Materials
D-Glucose was purchased from Sigma-Aldrich (MO, USA), and Bacto-yeast extract, peptone, and agar were purchased from Difco Laboratories Inc. (NJ, USA). Disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O) was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Citric acid, ampicillin sodium, glycerol, and lysozyme were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cellulase “ONOZUKA” R-10 was purchased from Yakult Pharmaceutical Industry Co., Ltd. (Tokyo, Japan). Sodium hyaluronate FCH-60 was purchased from Kikkoman Biochemifa Co. (Tokyo, Japan). All other chemicals were of reagent-grade quality or better. Gluconacetobacter hansenii (present name is Komagataeibacter hansenii) ATCC 23769 was purchased from the American Type Culture Collection.
Bacterial strains and transformation
All procedures were according to our previous report (Takahama et al. 2021). Briefly, plasmids were introduced into Gluconacetobacter hansenii ATCC23769 (Gh) by electroporation (Hall et al. 1992) to obtain Gh(HA), an HA-producing transformant, and Gh(pTI99), a negative control (Sunagawa et al. 2012; Fang et al. 2015) carrying a plasmid vector without genes for HA synthesis. Positive clones with a high cellulose-producing ability were selected. After incubation for 3 days, colonies on the HS–agar plates were transferred to liquid HS medium containing ampicillin sodium (Amp) and cellulase, and grown at 30 °C under continuous rotary shaking (125 rpm) for 22–24 h. This culture broth was also used as a seed culture for the following culture for nanocomposite production.
Nanocomposite production and purification
According to our previous report on nanocomposites production (Takahama et al. 2021), Gh transformants (Gh(HA) and Gh(pTI99)) were grown in two culture steps, namely, pre-culture and main culture. Briefly, the seed culture was inoculated to HS medium supplemented with cellulase (0.1%, w/v) and Amp, and then grown under shaking conditions (125 rpm) (pre-culture). The pre-culture was further grown for 5 days in total, with a culture medium exchange after the first 48 h. After the pre-culture, cells in each culture were pelleted and resuspended in fresh medium without cellulase (main culture), respectively. The main culture was grown statically for 36 or 72 h to produce pellicles. All culture processes were conducted at 30 °C.
The following three types of pellicle were used in this study: (i) In vivo HA/BC nanocomposite pellicles (in vivo HA/BC) produced by Gh(HA); (ii) in situ HA/BC nanocomposite pellicles (in situ HA/BC) secreted from Gh(pTI99) in a mixed culture comprising a normal BC production medium and commercial sodium hyaluronate (0.5%, w/v); and (iii) native BC pellicles (native BC) produced by Gh(pTI99) as a reference.
The pellicles were purified with a combination method of enzymatic treatments using lysozyme and NaOH/sodium dodecyl sulfate (SDS), as previously described (Takahama et al. 2021).
Biodegradation of nanocomposites, HA extraction, and quantitative HA assay
Lyophilized pellicles were immersed in a cellulase mixture (1 mL, cellulase “ONOZUKA” R-10, 0.5% (w/v) in 50 mM sodium acetate buffer). The initial enzyme/pellicle weight ratio was 1:3.5 ± 0.3. The mixtures were incubated in a water bath at 50 °C for 48 h to digest pellicles. The supernatants were separated from the residues by centrifugation (14,000 ⋅g, 10‒15 min). HA in each supernatant was purified by ethanol precipitation, and then quantified using a Hyaluronan Quantification Kit (PG Research Inc., Tokyo, Japan), which determines the HA concentration on the basis of competitive ELISA-like methods using hyaluronan binding protein as the detector. The amount of HA released from each pellicle (µg/g) was calculated by dividing the total HA in the supernatant (µg) by the weight of solubilized (digested by cellulase) pellicles (g). Cellulase treatment did not affect the HA assay (see Figs. S1b and S1c), as confirmed by measuring HA in HA solutions (100 ng/mL) with or without cellulase (Fig. S1b).
The residues separated from supernatants were washed once with 50 mM sodium acetate buffer and once with water, and then freeze-dried. The dry weight of each residue was measured to calculate the rate of solubilization with enzymatic digestion by cellulase. Each residue (approximately 2 mg) was mixed with fine potassium bromide (KBr) powder (200 mg) to prepare KBr pellets. Fourier-transform infrared spectroscopy (FT-IR) were recorded by a FT/IR-6700 spectrophotometer (JASCO Inc., Tokyo, Japan) equipped with a triglycine sulfate detector over the wavenumber range of 4000 to 400 cm−1, using 64 scans with a 2-cm−1 resolution. The spectra were normalized using the band at 1060 cm−1, corresponding to C–O stretching (Kondo 1997).
Enzymatic HA extraction was conducted by ionic liquid pretreatment, as follows. Lyophilized pellicles were dissolved (or dispersed) in 60% (w/v) tetrabutylphosphonium hydroxide (TBPH) under shaking at 240 rpm and 30 °C for 72 h, respectively. Cellulose was regenerated from each TBPH solution (or pellicle dispersion in TBPH) by adding 1 M NaCl solution, and HA was precipitated from the supernatant by adding ethanol. The regenerated cellulose and precipitated HA obtained from each pellicle were combined, treated with 1 mL of the cellulase mixture, and then purified by adding trichloroacetic acid and diethyl ether, followed by ethanol precipitation. The amount of HA extracted from each pellicle was measured using the ELISA-like HA assay method.
