Histamine augments collagen content via H1 receptor stimulation in cultures of myofibroblasts taken from wound granulation tissue.

The inflammatory reaction influences the deposition of collagen within wound granulation tissue. The aim of the present study is to determine whether histamine acting directly on myofibroblasts derived from wound granulation tissue may influence collagen deposition. It also identifies the histamine receptor involved in this process. The experiments were carried out on cells isolated from the granulation tissue of a wound model (a polypropylene net inserted subcutaneously to rats) or intact rat skin. Collagen content was measured following the addition of different concentrations of histamine and treatment with histamine receptor antagonists (ketotifen - H1 inhibitor, ranitidine - H2 inhibitor) and a histamine receptor H1 agonist (2-pyridylethylamine dihydrochloride).The cells were identified as myofibroblasts: alpha-smooth muscle actin, vimentin, and desmin positive in all experimental conditions. Histamine increased the collagen level within both cell cultures, i.e., those isolated from granulation tissue or intact skin. It did not, however, influence the expression of either the collagen type I or III genes within the cultured myofibroblasts. Histamine activity was reduced by ketotifen (the H1 receptor inhibitor) and increased by the H1 receptor agonist, as demonstrated by changes in the levels of collagen in the myofibroblast culture. Histamine increased collagen content within the cultures, acting directly on myofibroblasts via H1 receptor stimulation.

The stiffness-controlled release of interleukin-6 by cardiac fibroblasts is dependent on integrin α2ß1.

Cardiac fibroblasts are able to sense the rigidity of their environment. The present study examines whether the stiffness of the substrate in cardiac fibroblast culture can influence the release of interleukin-6 (IL-6), interleukin-11 (IL-11) and soluble receptor of IL-6 (sIL-6R). It also examines the roles of integrin α2ß1 activation and intracellular signalling in these processes. Cardiac fibroblasts were cultured on polyacrylamide gels and grafted to collagen, with an elasticity of E = 2.23 ± 0.8 kPa (soft gel) and E = 8.28 ± 1.06 kPa (stiff gel, measured by Atomic Force Microscope). Flow cytometry and ELISA demonstrated that the fibroblasts cultured on the soft gel demonstrated higher expression of the α2 integrin subunit and increased α2ß1 integrin count and released higher levels of IL-6 and sIL-6R than those on the stiff gel. Substrate elasticity did not modify fibroblast IL-11 content. The silencing of the α2 integrin subunit decreased the release of IL-6. Similar effects were induced by TC-I 15 (an α2ß1 integrin inhibitor). The IL-6 levels in the serum and heart were markedly lower in α2 integrin-deficient mice B6.Cg-Itga2tm1.1Tkun/tm1.1Tkun than wild type. Inhibition of Src kinase by AZM 475271 modifies the IL-6 level. sIL-6R secretion is not dependent on α2ß1 integrin. Conclusion: The elastic properties of the substrate influence the release of IL-6 by cardiac fibroblasts, and this effect is dependent on α2ß1 integrin and kinase Src activation.

Central European ethnomedical and officinal uses of peat, with special emphasis on the Tolpa peat preparation (TPP): An historical review.

