Strategies for periodontal ligament cell viability: An overview

Abstract

The long-term success of the reimplanted teeth is related to the maintenance of periodontal ligament (PDL) cell viability. Dental tissues are unique in comparison to most other tissues in the body due to their marked capacity for regeneration. Understanding the circumstances leading to repair and regeneration in oral tissues has been a formidable challenge. Numerous storage media have been introduced by many authors that help to maintain the PDL cell viability. To present an overview of the various available storage media. A literature search for the past 20 years was performed across the Internet database (Pubmed) and relevant citations using the keywords PDL cell viability, tooth avulsion, storage media, and the combination of all to retrieve around (n=225) citations. Articles that included follow-up of intervention for avulsed and re-implanted teeth were considered (n=44) and some literature review from well-known text books were considered. Literature supports that moist storage appears to be a more productive approach to optimize PDL cell survival. However, no medium is ideal and in vivo studies are inadequate.

INTRODUCTION

“Small tissue, Big Issue,” is all about the maintenance of the periodontal ligament and pulp vitality after injury, which is essential for the long-term success of the reimplanted teeth. Reimplantation may be after traumatic avulsion or may be intentional. Dental tissues are unique in comparison to most other tissues in the body due to their marked capacity for regeneration. Injuries to the pulp and PDL may sometimes regenerate or alternatively show repair with fibrous scar tissue or bone. Understanding the circumstances leading to repair and regeneration in oral tissues has been a formidable challenge. In this regard, damage to PDL, cementum, alveolar bone, gingival, and pulp tissues releases a variety of signals that induce neighbor cell populations to respond by proliferation, migration, or differentiation. A literature search was performed across the Internet database (Pubmed) for relevant citations using the keywords PDL cell viability, tooth avulsion, storage media, and the combination of all to retrieve around n = 225 citations. Articles that included follow-up of intervention for avulsed and re-implanted teeth were considered (n = 44) and some literature review from well-known text books were considered.

REQUISITES FOR HEALING

The prerequisites for healing include the presence and migration of specific cell populations with regenerative potential (eg, pulp or PDL progenitor cells) and the absence of contamination, foreign bodies, and/or bacteria.[1]

To describe in short, the types of healing that take place after the avulsion injury are as follows:

1. Favorable healing.

   1. Healing with a normal PDL (without root resorption)
   2. Healing with surface resorption (repair-related resorption)

2. Unfavorable healing.

   1. Healing with ankylosis (replacement)
   2. Healing with inflammatory resorption (infection-related resorption)

The progression of osseous replacement resorption is dependent on the age of the patient or the turnover rate of bone. It is very aggressive in young individuals and runs a protracted course in older patients. The life span of such an ankylosed tooth can vary from 10 to 20 years.[2]

Tooth avulsion is characterized by complete displacement of the tooth from its socket, accounts for about 0.5%–16% of all cases of dental trauma. After avulsion, the tooth should be repositioned in the socket in an attempt to re-establish normality. Immediate reimplantation or maintenance of the avulsed tooth in storage media compatible for survival of these cells before reimplantation is an imperious procedure.[3]

METHODS TO TEST PERIODONTAL LIGAMENT CELL VIABILITY

Available tests

The Cellomics Cell Viability HCS Reagent Kit(Fischer Scientific, Pittsburg PA,USA) provides high-content screening (HCS)-qualified fluorescence reagents, which help in screening cell viability.[4]

The MTT assay, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], has been used to test cytotoxicity of reagents and cell viability.

