*3.2. Physical Properties of Uncoated CT and Coated CT/CMC + CA*

### 3.2.1. Basis Weight and Thickness

As seen in Table 3, the basis weight and thickness of coated CT are directly proportional to the coating layer and concentration of solids in the coating formulation. Coated samples gained the basis weight and thickness as expected compared with uncoated samples, with double-coated samples accumulating the highest weight gain and thickness. CT coated with CMC increased its weight and thickness but not as much as CT coated with CMC + CA. The substantial increase in CMC + CA samples' weight proved that cross-linked CMC + CA has better coating efficiency due to the accumulation of CA attached to the -OH group of CMC after the esterification process. It was also found that the CT/CMC weight and average thickness increased significantly with CA concentration. This is because the higher the CA concentration, the higher the probability of CA to cross-link with CMC. At a higher crosslinking degree, CMC + CA becomes more viscous, allowing more intermolecular bonds to form between CMC and CA [35], thus increasing their weight and average thickness. For medical purposes, such as skin grafting, it is proposed that the dressing film should be thinner than 400 µm to allow vascularization for nutrient delivery to the epidermis [36]. Therefore, the coating thickness of the samples was fabricated within the desirable range except for the samples OIR-1 and OIR-2 at 4M CA.


**Table 3.** Physical measurement of uncoated and coated cotton thread samples.

For the curing process via several drying regimes, IR samples showed the smallest thickness, followed by OD and OIR samples in single- and double-coated CT. A higher sample thickness on OIR samples is due to the unequal heating energy imposed in the transitioning process between the samples from OD and IR in the sequential drying regimes of OIR. Meanwhile, samples dried via IR and OD provided more uniform heating, allowing moisture from the samples to be removed efficiently and causing a substantial difference reduction in sample size [15]. Nevertheless, based on sample thickness, IR is the most effective, followed by OD and, finally, OIR. OIR is not as effective as expected, particularly as a solitary drying regime. It is due to the uneven energy used to dry the samples, and the samples are prone to absorb moisture when transitioning from one drying regime to another. Therefore, the ineffectiveness of OIR will have to prolong the drying time or increase the heating energy for the sample to be dried effectively.

#### 3.2.2. Moisture Content

Moisture content was determined to evaluate the amount of moisture in the conditioned uncoated and coated CT. After the drying process, the extent of destruction to the lamellar structure of the macromolecule network will differ among different drying regimes. Hence, the ability to absorb the surrounding moisture will determine the severity of drying regimes to the CMC-CA polymer networks. The ability to retain moisture is crucial for the CMC + CA coating on CT to function as a functional wound dressing. According to the Gibbs–Donnan effect theory, in regard to the transportation across cell membranes, if the Gibbs–Donnan equilibrium between the exterior surroundings and the wound bed is achieved, the increase in intracellular ions would cause cells to swell due to the osmotic influx of water. Low ionic strength can lead to a high distribution of ion mobility, establishing the Gibbs–Donnan equilibrium between the external environment and wound bed, and thus providing a stable structure for the coated CT to retain moisture and/or for drug delivery systems for wound healing [37].

Based on Table 4, the moisture content of CT/CMC + CA gradually decreases with the increase in the coating layer in all drying regimes. Increases in coating layers resulted in an increase in ionic strength, which reduced the number of mobile ions within the CMC + CA coating matrix, thus reducing the CMC + CA coating structure and limiting the moisture content of CT/CMC + CA [38]. The result is similar to the findings of other biomaterials, such as membrane [39] and hydrogel films [29,40], that are cross-linked with CA. Meanwhile, IR-1 shows the highest moisture content among the single-coated samples, followed by OD-1 and OIR-1. The same trend can be observed for the double-coated samples. The increase in moisture content at a low concentration of 2M CA in all drying regimes shows that the CMC + CA coated on CT can retain moisture. This is caused by the hygroscopic nature of CT/CMC + CA with hydroxyl and polar groups available and the interfacial area between CT and the CMC + CA matrix [41]. On the other hand, at a higher CA concentration (>2M) and double-coated CT/CMC + CA samples, a low moisture content was obtained, and it correlates with the cross-linking effect, which reduces the number of free hydrophilic groups in the coating, thus limiting molecular mobility, and binds the polymer together tightly [40].


