FTIR spectroscopy analysis
The spectra based on the FTIR-attenuated total reflection (ATR) mode of BC, BG, samples containing HA are shown in Fig. 2. In general, all of the BC pellicles exhibited the characteristic of the absorption bands that were in accordance with the BC-based composite. On the FTIR spectra, the typical β-1,4-glycosidic linkages at 899 cm− 1 and CH2 stretching vibration at 2900 cm− 1 of BC are found in all samples. The spectra of hydrogen bonds and hydroxyl group were identified at 3200–3500 cm-1 [10]. The presence of these peaks indicated that the primary chemical structure of BC was maintained in the media containing GT and HA. According to the FITR spectra of BCA and BGA, the different vibration modes of phosphate groups (PO4)3− in hydroxyapatite could be seen at absorption bands 662 cm− 1, 610 cm− 1, 950–1000 cm− 1, and 1031 cm− 1 [24]. These relative peaks represented the HA content in the spectra of BCA and BGA samples. This indicated that HA particles were present in the BC network.
Cellulose fibrils morphology, composition, and porous structure analysis
Figure 3 presents the morphology of BC fiber in different culture media components. The analysis was conducted to reveal the character of the cellulose fibrils regarding size and shape. The BET was used to determine the porous structure of samples. In Fig. 3, BC fibrils experienced significant changes regarding the composition in culture media. In the culture media without HA, the size of BC fibrils was measured around 45.1 ± 8.4 nm and 48.4 ± 25.4 corresponding BC and BG, respectively. In the contrast, the diameter size of the fiber increased significantly ranging from 135.4 ± 20.2 to 147.5 ± 26.3 nm when there was HA. The fibril surface of samples containing HA was rough to what was observed from net BC and BG samples. Based on BET results in Table 3, the pore size of most of the samples was around 14–15 nm. It should be noted that although changes in fibril size were observed, these changes did not proportionate with changes in the porous structure of the samples. The BG sample showed the surface area, pore volume, and pore size at 48.1 m2/g, 0.17 cm2/g, and 13.77 nm, which is the lowest compared to the other sample. This can be a result of the colloidal structure of the culture media which reduced the movement of bacteria reaching the nutrient or forming a loose cellulose networking structure. These numbers only improved higher when there was the presence of HA in the media. The surface area of BGA 0.25 and BGA 0.5 was 52.8 and 59.7 m2/g, respectively. Data of pore volume and pore size of BGA membranes were close to the net BC.
Regarding BCA and BGA, although there was no significant difference in fiber size obtained, the EDS showed improvements in the elemental composition of that fiber growth in the media containing gum. In Table 3, the addition of gum in BC culture media provided a higher amount of Ca and P in the sample. The amount of Ca and P covered on the surface of the fibrils was about 2 times higher than samples at the same HA concentration but without GT. The percentage of Ca and P was normally used to prove the presence of elements specific to Hydroxyapatite in the material. In our previous study, hydroxyapatite was added to the BC culture media in the synthesized form on the CNC fiber surface to increase the dispersant ability [18]. The result showed a higher amount of Ca and P found in the BC matrix (1.02% and 0.59%, respectively) at the concentration of 0.5% of hydroxyapatite/CNC dispersants (HC) added in the culture media. According to [17], using solid agar shredded at 0.3% can trap the HA supernate which resulted in a higher amount of Ca and P found in the BC matrix. [16] reported the addition of 1% w/v CMC in the culture with 1% of HA during cultivation increased 23.7% of the inorganic phase of the total weight of the nanocomposite.
The interference of bacterial culture media by different additives returns various characteristics of HA incorporated into the BC matrix. In the presence of CMC, HA was found agglomerated in micrometric particles in different areas in the BC composite [16]. The incorporation of HA on BC fibrils using a CNC dispersant resulted in a spherical shape morphology of HA found in the BC matrix. HA particle in the BC matrix was found similar to their form in the synthesized particles scattered in the BC matrix [18]. Under the addition of GT, HA particles were found mostly in a size of around 64.3 ± 9.6 nm. The particles were found homogeneously covered on cellulose fibrils. In this experiment, the repeated results of SEM and EDS indicated the consistency of the approach to providing homogeneity of hydroxyapatite particle dispersion.
It can be seen that the attempt to attach HA particles to the BC matrix by increasing the colloidal ability of the bacterial culture media can be a selection. However, the matter is which substrate can be combined with the BC culture media. [16, 17, 22] reported a strong reduction of BC yield at the concentration of 1% w/v of agar shredded, xanthan gum, or CMC. When the colloidal structure of the culture media increased, the longer culture time was found. At 1% w/v of CMC, BC fibril size was found smaller comparing the net BC [16]. The spaces between the cellulose ribbons were also reported reduction with increasing CMC concentration due to the aggregation of CMC [25]. At 1% of agar shredded particles, there was no BC found. In the present study, although there was no significant difference in fiber size found, the addition of 1.8% w/v gum tragacanth experienced slower growth of BC with denser porosity. In the culture media containing only gum tragacanth, it took more than 10 days to get the goal thickness (Fig. 1 and Table 1). When there was the presence of HA, the BC took a shorter time. This might be due to a higher mineral component provided by the HA in the culture media.
