Evaluation of the In Vitro Oral Wound Healing Effects of Pomegranate (Punica granatum) Rind Extract and Punicalagin, in Combination with Zn (II).

Pomegranate (Punica granatum) is a well-established folklore medicine, demonstrating benefits in treating numerous conditions partly due to its antimicrobial and anti-inflammatory properties. Such desirable medicinal capabilities are attributed to a high hydrolysable tannin content, especially punicalagin. However, few studies have evaluated the abilities of pomegranate to promote oral healing, during situations such as periodontal disease or trauma. Therefore, this study evaluated the antioxidant and in vitro gingival wound healing effects of pomegranate rind extract (PRE) and punicalagin, alone and in combination with Zn (II). In vitro antioxidant activities were studied using DPPH and ABTS assays, with total PRE phenolic content measured by Folin-Ciocalteu assay. PRE, punicalagin and Zn (II) combination effects on human gingival fibroblast viability/proliferation and migration were investigated by MTT assay and scratch wounds, respectively. Punicalagin demonstrated superior antioxidant capacities to PRE, although Zn (II) exerted no additional influences. PRE, punicalagin and Zn (II) reduced gingival fibroblast viability and migration at high concentrations, but retained viability at lower concentrations without Zn (II). Fibroblast speed and distance travelled during migration were also enhanced by punicalagin with Zn (II) at low concentrations. Therefore, punicalagin in combination with Zn (II) may promote certain anti-inflammatory and fibroblast responses to aid oral healing.

Rhodoptilometrin, a Crinoid-Derived Anthraquinone, Induces Cell Regeneration by Promoting Wound Healing and Oxidative Phosphorylation in Human Gingival Fibroblast Cells.

Gingival recession (GR) potentially leads to the exposure of tooth root to the oral cavity microenvironment and increases susceptibility to dental caries, dentin hypersensitivity, and other dental diseases. Even though many etiological factors were reported, the specific mechanism of GR is yet to be elucidated. Given the species richness concerning marine biodiversity, it could be a treasure trove for drug discovery. In this study, we demonstrate the effects of a marine compound, (+)-rhodoptilometrin from crinoid, on gingival cell migration, wound healing, and oxidative phosphorylation (OXPHOS). Experimental results showed that (+)-rhodoptilometrin can significantly increase wound healing, migration, and proliferation of human gingival fibroblast cells, and it does not have effects on oral mucosa fibroblast cells. In addition, (+)-rhodoptilometrin increases the gene and protein expression levels of focal adhesion kinase (FAK), fibronectin, and type I collagen, changes the intracellular distribution of FAK and F-actin, and increases OXPHOS and the expression levels of complexes I~V in the mitochondria. Based on our results, we believe that (+)-rhodoptilometrin might increase FAK expression and promote mitochondrial function to affect cell migration and promote gingival regeneration. Therefore, (+)-rhodoptilometrin may be a promising therapeutic agent for GR.

Hyperbaric oxygen therapy (HBOT) and peridontal health.

This study has tested the effects of hyperbaric oxygen in periodontal structures in agreement with the theories supported by literature research. Eight patients, from 30 to 50 years-of-age, were tested with pure oxygen inhalation, at the 2.5 ATA absolute pressure. Main approved tests of periodontal health were evaluated before and after HBOT’s cycles. The results in all patients treated with HBOT, have founded clear improvement of clinical and instrumental parameters.

Proliferation, migration, and expression of oral-mucosal-healing-related genes by oral fibroblasts receiving low-level laser therapy after inflammatory cytokines challenge.

BACKGROUND AND OBJECTIVES: Increased expression of inflammatory cytokines in the oral cavity has been related to the etiopathogenesis of oral mucositis and to delayed oral mucosal repair. Low-level laser therapy (LLLT) stimulates proliferation and migration of gingival fibroblasts, but the effects of specific inflammatory cytokines on oral mucosal cells and the modulation of these effects by LLLT have not been fully investigated. Therefore, this study investigated the effects of LLLT on oral fibroblasts after being challenged by oral-mucositis-related inflammatory cytokines. METHODS: Human gingival fibroblasts were seeded in plain culture medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 hours. Then, cells were kept in contact with inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8) in serum-free DMEM for 24 hours. After this period, cells were subjected to LLLT with a diode laser device (LaserTABLE, InGaAsP, 780 nm, 25 mW) delivering energy doses from 0.5 to 3 J/cm2 . Irradiation was repeated for 3 consecutive days. Twenty-four hours after the last irradiation, cell migration (wound-healing and transwell migration assays), cell proliferation (BrdU), gene expression of COL-I and growth factors (real-time PCR), and synthesis of COL-I (Sirius Red assay) and VEGF (ELISA) were assessed. Data were subjected to two-way ANOVA and Tukey's tests or Kruskall-Walis and Mann-Whitney tests (P < 0.05). RESULTS: The inflammatory cytokines decreased the migration capacity of gingival fibroblasts. However, a statistically significant difference was observed only for IL-6, detected by transwell assay, where 30% less cells migrated through the pores (P < 0.05) and IL-8, with an increased wound area (116%; P < 0.05), detected by the wound healing method. Cell proliferation was not affected by contact with cytokines, while growth factors and COL-I expression (approximately 80%; P < 0.05), as well as VEGF synthesis (approximately 20%; P < 0.05), were decreased after contact to all tested cytokines. The opposite was seen for total collagen synthesis. LLLT promoted an acceleration of fibroblast migration (30%; P < 0.05) and proliferation (112%; P < 0.05) when delivering 0.5 J/cm2 to the cells previously in contact with the inflammatory cytokines. Gene expression of VEGF (approximately 30%; P < 0.05), and EGF (17%; P < 0.05), was stimulated by LLLT after contact with TNF-α and IL-6. CONCLUSION: LLLT can counteract the negative effects of high concentrations of inflammatory cytokines, especially IL-6 and IL-8 on gingival fibroblast functions directly related to the wound-healing process. Lasers Surg. Med. 48:1006-1014, 2016. © 2016 Wiley Periodicals, Inc.

