influence of dibutyrylchitin on histamine release … · minimize the scaring effect [6]. beschitin...

Progress on chemistry and application of chitin and its ... Volume XIII, 2008 95

1. Introduction1.1. Dibutyrylchitin as chitin derivative. short backgroundPoly(N-acetyl-glucosamine). commonly known as chitin drew scientists’ attention for the first time almost 200 years ago. It was discovered in 1811, and named as we know it today in 1984. In the first half of the 20th century three Nobel Prize winners in chemistry were carrying out experiments with its use. In 1903 Fischer synthesised glucosamine, in 1929 Karrer decomposed chitin with chitynaz and finally Haworth, who in 1939 determined the absolute configuration of glucosamine [1]. In 1970 Prudded [2] observed that glucosamine, being the main compound of sharks cartilage accelerates wound healing process. The same effect was observed for a glucosamine derivative, N-acetyl-D-glucosamine (GlcNAc) [3]. Nowadays poly(N-acetyl-D-glucosamine) is commonly known for its beneficial influence on wound healing process for both humans [4] and animals [5, 6]. Application of chitin and its derivatives to the wounds allows to influence the healing processes at molecular [7], cellular [8] and systemic levels [9]. In 1972 the Hokkaido Prefecture chose the supervision of prof. Tokura from the University of Hokkaido, of a research project on the utilization of chitin, which principal sources are shells of crabs, shrimps and other shellfish. Chemical structure of chitin resembles cellulose, and similarly requires a specific solvent, since in the commonly used ones (eg. ethanol, DMSO) it is not soluble. This significant drawback, whenever one considers chitin materials manufacturing, increases the complexity of technological process and as a direct consequence the overall production cost. For this reason chitin had not been considered to be an attractive compound until the development of active materials. Currently ready made products are available on the market, especially in Japan. They include mostly textile products such as fibers, chirurgical sutures, and nonowovens as well as chitin dressing materials, commercially known as Bestchitin (Unitika, Japan) or Chitipack (Eisai, Japan). These materials not only accelerate the healing process but also minimize the scaring effect [6]. Beschitin is the product manufactured by highly purified

INFLUENCE OF DIBUTYRYLCHITIN ON HISTAMINE RELEASE FROM MAST CELLS

Anna Błasińska, Tomasz Kun*

Technical University of Lodz, Department of Fibre Physics and Textile Metrology, Zeromskiego 116, 90-543 Lodz, Poland, e-mail: [email protected]

*Medical University of Lodz, Department of General and Experimental Pathology,Narutowicza 60, 90-136 Łódź, Poland, e-mail:[email protected]

Progress on chemistry and application of chitin and its ... Volume XIII, 200896

A. Błasińska, T. Kun

chitin. In April 1988, as a result of cooperation between Unitika and a branch of a French company Roussel Medica, chitin ‘artificial’ skin was introduced. This material, stimulating the growth of new skin, was priced at about 200 USD per 120 cm2. Currently Unitika is making efforts to locate Beschitin in the medical devices for animal use.

Up to now, many various research results concerning the applicability chitin, chitosan and its derivatives as materials for use in a wide range of applications (found healing, artificial tissue, antibacterial agents are just some examples) [10, 11]. Chitin and chitosan in the form of biocompatible materials for medicinal purposes were worked out using classical and non-classical techniques. The use of conventional techniques only suggest the availability of such materials. Whatever route was chosen they failed in commercialization.

For many years scientists from various research facilities all around the world have been working on the problem of chemical modification of chitin. The reaction of de-N-acetylation of chitin in the presence of hot alkali environment leads to development of a group of copolymers known collectively as chitosan. These group contain from 65% to 100% of D-glucosamine residues and can be used in materials manufacturing, which biological activity is similar to that of chitin.

