Potential Therapeutic Application of Fungal Filaments in Wound Management

by

Paul F Hamlyn1 & Richard J Schmidt2

1BTTG Biotechnology, Shirley House, Didsbury, Manchester M20 2RB
2Welsh School of Pharmacy, UWCC, King Edward VII Avenue, Cardiff CF1 3XF

(This article originally appeared in  Mycologist, 1994, 8(4), 147-152.)


Introduction

The use of fungal materials as styptics in folk medicine has been documented (Baker, 1989; Vaidya & Rabba, 1993) as has the use of mouldy bread, jam, etc. and, more recently, live mycelial pads for the prevention and treatment of wound infections - efficacy apparently being related to antibiotic (patulin rather than penicillin) content (Wainwright, 1989; Wainwright et al., 1992).

With the availability of haemostats (styptics) based on calcium alginate (a seaweed-derived polysaccharide), and the preferred use of systemic rather than local antibiotic therapy, it would appear that there is no place for fungal materials in modern wound management. It may seem curious, therefore, that novel fungal materials with the potential to be used in wound management are currently being developed.

The major components of fungal cell walls are polysaccharides which typically account for 80 - 90% of the dry weight, the remainder consisting of protein and lipids (Bartnicki-Garcia, 1968). Chitin is present in the majority of fungi as one of the skeletal polysaccharides responsible for maintaining the rigidity and shape of the cell wall. It is a b -1,4-linked linear homopolymer of N-acetyl-D-glucosamine similar to cellulose but with an acetamido group rather than an hydroxyl at C-2. Chitin is not the only structural polysaccharide present in fungal cell walls. The alkali-insoluble cell wall residue of ascomycetes and basidiomycetes has been reported to consist of a branched b -1,3- / b -1,6- glucan together with chitin (Rosenberger, 1976). In zygomycetes in particular, the chitin may be partially deacetylated (the fully deacetylated material being known as chitosan). The sugars fucose, galactose, mannose and xylose have been identified as minor components in hydrolysates of cell walls from various fungal species whilst galactosamine has also been identified in the cell walls of several fungi, though usually accounting for only a few percent of the total monomers. Furthermore, the chitin content varies between species and even growth forms (e.g. vegetative mycelium, sporangiophores) and can be greatly influenced by the composition of the growth medium and the conditions of culture.

Chitin is not unique to fungi, also being found in the shells of crustaceans and in the exoskeletons of insects (Austin et al., 1981). Indeed, chitin is currently obtained commercially from crab- and prawn- shells which are waste products of the food industry.

Scientific studies on the use of chitin in wound healing were first reported in the 1970s (Balassa, 1972; Balassa & Prudden, 1978). In rats, the bursting strengths of experimental wounds treated with chitin of crustacean or fungal origin, were found to have increased by up to 60% relative to controls. Since then, further evidence has been obtained to suggest that chitin has wound healing acceleration properties although the mechanism of action is still largely unknown (Olsen et al., 1989).

Filamentous fungi are an attractive source of chitin for medical applications because specific products can be manufactured under standard conditions and the chitin is produced in a fibrous form so that only relatively cheap chemical treatments are required during processing. This article summarises a programme of work carried out at BTTG and the Welsh School of Pharmacy to investigate the potential application of fungal filaments in wound management.

Experimental Materials

At BTTG a range of filamentous materials was prepared containing from about 20% (w/w) up to 90% (w/w) chitin (Table 1). It should be noted that the assay method we used was not able to differentiate chitin and chitosan. The assumption made for the purposes of calculation was, therefore, that only chitin was present. The fungal materials are more appropriately described as containing ‘chitin/chitosan’. In addition to mycelia from Fusarium graminearum, Rhizomucor miehei, Rhizopus oryzae and Mucor mucedo, sporangiophores of Phycomyces blakesleeanus and Agaricus bisporus stipe (a waste product from the cultivated mushroom industry) were also included in these investigations. All the products had been treated with 2M NaOH at 100oC for 1 h to remove protein and other alkali-soluble components. Further details about the fungal culture conditions and the preparation of the filamentous materials are given in Chung et al. (1994).

Cell Proliferation Studies

Mammalian cell culture systems were used for evaluating bioactivity. This work was carried out at the Welsh School of Pharmacy where comparative bioassay systems have been developed to assess and compare materials used in wound management (Turner et al., 1989; Schmidt et al., 1989; Schmidt et al., 1993). The objective of these studies was to establish whether any of the fungal materials can affect the rate of proliferation of fibroblasts, the cell type responsible for laying down new (granulation) tissue in a healing wound, and therefore whether the materials had the potential to increase the rate of wound healing. A second objective was to determine whether there is a correlation between the chitin content of the materials and their effects on fibroblast proliferation.

