, egg white, and Hanks’ balanced salt solution (HBSS) (12). These days, the best known storage medium is HBSS (13), but except for a few areas of the world, it is not easily available at the scene of the injury (14). In this study, oral submucosal tissue was tested for the ability to maintain the viability of PDL cells because, when avulsion occurs in a hospital setting, for example during intubation of endotracheal tube, in comatose 265
266
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patients or patients with low Glasgow Coma Scale (GCS), the easiest and fastest way for a physician is creating a stab incision in the oral mucosa, placing the avulsed tooth in the depth of incision, and finally suturing the incision with one or two stitches. Then, after improving the condition, for example recovery from general anesthesia (GA) or increasing the level of patient consciousness, the hidden tooth can be removed and replanted in its socket by that physician or a dentist. The purpose of this study was to evaluate the capability of oral submucosal tissue in comparison with HBSS (as the positive control) and water (as the negative control) in maintaining the viability of PDL cells. Materials and methods
This study was approved by the Ethics Committee of the Hamadan University of Medical Sciences (P/16/35/ 1/120151) prior to the initiation of the study. Thirty healthy cats were examined in this experiment. The animals were housed in individual cages and maintained on a commercial animal diet. After each surgical procedure, a soft diet was administered for 10 days. Each cat was operated twice. All surgical procedures were performed under general anesthesia, achieved by 2–3 mg kg 1 Xylazine (Eurovet Animal Health B.V., Bladel, The Netherlands) intramuscularly, followed by an intramuscular injection of ketamine at 15 mg kg 1 (Rotexmedica, Trittau, Germany). For reduction in pain, all incised sites were infiltrated with lidocaine 1% (Pasteur Institute of Iran, Tehran, Iran) before incision. Before the first surgery, the mouth was rinsed with chlorhexidine 0.2% (Behsa Pharmaceutical, Arak, Iran). Then, three lower incisors of each cat were extracted as atraumatically as possible. Teeth with moderate-to-severe periodontal disease or with caries were not included in this study. Following extractions, the teeth were held with forceps by the coronal region, and coronal 2 mm of PDL was scraped with a curette to remove cells that may have been damaged. One tooth was placed in tap water as a negative control, and the other one was put in HBSS as a positive control. Before extraction of third incisor, a stab incision was performed in the lower vestibule, and after creation of adequate space in the depth of submucosa, this tooth was extracted and immediately placed in this submucosal space. The incision was sutured with 4-0 silk suture (SUPA Medical Devices, Tehran, Iran). Totally, 90 permanent periodontal disease-free lower incisor teeth were extracted. Thirty teeth were placed in the water, 30 were placed in the HBSS, and 30 were inserted in the depth of oral submucosal tissue. All cats received intramuscular injection of penicillin G 800 000 U (20 000 U kg 1, BID) (Jaber Ebne Hayyan Pharmaceutical Co, Tehran, Iran) postsurgically for 5 days and were fed on a soft diet for 10 days. At the end of the first surgery, the cats were randomly divided into five groups and the teeth in groups 1–5 were removed from submucosa of each cat and its controls (HBSS and water) after 8, 24, 48, 72, and
