Viability of fibroblasts in a novel probiotic storage media

Abstract – A number of storage media have been investigated as to their ability to maintain the viability of the periodontal ligament (PDL) cells and thus to permit longer extra‐alveolar periods prior to replantation of avulsed teeth. The aim of the present in vitro study was to evaluate the number of viable PDL cells of avulsed teeth treated by Hank’s Balanced Salt Solutions (HBSS), saline, a novel probiotic solution and milk. Thirty‐six freshly extracted single‐rooted human teeth with closed apices were divided into one of the four experimental groups and two control groups (N = 6 each). The positive and negative controls corresponded to 0 min and an 8‐h dry time respectively. Following extraction, the coronal 3 mm of PDL tissue was scraped with a #15 scalpel to remove cells that might have been damaged. The experimental teeth were dried for 30 min followed by a 45 min immersion in one of the four experimental media. Each experimental tooth, after drying and soaking, was incubated for 30 min with a 2.5 ml solution of 0.2 mg ml^?1 of collagenase CLS II and a 2.4 mg ml^?1 solution of dispase grade II in phosphate buffer saline (PBS). The cells were then labelled with 0.4% Trypan blue for determination of viability. The teeth stored in positive control demonstrated the highest number of viable PDL cells followed in rank order by HBSS, saline, Lactobacillus reuteri solution and milk. There was no significant difference in the number of viable PDL cells between HBSS, milk, L. reuteri solution and saline. Within the parameters of this study, it appears that probiotic may be able to maintain PDL cell viability as HBSS, milk, or saline.     

(-)-Epigallocatechin-3-gallate: a novel storage medium for avulsed teeth

The purpose of the present study was to evaluate the efficacy of (-)-epigallocatechin-3-gallate (EGCG) in maintaining the vitality of human periodontal ligament (PDL) cells when used as a storage medium for avulsed teeth prior to replantation. Thirty freshly extracted single-rooted human teeth with closed apices were randomly assigned to three experimental groups with 10 samples per group and immersed in one of the storage media: EGCG, Hank's balanced salt solution (HBSS), or milk for 2 h. The PDL cells were dissociated by an enzyme treatment with collagenase and trypsin. The cells were then labeled with 0.4% Trypan blue for the determination of viability. The result showed that EGCG group had the highest percentage of cell viability, followed by HBSS and milk group, in descending order.     

A quantitative analysis of Propolis: a promising new storage media following avulsion

Abstract –  Both length of extra-alveolar time and type of storage media are significant factors that can affect the long-term prognosis of replanted teeth. Numerous studies have examined various media in an attempt to determine the ideal material for storage of the avulsed tooth. The purpose of this study was to use a Collagenase–Dispase assay to investigate the potential of a new storage media, Propolis, in maintaining viable periodontal ligament (PDL) cells on simulated avulsed teeth. Seventy freshly extracted human teeth were divided into five experimental groups and two control groups. The positive and negative controls corresponded to 0-min and an 8-h dry time, respectively. The experimental teeth were stored dry for 30 min and then immersed in one of the five media (Hank's balanced salt solution (HBSS), milk, saline, Propolis 50%, and Propolis 100% for 45 min). The teeth were then treated with dispase grade II and collagenase for 30 min. The number of viable PDL cells were counted with a hemocytometer and analyzed. Statistical analysis demonstrated that both Propolis groups kept significantly more PDL cells viable compared to either milk, saline, or HBSS. Within the parameters of this study, it appears that Propolis may be a better alternative to HBSS, milk, or saline in terms of maintaining PDL cell viability after avulsion and storage.     

Comparison of coconut water, propolis, HBSS, and milk on PDL cell survival

Coconut water is biologically pure and sterile, with a rich presence of amino acids, proteins, vitamins, and minerals. The purpose of this study was to use a collagenase-dispase assay to investigate the potential of a new storage medium, coconut water, in comparison with propolis, Hank's balanced salt solution (HBSS), and milk in maintaining viable periodontal ligament (PDL) cells on simulated avulsed teeth. Seventy freshly extracted human teeth were divided into 4 experimental groups and 2 control groups. The positive and negative controls corresponded to 0-minute and 8-hour dry times, respectively. The experimental teeth were stored dry for 30 minutes and then immersed in 1 of the 4 media (coconut water, propolis, HBSS, and milk). The teeth were then treated with dispase grade II and collagenase for 30 minutes. The number of viable PDL cells was counted with a hemocytometer and analyzed. Statistical analysis 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.     

