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The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro
Biomaterials 22 (2001) 2959}2966
The e!ect of chitin and chitosan on the proliferation of human skin "broblasts and keratinocytes in vitro Graeme I. Howling , Peter W. Dettmar, Paul A. Goddard, Frank C. Hampson, Michael Dornish, Edward J. Wood * Leeds Skin Research Centre, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK Reckitt & Colman Products, Dansom Lane, Hull HU8 7DS, UK Pronova Biomedical, Gaustadalleen 21, N-0349, Oslo, Norway Received 29 August 2000; accepted 24 January 2001
Abstract The e!ects of chitin [(1P4)-2-acetamido-2-deoxy--D-glucan] and its partially deacetylated derivatives, chitosans, on the proliferation of human dermal "broblasts and keratinocytes were examined in vitro. Chitosans with relatively high degrees of deacetylation strongly stimulated "broblast proliferation while samples with lower levels of deacetylation showed less activity. Fraction, CL313A, a shorter chain length, 89% deacetylated chitosan chloride was further evaluated using cultures of "broblasts derived from a range of human donors. Some "broblast cultures produced a positive mitogenic response to CL313A treatment with proliferation rates being increased by approximately 50% over the control level at an initial concentration of 50 g/ml, whilst others showed no stimulation of proliferation or even a slight inhibition ((10%). The stimulatory e!ect on "broblast proliferation required the presence of serum in the culture medium suggesting that the chitosan may be interacting with growth factors present in the serum and potentiating their e!ect. In contrast to the stimulatory e!ects on "broblasts, fraction CL313A inhibited human keratinocyte mitogenesis with up to 40% inhibition of proliferation being observed at 50 g/ml. In general highly deacetylated chitosans were more active than those with a lower degree of deacetylation. These data demonstrate that highly deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Analysis of their e!ects on cells in culture may be useful as a screen for their potential activity in vivo as wound healing agents, although in the case of "broblasts it is important to select appropriate strains of cells for use in the screen. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Chitosan; Chitin; Fibroblast; Keratinocyte; Skin; Wound healing
1. Introduction Wound repair is a complex process involving an integrated response by many di!erent cell types controlled by a variety of growth factors. During the initial in#ammatory phase "broblasts start to enter the wound where they synthesise and later remodel new extracellular matrix material, of which collagen is the main component [1]. The dermal response is only one aspect of cutaneous wound repair however, the outermost and vital barrier layer, the epidermis which is composed of several layers of keratinocytes, must also be restored. In injured skin,
* Corresponding author. Tel.: #44-(0)113-233-3100; fax: #44(0)113-233-3167. E-mail address: [email protected] (E.J. Wood).
basal layer keratinocytes migrate from the wound edge and from injured epidermal appendages (hair follicles and sweat glands) into the defect, moving over the newly formed dermal sca!olding. They proliferate, stratify and di!erentiate to produce a neoepidermis to cover the wound and restore the skin's barrier function [1]. Various in vitro cell culture systems have been used to investigate the cellular processes, such as "broblast and keratinocyte proliferation and migration in response to the growth factors that are present in a wound [2}4]. Such models simplify and standardise the system compared with the in vivo situation and also enable materials to be assessed for their potential, at least in a preliminary way, to promote wound repair by stimulating cell proliferation, and for their biocompatibility. Chitin [(1P4)-2-acetamido-2-deoxy--D-glucan], a linear, unbranched structural polysaccharide from the
0142-9612/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 0 4 2 - 4
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Table 1 Chitin/chitosan samples No.
Sample
Degree of polymerisation (DP )
Molecular mass (MM )
% deacetylation
1 2 3 4 5 6
Chitin 50 Chitin 50 fraction A Seacure CL311 Seacure CL311 fraction A Seacure CL313 Seacure CL313 fraction A
900 170 890 55 1220 60
197,200 37,300 194,000 12,000 263,800 13,000
37 37 58 58 89 89
Seacure is a trade name for chitosans produced by Pronova Biomedical. Number of N-acetylglucosamine or glucosamine units. DP and M M determined by intrinsic viscosity (MM ) and light scattering methods (M M ). E X % deacetylation determined by NMR spectroscopy.
shells of crustaceans and fungal mycelia, and its deacetylated derivative, chitosan, have previously been shown to possess both material and bioactive properties that may be bene"cial to enhancing wound repair (reviewed in [5]). In experimental animal models, chitin/chitosan were shown to in#uence all stages of wound repair [6,7]. In the in#ammatory phase chitosan has unique haemostatic properties that are independent of the normal clotting cascades [8]. In vivo these polymers can also interact with and modulate the migration behaviour of neutrophils and macrophages modifying subsequent repair processes such as "broplasia and reepithelialisation [6,9]. In vitro, the e!ects of chitin and chitosan on "broblasts have been studied but as in in vivo studies, both stimulatory [10] and inhibitory [11] actions have been reported. These contradictory data appear at least in part to result from the di!erent chemical compositions and physical forms of the biopolymer samples investigated, making it di$cult to be clear about the relationship between chitosan structure and its e!ect on "broblast behaviour. The present study examines the e!ect of chitin and chitosan samples with various deacetylation levels and polymer lengths, on the proliferation of human dermal "broblasts in vitro. Chitosan has also been shown to interact with epidermal cells; it appeared to stimulate reepithelialisation in dog and rat experimental wounds [7,12] but direct e!ects on keratinocyte proliferation have not been reported. Therefore, we also investigated the e!ect of chitin and chitosans on keratinocyte proliferation in vitro using both an immortalised human keratinocyte cell line (HaCaT), and primary human keratinocytes cultured with and without an irradiated "broblast feeder layer.