Dibutyrylchitin (DBC) is very interesting ester derivative of chitin from manufacturing point of view as well as its biological activity. In the year 1996, L. Szosland from the Technical University of Łódź created a method which lead to obtaining the DBC, in heterogeneous conditions [12]. Large butyryl groups are being introduced to the chitin macromolecule next to C-3 and C-6 carbon atoms. In this case the degree of substitution becomes close to 2 and the product of estrification is dibutyrylchitin (DBC). It is believed that the introduction of large and voluminal groups results in extending the distance between polymer chains and leads to a dramatic improvement of solubility, making it possible to use common organic solvents. If the degree of substitution of ester groups is below 2 then the result of the estrification reaction is a family of copolymers also known as butyrylchitin (BC). The butyrylchitin having the maximal degree of substitution of 1.8, was synthesized and described by a Japanese researcher K. Kaifu [13]. These works have inspired the research on the DBC.

Numerous scientific projects have shed some light on many interesting features of DBC [14, 15] that make this compound worth considering for various kinds of applications. Among them are: very good solubility in common organic solvents, thin transparent films forming ability [14, 16], fibre forming via dry and wet spinning[14, 17], textile materials formation directly from polymer solution via state of the art techniques such as electrospinning. In the last case, the cross-sectional diameter of a fibre is expressed not in micro- but in nanometers [15]. DBC can also form microspheres encapsulating a proper biocyd, which will be then released in time in a controlled manner [14, 18]. All materials obtained from dibutyrylchitin can undergo an alkaline hydrolysis, however depending on the conditions this process may result in detaching all of butyryl groups, in this way we will obtain regenerated chitin (RC), or only a part of them in which case we will obtain a family of copolymers, butyrylchitin (BC). The hydrolysis process does not cause any damage to the structure of the textile material [19].


Influence of dibutyrylchitin on histamine release from mast cells

Initial biological tests of dibutyrylchitin and regenerated chitin made materials have shown that they are biodegradable and fulfill all biological requirements for medical products described in the European standards PN EN ISO 10993. These works [20 - 23] formed the basis for a series of pilot clinical trials, which have confirmed the fact that DBC accelerates the wound healing process of thermally or mechanically induced wounds [24]. The goal of the next series of experiments [25] was to determine the mechanisms of DBC action on a living organism which was previously observed in clinical conditions. The study documents the beneficial influence of dibutyrylchitin on the repair, which could be explained by the modification of the extracellular matrix (total and soluble collagen, glycosaminoglycans) and cells number. DBC action cannot be limited to the wound because the influence of that material on the whole body homeostasis (thermoregulation and body weight) was shown. These phenomena were compared with other dressing materials made of butyrylchitin, regenerated chitin, and chitosan. Researchers attempted the clear determination of indications and contraindications to its applicability and what may be the potential dangers associated with DBC use.

1.2. Mast cellsMast cells (mastocytes) can be found in almost all organs and tissues throughout human organism, but the distribution of those cells is irregular. Mast cells appear to play a pivotal role in both physiology and pathology, especially in regulation of inflammatory reaction [27, 28].

Mast cells are usually round-shaped or slightly elongated cells which have 10 - 20 mcm in diameter. The typical organelles were found in cytoplasm of mastocytes, but unique characteristic of those cells is presence of multiple cytoplasmic granules which exhibit exceptional histochemical features in comparison to other cells [29].

The general population of mast cells exhibits morphological and functional heterogeneity. The differences between mastocytes derived from various tissues were first described by Enerback [32]. Mast cells isolated from rat peritoneal fluid are considered to belong to group of connective tissue mast cells (CTMC), also described as “typical” mastocytes. The similar cells can be found in skin and subcutaneous connective tissue. Differences between

Figure 1. Mast cells with granules containing histamine: A) drawn in the literature [26], B) isolated during experiment, from rat peritoneal fluid.



various populations of mast cells include size, histamine content, and different reactivity to histamine liberators and degranulation inhibitors. Also functional and morphological differences between rat an human must cells should be taken under consideration regarding conclusions from any animal-based laboratory model.

