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

Mei-Yin Chien,Ying-Chen Chen,Ming-Thau Sheu,Ling-Chun Chen,Ya-Chi Tsai,Ching-Hua Su,Der-Zen Liu,Hsiu-O Ho,Robert W Dettman

doi:10.1371/journal.pone.0134090

Mei-Yin Chien, Ying-Chen Chen + Show 7 more

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https://doi.org/10.1371/journal.pone.0134090

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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.

Highlights

Chitin and the deacetylated derivative of chitosan are principal parts of fungi’s cell walls.[1]
In order to determine suitable culture conditions for R. stolonifer to obtain mycelia mattresses formed with the highest amount of dried and deproteinized mattress weights, the sporangium in the spore suspension (1.5x107/ flask) inoculated into 250-ml flasks and No sporangium/12 mm2 (Fig 2) and dried and deproteinized mattress weights for mycelia mattress formation (Fig 3) were first compared by culturing R. stolonifer in varying combinations of glucose (PDB surplus 1%~4%, Figs 2A and 3A) or peptone (PDB surplus 1%~4%, Figs 2B and 3B)
Rhizochitin is a chitin-like biomaterial with some beneficial properties, e.g., biocompatible, biodegradable, and nontoxic, and it accelerates wound healing

Summary

Methods

CultureRhizopus stolonifer var. stolonifer (Ehrenberg; Fries)Vuillemin BCRC 32002 was originally isolated fromfermented rice grains bought from Taiwan Tobacco & Liquor Corporation byProf. Stolonifer (Ehrenberg; Fries)Vuillemin BCRC 32002 was originally isolated fromfermented rice grains bought from Taiwan Tobacco & Liquor Corporation byProf. Su. The culture was stored at 4°C on potato dextrose agar (PDA, BD Difco, Franklin Lakes, NJ, USA). Sporangiospore suspensions with a spore concentration of 107/ml were prepared by adding a sterilized 0.1% Tween 80 solution to agar slants followed by sonication for 3 min. Spore suspensions (1.5 x 107/flask) were inoculated into 250-ml flasks containing 100 ml potato dextrose broth (PDB, BD Difco), and PDB with surplus glucose of 1.0%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, and 4.0%. PDB was added with a surplus peptone of 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, and 4.0%.

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