**Table 4.** Moisture content (%) of CMC cross-linked with different concentrations of CA-coated cotton thread samples.

The findings are consistent with the basis weight of coated samples portrayed in Table 2. A higher moisture content by IR showed that IR is more effective in removing the moisture than OD and OIR. At a similar temperature and time, unlike OD, IR minimized the capillary force driven by the surface tension of water, hindering the destruction of its lamellar structure. For OIR, the lowest moisture content absorbed by them is due to the sequential drying regime unable to effectively remove moisture during the drying process, resulting in high capillary force and the destruction of OIR samples' lamellar structure, rendering them unable to retain moisture.

#### *3.3. Surface Morphology of Uncoated CT and Coated CT/CMC + CA*

The surface morphology of uncoated and coated samples is shown in Figures 4 and 5, respectively. In Figure 4, the uncoated samples (CT-A, CT-B, and CT-C) showed no significant difference in terms of morphology and embodied plenty of empty spaces among the cotton fibers, making them porous. This will allow CMC + CA solutions to penetrate easily during the coating process.

**Table 4.** Moisture content (%) of CMC cross-linked with different concentrations of CA-coated cot-

CT-B n.a IR 3.05 ± 0.03 100 ± 0.02 CT-C n.a Oven+IR 3.11 ± 0.03 84.6 ± 0.03

CMC

CMC

CMC

CMC

CMC

CMC

*3.3. Surface Morphology of Uncoated CT and Coated CT/CMC+CA* 

*Polymers* **2022**, *14*, x 14 of 23

**gime** 

Oven

IR

Oven+IR

Oven

IR

Oven+IR

The surface morphology of uncoated and coated samples is shown in Figures 4 and 5, respectively. In Figure 4, the uncoated samples (CT-A, CT-B, and CT-C) showed no significant difference in terms of morphology and embodied plenty of empty spaces among the cotton fibers, making them porous. This will allow CMC+CA solutions to pen-

Figure 5 shows CT morphology after coating (single and double coat) and the respective drying process. In comparison with Figure 4, Figure 5 shows that the empty spaces

n.a Oven 3.08 ± 0.04 92.3 ± 0.02

2 3.43 ± 0.06 95.0 ± 0.03 3 3.29 ± 0.06 45.8 ± 0.04 4 3.17 ± 0.03 19.0 ± 0.05

2 3.39 ± 0.03 120.9 ± 0.04 3 3.28 ± 0.02 70.9 ± 0.02 4 3.15 ± 0.04 34.1 ± 0.03

2 3.45 ± 0.04 90.3 ± 0.02 3 3.38 ± 0.03 44.6 ± 0.03 4 3.33 ± 0.04 18.0 ± 0.06

2 3.38 ± 0.03 36.1 ± 0.05 3 3.22 ± 0.04 32.1 ± 0.06 4 3.11 ± 0.03 12.5 ± 0.03

2 3.31 ± 0.04 71.9 ± 0.04 3 3.21 ± 0.03 50.0 ± 0.05 4 3.11 ± 0.06 34.0 ± 0.03

2 3.43 ± 0.03 24.4 ± 0.02 3 3.37 ± 0.06 17.2 ± 0.06 4 3.31 ± 0.03 7.1 ± 0.05

**Moisture Content (%) Dry Wet** 

3.15 ± 0.04 88.9 ± 0.06

3.14 ± 0.02 63.2 ± 0.03

3.33 ± 0.06 33.3 ± 0.03

3.12 ± 0.02 19.5 ± 0.02

3.09 ± 0.06 70.6 ± 0.03

3.31 ± 0.04 20.0 ± 0.06

**Samples Coating Layer (CA) (M) Drying Re-**

0

1

2

etrate easily during the coating process.

ton thread samples.