Light-emitting diode irradiation promotes donor site wound healing of the free gingival graft.

BACKGROUND: This study aims to evaluate the effect of light-emitting diode (LED) light irradiation on the donor wound site of the free gingival graft. METHODS: Rat gingival fibroblasts were chosen to assess the cellular activities and in vitro wound healing with 0 to 20 J/cm(2) LED light irradiation. Seventy-two Sprague-Dawley rats received daily 0, 10 (low-dose [LD]), or 20 (high-dose [HD]) J/cm(2) LED light irradiation on the opened palatal wound and were euthanized after 4 to 28 days; the healing pattern was assessed by histology, histochemistry for collagen deposition, and immunohistochemistry for tumor necrosis factor (TNF)-α infiltration. The wound mRNA levels of heme oxygenase-1 (HO-1), TNF-α, the receptor for advanced glycation end products, vascular endothelial growth factor, periostin, Type I collagen, and fibronectin were also evaluated. RESULTS: Cellular viability and wound closure were significantly promoted, and cytotoxicity was inhibited significantly using 5 J/cm(2) LED light irradiation in vitro. The wound closure, reepithelialization, and collagen deposition were accelerated, and sequestrum formation and inflammatory cell and TNF-α infiltration were significantly reduced in the LD group. HO-1 and TNF-α were significantly upregulated in the HD group, and most of the repair-associated genes were significantly upregulated in both the LD and HD groups at day 7. Persistent RAGE upregulation was noted in both the LD and HD groups until day 14. CONCLUSION: LED light irradiation at 660 nm accelerated palatal wound healing, potentially via reducing reactive oxygen species production, facilitating angiogenesis, and promoting provisional matrix and wound reorganization.

Epithelial TRPV1 signaling accelerates gingival epithelial cell proliferation.

Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43°C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca(2+) levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation.

[The role of retinal-hypotalamic-pineal system in pro- and antioxidant processes in the gingival tissues of adult male albino rats].

The aim of this study is to evaluate the effects of pineal gland functional state on the prooxydant processes and antioxidant system in the gingival tissues. Male rats were assigned into one of the following groups in accordance with the duration of photoperiod: 1) control--natural daylight; 2) permanent darkness for 14 days; 3) permanent light for 14 days. The following parameters were measured in gingival tissues and the blood serum: 1) prooxidant factors (dieneconjugates--DC and malonic dialdehyde--MD); 2) antioxidant enzymes (superoxide dismutase--SOD and catalase). The present findings indicate that the gingival tissue of rats reacts to the changes in the duration of photoperiod by peroxidation and activity of antioxidant enzymes. The antioxidant-prooxidant index under dark conditions (high function of the pineal gland) was lower than under condition of permanent light ("physiological" pinealectomy). Different durations of photoperiod change the intensity of free radical oxidation and the activity of antioxidant enzymes at the systemic level (blood serum) and much more at the organ level (tissues of gingiva). Our data suggest that the gingival tissues possess rather powerful protective antioxidant system, which depends on the functional state of the pineal gland.

Responses of human gingival and periodontal fibroblasts to a low-zinc environment.

