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ORIGINAL ARTICLE
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The effect of levodopa in combination with Hank's balanced salt solution in enhancing periodontal ligament cell viability: An in vitro study


1 Department of Pedodontics and Preventive Dentistry, S. Nijalingappa College of Dental Sciences and Research, Gulbarga, Karnataka, India
2 Department of Oral Medicine and Radiology, Navodaya Dental College, Raichur, Karnataka, India
3 Department of Public Health Dentistry, Pandit Deendayal Upadhyay Dental College, Solapur, Maharastra, India

Date of Submission19-Nov-2019
Date of Decision10-Jan-2020
Date of Acceptance03-Feb-2020

Correspondence Address:
Prashant B Patil,
Department of Oral Medicine and Radiology, Navodaya Dental College, Raichur, Karnataka
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/sjos.SJOralSci_88_19

  Abstract 


Aim: This study aims to study the viable property of periodontal ligament (PDL) cells in the avulsed teeth using Hank's balanced salt solution (HBSS) storage medium with levodopa (L-dopa) and without L-dopa.
Materials and Methods: The study samples included 40 freshly extracted, noncarious, non-impacted human teeth with closed apices. The teeth were then randomly divided into four groups containing four different experimental storage solutions, namely Group 1: Deionized water; Group 2: Deionized water with L-dopa (1 μg/μl); Group 3: HBSS; and Group 4: HBSS with L-dopa (1 μg/μl). All the teeth were stored in different test solutions for 60 min. Collagenase assay was performed followed by analyses of the number of viable PDL cells using a hemocytometer under a light microscope at ×20 magnification. The mean and standard deviation (SD) of viable PDL cells of all the four groups obtained were evaluated using Kruskal–Wallis test. P < 0.001 was considered statistically significant. Comparison between the four groups was done using Mann–Whitney test and U <23 and P < 0.05 was considered statistically significant.
Results: Comparison of mean and SD of viable PDL cells showed a significant difference between the four groups (P < 0.001). Group 4 showed the highest mean number of PDL cells, followed by Group 3, Group 2, and Group 1, respectively. These findings indicate that the viability of PDL cells was highest when teeth were stored in HBSS with L-dopa, followed by HBSS, than de-ionized water with L-dopa and least in de-ionized water.
Conclusion: The results of the study showed that the PDL cells were rendered more viable by the synergistic effect of L-dopa along with HBSS as a storage medium for an avulsed tooth.

Keywords: Hank's balanced salt solution, levodopa, periodontal ligament cells, tooth avulsion



How to cite this URL:
Patil SP, Patil PB, Kashetty MV. The effect of levodopa in combination with Hank's balanced salt solution in enhancing periodontal ligament cell viability: An in vitro study. Saudi J Oral Sci [Epub ahead of print] [cited 2020 Aug 4]. Available from: http://www.saudijos.org/preprintarticle.asp?id=281109




  Introduction Top


Dental avulsion or exarticulation is the most serious emergencies in dental hospitals. Tooth avulsion is the most severe type of traumatic tooth injuries because it causes damage to several surrounding structures and results in the complete dislodgement of the tooth from its socket destroying the alveolar bone.[1] The incidence of avulsion ranges from 1% to 16% of all the traumatic injuries to the permanent dentition.[2] The anterior teeth, especially the central incisors, are the most commonly avulsed permanent teeth.[3] Avulsion of incisors is maximum in malocclusions with proclined dentition.[3] Children in the age range of 7–11 years are predisposed to avulsion with a higher prevalence among males.[4],[5]

The ideal clinical situation demands immediate re-implantation of exarticulated tooth because the extraoral time is a determinant factor for the success of treatment and better prognosis. Immediate reimplantation establishes the natural nutrient supply to the periodontal ligament (PDL) cell thereby minimizing further damage and also enhances the healing process.[6] Following avulsion, the number of viable cells on the root surface decreases with increasing drying time. Studies have also noticed that cell viability under a microscope could not be appreciated after 2 h.[6] The viability of PDL cells relies on three critical factors: The duration of extra-alveolar time, preservation of the root and storage media for the tooth, all of which determine the prognosis of dental reimplantation.[6],[7],[8],[9],[10]

Inflammatory resorption, replacement resorption, and dental alveolar ankylosis are the most common and significant complications after reimplantation of avulsed teeth.[8],[9]

The success of reimplantation was initially thought to be mainly associated with the speed with which the tooth is reimplanted, i.e., shorter the extra alveolar time better is the prognosis. However, this concept has changed with time and researchers have now demonstrated that the storage medium is one of the most important factors than the extraoral time. Short period of dry storage shows a comparatively poor prognosis than the prolonged storage in a suitable medium.[11]