Periodontal healing of replanted dog teeth stored in Viaspan, milk and Hank's balanced salt solution.

This study examined, histologically, periodontal healing and root resorption of replanted dogs teeth stored in Viaspan for different time periods and compared these healing patterns to those after storage in milk or Hank's balanced salt solution (HBSS). Seventy-two beagle incisors were endodontically treated, extracted, randomly divided and placed in Viaspan or milk for 6, 12, 24 and 36 h, and Viaspan or HBSS for 36, 48, 72 and 96 h, after which they were replanted. Four negative control teeth were immediately replanted while four positive controls were allowed to dry for one hour before replantation. All replanted teeth were splinted for 14 days. Two months after replantation the dogs were killed and the teeth histologically examined for healing of the supporting tissues. For Viaspan neither replacement nor inflammatory root resorption was seen after 6 and 12 h storage. A statistically significant rise in the incidence of replacement resorption was seen at 24, 36 and 48 h which decreased again at 72 and 96 h to levels equal to storage for 6 and 12 h. The occurrence of inflammatory root resorption was low and significantly increased only at 48 h after which it decreased significantly again. Viaspan proved superior to milk as a storage medium. Teeth stored in HBSS showed healing results similar to those stored in Viaspan.     

Periodontal healing of replanted dog teeth stored in Viaspan, milk and Hank's balanced salt solution

Abstract This study examined, histologically, periodontal healing and root resorption of replanted dogs teeth stored in Viaspan for different time periods and compared these healing patterns to those after storage in milk or Hank's balanced salt solution (HBSS). Seventy-two beagle incisors were endodontically treated, extracted, randomly divided and placed in Viaspan or milk for 6, 12, 24 and 36 h, and Viaspan or HBSS for 36, 48, 72 and 96 h, after which they were replanted. Four negative control teeth were immediately replanted while four positive controls were allowed to dry for one hour before replantation. All replanted teeth were splinted for 14 days. Two months after replantation the clogs were killed and the teeth histologically examined for healing of the supporting tissues. For Viaspan neither replacement nor inflammatory root resorption was seen after 6 and 12 h storage. A statistically significant rise in the incidence of replacement resorption was seen at 24, 36 and 48 h which decreased again at 72 and 96 h to levels equal to storage for 6 and 12 h. The occurrence of inflammatory root resorption was low and significantly increased only at 48 h after which it decreased significantly again. Viaspan proved superior to milk as a storage medium. Teeth stored in HBSS showed healing results similar to those stored in Viaspan.     

不同离体时间和保存液对犬离体牙牙周膜细胞活力影响的实验研究

目的:探讨完全脱位牙不同离体时间和保存液对牙周膜细胞活力的影响。方法:麻醉拔除犬牙35个,首先将20个牙随机分为5组,分别为室温干燥放置0、30、60、120、240 min组,另15个牙室温干燥放置30 min后,随机分为3组,分别放入牛奶、HBSS液,100 g/L蜂胶液中浸泡2 h。各组处理完成后,采用全牙消化法获得牙周膜细胞,并通过4 g/L台盼蓝染色法检测各组牙周膜活细胞数和存活率。结果:室温干燥放置30、60、120、240 min后,牙周膜细胞存活率依次为33.6%、23.6%、18.5%、0.8%,而0 min的牙周膜细胞存活率可达95.5%。拔后30 min,经牛奶、HBSS液和100 g/L蜂胶液中保存2 h后,牙周膜细胞均有活力,其细胞存活率大小依次为100 g/L蜂胶液、HBSS液和牛奶,其中100 g/L蜂胶液与HBSS液相比无统计学差异(P>0.05),但与牛奶组相比,均有统计学差异(P<0.05)。结论:随着离体时间延长,完全脱位牙根面牙周膜细胞活力明显下降。100 g/L蜂胶液和HBSS液保存犬牙牙周膜细胞活力优于牛奶液。     

Potential of the propolis as storage medium to preserve the viability of cultured human periodontal ligament cells: an in vitro study