ETHNOPHARMACOLOGICAL RELEVANCE: Medical or hygienic uses of peat mosses dates back to the 18th century. Peat was used externally (as poultices) in the early 19th century. The peat preparation invented by Stanislaw Tolpa (Tolpa peat preparation, TPP) was patented in Poland in 1991; its concept had emerged in the 1980s. It raised high therapeutic expectations still being researched in the early 1990s. Profound expectations for peat, a natural product well known in Central European (and Polish) spas (for medicated baths and poultices), earned Tolpa's preparation great renown before any actual benefits (internal actions) were scientifically confirmed. AIM OF THE REVIEW: We study the origins of medical interest in peat in Polish science against the background of the historical ethnopharmacy of peat and Sphagnum moss in Central Europe. It is aimed at shedding a new light on the history of TPP, its connections with local ethnopharmacological traditions and inspirations for local medical studies on peat products and peat-derived drugs of the 1980s and early 1900s. MATERIALS AND METHODS: The literature on peat baths was found and reviewed including the information and data about the studies of TPP from published though unknown sources as well as from Polish patents, unpublished typescripts, press interviews and reports. RESULTS: Tolpa's research team missed the historical data about external and topical actions of Sphagnum peat or its preparations which were published in the 19th- and early 20th-century. This is strange because folk medicine based on peat emanated eastwards from ethnic Austria along the Vistula river and the Carpathians. Tolpa ignored balneotherapeutic (external) applications as well as the action of sphagnan from Sphagnum herb, and rejected this kind of peat as scientifically not promising, based on a single biological test on plants. The concept of an active principle in peat or its preparations evolved, and speculation concerning its nature was not followed by adequate basic research. The active principle was not found. Results concerning plant meristem growth were too readily applied in animal production and finally human medicine. The natural ingredient in TPP production was never defined botanically. Anti-cancer properties ascribed to the TPP on the basis of bio-stimulation tests stirred powerful social emotions. CONCLUSIONS: Topical peat cure originated in Austria in about 1820. It evolved as a whole branch of Central European balneotherapy which had been completely scientifically described by the 1950s. At that time an undefined peat extract was once successfully used in ear infections in paediatrics. Stanislaw Tolpa's research project to find any internal application of peat ignored the achievements of ethnobiology, balneotherapy, surgery and otorhinolaryngology known at that time. His strenuous and insistent efforts, carried out in isolation, crucially failed pre-clinical and clinical tests in any branch of his therapy. Three commercial drugs were allowed for 3 years before substantial clinical proofs of peat efficacy were achieved. Social impact was high and resulted in the birth of the Polish legend of Tolpa's marvellous drug.

Collagenous scaffolds supplemented with hyaluronic acid and chondroitin sulfate used for wound fibroblast and embryonic nerve cell culture.

BACKGROUND: Tissue engineering is a strategy aimed at improving the regeneration of injured tissues. OBJECTIVES: The aim of the present study was to determine whether a tri-copolymer composed of crosslinked collagen, chondroitin sulfate and hyaluronic acid (Col + CS + HA) provides a better environment for fibroblast and embryonic nerve cell culture than a collagenous scaffold (Col). MATERIAL AND METHODS: The porosity of each of the matrices was characterized with a scanning electron microscope. Fibroblasts were isolated from rat wound granulation tissue (polypropylene net implanted subcutaneously). Embryonic nerve cells were obtained from the brains of rat embryos. The cells were applied to scaffolds and then stained with bisbenzimide to calculate cell entrapment within the material. The metabolic activity of the cells cultured within the scaffolds was tested using the 3-(4,5-dimethythiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: The Col scaffolds had a homogenously porous structure with a pore diameter of 50 µm for 70% of pores. The pore diameter in the tri-copolymer (Col + HA + CS) ranged from 24 to 160 µm (95% of total pore volume). Four times more cells (fibroblasts and embryonic nerve cells) were trapped within the superficial part of the collagenous scaffold than that of the tri-copolymer. On the third day of culture the metabolic activity of the fibroblasts within the 2 tested scaffolds was significantly higher than in the control conditions (cell culture on a laminin-coated surface). Also, the embryonic nerve cells demonstrated increased metabolic activity in Col + CS + HA scaffolds than the Col scaffolds. CONCLUSIONS: Both fibroblasts and embryonic nerve cells could be seeded within the 2 tested scaffolds. Both the scaffolds provide good conditions for fibroblast culture. However, the Col + CS + HA tri-copolymer is preferable for embryonic nerve cell engineering.

Tangled history of the European uses of Sphagnum moss and sphagnol.