At high concentrations, the purified fungal preparations had anti-proliferant activity. This did not correlate with chitin content and may have been the result of a direct chitosan / plasma membrane interaction (Chung et al., 1993) At lower concentrations, pro-proliferant effects which appeared to correlate with chitin content were observed. These effects were most marked with material derived from sporangiophores of Phycomyces blakesleeanus which had the highest chitin content of all the fungal preparations examined (Tables 1 & 2). Material derived from sporangiophores of P. blakesleeanus or mycelium of Mucor mucedo also exhibited cell attractant and cell binding properties in fibroblast cultures (results not shown), effects that could conceivably assist movement and attachment of fibroblasts in the healing wound. Fibroblasts are responsible for laying down collagen in the wound and hence rebuilding tissue.

In order to explain the pro-proliferant effects of the fungal materials, a mechanism involving auto-oxidatively generated hydrogen peroxide has been proposed (Chung et al., 1993; Schmidt et al., 1994). Auto-oxidation is the process of spontaneous oxidative degradation in air; this process has long been known to lead to hydrogen peroxide formation and requires the presence of traces of transition metal ions (such as iron or copper) to proceed. Hydrogen peroxide acts as a fibroblast cell proliferant at the very low concentrations generated by auto-oxidation of the materials in the culture medium (Burdon et al., 1989; Schmidt et al., 1992). Addition of catalase (an enzyme which specifically destroys hydrogen peroxide) to the culture media completely abolishes the pro-proliferant effects of the fungal materials. It was also found that the hydrogen peroxide generating capacity did not correlate with chitin content in these fungal materials. Our more recent work has demonstrated that hydrogen peroxide generating capacity appears to be a property of chitosan rather than chitin, at least in crustacean-derived materials (Chung et al., 1993).

Fabrication

It is envisaged that two basic types of dressing could be directly manufactured from fungal filaments: an absorbent freeze-dried pad for deeper wounds and a thin wet-laid surface dressing (Hamlyn, 1991). The two types of product could be used in conjunction with other materials to form a multi-layered structure (Sagar et al., 1990).

In comparison with conventional natural fibres, fungal filaments have the advantage that the biomass is available in days rather than months and that different kinds of filamentous structures are available, ranging from straight fibres several centimetres in length (sporangiophores) to branched microscopic filaments (mycelium). Fungal filaments are also hollow leading to the possibility of their development as carriers of micro-encapsulated drugs.

Sporangiophores are only obtained on solid substrates similar to those employed for mushroom cultivation, whereas vegetative mycelium can be produced most economically in a liquid growth medium inside a bioreactor (Fig 1). From a commercial point of view the technology for growing a fungus in a bioreactor is well established. Industrial bioreactors of over 1,000,000 litres in volume are now used for the production of biomass.

After growing a fungus in a bioreactor, the resulting broth contains very fine branched filaments which, after suitable processing, can be fabricated in several different ways. The broth is first passed through a fine mesh filter and the solid material thoroughly washed with water to remove residual medium. Depending on the end-use, alkali treatment can be employed to remove proteins and other impurities and the filaments can be bleached to give a white product. Such bleaching can, however, modify the bioactivity of the material (Schmidt et al., 1994) The treated filaments are again thoroughly washed and finally re-suspended in water.

At this stage the filaments can be wet-laid, either on their own or mixed with conventional fibres such as wood pulp and polyester using normal laboratory paper-making equipment (Fig 2). When mixed with other fibres, the resulting composite mats are coherent but paper-like. On their own, the microfungal filaments are very brittle. However, these can be plasticised (for instance with glycerol) to form flexible, membrane-like materials. Another type of novel material can be made by freeze-drying a thick slurry of the filaments to produce an absorbent pad. In the case of sporangiophores (Fig 3), attempts to spin a yarn from these long straight filaments have, so far, proved unsuccessful because of their brittle nature in the dried state. Sporangiophores suffer from the dual drawback of having both low tensile strength and low breaking extension in comparison with conventional textile fibres (Table 3). Therefore, it has only been possible to fabricate these filaments by the methods already described after they have been cut into suitable short lengths to aid dispersion.

Following alkali treatment and bleaching, highly absorbent products can be made from the sporangiophores. The ultrastructure of these novel fungal products as revealed by the scanning electron microscope is shown in Figs 4 & 5.

Conclusions

The next generation of wound management products are expected to participate ‘actively’ in the wound healing process by releasing stimulatory molecules to the wound surface (Turner, 1986). These products are being developed, in particular, for the treatment of chronic wounds such as leg ulcers and bed sores which can take many months to heal by traditional means, thereby placing a considerable burden on hospital and nursing resources.