CT-A

OD-1

IR-1

OIR-1

OD-2

IR-2

OIR-2

**Figure 4.** Uncoated cotton thread surface of (**a**) CT-A, (**b**) CT-B, and (**c**) CT-C. **Figure 4.** Uncoated cotton thread surface of (**a**) CT-A, (**b**) CT-B, and (**c**) CT-C. dressing.

**Figure 5.** Observation under an optical microscope of a single coat (OD-1, IR-1, and OIR-1) and double coat (OD-2, IR-2, and OIR-2) cross-linked with (**a**) 2M CA, (**b**) 3M CA, and (**c**) 4M CA under different drying regimes with a scale at 0.1 mm. **Figure 5.** Observation under an optical microscope of a single coat (OD-1, IR-1, and OIR-1) and double coat (OD-2, IR-2, and OIR-2) cross-linked with (**a**) 2M CA, (**b**) 3M CA, and (**c**) 4M CA under different drying regimes with a scale at 0.1 mm.

As shown in Figure 5, the cotton threads coated with CMC cross-linked with CA presented textured surfaces, particularly when a high concentration of CA (4M CA) was used, which affected their physical properties. CA-cross-linked CT/CMC thread had more protrusions than uncoated cotton threads, especially at a high CA concentration, indicating increased surface roughness. On the other hand, double-coated samples (OD-2, IR-2, and OIR-2) showed excessive citric acid crystal formations due to the high CA amount present in the coating, promoting a coarser surface on cotton thread. The enhancement of surface roughness in a medical application provides a better interaction zone between the dressing with the cells and tissue surface, allowing dermal fibroblast adhesion [42], which enhances cell adhesion and cell proliferation [43]. This aids in strong protein adhesion to the wound dressing surfaces, implying that a rough surface can stimulate cell signaling Figure 5 shows CT morphology after coating (single and double coat) and the respective drying process. In comparison with Figure 4, Figure 5 shows that the empty spaces between the cotton thread fibers of single- and double-coated samples are filled with coated materials. In Figure 5, all samples show that the CMC-CA were homogeneously and evenly coated on the CT surfaces. The cross-linking between CT/CMC + CA involves the interaction of covalent esters composited as a coating layer that enhances the physicochemical properties of the CT. This is a crucial feature for CT/CMC + CA to achieve a stable structure. The stable structure permits CT/CMC + CA moisture retention enhancement, as shown in Section 3.2.2, and further improvement of CT/CMC + CA mechanical properties and absorption capability. Therefore, CT/CMC + CA can be utilized as a functional wound dressing.

by imitating similar cues as the extracellular matrix network [44–46]. IR-1 and IR-2 samples visually showed a smoother coating topography than OD and OIR drying samples, which exhibited rougher surface coverage. This was due to IR drying involving a more uniform heating mechanism where the temperature of the samples was As shown in Figure 5, the cotton threads coated with CMC cross-linked with CA presented textured surfaces, particularly when a high concentration of CA (4M CA) was used, which affected their physical properties. CA-cross-linked CT/CMC thread had more protrusions than uncoated cotton threads, especially at a high CA concentration, indicating increased surface roughness. On the other hand, double-coated samples (OD-2, IR-2, and OIR-2) showed excessive citric acid crystal formations due to the high CA amount present

in the coating, promoting a coarser surface on cotton thread. The enhancement of surface roughness in a medical application provides a better interaction zone between the dressing with the cells and tissue surface, allowing dermal fibroblast adhesion [42], which enhances cell adhesion and cell proliferation [43]. This aids in strong protein adhesion to the wound dressing surfaces, implying that a rough surface can stimulate cell signaling by imitating similar cues as the extracellular matrix network [44–46].