1.3. Mast cells’ mediatorsAbundance of mast cells functions in physiological and pathological processes is closely related to diversity of mediators released from those cells. Mediators produced and stored by mast cells can be categorized into three main groups [32]:n preformed mediators – stored in secretory granules, ex. Histaminen mediators synthesized “de novo” from plasmatic membrane phospholipids after mast

cell activationn cytokines.

One of the most important factor released from mast cells after activation is histamine – amine formed by decarboxylation of amino-acid histidine. Human mast cells store in granules only histamine, but for example rodent mastocytes contains also other amines (ex. serotonin).

Histamine mediate wide range of biological functions by interactions with specific receptors located on target cells membranes. The role of histamine in inflammation and wound healing is described in chapter 1.5.

1.4. Mechanisms of mast cells activation and degranulationResponse of mast cell to direct stimulus can be proceeded as a degranulation or augmentation of cell quantity. Degranulation lies at the bottom of early-type hypersensitivity reaction, while augmentation of cells number is rather characteristic for late-type hypersensitivity reactions.

Cross-linkage of immunoglobulins IgE attached to cell membrane via its high affinity receptor (FcERI) by specific multivalent allergen is a key mechanism of must cell activation. Such a “immunological” activation lies behind the process called: anaphylactic reaction. Mast cell activation may be also initiated by factors which do not involve IgE or FcERI. This process is described as “anaphilactoid” reaction. Non-immunologic stimuli which may lead to mast cell degranulation include multiple endogenous end exogenous substances (ex. Toxins, certain drugs) [33]. Probably one of the most widely known, nonspecific histamine liberator is compound 48/80.

1.5. Influence of histamine on wound healingThe development and maturation of connective tissue (seen during process of wound healing) are consequences of complex transformations of connective tissue cells, fibers and intercellular substance [34]. Histamine – the main mediator of mast cells seems to affect mainly two processes crucial for normal wound healing: collagenogenesis and angiogenesis.



The most significant observation coming from studies on collagenogenesis is that during reparation and regeneration histamine content in healing tissue rises. The concentration of histamine is ten fold greater in wound associated connective tissue than in normal, resting connective tissue. Increased histamine concentration in healing tissue is connected with increase in collagen synthesis. This fact leads to the conclusion that histamine play an important role in regulation of collagen synthesis and wound healing.

Another important observation is that function of histamine depends on its concentration: slight increase in concentration of histamine stimulate synthesis of GAG and collagen, while high concentration of histamine may inhibit collagenogenesis and polymerization of collagen fibers. Both effect are probably mediated by H2 histamine receptors [35, 36]. The conclusion is that histamine exert bidirectional influence on fibroblast metabolism and wound healing.

Ambivalent action of histamine can be seen also in process of angiogenesis, which has a fundamental importance for wound healing [37]. In slight concentrations histamine induces formation of capillaries. Moreover, histamine increases permeability of endothelium leading to augmentation of tissue supply with required metabolites. However in high concentration histamine may damage capillary endothelium and thus may impair tissue perfusion.

Observations described previously lead to conclusion that mast cells, which are the main source of histamine, can control connective tissue metabolism and may regulate reparation and wound healing. Excessive activation of mast cells, connected with excessive histamine release to surrounding tissues, may induce inflammatory reaction and thus may reduce the rate of wound healing. While limited (not maximal?) activation of mastocytes in healing tissues may directly and indirectly stimulate development of cellular and fibrous elements of connective tissue leading to facilitation of wound healing.

Influence of chitin and its derivatives on wound healing is not profoundly studied. The main aim of our research was to test the effect of chitin polymers on mast cells activation, which can than modulate process of reparation (as previously described).

To estimate the influence DBC on mastocytes we used laboratory model based on mast cells purified from rat peritoneal fluid. The results for DBC were compared with histamine release induced by chitin and chitosan.