The results of the tissue culture studies reviewed in this article have been encouraging, indicating that fungal filaments exhibit biological activities (cell attractant, cell-binding, and pro-proliferant) of relevance to the wound healing process and that the pro-proliferant activity may be a feature of the chitosan present in the filaments, and more specifically the outcome of hydrogen peroxide generation by auto-oxidation of the fungal filaments. If these fungal materials were developed for use in wound management, it is conceivable that they would promote the growth of fibroblasts into the wound cavity and provide a matrix for their anchorage. This would lead to a more rapid deposition of new collagen and hence granulation tissue.

Acknowledgements

This work was supported by the UK Department of Trade and Industry and a group of industrial co-sponsors. The authors also thank Gladys Hadfield for carrying out the scanning electron microscopy and Sue Ramsbottom for technical assistance.

Copyright Note

The authors thank Dr Geoff Hadley, Senior Editor of the Mycologist, for granting permission to republish this article on-line. Mycologist is a quarterly journal, published for the British Mycological Society by Cambridge University Press.

Change of Address

Richard Schmidt  is now at Wound Care Research Group, Johnson & Johnson Medical, Airebank Mill, Gargrave BD23 3RX.


References

Austin, P.R., Brine, C.J., Castle, J.E. & Zikakis, J.P. (1981) Chitin: new facets of research. Science 212: 749-753.

Baker, T. (1989) Fungal styptics. Mycologist 3: 19-20.

Balassa, L.L. (1972) Use of chitin for promoting wound healing. United States Patent 3,632,754.

Balassa, L.L. & Prudden, J.F. (1978) Applications of chitin and chitosan in wound-healing acceleration. In Proc. 1st Internat. Conf. on Chitin/Chitosan. (R.A.A. Muzzarelli & E.R. Pariser, eds): 296-305. MIT Sea Grant Report MITSG 78-7.

Bartnicki-Garcia, S. (1968) Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annual Review of Microbiology 22: 87-108.

Burdon, R.H., Gill, V. & Rice-Evans, C. (1989) Cell-proliferation and oxidative stress. Free Radical Research Communications 7: 149-159.

Chung, L.Y., Schmidt, R.J., Andrews, A.M. & Turner, T.D. (1993) Modulation of the rate of proliferation of murine L929 fibroblasts by a chitosan/plasma membrane interaction and by hydrogen peroxide formed by auto-oxidation of chitin/chitosan in cell culture medium. In Proc. 2nd Europ. Conf. on Advances in Wound Management. (K.G. Harding, G. Cherry, C. Dealey & T.D. Turner, eds): 144-149. Macmillan Magazines, London.

Chung, L.Y., Schmidt, R.J., Hamlyn, P.F., Sagar, B.F., Andrews, A.M. & Turner, T.D. (1994) Biocompatibility of potential wound management products: fungal mycelia as a source of chitin/chitosan and their effect on the proliferation of human F1000 fibroblasts in culture. Journal of Biomedical Materials Research 28: 463-469.

Hamlyn, P.F. (1991) Fabricating Fungi. In Textile Technology International (I. Glasman & P. Lennox-Kerr, eds): 254-257. Sterling Publications Ltd, London.

Olsen, R., Schwartzmiller, D., Weppner, W. & Winandy, R. (1989) Biomedical applications of chitin and its derivatives. In Proceedings of the 4th International Conference on Chitin and Chitosan (G. Skjak-Braek, T. Anthonsen & P. Sandford, eds): 813-828. Elsevier Science Publishers Ltd., London.

Rosenberger, R.F. (1976) The cell wall. In The Filamentous Fungi. Vol. 2. (J.E. Smith & D.R. Berry, eds): 328-344. Edward Arnold, London.

Sagar, B.F., Hamlyn, P.F. & Wales, D.S. (1990) Wound dressing. United States Patent 4,960,413.

Schmidt, R.J., Spyratou, O. & Turner, T.D. (1989) Biocompatibility of wound management products: the effect of various monosaccharides on L929 and 2002 fibroblast cells in culture. Journal of Pharmacy and Pharmacology 41: 781-784.

Schmidt, R.J., Chung, L.Y., Andrews, A.M. & Turner, T.D. (1992) Hydrogen peroxide is a murine (L929) fibroblast cell proliferant at micro- to nano-molar concentrations. In Proceedings of the 1st European Conference on Advances in Wound Management (K. G. Harding, D. L. Leaper & T. D. Turner, eds): 117-120. Macmillan Magazines, London.

Schmidt, R.J., Chung, L.Y., Andrews, A.M., Spyratou, O. & Turner, T.D. (1993) Biocompatibility of wound management products: a study of the effects of various polysaccharides on murine L929 fibroblasts proliferation and macrophage respiratory burst. Journal of Pharmacy and Pharmacology 45: 508-513.