ETHNOPHARMACOLOGICAL RELEVANCE: Sphagnum mosses and peat could have been utilized as wound dressings for centuries, however reliable data on this subject are ambiguous; sometimes even no distinction between peat moss (Sphagnum spp.) and peat is made or these terms become confused. The first scientific account on surgical use of peat comes from 1882: a peat digger who successfully, by himself and in the way unknown to the then medicine, cured an open fracture of his forearm with peat. The peat, and very soon the peat moss itself (which is the major constituent of peat) drew attention of the 19th-century surgeons. AIMS OF THE WORK: We search for reliable information on: (1) inspirations for Sphagnum usage for medical purposes and its beginnings in the 19th century, (2) substances or products named sphagnol and their connections with (1); (3) on the origin of this name, (4) and on the occurrence of this name in medical sources. MATERIALS AND METHODS: We have identified and studied published sources on the uses of peat-based and Sphagnum-based preparations and products of any processing level (including herbal stock, distillate, isolated pure or impure active principle, or a mixture of such) in surgery, pharmacy or cosmetics. A special attention was paid to the name sphagnol, which appeared many a time, in more than one context since 1899. Source publications were critically analysed from the taxonomical, pharmacognostical and ethnopharmacological points of view. Gathered data were cross-checked with the modern knowledge of the biologically active principles of Sphagnum and the prospects of their medical use. RESULTS: The application of peat in surgery started 1882. The use of peat moss as dressings was developed in the 1880's. It returned to surgical practice during WW1. The name sphagnol has two meanings: (1) A chemical substance isolated from the cell walls of Sphagnum mosses in 1899. A post-1950 research showed it to be a mixture of phenols dominated by sphagnum acid. (2) A product of dry distillation of peat contains solid and liquid fractions and was applied in skin diseases due to antiseptic properties. It was added to ointments and medicated soaps manufactured up to the late 1960's. Today none of these two sphagnols is in use. CONCLUSIONS: Surgical application of peat had an ethnopharmacological origin: a case of wound treatment with peat as a remedy rather than a dressing (1880, published 1882) shortly shifted the surgeons' attention to peat moss as an absorptive dressing. The 1880's tests of antiseptic properties of peat and peat moss failed, the sterilization methods overrode the physiological effects of Sphagnum dressings. Sphagnan, a polysaccharide from Sphagnum cell walls, discovered 1983, inhibits microbial growth, tans the collagen and removes ammonia from microbial environments. Portions of raw peat could be sterile. The isolation of sphagnol (1899) from Sphagnum cell walls was not inspired by old surgery. Main component of sphagnol, the sphagnic acid, was used clinically during WW2, but was proved a weak antimicrobial agent. A homonymous name sphagnol appeared independently for a product of dry distillation of peat, introduced commercially probably about 1899, too, which gave rise to confusions: a) the commercial, "distilled" sphagnol was not the crystalline principle of Sphagnum cell walls. 2) the "distilled" sphagnol was hardly defined technologically or pharmacologically, never standardized in terms of the substrate (a variety of peat rather than Sphagnum herb) and the production process. This sphagnol, resembling pitch or tar, was an additive to medicated soaps and ointments for skin treatment and care. It must have been a low-scale product although advertised worldwide. Neither sphagnum acid nor sphagnan are used medicinally today.

Effects of electrospun scaffolds of di-O-butyrylchitin and poly-(ε-caprolactone) on wound healing.

BACKGROUND: We sought to determine the usefulness of electrospun dibutyrylchitin (DBC) or poly-(ε-caprolactone [PCL]), in wound treatment. We investigated the mechanisms of action of these polymers on wound healing. METHODS: We synthesized DBC, a newly identified ester derivative of chitin, using a patented method comprising the substitution of butyryl groups at positions C-3 and C-6 in chitin molecules. We confirmed the double substitution by the butyric groups using infrared spectrometry. The fibrous scaffolds were obtained using the electrospinning method. A polypropylene net was implanted subcutaneously in the rat and served as a wound model. RESULTS: Both DBC and PCL increased granulation tissue weight in the wound. In contrast to PCL, DBC did not abolish glycosaminoglycan changes in wounds. The tested samples did not impair total collagen synthesis or induce excessive fibrosis. In both PCL- and DBC-treated wounds, we observed a lower level of soluble collagen (compared with controls). The results show better hydration of the wounds in both the DBC and PCL groups. No induction of large edema formation by the tested materials was observed. These polymers induced almost identical macrophage-mediated reactions to foreign-body implantation. The implants increased the blood vessel number in a wound. CONCLUSION: Both PCL and DBC could be used as scaffolds or dressings for wound treatment. The materials were safe and well tolerated by animals. As DBC did not disturb glycosaminoglycan accumulation in wounds and absorbed twice as much liquid as PCL, it can be considered superior.

