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Microbially-derived nanofibrous cellulose polymer for connective tissue regeneration
Materials Science & Engineering C 99 (2019) 96–102
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Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Microbially-derived nanofibrous cellulose polymer for connective tissue regeneration Mousa Younesia, Anna Akkusb,c, Ozan Akkusa,d,e,
T
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a
Department of Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, OH 44106, USA Department of Macromolecular Science and Engineering, School of Engineering, Case Western Reserve University, Cleveland, OH 44106, USA c Department of Comprehensive Care, School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44106, USA d Department of Orthopedics, Case Western Reserve University, Cleveland, OH 44106, USA e Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial cellulose Mechanical properties Glucose concentration Crystallinity Tissue engineering
Among a vast array of biomaterials investigated for tissue engineering applications, bacterial cellulose (BC) has not been evaluated in depth, despite the material's strong potential of applicability in the field of biotechnology. In this study we investigate the effect of sugar concentration and culture duration on physical and mechanical properties of BC. BC was grown in culture media with different glucose concentrations (weight percent) of 1.25%, 2.50%, 5.00%, 10.00%, 15.00% and also in media with fructose concentration of 5.00%. The swelling ratio of harvested BC sheets did not change significantly with concentration of glucose or the type of sugar (fructose vs glucose). Swelling ratio did not change significantly with culture duration either. Cellulose production rate was significantly higher (p < 0.05) at 5.00%wt. glucose concentration compared to other groups. Ultimate tensile strength (309.3 ± 32.8 MPa) and Young's modulus (3.1 ± 0.6 GPa) of BC sheets harvested from the medium with 5.00%wt. glucose concentration were the highest among all treatment groups. Bacterial removal process and testing condition (wet/dry) did not affect the mechanical performance of the bacterial cellulose significantly. X-ray diffraction data demonstrated higher crystallinity for samples cultured in media with 5.00%wt. glucose concentration. Viability/cytotoxicity, proliferation, and cells' metabolic activities demonstrated BC to be biocompatible. Cells attached, spread, and proliferated with time on bacterial cellulose. Results of this study showed 5.00 wt% glucose concentration is the optimum concentration of sugar in media to produce BC with highest strength and modulus compared to other concentration. High mechanical strength along with biocompatibility present bacterial cellulose as an invaluable material for use in tissue engineering of load bearing connective tissues such as tendons and ligaments.
1. Introduction Bacterial cellulose (BC) is a byproduct of sweet tea fermentation process that results in the beverage that is known as kombucha. The beverage has more than two thousand years of history and it originated in China [1]. Later, the drink was brought to Japan from Korea and eventually made its way to Russia and to Eastern Europe. Currently the beverage is gaining popularity in the West [2,3]. Bacterial cellulose (BC) has not been evaluated in depth as a biomaterial despite the material's strong potential to become a high value product in the field of biotechnology [3–5]. The unique physical and mechanical properties of BC provided a wide range of applications such as high quality audio membranes [6], optically functional materials [7], dental cement composite [8], fuel cells [9], and novel materials for
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medical applications. Bacterial cellulose has been used as scaffolds for a variety of tissue engineering applications such as blood vessel repair [10,11] and urinary reconstructions [12]. Other researchers used BC for bone [13,14], cartilage [15], wound healing [16] and meniscal repair [17]. However, there are no reports addressing potential fabrication and viability of the kombucha generated BC for connective tissue repair, such as tendons and ligaments. This study examines the effect of sugar concentration in the growth media on the yield, mechanical and physical properties of the BC which has not been investigated before to the best of our knowledge. Besides physical properties viability, proliferation and morphology of mesenchymal stem cells that were seeded on BC were studied.
Corresponding author at: Department of Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, OH 44106, USA. E-mail address: [email protected] (O. Akkus).
https://doi.org/10.1016/j.msec.2019.01.090 Received 6 September 2017; Received in revised form 4 January 2019; Accepted 18 January 2019 Available online 22 January 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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2. Materials and methods 2.1. Bacterial cellulose culture The BC pellicles, a symbiotic colony of Acetobacter and Saccharomyces, were provided by the R.P. Dressel dental materials laboratory, Department of Comprehensive Care, School of Dental Medicine, Case Western Reserve University, Cleveland, US. Glucose and fructose were purchased from Sigma-Aldrich (St. Louis, MO, US). BC pellicles were cultured in culture media consisting of deionized water with different glucose weight percentage of 1.25%, 2.5%, 5.00%, 10.00%, and 15.00%. BC pellicles were also cultured in media with 5.00%wt. fructose to compare the effect sugar type on BC production rate and its mechanical and physical properties. BC sheets were harvested at 2, 4, and 6 weeks. Samples were washed with deionized water and dried at room temperature overnight. 2.2. Swelling ratio 10 mm diameter discs were cut from the BC sheets using a biopsy punch. Dry and wet weights of samples were measured and differences were normalized to dry weights of samples and were reported as swelling ratio. 5 samples per group were tested and means and standard deviations were reported. 2.3. Mechanical testing Rectangular strips were cut from the BC sheet with a razor blade. Samples dimensions were measured with a micrometer. Tensile tests were performed using a materials test machine (Test Resource 800LE32, Test Resources Inc., MN, USA). Samples were clamped in the testing machine at a gauge length of 10 mm and the tests were performed at a strain rate of 10 mm/min. Stress and strain values were calculated from the load-displacement curve using cross sectional area and gage length, respectively. Ultimate tensile stress and Young's modulus of the samples were the calculated from the stress strain curves.
