Biomaterials 21 (2000) 1797}1802

Alginates from wound dressings activate human macrophages to secrete tumour necrosis factorA. Thomas, K.G. Harding, K. Moore*
Wound Healing Research Unit, Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiw CF14 4XN, UK Received 23 August 1999; accepted 16 January 2000

Abstract Alginates are used to manufacture a number of wound dressings. Clinical observations indicate that they may initiate or accelerate healing of chronic wounds after treatment of underlying pathology. Wound granulation tissue contains large numbers of macrophages and they are thought to regulate the healing process. As puri"ed alginates have been demonstrated to activate macrophages this study was initiated to determine whether alginates present within wound dressings may interact with wound macrophages. Alginate "bres taken from four commercially available dressings were co-cultured with the human histiocytic lymphoma cell line U937 following its di!erentiation with PMA. Activation was assessed by measurement of TNF production. Two of the dressings, Seasorb and Tegagen, had a minimal e!ect whilst Sorbsan at 1 mg/ml induced 302#19 pg/ml TNF . This e!ect was inhibited by polymyxin B indicating that activation was due to endotoxin contamination. Kaltostat induced production of 839#36 pg/ml TNF . This e!ect was induced both by polymyxin inhibitable endotoxin and a direct interaction with the alginate "bres. These data indicate that some alginate containing dressings have the potential to activate macrophages within the chronic wound bed and generate a pro-in#ammatory signal which may initiate a resolving in#ammation characteristic of healing wounds. 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Alginate; Wound healing; Macrophage; Activation; TNF ; Endotoxin

1. Introduction Alginate dressings are widely used in the treatment of exuding wounds and there have been anecdotal reports that they may enhance the healing process. Whilst this may in part be due to the moisture handling properties of alginate "bres it is known that alginates can exert bioactivity and induce cytokine production by human monocytes [1]. A central tenet of the understanding of the healing process is that macrophages play a key regulatory role in healing [2] and this gives rise to the concept that alginates applied to the surface of wound granulation tissue may modulate cell function in the wound environment and thus in#uence the healing process. The sodium and calcium salts of alginic acid which comprise the alginates used in dressing manufacture are

* Correspondence address: Wound Healing Research Unit, Cardi! Medicentre, Heath Park, Cardi! CF14 4UJ, UK. Fax: #44-02920754217. E-mail address: wsrkm@cf.ac.uk (K. Moore).

prepared by alkaline extraction of seaweed cell walls. The resultant colloidal solution of sodium alginate is precipitated by addition of calcium chloride (for review of alginate manufacture see [3]). Following precipitation the calcium alginate is redissolved using sodium carbonate to generate sodium alginate prior to "nal preparation of either calcium alginate or sodium/calcium alginate for dressing manufacture. The alginic acid component is a polysaccharide consisting of mannuronic and guluronic acid residues. Various alginate dressings are available which possess di!erent chemical and physical properties dependent upon the proportion and arrangement of mannuronic and guluronic acid residues and the content of calcium and sodium ions. When the alginate dressing comes into contact with wound exudate, ion exchange occurs between the calcium ions of the dressing and the sodium ions in the exudate resulting in the formation of a gel on the surface of the wound. This gel absorbs moisture and maintains an appropriately moist environment which is considered to promote optimal healing [4]. Conventionally, the value of treating wounds with alginates has been considered to be a consequence of

0142-9612/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 0 7 2 - 7

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maintaining an optimally moist environment. With increasing knowledge of the cellular interactions that occur during healing the possibility arises that alginates may promote healing via a direct modulatory e!ect on wound macrophages. This study was initiated to determine whether alginate containing dressings may induce macrophage activation in vitro and thus provide an additional mechanism to account for their observed healing promoting properties.