2. Materials and methods 2.1. Polymer samples The chitin/chitosan samples used (Table 1) were dissolved in 17 mM acetic acid in a range of 10; stock
concentrations, "lter sterilised though a 0.2 m polycarbonate "lter (Millipore, Bedford MA, USA) and diluted for use with "broblast growth medium (Dulbecco's modi"ed Eagle's medium (DMEM), 5% (v/v) newborn calf serum, 1% (v/v) penicillin/streptomycin [10,000 U/ml penicillin, 10,000 g/ml streptomycin; Gibco, Paisley, Scotland]) to produce nominal 0, 2.5, 5, 50, 500 g/ml concentrations. Chitosan chloride samples CL313 and CL313A slowly precipitated in pH 7.4 medium over a period of 3 days. To determine the concentration of chitosan remaining in solution, a colorimetric assay was developed from a method by described by Muzzarelli [13]. Cibacron brilliant red 3B-A (also known as Reactive red 4, Sigma, Poole, UK) was prepared by dissolving 150 mg dye in 100 ml deionised H O; 5 ml of this solution was diluted to 100 ml with 0.1 M glycine}HCl bu!er, pH 3.2, to give a "nal concentration of 75 mg/l. Standards or test samples (300 l) were added to 3 ml dye solution and the absorbance read at 575 nm after 10 min. The test samples of nominal concentration 5, 50 and 500 g/ml chitosan CL313A made up in DMEM were left for di!erent times at 373C in a 5% CO /95% air atmosphere. At each time point the medium was centrifuged on a bench top centrifuge for 5 min, at 13,000 rpm, to remove solid debris and precipitated chitosan, after which the supernatant was examined under microscopy to check that all visible insoluble aggregates had been pelleted. The supernatant was then removed and used in the assay. 2.2. Cell culture Human dermal "broblasts were obtained from specimens of skin from healthy donors undergoing routine minor elective surgery. Cultures were established by a single-cell suspension technique following enzymatic digestion of the skin samples. Brie#y, skin specimens were incubated with dispase (1.5 mg/ml grade II, Boehringer Mannheim, Germany) in DMEM to facilitate separation of the epidermis from the dermis. Fibroblasts
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were liberated from the dermis by subsequent treatment with Clostridium histolyticum collagenase A (1 mg/ml, Boehringer Mannheim, Germany) and cultured in "broblast medium at 373C in a 5% CO /95% air atmo sphere with medium changes twice weekly. Upon reaching con#uence the "broblast cultures were split 1 : 3 to give approximately 2.5;10 cells per 25 cm culture #ask. Cells were utilised for experimental work between passages 2 and 10. HaCaT cells, an immortalised but non-tumorigenic keratinocyte cell-line which retain their di!erentiation potential [14], were cultured in `HaCaT mediuma (DMEM, 10% (v/v) foetal calf serum, 1% (v/v) penicillin/streptomycin (10,000 U/ml penicillin, 10,000 g/ml streptomycin, Gibco, Paisley, Scotland)). Human epidermal keratinocytes were isolated and cultured on a lethally irradiated 3T3 "broblast feeder layer according to Rheinwald and Green [15] as modi"ed by Navasaria et al. [16]. Brie#y, keratinocytes were liberated from epidermis by tryptic digestion (2.5 mg/ml trypsin, 200 g/ml EDTA, in PBS) and the cells collected and seeded at 1;10 cells per 25 cm #ask which contained an irradiated 3T3 "broblast feeder layer (5;10 cells/#ask). Keratinocyte cultures were maintained in specialised keratinocyte medium (110 ml Ham's F12 (Gibco, Paisley, Scotland), 330 ml DMEM (Gibco), 50 ml foetal calf serum (Gibco, Paisley, Scotland), with the addition of 5 ml penicillin/streptomycin (10,000 U/ml penicillin, 10,000 g/ml streptomycin, Gibco, Paisley, Scotland), 2.5 ml epidermal growth factor (EGF) (2 g/ml stock, Sigma, Poole, UK), 500 l hydrocortisone (400 g/ml), 1 ml transferrin (5 mg/ml)/3,3,5 triiodo Lthyronine (136 g/ml) combined stock, 500 l insulin (Humalin-S威 100 U/ml, Lilly, Indianapolis, USA), 5 l cholera toxin (1 mg/ml stock, Sigma, Poole, UK)) at 373C in a 5% CO /95% air atmosphere with medium changes every 3 days.
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Keratinocyte proliferation was assayed using a similar method except that for primary cells a lethally irradiated 3T3 feeder layer was present in each well (4; 10 cells/well) (these cells had previously been shown not to interfere with the assay). 2.4. Statistical analysis Results were analysed using 1-way ANOVA or a paired student's t-test where indicated.
3. Results 3.1. Ewect of chitin/chitosan samples on xbroblast proliferation in vitro The e!ect of chitin and chitosans on "broblast proliferation was investigated over a range of concentrations (initially 5}500 g/ml). The biopolymer samples (Table 1) were initially screened to identify samples that strongly stimulated "broblast proliferation in vitro using H thymidine incorporation. Chitosan CL313 and its shorter chain length fraction, CL313A, had the strongest stimulatory e!ects on cell proliferation (Fig. 1) at all the concentrations tested. Four other highly deacetylated chitosans all showed a stimulatory e!ect on "broblast proliferation (data not shown). In general chitosan samples with the higher levels of deacetylation stimulated "broblast proliferation more than chitin and chitosan samples deacetylated to a lesser degree. The lower molecular weight form, CL313A, displayed higher proliferative activity and maximal stimulation was observed in "broblasts cultured in medium containing 5 g/ml CL313A. Other chitosan/chitin had lesser e!ects on proliferation, and chitin-50A had antiproliferative e!ects on "broblasts at higher doses, with maximal inhibition
2.3. Methyl-[3H]-thymidine cell proliferation assay Fibroblasts were seeded into 24 well culture plates at 20,000 cells/well and incubated overnight to allow attachment. The medium was then replaced with the appropriate test medium containing the polymer samples and the plates incubated at 373C in a 5% CO /95% air atmosphere for the speci"ed time. The test medium was then discarded and the cells pulse-labelled for 3 h with 1 Ci/well H thymidine (5 Ci/mmol; ICN, Basingstoke, UK) in "broblast medium. After labelling, cells were washed twice with ice-cold PBS and acid-soluble radioactivity removed by 2;10 min 10% (v/v) trichloroacetic acid incubations. The cells were then washed in 70% ethanol and solubilised in 0.1 M NaOH, 2% (w/v) Na CO , 1% (w/v) SDS. The radioactivity incorporated into acid-insoluble material was determined by liquid scintillation counting.
Fig. 1. Initial screening of chitin/chitosan samples for e!ect on "broblast proliferation in vitro. Human dermal "broblasts (C520) were treated with various polymer samples for 3 days with one change of medium on day 2. The H thymidine cell proliferation assay was then performed. Data (n"3$SEM) are presented as percentage of the controls (no polymer present) (*P(0.05, **P(0.01, ANOVA).