Fig. 1. (a) Glucose concentration and culturing time do not affect swelling ratio of bacterial cellulose. (b) Bacterial cellulose has the maximum production rate at 5.00%wt. glucose at all time points. Bacterial cellulose has the highest production rate (the volume of produced cellulose) at 5.00%wt. glucose concentration.
stem cells (hMSCs) at passage 2 were cultured on samples in growth media (low glucose Dulbecco's modified eagles medium, 10% fetal bovine serum, 1% penicillin/streptomycin) to investigate cell viability on BC sheets. Cells were cultured at a density of 10,000 cells/cm2. After 24 h samples were stained with live/dead assay kit (life technologies) and imaged with fluorescent microscopy. Number of live and dead cells were quantified by counting the cells in four separate field of view from above, below, left, and right of part of the samples. Cells number was divided by the total area of the field view to obtain the number of cells per mm2. Average and standard deviation for live and dead cells were reported (n = 5).
2.4. X-ray diffraction analysis X-ray diffraction patterns of cellulose samples obtained via a Bruker Discover D8 X-ray diffractometer with a monochromatic X-ray source (with a Co K-alpha X-ray tube) at room temperature from 10° to 40° at 45 kV and 40 mA. The scan speed was set at 0.05°/s with a step size of 0.025°. Crystallinity of samples was calculated using the peak intensity method [18,19]:
Crystallinity = (ICr − Iam)/ ICr × 100 where ICr is the intensity of the peak at 2θ = 26.3° and Iam is the minimum intensity corresponding to the amorphous content at 2θ = 21.2°.
2.7. Cell proliferation and metabolic activity Disc shaped BC samples were placed in low attachment 24 wellplates and seeded with MSCs as elucidated in live/dead assays. After 4 h, non-attached cells were collected by changing the media and counted to calculate the number of attached cells at days 4 and 7 as follows. Samples were stained with Alexa Fluor 488 (life technologies) for actin staining and with DAPI (4′,6‑diamidino‑2‑phenylindole) for nuclei. Cell numbers on samples were counted at each time points (n = 5). Furthermore, alamar blue assay (life technologies) was performed on another set of samples at the same time points of days 1, 4, and 7 of culture to investigate the effect of BC on metabolic activities of cells (N = 3 per group, averages and standard deviations were reported). Results were presented for individual cells at each time point (normalized with cell number at each time point) as well as samples at each time point (not normalized by cell number).
2.5. Decellularization of BC Bacteria are trapped in the BC sheets during synthesis and they should be removed from the samples for biomaterials applications. For this purpose, samples were incubated in 2 wt% NaOH solution for 4 h at 80 °C. After this step samples were rinsed thoroughly with deionized water for 3 times each 30 min. Dried samples were sputter coated and analyzed with scanning electron microscope (FEI Helios Nanolab 650) to investigate the efficacy of the process in removing the bacterial mass. 2.6. Cytotoxicity assay BC samples were cut into 10 mm diameter discs with a biopsy punch. Samples were disinfected in 70% v/v ethanol solution overnight and washed with 1 × PBS 3 times each 30 min. Human mesenchymal 97
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Fig. 2. Bacterial cellulose has the maximum ultimate tensile strength (UTS) (a) and Young's modulus at 5.00%wt. glucose concentration (b). Duration of the culture does not affect the ultimate tensile strength and Young's modulus of the bacterial cellulose. Bacterial cellulose harvested from culture media with 5.00% wt. glucose has significantly (p < 0.05) higher ultimate tensile strength than samples from culture media with 5.00%wt. fructose.
Fig. 3. X-ray diffraction analysis of bacterial cellulose cultured in media at different glucose concentrations. X-ray diffraction analysis demonstrated a peak with higher intensity at 26.3° and lower intensity at 21.2° for samples harvested from media with 5.00%wt. glucose concentration (a). Data calculated from the formula presented in similar works demonstrate higher degree of crystallinity for cellulose harvested from media with 5.00%wt. glucose concentration (b).
2.8. Statistical analysis Swelling ratio, thickness of the cellulose samples harvested from media with different sugar concentrations, mechanical test data, and Xray diffraction analysis data were analyzed with one-way ANOVA with Tukey's pairwise comparison to determine significant differences between physical and mechanical properties of samples cultured in different sugar concentrations and different culture durations. The same analysis was used for cell proliferation assay data to find the significant differences in cells number at different time points. Data are reported as mean and standard deviation. Significance is reported at the level of p < 0.05.
the swelling ratio of the BC samples significantly (p > 0.05). Moreover, there was not any significant effect of culture duration on swelling ratio of samples harvested at different time points. In aggregate, samples had the swelling ratios in the range of 5673 ± 2842 wt% (2 weeks time point at 10.00%wt. sugar conc.) to 11,580 ± 5913 wt% (6 weeks time point at 1.25% sugar conc.). Samples harvested from fructose culture media showed no differences in swelling ratio compared to those made in glucose supplemented culture media. Yield of BC was affected by glucose concentration. Volume of the BC sheet made by bacteria in each of the culture conditions consider as criteria for production rate. Volume measurement data from produced cellulose showed the rate of cellulose production at glucose
3. Results Increasing concentration of glucose in culture media didn't affect
Table 1 Ultimate tensile strength and Young's modulus of BC samples as function of culture duration and glucose concentration in culture media. Ultimate tensile strength (MPa)
Young's modulus (GPa)
Sugar conc. wt. %
1.25
2.50
5.00
10.00
15.00
5.00 fruc.