IR-1 and IR-2 samples visually showed a smoother coating topography than OD and OIR drying samples, which exhibited rougher surface coverage. This was due to IR drying involving a more uniform heating mechanism where the temperature of the samples was increased internally [15]. When the samples were heated internally via the IR drying process, disturbance to the polymer interactions scarcely occurred, facilitating a uniform solidification of coating materials interpreted by a smoother topography as depicted in Figure 5. Meanwhile, for OD and OIR samples in Figure 5, nonuniform topography is triggered by the sample's placement during the drying process. Samples' positioning promotes temperature variations in the oven environment, which subsequently cause propagation of polymer interactions during the solidification process of coating materials [15,16]. Besides, a sequential drying process of OIR is inefficient due to the change of heating energy between the two drying regimes. In addition, the surrounding factor during the transition of OD to IR drying is exacerbated by moisture absorption and the sudden temperature drop and, thereupon, disturbs the solidification process of coating materials. In essence, due to the uneven heating energy, OD- and OIR-dried samples exhibit a heterogeneous structure and texture. With this in mind, different drying regimes gives different morphology depending on the drying mechanism efficiency [47].

#### *3.4. Mechanical Properties of Uncoated CT and Coated CT/CMC + CA*

The tensile test was performed to investigate the effects of coating layers and drying regimes on the mechanical performances of CT/CMC + CA. The samples' tensile strength and elongation percentage (%) are presented in Figures 6 and 7. *Polymers* **2022**, *14*, x 16 of 23

**Figure 7.** Elongation percentage of CT/CMC samples cross-linked with different CA concentrations

dried by (**a**) OD, (**b**) IR, and (**c**) OIR.

OD, (**b**) IR, and (**c**) OIR

**Figure 7.** Elongation percentage of CT/CMC samples cross-linked with different CA concentrations dried by (**a**) OD, (**b**) IR, and (**c**) OIR. **Figure 7.** Elongation percentage of CT/CMC samples cross-linked with different CA concentrations dried by (**a**) OD, (**b**) IR, and (**c**) OIR.

**Figure 6.** Tensile strength of CT/CMC cross-linked with different CA concentrations dried by (**a**)

Figures 6 and 7 show that the tensile strength and elongation percentage of CT/CMC + CA are higher than those of uncoated samples. It is well understood that as tensile strength increases, the elongation percentage decreases and vice versa [48]. However, CA, as the crosslinker in the coating system, increases tensile strength and elongation due to a higher potential of interaction between material and matrix polymer, resulting in increased of intra- and intermolecular cross-linking between polymers [49]. The CT fiber twisting orientation also plays a role in the increase in tensile strength and elongations percentage [50] as the twisting provides extra length, allowing CT to stretch more when a load is applied. The extension increased CT lateral pressure and compaction and, as a result, CT became denser and more coherent to strain. CT has a slightly loose twisting orientation that allows CMC + CA to penetrate and coat individual fiber surfaces, which further improves CT load bearing capacity by intercepting higher loads, thus leading to the improvement of elongation and avoiding early breakage [51].

The interactions contributed to a more coherent structure, improving the tensile strength and elongation percentage of CT/CMC + CA. When comparing single- and double-coated samples, single-coated CT had higher tensile strength than double-coated samples. Generally, the tensile strength of composites increases with composite content until a maximum or optimum value is achieved. Once the maximum value is reached, the tensile strength will decrease. A higher tensile strength for single-coated CT for all drying regimes is due to the penetration of the coating solution between the fibers. CMC + CA acts as a space filler, which also contributes to the load distribution, increasing the tensile strength and elongation percentage [52]. However, the reversed scenario was observed for double-coated CT/CMC + CA samples. This is due to the high amount of CMC + CA content in the double-coating layer that reduces the matrix mobility, making it stiffer, hence indicating that the amount of CMC + CA in double-coated CT might have exceeded the optimum composite content of CT/CMC + CA strength [53].

