The role of LL-37 in periodontal wound healing

Introduction: As a result of chronic inflammation during periodontal disease the junctional epithelium becomes micro-ulcerated. The inflammatory process is mediated by both bacterial and host cell products. Host defence peptides such as defensins, secretory leucocyte protease inhibitor (SLPI) and the sole human cathelicidin, LL-37, are secreted by both periodontal cells and neutrophils into gingival crevicular fluid (GCF). They have the ability to modulate the immune response in periodontitis and are thought to have a potential role in periodontal wound healing. Objectives: The aims of this study were to determine the role of LL-37 in the production of Interleukin (IL)-8, IL-6, hepatocyte growth factor (HGF) and basic-fibroblast growth factor (bFGF) by gingival fibroblasts. The role of LL-37 in modulating total matrix metalloproteinase (MMP) activity and expression of tissue inhibitors of metalloproteinase (TIMP)-1 and -2 by gingival fibroblasts was also investigated. Methods: Primary gingival fibroblasts were co-cultured with concentrations of LL-37 (1, 5 and 10µg/ml) for 24 hours and their supernatants tested for levels of IL-8 and IL-6, HGF, bFGF, TIMP-1 and TIMP-2 by ELISA. Rates of MMP turnover in the supernatants were tested by fluorogenic assay using fluorescence resonance energy transfer (FRET) peptide substrates. Cytotoxicity was measured by MTT assay. Statistical significance was measured using the independent t-test and p<0.05 was considered significant. Results: LL-37 significantly upregulated levels of IL-8, IL-6, HGF, bFGF and TIMP-1 (p<0.05) in a dose-dependent fashion. LL-37 significantly decreased the total MMP activity (p<0.05). None of the LL-37 concentrations tested were cytotoxic to gingival fibroblasts. Conclusion: These results indicate that LL-37 is involved in periodontal wound healing. LL-37 increased levels of proinflammatory cytokines and increased levels of growth factors involved in re-epithelialisation. LL-37 has the ability to regulate remodelling of the periodontium by controlling MMP overactivity both directly and by stimulating production of inhibitors by gingival fibroblasts.

Nuclear factor I-C links platelet-derived growth factor and transforming growth factor beta1 signaling to skin wound healing progression.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor A (PDGFAlpha) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-beta signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C(-/-) and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-beta1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C(-/-) mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C(-/-) mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-beta in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Role of intracellular signaling pathways activated in fibroblasts in response to lipoproteins