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Table 2 Chitosan CL313A concentration in "broblast medium. Fibroblast medium (pH 7.4) containing CL313A at initial concentrations 50 and 500 g/ml were incubated for 1, 2, and 3 days at 373C in a 5% CO /95% air atmosphere to duplicate conditions of media in "broblast cultures. Chitosan concentration was determined at 575 nm
Table 3 Donor dependent variation in the e!ect of chitosan CL313A on "broblast populations isolated from di!erent donors. Two distinct populations of human dermal "broblasts, one responsive to stimulation of proliferation by chitosan CL313A and the other non-responsive ID Number
Donor age
Location
Passage number
CL313A responsive
A534 A655 A665 A693 A695 A701 C488 C495 C498 C520 A377 A407 A408 A418 C400 C434 C491 C516
47(f) 31 28(f) 38(f) 38(f) 15 13 3 6 6 32(f) 48(f) 27(f) 36(f) 3 4 7 2
Abdomen Foreskin Breast Abdomen Abdomen Foreskin Foreskin Foreskin Foreskin Foreskin Abdomen Breast Abdomen Breast Foreskin Foreskin Foreskin Foreskin
7 4 4, 3 4, 2 8 6, 4 2, 8 5 6 7 4, 5, 4, 5
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No No No
CL313A concentration (g/ml), mean$SEM Initial concentration
Day 1
Day 2
Day 3
50 500
27.6$1.1 80.4$1.7
24.3$0.5 59.3$0.5
22.5$0.63 57.8$0.31
occurring at the initial concentration 500 g/ml, where proliferation was 60% of the controls. In culture medium, when incubated at 373C in 5% CO /95% air atmosphere, the higher concentrations of chitosans CL313 and CL313A gradually formed insoluble aggregates, which could be removed by centrifugation. Highly deacetylated chitosans have low solubility at pH 7.4, and other researchers have used chitosan in suspension or slurries for wound repair studies in vivo and in vitro [17,18]. In the present work, precipitation of chitosan was quanti"ed in order to assess the amount remaining in solution. Table 2 shows that the concentration of CL313A initially at 50 g/ml slowly decreased over 3 days to give a steady concentration of 22.5 g/ml with most insoluble aggregates being formed within the "rst 24 h. In the 500 g/ml solution, 24 h after preparing the solution only approximately 80 g/ml was still in solution, the dissolved concentration fell further over the following 24 h to 59.3 g/ml (Table 2). Chitosan sample CL313A which exhibited the strongest stimulation of proliferation was selected for further study. Fibroblasts isolated and cultured from di!erent donors were used to assess the e!ect of CL313A on proliferation in di!erent cell populations (Table 3). Not all "broblast cultures responded and indeed they appeared to fall into two categories: cells were reproducibly either `respondersa or `nonrespondersa to CL313A-induced proliferation (Table 3). There was no obvious correlation between responsiveness and age or anatomical site of the donor skin and this e!ect persisted even after subculturing numerous times. Fig. 2 shows representative results against a background of the average for 10 responsive cultures and 8 nonresponsive cultures: culture A695 was strongly responsive to CL313A-induced proliferation, while A418 was shown to be nonresponsive. The mechanism by which chitin/chitosan interacts with "broblast cells is unknown but chitosan has been reported to bind serum factors, such as growth factors, perhaps protecting them from degradation or perhaps having activating e!ects that would stimulate cell prolif-
6 5
10 7, 8
7 7 7, 8
(f )"female. Indicates samples from same donor but non-adjoining areas.
Fig. 2. Examples of donor dependent variation in "broblast proliferation induced by chitosan. CL313A "broblasts A695 P4 were representative of cultures that were responsive to chitosan CL313A stimulated proliferation, while A418 P7 were representative of nonresponders. Data (n"3$SEM) are presented as percentage of controls. The mean results of all the responders (n"10$SEM) and nonresponders (n"8$SEM) are also shown.
eration more [11,19]. Therefore, it was decided to examine whether the chitosan-stimulated "broblast proliferation was dependent on the presence of serum, which supplies the cells with growth factors, using "broblasts that had previously been de"ned as `responsivea or `nonresponsivea. C520 "broblasts (CL313A responsive) required the presence of newborn calf serum (NBCS) for
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Fig. 4. E!ect of chitosan CL313A and chitin-50A on human keratinocyte HaCaT cell proliferation in vitro. HaCaT cells were treated with CL313A and chitin-50A for 3 days, with one change of medium on day 2. H thymidine incorporation was performed. Data (n"4$SEM) are presented as percentage of control (without polymer) (*P(0.01, ANOVA).
Fig. 3. The dependence of chitosan CL313A-stimulated "broblast proliferation on serum concentration. The e!ect of serum concentration was examined in responders (A: C520) and nonresponders (B: C491) to CL313A stimulation of "broblast proliferation. Cells were treated with $5 g/ml CL313A at various serum concentrations indicated and H thymidine incorporation assay performed after 3 days. Data (n"3$SEM) are presented as mean counts per minute (C5"treatment with CL313A at 5 g/ml, *P(0.05, **P(0.005, students t-test).
the chitosan to be able to stimulate proliferation (Fig. 3A). At 5% serum, chitosan stimulated proliferation by 27% and at 2.5% serum by 56%. C520 "broblasts cultured with lower serum concentrations (1% and 0%) with and without chitosan had similar proliferation rates. Chitosan had no signi"cant e!ect on the proliferation of nonresponsive "broblasts additional to that produced by serum factors (Fig. 3B). 3.2. Ewect of Chitosan CL313A and Chitin-50A on human keratinocyte proliferation in vitro The e!ects of chitosan CL313A and chitin-50A on human keratinocytes were initially investigated using HaCaT cells, which are a keratinocyte cell-line that require no feeder layer or complex media for growth and are generally easier to handle than primary keratinocyte cultures. Fig. 4 shows that at initial concentrations of 5 and 50 g/ml chitosan CL313A inhibited HaCaT
Fig. 5. E!ect of CL313A on primary human keratinocyte proliferation in vitro. Keratinocytes were cultured in keratinocyte medium containing CL313A at 0, 5, 50 g/ml with and without an irradiated 3T3 feeder layer. The H thymidine proliferation assay was performed after 3 days. Data (n"3$SEM) are presented as counts per minute (*P(0.05, **P(0.01, ANOVA).
proliferation by approximately 26% and 20%, respectively, while Chitin-50A, on the other hand, had no e!ect on HaCaT proliferation at either concentration tested. Chitosan CL313A was also tested on three primary human keratinocyte cultures, derived from di!erent donors and similar results were obtained in all the experiments. No donor dependent phenotypic variation was found as is observed for "broblasts. Fig. 5 displays representative data showing the e!ect of CL313A at 5 and 50 g/ml initial concentrations on human primary keratinocytes, with and without a lethally irradiated 3T3 "broblast feeder layer. Keratinocytes proliferate at a much higher rate when cultured on a feeder layer but in both keratinocyte culture systems ($feeder layer) cell proliferation was dose-dependently inhibited by chitosan CL313A. For example, keratinocytes cultured with
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Table 4 E!ect of CL313A on primary human keratinocyte proliferation in vitro. Results from Fig. 5 are displayed as percentage of corresponding controls (0 g/ml) for culture system used ($feeder layer) to allow direct comparison of the data Treatment
No feeder layer % of control (SEM)
With feeder layer % of control (SEM)
0 C5 C50
100 ($11.8) 59 ($5.9) 19 ($0.9)
100 ($7.4) 78 ($3.1) 58 ($3.4)
a feeder layer proliferated at 78% and 58% when treated with 5 and 50 g/ml (initial concentrations) CL313A, respectively compared with the controls. Table 4 shows that the irradiated "broblast feeder layer protected the keratinocytes from the chitosan induced inhibition of proliferation. For example, keratinocytes treated with CL313A at an initial concentration of 50 g/ml had a proliferation rate of 19% of control without a feeder layer but with a feeder layer present the rate was 58% of control.