1.25%
2.50
5.00
10.00
15.00
5.00 fruc.
Week 2 Week 4 Week 6
198 ± 43 148 ± 47 135 ± 20
172 ± 38 92 ± 31 153 ± 39
309 ± 33 216 ± 31 269 ± 70
201 ± 23 159 ± 20 156 ± 42
131 ± 53 105 ± 39 184 ± 51
142 ± 44 138 ± 23 217 ± 59
2.24 ± 0.67 2.85 ± 0.94 2.59 ± 0.70
1.92 ± 0.43 2.37 ± 0.47 1.66 ± 0.69
3.13 ± 0.56 3.21 ± 0.92 3.09 ± 0.37
2.06 ± 0.29 2.30 ± 0.67 1.71 ± 0.38
1.97 ± 0.37 2.08 ± 0.59 2.05 ± 0.83
2.19 ± 0.57 1.95 ± 0.56 2.35 ± 0.60
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Fig. 4. Electron microscope images show BC before (a) and after (b) removal of bacteria. Images with higher magnification show no evidence of bacterial body in cellulose samples after the removal process (c). Mechanical test results show the ultimate tensile strength (UTS) and Young's modulus before and after bacterial removal are not significantly different (p > 0.05).
(Fig. 2a & b). The modulus of samples harvested from media containing 5.00%wt. glucose was significantly higher (p < 0.05) than those cultured in 2.50, 10.00, and 15.00%wt. but not 1.25%wt. Fig. 3a demonstrates the X-ray diffraction pattern from cellulose samples harvested from culture media with different glucose concentrations. X-ray diffraction results (Fig. 3a) illustrated that the cellulose samples harvested from culture media with 5.00%wt. glucose had higher intensity at 26.3° compared to the cellulose samples harvested from media with lower or higher glucose concentration. Using the formulation from other works [18] degree of crystallinity was calculated for all samples. Results (Fig. 3b) demonstrated significantly (p < 0.05) higher crystallinity for cellulose harvested from media containing 5.00%wt. glucose compared with other groups (except for the group with 2.50%wt. glucose concentration difference is not significant). Electron microscopy imaging of the samples (Fig. 4a & b) confirmed the complete removal of bacterial bodies from cellulose samples. Furthermore, high magnification electron microscope images demonstrated cellulose fibers to remain intact following bacterial removal process (Fig. 4c). Results of tensile tests demonstrated no significant (p > 0.05) changes in ultimate tensile strength and Young's modulus of the samples after bacterial removal process (Fig. 4d). Moreover, mechanical properties (ultimate tensile strength and Young's modulus) of bacterial cellulose were not different in wet and dry states (Fig. 5). 98% of the cells cultured on BC cultured on samples harvested from media with 5.00%wt. glucose concentration were alive after 24 h of culture (Fig. 6a & b). Morphologically, cells attached and spread over the BC samples. Cell number increased by 3- and 10-fold from 79.6 ± 46.0 cell/mm2 at day 1 to 244 ± 73.3 cell/mm2 and 785 ± 230.8 cell/mm2 at day 4 and 7 of culture (Fig. 7a–d). Cell metabolic activities significantly increased 5 and 10 fold from day 1 to day 4 and 7 of culture, respectively (Fig. 7e). However, when these data were normalized by cell number at each time point, metabolic activity of individual cells did not change significantly over time from day 1 to day 7 (Fig. 7e).
Fig. 5. Ultimate tensile strength and Young's modulus of the bacterial cellulose are not significantly different (p > 0.05) at dry condition compared to wet condition.