Summary The best described physiological function of low-density lipoproteins (LDL) is to transport cholesterol to target tissues. LDL deliver their cholesterol cargo to cells following their interaction with the LDL receptor. LDL, when their vascular concentrations increase, have also been implicated in pathologies such as atherosclerosis. Among the cell types that are found in blood vessels, endothelial and smooth muscle cells have dominated cellular research on atherosclerotic mechanisms and LDL activation of signaling pathways, while very little is known about adventitial fibroblast activation caused by elevated lipoprotein levels. Since fibroblasts participate in wound repair and since it has recently been recognized that fibroblasts may play pivotal roles in vascular remodeling and repair of injury, we assessed whether lipoproteins affect fibroblast function. We have found that LDL specifically mediate the activation of a class of mitogen-activated protein kinases (MAPKs): the p38 MAPKs. The activation of this pathway in turn modulates cell shape by promoting lamellipodia formation and extensive cell spreading. This is of particular interest because it provides a mechanism by which LDL can promote wound healing or vessel wall remodeling as observed during the development of atherosclerosis. In order to understand the molecular mechanisms by which LDL induce p38 activation we searched for the component in the LDL particle responsible for the induction of this pathway. We found that cholesterol is the major component of lipoprotein particles that mediates their ability to stimulate the p38 MAPK pathway. Furthermore, we investigated the cellular mechanisms underlying the ability of LDL to induce cell shape changes and whether this could participate in wound repair. Our recent data demonstrates that the capacity of LDL to induce fibroblast spreading relies on their ability to stimulate IL-8 secretion, which in turn leads to accelerated wound healing. LDL-induced IL-8 production and subsequent wound closure are impaired upon inhibition of the p38 MAPK pathway indicating that the LDL-induced spreading and accelerated wound sealing rely on the ability of LDL to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Therefore, regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL-cholesterol levels, IL-8 production and extensive remodeling of the vessel wall. Résumé: La fonction physiologique des lipoprotéines à faible densité (LDL) la mieux décrite est celle du transport du cholestérol aux tissus cibles. Les LDL livrent leur cargaison de cholestérol aux cellules après leur interaction avec le récepteur au LDL. Une concentration vasculaire des LDL augmenté est également impliquée dans le développement de l'athérosclérose. Parmi les types de cellule présents dans les vaisseaux sanguins, les cellules endothéliales et les cellules du muscle lisse ont dominé la recherche cellulaire sur les mécanismes athérosclérotiques et sur l'activation par les LDL des voies de signalisation intracellulaire. A l'inverse peu de choses sont connues sur l'activation des fibroblastes de l'adventice par les lipoprotéines. Puisqu'il a été récemment reconnu que les fibroblastes peuvent jouer un rôle central dans la remodélisation vasculaire et la réparation tissulaire, nous avons étudié si les lipoprotéines affectent la fonction des fibroblastes. Nous avons constaté que les LDL activent spécifiquement une classe de protéines kinases: les p38 MAPK (mitogen-activated protein kinases). L'activation de cette voie module à son tour la forme de la cellule en favorisant la formation de lamellipodes et l'agrandissement des cellules. Cela a un intérêt particulier car il fournit un mécanisme par lequel les LDL peuvent promouvoir la cicatrisation ou la remodélisation des parois vasculaires comme observés lors du développement de l'athérosclérose. Pour comprendre les mécanismes moléculaires par lesquels les LDL provoquent l'activation des p38 MAPK, nous avons cherché à identifier les composants dans la particule de LDL responsables de l'induction de cette voie. Nous avons constaté que le cholestérol est l'élément principal des particules de lipoprotéine qui contrôle leur capacité à stimuler la voie des p38 MAPK. En outre, nous avons examiné les mécanismes cellulaires responsables de la capacité des LDL à induire des changements dans la forme des cellules. Nos données récentes démontrent que la capacité des LDL à induire l'agrandissement des cellules, ainsi que leur aptitude à favoriser la cicatrisation, reposant sur leur capacité à stimuler la sécrétiond'IL-8. La production d'IL-8 induite par les LDL est bloquée par l'inhibition de la voie p38 MAPK, ce qui indique que l'étalement des cellules induit par les LDL ainsi que l'accélération de la cicatrisation sont liés à la capacité des LDL à stimuler la sécrétion d'IL8 via l'activation des p38 MAPK. La régulation de la forme et de la migration des fibroblastes par les lipoprotéines peuvent donc participer au développement de l'athérosclérose qui est caractérisée par l'augmentation des niveaux de production de LDL-cholestérol et d'IL-8 ainsi que par une remodélisation augmentée de la paroi du vaisseau.

Apatite-Polymer Composite Particles for Controlled Delivery of BMP-2: In Vitro Release and Cellular Response

Bone morphogenetic protein-2 (BMP-2) has the ability to induce osteoblast differentiation of undifferentiated cells, resulting in the healing of skeletal defects when delivered with a suitable carrier. We have applied a versatile delivery platform comprising a novel composite of two biomaterials with proven track records – apatite and poly(lactic-co-glycolic acid) (PLGA) – to the delivery of BMP-2. Sustained release of this growth factor was tuned with variables that affect polymer degradation and/or apatite dissolution, such as polymer molecular weight, polymer composition, apatite loading, and apatite particle size. The effect of released BMP-2 on C3H10T1/2 murine pluripotent mesenchymal cells was assessed by tracking the expression of osteoblastic makers, alkaline phosphatase (ALP) and osteocalcin. Release media collected over 100 days induced elevated ALP activity in C3H10T1/2 cells. The expression of osteocalcin was also upregulated significantly. These results demonstrated the potential of apatite-PLGA composite particles for releasing protein in bioactive form over extended periods of time.