A storage medium may be defined as a physiological solution that closely replicates the oral environment to help preserve the viability of PDL cells following tooth avulsion.[12] The ability of a storage medium to support cell viability is more important than the extraoral time to prevent ankylosis and replacement resorption.[6],[13] A wide array of wet storage media have been evaluated in laboratory studies and clinical reports including cell and tissue culture solutions such as Hank's balanced salt solution (HBSS); medical/hospital products developed specifically for organ storage purposes such as Viaspan and Euro-Collins; culture media like minimum essential medium, saline, natural products such as water, saliva, bovine milk and its variations, propolis, green tea, Morus rubra (red mulberry), egg white, and coconut water; rehydrating solutions, such as Gatorade and Ricetral, and even contact lens solutions.[1]

Krasner and Person introduced a commercially available form of HBSS marketed as Save-A-Tooth, which was proven to maintain PDL cell vitality.[14] In addition to preserving the cell vitality, a storage media that has the potential to increase the cell viability is highly desired. Hence levodopa (L-dopa) is a new storage media that has been developed along these lines. L-dopa a precursor of central nervous system catecholamines is a drug with a possible mitogenic effect in the human body. L-dopa changes to dopamine, which stimulates the anterior part of hypophysis to secrete growth hormone which is a promoter of the healing process.[15] L-dopa has been the drug of choice for the treatment of Parkinson's disease.[15] It is also used for managing fractured long bones with delayed healing or nonunion healing.[15],[16] L-dopa also has an effect on endochondral bone repair and intramembranous bony repair in rats.[17] Partovi et al.[18] and Zarabian et al.[19] observed the effect of L-dopa on human PDL fibroblasts and concluded that L-dopa due to its mitogenic activity can be used for preserving the viability of PDL cells. The purpose of the present study is to evaluate the efficacy of L-dopa with HBSS as a new storage media for avulsed teeth. The ability of HBSS in maintaining the PDL cell vitality is established through available literature. However, through this study, we intend to observe the effect of the addition of L-dopa to HBSS on the viability of PDL cells. Hence, the aim of this study is to investigate the viability of PDL cells of an avulsed tooth preserved in a storage medium with and without L-dopa and also to check the effect of L-dopa in enhancing the efficacy of HBSS in maintaining the viability of PDL cells.


  Materials and Methods Top


The study was carried out in the Department of Pediatric and Preventive Dentistry, S. Nijalingappa College of Dental Sciences, Kalaburgi, Karnataka. Institutional Ethical Committee approval was obtained prior to the start of the study (Ref. no. HKES/SNIDSR/IEC/21/18).

Forty freshly extracted nonimpacted human premolars indicated for orthodontic treatments were obtained for this study. Patients consent forms were obtained after explaining the orthodontic treatment plan before extraction. The average age of the patients was 21 years (age range 18–25 years). Only teeth that were healthy, noncarious, without periodontal infections and with apices closed were selected for the study. All extractions were performed as atraumatically as possible by a single clinician.

Preparation of the specimen

After extraction, the teeth were held with forceps by the coronal region, and the coronal 3 mm of PDL was scraped with a spoon excavator to remove cells that might have been damaged during extraction [Figure 1]. All the teeth were left to dry for 30 min. This process was done to simulate the clinical situation wherein the teeth might have been left dry for at least 30 min (extraoral time period) before being taken to dental operatory prior to carrying out definitive clinical procedure.
Figure 1: Scrapping of coronal 3 mm of periodontal ligament with a spoon excavator

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Sample categorization

The teeth were then randomly divided into 1 of the 4 experimental storage solution groups [Figure 2], namely:
Figure 2: Storage of tooth in the four experimental storage solutions

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  • Group 1 - De-ionized water (Krishna Enterprises, Bengaluru)
  • Group 2 - De-ionized water with L-dopa (1 mg/ml)
  • (L-dopa - Sun Pharma INDS. 110 mg tabs)
  • Group 3 - HBSS (L0606 – Biowest Distributor, Jain Biological Private Ltd., Panchkula, Haryana)
  • Group 4 - HBSS with L-dopa (1 mg/ml).


Ten samples per group from 10 different patients were studied. The positive control is Group 3, and the negative control is Group 1. The teeth were then stored in the different test solutions for 60 min.