Table 2
Fiber diameter and pore size analysis by BET
|
Fiber diameter (nm)
|
Surface area
(m2/g)
|
Pore volume
(cm3/g)
|
Pore size
(µm)
|
BC
|
45.1 ± 8.4
|
62.4
|
0.23
|
14.96
|
BG
|
48.4 ± 25.4
|
48.1
|
0.17
|
13.77
|
BCA 0.25
|
136.6 ± 25.8
|
62.5
|
0.22
|
14.36
|
BCA0.5
|
139.0 ± 7.1
|
58.4
|
0.20
|
14.00
|
BGA 0.25
|
147.5 ± 26.3
|
52.8
|
0.20
|
15.2
|
BGA 0.5
|
135.4 ± 20.2
|
59.7
|
0.22
|
14.7
|
Note: the means ± SD represents the standard deviation of at least three independent experiments. |
Table 3
Elemental composition of samples by EDS
|
Elemental composition (% Atomic)
|
C K
|
O K
|
P K
|
Ca K
|
BC
|
55.81 ± 1.03
|
44.15 ± 0.95
|
-
|
-
|
BG
|
55.32 ± 0.32
|
44.62 ± 0.32
|
-
|
-
|
BCA 0.25
|
54.99 ± 0.2
|
44.78 ± 0.37
|
0.09 ± 0.05
|
0.15 ± 0.11
|
BCA0.5
|
54.37 ± 0.46
|
44.85 ± 0.36
|
029 ± 0.03
|
0.5 ± 0.07
|
BGA 0.25
|
53.83 ± 0.35
|
45.48 ± 0.3
|
0.26 ± 0.03
|
0.43 ± 0.05
|
BGA 0.5
|
52.48 ± 0.32
|
45.97 ± 0.07
|
0.57 ± 0.1
|
0.98 ± 0.18
|
*Note: the means ± SD represents the standard deviation of at least three independent experiments.
|
XRD
The interaction between HA and GT on the crystallinity behavior of BC composites is revealed in the XRD patterns. The XRD pattern and the crystallinity index of all samples are shown in Fig. 4 and Table 4, respectively. Typically, BC origin samples show three diffraction peaks at values around 14.5, 16.8, and 22.4, which correspond to the Miller indices of diffraction crystallographic planes of (100), (010), and (110), can be assigned to Iα cellulose [26]. It can be seen that HA expresses a weaker peak on the XRD spectra of BGA samples than those corresponding to pure HA. However, in different culture media, the cellulose fibrils exposed the distinct characteristic of a crystalline structure. The net BC had around 57.1 ± 1.2 CrI (%). In the presence of GT, the CrI reduced a bit lower to number around 52%. This number was witnessed to increase significantly when there was about 0.5% of HA in the culture media which showed in BGA 0.5 at 68.4 ± 1%.
According to [16, 27], a decrease in crystallite size is proportional to the decrease in average microfibril size (which was determined by TEM). In our study, the result may be in agreement when comparing the CrI of BC and BG with the CrI of BGA 0.5. Changes in CrI in the incorporation of HA on BC fiber can strongly relate to the amount of the mineral component attached to microfibrils. The results of CrI in the same group of samples with different amounts of HA in the culture media support this idea. Samples of 0.25% of HA exhibited a significantly lower CrI although fiber size was measured similarly to those of 0.5%. Although most of the main factors are listed, this cannot be ruled out that those factors such as incubation time and thickness of BC pellicles may alter more or less in the CrI of samples due to all samples showing specific yield and growth rate in culture media researched in the study. As observed in BET results, GT could have interfered self-assembly process of BC fibers. The presence of GT on the surface of the cellulose fibrils may prevent the formation of hydrogen bonds between the fibrils [22, 28]. This led to a reduction in crystallinity.
Table 4
Mechanical properties and CrI (%) of samples
|
Tensile strength
(N/mm2)
|
Young modulus
(N/mm)
|
Train at break (%)
|
CrI (%)
|
BC
|
3.5 ± 0.3c
|
21.8 ± 0.8c
|
4.9 ± 0.6a
|
57.1 ± 1.2a
|
BG
|
0.8 ± 0.03a
|
1.8 ± 0.1a
|
8.6 ± 1.0b
|
52.7 ± 0.8a
|
BGA 0.25
|
1.7 ± 0.25b
|
10.4 ± 2.0b
|
4.6 ± 1.1a
|
56.1 ± 0.7b
|
BGA 0.5
|
1.6 ± 0.1b
|
13.9 ± 3.2b
|
4.1 ± 0.2a
|
68.4 ± 1c
|
Means in the same column with different letters are significantly different (p ≤ 0.05).