The HCS reagent kits may be more sensitive than traditional assays, such as MTT assays, which rely on late-stage cytotoxic endpoints and can detect neurotoxic effects at lower concentrations than these biochemical assays.[5]

Fluorescein diacetate staining of viable cells

Cell viability can be assessed directly through the presence of cytoplasmic esterases that cleave moieties from a lipid-soluble nonfluorescent probe to yield a fluorescent product. The product is charged and thus is retained within the cell if membrane function is intact.[6]

Strategies to preserve the periodontal ligament cell viability

Avulsion of permanent teeth is the most serious of all dental injuries and one of the few real emergency situations in dentistry. The prognosis depends on the measures taken at the place of accident or the time immediately after the avulsion because the preservation of the cell viability would be significantly higher.[7,8]

Emergency treatment outside the dental office

On telephonic call by the parent to the dentist reporting that a tooth has been avulsed and it can be determined that the injury is without other oral, neurologic, or higher priority physical complications, the dentist can give the following instructions:

• Replace the tooth in the socket immediately and hold it in place with light finger pressure, while the patient is brought to the dental office.

• If the avulsion has occurred in a clean environment, nothing should be done to the tooth before the parent replants it.

• If the tooth is dirty, an attempt should be made to clean the root surface, but it is very important to preserve any remnants of the PDL that are still attached to the root. So the parent should be instructed to keep the tooth immersed in a suitable storage medium and bring the child and the tooth for immediate care.[9]

Emergency treatment in the dental office

Physical

Gently wash the root surface off gross debris with tap water or sterile saline if the tooth is to be reimplanted immediately at the accident site.[10]

Chemical

If the tooth cannot be reimplanted immediately, it should be placed in an appropriate storage medium until it can be reimplanted. Dry time must be avoided.

There has been extensive research done in order to chemically prepare the root to promote favorable healing or in situations where favorable healing is not possible to slow down the osseous replacement of the root.

• After an avulsion injury, deleterious effects occur to the PDL very quickly. Recent clinical research has shown that after approx. 20 min dry time some degree of unfavorable healing is expected.[11]

• However, most of the research into the beneficial effects of anti-inflammatory or anti-resorptive procedures has been performed on roots that have been dry for 60 min or more.

• Depending on the Extra Oral Dry Time [EODT] avulsed teeth can be categorized as below and treated accordingly:

Mature tooth short (<20 min) EODT

After the tooth has been soaked in Hank's balanced salt solution (HBSS) or physiological saline teeth should be replaced and splinted as soon as possible.[2]

Mature tooth—intermediate (20–60 min) EODT

A number of medications have been studied in order to minimize the initial inflammation after replacement by soaking the root surface.[2]

Tetracyclines

They are substantive due to their hard tissue binding capability and also have an innate antiresorptive action. They have the ability to bind to the tooth surface and then be slowly released in active form.[12] Tetracycline also promotes fibroblast and connective tissue attachment, enhancing regeneration of periodontal attachment lost to pathologic process.[12] Tetracycline inhibits collagenase activity and osteoblast function, which could be beneficial to a replanted tooth. Despite these effects, Bryson et al studied the effects of minocycline on healing of replanted dog teeth after extended dry times and reported no beneficial effect.[13]

Corticosteroids

Local administration has been shown to reduce osteoclastic bone resorption. Several mechanisms have been reported for this effect, including reduction of the number of osteoclasts by direct receptor-mediated and specific toxicity of these cells, making them more responsive to the existing systemic concentration of the hormone whose main function is to reduce bone resorptive activity. Sac-Lim et al (1998) found that in dogs, the topical (not systemic) application of corticosteriods enhanced a favorable healing.[14]

Alendronate

It has an inhibitory effect on osteoclasts and is being used therapeutically in osteoclast-mediated diseases.[2] Sodium alendronate has shown to reduce the incidence of radicular resorption when used both as an intracanal medicament and on topical application.[15,16] Dog's roots soaked in HBSS followed by alendronate had statistically significantly more healing than the roots soaked in HBSS without alendronate (30% vs 11%).[17]

Emdogain^® (enamel matrix protein)