The cell free supernatants were stored at !203C for subsequent analysis. Further experiments were performed in the same manner, using polymyxin B (PMB) supplemented alginate preparations at a "nal concentration of 25 g PMB/ml to inhibit the e!ects of any endogenous endotoxin eluted from the alginate "bres. Within every experiment each test situation was performed in quadruplicate and each experiment repeated four times. 2.3. Tumour necrosis factor- bioassay Our use of this assay has been described in detail previously [5]. Brie#y, murine L929 "broblasts were plated at a concentration of 1.2;105 cells/ml in RPMI10% in aliquots of 75 l/well in a 96-well tissue culture plate. The plates were incubated overnight at 373C and test samples diluted 1 : 50 in medium containing actinomycin-D were added in quadruplicate to the L929 cells. A negative control of RPMI alone, and positive controls of 1.0, 2.5, 5.0 and 10.0 pg/ml of recombinant tumour necrosis factor- (TNF ) were also included in each assay. The plates were incubated for a further 21 h at 373C, the supernatant from each well was discarded, the cells washed once in Dulbecco's saline solution (DSS), and the monolayer "xed in neutral bu!ered formalin for 10 min. The cells were washed again in DSS and 100 l of 0.5% w/v aqueous crystal violet added to each well for 20 min. Each well was washed 10; in tap water and the plates allowed to air dry. One-hundred microliters of 0.1% v/v acetic acid was added to each well and the stain eluted from the cells with gentle agitation. The absorbance of each well was measured at 540 nm using a microplate reader to determine the relative number of cells surviving in each well. The TNF content of each test solution was quanti"ed using a standard curve constructed using the recombinant TNF standards between 1 and 10 pg/ml. 2.4. Data analysis The mean results of each quadruplicate culture were determined within each individual experiment. The values from each set of four repeated experiments was then used to derive mean values for TNF production and signi"cance was assessed by use of a t-test.

2. Materials and methods 2.1. Dressings Four commercially available dressings were assessed in this study; Kaltostat (Convatec), Tegagen HG (3M Healthcare), Comfeel: Seasorb "ller (Coloplast) and Sorbsan (Braun, Maersk Medical). 2.2. Cell culture Cells of the human histiocytic lymphoma cell line U937 were cultured in RPMI 1640 medium supplemented with 10% v/v foetal calf serum (FCS,(10 EU/ml endotoxin), 100 units/ml penicillin, 100 g/ml streptomycin and 0.25 g/ml amphotericin B (RPMI-10%). U937 cells were suspended at 2;10 cells/ml in RPMI-10% containing 2.5;10\ M phorbol myristate acetate (PMA, Sigma, UK) and 1 ml aliquots added to each well of a 24-well tissue culture plate for incubation at 373C for 48 h to induce di!erentiation to an adherent macrophage phenotype (DU937). Non adherent cells were removed by washing the wells twice with 1 ml of RPMI-10%. This produced monolayers of cells which demonstrated greater than 98% viability assessed by Trypan Blue exclusion. Previous work had established that the PMA concentration and the incubation period were optimal for the production of maximal numbers of adherent cells. Alginate suspensions and lipopolysaccharide from E. coli serotype 026:B6 (LPS, Sigma) solutions were prepared in the same sterile RPMI medium supplemented with 15% FCS. Alginate "bres taken from the dressings were added to the medium and left overnight at room temperature with shaking in sealed sterile universal containers. The supernatent medium was removed from the DU937 monolayers and replaced with the appropriate test alginate suspension contained in 1 ml RPMI-15% FCS. For each experiment negative control cultures of RPMI-15% FCS alone and positive cultures of RPMI15% FCS containing 10 ng/ml LPS were included. After incubation for 24 h to allow cytokine production supernatant medium was removed and centrifuged at 13000 rpm for 5 min to remove any contaminating cells.