The collagenase assay was then performed as follows

From each group, the teeth were incubated at 36.5°C for 30 min in a test tube containing 2.5 ml of 0.2 mg/ml of collagenase [Figure 3]. After incubation 1 ml of fetal bovine serum was added to the test tube and centrifuged for 4 min at 1000 rpm. The supernatant fluid was removed with a sterile micropipette, and the cells were labeled with 0.4% trypan blue for the determination of cell viability. Twenty microliters of cell suspension were added tp 20 ml of trypan blue (0.4% w/v), and the cells that were stained by the dye suggestive of viability were counted. The number of viable PDL cells was counted under a light microscope using a hemocytometer at ×20 magnification.
Figure 3: Incubation of tooth for 30 min in a test tube containing 2.5 ml of 0.2 mg/ml of collagenase

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The data were entered into the computer and further tabulated and analyzed using SPSS software version 20 (IBM, USA, Armonk, New York). The mean and standard deviation (SD) of viable PDL cells of all the four groups obtained was evaluated using Kruskal–Wallis test. P <0.001 was considered statically significant. Comparison between the four groups was done using Mann–Whitney test and U <23 and P < 0.05 was considered statistically significant.


  Results Top


Mean and SD of viable PDL cells of all the four groups was compared using Kruskal–Wallis test (H value) that showed a significant difference between the four groups (P < 0.001). These findings indicate that the viability of PDL cells was highest when teeth were stored in HBSS with L-dopa, followed by HBSS, than de-ionized water with L-dopa and least in de-ionized water [Table 1].
Table 1: Comparison of viable periodontal ligament cells among four groups

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Intergroup comparison was also made using Mann–Whitney test (U value), and significant differences were found between all the groups at P < 0.05 [Table 2].
Table 2: Comparsion of viable periodontal ligament cells between the groups by Mann-Whithey test (U value)

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  Discussion Top


The primary aim of treating the avulsed teeth is to preserve the vitality and viability of PDL fibroblast cells.[20] Teeth from healthy individuals without periodontal disease were selected in this study since fibroblast function can be affected by age, trauma, and inflammation. In the current study, a 30 min drying time was chosen to simulate a typical clinical scenario during which the avulsed tooth may remain dry before being placed into a storage medium. These clinical situations may be the presence of more serious injuries needing emergency medical aid, unavailability of immediate dental aid that can result in drying of avulsed teeth. Cvek et al.[21] demonstrated that 13% of teeth kept in a dry state for 15 min, 40% of those kept in a dry state for 20–40 min and 100% of those stored dry for >60 min showed signs of ankylosis. Therefore, it is important to prevent the PDL cells from drying. Although 30 min of drying results in damage to many PDL cells yet some cells remain for assessment.

Two methods have been followed for evaluating the efficacy of different storage media in preserving the viability of dental fibroblast cells. The first method involves the removal of fibroblasts from the root surface and added to storage medium for culturing. The main benefit of this method is that only few teeth are required for the study since large number of fibroblasts can be derived from those teeth. The biggest drawback of this method is that it does not replicate clinical scenario as the cells in the proliferative phase are placed directly in the medium rich in nutrients.[22] In the present study, the extracted tooth was placed directly in the storage medium. After a pre-determined time, the PDL cells were isolated using enzymes and the stained fibroblast cells were micropipetted out of the medium to evaluate cell viability.[23] This method is identical to primary cell culture and closely replicates the actual clinical situation. In the current study to preserve maximum cell viability, the root surface was treated with enzyme collagenase. This results in rapid cell retrieval and maintains maximum cellular integrity.[24] Trypan blue exclusion staining technique was used because it is quick easily performed and distinctively differentiates viable from non-viable PDL cells.

The ability to proliferate is critical for the fibroblast cells when stored in storage media to regenerate the oral tissues after damage. HBSS, as a storage medium, has an excellent ability in maintaining the viability of the periodontal cells and also prevents the morphological distortion of the cells. HBSS can preserve the viability of 70% of fibroblasts for 96 h. In this study, HBSS, deionized water and L-dopa were tested for their effectiveness in maintaining the viability of PDL cells. From the results of the study [Table 1], it can be inferred that the number of viable PDL cells is highest when teeth were stored in HBSS with L-dopa, followed by HBSS, than de-ionized water with L-dopa and least in de-ionized water. Our results are in accordance with the results obtained in the previous studies suggesting that the addition of L-dopa to HBSS storage media would enhance the maintenance of cell viability of PDL cells.[25] This may be due to the synergistic effects of HBSS which has all nutrients, pH and osmolarity for cellular growth and L-dopa which contributed in the mitogenic activity of these viable PDL cells.