2. Materials and methods2.1.ObjectsKrill chitin with average viscosity molecular weight value Mv = 454.6 kDa and degree of acetylation 0.97 was purchased from the Sea Fisheries Institute, Gdynia, Poland. Chitin was purified from proteins and calcium carbonate

Dibutyrylchitin (DBC) was syntheses from krill chitin in heterogeneous conditions according to the method described in the patent [1]. Its molecular weight, expressed by the value of intrinsic viscosity measured in N,N-dimethylacetamide (DMAc) at 25 °C. Intrinsic viscosity values were assessed as [η]DBC = 1.75 dl/g.



Chitosan. Fibers were purchased from Dayang Chemical Co.Ltd, and finely cut up. The molecular weight of chitosan polymer was comparable to the molecular weight of the DBC polymer. The physico-mechanical characteristics of fibres were as follows: tenacity 16.22 cN/tex, elongation 4.89% and linear mass 0.158 tex.

2.2. AnimalsMale Wistar rats weighting between 300 - 400 g were used as a source of mast cells. Before laboratory procedure animals were kept in local animal quarters under standardized conditions (temperature 20 °C, 12 hours day-night cycle).

2.3. BufersPhysiological buffer composition used in our experiment was: 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 0.4 mM NaH2PO4, 10 mM HEPES, 1 mg/ml of bovine serum albumin, 10 mM glucose. The buffer pH was adjusted to 6.9 using 0.01M solution of NaOH. After pH adjustment glucose and albumins were added.

2.4. Isolation of mast cells:Laboratory animals were anesthetized with carbon dioxide and killed by decapitation. 10 ml of cold (4 °C) buffer was injected into rat peritoneal cavity. Abdominal integuments were than gently massaged for about 90 seconds. After incision of abdominal skin and muscles peritoneal fluid was collected from peritoneal cavity using Pasteur pipette. Cellular content of peritoneal cavity was then purified by centrifugation on continous density gradient of Percoll (600 g; 20 minutes). After centrifugation the thin layer of purified mast cells was observed at the bottom of test tube. Mast cell layer was washed twice with cold buffer and centrifugated (200 g, 7 minutes). The number of mastocytes in cellular suspension was counted in Burker camera after histochemical staining with toluidine blue.

2.5. Cell incubationSuspension of purified mast cells was diluted with buffer to obtain on average 5000 of mast cells in every 180 mcl of suspension and then distributed to test tubes (180 mcl to each tube). Than mastocytes samples were preincubated in water bath for 5 minutes in temperature 37 °C.

After initial preincubation 20 mcl of tested substances suspensions were added to each tube. We used three different concentrations of each tested substance (chitin, chitosan and DBC): 5, 50 and 500 mcg/ml. Addition of pure buffer was used as negative control, while addition of compound 48/80 in concentration 1 mcg/ml served as positive control. Two periods of incubations were chosen: 30 and 120 minutes. Incubation was stopped by immersing the tubes in cold ice bath and by adding of 1.80 ml of cold (4 °C) buffer to each tube. After centrifugation (200 g, 7 min.) the supernatants were collected into other test tubes containing 50 mcl of 3N HCl, 50 mcl of 3 N HCl and 2.0 ml o buffer where then added to each test tube containing sediment. Samples where then frozen in -20 °C to obtain total lysis of mast cells remained in sediment.



2.6. Histamine release assayHistamine concentration was assayed spectrofluorometrically both in the sediment and supernatant (released histamine) after condensation with orthophthalic aldehyde (OPT). Results were expressed as a percentage of histamine released (supernatant) with respect to the total histamine content (supernatant plus sediment).

2.7. Statistical analysisResults were analyzed using Wilcoxon non-parametric test. A probability level of 0.05 or smaller was used for statistical significance. The “Statistica” licenced software was used to calculate all tests.