3. Results The FCS used to supplement culture media used in these experiments was certi"ed by the manufacturer to contain less than 10 endotoxin units (EU)/ml. Previous work in our laboratories has demonstrated that endotoxin levels below 10 EU/ml do not induce activation

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Fig. 1. Induction of TNF secretion by alginates from wound dressings: "bres from each dressing were extracted overnight in RPMI-15% and then co-cultured with adherent cells derived from 2;10 PMA di!erentiated U937. After 24 h culture at 373C cells and "bres were removed from the supernatant and TNF concentration determined by L929 bioassay. Each bar is the mean of four repeat experiments each performed as quadruplicate 1 ml cultures. Error bars"SEM. Kaltostat: mean control culture (no alginate present)"63$9 pg TNF /ml. P(0.001, (0.001 and (0.01 for 1000, 100 and 10 g/ml vs. control. 1 g/ml vs. control*not signi"cant. Sorbsan: mean control culture (no alginate present)"39$7 pg TNF /ml. P(0.001 and (0.001 for 1000 and 100 g/ml vs. control. 10, 1 and 0.1 g/ml vs. control*not signi"cant. Seasorb: mean control culture (no alginate present)"43$5 pg TNF /ml. P(0.05 for 1000 g/ml vs. control. 100, 10, 1 and 0.1 g/ml vs. control*not signi"cant. Tegagen: mean control culture (no alginate present)"53$9 pg TNF /ml. P(0.05 for 1000 g/ml vs. control. 100, 10, 1 and 0.1 g/ml vs. control*not signi"cant.

of PMA di!erentiated DU937 cells resulting in low background levels of TNF production in control cultures containing no added LPS. Thus any induction of TNF secretion can be concluded to be due to a direct e!ect of test agents added into the culture system. In comparison to the other dressing components under test addition of Kaltostat elicited the most marked enhancement of TNF production from the DU937 cells with a mean production of 839$36 pg/ml in the presence of 1 mg/ml alginate "bres (Fig. 1). A dose response relationship was observed over the range of 1}1000 g added Kaltostat with the increased TNF production being signi"cant over the control baseline levels at 1000, 100, and 10 g/ml. Addition of Sorbsan at 1000 and 100 g/ml induced signi"cant TNF production, whilst Seasorb and Tegagen had a limited but statistically signi"cant e!ect only at the 1000 g/ml concentration.

To explore the possibility that TNF secretion may be induced by endotoxin contaminating the alginates Polymyxin B (PMB) was used to inhibit the e!ects of any endotoxin present. PMB is a cationic polypeptide which at a concentration of 25 g/ml could be clearly demonstrated to inhibit endotoxin induced activation of DU937 cells and release of TNF in our system (columns 2 and 3, Fig. 2) whilst PMB alone had no demonstrable e!ect on DU937 cells (column 4, Fig. 2). Addition of 25 g PMB to co-cultures of either Kaltostat or Sorbsan with DU937 cells demonstrated markedly di!erent e!ects. The activating e!ect of Sorbsan was eliminated by PMB whereas the e!ect of Kaltostat was reduced only by 61% leaving a residual level of 296$50 pg/ml TNF secretion. The small enhancement of TNF secretion induced by Seasorb and Tegagen at 1000 g/ml was totally abrogated by PMB (data not presented).

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overnight incubation in culture medium. This resulted in a decrease in TNF secretion from 545$9 to 249$17 pg/ml (Fig. 3). The bioactivity associated with the "ltrate was signi"cantly reduced by PMB treatment.