It is inductive for acellular cementum formation on traumatized root surface. Emdogain^® was shown in a study by Iqbal and Bamaas (2001) to almost double the favorable healing in dogs teeth dried for 60 min.[18] Numerous case reports have shown that the use of Emdogain^® slowed down the osseous replacement and was not able to reconstitute the PDL completely.[19] In a recent study by Wiegand et al, no firm conclusion regarding the efficacy of Emdogain^® application on healing of replanted or autotransplanted permanent teeth could be drawn because of lack of randomized controlled trials (RCTs) and clinical controlled trials (CCTs).[20]

Calcitonin

Calcitonin is a hormone secreted by the parafollicular cells of the thyroid gland and is known to cause contraction of osteoclasts and inhibit activity. Topical use of calcitonin (placed in the root canal) has been found to be effective in controlling inflammation related to root resorption.[21]

Ledermix

Ledermix is a paste containing triamcinolone and demeclocycline with demonstrated anti-inflammatory activity that may slow down resorptive processes after severe traumatic injuries to the dentition. It is used as a slow release anti-inflammatory drug using the root canal as a reservoir. Bryson et al have shown that immediate intracanal placement of Ledermix Paste at the emergency visit after an avulsion injury appears to decrease resorption and increase favorable healing.[22]

Antiresorptive–regenerative therapy

Antiresorptive–regenerative therapy (ART) with the local application of glucocorticoids and enamel matrix derivative and the systemic administration of doxycycline was used and has been found that the use of ART might have the potential for enhancing the prognosis of teeth with a PDL that is compromised but not in a hopeless condition when compared with Ca(OH)₂.[23]

Mature teeth—extraoral dry time > 60 min

The prognosis of the tooth reimplantation depends on the existence of feasible cells in the PDL and also depends on those which are able to proliferate on the damaged areas of the root. The portion of the PDL attached to the alveolar walls remains alive and does not need treatment while those attached to the tooth relate to the repairing process after reimplantation.[24]

Besides the promptness of reimplantation, the storage medium in which the tooth is placed is also a determining factor to extend the life of the tooth. The maintenance of vitality of PDL attached to the tooth is lower in a dry environment.[24] There are solutions which are capable of preserving the vitality of PDL during the time they are out of their alveolar socket. These solutions must be used when immediate reimplantation cannot be done.[25]

It is evident through literature that there are numerous storage media introduced by many authors. But before the media are acceptable, they have to undergo various experimental studies. Tap water, Saliva, Normal Saline, Gatorade, Contact lenses solutions, Propolis, Emdogain^®, Egg white, Milk, HBSS, Viaspan^®, Eagle's Medium, Conditioned medium, α-MEM (α-Minimal Essential Medium), Long shelf milk (milk products), and Coconut water are some of the storage media available.

Tap water

Among the studied environments, tap water has shown to be the one with least desirable results, although it protects the tooth from dehydration for being a hypotonic medium, it causes rapid cellular lyses of the PDL, similar to a dry storage.[26]

Saliva

Saliva can be used as a storing medium for a short period of time, for it can damage the cells of the PDL if used for longer than an hour.[26] Its osmolality is much lower than the physiologic (60–70 mOsm/kg), thus it boosts the harming effects of bacterium contamination. Its only advantage is its availability.

Normal saline

The saline solution provides osmolality of 280 mOsm/kg and despite being compatible to the cells of the PDL, it lacks essential nutrients, such as magnesium, calcium, and glucose; necessary to the normal metabolic needs of the cells of the PDL.[27]

Krasner[25] stated that saline solution was harmful to the cells of the PDL in avulsed teeth if it is used for longer than 2 h.

Gatorade

Gatorade, according to Harkacz et al[28] did not turn to be an adequate storing medium for avulsed teeth due to its pH around 2.91 and its osmolality of 407 mOsm/kg. According to Chamorro et al. when cells are exposed to Gatorade, it is possible that the delicate cellular membrane gets damaged because of the low pH, which makes it impossible for the cells to grow.[29] As for osmolality, because Gatorade is hypertonic, it can make cells lose water.