concentration of 5.00%wt. was the maximum (152.3 ± 7.7 mm3) and significantly higher compared to volume of cellulose made at other concentrations (Fig. 1b). However, the culture duration didn't affect the amount of cellulose at different glucose concentration significantly (p > 0.0.5) (Fig. 1b). Fig. 2 and Table 1 demonstrated visual and numerical comparison of ultimate tensile strength and Young's modulus of harvested BC samples at different time point of 2, 4, and 6 weeks from culture media with different glucose weight percentage of 1.25%, 2.5%, 5.00%, 10.00%, and 15.00% and media with 5.00%wt. fructose. Tensile tests demonstrated the cellulose samples produced in media with 5.00%wt. glucose concentration had the highest (p < 0.05) ultimate tensile strength (309.3 ± 32.8 MPa) among all groups with different glucose concentrations. Furthermore, culture duration didn't have any meaningful effect on mechanical performance of the samples in culture media with different glucose concentrations as the results showed in some of the glucose concentrations (such as 1.25%wt. glucose concentration) the samples strength reduced with increasing the culture duration and in some glucose concentrations it improved with increasing the culture time (15.00%wt. glucose concentration) (Fig. 2a). Ultimate tensile strength of the samples produced at 5.00%wt. glucose concentration were greater than that of the samples cultured in 5.00%wt. fructose
4. Discussion Bacterial cellulose cannot produce adequate amount of cellulose 99
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amount of cellulose, and increasing the glucose concentration from 5.00%wt. to 15.00%wt. (50 g/l to 150 g/l) resulted in gradual decrease in the yield. This finding agrees with an earlier study by Embuscado et al. [23], where a decrease in cellulose production was observed as sugar concentration was elevated beyond an optimal level. Therefore, an appropriate level of sugar is necessary for maximizing the bacterial cellulose production. The glucose concentration of 1.25%wt. (12.5 g/l) and 15.00%wt. (150 g/l) produced the lowest amount of bacterial cellulose. Shortage of the glucose in media with 1.25%wt. (12.5 g/l) glucose is the reason of lower cellulose production. Frank et al. [20] showed that the production of more metabolic products during the course of fermentation when an excess amount of sugar is present in the culture media might explain the decrease in cellulose production. Increasing the amount of glucose in media > 5.00%wt. results in a sharp increase in metabolic byproducts originating from bacteria which leads to inhibition of cellulose production. Caldwell et al. [24] showed the excessive generation of harmful metabolic byproducts could be the reason for decreasing the cellulose production with increasing the glucose concentration at elevated concentration levels. In general, increasing the culture period from 2 weeks to 4 and 6 weeks did not significantly increase the cellulose production in all glucose and fructose concentrations. Similar profiles have been observed in static fermentation experiments conducted by other researchers [22,25]. Other researchers similarly reported that the yield of bacterial cellulose increased sharply after a few days of induction until the rate reached a maximum. Yamanaka et al. [26] and Yoshinaga et al. [27] described the process as in the start of culture, bacteria began to proliferate by using the dissolved oxygen in the media and produce cellulose in soluble form. Following the consumption of the dissolved oxygen in the media only the bacteria living close to the surface can continue to produce cellulose. After sometime the bacteria on the surface become embedded in the cellulose pellicle they produce. As a result of this process the rate of cellulose production will decrease over time and stagnate at some point. In cellulose, crystalline structure is where the molecular chains are largely locked in place against one another in an oriented form. This crystalline structure under load has more resistance to deformation and failure. However, in amorphous form, randomly oriented molecular chains slide more easily on each other under load which results in lower strength and higher deformability. Most polymers comprise crystalline and amorphous structures. The proportion of these two phases is determined in part by processing conditions. An amorphous polymer might still contain 10% crystalline structures while one that's crystalline might actually only have 80% of its structure truly ordered in a crystalline manner. While culture medium with 5.00 wt% glucose provided the optimum condition for synthesizing the highest amount of BC, the X-ray diffraction analysis [18,19,28] demonstrated that BC produced in this culture medium to have the highest crystalline content compared to other medium with different glucose concentrations. These results supported the mechanical data where cellulose with higher degree of crystallinity (cellulose made in 5% glucose) has higher ultimate tensile strength and Young's modulus. While our results agreed with previous studies [29–31] with regard to effect of crystallinity on mechanical properties, the data showed that glucose concentration in media has significant effect on crystallinity of the cellulose synthesized by bacteria as the cellulose and by adjusting the glucose concentration in media degree of BC crystallinity and consequently mechanical properties of BC can be controlled to some extent. The results of this study demonstrated bacterial cellulose as a biocompatible material which cells can attach, spread and proliferate on its surface without any adverse effect on cell viability. Slow rate of degradation with high mechanical strength make this bacterial cellulose an ideal choice for fabrication of scaffolds for tissue engineering of slow regenerating connective tissues such as tendons and ligaments.