3. ResultsBefore the procedure of purification cell suspension collected from rat peritoneal cavity contained 579+/-220 mast cells per 1 mcl. Mastocytes constituted 7.4% of all cells in collected suspension. Cellular suspension containing 93.8% of mastocytes was obtained after the purification in density gradient of Percoll. Contaminating cells (epithelial cells, fibroblasts, leucocytes and erytrocytes) were almost totally eliminated from peritoneal lavage and could not significantly modify the reaction of mast cells to tested chitin polymers in vitro.

After purification 1 mcl of suspension contained 446+/-174 mast cells. Thus mastocyte recovery was 77% - it means that only 23% of primary mast cell number was lost during the procedure of purification.

Spontaneous histamine secretion was between 2.94 - 8.92% (mean 5.55%) after 30 minutes of incubation in 37 °C (Table 1).

Table 1. Percentage of released histamine from chitin, chitosan, DBC, negative and positive reference samples after 30 min.

ControlChitin Chitosan DBC 48/80

com-pound

5 mcg/ml

50 mcg/ml

500 mcg/ml

5 mcg/ml

50 mcg/ml

500 mcg/ml

5 mcg/ml

50 mcg/ml

500 mcg/ml

2.94 3.25 4.59 4.15 6.43 10.96 11.97 4.34 3.06 3.97 53.68 4.01 2.85 9.21 4.33 8.54 10.13 14.86 3.63 3.23 3.63 55.93 3.04 5.93 4.39 8.16 8.43 8.99 8.35 2.94 3.91 3.32 54.58 4.77 6.64 4.21 8.57 7.68 8.36 8.57 7.18 5.78 6.47 56.97 5.22 5.46 5.78 3.84 2.62 1.18 2.33 4.2 4.52 5.77 36.87 7.29 7.15 7.68 7.58 1.24 0.88 1.94 10.53 9.26 10.59 40.69 8.92 6.4 6.48 7.36 1.48 1.57 2.1 8.91 9.35 10.04 40.53 1.29 1.17 2.15 3.03 7.99 8.72 10.42 6.64 1.15 9.43 62.79 1.60 0.77 0.03 6.49 6.31 8.87 6.89 1.92 1.90 3.99 64.66

8.15 8.27 9.23 10.8 8.75 8.47 7.44 9.08 9.79 16.33 61.038.91 9.14 8.92 7.81 7.29 9.28 7.25 8.65 12.75 18.92 53.598.24 6.82 7.88 7.59 6.22 12.78 6.12 8.29 6.06 9.01 51.947.78 9.88 7.59 13.48 7.51 9.09 6.68 6.68 6.29 8.81 57.32

Average 5.55 5.67 6.01 7.17 6.19 7.64 7.30 6.38 5.93 8.48 53.12SD 2.81 2.88 2.84 2.92 2.66 3.86 3.81 2.72 3.48 4.84 8.71



Those results confirm that mastocytes collected and purified from rat peritoneal fluid are morphologically and functionally intact and were not stimulated to degranulate during the procedure of purification.

Moreover mast cells used for future tests were properly reactive to stimulation by compound 48/80. Mean histamine release after activation with 48/80 in concentration 1 mcg/ml was 53.12% (after 30 minutes of incubation), which additionally confirms functional stability of purified cells and ability to secrete mediators after stimulation (Table 1).

Our results revealed that both chitin and chitosan regardless of concentration and time of incubation were not able to stimulate mast cells to secrete histamine. Admittedly, secretion of histamine from mastocytes incubated with chitin in concentration 500 mcg/dl for 120 minutes was higher than control, but difference was not statistically significant (Figure 2 and Figure 3).

However DBC demonstrated ability to activate mast cells in concentration dependent manner.

This action is especially visible for samples incubated with DBC for 120 minutes. It was proved that DBC released 11.41% and 14.75% of histamine from mast cells in concentrations 50 and 500 mcg/ml respectively, after 120 minutes of incubations. In samples incubated for 120 minutes increase in spontaneous histamine release (8.64%) was also observed, in comparison to 30 minutes group – Table 1 and Table 2. This phenomenon can be explained by growing number of cells directly damaged by non-physiological in vitro conditions. But statistical analysis shows significance between control (spontaneous histamine release) and samples of cells stimulated by DBC in concentration 50 and 500 mcg/ml (Figure 3).