4. Discussion The initial aim of this work was to evaluate the various chitin and chitosans described in Table 1, for their ability to in#uence the proliferation of human "broblasts and keratinocytes in vitro as an index for their potential as wound healing agents. The screening of the biopolymers for their e!ects on "broblast proliferation showed that chitosan `Seacure CL313a, and its shorter chain length fraction, CL313A, had the greatest mitogenic activity at all concentrations tested. The other chitosan samples showed lesser degrees of mitogenic activity and some samples, such as chitin 50A, had antiproliferative e!ects. The deacetylation level of chitosan seems to be a key factor in the mitogenic activity on "broblasts with the molecular mass of the biopolymer being somewhat less important. This notion was supported by work with other highly deacetylated chitosan samples with di!erent average molecular masses and which also stimulated the proliferation of "broblasts (data not shown). These samples had deacetylation levels of 84% and 86%, and ranged in average molecular mass from 23,800 to 145,000 Da, again suggesting that polymer chain length was not particularly important for biological activity, at least in the range tested. Chitosan CL313A was chosen for further evaluation. Subsequent studies to investigate the e!ects of CL313A on dermal "broblast proliferation revealed that some "broblast cultures showed a mitogenic response to treatment (termed &responders'), while other cultures showed
no stimulation of proliferation (&nonresponders'). In a very few cases there was a slight inhibition of proliferation (see Fig. 2), for example, CL313A inhibited A418 "broblast proliferation rates by approximately 10%. This phenomenon has not been previously reported, possibly due to the fact that much of the work describing the e!ect of chitosan used speci"c cell lines such as L929 mouse "broblasts [17,11], F1000 embryonic skin and muscle "broblasts [10], or "broblasts from animals, such as rats or mice [11]. Fibroblasts isolated from various human donors of di!erent age and sex and from di!erent anatomical sites were investigated but there appeared to be no correlation between responsiveness to CL313A and age, sex or anatomical site. It was also established that the responsive/nonresponsive characteristic was retained over numerous passages and seemed to be a stable phenotype in the particular cell population. The mechanism by which chitosan CL313A stimulates "broblast cell growth is unknown although it has been postulated that it may function in similar way to hyaluronan [5]. Chitosan may accelerate "broblast proliferation indirectly, possibly through forming polyelectrolyte complexes with serum components such as heparin [11], or potentiating growth factors such as platelet derived growth factor (PDGF) [19]. To investigate the e!ects of serum components on chitosan induced "broblast mitogenesis, responder and nonresponder cultures were treated with 5 g/ml CL313A in medium containing varying serum concentration (0%, 1%, 2.5%, 5% NBCS). The mitogenic response induced by CL313A was found to be dependent on the presence of serum in the medium (Fig. 3). Inui et al. [19] suggest that chitosan oligosaccharides interact with a receptor on the surface of vascular smooth muscle cells (VSMC) and their data indicate that four or more D-glucosamine units are required for activation. They also suggest that chitooligosaccharides act as a &progression factor' in mitogenesis induced by PDGF in VSMC. PDGF is regarded as a &competence factor' that requires the presence of progression factors, such as insulin, to induce cell proliferation. Inui et al. [19] also demonstrated that cell proliferation induced by PDGF was stimulated by both insulin and chitosan but that there was no synergistic e!ect: thus chitosan may mimic insulin, acting as a progression factor. The data presented here support this although clearly further study is required to identify possible receptors involved. The requirement of serum for chitosan stimulated "broblast proliferation may, in addition to mimicking progression factors, be due to interaction with components in the serum such as heparin or growth factors. Chitosan may bind these components and in the process stabilise and activate them, thus stimulating proliferation and hence wound healing indirectly [11,20,21]. The results present here support the hypothesis that chitosan may bind serum components and either protect them
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from enzymatic degradation or present them to the cells in an activated form. Bound growth factors could be slowly released by the action of lysozyme on chitosan, supplying the cells with a sustained level of mitogenic signals. Chitosan may also localise these factors near the cells by binding to both and therefore increasing the chance of the growth factor binding to its receptor on the cell surface. The "broblast response to injury is only one aspect of the wound healing process: for complete restoration of the integrity of the skin other cell-types, especially keratinocytes, must be involved. The e!ect of chitosan CL313A and chitin-50A on keratinocyte proliferation was initially studied using HaCaT cells. When cultured with CL313A at initial concentrations of 2.5, 5 and 50 g/ml these cells showed a reduced proliferation rate compared to controls, whilst chitin-50A appeared to have no signi"cant e!ect on HaCaT cell proliferation. These results seem to support the work on "broblasts which indicated that highly deacetylated chitosan samples, in contrast to chitin, possess bioactive properties that may in#uence wound repair, although CL313A stimulated "broblast proliferation but inhibited keratinocyte proliferation. The mechanism by which chitosan CL313A may interfere with HaCaT cell proliferation is unclear. It may interact with growth factors in the serum or metal ions (e.g. calcium), reducing the availability of these to cells. However, there are currently very few published data on the e!ects of chitosan on HaCaT cells or primary human keratinocytes. With respect to primary keratinocytes an irradiated 3T3 feeder layer is normally used to support keratinocyte growth and stimulate cell proliferation in vitro [15]. Therefore in the presence and absence of a "broblast feeder layer CL313A appeared to in#uence keratinocyte proliferation in a similar manner to which it a!ected HaCaT cell proliferation: namely it had an inhibitory e!ect on the proliferation of primary keratinocytes cultured with feeder cells. In the absence of a feeder layer, CL313A more strongly inhibited proliferation, possibly indicating that the feeder layer may confer some protective e!ect. Again, the mechanism for this is not clear, although the feeder layer may supply growth factors, such as EGF, or possibly extracellular matrix components that are conducive to keratinocyte proliferation and antagonistic to the chitosan growth limiting e!ect. These results are in accord with the "nding of Denuziere et al. [22] who examined cytocompatibilty of "lms of chitosan and chitosan associated with glycosaminoglycans (GAGs) with human keratinocytes. When human keratinocytes were cultured on chitosan "lms cell growth was consistently approximately 60% of that of controls. In summary, chitosan CL313A demonstrated stimulatory e!ects on "broblast proliferation which appears to be dependent on it being highly deacetylated. However this mitogenic property was not universal for all "broblast cultures tested. Fibroblast cultures could be divided
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into two phenotypes (responders and nonresponders), which were stable and appeared to be donor-dependent although not correlated to sex, age or anatomical site. The mitogenic stimulation by CL313A in responsive "broblasts was dependent on the presence of serum, implying that chitosan may interact with serum components or act as a progression factor. The e!ect of chitosan on keratinocytes also appeared to be dependent on the degree of deacetylation. Chitin-50A (37% deacetylated) showed no e!ect while CL313A (89%) inhibited proliferation. These results indicate that highly deacetylated chitosan samples are more biologically active than chitin and less deacetylated chitosans and therefore possibly have more potential as wound healing agents or dressing materials. This work also shows the importance of using primary human cells derived from a range of di!erent donors in the in vitro screening of new potential therapeutic agents, such as chitosan/chitin, to identify whether donor-dependent phenotypic variation occurs.
Acknowledgements GIH was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) CASE award with Reckitt & Colman Products Ltd., Hull, UK.