Abstract – Aim: In vitro experiments were carried out to evaluate the potential of propolis, a natural resin known for its wide therapeutic window, as storage medium to preserve the viability of cultured human periodontal ligament (PDL) cells. Materials and Methods: Primary cultures of human PDL cells were subjected to either independent exposure of propolis (2.5%, 5.0%, 10.0%, and 20.0%), Hank’s balanced salt solution (HBSS), milk (0.5%), artificial saliva, Dulbecco’s modified Eagle’s medium (DMEM) or combination of propolis 10% + DMEM, propolis 20% + DMEM for 30 min to 24 h at 37°C. Cell viability was assessed using standard endpoints i.e., tetrazolium bromide salt (MTT), neutral red uptake, and trypan blue dye exclusion assay. Results: In general, combinations of propolis 10% + DMEM, propolis 20% + DMEM, and DMEM alone were found to be better than other media used in this study. The difference in the potentials of these media to maintain the cell viability reached to the statistically significant levels by 24 h, when compared with other media used viz., propolis 2.5% (P < 0.01), propolis 5.0% (P < 0.05), propolis 10.0% (P < 0.05), propolis 20.0% (P < 0.001), HBSS (P < 0.001), and milk (P < 0.01). Trypan blue dye exclusion assay could be recorded the most sensitive among all the assays selected to study the cell viability of PDL cells. Conclusions: Study indicates that combinations of propolis 10% + DMEM, propolis 20% + DMEM, and DMEM alone are equally good as storage media of choice to keep PDL cells viable during extra‐alveolar period up to 24 h. Other more readily available medium such as milk may serve as appropriate alternative storage medium for shorter time periods i.e., up to 12 h.     

Effect of preservation media on proliferation and collagen biosynthesis of periodontal ligament fibroblasts

Abstract The viability of periodontal ligament (PDL) cells, which are responsible for the production of ligament proteins, is essential for clinical healing of replanted exarticulated teeth. Cultured human PDL fibroblast-like cells were used to study the effects of various transport media on cell proliferation, collagen synthesis and total protein synthesis. The PDL cells were obtained from intact premolars of a healthy young male patient by trypsinization and scraping the PDL tissue. Proliferation of the cells, the activity of prolyl 4-hydroxylase, total protein and collagen synthesis were studied after 60 min incubation in Dulbecco's Modified Eagle's Medium (DMEM), milk, saliva and tap water. Proliferation capacity was also investigated after prolonged incubation in milk. The results indicated that milk and saliva were superior to water in maintaining these cell functions, but as the saliva used in this study does not resemble that in the clinical situation, we recommend milk as a temporary storage medium for exarticulated teeth before replantation.     

Storage media enhance osteoclastogenic potential of human periodontal ligament cells via RANKL‐independent signaling

Abstract – Background: Hank’s balanced salt solution (HBSS) and milk have gained wide acceptance as storage media for avulsed tooth. However, the effect of the media and storage time on the periodontal ligament (PDL) cells involvement in the development of root resorption is still unclear. The purpose of this study was to evaluate whether precultured PDL cells in HBSS, milk, or modified Eagle’s medium alpha (α‐MEM) would affect osteoclastogenesis. Materials and methods: PDL cells were precultured in HBSS, milk, or α‐MEM for 1 h or 6 h before being co‐cultured with RAW 264.7 cells for an additional 3 days for mRNA analysis and 11 days for osteoclastogenesis assay. Results: Cyclooxygenase‐2 (COX‐2) mRNA was detected immediately in PDL cells precultured in the three storage media. The expression was up‐regulated markedly in all co‐cultures when compared with RAW cells alone. As a result of the co‐culture, interleukin‐1β (IL‐1β) expression was detectable in both PDL and RAW cells. TRAP+ multinucleated, osteoclast‐like cells developed in all co‐cultures; the number of TRAP+ cells was highest (P < 0.05) in the co‐cultures that PDL cells precultured in milk for 6 h. The mRNA level of receptor activator of nuclear factor‐kappa B ligand (RANKL) was not detected in PDL cells. Osteoprotegerin (OPG) mRNA expression reduced with increased preculture time, but the difference was not significant (P > 0.05). Conclusions: PDL cells kept in the three storage media led to TRAP+ multinucleated, osteoclast‐like cells formation via RANKL‐independent signaling. The ability to induce osteoclastogenesis may be considered as one of the factors to evaluate the ability of storage medium to maintain PDL viability after tooth avulsion.     

