Currently, most cases of organ failure or severe injury are treated with organ transplants. However, considering both the lack of available donors and occurrences of organ injury and transplant failure, there is a need for alternative solutions. Tissue engineering attempts a better solution. There are two main types of tissue engineering: seeding a scaffold with human cells or implanting healthy cells into failing tissue. This study explores the newly expanding method of creating scaffolds using biomaterials, analyzing the growth of a unique mycelium strain-growth medium combination. Mycelium, the vegetative root of fungi, is emerging as a promising alternative to synthetic materials. Mycelium, which is composed of well-organized interconnected fibers, has been shown to be a cost-effective, all-natural bio-scaffold whose properties are tunable based on the strain-substrate combination. Recent research suggests that the entire fibrous structure can be used as a bioscaffold with just the one-step process of inactivation with an autoclave. This study uses the one-step process of inactivation with autoclave. The viability of combinations of Lentinula edodes and Pholiota nameko cultured in either Potato Dextrose Broth (PDB) or d-glucose enriched PDB was assessed by using scanning electron microscope imaging (SEM) and attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. Analysis of the SEM images revealed a wide distribution of pore sizes, but the majority of these strain-medium combinations demonstrated a porosity range with some potential to facilitate cell migration, adhesion, and ECM production. Additionally, the SEM imaging revealed a substance that the mycelium secreted which might provide an explanation for why some samples were much denser or more porous than others. The spectra gathered from the ATR-FTIR spectrometer was almost exactly identical to the spectra of two well documented strains of mycelium, and one of which has been shown to be a viable bio-scaffold. Given the tunable properties of mycelium and the cheap cost of growth, mycelium has the potential to become the next go-to material for creating bio scaffolds for tissue engineering.
Influence of Two Strain and Growth Medium Combinations on the Chemical Composition and Morphology of Mycelium Bio-Scaffolds and its Implications SEM Analysis of Samples At University of Connecticut’s Biosciense Electron Microscopy Lab, the samples were sputtered with a 20 nm gold-palladium (80/20) layer and analyzed using a FEI Nova NanoSEM 450 Scanning Electron Microscope (SEM) operating at 2 kV of accelerating voltage. Four images were taken per sample (two different magnifications at two different sites in the same sample), and three samples from each combination were sampled to ensure consistent results. Figure 1 shows that P. nameko has hyphae that are much stringier and more elongated than those of L. edodes, and P. nameko is also much more porous than the L. edodes strain. (See Figure 2). Indeed, Figure 1 (e-g) show how dense the L. edodes strain is compared to P. namiko, with the cross-sectional image displaying the condensed layers stacked on top of one another. Furthermore, Figure 1 (b) and (d) show the shrinkage undergone by the mycelium, which means that the samples were definitely dehydrated post autoclave.
(a) (b)
Project Purpose
(c)
(d)
(e) (f )
(h)
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L. edodes
To analyze the properties of P. nameko and L. edodes when cultured in PDB or d-glucose enriched PDB to determine the sample’s potential as a bioscaffold after only inactivation. These data will be compared with data from other strain-medium combinations to provide a more comprehensive prediction for the viability of mycelium as a bioscaffold.
Potato Dextrose Broth
D-glucose enriched Potato Dextrose Broth
P. nameko
Summary
Hypothesis Figure 1. (a-g) SEM images of autoclaved mycelia.
P. nameko and L. edodes cultured in either PDB or d-glucose enriched PDB will have similar hydrodynamic properties, morphologies, and chemical compositions to those of human extracellular matrices (ECMs).
(h) (i)
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Strain, Media, and Growth Conditions Pholiota nameko NSPN1 and Lentinula edodes NSLE2 plate cultures were purchased from North Spore and were left incubating in their cultures. A maintained in 90 mm petri dish with either Potato Dextrose Broth (PDB) or d-glucose enriched PDB as growth medium. A piece of mycelium was inoculated in 90 mm Petri dishes containing either 30 mL of PDB at 24 g/L in water or PDB 24g/L with d-glucose 30 g/L. The PDB medium composition used is the standard formulation suggested by Merck. All media were autoclaved before use, at 120 °C for 20 min. Mycelia were incubated in the dark in an incubator at 27 °C, and bowls of water were placed inside the incubator to achieve approximately 78% relative humidity.
Figure 2. (h-i) SEM images of autoclaved mycelia. Strain-medium combinations from left to right: P. nameko in d-glucose enriched PDB, L. edodes in PDB, L. edodes in PDB (zoomed in image of (i)), P. nameko in PDB.
Water Absorption Measurements To measure the water retention capabilities of these combinations of mycelium and growth media, dry samples of mycelium were weighed on a sensitive electronic balance and then placed into a incubator with bowls filled with water. After 24 h of air drying, samples were weighed, and then transferred to 86% humidity conditions for approximately 21 h, before being weighed again. The amount of adsorbed water was calculated based on the initial dry weight. The percent by mass of water in the hydrated samples of mycelium was measured using the formula MW/MM, where MW is the mass of the water and MM is the mass of the hydrated mycelium. Only one strain-medium combination--P. nameko cultured in d-glucose enriched PDB--had consistent results, which was 5.54% water by mass after 21 h in 86% RH. Future research on this area may be fruitful.