Mechanical properties of BGA membranes
In Table 4, it is evident that the tensile young modulus of BGA membranes decreased in the presence of GT. BG showed the lowest tensile strength and Young’s modulus but the deformability was a bit greater comparing the other samples. When there was HA, the rheology of the composites improved higher in which the young modulus of BGA 0.25 and BGA 0.5 was at 10.4 and 13.9, respectively. This can be noted that the presence of foreign substrates in the BC membrane required a higher attempt for cellulose fibrils to form bonding with the additives. The colloidal texture of culture media altered the interaction between the chain, followed by a reduction in the intermolecular hydrogen bonds. This was observed in the physiochemical characteristics of the composite. In the presence of HA, the homogenized distribution of the mineral particles stabilized the BC fibrils. This improved the mechanical integrity of the composite when there was a higher amount of HA incorporated in the matrix [24, 29].
Cell viability
Before the main assay was conducted, cell biocompatibility by SEM and cell cytotoxicity were performed as preliminary tests to determine the possibility of culturing cells of the membranes. Generally, the indirect cell test (Fig. 5B) showed that BC membranes containing HA in this study caused no toxicity to the pre-osteoblast cell. All of the samples possessed a number of more than 90% cell survival after one-day incubation with the cells. Interestingly, the biocompatibility test by SEM showed that the morphology of MC 3T3 cells flattened out on the surface like the BC membranes. On day 3rd of the incubation, cells displayed an extracellular matrix of filamentous fibers formed with large lamellipodia that anchored and pulled the cell body by grasping onto the surrounding area. The cells adhered and spread to the surface of the membranes which maintained an extended fibroblast membrane morphology. This confirmed a well-adapted cell on the membranes.
Cell viability of MC3T3 on bacterial cellulose-based membranes was analyzed by using MTS assay and photometric observation by live/dead cell assay under confocal. It is shown in Fig. 5C that the BC, BG, and BGA membranes could support osteoblast cells to proliferate as a scaffold. The number of cell survival on BC and BGA membranes on day 1 had a similar number in which more than 90% of cells survived detected. After 7 days of incubation, the cells on BGA membranes expressed a significant number compared to the others. The trend of data was predictable since the presence of HA has been well reported to support the cell attachment on the membrane [23]. Noticeably, the data obtained from the BG membrane showed a slight reduction in cell survival and proliferation after 7 days of incubation. In previous studies, several materials took benefit from gum to providing a biocompatible material for cells [21, 30–32]. It is well known that GT contains two main fractions of bassorin (water-swellable portion) and a water-soluble portion named tragacanthin [20]. After the purification process, the soluble fractions of GT may be washed out remain insoluble fractions on the BC fibrils. The hydrophilic character of the membrane is a factor that facilitated the adherence of cells over the surface through hydrogen bonding [33]. When GT covers BC fibrils, GT may change the ability of protein absorption or hydrophilic of the membrane. This led to the changes in cell numbers found on the membrane. Although the number was slightly lower than observed on the BG, all of the data exhibited no significantly different number on day 3rd of incubation. In the presence of HA, these problems caused by GT were minimal because the finding result proved that the cells grew well on BGA composites. At different levels of HA, there were no significant differences observed in the cell survival by MTS assay in the first 3 days of culturing. A significant difference was only obtained when the cells had a longer time staying on the material. At the time point of day 7th, the viability of cell growth on the BGA 0.5 membrane was recorded at the highest level, around 200%, which showed a significant leap from the others.
Figure 6 presents the growth of MC3T3 cells under 3-time points. It is well noted that BC has high hydrophilic OH- groups and excellent liquid adsorption performance that expressed good biocompatibility with the cells [34]. As the feature of a good network structure, the BC-based membrane absorbs good ECM in the culture media, becoming a suitable environment for cell attachment and proliferation. At the first day point, it can be seen that cells on the BC membrane had elongated shapes. There were just a few cells recorded with rounded morphology. This elongated morphology of the cell was even recorded more on the BGA membranes. On BGA membranes, cells were faster becoming a group with the aggregation of many cells. Cell-to-cell contacts were established via long and thin projections. After 7 days of culturing, pellets of cells were found on membranes.
Alkaline phosphate enzyme determination
Besides quantities and morphological criteria, the ability to produce ALP enzyme inner cells was used in order to further understand the effect of the BGA membranes on the growth and differentiation of the MC3T3. ALP is known as an early marker for osteogenic differentiation [35]. In Fig. 7, the changes in the ratio of ALP enzyme/total protein during 21 days of culture is presented. The ratio of ALP/total protein produced was used despite the effect of the number of cells on the amount of enzyme recorded. In the control of cells grown on a well-plate, the ratio of ALP enzymes was the highest on the 7th day of cultivation with the number around 5.2 nMol/Min/µgProtein. The peak of this ratio in BC samples was on the 21st day of cultivation. It can be observed that the higher the amount of HA in the composite, the faster the ALP enzyme secrete in cells. All BGA samples showed a higher amount of ALP enzyme/total protein compared to the other controls. Cells on BGA 0.5 membrane peaked at the ALP enzyme on the 3rd day of cultivation and then reduced to the number 4.4 nMol/Min/µgProtein on day 21st. In BGA 0.25, the highest ALP enzyme was recorded on day 7th with the amount around 7.9 nMol/Min/µgProtein. The changes in the ALP enzyme recorded in this study proved that the BGA membrane can support osteoblast cells in differentiation and mineralization.