Periodontal healing of replanted monkey teeth prevented from drying

Root resorption of replanted teeth is dependent on the duration of the extra-alveolar period and on the storage environment. In the present investigation the significance of preserving the humidity of the periodontal ligament (PDL) during the extra-alveolar period was tested on isolated PDL cells and on replanted monkey teeth. The isolated PDL cells were tested with respect to cell viability (trypan blue exclusion test) and to cell recovery (number of cells after additional cultivation). About 70% of the cells were viable and 44% recovered after 1 h in a humid atmosphere. Practically no cells were viable or recovered after 1 h of drying. Replanted teeth that had been wrapped in plastic foil for 1 h before replantation showed no more resorption than immediately replanted teeth. This is in contrast to teeth dried in air for 1 h before replantation. They showed extensive root resorption on almost all root surfaces. Thus, prevention of evaporation of tissue fluid from the PDL must be considered a primary goal if the tooth cannot be replanted immediately.     

Effect of propolis on proliferation and apoptosis of periodontal ligament fibroblasts

The most critical factors affecting the prognosis of an avulsed tooth are extraoral dry time and storage media used before replantation. Studies have analyzed different storage media to determine the ideal solution to preserve periodontal ligament (PDL) cell viability. Propolis has anti-inflammatory and antimicrobial properties, and has been previously suggested as a storage medium. The purpose of this study was to assess not only cell viability but also physiological health of PDL cells after exposure to propolis. PDL cells were exposed to different concentrations of propolis or Hanks balanced salt solution, and the apoptotic levels were determined using apoptosis assay and flow cytometry. Additional cell viability and proliferation were analyzed by XXT assay in dry and wet conditions. Propolis not only decreased apoptosis but also increased the metabolic activity and proliferation of PDL cells. This study suggests that propolis is a suitable storage medium for avulsed teeth.     

Assessment of post-traumatic PDL cells viability by a novel collagenase assay

Abstract – Both length of extra-alveolar time and type of storage media are significant factors that can affect the long-term prognosis for replanted teeth. Numerous studies have examined various media in an attempt to determine the ideal material for storage of the avulsed tooth. The purpose of this study was to compare the number of viable periodontium ligament (PDL) cells in different storage media using a collagenase assay. Thirty-three freshly extracted human teeth were divided into four experimental and two control groups. The positive and negative controls corresponded to 0 min and an 8-h dry time, respectively. The experimental teeth were stored dry for 30 min and then immersed in one of four media (Hank's balanced salt solution (HBSS), milk, saline, water) for 45 min. The teeth were then treated with dispase grade II and collagenase for 30 min. The number of viable and nonviable PDL cells was counted with a hemocytometer and analyzed. An anova demonstrated no statistically significant differences in the viability of PDL cells among saline, HBSS and milk. Within the parameters of this study, it appears that milk or saline is an equally viable alternative to HBSS for storage of avulsed teeth.     

Periodontal healing of replanted dog teeth stored in milk and egg albumen

Abstract – The type of storage medium used to store avulsed teeth prior to replantation has been shown to be a decisive factor in periodontal ligament (PDL) healing. The aim of the present study was to investigate the effect of storage medium on periodontal healing. Thirty teeth from three dogs were endodontically treated to prevent subsequent inflammatory root resorption. The teeth were atraumatically extracted and randomly stored in milk or egg albumen for 3, 6 and 10 h at 4°C. All teeth were splinted for 1 weeks after replanting. After 2 months animals were sacrificed using vital perfusion‐fixation and teeth were histologically prepared and evaluated following Andreasen’s method. It was found that teeth stored in egg albumen for 6 and 10 h had significantly higher incident of PDL healing than those treated with milk for the same period (P < 0.05). . The highest incidence of PDL healing was observed in teeth stored in egg albumen for 6 h. The least surface resorption was also evident in this group (P < 0.05). The result of this study shows that egg albumen is an excellent storage media for up to 10 h considering its likely availability at most accident sites.     