Morphology, motility, proliferation rate and markers of oxidative stress in primary human gingival fibroblasts (GF) and periodontal ligamental fibroblasts (PDL-F) grown in zinc-deficient cultivation medium (ZDM), were studied over a 5-week culture period. A low-zinc environment effectively reduced the total, as well as the free, intracellular zinc content in both cell types, over the course of the experiment. Decreased intracellular zinc content resulted in altered cellular morphology, reduced motility, and rearrangement of actin and tubulin in the cytoskeleton. In addition, fibroblasts with low zinc content exhibited decreased proliferation, accompanied by changes in cell cycle distribution, expression of specific biochemical markers, increased oxidative stress and the activation of caspase-3. Supplementation of ZDM with exogenous zinc prevented the loss of intracellular zinc, while also restoring the morphology, cell proliferation and mitogenic signalling of the cultured cells. Moreover, such supplemented cells were protected against oxidative stress and cell death. Of the two primary cell cultures examined, GF were more sensitive to decreased intracellular zinc content, when compared to PDL-F. The results obtained suggest that the human primary cell cultures can be useful for the longer-term evaluation of the effects of nutritional factors originating from the environment.

On a path to unfolding the biological mechanisms of orthodontic tooth movement.

Orthodontic forces deform the extracellular matrix and activate cells of the paradental tissues, facilitating tooth movement. Discoveries in mechanobiology have illuminated sequential cellular and molecular events, such as signal generation and transduction, cytoskeletal re-organization, gene expression, differentiation, proliferation, synthesis and secretion of specific products, and apoptosis. Orthodontists work in a unique biological environment, wherein applied forces engender remodeling of both mineralized and non-mineralized paradental tissues, including the associated blood vessels and neural elements. This review aims at identifying events that affect the sequence, timing, and significance of factors that determine the nature of the biological response of each paradental tissue to orthodontic force. The results of this literature review emphasize the fact that mechanoresponses and inflammation are both essential for achieving tooth movement clinically. If both are working in concert, orthodontists might be able to accelerate or decelerate tooth movement by adding adjuvant methods, whether physical, chemical, or surgical.

The influence of surface topography of ceramic abutments on the attachment and proliferation of human oral fibroblasts.

As different implant abutments are introduced to obtain a sufficient soft tissue barrier, the aim of this study was to determine the effect of three different surface modifications of densely sintered high-purity aluminium oxide on morphology, attachment and proliferation of human gingival fibroblasts. Fibroblasts were cultured on pressed aluminium oxide, milled, and then sintered to full density (1), on pressed, densely sintered (2), and on pressed, densely sintered and then polished surfaces (3). The different surfaces were analyzed using a confocal laser scanner, an atomic force microscope and a scanning electron microscope. The cell profile areas were measured using a semiautomatic interactive image analyzer and the figures were expressed as percent of attachment. The polished specimens had the smoothest surfaces and the roughest were the milled surfaces in terms of height deviation. No difference was found in the spacing between the peaks on the polished surfaces compared to the milled surfaces. Fibroblasts on the milled ceramic appeared to follow the direction of the fine irregularities on the surface. The analyses showed the polished surfaces had significantly higher percentages of initial cell attachment than the other surfaces (P < 0.05). After 3 days of cell culture, significantly more cells were attached to the milled and sintered surfaces than to the polished one, possibly indicating higher proliferation capacity on those types of surfaces.

Mineralization of basement membrane mediates dentogingival adhesion in mammalian and nonmammalian vertebrates.

In order to elucidate the mechanism of adhesion between the gingiva and the tooth, detailed comparative ultrastructural studies of the dentogingival border were done in the monkey and shark. The tissues were prepared with or without demineralization for the ultrastructural observations. At the border, the internal basement membrane, which is firmly bound to the junctional epithelium through hemidesmosomes, was specialized differently in these species. In the monkey, the lamina densa was closely associated at its enamel side with an additional layer which had characteristics of the lamina densa and was referred to as the supplementary lamina densa. In the shark, the lamina densa showed a unique, hemidesmosome-related specialization in the form of the intermittent occurrence of bulges along its surface facing the epithelium. In nondemineralized tissues a part of the basement membrane, that is, the supplementary lamina densa (monkey) and the main lamina densa but not bulges (shark), was preferentially mineralized. The mineral deposit was continuous with that in the enamel and enameloid/dentine, thus constituting an advancing edge of mineralization. The network arrangement of the mineral crystals in the monkey basement membrane resembled the pattern of the cord network of the basement membrane, suggesting the presence of a delicate mutual basement membrane-mineral interaction. Thus, the organic phase and the mineral phase are allowed to make contact at this mineralized area of the basement membrane and firmly bind to one another. Therefore, strong gingiva-tooth adhesion is established by partial mineralization of the internal basement membrane, in a way similar to that found in the previously reported association of maturation stage ameloblasts with the enamel.

Topical application of 5% eutectic mixture of lignocaine and prilocaine (EMLA) before removal of arch bars.

