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. 2015 Aug 14;10(8):e0134090.
doi: 10.1371/journal.pone.0134090. eCollection 2015.

Mycelial Mattress from a Sporangia Formation-Delayed Mutant of Rhizopus stolonifer as Wound Healing-Enhancing Biomaterial

Mei-Yin Chien  1 , Ling-Chun Chen  2 , Ying-Chen Chen  2 , Ming-Thau Sheu  3 , Ya-Chi Tsai  4 , Hsiu-O Ho  2 , Ching-Hua Su  5 , Der-Zen Liu  6
Affiliations

Mycelial Mattress from a Sporangia Formation-Delayed Mutant of Rhizopus stolonifer as Wound Healing-Enhancing Biomaterial

Mei-Yin Chien et al. PLoS One. .

Abstract

A mycelial mattress of Rhizopus stolonifer obtained from a liquid static culture was utilized for wound dressing and biomedical use. Following screening of mutants induced by UV radiation, F6, exhibiting delayed sporangium formation was selected because its sporangium maturation exhibited a 5-day delay without significant loss of mycelial weight compared to the wild type. The sporangium-free mycelial mattress from the sporangiospore culture of F6 was treated with 1N sodium hydroxide NaOH at 85°C for 2 h to produce a sponge-like membrane named Rhizochitin. The trifluoroacetic acid hydrolysate of Rhizochitin contained 36% N-acetylglucosamine and 53% hexose respectively detected by the Elson-Morgen and phenol-sulfuric acid methods. Results indicated the wound area in rats covered with Rhizochitin was 40% less than that of the uncovered group. Rhizochitin decreased the expression of PDGF in the proliferation stage, increased the expression of TGF-β in the inflammation and proliferation stages, and increased the expression of VEGF in the inflammation and proliferation stages. Rhizochitin inhibited secretion of matrix metalloproteinase-9 on days 1, 7, 9, and 12 and matrix metalloproteinase-2 on days 3, 7, 9, and 12. It was concluded that Rhizochitin has beneficial properties of biocompatible, biodegradable, and wound healing.

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Conflict of interest statement

Competing Interests: Although one author is employed by Ko Da Pharmaceutical Co., this does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Rhizopus stolonifer.
(a) Cultured at 29°C on the surface of potato dextrose agar (PDA) for 7 days; (b) sporangiophore (100x); (c) sporangia (100x); (d) sporangia (200x).
Fig 2
Fig 2. Sporangium with respect to the time in the spore suspension (1.5x107/flask) inoculated in 250-ml flasks containing 100 ml potato dextrose broth (PDB), with 1%~4% surplus glucose (a) and 1%~4% surplus peptone (b). G: surplus glucose; P: surplus peptone
Fig 3
Fig 3. (a)1%~4% surplus glucose and (b)1%~4% surplus peptone.
Dried mattress weight with glucose (GA) or peptose (PA); deproteinized mattress weight with glucose (GB) or peptose (PB) of mycelia mattresses formed with respect to the time cultured in potato dextrose broth with glucose (PDBA) or peptose (PDBB).
Fig 4
Fig 4. Dried mattress weight (right column) and number of sporangium (left column) of mycelia mattresses formed with respect to the time cultured in potato dextrose broth (PDB), with 2% surplus glucose at three different temperatures of 25 (a,b), 29 (c,d), 37°C (e,f).
Fig 5
Fig 5. Number of sporangium (a) and dried mattress weight (b: Dried mattress weight; c: deproteinized dried mattress weight) for the wild type and its 16 mutants induced by UV radiation cultured in potato dextrose broth (PDB) with 2% surplus glucose at an incubation temperatures of 29°C for four different time periods.
Fig 6
Fig 6. Dry mattress weight for mutant no. 6 cultured in a flask or tray with three different inoculation densities (a), and non-deproteinized (A) and deproteinized (B) dried mattress weight (b) for mutant no. 6 cultured in potato dextrose broth (PDB) or PDB with 2% surplus glucose (G) for different time periods. Number of sporangium (c) of mutant no. 6 cultured in PDB or PDB with 2% surplus glucose for different time periods.
Fig 7
Fig 7. Mutant no. 6 cultured in a flask or tray (a); the appearance of the dried mattress (right column) and deproteinized dried mattress (left column) for mutant no. 6 (b) and wild type (c) cultured in potato dextrose agar (PDA) with 2% surplus glucose at 29°C for 8 days.
Fig 8
Fig 8. a: Thin layer chromatography (visualized with Elson-Morgan reagent) after 16 hr digestion of trifluoroacetic acid for Mutant F6 cultured in PDB or surplus (from left to right) 1.0 or 4% glucose, 1.0 or 4% peptone (lane 1–5, lane 6: N-acetyl glucosamine, lane 7: chitin, lane 8: chitosan, lane 9: glucosamine); b: Thin layer chromatography (visualized with Naphthoresorcinol/ethanol/sulphuric acid reagent) after 16 hr digestion of trifluoroacetic acid for Mutant No. 6 cultured in PDB or surplus (from left to right) 1.0 or 4% glucose, 1.0 or 4% peptone (lane 1–5, lane 6: galactose, lane 7: chitin, lane 8: chitosan, lane 9: glucose).
Fig 9
Fig 9. Plots of changes in the wound area versus time for those covered with cotton gauze as the control.
Fig 10
Fig 10. Changes in the amounts of growth factor (PDGF) (a), transforming growth factor (TGF)-β (b), and vascular endothelial growth factor (VEGF) (c) versus time in the wound area covered with cotton gauze, Sacchachitin, Rhizochitin, or BESCHITIN-W.
Fig 11
Fig 11. Histological staining with hematoxylin and eosin (H&E) for the wound area covered for 3 days with the control (C), cotton gauze, Sacchachitin (S), Rhizochitin (R), or BESCHITIN (B).
Fig 12
Fig 12. Detection of matrix metalloproteinase (MMP)-9 (92 KDa) and -2 (72 KDa) by gelatin zymography in extracts from the wound area covered for different time periods with the control, cotton gauze (C), Rhizochitin (R), Sacchachitin (S), BESCHITIN-W (B).
(M, marker; P, Protease standard).
See this image and copyright information in PMC

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Financial funding from the Center of Excellence for Clinical Trials and Research in Neuroscience (DOH 100-TD-B-111–003) and Ministry of Science and Technology of ROC (NSC 100-2320-B-038-004-MY3) is highly appreciated. Ko Da Pharmaceutical Co., Taoyuan, Taiwan provided support in the form of salaries for author MYC, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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