The influence of storage conditions on the clonogenic capacity of periodontal ligament cells: implications for tooth replantation

Viable periodontal ligament (PL) cells are required for PL healing of avulsed teeth following replantation. If immediate replantation cannot be accomplished, the ability of PL progenitor cells to reproduce (clonogenic capacity) and recolonize the wound may be extended by prevention of desiccation and storage in physiological media. This investigation examined the effects of storage in saliva, milk, Hank's balanced salt solution (HBSS) and Eagle's medium (αMEM) on the clonogenic capacity of human PL progenitor cells at 30 and 60 min extra-alveolar time. Twenty erupted human premolar teeth extracted as atraumatically as possible for orthodontic purposes were used in the present study. Fifteen premolars were placed immediately in freshly collected autologous saliva at room temperature, (+ 23°C) for 15 min. These 15 premolars were next divided into three groups of five and stored in either saliva, milk or HBSS at + 4°C in plastic cups surrounded by ice. The remaining five teeth served as positive controls and were immediately placed in αMEM at + 4°C. PL tissue was scraped from one-half of the root surface with a scalpel at 30 and 60 min total extra-alveolar duration. Cells were released from the tissue sample with a 30 min enzymatic digestion procedure and the cells from the tissue samples analyzed for clonogenic capacity. There was a reduction in clonogenic capacity with time for all protocols. Periodontal ligament cells stored in αMEM showed the least reduction between 30 and 60 min and the greatest reduction was observed for PL cells stored in saliva. The difference in clonogenic capacity following transfer from saliva to milk or HBSS was not significant at 30 min. At 60 min, cells transferred from saliva to HBSS had a statistically higher percentage of clonogenic cells than those transferred to milk (5.9% vs. 3.5%; P < 0.05). We conclude that immediate storage of avulsed teeth in autologous saliva, followed by transfer to chilled milk, preserves the presence of sufficient progenitor cells in the PL to warrant replantation and the possibility of PL healing at 60 min extra-alveolar duration.     

Periodontal healing of exarticulated monkey teeth stored in milk or saliva

Abstract – Saliva has usually been recommended as a storage medium for exarticulated teeth. Recent tissue culture studies have, however, shown that milk has a more suitable osmolaliry than saliva and that milk is a bettter storage medium for human periodontal ligament cell. In the present investigation periodontal healing of replanted monkey teeth has been studied after storage of teeth in milk or saliva before replantation. There was much less root resorption, especially inflammatory resorption, after storage in milk thatn in saliva. Storage of teeth in milk for 3 h before replantation resulted in the same low frequence of root resorptions as was seen after immediate replantation. There was hardly any replacement resorption (ankylosis) seen in the replanted teeth.     

Effects of temperature, storage time and media on periodontal and pulpal healing after replantation of incisors in monkeys

Abstract – The effect of temperature of various storage media and at varying storage periods upon periodontal ligament (PDL) and pulpal healing after tooth replantation was examined in green vervet monkeys (Cercopithecus aethiops). Mandibular lateral incisors with mature root formation were extracted and kept in dry storage at 22, 4 and ?18 °C; in saline at 37, 22, 4 and ?18 °C; or in saliva (i.e. in the buccal vestibule) at 37 °C for either 60 or 120 min prior to replantation. The animals were sacrificed 8 weeks after replantation and the replanted teeth examined histometrically. The following histological parameters were registered for each tooth: normal PDL, surface resorption, inflammatory resorption, replacement resorption (ankylosis), downgrowth of pocket epithelium, periapical inflammatory changes, and the extent of vital pulp. A total of 125 replanted teeth were examined. Storage in saliva at 37 °C showed a similar amount of normal PDL compared to saline storage for both 60 and 120 min. Saline storage for 60 or 120 min showed no difference in the extent of normal PDL when storage was compared at 37, 22 and 4 °C. However, storage at ?18 °C resulted in significantly less normal PDL than storage at other temperatures. Dry storage for 60 min showed significantly less root resorption at 4 °C compared to 22 °C. Furthermore, dry storage at ?18 °C showed significantly less normal PDL than storage at 4 °C. When the dry storage period was extended to 120 min, no difference was found between 22, 4 and ?18 °C. It is concluded that the temperature (above 0 °C) of the storage medium is of importance only for dry storage and in such a situation only for shorter extra‐alveolar periods, i.e. for 60‐min storage and not for 120 min, where extensive destruction of the PDL always takes place. It is suggested that the temperature effect of 4 °C could be related to less evaporation from the PDL and thereby less damage to the PDL cells or a strict temperature effect upon cell metabolism. Pulp healing in all the cases was limited to the entrance of the pulp canal, and no significant pattern was found between storage media, time and temperature.     