Contact lens solutions

Contact lens solution comprises a fatty acid monoester and a cationic antimicrobial component. The solutions for keeping contact lenses were worse than saline solution, milk, and HBSS.[29] The presence of preservatives in its formula was harmful to the cells of the PDL.

Propolis

Propolis has been a promising storage medium for the maintenance of cellular viability of the PDL of avulsed teeth. Authors have concluded that it can be considered as a favorable storing medium for the fact that it maintains the cellular viability of the PDL, besides being anti-microbiotic, anti-inflammatory, and anti-oxidant.[30,31]

Egg white

Khademi et al.[32] compared milk and egg white as solutions for storing avulsed teeth, and the results have shown that teeth stored in egg white for 6–10 h had a better incidence of repair than those stored in milk for the same amount of time (P < 0.05). Cellular growth occurs at an osmolality of 230–400 mOsm/kg and a pH of 6.6–7.8, but its optimal growth happens at an osmolality of 290–300 mOsm/kg and pH of 7.2–7.4. The osmolality of the egg white is between 251 and 298 mOsm/kg.

Milk

The American Association of Endodontics indicates milk as a solution for avulsed teeth, for keeping the viability of the human cellular PDL.[25] Milk is significantly better than other solutions for its physiological properties, including pH and osmolality compatible to those of the cells from the PDL; the easy way of obtaining it and for being free of bacteria[33] but it is important that it is used in the first 20 min after avulsion. The favorable results of milk probably occur due to the presence of nutritional substances, such as amino acids, carbohydrates, and vitamins.[34]

Milk formulas

Long shelf-life milk, which has the advantage of not requiring refrigeration, is as effective a storage medium for avulsed teeth as regular pasteurized milk and more effective than Save-A-Tooth medium. Pearson et al compared the efficacy of several milk substitutes, which included reconstituted powdered milk, evaporated milk or one of two baby formulas: Similac or Enfamil; compared to whole milk and reported that Enfamil does not require special storage, has a shelf-life of 18 months and is a more effective storage medium in maintaining the viability of human PDL cells than pasteurized or whole milk for at least 4 h.[35]

Probiotic solution

Caglar et al tested the viability of fibroblasts using HBSS, saline, Lactobacillus reuteri solution, and milk and concluded that there was no significant difference in the number of viable PDL cells between HBSS, milk, L. reuteri solution, and saline. Within the parameters of their study, it appears that probiotic may be able to maintain PDL cell viability as HBSS, milk, or saline.[36]

Hank's balanced salt solution

HBSS is a standard saline solution, which is widely used in biomedical research to support the growth of many cells types.[37] This solution is nontoxic; it is biocompatible with PDL cells, pH balanced at 7.2, and has an osmolality of 320 mOsm/kg. It is composed of 8 g/L sodium chloride; 0.4g/L of d-glucose; 0.4g/L potassium chloride; 0.35g/L sodium bicarbonate; 0.09g/L sodium phosphate; 0,14g/L potassium phosphate; 0.14g/L calcium chloride; 0.1g/L magnesium chloride; and 0.1g/L magnesium sulfate (Biological Industries, Beit Haemek,Israel). It contains ingredients, such as glucose, calcium, and magnesium ions, which can sustain and reconstitute the depleted cellular components of the PDL cells.[38,39]

According to Ashkenazi et al, HBSS was the most effective medium for preserving viability, mitogenicity, and clonogenic capacities of PDL cells for up to 24 h at 4°C when compared with other solutions. References[38,39,40] describe how to save a tooth in detail. HBSS is commercially available as Save-A-Tooth. The Save-A-Tooth^® system was designed to suspend the tooth in the solution to prevent damage to the cells. It has an inner basket and net which help wash debris off the tooth without scrubbing or touching the cells. This basket also lets a dentist remove the tooth from the system without having to try to grab it with fingers or forceps, thus causing minimum trauma to the viable cells.