168 h in order. Therefore, for each tooth that was maintained in submucosal tissue for the determined time, there was one tooth that was preserved in HBSS for the same time (as positive control) and there was one tooth that was preserved in water for the same time (as negative control). The teeth that were removed from submucosa were washed in sterile saline solution to wash out the residual blood. Then, the specimens were transferred to the laboratory in 15-ml Falcon tubes with 10 ml of HBSS. The method used in this study for preparation and counting of the viable PDL cells was the same as the method used by Doyle et al. (15). To obtain the PDL cells, the teeth were trypsinized using a 2.5% trypsin solution (Sigma-Aldrich, St. Louis, MO, USA) in normal saline at 37°C for 10 min with occasional shaking, and then, the supernatant was removed. Each specimen tooth, after washing, was incubated for 40 min in 10 ml of 0.1% collagenase (SigmaAldrich) in normal saline at 37°C with occasional shaking. After incubation, the enzyme activity was stopped by the addition of 5 ml of RPMI 1640 culture medium (Sigma-Aldrich) with 10% fetal calf serum. Then, the tooth was drawn out and the suspension was centrifuged at 172 g for 5 min at 4°C. Finally, after removing the supernatant , the precipitate was re-suspended in 10 ml of HBSS at 4–6°C and filtered through a 100lm-size-pore filter. Cell viability was determined by adding trypan blue (1 volume per 2.5 volumes of medium; Sigma-Aldrich) to the suspension (16). Then, cells were examined for staining under light microscope with 400% magnification. An average of 100 cells per field was counted in four different fields per culture. Data were statistically analyzed using a two-way repeated measurement complemented by the Bonferroni test. Cell viability was transformed by Arcsin. The level of statistical significance was lower than 5% (P < 0.05). Results
Table 1 shows the mean percentage and standard deviation values of cell viability in different examined times. The percentage of viable PDL cells in submucosal tissue were significantly more than in tap water (negative control) in all tested times (P < 0.001). According to these results, there is no statistically significant difference between oral submucosal tissue and HBSS (Table 1, Fig. 1). Discussion
The prognosis of an avulsed tooth is largely dependent on the number of viable PDL cells at the moment of replantation (17). On the other hand, the viability of these cells is highly dependent on the extra-alveolar time (3, 4). Hence, the ideal treatment of choice at the time of avulsion is immediate replantation (9). But some conditions may occur that delay immediate replantation. Usually, there are concomitant injuries with tooth avulsion, as facial lacerations or fractures (3), or even more serious injuries as intracranial or abdominal traumas. Obviously, these injuries should be © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Avulsed tooth preservation in oral submucosa Table 1. Mean percentage and standard CRUD values of cell viability in different times Time (h) Material 8
HBSS Submucosal tissue HBSS Submucosal tissue HBSS Submucosal tissue HBSS Submucosal tissue
24 48 72
Mean SD
P (95% CI)
85.56 9.27 86.67 8.34
0.731 ( 7.42, 5.20)3
30.87 4.71 35.54 4.99
0.134 ( 6.14, 1.19)3
18.81 4.7 16.44 3.92
0.146 ( 0.73, 0.93)3
9.34 1.87 8.42 2.79
0.304 ( 0.78, 2.62)3
1
P2 (95% CI) 0.126
1 The P values to compare cell viability mean between HBSS and submucosal clefts at 8, 24, 48, and 48 h separately based on the Bonferroni test. 2 The P value of repeated measurement analysis to compare cell viability mean between HBSS and submucosal clefts at 8, 24, 48, and 48 h. 3 95% confidence interval for mean difference in cell viability between HBSS and submucosal clefts at 8, 24, 48, and 48 h.
Materials: HBSS Submucosal tissue Water
100.00
Means
80.00
60.00
40.00
20.00
0.00 8.00
24.00
48.00 Hours
72.00
Fig. 1. Time-dependent results according to the experimental solutions and cell viability.