Intergroup comparison was also carried out [Table 2] where comparison between Group 1 and Group 3 showed that HBSS was the effective medium for maintaining the vitality as well as viability of PDL cells. Results obtained in our study correlated with results obtained by Ashkenazi et al.[26] who concluded that HBSS was the most effective medium for preserving viability as well as maintaining mitogenicity and clonogenic capacities of PDL cells up to 24 h. Hwang et al.[27] reported 94% PDL cell viability after storing in HBSS media for 24 h while Pileggi et al.[24] observed approximately 90% PDL cell viability. The property of HBSS storage medium to maintain the viability and vitality of PDL cells is attributed to low Ph (7.4) and osmolarity (280 mOsmol/kg) which are ideal requisites to preserve cells and tissues up to 24 h.[14]

Results obtained from the comparison between Group 1 and 2, Group 3 and 4 suggests that the addition of L-dopa to the storage media enhances the viability of PDL cells. Similar results were also obtained in the studies by Partovi et al.,[18] Zarabian et al.,[19] and Ebenezar et al.[25] This may be due to the mitogenic effect of L-dopa on PDL cells.[18] The addition of L-dopa to HBSS (Group IV) resulted in a marked increase in the viability of PDL cells when compared with HBSS alone (Group III). This may be due to the synergistic effects of HBSS which has all nutrients, pH and osmolarity for cellular growth and L-dopa which accelerates the mitogenic activity of these viable PDL cells.[19] Our study further substantiates the available literature about the use of L-dopa with HBSS as a storage media. Ideally, anin vivo study should be designed to test the same solutions in the manner that would be clinically more relevant. This is a limitation of the present study. In addition to the trypan blue exclusion staining technique, the cell recovery assay and cell apoptosis assay could be used to more accurately determine the condition of cells after exposure to different solutions.


  Conclusion Top


Within the limitations of this study, it can be concluded that the synergistic effect of L-dopa and mitogenic activity of HBSS paves way for its usage as an effective storage medium in increasing the viability of PDL cells in an avulsed tooth. Hence, PDL cells have been proved to be more viable when stored in HBSS with L-dopa than HBSS alone. Further large scale clinical research is essential before its wider application in clinical dentistry.

Acknowledgment

We would like to acknowledge Dr. Shrikala Puranik, Senior Lecturer Department of Oral Pathology and microbiology, and Mrs. Jyoti (statistician) for their help and technical support for the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Poi WR, Sonoda CK, Martins CM, Melo ME, Pellizzer EP, de Mendonça MR, et al. Storage media for avulsed teeth: A literature review. Braz Dent J 2013;24:437-45.  Back to cited text no. 1
    
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Marino TG, West LA, Liewehr FR, Mailhot JM, Buxton TB, Runner RR, et al. Determination of periodontal ligament cell viability in long shelf-life milk. J Endod 2000;26:699-702.  Back to cited text no. 2
    
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Andersson L, Al-Asfour A, Al-Jame Q. Knowledge of first-aid measures of avulsion and replantation of teeth: An interview of 221 Kuwaiti schoolchildren. Dent Traumatol 2006;22:57-65.  Back to cited text no. 5
    
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Flores MT, Andersson L, Andreasen JO, Bakland LK, Malmgren B, Barnett F, et al. Guidelines for the management of traumatic dental injuries. II. Avulsion of permanent teeth. Dent Traumatol 2007;23:130-6.  Back to cited text no. 7
    
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Sharma M, Sharma S, Reddy YG, Mittal R, Agarwal V, Singh C, et al. Evaluation of periodontal ligament cell viability in three different storage media: Anin vitro Study. J Dent (Tehran) 2015;12:524-31.  Back to cited text no. 11
    
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Ingle JI, Bakland LK, Baumgartner JC. Endodontic considerations in Denatl Trauma. Ingle's Endodontics. 6th ed. Hamilton, Ontario: B.C. Decker Inc,; 2008. p. 1348-9.  Back to cited text no. 12
    
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Krasner P, Person P. Preserving avulsed teeth for replantation. J Am Dent Assoc 1992;123:80-8.  Back to cited text no. 14
    
15.
Pritchett JW. L-dopa in the treatment of nonunited fractures. Clin Orthop Relat Res 1990. p. 293-300.  Back to cited text no. 15
    
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18.
Partovi M, Sadeghein A, Azizi E, Ostad SN. Mitogenic effect of L-dopa on human periodontal ligament fibroblast cells. J Endod 2002;28:193-6.  Back to cited text no. 18
    
19.
Zarabian M, Salehipour F, Ostad SN. The study of dose-response mitogrnic effect of L-dopa on the human periodontal ligament fibroblast cells. Acta Medica Iranica 2004;42:363-66.  Back to cited text no. 19
    
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21.
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22.
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.  Back to cited text no. 22
    
23.
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24.
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25.
Ebenezar AV, Mahendran T, Priya J. Addition of L-dopa to HBSS in enhancing the maintenance of cell viability of Periodontal Ligament (PDL) cells: An in-vitro Study. J Clin Diagn Res 2014;8:ZC79-80.  Back to cited text no. 25
    
26.
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;15:149-56.  Back to cited text no. 26
    
27.
Hwang JY, Choi SC, Park JH, Kang SW. The use of green tea extract as a storage medium for the avulsed tooth. J Endod 2011;37:962-7.  Back to cited text no. 27
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

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