Increase of histamine release in samples stimulated with chitin and chitosan was not significantly different from control.

Similarly statistically significant increase in histamine release from mast cells was observed in samples stimulated by DBC in concentration 500 mcg/ml for 30 minutes. For this time of

Table 2. Percentage of released histamine from chitin, chitosan, DBC, negative and positive reference samples after 120 min.

ControlChitin Chitosan DBC 48/80

compo-und

5 mcg/ml

50 mcg/ml

500 mcg/ml

5 mcg/ml

50 mcg/ml

500 mcg/ml

5 mcg/ml

50 mcg/ml

500 mcg/ml

12.75 10.4 16.24 13.62 4.51 9.51 1.98 11.48 12.08 9.71 41.35 7.83 9.85 11.01 13.26 4.23 2.28 6.23 14.61 10.45 11.3 43.62 10.33 14.73 11.57 13.44 3.17 5.88 3.63 16.02 13.02 11.67 50.76 5.1 2.46 4.05 4.26 5.65 5.44 8.46 3.51 5.81 10.2 67.94 3.27 4.21 3.76 6.16 12.28 12.61 12.24 0.48 5.43 8.53 70.69 7.54 10.3 17.32 6.22 4.69 7.62 12.03 10.41 11.27 20.02 65.62 10.27 6.59 4.84 14.31 16.27 9.41 14.138 10.14 11.45 24.094 59.47 7.97 13.78 13.82 12.48 13.61 11.08 15.35 19.61 14.96 20.69 62.71 12.72 18.25 19.78 15.15 10.32 12.84 15.61 12.02 18.26 16.58 66.84

Average 8.64 10.06 11.38 10.99 8.30 8.52 9.96 10.92 11.41 14.75 58.78SD 3.22 5.09 6.02 4.18 4.86 3.53 5.10 5.93 4.04 5.70 10.91



incubation we did not find significant difference in histamine release in samples incubated with DBC in concentration 5 and 50 mcg/ml (Figure 2).

Concomitantly we observed that histamine excretion from mast cells stimulated with typical histamine liberator: compound 48/80 were considerably higher than histamine release after

Figure 2. Percentage of released histamine for different concentrations of chitin, chitosan and DBC, for 30 minutes incubation time.

Figure 3. Percentage of released histamine for different concentrations of chitin, chitosan and DBC, for 120 minutes incubation time.



stimulation with DBC, even in higher concentration. This fact lead to conclusion that histamine release from mast cells after stimulation with DBC cannot be described as a prototypic “anaphylactoid” reaction (as it is observed for compound 48/80). Elongation of incubation time for DBC to 120 minutes led to almost twofold increase in histamine excretion from stimulated mast cells. In comparison, for compound 48/80 we observed only slight increase in histamine release (from 53.12% to 58.78% - Table 1 and Table 2). Our results suggest that time of incubation with DBC suspensions has a great importance for rate of histamine release from mast cells.

4. ConclusionsBasing on statistical analysis of results we can initially conclude:n DBC induces histamine release from mast cells and can modulate function of mast cells

in vitro.n Histamine excretion from mast cells activated with DBC is considerably lower than for

model of anaphylactoid reaction.n Modulation of mast cell activity by DBC in healing tissue may modify inflammatory

reaction and favourably influence on process of wound healing.n Results described above require future studies involving in vivo model.

5. Acknowledgmentsn This study was partly financed by the European Union within the scope of the project CHITOMED

No QLK5-2002-01330 and Ministry of Science and Higher Education, project No 4 T08E 001 24.n Participation in the XIII Workshop of Polish Chitin Society (Wroclaw, September 17-19nd , 2007),

was supported by Polish Textiles & Health Scientific Network

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influence of dibutyrylchitin on histamine release … · minimize the scaring effect [6]. beschitin...

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