Schmidt, R.J., Chung, L.Y., Andrews, A.M., Sagar, B.F., Hamlyn, P.F. & Turner, T.D. (1994) Hydrogen peroxide generation by chitin/chitosans of fungal origin and its relationship to their pro- and anti-proliferative effects on fibroblasts in culture. In Proceedings of the 3rd European Conference on Advances in Wound Management (K. G. Harding, C. Dealey, G. Cherry & F. Gottrup, eds): 80-81. Macmillan Magazines, London.

Turner, T.D. (1986) Recent advances in wound management products. In Advances in Wound Management. (T.D. Turner, R.J. Schmidt & K.G. Harding, eds): 3-6. John Wiley, Chichester.

Turner, T.D., Spyratou, O. & Schmidt, R.J. (1989) Biocompatibility of wound management products: standardization of and determination of cell growth rate in L929 fibroblast cultures. Journal of Pharmacy and Pharmacology 41: 775-780.

Vaidya, J.G. & Rabba, A.S. (1993) Fungi in folk medicine. Mycologist 7: 131-133.

Wainwright, M. (1989) Moulds in ancient and more recent medicine. Mycologist 3: 21-23.

Wainwright, M., Rally, L. & Ali, T.A. (1992) The scientific basis of mould therapy. Mycologist 6: 108-110.


Table 1  Effect of fungal materials (0.01% w/v) on the proliferation of murine L929 fibroblasts in culture after 6 days

Organism

Growth form

Chitina %

Cell no. x 104 cells ml-1
Mean S.D.  (n = 3)

% Change

Control

-

-

138.95 4.02

-

Fusarium graminearum

mycelium

19

135.55 2.75

-2

Agaricus bisporus

stipe

42

144.00 4.41

+4

Rhizomucor miehei

mycelium

70

154.94 4.86*

+11

         

Control

-

-

152.17 7.34

-

Rhizopus oryzae

mycelium

72

171.34 1.21*

+13

Phycomyces blakesleeanus

sporangiophores

91b

173.79 2.16*

+14

   

94c

178.78 6.30*

+17


% Change:  % Change in cell yield relative to the control
* p <0.05 compared to the control (Student's t-test)
a Estimated chitin content. Some of the chitin could be deacetylated.
b Unbleached.
c Bleached.

Results taken from Schmidt et al., 1994.


Table 2  Effect of prepared sporangiophores (0.05% w/v) from Phycomyces blakesleeanus on the proliferation of human F1000 fibroblasts in culture.

 

Day 3

 

Day 6

 

Day 9

 

Day13

 
  Cell no. x 104 cells ml-1
Mean S.D.
(n = 3)
%
Change
Cell no. x 104 cells ml-1
Mean S.D.
(n = 3)
%
Change
Cell no. x 104 cells ml-1
Mean S.D.
(n = 3)
%
Change
Cell no. x 104 cells ml-1
Mean S.D.
(n = 3)
%
Change

Control

14.0 0.50

-

16.93 0.90

-

20.11 0.74

-

24.09 0.23

-

P.blakes-
leeanus

14.49 0.66

+3

20.22 1.59*

+18

24.53 1.31*

+22

34.22 2.10*

+42


% Change:  % Change in cell yield relative to the control
* p <0.05 compared to the control (Student's t-test)

Results taken from Chung et al., 1994.


Table 3  Tensile properties of sporangiophores from Phycomyces blakesleeanus and some conventional textile fibres

Fibre

Tenacity (cN/tex)

Breaking extension (%)

Sporangiophores
(P.blakesleeanus)

7*

3*

Cotton

19-45

6-9

Wool

11-14

30-43

Flax

57

3

Jute

31-70

2

Viscose rayon

7-27

16-27

Casein

10

63


Tenacity:  The strength of textile fibres is referred to as their tenacity determined by measuring the force required to break the fibre.
Breaking Extension:  Measure of the extension of the fibre before failure.
* Mean of 20 determinations on individual filaments. The sporangiophores were conditioned at 65% Relative Humidity prior to testing.

Results taken from Hamlyn, 1991.


Click on each figure number to see the corresponding picture then click the back button on your browser to come back to this page

Figure 1  Pilot scale 20 litre bioreactor.

Figure 2  Preparation of wet-laid mats from fungal filaments using a British Standard Sheet Paper-making machine.

Figure 3  Sporangiophores of Phycomyces blakesleeanus produced after 10 days.

Figure 4  Scanning electron micrograph showing the ultrastructure of a composite mat composed of fungal and wood pulp fibres. Bar = 36mm.

Figure 5  Scanning electron micrograph illustrating the cross-section of a pad composed of sporangiophores. Note the hollow nature of these filaments. Bar = 250mm.


Copyright 1994:  Hamlyn & Schmidt / Mycologist

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