Discussion From Figure 1 (e) and Figure 2 (h-j), the SEM reveals that there are some interfibrous substance that the mycelium excreted, perhaps acting like a glue for the fibers. Figure 2 (i) and (j) show that this flatter substance attempts to repair holes torn into it, which further shows that it could be a supportive structure for the fibers. The L. edodes samples grown in d-glucose enriched PDB are particularly dense, as evidence by Figure 1 (e). Prior to autoclaving, L. edodes was predominantly in a gel-like form, while the more developed P. nameko strain appeared to be composed mostly of its hyphae (the elongated cells that compose the mycelium). Therefore, the substance that gave L. edodes its volume could have potentially evaporated while it was in the autoclave or while it was air drying. In addition to the high pressure environment inside the autoclave, this potential evaporation would leave condensed layers of what remained. Further research needs to be done on the gel-like substance that is dominant in L. edodes cultured in d-glucose enriched PDB, and somewhat present in all other combinations. Figure 1 (e) and (h) show the outlines of fibers emerging in the gel-like substance, suggesting that this substance could potentially be undeveloped fibers. In culture prior to imaging, the substance was significantly lighter in color and more gelatinous than the fibrous structure present in P. nameko. Additionally, L. edodes takes longer to grow than P. namiko, so one hypothesis could be that this substance is simply premature hyphae, pooling nutrients and preparing to expand into the area the premature part occupies. Furthermore, because of the shrinkage displayed in Figure 1 (b) and (d), more work will need to be done to ensure that the structure of the mycelium remains unchanged during and post-autoclave. Finally, Figure 2 (k) shows a crystal attached to the structure of the mycelium, which means that the samples were not rinsed thoroughly enough prior to autoclaving and SEM imaging. Measures should be taken in the future to correct this error. Applications. On average, 16 people die every day because of organ failure, and the gap between the amount of organs available for transplant and the amount of organs needed grows steadily each year. Tissue engineering has been a robust field for the past 30 years, attempting to solve exactly this problem. However, many synthetic polymers that have been created to date (e.g. PGA or PLA) require follow-up treatment to display all the necessary hydrodynamical and chemical properties of a bio-scaffold, not simply just the proper structure. Furthermore, because these compounds rely on synthetic compounds, they are not sustainable. With the recent mycelium research suggesting that it can potentially be a biomedical scaffold after a simple one-step autoclaving process, there now exists a sustainable, low-cost, and effective solution to the problem. Mycelium has already been shown to be a successful and scalable alternative to meat and animal clothing products, potentially alleviating two significant contributors to global warming.These paths serve to show the promise mycelium has for scaffolding animal cellular growth and its many applications. Future research in the field should work towards a predictive model, in which one can request what tissue they want to replicate, and the model outputs what substrate-strain.
Conclusions Figure 3. Distributions of the pore sizes in the four samples of mycelium
The majority of pores in the distributions do not favor cell growth, though some previous literature suggests that smaller pores favor cell adhesion, bridging, and ECM production. Except for the L. ed culture in d-glucose enriched PDB, these samples have some pores that are in the “optimal” range for cell growth (>100μm).
ATR FTIR Spectroscopy Analysis Previous literature has laid out this procedure for analyzing the spectra of samples of mycelium. Infrared spectra of samples are obtained with an attenuated total reflection (ATR) accessory coupled to a Fourier transform infrared spectrophotometer FTIR. All spectra are recorded in the range from 3800 to 600 cm−1, with 4 cm−1 resolution, accumulating 64 scans. The sample was gently placed on a spot of ATR accessory and slowly pressed, with the part grown in contact with the substrate on the ATR crystal. To ensure the reproducibility of the obtained spectra, three samples of each type are measured. Spectra analysis is performed with the OMNIC™ Spectra Analysis software that comes with the Nicolet™ Summit Spectrometer. These spectra align almost entirely with those of Pleurotus ostreatus and Ganoderma lucidum cultured in PDB. P. ostreatus was reported to be biocompatible After 20 days of growth, when the majority of the plate is covered, mycelium was with human keratinocytes, which provides a favorable collected from the substrate with a spatula and rinsed with hot deionized water. outlook on the potential of the samples presented in Mycelia are then autoclaved at 120 °C for 20 min. Autoclaved mycelia are then this study. Figure 4 displays how P. na cultured in PDB has the highest absorbance at all points along the dried under a laminar fume hood and illuminated for approximately 100 min with spectrum, followed by L. ed cultured in d-glucose UV light. enriched PDB, L. ed cultured in PDB, and then P. na cultured in d-glucose enriched PDB. The one main difference between the samples’ spectra is that every sample but P. na cultured in PDB has a peak at 1550 cm-1 (amide II stretching region). Previous literature has observed that differences in this region are likely related to variations in chitin content, which can influence mycelial properties such as hydrophobicity and mechanical resistance. The presence of all of these functional groups in the ATR-FTIR analysis shows promise for the potential of these samples to be a bio-scaffold.
Material Preparation
Figure 3. ATR-FTIR of the four samples of mycelium. “p” means cultured in PDB, and “p and d” means cultured in d-glucose enriched PDB.
- Analysis of the SEM images revealed a wide distribution of pore sizes, but the majority of these strain-medium combinations demonstrated a porosity range with potential to facilitate cell migration, adhesion, and ECM production. - All combinations aside from L. ed cultured in d-glucose enriched PDB have pore sizes big enough that are “optimal” for cell growth (>150𝜇m2) - An unknown, self-repairing substance was discovered in the scaffold - ATR-FTIR revealed that these combinations are almost exactly identical to the spectra of two well documented strains of mycelium, and one of which has been shown to be a viable bio-scaffold.
Future Work - Determine the best procedure to use when autoclaving the mycelia to preserve their structure. - Gather more data on a selection of strain-medium combinations and analyze their viability as a bioscaffold. - Inquire further into the unknown, self-repairing gel-like substance that is very present in L. edodes and somewhat present in P. nameko . - Identify which bodily tissues and organs are most supported by the presented scaffolding combinations.
All graphs and images aside from the SEM images were created by the student researcher. SEM images were taken by the Bioscience Electron Microscopy Laboratory at the University of Connecticut.