Fig. 6. Cytotoxicity (live/dead) assay images show the live ((a), green) and dead cells ((b), Red) on a cellulose sample. Quantifying the live/dead assay results show that > 98% of the cells are viable after 24 h (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
without the presence of carbon source in culture media [20]. Reis et al. [21] investigated the effect of sugar type (sucrose, lactose, glucose and fructose) at various concentrations (50–150 g/l) on the metabolism of the tea fungus and on the formation of ethanol and lactic acid. Their results showed that ethanol production is maximum at certain concentrations and higher concentrations of sugar decrease the yield of ethanol. In the present study, we investigated the effect of sugar concentration and type on mechanical and physical properties of the produced cellulose. Results revealed that the concentration of glucose in the media affects the yield of bacterial cellulose and these results are similar to the previous report [22]. Our results showed at glucose concentration of 5.00%wt. (50 g/l) bacteria produced the highest 100
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Fig. 7. Actin filament staining of cells (day 1(a), day 4 (b), and day 7 (c)) and demonstrate that cells adhere, spread, and proliferate on surface of bacterial cellulose. Cells number at day 7 is significantly higher than day 1, and 4 (d) (p < 0.05). Results of cells metabolic activities at different time point of day 1, 4, and 7 quantified via Alamar blue assay showed while cells metabolic activity on each samples changed significantly over time (p < 0.05), however, individual cell metabolic activity didn't change significantly over time on each sample (e) (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5. Conclusion
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Microbially-derived nanofibrous cellulose polymer for connective tissue regeneration Mousa Younesia, Anna Akkusb,c, Ozan Akkusa,d,e,
T
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a
Department of Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, OH 44106, USA Department of Macromolecular Science and Engineering, School of Engineering, Case Western Reserve University, Cleveland, OH 44106, USA c Department of Comprehensive Care, School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44106, USA d Department of Orthopedics, Case Western Reserve University, Cleveland, OH 44106, USA e Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial cellulose Mechanical properties Glucose concentration Crystallinity Tissue engineering
Among a vast array of biomaterials investigated for tissue engineering applications, bacterial cellulose (BC) has not been evaluated in depth, despite the material's strong potential of applicability in the field of biotechnology. In this study we investigate the effect of sugar concentration and culture duration on physical and mechanical properties of BC. BC was grown in culture media with different glucose concentrations (weight percent) of 1.25%, 2.50%, 5.00%, 10.00%, 15.00% and also in media with fructose concentration of 5.00%. The swelling ratio of harvested BC sheets did not change significantly with concentration of glucose or the type of sugar (fructose vs glucose). Swelling ratio did not change significantly with culture duration either. Cellulose production rate was significantly higher (p < 0.05) at 5.00%wt. glucose concentration compared to other groups. Ultimate tensile strength (309.3 ± 32.8 MPa) and Young's modulus (3.1 ± 0.6 GPa) of BC sheets harvested from the medium with 5.00%wt. glucose concentration were the highest among all treatment groups. Bacterial removal process and testing condition (wet/dry) did not affect the mechanical performance of the bacterial cellulose significantly. X-ray diffraction data demonstrated higher crystallinity for samples cultured in media with 5.00%wt. glucose concentration. Viability/cytotoxicity, proliferation, and cells' metabolic activities demonstrated BC to be biocompatible. Cells attached, spread, and proliferated with time on bacterial cellulose. Results of this study showed 5.00 wt% glucose concentration is the optimum concentration of sugar in media to produce BC with highest strength and modulus compared to other concentration. High mechanical strength along with biocompatibility present bacterial cellulose as an invaluable material for use in tissue engineering of load bearing connective tissues such as tendons and ligaments.
1. Introduction Bacterial cellulose (BC) is a byproduct of sweet tea fermentation process that results in the beverage that is known as kombucha. The beverage has more than two thousand years of history and it originated in China [1]. Later, the drink was brought to Japan from Korea and eventually made its way to Russia and to Eastern Europe. Currently the beverage is gaining popularity in the West [2,3]. Bacterial cellulose (BC) has not been evaluated in depth as a biomaterial despite the material's strong potential to become a high value product in the field of biotechnology [3–5]. The unique physical and mechanical properties of BC provided a wide range of applications such as high quality audio membranes [6], optically functional materials [7], dental cement composite [8], fuel cells [9], and novel materials for
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medical applications. Bacterial cellulose has been used as scaffolds for a variety of tissue engineering applications such as blood vessel repair [10,11] and urinary reconstructions [12]. Other researchers used BC for bone [13,14], cartilage [15], wound healing [16] and meniscal repair [17]. However, there are no reports addressing potential fabrication and viability of the kombucha generated BC for connective tissue repair, such as tendons and ligaments. This study examines the effect of sugar concentration in the growth media on the yield, mechanical and physical properties of the BC which has not been investigated before to the best of our knowledge. Besides physical properties viability, proliferation and morphology of mesenchymal stem cells that were seeded on BC were studied.
Corresponding author at: Department of Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, OH 44106, USA. E-mail address: [email protected] (O. Akkus).
https://doi.org/10.1016/j.msec.2019.01.090 Received 6 September 2017; Received in revised form 4 January 2019; Accepted 18 January 2019 Available online 22 January 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.
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2. Materials and methods 2.1. Bacterial cellulose culture The BC pellicles, a symbiotic colony of Acetobacter and Saccharomyces, were provided by the R.P. Dressel dental materials laboratory, Department of Comprehensive Care, School of Dental Medicine, Case Western Reserve University, Cleveland, US. Glucose and fructose were purchased from Sigma-Aldrich (St. Louis, MO, US). BC pellicles were cultured in culture media consisting of deionized water with different glucose weight percentage of 1.25%, 2.5%, 5.00%, 10.00%, and 15.00%. BC pellicles were also cultured in media with 5.00%wt. fructose to compare the effect sugar type on BC production rate and its mechanical and physical properties. BC sheets were harvested at 2, 4, and 6 weeks. Samples were washed with deionized water and dried at room temperature overnight. 2.2. Swelling ratio 10 mm diameter discs were cut from the BC sheets using a biopsy punch. Dry and wet weights of samples were measured and differences were normalized to dry weights of samples and were reported as swelling ratio. 5 samples per group were tested and means and standard deviations were reported. 2.3. Mechanical testing Rectangular strips were cut from the BC sheet with a razor blade. Samples dimensions were measured with a micrometer. Tensile tests were performed using a materials test machine (Test Resource 800LE32, Test Resources Inc., MN, USA). Samples were clamped in the testing machine at a gauge length of 10 mm and the tests were performed at a strain rate of 10 mm/min. Stress and strain values were calculated from the load-displacement curve using cross sectional area and gage length, respectively. Ultimate tensile stress and Young's modulus of the samples were the calculated from the stress strain curves.