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[17] Schmidt RJ, Chung LY, Andrews AM, Spyratou O, Turner TD. Biocompatibility of wound management products: a study of the e!ect of various polysaccharides on murine L929 "broblast proliferation and macrophage respiratory burst. J Pharm Pharmacol 1993;45:508}13. [18] Su CH, Sun CS, Juan SW, Ho HO, Hu CH, Sheu MT. Development of fungal mycelia as skin substitutes: e!ect on wound healing and "broblast. Biomaterials 1999;20:61}8. [19] Inui H, Tsujikubo M, Hirano S. Low molecular weight chitosan stimulation of mitogenic response to platelet-derived growth factor in vascular smooth muscle cells. Biosci Biotech Biochem 1995;59:2111}4. [20] Kratz G, Arnander C, Swedenborg J, Back M, Falk C, Gouda I, Larm O. Heparin-chitosan complexes stimulate wound healing in human skin. Scand J Plast Reconstr Hand Surg 1997;31:119}32. [21] Kratz G, Back M, Arnander C, Larm O. Immobilised heparin accelerates the healing of human wounds in vivo. Scand J Plast Reconstr Hand Surg 1998;32:381}5. [22] Denuziere A, Ferrier D, Damour O, Domard A. Chitosanchondroitin sulphate and chitosan-hyaluronate polyelectrolyte complexes: biological properties. Biomaterials 1998;19: 1275}85.
The e!ect of chitin and chitosan on the proliferation of human skin "broblasts and keratinocytes in vitro Graeme I. Howling , Peter W. Dettmar, Paul A. Goddard, Frank C. Hampson, Michael Dornish, Edward J. Wood * Leeds Skin Research Centre, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK Reckitt & Colman Products, Dansom Lane, Hull HU8 7DS, UK Pronova Biomedical, Gaustadalleen 21, N-0349, Oslo, Norway Received 29 August 2000; accepted 24 January 2001
Abstract The e!ects of chitin [(1P4)-2-acetamido-2-deoxy--D-glucan] and its partially deacetylated derivatives, chitosans, on the proliferation of human dermal "broblasts and keratinocytes were examined in vitro. Chitosans with relatively high degrees of deacetylation strongly stimulated "broblast proliferation while samples with lower levels of deacetylation showed less activity. Fraction, CL313A, a shorter chain length, 89% deacetylated chitosan chloride was further evaluated using cultures of "broblasts derived from a range of human donors. Some "broblast cultures produced a positive mitogenic response to CL313A treatment with proliferation rates being increased by approximately 50% over the control level at an initial concentration of 50 g/ml, whilst others showed no stimulation of proliferation or even a slight inhibition ((10%). The stimulatory e!ect on "broblast proliferation required the presence of serum in the culture medium suggesting that the chitosan may be interacting with growth factors present in the serum and potentiating their e!ect. In contrast to the stimulatory e!ects on "broblasts, fraction CL313A inhibited human keratinocyte mitogenesis with up to 40% inhibition of proliferation being observed at 50 g/ml. In general highly deacetylated chitosans were more active than those with a lower degree of deacetylation. These data demonstrate that highly deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Analysis of their e!ects on cells in culture may be useful as a screen for their potential activity in vivo as wound healing agents, although in the case of "broblasts it is important to select appropriate strains of cells for use in the screen. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Chitosan; Chitin; Fibroblast; Keratinocyte; Skin; Wound healing
1. Introduction Wound repair is a complex process involving an integrated response by many di!erent cell types controlled by a variety of growth factors. During the initial in#ammatory phase "broblasts start to enter the wound where they synthesise and later remodel new extracellular matrix material, of which collagen is the main component [1]. The dermal response is only one aspect of cutaneous wound repair however, the outermost and vital barrier layer, the epidermis which is composed of several layers of keratinocytes, must also be restored. In injured skin,
* Corresponding author. Tel.: #44-(0)113-233-3100; fax: #44(0)113-233-3167. E-mail address: [email protected] (E.J. Wood).
basal layer keratinocytes migrate from the wound edge and from injured epidermal appendages (hair follicles and sweat glands) into the defect, moving over the newly formed dermal sca!olding. They proliferate, stratify and di!erentiate to produce a neoepidermis to cover the wound and restore the skin's barrier function [1]. Various in vitro cell culture systems have been used to investigate the cellular processes, such as "broblast and keratinocyte proliferation and migration in response to the growth factors that are present in a wound [2}4]. Such models simplify and standardise the system compared with the in vivo situation and also enable materials to be assessed for their potential, at least in a preliminary way, to promote wound repair by stimulating cell proliferation, and for their biocompatibility. Chitin [(1P4)-2-acetamido-2-deoxy--D-glucan], a linear, unbranched structural polysaccharide from the
0142-9612/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 0 4 2 - 4
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Table 1 Chitin/chitosan samples No.
Sample
Degree of polymerisation (DP )
Molecular mass (MM )
% deacetylation
1 2 3 4 5 6
Chitin 50 Chitin 50 fraction A Seacure CL311 Seacure CL311 fraction A Seacure CL313 Seacure CL313 fraction A
900 170 890 55 1220 60
197,200 37,300 194,000 12,000 263,800 13,000
37 37 58 58 89 89
Seacure is a trade name for chitosans produced by Pronova Biomedical. Number of N-acetylglucosamine or glucosamine units. DP and M M determined by intrinsic viscosity (MM ) and light scattering methods (M M ). E X % deacetylation determined by NMR spectroscopy.
shells of crustaceans and fungal mycelia, and its deacetylated derivative, chitosan, have previously been shown to possess both material and bioactive properties that may be bene"cial to enhancing wound repair (reviewed in [5]). In experimental animal models, chitin/chitosan were shown to in#uence all stages of wound repair [6,7]. In the in#ammatory phase chitosan has unique haemostatic properties that are independent of the normal clotting cascades [8]. In vivo these polymers can also interact with and modulate the migration behaviour of neutrophils and macrophages modifying subsequent repair processes such as "broplasia and reepithelialisation [6,9]. In vitro, the e!ects of chitin and chitosan on "broblasts have been studied but as in in vivo studies, both stimulatory [10] and inhibitory [11] actions have been reported. These contradictory data appear at least in part to result from the di!erent chemical compositions and physical forms of the biopolymer samples investigated, making it di$cult to be clear about the relationship between chitosan structure and its e!ect on "broblast behaviour. The present study examines the e!ect of chitin and chitosan samples with various deacetylation levels and polymer lengths, on the proliferation of human dermal "broblasts in vitro. Chitosan has also been shown to interact with epidermal cells; it appeared to stimulate reepithelialisation in dog and rat experimental wounds [7,12] but direct e!ects on keratinocyte proliferation have not been reported. Therefore, we also investigated the e!ect of chitin and chitosans on keratinocyte proliferation in vitro using both an immortalised human keratinocyte cell line (HaCaT), and primary human keratinocytes cultured with and without an irradiated "broblast feeder layer.