4. Discussion Alginate dressings are used clinically to absorb excess exudate at the wound surface. A "brous gel is formed at the wound surface when ion exchange occurs as the calcium alginate portion of the dressing comes into contact with Sodium ions in the exudate. The resultant gel creates a moist environment which promotes cellular regeneration [6]. Some dressings are also used for their haemostatic properties. Kaltostat has approval for use as a haemostat, whilst Sorbsan has also been shown to have signi"cant haemostatic properties when utilised in the control of blood loss from skin graft donor sites [7]. Experiments with puri"ed alginates suggests that some alginate dressings may enhance wound healing through additional bioactive mechanisms. For example, alginates have been shown to induce the stimulation of human monocytes to produce elevated levels of TNF , IL-6 and IL-1 with the active portion of the alginate being identi"ed as the mannuronic acid residues [1]. Induction of production of these cytokines at the wound site would result in delivery of a pro-in#ammatory stimulus which may be advantageous in some clinical situations but contra-indicated in other clinical situations. Alginates are under investigation as a carrier for cell immobilisation and subsequent implantation. One example being the implantation of insulin secreting cells for the treatment of diabetes mellitus [8]. A problem with such alginate capsules has been the induction of "broblast overgrowth following in vivo injection. This may well be the consequence of an in#ammatory signal delivered by the alginate carrier resulting in the production of "brogenic cytokines such as IL-1 and TNF which have the ability to promote "broblast growth. The reverse may hold in the treatment of wounds with alginates. A localised in#ammatory reaction caused by an alginate dressing may stimulate cellular activity at the chronic wound site and thereby enhance the healing process or initiate healing in a recalcitrant chronic wound. Macrophages play a central role in the regulation of the wound healing process and to achieve this they secrete an array of cytokines and growth factors. It is possible to hypothesise that in a chronic wound the macrophages present have not achieved an appropriate di!erentiation state and may therefore be receptive to an exogenous pro-in#ammatory stimulus. In this study we have demonstrated that all the dressings, to varying degrees, induced secretion of TNF . The dressing marketed as Kaltostat induced the most profound e!ect demonstrating a signi"cant increase in

Fig. 2. E!ect of polymyxin B on TNF induction by Kaltostat and Sorbsan: "bres from Sorbsan and Kaltostat were cultured and analysed as for Fig. 1 except that Polymyxin B was added to those cultures marked PMB and bacterial lipopolysaccharide to those marked LPS. Control"cells cultured in RPMI-15% alone: LPS"positive control of cells cultured in RPMI-15% plus 10 ng/ml bacterial LPS: LPS#PMB"cells cultured in 10 ng/ml LPS#25 g/ml PMB: PMB"cells cultured in RPMI-15%#25 g/ml PMB. Each bar is the mean of four repeat experiments each performed as quadruplicate 1 ml cultures. Error bars"S.E.M. P(0.001 for LPS vs. LPS#PMB. P(0.001 for Kaltostat vs. Kaltostat#PMB. P(0.001 for Sorbsan vs. Sorbsan#PMB.

Fig. 3. Separation of e!ect mediated by Kaltostat "bres and soluble factor: as for Fig. 1 except "bres from Kaltostat were extracted overnight and either incubated with U937 cells with "bres present (K-FIB) or with "bres removed by "ltration (K-FIL). Control"cells cultured in RPMI-15% alone: LPS"positive control of cells cultured in RPMI15% plus 10 ng/ml bacterial LPS. Each bar is the mean of 4 repeat experiments each performed as quadruplicate 1 ml cultures. Error bars"SEM. P(0.001 for control vs. K-FIB, (0.01 for control vs. K-FIL and (0.001 for K-FIB vs. K-FIL.

These data raised the possibility that the bioactivity exerted by Kaltostat may have at least two components. A PMB inhibitable component, presumably endotoxin which would be water soluble and a non-PMB inhibitable component possibly residing in the insoluble portion of the alginate "bres. The dual mode of action was demonstrated by removal of the "bres by "ltration after