Fig. 1. (a) Glucose concentration and culturing time do not affect swelling ratio of bacterial cellulose. (b) Bacterial cellulose has the maximum production rate at 5.00%wt. glucose at all time points. Bacterial cellulose has the highest production rate (the volume of produced cellulose) at 5.00%wt. glucose concentration.
stem cells (hMSCs) at passage 2 were cultured on samples in growth media (low glucose Dulbecco's modified eagles medium, 10% fetal bovine serum, 1% penicillin/streptomycin) to investigate cell viability on BC sheets. Cells were cultured at a density of 10,000 cells/cm2. After 24 h samples were stained with live/dead assay kit (life technologies) and imaged with fluorescent microscopy. Number of live and dead cells were quantified by counting the cells in four separate field of view from above, below, left, and right of part of the samples. Cells number was divided by the total area of the field view to obtain the number of cells per mm2. Average and standard deviation for live and dead cells were reported (n = 5).
2.4. X-ray diffraction analysis X-ray diffraction patterns of cellulose samples obtained via a Bruker Discover D8 X-ray diffractometer with a monochromatic X-ray source (with a Co K-alpha X-ray tube) at room temperature from 10° to 40° at 45 kV and 40 mA. The scan speed was set at 0.05°/s with a step size of 0.025°. Crystallinity of samples was calculated using the peak intensity method [18,19]:
Crystallinity = (ICr − Iam)/ ICr × 100 where ICr is the intensity of the peak at 2θ = 26.3° and Iam is the minimum intensity corresponding to the amorphous content at 2θ = 21.2°.
2.7. Cell proliferation and metabolic activity Disc shaped BC samples were placed in low attachment 24 wellplates and seeded with MSCs as elucidated in live/dead assays. After 4 h, non-attached cells were collected by changing the media and counted to calculate the number of attached cells at days 4 and 7 as follows. Samples were stained with Alexa Fluor 488 (life technologies) for actin staining and with DAPI (4′,6‑diamidino‑2‑phenylindole) for nuclei. Cell numbers on samples were counted at each time points (n = 5). Furthermore, alamar blue assay (life technologies) was performed on another set of samples at the same time points of days 1, 4, and 7 of culture to investigate the effect of BC on metabolic activities of cells (N = 3 per group, averages and standard deviations were reported). Results were presented for individual cells at each time point (normalized with cell number at each time point) as well as samples at each time point (not normalized by cell number).
2.5. Decellularization of BC Bacteria are trapped in the BC sheets during synthesis and they should be removed from the samples for biomaterials applications. For this purpose, samples were incubated in 2 wt% NaOH solution for 4 h at 80 °C. After this step samples were rinsed thoroughly with deionized water for 3 times each 30 min. Dried samples were sputter coated and analyzed with scanning electron microscope (FEI Helios Nanolab 650) to investigate the efficacy of the process in removing the bacterial mass. 2.6. Cytotoxicity assay BC samples were cut into 10 mm diameter discs with a biopsy punch. Samples were disinfected in 70% v/v ethanol solution overnight and washed with 1 × PBS 3 times each 30 min. Human mesenchymal 97
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Fig. 2. Bacterial cellulose has the maximum ultimate tensile strength (UTS) (a) and Young's modulus at 5.00%wt. glucose concentration (b). Duration of the culture does not affect the ultimate tensile strength and Young's modulus of the bacterial cellulose. Bacterial cellulose harvested from culture media with 5.00% wt. glucose has significantly (p < 0.05) higher ultimate tensile strength than samples from culture media with 5.00%wt. fructose.
Fig. 3. X-ray diffraction analysis of bacterial cellulose cultured in media at different glucose concentrations. X-ray diffraction analysis demonstrated a peak with higher intensity at 26.3° and lower intensity at 21.2° for samples harvested from media with 5.00%wt. glucose concentration (a). Data calculated from the formula presented in similar works demonstrate higher degree of crystallinity for cellulose harvested from media with 5.00%wt. glucose concentration (b).
2.8. Statistical analysis Swelling ratio, thickness of the cellulose samples harvested from media with different sugar concentrations, mechanical test data, and Xray diffraction analysis data were analyzed with one-way ANOVA with Tukey's pairwise comparison to determine significant differences between physical and mechanical properties of samples cultured in different sugar concentrations and different culture durations. The same analysis was used for cell proliferation assay data to find the significant differences in cells number at different time points. Data are reported as mean and standard deviation. Significance is reported at the level of p < 0.05.
the swelling ratio of the BC samples significantly (p > 0.05). Moreover, there was not any significant effect of culture duration on swelling ratio of samples harvested at different time points. In aggregate, samples had the swelling ratios in the range of 5673 ± 2842 wt% (2 weeks time point at 10.00%wt. sugar conc.) to 11,580 ± 5913 wt% (6 weeks time point at 1.25% sugar conc.). Samples harvested from fructose culture media showed no differences in swelling ratio compared to those made in glucose supplemented culture media. Yield of BC was affected by glucose concentration. Volume of the BC sheet made by bacteria in each of the culture conditions consider as criteria for production rate. Volume measurement data from produced cellulose showed the rate of cellulose production at glucose
3. Results Increasing concentration of glucose in culture media didn't affect
Table 1 Ultimate tensile strength and Young's modulus of BC samples as function of culture duration and glucose concentration in culture media. Ultimate tensile strength (MPa)
Young's modulus (GPa)
Sugar conc. wt. %
1.25
2.50
5.00
10.00
15.00
5.00 fruc.