2. Materials and methods 2.1. Polymer samples The chitin/chitosan samples used (Table 1) were dissolved in 17 mM acetic acid in a range of 10; stock
concentrations, "lter sterilised though a 0.2 m polycarbonate "lter (Millipore, Bedford MA, USA) and diluted for use with "broblast growth medium (Dulbecco's modi"ed Eagle's medium (DMEM), 5% (v/v) newborn calf serum, 1% (v/v) penicillin/streptomycin [10,000 U/ml penicillin, 10,000 g/ml streptomycin; Gibco, Paisley, Scotland]) to produce nominal 0, 2.5, 5, 50, 500 g/ml concentrations. Chitosan chloride samples CL313 and CL313A slowly precipitated in pH 7.4 medium over a period of 3 days. To determine the concentration of chitosan remaining in solution, a colorimetric assay was developed from a method by described by Muzzarelli [13]. Cibacron brilliant red 3B-A (also known as Reactive red 4, Sigma, Poole, UK) was prepared by dissolving 150 mg dye in 100 ml deionised H O; 5 ml of this solution was diluted to 100 ml with 0.1 M glycine}HCl bu!er, pH 3.2, to give a "nal concentration of 75 mg/l. Standards or test samples (300 l) were added to 3 ml dye solution and the absorbance read at 575 nm after 10 min. The test samples of nominal concentration 5, 50 and 500 g/ml chitosan CL313A made up in DMEM were left for di!erent times at 373C in a 5% CO /95% air atmosphere. At each time point the medium was centrifuged on a bench top centrifuge for 5 min, at 13,000 rpm, to remove solid debris and precipitated chitosan, after which the supernatant was examined under microscopy to check that all visible insoluble aggregates had been pelleted. The supernatant was then removed and used in the assay. 2.2. Cell culture Human dermal "broblasts were obtained from specimens of skin from healthy donors undergoing routine minor elective surgery. Cultures were established by a single-cell suspension technique following enzymatic digestion of the skin samples. Brie#y, skin specimens were incubated with dispase (1.5 mg/ml grade II, Boehringer Mannheim, Germany) in DMEM to facilitate separation of the epidermis from the dermis. Fibroblasts
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were liberated from the dermis by subsequent treatment with Clostridium histolyticum collagenase A (1 mg/ml, Boehringer Mannheim, Germany) and cultured in "broblast medium at 373C in a 5% CO /95% air atmo sphere with medium changes twice weekly. Upon reaching con#uence the "broblast cultures were split 1 : 3 to give approximately 2.5;10 cells per 25 cm culture #ask. Cells were utilised for experimental work between passages 2 and 10. HaCaT cells, an immortalised but non-tumorigenic keratinocyte cell-line which retain their di!erentiation potential [14], were cultured in `HaCaT mediuma (DMEM, 10% (v/v) foetal calf serum, 1% (v/v) penicillin/streptomycin (10,000 U/ml penicillin, 10,000 g/ml streptomycin, Gibco, Paisley, Scotland)). Human epidermal keratinocytes were isolated and cultured on a lethally irradiated 3T3 "broblast feeder layer according to Rheinwald and Green [15] as modi"ed by Navasaria et al. [16]. Brie#y, keratinocytes were liberated from epidermis by tryptic digestion (2.5 mg/ml trypsin, 200 g/ml EDTA, in PBS) and the cells collected and seeded at 1;10 cells per 25 cm #ask which contained an irradiated 3T3 "broblast feeder layer (5;10 cells/#ask). Keratinocyte cultures were maintained in specialised keratinocyte medium (110 ml Ham's F12 (Gibco, Paisley, Scotland), 330 ml DMEM (Gibco), 50 ml foetal calf serum (Gibco, Paisley, Scotland), with the addition of 5 ml penicillin/streptomycin (10,000 U/ml penicillin, 10,000 g/ml streptomycin, Gibco, Paisley, Scotland), 2.5 ml epidermal growth factor (EGF) (2 g/ml stock, Sigma, Poole, UK), 500 l hydrocortisone (400 g/ml), 1 ml transferrin (5 mg/ml)/3,3,5 triiodo Lthyronine (136 g/ml) combined stock, 500 l insulin (Humalin-S威 100 U/ml, Lilly, Indianapolis, USA), 5 l cholera toxin (1 mg/ml stock, Sigma, Poole, UK)) at 373C in a 5% CO /95% air atmosphere with medium changes every 3 days.
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Keratinocyte proliferation was assayed using a similar method except that for primary cells a lethally irradiated 3T3 feeder layer was present in each well (4; 10 cells/well) (these cells had previously been shown not to interfere with the assay). 2.4. Statistical analysis Results were analysed using 1-way ANOVA or a paired student's t-test where indicated.
3. Results 3.1. Ewect of chitin/chitosan samples on xbroblast proliferation in vitro The e!ect of chitin and chitosans on "broblast proliferation was investigated over a range of concentrations (initially 5}500 g/ml). The biopolymer samples (Table 1) were initially screened to identify samples that strongly stimulated "broblast proliferation in vitro using H thymidine incorporation. Chitosan CL313 and its shorter chain length fraction, CL313A, had the strongest stimulatory e!ects on cell proliferation (Fig. 1) at all the concentrations tested. Four other highly deacetylated chitosans all showed a stimulatory e!ect on "broblast proliferation (data not shown). In general chitosan samples with the higher levels of deacetylation stimulated "broblast proliferation more than chitin and chitosan samples deacetylated to a lesser degree. The lower molecular weight form, CL313A, displayed higher proliferative activity and maximal stimulation was observed in "broblasts cultured in medium containing 5 g/ml CL313A. Other chitosan/chitin had lesser e!ects on proliferation, and chitin-50A had antiproliferative e!ects on "broblasts at higher doses, with maximal inhibition
2.3. Methyl-[3H]-thymidine cell proliferation assay Fibroblasts were seeded into 24 well culture plates at 20,000 cells/well and incubated overnight to allow attachment. The medium was then replaced with the appropriate test medium containing the polymer samples and the plates incubated at 373C in a 5% CO /95% air atmosphere for the speci"ed time. The test medium was then discarded and the cells pulse-labelled for 3 h with 1 Ci/well H thymidine (5 Ci/mmol; ICN, Basingstoke, UK) in "broblast medium. After labelling, cells were washed twice with ice-cold PBS and acid-soluble radioactivity removed by 2;10 min 10% (v/v) trichloroacetic acid incubations. The cells were then washed in 70% ethanol and solubilised in 0.1 M NaOH, 2% (w/v) Na CO , 1% (w/v) SDS. The radioactivity incorporated into acid-insoluble material was determined by liquid scintillation counting.
Fig. 1. Initial screening of chitin/chitosan samples for e!ect on "broblast proliferation in vitro. Human dermal "broblasts (C520) were treated with various polymer samples for 3 days with one change of medium on day 2. The H thymidine cell proliferation assay was then performed. Data (n"3$SEM) are presented as percentage of the controls (no polymer present) (*P(0.05, **P(0.01, ANOVA).
