Milk as an interim storage medium for avulsed teeth.

In this study, milk was evaluated as a potential storage medium for avu]sed teeth. In various experiments, storage in milk was compared to storage in saliva, in water, and to air drying. The results demonstrate that milk is significantly superior to saliva, water, and air in preserving periodontal ligament fibroblast viability. Based on these results, it is recommended that when an avulsed tooth cannot be reimplanted immediately, it should be placed in milk and brought to the dentist for reimplantation. Several techniques have been suggested for interim preservation of avulsed teeth until reimplantation can be accomplished. Immediate replacement of the avulsed tooth into the socket is the soundest biological approach,1 but replacement rarely is accomplished at the time of the traumatic injury. Because nonprofessionals hesitate to implement this technique, several procedures for interim storage have been suggested. Some investigators have recommended placing the avulsed tooth into tap water while others suggest placing it in the buccal vestibule where is is bathed by saliva.2 Recently, Blomlof and coworkers recommended commercial bovine milk as an interim storage medium.3,, Advantages of milk as a preservative fluid are that it is nearly isotonic and is relatively free of bacterial contamination. Other investigators have demonstrated the presence of metabolically active and viable cells that are secreted with human colostrum.5 These data indicated that milk may be an effective temporary storage medium for periodontal ligament (PDL) fibroblasts associated with the roots of avulsed teeth. The purpose of this study is to compare the efficacy of commercial bovine milk to other suggested storage media in the maintenance of PDL fibroblast viability, human skin fibroblast viability, and the proliferative capacity of human skin fibroblasts. Methods and Materials Viability of PDL Cells Extracted premolars and third molars, were used to evaluate PDL cell viability. They were free of caries and advanced periodontal disease. Immediately after extraction, the teeth were placed in Hank’s buffered saline a (HBS) at a temperature of 4°C. All remnants of blood, gingiva, and osseous tissue were removed under sterile conditions with HBS washes, tissue forceps, and a surgical blade. Teeth were kept immersed in HBS during this procedure. These procedures were accomplished within 30 minutes of tooth extraction. Media tested for their ability to preserve the viability of PDL fibroblasts were: (1) HBS as a positive control, (2) pooled whole human saliva, (3) tap water, (4) commercial pasteurized whole bovine milk. Treatment of the extracted teeth consisted of immersion in one of the experimental media for i hour at 20°C. Additionally, one group of teeth was allowed to air-dry I hour at 20°C. A total of five teeth were used in each group. After this incubation period, fibroblasts associated with the PDL were removed enzymatically by collagenase (0.15% collagenase for 2 hours at 37°C). b (The teeth were placed in culture tubes containing 5 ml of collagenase during the treatment.) After the collagenase treatment the culture tubes were vortexed and the teeth removed. The remaining cell suspensions of fibroblasts then were centrifuged (800 g for 10 minutes) and washed three times in HBS. Next, the fibroblasts were suspended in 0.5 ml of HBS and diluted 1:10 in 0.05% trypan blue diluted with 0.9 N NaC1. The percentage of viable and nonviable cells were determined using hemocytometer counts. Fibroblasts were considered nonviable if they could not exclude trypan blue or if they exhibited ballooning degeneration. Significant differences in each group were determined using Student’s t-distribution. a Gibco, Grand Island, N.Y. b Collagenase #C-0130 Sigma, St. Louis, Mo. PEDIATRIC DENTISTRY: Volume 5, Number 3 183 Viability of Human Skin Fibroblasts Because of difficulties in PDL fibroblast quantitation and viability determination, human foreskin fibroblasts were used in the following two groups of experiments. Human foreskin fibroblasts have been shown to be analogous to PDL fibroblasts and easily can be quantitated. The cells were obtained and maintained in culture as previously described. 6 All fibroblasts were used beo tween the second and fifth serial passage and were proliferating actively (noncontact inhibited). A total of 5 105 human fibroblasts in 2.5 ml of tissue culture medium (RPMI 1640 supplemented with 10% fetal calf serum, 50 units of L-glutamine, and 10 ug/ml of gentamycin) were placed in 5 ml culture tubes. The cells were pelleted (800 g, 10 minutes) and the supernatant aspirated. The various preservation media were layered gently over cell pellets that were approximately 0.2 mm in depth. The preservation media then were incubated with the cell pellets for I hour at 20°C. After this incubation the test media carefully were removed and the cell pellet resuspended in HBS. Cell viability and recovery rates were determined by the trypan blue dye exclusion method previously described. Significant differences between experimental groups were determined using Student’s t-distribution. Proliferative Ability of Human Foreskin Fibroblast The proliferative ability of experimentally treated human foreskin fibroblasts was determined by a tritiated thymidine (3H-TdR) incorporation technique. 6 Briefly, fibroblasts in these assays were prepared and treated with experimental media as in the assay for fibroblast viability. In these experiments both actively proliferating and sessile (contact inhibited) fibroblasts were used. The saliva, milk, and tap water were sterilized by ultrafiltration through a 0.45 m filter apparatus, c After incubations with the experimental media the fibroblasts were resuspended in 5 ml of tissue culture media, and 0.2 ml aliquots were placed in cells of a microtiter tissue culture plate, d Each well was immediately pulsed with 0.2/~ Ci of 3H-TdRe and the fibroblasts cultured 24 hours at 37°C in a 5% CO2 atmosphere. After the culture period, the fibroblasts were collected and washed with an automatic cell harvester (MASH). Fibroblast proliferative capacity was determined by 3HTdR incorporation into DNA as measured by liquid scintillation counting. Statistical differences between experimental groups were determined using Student’s t-distribution.