1.25%
2.50
5.00
10.00
15.00
5.00 fruc.
Week 2 Week 4 Week 6
198 ± 43 148 ± 47 135 ± 20
172 ± 38 92 ± 31 153 ± 39
309 ± 33 216 ± 31 269 ± 70
201 ± 23 159 ± 20 156 ± 42
131 ± 53 105 ± 39 184 ± 51
142 ± 44 138 ± 23 217 ± 59
2.24 ± 0.67 2.85 ± 0.94 2.59 ± 0.70
1.92 ± 0.43 2.37 ± 0.47 1.66 ± 0.69
3.13 ± 0.56 3.21 ± 0.92 3.09 ± 0.37
2.06 ± 0.29 2.30 ± 0.67 1.71 ± 0.38
1.97 ± 0.37 2.08 ± 0.59 2.05 ± 0.83
2.19 ± 0.57 1.95 ± 0.56 2.35 ± 0.60
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Fig. 4. Electron microscope images show BC before (a) and after (b) removal of bacteria. Images with higher magnification show no evidence of bacterial body in cellulose samples after the removal process (c). Mechanical test results show the ultimate tensile strength (UTS) and Young's modulus before and after bacterial removal are not significantly different (p > 0.05).
(Fig. 2a & b). The modulus of samples harvested from media containing 5.00%wt. glucose was significantly higher (p < 0.05) than those cultured in 2.50, 10.00, and 15.00%wt. but not 1.25%wt. Fig. 3a demonstrates the X-ray diffraction pattern from cellulose samples harvested from culture media with different glucose concentrations. X-ray diffraction results (Fig. 3a) illustrated that the cellulose samples harvested from culture media with 5.00%wt. glucose had higher intensity at 26.3° compared to the cellulose samples harvested from media with lower or higher glucose concentration. Using the formulation from other works [18] degree of crystallinity was calculated for all samples. Results (Fig. 3b) demonstrated significantly (p < 0.05) higher crystallinity for cellulose harvested from media containing 5.00%wt. glucose compared with other groups (except for the group with 2.50%wt. glucose concentration difference is not significant). Electron microscopy imaging of the samples (Fig. 4a & b) confirmed the complete removal of bacterial bodies from cellulose samples. Furthermore, high magnification electron microscope images demonstrated cellulose fibers to remain intact following bacterial removal process (Fig. 4c). Results of tensile tests demonstrated no significant (p > 0.05) changes in ultimate tensile strength and Young's modulus of the samples after bacterial removal process (Fig. 4d). Moreover, mechanical properties (ultimate tensile strength and Young's modulus) of bacterial cellulose were not different in wet and dry states (Fig. 5). 98% of the cells cultured on BC cultured on samples harvested from media with 5.00%wt. glucose concentration were alive after 24 h of culture (Fig. 6a & b). Morphologically, cells attached and spread over the BC samples. Cell number increased by 3- and 10-fold from 79.6 ± 46.0 cell/mm2 at day 1 to 244 ± 73.3 cell/mm2 and 785 ± 230.8 cell/mm2 at day 4 and 7 of culture (Fig. 7a–d). Cell metabolic activities significantly increased 5 and 10 fold from day 1 to day 4 and 7 of culture, respectively (Fig. 7e). However, when these data were normalized by cell number at each time point, metabolic activity of individual cells did not change significantly over time from day 1 to day 7 (Fig. 7e).
Fig. 5. Ultimate tensile strength and Young's modulus of the bacterial cellulose are not significantly different (p > 0.05) at dry condition compared to wet condition.