The presence of CA caused significant differences (*p* < 0.05) in the tensile strength. In all drying regimes, regardless of coating layers, the tensile strength and elongation percentage are the highest at 2M CA and continuously decrease afterward. At higher

concentrations of CA, namely, 3M and 4M, clumping is prone to occur due to the quick cross-linking process between polymers. When cross-linking occurs at a higher rate, it causes a higher number of CA that might have cross-linked with CMC in the matrix, causing CMC + CA agglomeration. With that being said, a higher concentration of CA leads to clumping [49,54], thus acting as stress concentrators, causing CT to be stiff and resulting in lower mechanical performances [55]. Based on the results obtained, the mixture performed best at a lower concentration of CA (2M) to improve the tensile properties of CT because of the intramolecular and intermolecular interaction forces, which enhanced the coated CT strength due to the formation of a stronger network force in the matrices. In addition, at higher CA concentrations (>2M) and coating layers, high CA content results in the formation of CMC + CA crystals (see Figure 6). This causes the CT/CMC + CA structure to be brittle [56]. Brittleness degrades the mechanical performances of CT by disrupting the elasticity and intermolecular force network in the CMC + CA matrices. This explains the low tensile properties of the above-stated determinant. CA concentrations of more than 2M were shown to be unsatisfactory when conjoined with the double coating process.

Apart from the effect of the coating layer, an apparent distinction between different drying regimes can also be seen in Figure 6. Overall, IR-dried single- and double-coated CTs showed the highest increment in tensile strength and elongation percentage, followed by OD and OIR. The tensile strength of single-coat samples OD and IR improved by 33% and 36%, respectively. In contrast, for double-coated samples, the tensile strength increased by 24% and 30%, respectively, compared with the uncoated samples. As regards OIR, it showed the lowest tensile strength improvement at 26% for single coat and 18% for double coat. The tensile strength between the single- and double-coated samples had a significant difference of *p* < 0.05 through all drying regimes. The significant differences were attributed to a uniform heating pattern of OD and IR drying in the single-coated samples, which allowed moisture to evaporate efficiently [15] compared with the doublecoated samples. However, for OIR, despite the significant differences, the regime did not effectively remove moisture from the coated samples, both single and double, as shown in Table 4, under the dry moisture content percentage. A rapid removal of water induced by IR is more focused than the OIR technique, where the differences in heating energy during OIR transitioning affect samples' tensile properties. In OIR, environmental factors, such as moisture absorption that ensued with a temperature drop during the samples' transitioning process, should also be considered. Due to this matter, incomplete drying can cause the formation of a void of a channel structure in the coating matrix, which also leads to the presence of moisture during OIR sample fabrication, which disrupts the solidification of CMC + CA due to air entrapment in the coating structure [57]. A heated CT via OD prior samples transferred to IR creates osmotic differences between samples and the environment. Thus, moisture will be absorbed by the CT, which leads to a temperature drop. Consequently, OIR CT/CMC + CA samples have irregularities in their chemical and physical interactions due to the fluctuating sample temperature, which were later signified by the OIR sample morphological structure and tensile strength.

#### *3.5. Effects of Drying and Coating Layers on Water Absorption*

The average water absorption of CT/CMC + CA samples versus time is plotted in Figure 8. It can be observed that the water absorption of CT/CMC + CA samples increased with an increasing time and equilibrium after 5 min of immersion for single-coated samples and 3 min of immersion for double-coated samples in all drying regimes. Due to the relatively higher CMC + CA content in double-coated samples than single-coated samples, their water absorption capability is reduced, causing it to reach its maximum capacity faster than single-coated samples. When CMC + CA double-coats the CT, the solid content increases due to the cross-linking interactions, creating more networks between CMC and CA, thus reducing the mobility of the ionized ionic group in the CMC + CA matrix due to a stiffer and denser structure [31,56]. On the other hand, in a single CMC + CA coating, the

carboxyl groups (ionic group) in CMC are known to be highly hydrophilic. When ionized (COO-), it increases the electrostatic repulsion between intramolecular and intermolecular interaction forces to open the polymer matrix, increasing the water absorption properties to the materials [58]. However, by adding double layers, the coating structure became stiffer and denser. This resulted in moderate changes in the CT/CMC+CA properties, such as tensile strength, moisture absorption, and water absorption, when a single layer was applied, and a significant impact when double layers were added, regardless of the drying regimes used.