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Table 2 Chitosan CL313A concentration in "broblast medium. Fibroblast medium (pH 7.4) containing CL313A at initial concentrations 50 and 500 g/ml were incubated for 1, 2, and 3 days at 373C in a 5% CO /95% air atmosphere to duplicate conditions of media in "broblast cultures. Chitosan concentration was determined at 575 nm
Table 3 Donor dependent variation in the e!ect of chitosan CL313A on "broblast populations isolated from di!erent donors. Two distinct populations of human dermal "broblasts, one responsive to stimulation of proliferation by chitosan CL313A and the other non-responsive ID Number
Donor age
Location
Passage number
CL313A responsive
A534 A655 A665 A693 A695 A701 C488 C495 C498 C520 A377 A407 A408 A418 C400 C434 C491 C516
47(f) 31 28(f) 38(f) 38(f) 15 13 3 6 6 32(f) 48(f) 27(f) 36(f) 3 4 7 2
Abdomen Foreskin Breast Abdomen Abdomen Foreskin Foreskin Foreskin Foreskin Foreskin Abdomen Breast Abdomen Breast Foreskin Foreskin Foreskin Foreskin
7 4 4, 3 4, 2 8 6, 4 2, 8 5 6 7 4, 5, 4, 5
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No No No
CL313A concentration (g/ml), mean$SEM Initial concentration
Day 1
Day 2
Day 3
50 500
27.6$1.1 80.4$1.7
24.3$0.5 59.3$0.5
22.5$0.63 57.8$0.31
occurring at the initial concentration 500 g/ml, where proliferation was 60% of the controls. In culture medium, when incubated at 373C in 5% CO /95% air atmosphere, the higher concentrations of chitosans CL313 and CL313A gradually formed insoluble aggregates, which could be removed by centrifugation. Highly deacetylated chitosans have low solubility at pH 7.4, and other researchers have used chitosan in suspension or slurries for wound repair studies in vivo and in vitro [17,18]. In the present work, precipitation of chitosan was quanti"ed in order to assess the amount remaining in solution. Table 2 shows that the concentration of CL313A initially at 50 g/ml slowly decreased over 3 days to give a steady concentration of 22.5 g/ml with most insoluble aggregates being formed within the "rst 24 h. In the 500 g/ml solution, 24 h after preparing the solution only approximately 80 g/ml was still in solution, the dissolved concentration fell further over the following 24 h to 59.3 g/ml (Table 2). Chitosan sample CL313A which exhibited the strongest stimulation of proliferation was selected for further study. Fibroblasts isolated and cultured from di!erent donors were used to assess the e!ect of CL313A on proliferation in di!erent cell populations (Table 3). Not all "broblast cultures responded and indeed they appeared to fall into two categories: cells were reproducibly either `respondersa or `nonrespondersa to CL313A-induced proliferation (Table 3). There was no obvious correlation between responsiveness and age or anatomical site of the donor skin and this e!ect persisted even after subculturing numerous times. Fig. 2 shows representative results against a background of the average for 10 responsive cultures and 8 nonresponsive cultures: culture A695 was strongly responsive to CL313A-induced proliferation, while A418 was shown to be nonresponsive. The mechanism by which chitin/chitosan interacts with "broblast cells is unknown but chitosan has been reported to bind serum factors, such as growth factors, perhaps protecting them from degradation or perhaps having activating e!ects that would stimulate cell prolif-
6 5
10 7, 8
7 7 7, 8
(f )"female. Indicates samples from same donor but non-adjoining areas.
Fig. 2. Examples of donor dependent variation in "broblast proliferation induced by chitosan. CL313A "broblasts A695 P4 were representative of cultures that were responsive to chitosan CL313A stimulated proliferation, while A418 P7 were representative of nonresponders. Data (n"3$SEM) are presented as percentage of controls. The mean results of all the responders (n"10$SEM) and nonresponders (n"8$SEM) are also shown.
eration more [11,19]. Therefore, it was decided to examine whether the chitosan-stimulated "broblast proliferation was dependent on the presence of serum, which supplies the cells with growth factors, using "broblasts that had previously been de"ned as `responsivea or `nonresponsivea. C520 "broblasts (CL313A responsive) required the presence of newborn calf serum (NBCS) for
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Fig. 4. E!ect of chitosan CL313A and chitin-50A on human keratinocyte HaCaT cell proliferation in vitro. HaCaT cells were treated with CL313A and chitin-50A for 3 days, with one change of medium on day 2. H thymidine incorporation was performed. Data (n"4$SEM) are presented as percentage of control (without polymer) (*P(0.01, ANOVA).
Fig. 3. The dependence of chitosan CL313A-stimulated "broblast proliferation on serum concentration. The e!ect of serum concentration was examined in responders (A: C520) and nonresponders (B: C491) to CL313A stimulation of "broblast proliferation. Cells were treated with $5 g/ml CL313A at various serum concentrations indicated and H thymidine incorporation assay performed after 3 days. Data (n"3$SEM) are presented as mean counts per minute (C5"treatment with CL313A at 5 g/ml, *P(0.05, **P(0.005, students t-test).
the chitosan to be able to stimulate proliferation (Fig. 3A). At 5% serum, chitosan stimulated proliferation by 27% and at 2.5% serum by 56%. C520 "broblasts cultured with lower serum concentrations (1% and 0%) with and without chitosan had similar proliferation rates. Chitosan had no signi"cant e!ect on the proliferation of nonresponsive "broblasts additional to that produced by serum factors (Fig. 3B). 3.2. Ewect of Chitosan CL313A and Chitin-50A on human keratinocyte proliferation in vitro The e!ects of chitosan CL313A and chitin-50A on human keratinocytes were initially investigated using HaCaT cells, which are a keratinocyte cell-line that require no feeder layer or complex media for growth and are generally easier to handle than primary keratinocyte cultures. Fig. 4 shows that at initial concentrations of 5 and 50 g/ml chitosan CL313A inhibited HaCaT
Fig. 5. E!ect of CL313A on primary human keratinocyte proliferation in vitro. Keratinocytes were cultured in keratinocyte medium containing CL313A at 0, 5, 50 g/ml with and without an irradiated 3T3 feeder layer. The H thymidine proliferation assay was performed after 3 days. Data (n"3$SEM) are presented as counts per minute (*P(0.05, **P(0.01, ANOVA).
proliferation by approximately 26% and 20%, respectively, while Chitin-50A, on the other hand, had no e!ect on HaCaT proliferation at either concentration tested. Chitosan CL313A was also tested on three primary human keratinocyte cultures, derived from di!erent donors and similar results were obtained in all the experiments. No donor dependent phenotypic variation was found as is observed for "broblasts. Fig. 5 displays representative data showing the e!ect of CL313A at 5 and 50 g/ml initial concentrations on human primary keratinocytes, with and without a lethally irradiated 3T3 "broblast feeder layer. Keratinocytes proliferate at a much higher rate when cultured on a feeder layer but in both keratinocyte culture systems ($feeder layer) cell proliferation was dose-dependently inhibited by chitosan CL313A. For example, keratinocytes cultured with
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Table 4 E!ect of CL313A on primary human keratinocyte proliferation in vitro. Results from Fig. 5 are displayed as percentage of corresponding controls (0 g/ml) for culture system used ($feeder layer) to allow direct comparison of the data Treatment
No feeder layer % of control (SEM)
With feeder layer % of control (SEM)
0 C5 C50
100 ($11.8) 59 ($5.9) 19 ($0.9)
100 ($7.4) 78 ($3.1) 58 ($3.4)
a feeder layer proliferated at 78% and 58% when treated with 5 and 50 g/ml (initial concentrations) CL313A, respectively compared with the controls. Table 4 shows that the irradiated "broblast feeder layer protected the keratinocytes from the chitosan induced inhibition of proliferation. For example, keratinocytes treated with CL313A at an initial concentration of 50 g/ml had a proliferation rate of 19% of control without a feeder layer but with a feeder layer present the rate was 58% of control.