Delayed replantation of rat teeth after use of reconstituted powdered milk as a storage medium

Abstract – Minimal extraoral dry storage period and moist storage for the avulsed tooth are identified as key steps for the treatment protocol of tooth replantation. Among the possible moist storage media, bovine milk has stood out because of its capacity of preserving the integrity of the periodontal ligament (PDL) fibers. This condition has attracted the attention to investigate the use of powdered milk, which is one of the presentation forms of bovine milk, as a feasible storage medium in cases of delayed tooth replantation. The aim of this study was to evaluate the healing process after delayed replantation of rat teeth stored in reconstituted powdered milk and long shelf‐life (ultra high temperature) whole milk. Forty maxillary right rat incisors were assigned to four groups (n = 10): group I – the teeth were extracted and immediately replanted into theirs sockets; group II – the teeth were stored for 60 min in 200 ml of freshly reconstituted powdered milk; group III – the teeth were stored for 60 min in 200 ml of long shelf‐life whole milk; group IV – the teeth were kept dry for the same time. All procedures were performed at room temperature. Next, the root canals of teeth in groups II, III, and IV were instrumented, filled with a calcium hydroxide‐based paste, and replanted into their sockets. All animals received systemic antibiotic therapy and were killed by anesthetic overdose 60 days after replantation. The pieces containing the replanted teeth were removed, fixed, decalcified, and paraffin‐embedded. Semi‐serial 6‐μm‐thick sections were obtained and stained with hematoxylin and eosin for histomorphological analysis. There was statistically significant difference (P < 0.05) between groups I and IV regarding the presence of replacement resorption and PDL remnants on root surface. The powdered milk and long shelf‐life whole milk presented similar results to each other and may be indicated as storage media for avulsed teeth.     

Current developments in interim transport (storage) media in dentistry: an update

Healing following avulsion and replantation is dependent on the extent of pulpal and periodontal ligament (PDL) tissue damage. Therefore, immediate replantation is the recommended treatment of choice for an avulsed permanent tooth. To achieve a more favourable prognosis following tooth replantation, use of an appropriate interim transport medium is usually advocated. Numerous studies have researched and advocated the use of media like saliva, milk, Hank's Balanced Salt Solution (HBSS) and ViaSpan. However, current research has indicated the use of few newer media as promising interim transport media for an avulsed tooth. This review summarises the current developments regarding the introduction of newer interim transport media for the treatment of avulsed teeth.     

Which is the most recommended medium for the storage and transport of avulsed teeth? A systematic review

Background/Aims

A wide variety of materials has been researched for their use as potential storage media for avulsed teeth, but it is essential to recognize the medium most recommended for improvement of the prognosis of avulsed teeth. The aim of this systematic review was to identify the most recommended medium to store and transport avulsed teeth based on the survival of periodontal ligament (PDL) cells as determined by in vitro studies.

Methods

Only laboratory‐based experimental studies on PDL cells found on adult permanent teeth were included. Data were collected using PubMed, CINAHL plus (EBSCO host), and the Cochrane Library, along with Google Scholar and a hand search. The key terms employed were permutations of [avulsed permanent teeth* OR dental avulsion* OR knocked out teeth*] AND [storage media* OR transport media* OR biological transport* OR PDL cell viability* OR PDL cell survival*]. A customized data extraction pro forma was used to extract the data and to evaluate the quality and risk of bias.

Results

The initial search yielded 978 articles, but only 67 were selected. Milk was the most recommended individual medium followed by Hank's balanced salt solution. Among natural products other than milk, propolis and coconut water were most frequently recommended. Recommendations were based on maintenance of PDL cell viability followed by ease of availability, low cost, and long shelf life.

Conclusions

Natural products are more effective in maintaining the PDL cell viability compared to synthetic products. Some storage media recommendations were also based upon practical aspects. Although natural products other than milk have more recommendations as a group, milk is the most recommended storage medium individually, based not only on PDL cell viability, but also practical considerations.