A novel self-healing supramolecular polymer system

Utilising supramolecular pi-pi stacking interactions to drive miscibility in two-component polymer blends offers a novel approach to producing materials with unique properties. We report in this paper the preparation of a supramolecular polymer network that exploits this principle. A low molecular weight polydiimide which contains multiple pi-electron-poor receptor sites along its backbone forms homogeneous films with a siloxane polymer that features pi-electron-rich pyrenyl end-groups. Compatibility results from a complexation process that involves chain-folding of the polydiimide to create an optimum binding site for the pi-electron-rich chain ends of the polysiloxane. These complementary pi-electron-rich and -poor receptors exhibit rapid and reversible complexation behaviour in solution, and healable characteristics in the solid state in response to temperature. A mechanism is proposed for this thermoreversible healing behaviour that involves disruption of the intermolecular pi-pi stacking cross-links as the temperature of the supramolecular film is increased. The low T-g siloxane component can then flow and as the temperature of the blend is decreased, pi-pi stacking interactions drive formation of a new network and so lead to good damage-recovery characteristics of the two-component blend.

Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1 beta (IL-1 beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alphalbeta (CINC-2 alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process. Copyright (c) 2007 John Wiley & Sons, Ltd.

Delayed local inflammatory response induced by Thalassophryne nattereri venom is related to extracellular matrix degradation

Symptoms evoked by Thalassophryne nattereri fish envenomation include local oedema, severe pain and intense necrosis with strikingly inefficient healing, continuing for several weeks or months. Investigations carried out in our laboratory showed that, in the venom-induced acute inflammation, thrombosis in venules and constrictions in arterioles were highly visible, in contrast to a notable lack of inflammatory cell. Nevertheless, the reason that the venom toxins favour delayed local inflammatory response is poorly defined. In this study, we analysed the movement of leucocytes after T. nattereri venom injection in the intraplantar region of Swiss mice, the production of pro-inflammatory mediators and the venom potential to elicit matrix metalloproteinase production and extracellular matrix degradation. Total absence of mononuclear and neutrophil influx was observed until 14 days, but the venom stimulates pro-inflammatory mediator secretion. Matrix metalloproteinases (MMP)-2 and MMP-9 were detected in greater quantities, accompanied by tissue degradation of collagenous fibre. An influx of mononuclear cells was noted very late and at this time the levels of IL-6, IL-1 beta and MMP-2 remained high. Additionally, the action of venom on the cytoskeletal organization was assessed in vitro. Swift F-actin disruption and subsequent loss of focal adhesion was noted. Collectively these findings show that the altered specific interaction cell-matrix during the inflammatory process creates an inadequate environment for infiltration of inflammatory cells.

Tissue response around silicon nitride implants in rabbits

The chemical and dimensional stability associated with suitable fracture toughness and propitious tribological characteristics make silicon nitride-based ceramics potential candidates for biomedical applications, mainly as orthopedic implants. Considering this combination of properties, silicon nitride components were investigated in relation to their biocompatibility. For this study, two cylindrical implants were installed in each tibia of five rabbits and were kept in the animals for 8 weeks. During the healing time, tissue tracers were administrated in the animals so as to evaluate the bone growth around the implants. Eight weeks after the surgery, the animals were euthanized and histological analyses were performed. No adverse reactions were observed close to the implant. The osteogenesis process occurred during the entire period defined by the tracers. However, this process occurred more intensely 4 weeks after the surgery. In addition, the histological analyses showed that bone growth occurred preferentially in the cortical areas. Different kinds of tissue were identified on the implant surface, characterized by lamellar bone tissue containing osteocytes and osteons, by a noncalcified matrix containing osteoblasts, or by the presence of collagen III, which may change to collagen I or remain as a fibrous tissue. The results demonstrated that silicon nitride obtained according to the procedure proposed in this research is a biocompatible material. (c) 2007 Wiley Periodicals, Inc.

Effects of Purified Saccharomyces cerevisiae (1→3)-β-Glucan on Venous Ulcer Healing

Water-insoluble glucan was isolated from the baker’s yeast Saccharomyces cerevisiae. The yeast cells were treated with alkali and the residue then with acid. Chemical and NMR (1D and 2D) analyses showed that a linear (1→3)-β-glucan was purified that was not contaminated with other carbohydrates, proteins or phenolic compounds. The effects of the glucan on wound healing were assessed in human venous ulcers by histopathological analysis after 30 days of topical treatment. (1→3)-β-glucan enhanced ulcer healing and increased epithelial hyperplasia, as well as increased inflammatory cells, angiogenesis and fibroblast proliferation. In one patient who had an ulcer that would not heal for over 15 years, glucan treatment caused a 67.8% decrease in the area of the ulcer. This is the first study to investigate the effects of (1→3)-β-glucan on venous ulcer healing in humans; our findings suggest that this glucan is a potential natural biological response modifier in wound healing