CONTEXTE: Nous avons cherché à déterminer l'utilité du dibutyryl-chitine (DBC) ou du poly-(ε-caprolactone [PCL]) électrofilés dans le traitement des plaies. Nous avons étudié les mécanismes d'action de ces polymères sur la cicatrisation des plaies. MÉTHODES: Nous avons synthétisé le DBC, un dérivé ester récemment identifié de la chitine, à l'aide d'une méthode brevetée incluant la substitution des groupes butyryl aux positions C-3 et C-6 des molécules de chitine. Nous avons confirmé la substitution double par les groupes butyriques à l'aide de la spectrométrie infrarouge. Les échafaudages fibreux ont été obtenus grâce à la méthode de filage électrostatique. Un filet en polypropylène a été implanté par voie sous-cutanée dans le rat et a servi de modèle de plaie. RÉSULTATS: Le DBC et le PCL ont tous deux augmenté le poids du tissu de granulation dans la plaie. Contrairement au PCL, le DBC n'a pas supprimé les changements des glycosaminoglycanes des plaies. Les échantillons examinés n'ont pas perturbé la synthèse totale de collagène ni entraîné une fibrose excessive. Nous avons observé un niveau inférieur de collagène soluble (par rapport aux témoins) tant dans les plaies traitées par PCL que par DBC. Les résultats montrent une amélioration de l'hydratation des plaies tant pour les groupes DBC que PCL. Les matériaux à l'étude n'induisaient pas d'œdème étendu. Ces polymères ont induit des réactions macrophagiques presque identiques à l'implantation d'un corps étranger. Les implants ont accru le nombre de vaisseaux sanguins de la plaie. CONCLUSION: Tant le PCL que le DBC pourraient être utilisés comme échafaudages ou pansements pour le traitement des plaies. Les matériaux étaient sécuritaires et ont été bien tolérés par les animaux. Comme le DBC n'a pas perturbé l'accumulation des glycosaminoglycanes des plaies et a absorbé 2 fois plus de liquide que le PCL, il peut être considéré comme étant supérieur.

Melatonin-induced augmentation of collagen deposition in cultures of fibroblasts and myofibroblasts is blocked by luzindole--a melatonin membrane receptors inhibitor.

BACKGROUND: Melatonin has been proven to have a regulatory influence on collagen accumulation in different types of wound. It was found to inhibit collagen accumulation in the superficial wound model but increase it in the myocardial infarction scar. The aim of the study is to determine the mechanism of melatonin action in the two wound types in rats. METHODS: Cells were isolated from both the superficial wound (subcutaneously inserted polypropylene net) and myocardial infarction scar (induced by ligation of the left coronary artery) and were identified by electron microscopy. RESULTS: Long-shaped cells forming whirl-like structures in culture (mainly identified as fibroblasts) were isolated from the superficial wound model, while myofibroblasts growing in a formless manner were acquired from the infarcted heart scar. Melatonin (10(-7) M) increased collagen accumulation in both fibroblast and myofibroblast cultures. Luzindole (10(-6) M), the blocker of both MT1 and MT2 melatonin membrane receptors, inhibited the effect of melatonin on the two types of cells. CONCLUSION: Regardless of various healing potentials demonstrated by the tested cells (different cell composition, growth and organization), their response to melatonin was similar. Moreover, in the two investigated cultures, augmentation of the collagen content by melatonin was reversed by luzindole, which indicates the possibility of melatonin membrane receptor involvement in that process. The present results suggest that the increased melatonin-stimulated deposition of collagen observed in the infarcted heart of rats could be dependent on activation of the melatonin membrane receptors on scar myofibroblasts.

The opposite effect of morning or afternoon application of melatonin on collagen accumulation in the sponge-induced granuloma.

OBJECTIVES: Collagen accumulation in the sponge-induced granuloma is under inhibitory influence of melatonin. This effect was observed when the pineal hormone was injected late in the afternoon. DESIGN: The present study was conducted to compare the afternoon and morning melatonin application and to answer the question whether melatonin effect on the collagen accumulation in wounds is dependent on tissue circadian sensitivity to the pineal hormone. METHODS: In order to induce granulation tissue development, the Ivalon sponge was implanted subcutaneously in the Wistar rats. Then the animals were divided into 5 groups: intact controls, rats injected with vehicle in the morning or in the late afternoon, as well as animals receiving melatonin (30 &mgr;g/100g body wt) in the morning or late afternoon. After 4 weeks, the sponges were removed for the analysis of both soluble and total collagen content. Collagen was analyzed as hydroxyproline content in the tissue. Insoluble collagen was calculated. RESULTS: The afternoon application of melatonin was seen to decrease the total and insoluble collagen content in the sponge-induced granuloma (p<0.05). However, morning administration of the pineal hormone increased collagen capacity in the granulation tissue (p<0.01). CONCLUSION: Therefore, contradictory effect of morning or afternoon melatonin application on the collagen content in the granulation tissue has been shown. This phenomenon is supposed to be due to various circadian sensitivity of tissues to melatonin and may suggest that the mechanism of melatonin regulatory action on collagen accumulation in wounds is complex.