The Effects of Continual Intermittent Parathyroid Hormone Treatment on Human Periodontal Ligament Fibroblasts in an In Vitro Wound Repopulation Model.

INTRODUCTION: Periodontal ligament fibroblasts (PDLFs) play a vital role in periodontal regeneration. Parathyroid hormone (PTH) is important in catabolic regulation on osteoclasts; it also has anabolic effects on hard tissue formation. Using an in vitro wound repopulation model, this study investigated the effect of continual intermittent administration of PTH on PDLFs wound repopulation. Methods and Materials: PDLFs were grown in 12-well plates and divided into 0 (control), 5, 10, 20, 40, and 80 nM of PTH treatments. A 3-mm wound was created on confluent and synchronized cells. Six PTH treatments were initiated using serum-free medium with supplements. Cell repopulation was measured at four time points: 5, 10, 15, and 20 days. RESULTS: A 5% increase wound repopulation showed an enhancement on day 10 for all treatment groups as compared to control groups. On days 15 and 20, treatment groups showed a decrease in proliferation and migration compared to controls with significant decreases at concentrations of 40 and 80 nM. CONCLUSION: Continual intermittent treatment with PTH has the potential to enhance proliferation and migration of PDLFs for wound repopulation at early time points. A dose-dependent correlation was seen with a positive trend on day 10 while a significant decrease on day 20.

Effect of Sodium Lauryl Sulfate (SLS) on Primary Human Gingival Fibroblasts in an In Vitro Wound Healing Model.

OBJECTIVES: Sodium lauryl sulfate (SLS) is a surfactant used to decrease the surface tension of water. Most commercially available dentifrices contain 0.5-2.0% SLS. This study investigated the potential effect of SLS on oral wound healing using primary human gingival fibroblasts (HGFs). METHODS: HGFs cells were grown in12-well culture plates in DMEM medium. A 3 mm wound was created on confluent HGFs. The cells were challenged with 0 (the control group), 0.01, 0.02, 0.03, 0.04, or 0.05% SLS-containing media once daily for 2 minutes. The cells were stained on day 0, 2, 4, 6 and 8. The percent of wound fill area was measured. RESULTS: On day 2, 4, 6, and 8, the wound fill of the control group (0% SLS) was 15, 35, 67 and 98%, respectively; at 0.01% SLS, it was 10, 20, 65 and 84%; at 0.02%, it was 7, 10, 15 and 25%; at 0.03% SLS, it was only 5% and 8% on day 2 and 4. CONCLUSION: Our results showed a dose- and time-dependent inhibition on HGFs wound fill by SLS; however, future in vivo studies are needed to validate if our in vitro findings using SLS-free dentifrices to avoid the potential delay of wound healing.

Chondrotoxicity of Liposomal Bupivacaine in Articular Chondrocytes: Preliminary Findings.

OBJECTIVE: The chondrotoxicity of local anesthetics has been previously recognized. Recent introduction of a liposomal formulation of bupivacaine has been found to significantly improve postoperative pain control but its effect on chondrocyte viability has yet to be investigated with this new formulation. We sought to assess the in vitro chondrotoxicity of liposomal bupivacaine. METHODS: Chondrocytes were isolated from articular cartilage from fresh stifle joints and grown in culture medium. Cultured chondrocyte-derived cells (CDCs) were treated with 0.9% normal saline solution, 0.5%, 0.25%, and 0.13% bupivacaine and ropivacaine, 1.3% liposomal bupivacaine for 1 hour. Following treatment, cells were washed and incubated in media for 23 hours. The CDCs were then harvested and viability was assessed by flow cytometry using SYTOX green dead cell stain. RESULTS: Treated CDCs demonstrated a dose-response effect for chondrocyte viability when treated with bupivacaine, ropivacaine, and liposomal bupivacaine. Liposomal bupivacaine demonstrated the highest chondrocyte viability following treatment. Ropivacaine demonstrated higher chondrocyte viability than bupivacaine. CONCLUSION: Following 1 hour of treatment, liposomal bupivacaine demonstrated the highest chondrocyte viability. Chondrocyte viability was inversely proportional to anesthetic concentration.

In vitro study of the adverse effect of nicotine and physical strain on human gingival fibroblasts as a model of the healing of wounds commonly found in the military.

BACKGROUND: Significant adverse effects on fibroblast growth and metabolism are observed with nicotine. We investigated the synergistic effects of nicotine and cyclical mechanical strain (CMS) on human gingival fibroblasts (HGFs) in a wound-healing model. METHODS: HGFs were isolated and grown in Dulbecco's modified Eagle's medium. Three-millimeter wounds were created on a confluent cell monolayer grown in a media containing 0, 1, 2, or 4 mM nicotine, with or without CMS. The applied deformation regimen remains constant for 6 days. On days 1, 2, 4, and 6, the cells were stained with hematoxylin and eosin Y for the evaluation of wound repopulation. RESULTS: The application of CMS alone demonstrates a biphasic response, with an initial stimulatory effect on wound repopulation (days 1-2) and less repopulation during the later phase (days 4-6). The addition of nicotine clearly demonstrated a time and inverse dose-dependent relationship on wound repopulation, with no effect during the early phase and reduced wound repopulation during the later phase. CONCLUSIONS: Initial treatment of HGF wounds with CMS resulted in faster wound repopulation regardless of nicotine presence. By day 6, wound healing of HGF exposed to both nicotine and CMS is delayed. These findings suggest that CMS and nicotine may affect fibroblasts and delay wound healing at other sites in the body as well.