Confocal laser scanning microscopy (CLSM)
Purified pellicles were fluorescently labelled with Calcofluor White M2R (Sigma-Aldrich) (CW) by immersing pellicles in 10 µg/mL CW solution. After staining, pellicles were washed with DI water, cut into square pieces with sides of approximately 10 mm, and placed in glass dishes for microscopic imaging.
Three-dimensional images of the pellicles were obtained using a confocal laser scanning microscope (TCS SP8, Leica Microsystems, Germany) with 100⋅/1.4 NA oil immersion objective. The fluorophore was excited by a 405-nm diode laser and fluorescence was detected at 415−470 nm. The voxel size was set as 28 nm ⋅ 28 nm ⋅ 110 nm based on the ideal Nyquist sampling criterion. 29.06 ⋅ 29.06 ⋅ 5.07 µm3 regions were imaged at depths a few micrometers from the surface.
The thicknesses of never-dried pellicles were measured by CLSM before dynamic mechanical analysis (described later). CW-stained pellicle test pieces, which were made from the same culture batch, were observed with a 10⋅ objective, and the thicknesses were calculated at three positions and averaged for each test piece based on distances between the z-positions of the bottom and top surfaces for each pellicle of in vivo HA/BC, in situ HA/BC, and native BC.
Normal human epidermal keratinocytes grown on the pellicles were observed with the following laser settings for excitation (lex) and detection (lem): lex = 486 nm and lem = 498–557 nm for phalloidin (F-actin); lex = 405 nm and lem = 410–478 nm for Hoechst 33342 (nucleus). The methods for sample staining and image analysis are described later.
Network structure analysis of CLSM images of pellicles
Quantitative analysis of the network structure was performed using open-source program SOAX (Xu et al. 2015) following Mohan’s procedure (Mohan et al. 2017). Prior to image analysis, 3D fluorescent images were deconvoluted using commercial deconvolution program Huygens Professional (Scientific Volume Imaging, The Netherlands) with a theoretical point spread function. The deconvoluted images were then preprocessed using a gaussian blur filter and enhanced contrast by ImageJ Fiji (Schindelin et al. 2012), and resized to 256 ⋅ 256 pixels for image analysis. 3D reconstructions using SOAX were performed by detecting and tracing the centerlines of the fibrils based on the fluorescent intensity distribution.
Dynamic mechanical analysis (DMA)
Never-dried purified pellicles were cut into dumb-bell-shaped test pieces (ISO 37, type 4) and the thickness value described above was employed. The dynamic viscoelasticity of samples was measured underwater using a DVA-200 instrument (IT Keisoku Co. Ltd., Osaka, Japan) equipped with a temperature control system in stretch deformation mode at a dynamic strain of 0.1% with multiple frequencies (0.1, 1, and 10 Hz). Elastic moduli (storage modulus (E’) and loss modulus (E”)) of pellicles of in vivo HA/BC, in situ HA/BC, and native BC were measured at 37 °C and averaged for five individual test pieces. To examine temperature-dependency, elastic moduli were measured with increasing water temperature from 30 to 80 °C at a heating rate of 3 °C. The loss tangent (tand = E”/E’), as an index of the viscous characteristics, was also obtained.
Human epidermal cell culture on nanocomposite sheets
Aseptically purified pellicles were placed into glass dishes (diameter, 35 mm; IWAKI Co., Ltd. Tokyo Japan) and air-dried at 25 °C on a clean bench to become fixed to the bottom of the dishes. The as-prepared pellicle templates were sterilized under UV light for 1 h, and then soaked in growth medium (HuMedia-KG2, Kurabo Industries Ltd.). Normal human epidermal keratinocytes (NHEK) (Epidercell Human Epidermal Keratinocyte, Kurabo Industries Ltd., 0.8 ⋅ 105 cells/mL) were seeded onto all pellicle samples (0.5 ⋅ 104 cells/well) simultaneously. The samples were incubated at 37 °C under 5% CO2 in a humidified chamber for 48 and 96 h, respectively. The growth medium was replaced every 24 h during the culture period.
The pellicles on dishes were washed once with phosphate buffered saline (PBS, pH 7.4), fixed with 3.7% (w/v) paraformaldehyde in PBS (pH 7.0) for 10 min at 25 °C, and permeabilized with 0.5% Triton X-100 in PBS for 5 min at 25 °C. The fixed cells were then incubated with fluorescent phalloidin conjugate (Acti-stain 488 phalloidin, Cytoskeleton Inc. Denver, CO, USA) and nucleic acid staining reagent (Hoechst 33342, Thermo Fisher Scientific Inc., Tokyo, Japan). The stained cells were washed three times with PBS and ProLong Antifade Reagent was added before observations.
The stained samples were observed by CLSM and the images were processed using LAS X software (construction of three-dimensional images) and ImageJ/Fiji software. F-actin images visualized with fluorescent phalloidin were used to estimate cell spreading areas on substrates (Trappmann et al. 2012). Three individual F-actin images (1.16 mm ⋅ 1.16 mm) of each group were analyzed using ImageJ/Fiji software for quantification of the cell count (cells/mm2), total cell area (%), and projected cell area (µm2) using the following procedures. After the range of pixel values was normalized (contrast stretching with 1% float), the 8-bit images were filtered (smoothing with a median filter) and binarized using a threshold of fixed value. White areas in the binary images were then segmented by applying the watershed algorithm, followed by particle analysis. The selected particles were compared with the original images and then incorrectly segmented areas were removed from the selections prior to calculations. The calculated values were compared statistically between groups using the Tukey–Kramer test (cell count and total cell area) and Steel–Dwass test (projected cell area), with p < 0.05 considered statistically significant (*) in both cases.
All statistical analyses were performed using the programs in R software (The R Foundation).