4. Discussion The initial aim of this work was to evaluate the various chitin and chitosans described in Table 1, for their ability to in#uence the proliferation of human "broblasts and keratinocytes in vitro as an index for their potential as wound healing agents. The screening of the biopolymers for their e!ects on "broblast proliferation showed that chitosan `Seacure CL313a, and its shorter chain length fraction, CL313A, had the greatest mitogenic activity at all concentrations tested. The other chitosan samples showed lesser degrees of mitogenic activity and some samples, such as chitin 50A, had antiproliferative e!ects. The deacetylation level of chitosan seems to be a key factor in the mitogenic activity on "broblasts with the molecular mass of the biopolymer being somewhat less important. This notion was supported by work with other highly deacetylated chitosan samples with di!erent average molecular masses and which also stimulated the proliferation of "broblasts (data not shown). These samples had deacetylation levels of 84% and 86%, and ranged in average molecular mass from 23,800 to 145,000 Da, again suggesting that polymer chain length was not particularly important for biological activity, at least in the range tested. Chitosan CL313A was chosen for further evaluation. Subsequent studies to investigate the e!ects of CL313A on dermal "broblast proliferation revealed that some "broblast cultures showed a mitogenic response to treatment (termed &responders'), while other cultures showed
no stimulation of proliferation (&nonresponders'). In a very few cases there was a slight inhibition of proliferation (see Fig. 2), for example, CL313A inhibited A418 "broblast proliferation rates by approximately 10%. This phenomenon has not been previously reported, possibly due to the fact that much of the work describing the e!ect of chitosan used speci"c cell lines such as L929 mouse "broblasts [17,11], F1000 embryonic skin and muscle "broblasts [10], or "broblasts from animals, such as rats or mice [11]. Fibroblasts isolated from various human donors of di!erent age and sex and from di!erent anatomical sites were investigated but there appeared to be no correlation between responsiveness to CL313A and age, sex or anatomical site. It was also established that the responsive/nonresponsive characteristic was retained over numerous passages and seemed to be a stable phenotype in the particular cell population. The mechanism by which chitosan CL313A stimulates "broblast cell growth is unknown although it has been postulated that it may function in similar way to hyaluronan [5]. Chitosan may accelerate "broblast proliferation indirectly, possibly through forming polyelectrolyte complexes with serum components such as heparin [11], or potentiating growth factors such as platelet derived growth factor (PDGF) [19]. To investigate the e!ects of serum components on chitosan induced "broblast mitogenesis, responder and nonresponder cultures were treated with 5 g/ml CL313A in medium containing varying serum concentration (0%, 1%, 2.5%, 5% NBCS). The mitogenic response induced by CL313A was found to be dependent on the presence of serum in the medium (Fig. 3). Inui et al. [19] suggest that chitosan oligosaccharides interact with a receptor on the surface of vascular smooth muscle cells (VSMC) and their data indicate that four or more D-glucosamine units are required for activation. They also suggest that chitooligosaccharides act as a &progression factor' in mitogenesis induced by PDGF in VSMC. PDGF is regarded as a &competence factor' that requires the presence of progression factors, such as insulin, to induce cell proliferation. Inui et al. [19] also demonstrated that cell proliferation induced by PDGF was stimulated by both insulin and chitosan but that there was no synergistic e!ect: thus chitosan may mimic insulin, acting as a progression factor. The data presented here support this although clearly further study is required to identify possible receptors involved. The requirement of serum for chitosan stimulated "broblast proliferation may, in addition to mimicking progression factors, be due to interaction with components in the serum such as heparin or growth factors. Chitosan may bind these components and in the process stabilise and activate them, thus stimulating proliferation and hence wound healing indirectly [11,20,21]. The results present here support the hypothesis that chitosan may bind serum components and either protect them
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from enzymatic degradation or present them to the cells in an activated form. Bound growth factors could be slowly released by the action of lysozyme on chitosan, supplying the cells with a sustained level of mitogenic signals. Chitosan may also localise these factors near the cells by binding to both and therefore increasing the chance of the growth factor binding to its receptor on the cell surface. The "broblast response to injury is only one aspect of the wound healing process: for complete restoration of the integrity of the skin other cell-types, especially keratinocytes, must be involved. The e!ect of chitosan CL313A and chitin-50A on keratinocyte proliferation was initially studied using HaCaT cells. When cultured with CL313A at initial concentrations of 2.5, 5 and 50 g/ml these cells showed a reduced proliferation rate compared to controls, whilst chitin-50A appeared to have no signi"cant e!ect on HaCaT cell proliferation. These results seem to support the work on "broblasts which indicated that highly deacetylated chitosan samples, in contrast to chitin, possess bioactive properties that may in#uence wound repair, although CL313A stimulated "broblast proliferation but inhibited keratinocyte proliferation. The mechanism by which chitosan CL313A may interfere with HaCaT cell proliferation is unclear. It may interact with growth factors in the serum or metal ions (e.g. calcium), reducing the availability of these to cells. However, there are currently very few published data on the e!ects of chitosan on HaCaT cells or primary human keratinocytes. With respect to primary keratinocytes an irradiated 3T3 feeder layer is normally used to support keratinocyte growth and stimulate cell proliferation in vitro [15]. Therefore in the presence and absence of a "broblast feeder layer CL313A appeared to in#uence keratinocyte proliferation in a similar manner to which it a!ected HaCaT cell proliferation: namely it had an inhibitory e!ect on the proliferation of primary keratinocytes cultured with feeder cells. In the absence of a feeder layer, CL313A more strongly inhibited proliferation, possibly indicating that the feeder layer may confer some protective e!ect. Again, the mechanism for this is not clear, although the feeder layer may supply growth factors, such as EGF, or possibly extracellular matrix components that are conducive to keratinocyte proliferation and antagonistic to the chitosan growth limiting e!ect. These results are in accord with the "nding of Denuziere et al. [22] who examined cytocompatibilty of "lms of chitosan and chitosan associated with glycosaminoglycans (GAGs) with human keratinocytes. When human keratinocytes were cultured on chitosan "lms cell growth was consistently approximately 60% of that of controls. In summary, chitosan CL313A demonstrated stimulatory e!ects on "broblast proliferation which appears to be dependent on it being highly deacetylated. However this mitogenic property was not universal for all "broblast cultures tested. Fibroblast cultures could be divided
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into two phenotypes (responders and nonresponders), which were stable and appeared to be donor-dependent although not correlated to sex, age or anatomical site. The mitogenic stimulation by CL313A in responsive "broblasts was dependent on the presence of serum, implying that chitosan may interact with serum components or act as a progression factor. The e!ect of chitosan on keratinocytes also appeared to be dependent on the degree of deacetylation. Chitin-50A (37% deacetylated) showed no e!ect while CL313A (89%) inhibited proliferation. These results indicate that highly deacetylated chitosan samples are more biologically active than chitin and less deacetylated chitosans and therefore possibly have more potential as wound healing agents or dressing materials. This work also shows the importance of using primary human cells derived from a range of di!erent donors in the in vitro screening of new potential therapeutic agents, such as chitosan/chitin, to identify whether donor-dependent phenotypic variation occurs.
Acknowledgements GIH was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) CASE award with Reckitt & Colman Products Ltd., Hull, UK.
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