The analgesic effect of topical application of a 5% eutectic mixture of lignocaine and prilocaine (EMLA) was studied in 45 patients undergoing removal of oral arch bars used for the treatment of mandibular fractures. Employing a double-blind technique, either 4 g of the eutectic mixture (EMLA group, n = 15) or 4 g of a similar emulsion containing no local anaesthetic (placebo group, n = 15) was applied to the gingivae using a toothbrush and a standardised technique. In the control group (n = 15), infiltration anaesthesia with lignocaine was used only if requested by the patient during the removal of the arch bars. The patients in the EMLA group had significantly better analgesia (P less than 0.005) of the gingivae just before removal of the arch bars than patients in the placebo group, but by the end of the procedure the difference in analgesia was not significant. The number of patients who found the procedure pain-free was significantly higher in the EMLA group (7/14) than in the placebo group (2/15) (P less than 0.005). The plasma concentrations of both lignocaine and prilocaine were well below the toxic levels. Topical application of EMLA can be recommended for short procedures as an alternative to infiltration.

Single-crystal sapphire endosseous dental implant loaded with functional stress--clinical and histological evaluation of peri-implant tissues.

Peri-implant tissues of the single-crystal sapphire implant connected with neighbouring teeth by a metal bridge-work were examined clinically, radiographically, and histologically in ten monkeys. Professional tooth cleaning was performed during the study. At 3-12 months after insertion, most of the implants were firmly connected to the surrounding tissues and peri-implant gingiva was regarded as healthy, based on various periodontal parameter scores. Destructive changes of the peri-implant bone were not found radiographically. Histologically, peri-implant gingiva was revealed to show similar structure to that of the gingiva around natural teeth. Direct bone-implant interface was observed at 3 months after insertion, while a thin loose fibrous connective tissue layer was present between bone and implant at 6 and 12 months. Such different interrelationship between bone and implant might be attributable to the difference in distribution of functional stress.

Endosseous dental implantology from the periodontist's viewpoint.

Oral implantology has been a controversial dental therapeutic procedure for many years. Periodontics, as a specialty, did not really get involved until the word, osseointegration, was placed in the implant nomenclature by Dr. Per-Ingvar Branemark and the attendant success rate for osseointegrated prostheses was presented in a fully documented format. In March 1985, the definition of the scope of periodontics was provisionally changed by the Executive Council of the American Academy of Periodontology to include the discipline of oral implantology. Requirements for Advanced Specialty Education Programs in Periodontics with an effective date of January 1, 1986, include a statement as to the desirability of including implantology in some form in the postdoctoral curriculum. Many implant materials and designs are presently being used in endosseous dental implantology; studies are in progress to evaluate the short- and long-term response of hard and soft tissues to these materials. Continuing, nonbiased research is needed to fully understand and use this promising modality.

Zinc concentration in normal and healing gingival tissues in beagle dogs.

Previous studies have shown that the administration of zinc (Zn) may enhance the healing of gingival and other wounds. This study was undertaken to determine if Zn concentration ([Zn]) is increased in healing gingival tissues and if oral supplementation of Zn would result in a local increase in [Zn] within these tissues. On Day 0, biopsies were obtained from the maxillary left buccal gingiva of each of 10 beagle dogs. Gingival biopsies were taken from the healing original biopsy sites on Day 14. On Day 15, oral supplementation of Zn gluconate (250 mg/day, equivalent to 32.5 mg of elemental Zn) was begun in seven dogs. Three dogs remained as unsupplemented controls. Two weeks later (Day 28), normal gingival biopsies were obtained from the right side of the maxilla and on Day 42 final biopsies were taken from the same healing sites. In addition, serum samples were obtained on Days 0, 14, 28 and 42. All samples were analyzed for Zn content using atomic absorption spectrophotometry. The [Zn] of healing tissues was significantly higher (P less than 0.0005) than normal tissues. This was also true when healing tissues were compared to normal tissues during the Zn supplementation phase (Day 28 vs. Day 42; P less than 0.005). Zn supplementation resulted in significant increases in Zn levels in normal (Day 0 vs. Day 28; P less than 0.05) and healing tissues (Day 14 vs. Day 42; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of vitamin E on the healing of gingival wounds in rats.

Forty male and female albino rats received a standardized gingival wound (gingivectomy) between the mandibular incisor teeth. One half of the animals received 60 I.U. of d-alpha-tocopheryl acetate daily, administered orally by pipette. An additional control group of 20 animals was not wounded and half of these animals received 60 I.U. of d-alpha-tocopheryl daily. Four animals in each of the two gingivectomy groups (Groups 1, 2) were sacrificed at periods of 1, 2, 4, 7 and 14 days following gingivectomy. Two animals in each of two control unwounded groups (Groups 3, 4) were sacrificed at similar times. Gingival healing was studied grossly and histologically. The animals receiving the vitamin E supplements healed more rapidly, with almost complete restoration of gingiva by 7 days. Complete healing was seen in both control and experimental groups by 14 days. Vitamin E was shown to accelerate gingival wound healing in experimental animals.