ViaSpan^®

The ViaSpan^® (Belzer VW-CSS, Du Pont Pharmaceuticals, Wilmington, DE, USA) is a medium used for the transportation of organs, which are going to be transplanted and it has been very effective for storing avulsed teeth. ViaSpan^® has osmolality of 320 mOsm/kg, which enables excellent cellular growth. Its pH is around 7.4 at room temperature, which is ideal for the cellular growth.[41]

Eagle's medium

Eagle's Minimal Essential Medium containing 4 mL of L-glutamine—105 IU/L of penicillin; 100 μg/mL of streptomycin, 10 μg/mL of Nystatin, and calf serum (10% v/v)—demonstrated that the cell culture medium (Eagle's medium at 37°C) can preserve PDL fibroblasts for extended periods before dental reimplantation.[18,39,41]

Coconut water

Coconut water is biologically pure and sterile, with a rich presence of amino acids, proteins, vitamins, and minerals. Gopikrishna investigated the potential of a new storage medium, coconut water, in comparison with propolis, HBSS, and milk in maintaining viable PDL cells on simulated avulsed teeth. Results showed that coconut water kept significantly more PDL cells viable compared with propolis, HBSS, or milk. Coconut water can be used as a superior transport medium for avulsed teeth.[42,43] Moreira-Neto et al observed that coconut water has an acidic pH of 4.1, which is deleterious to cell metabolism and concluded that the capacity of the storage media in maintaining human fibroblast cell viability in a decreasing order was milk > saline and coconut water with sodium bicarbonate > coconut water > still mineral water.[44]

If the root has been dry for more than 60 min the PDL is not expected to be viable. Thus osseous replacement of the root becomes inevitable. We should aim to prepare the tooth for as slow an osseous replacement of the root as possible. According to the guidelines by the American Academy of Pediatric Dentistry 2009 soaking in 3% citric acid for 3 min is a noninvasive approach for PDL removal followed by rinsing. Prior to replantation, soaking in a solution of NaF or APF for minimum 5 min and maximum 20 min is recommended, which delays ankylosis.[45]

Immature tooth—EODT < 60 min

The luxated or avulsed immature tooth is different from the mature tooth in that the pulp that is necrotic due to the traumatic injury has the possibility of revascularization. Revascularization of an immature tooth is of tremendous benefit in that the root canal walls continue to develop and strengthen and the apex closes, making future endodontic treatment if needed, a more predictable procedure. The less developed the root at the time of injury the greater the potential for revascularization. Doxycycline has antibacterial and antiresorptive properties. Cvek et al tested the effect of soaking extracted immature monkey teeth in doxycycline before replantation they found that the revascularization rate doubled (18%–41%) after the doxycycline soak.[46] Recently, Ritter et al were able to further increase the revascularization rate in dogs with the use of minocycline.[47]

Immature tooth—EODT > 60 min

The benefits of replanting an immature tooth that will definitely be lost due to osseous replacement have been long debated and some authors have concluded that such teeth should not be replanted.[2]

AUTHOR'S PERSPECTIVE

The most crucial factor for the success of reimplanted tooth is the maintenance of PDL cell viability. The risk of failure increases significantly with an EODT of more than 15 min. An EODT of 60 min is considered the point where survival of the root periodontal cells is unlikely. Literature supports that moist storage appears to be a more productive approach to optimize PDL cell survival. However, no medium is ideal and in vivo studies are inadequate. Commercial storage media are expensive, not easily available and have a limited shelf life. Coconut water, which has been shown to be a more promising storage medium than others is easily available, economical, has more shelf life and extended storage time, although more studies are needed to confirm this. Based on the current literature, milk seems to be the most ideal choice for a transport media for avulsed teeth. Dental practitioners should follow current literature and consider evidence-based recommendations that may enhance periodontal healing and limit complications of reimplantation.