concentration of 5.00%wt. was the maximum (152.3 ± 7.7 mm3) and significantly higher compared to volume of cellulose made at other concentrations (Fig. 1b). However, the culture duration didn't affect the amount of cellulose at different glucose concentration significantly (p > 0.0.5) (Fig. 1b). Fig. 2 and Table 1 demonstrated visual and numerical comparison of ultimate tensile strength and Young's modulus of harvested BC samples at different time point of 2, 4, and 6 weeks from culture media with different glucose weight percentage of 1.25%, 2.5%, 5.00%, 10.00%, and 15.00% and media with 5.00%wt. fructose. Tensile tests demonstrated the cellulose samples produced in media with 5.00%wt. glucose concentration had the highest (p < 0.05) ultimate tensile strength (309.3 ± 32.8 MPa) among all groups with different glucose concentrations. Furthermore, culture duration didn't have any meaningful effect on mechanical performance of the samples in culture media with different glucose concentrations as the results showed in some of the glucose concentrations (such as 1.25%wt. glucose concentration) the samples strength reduced with increasing the culture duration and in some glucose concentrations it improved with increasing the culture time (15.00%wt. glucose concentration) (Fig. 2a). Ultimate tensile strength of the samples produced at 5.00%wt. glucose concentration were greater than that of the samples cultured in 5.00%wt. fructose
4. Discussion Bacterial cellulose cannot produce adequate amount of cellulose 99
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amount of cellulose, and increasing the glucose concentration from 5.00%wt. to 15.00%wt. (50 g/l to 150 g/l) resulted in gradual decrease in the yield. This finding agrees with an earlier study by Embuscado et al. [23], where a decrease in cellulose production was observed as sugar concentration was elevated beyond an optimal level. Therefore, an appropriate level of sugar is necessary for maximizing the bacterial cellulose production. The glucose concentration of 1.25%wt. (12.5 g/l) and 15.00%wt. (150 g/l) produced the lowest amount of bacterial cellulose. Shortage of the glucose in media with 1.25%wt. (12.5 g/l) glucose is the reason of lower cellulose production. Frank et al. [20] showed that the production of more metabolic products during the course of fermentation when an excess amount of sugar is present in the culture media might explain the decrease in cellulose production. Increasing the amount of glucose in media > 5.00%wt. results in a sharp increase in metabolic byproducts originating from bacteria which leads to inhibition of cellulose production. Caldwell et al. [24] showed the excessive generation of harmful metabolic byproducts could be the reason for decreasing the cellulose production with increasing the glucose concentration at elevated concentration levels. In general, increasing the culture period from 2 weeks to 4 and 6 weeks did not significantly increase the cellulose production in all glucose and fructose concentrations. Similar profiles have been observed in static fermentation experiments conducted by other researchers [22,25]. Other researchers similarly reported that the yield of bacterial cellulose increased sharply after a few days of induction until the rate reached a maximum. Yamanaka et al. [26] and Yoshinaga et al. [27] described the process as in the start of culture, bacteria began to proliferate by using the dissolved oxygen in the media and produce cellulose in soluble form. Following the consumption of the dissolved oxygen in the media only the bacteria living close to the surface can continue to produce cellulose. After sometime the bacteria on the surface become embedded in the cellulose pellicle they produce. As a result of this process the rate of cellulose production will decrease over time and stagnate at some point. In cellulose, crystalline structure is where the molecular chains are largely locked in place against one another in an oriented form. This crystalline structure under load has more resistance to deformation and failure. However, in amorphous form, randomly oriented molecular chains slide more easily on each other under load which results in lower strength and higher deformability. Most polymers comprise crystalline and amorphous structures. The proportion of these two phases is determined in part by processing conditions. An amorphous polymer might still contain 10% crystalline structures while one that's crystalline might actually only have 80% of its structure truly ordered in a crystalline manner. While culture medium with 5.00 wt% glucose provided the optimum condition for synthesizing the highest amount of BC, the X-ray diffraction analysis [18,19,28] demonstrated that BC produced in this culture medium to have the highest crystalline content compared to other medium with different glucose concentrations. These results supported the mechanical data where cellulose with higher degree of crystallinity (cellulose made in 5% glucose) has higher ultimate tensile strength and Young's modulus. While our results agreed with previous studies [29–31] with regard to effect of crystallinity on mechanical properties, the data showed that glucose concentration in media has significant effect on crystallinity of the cellulose synthesized by bacteria as the cellulose and by adjusting the glucose concentration in media degree of BC crystallinity and consequently mechanical properties of BC can be controlled to some extent. The results of this study demonstrated bacterial cellulose as a biocompatible material which cells can attach, spread and proliferate on its surface without any adverse effect on cell viability. Slow rate of degradation with high mechanical strength make this bacterial cellulose an ideal choice for fabrication of scaffolds for tissue engineering of slow regenerating connective tissues such as tendons and ligaments.
Fig. 6. Cytotoxicity (live/dead) assay images show the live ((a), green) and dead cells ((b), Red) on a cellulose sample. Quantifying the live/dead assay results show that > 98% of the cells are viable after 24 h (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
without the presence of carbon source in culture media [20]. Reis et al. [21] investigated the effect of sugar type (sucrose, lactose, glucose and fructose) at various concentrations (50–150 g/l) on the metabolism of the tea fungus and on the formation of ethanol and lactic acid. Their results showed that ethanol production is maximum at certain concentrations and higher concentrations of sugar decrease the yield of ethanol. In the present study, we investigated the effect of sugar concentration and type on mechanical and physical properties of the produced cellulose. Results revealed that the concentration of glucose in the media affects the yield of bacterial cellulose and these results are similar to the previous report [22]. Our results showed at glucose concentration of 5.00%wt. (50 g/l) bacteria produced the highest 100
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Fig. 7. Actin filament staining of cells (day 1(a), day 4 (b), and day 7 (c)) and demonstrate that cells adhere, spread, and proliferate on surface of bacterial cellulose. Cells number at day 7 is significantly higher than day 1, and 4 (d) (p < 0.05). Results of cells metabolic activities at different time point of day 1, 4, and 7 quantified via Alamar blue assay showed while cells metabolic activity on each samples changed significantly over time (p < 0.05), however, individual cell metabolic activity didn't change significantly over time on each sample (e) (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5. Conclusion
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