Effects of Purified Saccharomyces cerevisiae (1→3)-β-Glucan on Venous Ulcer Healing

Water-insoluble glucan was isolated from the baker’s yeast Saccharomyces cerevisiae. The yeast cells were treated with alkali and the residue then with acid. Chemical and NMR (1D and 2D) analyses showed that a linear (1→3)-β-glucan was purified that was not contaminated with other carbohydrates, proteins or phenolic compounds. The effects of the glucan on wound healing were assessed in human venous ulcers by histopathological analysis after 30 days of topical treatment. (1→3)-β-glucan enhanced ulcer healing and increased epithelial hyperplasia, as well as increased inflammatory cells, angiogenesis and fibroblast proliferation. In one patient who had an ulcer that would not heal for over 15 years, glucan treatment caused a 67.8% decrease in the area of the ulcer. This is the first study to investigate the effects of (1→3)-β-glucan on venous ulcer healing in humans; our findings suggest that this glucan is a potential natural biological response modifier in wound healing

Periodontal tissue response to coverage of root cavities restored with resin materials: A histomorphometric study in dogs

Background: the purpose of this study was to histomorphometrically evaluate the response of periodontal tissues covering Class V resin restorations in dogs.Methods: After raising a mucoperiosteal flap, bony defects measuring 5 x 5 mm were created on the buccal aspect of the canines of five dogs followed by cavity preparations on the root surface measuring 3 x 3 x 1 mm. Before repositioning the flap to cover the bone defect, the cavities were restored with composite resin (CR) or resin-modified glass ionomer cement (RMGIC) or were left unrestored as control (C). The dogs were euthanized 90 days after surgery. Specimens comprising the tooth and periodontal tissues were removed, processed routinely, cut into longitudinal serial sections in the bucco-lingual direction, and stained with hematoxylin and eosin (H&E) or Masson's trichrome. The most central sections were selected for histomorphometric analysis.Results: Histomorphometric analysis revealed apical migration of epithelial tissue onto the restorative materials (RMGIC and CR). The C group presented significantly longer connective tissue attachment (P < 0.05) than the RMGIC and CR groups and significantly higher bone regeneration (P < 0.05) compared to the RMGIC group. Histologically, the cervical third (CT) of all groups had the most marked chronic inflammatory infiltrate.Conclusions: Within the limits of this study, it can be concluded that the restorative materials used exhibit biocompatibility; however, both materials interfered with the development of new bone and the connective tissue attachment process.

Evaluation of tissue response to periodontal dressings: Histological study in tooth sockets of rats

Although the use of periodontal dressings is currently limited, there are some indications for their use. Selection of any material that will have direct contact with live tissues, such as periodontal dressings, should be careful in order to allow surgical wound healing. The aim of this study was to evaluate the intensity of inflammatory response and bone formation in tooth sockets of rats after implantation of three periodontal dressings. After removal of the right maxillary incisors of 84 male rats, each tooth socket received implantation of a polyethylene tube, 63 of which were filled with non-eugenol periodontal dressing and the remaining 21 tubes remained empty (control group). Histological evaluation assessed the intensity of inflammatory response and presence and location of bone tissue formation at postoperative periods of 7, 14 and 28 days. Statistical analysis was performed by the Kruskal-Wallis test at 5% significance level. Regarding the inflammatory infiltrate, at 28 days, there was statistically significant difference between one of periodontal dressings and control group (p < 0.05). Analysis of postoperative periods, showed that the control group presented statistically significant reduction in the inflammatory infiltrate comparing the 14- and 28-day periods (p < 0.05). Regarding bone tissue formation, there was difference in control group between the 7- and 28-day periods (p < 0.05). Within the experimental conditions, it may be concluded that no differences were found in the inflammatory response among the groups at 7 and 14 days and that Voco pac (TM) dressing induced a more intensive inflammatory reaction at 28 days.