The effectiveness of endodontic solvents to remove endodontic sealers.

Dental emergencies negatively affect troop readiness, especially during combat. Endodontic retreatment, when required, is especially challenging when the removal of endodontic sealer is required. In this study, we investigated the effectiveness of synthetic endodontic solvents to remove endodontic sealers. Fifty capillary tubes (2.7 mm ID×22 mm L), each filled to 15 mm with either Roth 801, AH Plus, MetaSEAL, or gutta-percha, were stored at 75% humidity for 14 days at 37°C. Ten capillary tubes containing each sealer were treated with either chloroform, xylene, EndoSolv R, EndoSolv E, or no solvent, and then penetrated with D3 ProTaper Universal Retreatment file on the same day. The time for the file to penetrate the length of each sealer was recorded, and the data statistically analyzed. Roth 801 failed to set and was not tested. The file took 3.4±0.1, 4.8±0.3, 5.7±0.4, 4.5±0.2, and 10.6±1.0 seconds (mean±SD) to penetrate gutta-percha using chloroform, xylene, EndoSolv R, EndoSolv E, or no solvent, respectively, and was performed by one endodontic resident at one sitting. The time for penetration of gutta-percha with any solvent was significantly faster (p≤0.05) than for AH Plus or MetaSEAL.The time for AH Plus ranged from 23.1±1.0 to 81.5±4.5 seconds. The time for MetaSEAL ranged from 97.2±6.1 to >180 seconds. EndoSolv E was the most effective solvent for AH Plus. It took significantly more time to remove MetaSEAL than AH Plus, regardless of the solvent used. Our study indicated that the use of the proper endodontic solvent makes complete removal of a sealer much more effective during retreatment.

Addition of epidermal growth factor improves the rate of sulfur mustard wound healing in an in vitro model.

OBJECTIVE: Sulfur mustard (SM) causes blisters on the human skin. These blisters delay healing of the skin and make the victims more susceptible to infection. In vitro models have been used for protection studies against SM injury, but study on wound healing after SM exposure has not been explored. The purpose of this study was to test whether the addition of exogenous growth factors could improve the rate of SM wound healing. METHODS: The model consisted of normal human epidermal keratinocytes seeded into 6-well plates, exposed to SM, and wounded (disruption of the cell monolayer) with a sterile wounding instrument. Cells were then stained and images were captured to measure percentage wound fill. Epidermal growth factor (EGF) and keratinocyte growth factor (KGF) were tested in this model. RESULTS: EGF (1 ng/mL) significantly increased wound fill on all of the days tested (days 6, 9, and 12). KGF did not significantly improve wound healing. CONCLUSIONS: EGF showed promise as a potential therapy for SM-induced wounds. This in vitro model was a valuable tool for screening therapeutics before animal testing. These results will be used to develop a dressing that can slowly release EGF on to a debrided wound bed to help speed the healing process.

Nicotine stimulates osteoclast resorption in a porcine marrow cell model.

BACKGROUND: Combustible tobacco use is generally linked with accelerated periodontal bone loss and diminished post-surgical wound healing; however, the pathogenesis of this process at the cellular level remains unclear. Nicotine is known to affect human gingival fibroblast orientation, attachment, and beta1 integrin expression, yet little is known about its effects on osteoclasts, the cells most responsible for bone resorption. The purpose of this study was to determine the effects of physiologically relevant nicotine levels on porcine osteoclast function as measured by resorption of calcium phosphate. METHODS: Pure nicotine was diluted in medium to the following concentrations: 0.03 microM, 0.15 microM, 0.30 microM, 0.60 microM, and 1.50 microM. Porcine osteoclasts were seeded onto calcium phosphate multi-test slides and incubated at 37 degrees C with half media changes every 24 hours. Cells received 0, 0.15, 0.30, 0.60, and 1.50 microM nicotine, or 25 nM parathyroid hormone (PTH). Osteoclast resorption was quantified by measuring the resorbed surface area of the calcium phosphate substrate. RESULTS: Osteoclast cultures resorbed bone slices and calcium phosphate substrate. All nicotine concentrations and PTH resulted in statistically significantly greater mean percent resorptions than the control group (P < 0.05). However, no statistical difference was found between the various nicotine doses or PTH. The number of osteoclasts increased in a linear relationship to the increasing nicotine concentrations; however, no correlation was found between osteoclast number and the amount of resorption. CONCLUSIONS: Nicotine is non-toxic to osteoclasts at the clinically relevant levels tested. Nicotine appears to stimulate osteoclast differentiation and resorption of calcium phosphate, the major component of bone. Nicotine-modulated osteoclast stimulation may, in part, explain the increased rapidity of periodontal bone loss and refractory disease incidence in smokers.