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TNF at concentrations in excess of 10 g/ml or greater. Secretion of TNF provides a convenient marker of macrophage activation [9] and these observations indicate that certain alginate dressings have the potential to activate macrophages in the wound environment. Although these dressings are received as sterile packs the alginate component is derived from natural sources and may still contain levels of endotoxin which are not destroyed by commercial processing and sterilization procedures. The induction of TNF production by three of the dressings tested in this study, Seasorb, Sorbsan and Tegagen, was inhibited by PMB. This speci"c inhibition of endotoxin bioactivity [10] indicates that any macrophage activation induced by these dressings is the consequence of elution of LPS from them to allow interaction with the CD14 receptor for LPS on the macrophage cell surface. In contrast the activity of Kaltostat was only partially inhibited by PMB. In concordance with the observation that Kaltostat may activate macrophages by a mechanism additional to that induced by endotoxin it was found that removal of the "bres from the macrophage cultures decreased the production of TNF . Thus, it appears that the high levels of bioactivity of the alginate "bres comprising Kaltostat dressings are induced by a soluble component, presumably endotoxin, and an activity associated with the insoluble "bres. Although it has been suggested that alginates containing a high proportion of mannuronic acid can interact directly with the CD14 receptor [11] this is unlikely to be the case for Kaltostat as it is manufactured from a guluronic-rich calcium alginate [3]. During the course of this study microscopic examination of the dressing "bres indicated considerable di!erences in "bre dimensions between the dressings (Table 1). The dressing with the thickest "bres (Kaltostat) exerted the greatest bioactivity whilst the dressings with the "nest "bres (Seasorb and Tegagen) had the least e!ect on the DU937 cells. Sorbsan, which consisted of a mixture of "ne and thick "bres had an intermediate e!ect on the stimulation of TNF secretion. Induction of macrophage TNF production by asbestos "bres is related to both "bre length and opsonization with IgG [12]. It follows that the physical dimensions of the alginate "bres released from the dressings by shaking in medium during the preculture extraction period may be important in determining the outcome of the macrophage}alginate interaction. Alginates are polymers composed of mannuronic and guluronic acids and previous work has demonstrated that the proportion of these two components is crucial to determination of their bioactivity. Alginates containing a high proportion of mannuronic acid residues have been demonstrated to be the most potent as inducers of cytokine secretion by macrophages [1,13]. This relationship does not appear to be the case with the alginate

Table 1 Dressing bioactivity and "bre composition and structure Dressing Bioactivity (Induction of TNF ) ### # # $ Fibre microscopic appearance Thick Thick and "ne mixed Fine Fine Predominant uronic acid residues Guluronic Mannuronic Guluronic Mannuronic

Kaltostat Sorbsan Seasorb Tegagen HG

dressings tested in the present study (Table 1). However, another factor which may be important in determining the ability of the alginate to induce cytokine secretion is not just the relative proportions of guluronic to mannuronic acid residues but also their polymeric arrangement [14]. The role of TNF in healing is controversial. Systemic depletion of TNF by administration of TNF binding protein impairs healing in mice [15] and increases in TNF levels have been observed in exudate from human skin graft donor sites [16] and human tears after laser mediated corneal injury [17]. In contrast administration of TNF to experimental wounds impairs healing [18]. Within chronic non-healing non-infected leg ulcers there appears to be a selective de"cit of TNF in the presence of high levels of other pro-in#ammatory mediators such as IL-1 and IL-8 [19]. After treatment of the underlying pathology, initiation of healing in chronic wounds such as venous leg ulcers may require a conversion of a chronic non-resolving in#ammatory state to an acute in#ammatory phase that will resolve as the wound reepithelialises [20,21]. Macrophages are present within the granulation tissue of chronic non-infected wounds in large numbers [22] yet produce only low levels of TNF [23]. Induction of TNF production by application of a bioactive dressing may exert a transient pro-in#ammatory e!ect that will recruit fresh leucocytes from the blood [24] to re-initiate the cascade of events required for healing. In this study we have demonstrated that some alginate containing dressings are able to activate human macrophages to secrete pro-in#ammatory cytokines, possibly by a direct interaction with the CD14 receptor for LPS [25]. Such a mechanism would explain clinical observations that alginate containing dressings may accelerate healing [26] by delivering a pro-in#ammatory signal to the wound environment.

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