managed before tooth replantation. Also, avulsion may be seen in patients with decreased or lost consciousness. For example, some avulsions may be occurred during the management of the comatose patients (1) or nasotracheal intubation procedure (1, 18–20). Its importance will be more obvious if we know that perioperative tooth damages are the most common medicolegal claimsrelated anesthesia (21). And also the knowledge of the emergency management of tooth avulsion by emergency medical staffs and physicians is not adequate (22). It is clear that replantation of avulsed tooth in patients with decreased consciousness could be resulted in aspiration of tooth and it is better to delay replantation until increasing the level of patient consciousness. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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In these situations, the avulsed tooth should be preserved in a storage medium that can maintain the PDL cells’ viability for a few days until definite dental treatment is fulfilled. Although HBSS is the best known storage medium, its ability to maintain tooth viability is proven for only 4 days. Likewise, HBSS may not be found in some operating rooms or medical emergency departments. On the other hand, surgeons usually use subcutaneous or submucosal tissue for temporary preserving autografts. For example, during a maxillary osteotomy in cleft patients, the excised septal cartilage is easily banked in the vestibule under mucosa and used for subsequent rhinoplasty some months later (23). Are these tissues, which can maintain the viability of cartilage cells for so long time, capable of preserving PDL cells’ viability, too? If it is possible, when avulsion occurs in hospital setting and there is not suitable storage medium or the tooth have to be replanted after 4 days, the clinician can place the avulsed tooth in submucosal tissue and use it in appropriate time for replantation. Therefore, this study was conducted to compare the effectiveness of submucosal tissue in maintaining the viability of PDL cells in comparison with HBSS. This study was carried out considering storage periods from 8 h to 7 days to be clinically relevant. Even though extra-alveolar time and storage medium mainly determine the prognosis of an avulsed tooth (24), some studies have shown that storage medium is more determinant (6, 25). The storage medium must supply the optimal osmolality, cell nutrients, and pH (26). It has been indicated that the growth of cells mainly happens at an osmolality of 230–400 mosmol kg 1 and a pH of 6.6–7.8, but its optimal growth occurs at an osmolality 290– 300 mosmol kg 1 and a pH of 7.2–7.4(27, 28). Hanks’ balanced salt solution is the most extensively tested solution that was recommended by International Association of Dental Traumatology as a storage medium for avulsed tooth (5). The osmolality and pH of HBSS are 270–290 mosmol kg 1 and 7.2, respectively. Also, it contains all of the nutrients that are necessary to maintain regular cell metabolism (26). Therefore, in this study, HBSS was used as a positive control. On the other hand, tap water is a hypotonic solution whose use leads to rapid cellular lysis (26); hence, we used it as a negative control. It sounds that the PDL cells in the depth of oral submucosa are in close contact with interstitial fluid. This fluid is a favorable storage medium for these cells, because the interstitial fluid osmolality and pH are 281 mosmol l 1 and 7.35; also, it has many necessary nutrients (29). Based on the results of the present study, there was no significant difference between HBSS and oral submucosal tissue at storage times of 8, 24, 48, and 72 h. Both could not maintain viability of PDL cells at 7 days after extraction. Our results demonstrated that the oral submucosal tissue can maintain the PDL cells’ viability just similar to HBSS and it cannot preserve PDL cells’ viability longer than HBSS.
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As our study, Andreasen used submucosal tissue for storing the extracted teeth (30). The purpose of that study was to determine whether root resorption after replantation could be inhibited by delayed replantation of teeth after submucosal storage to regenerate injured PDL cells. The extracted monkeys’ teeth were preserved in submucosal sites for 2 or 6 weeks. Then, they were recovered and replanted. The results of Andreasen’s study indicated that storing the teeth in submucosa cannot prevent root resorption, or in the other words, submucosal tissue is not able to regenerate damaged PDL cells. The explanation for the difference between our results and Andreasen’s findings is that, on the basis of our study, there are not any vital PDL cells on the extracted root surface after storing tooth in submucosa for 1 week or more. Therefore, if the extracted monkeys’ teeth were replanted before 1 week, the results might be different. In the dental literature, various techniques have been used to quantitate the number of viable PDL cells. We chose the method used by Doyle et al. (15). In this technique, the extracted tooth is placed directly in the storage medium. After a predetermined time, the PDL cells are isolated using enzymes and the tooth is taken out of the medium to evaluate cell viability. This method replicates more closely what actually occurs in clinical practice. It should be emphasized that this study is a preliminary animal study, and we never suggest its application in clinical practice. It is clear that there is need to other studies that confirm these results. Our intention was to present and evaluate a new concept for storing avulsed teeth especially when immediate replantation is not allowed. Conclusion
Within the parameters of this study, it appears that the ability of oral submucosal tissue to maintain Cat PDL cells viability is statistically similar to HBSS. Acknowledgements
The authors would like to extend their gratitude to the Deputy of Research at Hamadan University of Medical Sciences and the Dental Research Center for the support provided. Conflict of interest statement
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