increases due to the cross-linking interactions, creating more networks between CMC and CA, thus reducing the mobility of the ionized ionic group in the CMC+CA matrix due to a stiffer and denser structure [31,56]. On the other hand, in a single CMC+CA coating, the carboxyl groups (ionic group) in CMC are known to be highly hydrophilic. When ionized (COO-), it increases the electrostatic repulsion between intramolecular and intermolecular interaction forces to open the polymer matrix, increasing the water absorption properties

According to Figure 8, an obvious pattern is seen between different concentrations of CA. A higher CA causes a continual reduction in water absorption, with the most conspicuous at 4M CA. The CT coated with CMC + CA at 4M CA water absorption is even lower than the CT itself. Even though CMC and CA are naturally hydrophilic, excessive cross-linking events between polymers can induce the hydrophobic character. The induction is triggered by physical changes, where the cross-linking effect of CA results in the coat being hydrophobic with a high-density structure [28]. A high CA concentration can increase the solid content of the CT/CMC + CA matrix, which causes the cross-linking interactions to create a stronger bond between CMC and CA, resulting in a compact structure and higher density [34,58]. Besides, the carboxyl groups (ionic group) in CMC are known to be highly hydrophilic. When ionized (COO-), it increases the electrostatic repulsion between intramolecular and intermolecular interaction forces to open the polymer matrix, increasing the water absorption properties to the materials [58]. As proven in the surface morphology analysis, the different drying methods affect the reinforcement of CMC+CA into the CT structure. For this reason, water absorption is also altered, and the hydrophilicity of CMC+CA is influenced by different drying methods. However, other factors, such as coating layers, contribute to the result and must be considered. In this study, a single layer of CMC+CA was sufficient to coat the CT and penetrate the CT fiber.

*Polymers* **2022**, *14*, x 18 of 23

to the materials [58].

**Figure 8.** Water absorption behavior of CT/CMC+CA based on different drying methods. (**a**) OD-1, (**b**) IR-1, (**c**) OIR-1, (**d**) OD-2, (**e**) IR-2, and (**f**) OIR-2. **Figure 8.** Water absorption behavior of CT/CMC + CA based on different drying methods. (**a**) OD-1, (**b**) IR-1, (**c**) OIR-1, (**d**) OD-2, (**e**) IR-2, and (**f**) OIR-2.

*3.6. Antibacterial Activity of Coated CT*  According to Figure 8, an obvious pattern is seen between different concentrations of CA. A higher CA causes a continual reduction in water absorption, with the most conspicuous at 4M CA. The CT coated with CMC + CA at 4M CA water absorption is even lower than the CT itself. Even though CMC and CA are naturally hydrophilic, excessive cross-linking events between polymers can induce the hydrophobic character. The induction is triggered by physical changes, where the cross-linking effect of CA results in the coat being hydrophobic with a high-density structure [28]. A high CA concentration can increase the solid content of the CT/CMC + CA matrix, which causes the cross-linking interactions to create a stronger bond between CMC and CA, resulting in a compact structure and higher density [34,58]. Besides, the carboxyl groups (ionic group) in CMC are known to be highly hydrophilic. When ionized (COO-), it increases the electrostatic repulsion between intramolecular and intermolecular interaction forces to open the polymer matrix, increasing the water absorption properties to the materials [58]. As proven in the surface morphology analysis, the different drying methods affect the reinforcement of CMC + CA into the CT structure. For this reason, water absorption is also altered, and the hydrophilicity of CMC + CA is influenced by different drying methods. However, other factors, such as coating layers, contribute to the result and must be considered. In this study, a single layer of CMC + CA was sufficient to coat the CT and penetrate the CT fiber. However, by adding double layers, the coating structure became stiffer and denser. This resulted in moderate changes in the CT/CMC + CA properties, such as tensile strength, moisture absorption, and water absorption, when a single layer was applied, and a significant impact when double layers were added, regardless of the drying regimes used.