Analysis of the healing process in delayed tooth replantation after root canal filling with calcium hydroxide, Sealapex and Endofill: a microscopic study in rats

The major concern in the therapeutics of tooth replantation refers to the occurrence of root resorption and different approaches have been proposed to prevent or treat these complications. The purpose of this study was to evaluate tissue response to delayed replantation of anterior rat teeth treated endodontically using calcium hydroxide, Sealapex, and Endofill without the placement of gutta-percha cones. Thirty rats had their right upper incisor extracted and maintained in dry storage for 60 min. After removal of the dental papilla, enamel organ, pulp tissue, and periodontal ligament remnants, the teeth were immersed in 2% sodium fluoride phosphate acidulated, pH 5.5, for 10 min. The root canals were dried with absorbent paper points and the teeth were assigned to three groups (n = 10) according to the filling material. Group I - calcium hydroxide and propyleneglycol paste, Group II - Sealapex, and Group III - Endofill. The sockets were irrigated with saline and the teeth were replanted. Replacement resorption, inflammatory resorption and ankylosis were observed in all groups. Although the occurrence of inflammatory resorption was less frequent in Group I, there were no statistically significant differences among the groups. It may be concluded that compared to the paste, filling the root canals with Sealapex and Endofill sealers without the placement of gutta-percha cones did not provide better results.

Evaluation of Rat Alveolar Bone Response to Angelus MTA or Experimental Light-cured Mineral Trioxide Aggregate Using Fluorochromes

Introduction: The aim of this study was to evaluate the rat alveolar bone response after the implantation of experimental light-cured mineral trioxide aggregate (MTA) or Angelus MTA (Angelus, Londrina, Parana, Brazil) by histological and fluorescence analysis. Methods: Thirty Wistar Albino rats were divided into three groups. In the control group, empty polyethylene tubes were inserted into the rat alveolar sockets immediately after extraction. In the other groups, the tubes were filled with light-cured MTA or Angelus MTA. Five animals from each group were injected with calcein on day 7, alizarin on day 14, and oxytetracycline on day 21. on day 30, these animals were killed, and the right hemimaxillas were removed and histologically processed. Half of the maxillas were processed and stained with hematoxylin and eosin. The remaining maxillas were processed for fluorescence analysis and stained with Stevenel blue and alizarin red. New bone was histomorphometrically evaluated using a Merz grid. Results: The light-cured MTA presented a similar response when compared with Angelus MTA; it was characterized by a mild inflammatory response and complete bone healing. In the light-cured MTA group, the fluorescence areas were more evident at 21 days, showing an increase in bone formation. However, dystrophic mineralization was observed only with Angelus MTA. Conclusions: It was concluded that both materials present a similar inflammatory response and bone healing, but dystrophic mineralization was observed only with Angelus MTA. (J Endod 2011;37:250-254)

Effect of Smoking on Early Bone Healing Around Oxidized Surfaces: A Prospective, Controlled Study in Human Jaws

Background: This prospective and controlled histologic study evaluates the impact of smoking on bone-to-implant contact, the bone density in the threaded area, and the bone density outside the threaded area around microimplants with anodized surface retrieved from human jaws.Methods: A total of 24 subjects (mean age 51.32 +/- 7.5 years) were divided in two groups: smokers (n = 13 subjects) and non-smokers (n = 11 subjects). Each subject received one microimplant with oxidized surface during conventional mandible or maxilla implant surgery. After 8 weeks, the microimplants and the surrounding tissue were removed and prepared for histomorphometric analysis.Results: Three microimplants placed in smokers showed no osseointegration. The newly formed bone showed early stages of maturation, mainly in the non-smokers. Marginal bone loss, gap, and fibrous tissue were present around implants retrieved from smokers. Histometric evaluation indicated that the mean bone-to-implant contact ranged between 25.97% +/- 9.02% and 40.01% +/- 12.98% for smokers and non-smokers, respectively (P <0.001). Smokers presented 28.17% +/- 10.32% of bone density in the threaded area, whereas non-smokers showed 46.34% +/- 19.12%. The mean of bone density outside the threaded area ranged between 18.76% and 25.11% for smokers and non-smokers, respectively (P>0.05).Conclusion: The present data obtained in human subjects confirm that smoking has a detrimental effect on early bone tissue response around oxidized implant surfaces. J Periodontol 2010;81:575-583.