polymers
Article
Physicochemical Characterization, Biocompatibility, and
Antibacterial Properties of CMC/PVA/Calendula officinalis
Films for Biomedical Applications
Wen-Hsin Huang 1,† , Chia-Yi Hung 2,3,† , Pao-Chang Chiang 3 , Hsiang Lee 4 , I-Ting Lin 3 , Pin-Chuang Lai 5 ,
Ya-Hui Chan 6, * and Sheng-Wei Feng 3,6,7, *
Citation: Huang, W.-H.; Hung, C.-Y.;
Chiang, P.-C.; Lee, H.; Lin, I.-T.; Lai,
P.-C.; Chan, Y.-H.; Feng, S.-W.
Physicochemical Characterization,
Biocompatibility, and Antibacterial
Properties of CMC/PVA/Calendula
officinalis Films for Biomedical
Applications. Polymers 2023, 15, 1454.
https://doi.org/10.3390/
polym15061454
Academic Editor: Bruno Medronho
Received: 21 February 2023
Revised: 11 March 2023
Accepted: 12 March 2023
Published: 14 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Polymers 2023, 15, 1454. https://doi.org/10.3390/polym15061454
1 Department of Stomatology, MacKay Memorial Hospital, Taipei 104, Taiwan
2 School of Dentistry and Graduate Institute of Dental Science, National Defense Medical Center,
Taipei 114, Taiwan
3 School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan
4 Pro Hallmarks Taiwan Ltd., Taipei 114, Taiwan
5 Department of Periodontics, University of Missouri-Kansas City School of Dentistry,
Kansas City, MO 64108, USA
6 School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan
7 Division of Prosthodontics, Department of Dentistry, Taipei Medical University Hospital, Taipei 110, Taiwan
* Correspondence: babypg@hotmail.com (Y.-H.C.); shengwei@tmu.edu.tw (S.-W.F.);
Tel.: +886-2-2736-1661 (ext. 5104) (Y.-H.C.); +886-2-2736-1661 (ext. 5107) (S.-W.F.)
† These authors contributed equally to this work.
Abstract: This study reports a carboxymethyl cellulose (CMC)/polyvinyl alcohol (PVA) composite
film that incorporates Calendula officinalis (CO) extract for biomedical applications. The morphological,
physical, mechanical, hydrophilic, biological, and antibacterial properties of CMC/PVA composite
films with various CO concentrations (0.1%, 1%, 2.5%, 4%, and 5%) are fully investigated using
different experiments. The surface morphology and structure of the composite films are significantly
affected by higher CO concentrations. X-ray diffraction (XRD) and Fourier transform infrared spec-
trometry (FTIR) analyses confirm the structural interactions among CMC, PVA, and CO. After CO is
incorporated, the tensile strength and elongation upon the breaking of the films decrease significantly.
The addition of CO significantly reduces the ultimate tensile strength of the composite films from 42.8
to 13.2 MPa. Furthermore, by increasing the concentration of CO to 0.75%, the contact angle is de-
creased from 15.8◦ to 10.9◦ . The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
assay reveals that the CMC/PVA/CO-2.5% and CMC/PVA/CO-4% composite films are non-cytotoxic
to human skin fibroblast cells, which is favorable for cell proliferation. Remarkably, 2.5% and 4% CO
incorporation significantly improve the inhibition ability of the CMC/PVA composite films against
Staphylococcus aureus and Escherichia coli. In summary, CMC/PVA composite films containing 2.5%
CO exhibit the functional properties for wound healing and biomedical engineering applications.
Keywords: carboxymethyl cellulose; polyvinyl alcohol; antibacterial property
1. Introduction
Wounds in skin tissues are usually caused by thermal, chemical, and traumatic injuries.
Wound healing is one of the most complex and dynamic processes in the human body,
involving homeostasis, inflammation, proliferation, and the remodeling of skin tissue [1].
However, wound healing is easily affected and delayed in patients with diabetes mellitus
and complicated systematic health conditions [2,3]. In addition, delays to wound healing
significantly increase the risk of wound infection, which results in immune responses, tissue
inflammation, and damage. Thus, the development of wound dressings with biocompatible
and antimicrobial properties is important to accelerate the wound healing process [4].
https://www.mdpi.com/journal/polymers
Polymers 2023, 15, 1454 2 of 13

Recently, biopolymer-based wound dressings prepared by combining natural and synthetic

polymers have attracted considerable attention.
Carboxymethyl cellulose (CMC) is a natural polymer and water-soluble derivative
of cellulose. CMC has been widely used in the pharmaceutical, cosmetic, and food indus-
tries because of its excellent film-forming ability, high swelling ability, biocompatibility,
biodegradability, hydrophilicity, cost-effectiveness, and stable internal network structure
properties [5,6]. The hydrophilic groups (CH2 COO−) of CMC enhance hydrogen bonding
and bind to the hydroxyl groups of the glucopyranose chain of cellulose that constitutes
its backbone [6,7]. Furthermore, CMC is a pH- and ionic strength-sensitive polysaccha-
ride. As a polyanionic polymer, CMC has been found to possess bioadhesive properties
and can strongly attach to the mucosal surfaces of the oral cavity and gastrointestinal
tract [7]. The mucoadhesive properties of CMC allow for prolonged contact time in the
specific tissues, thus enhancing bioavailability and preventing degradation for drug de-
livery applications [8]. In addition, CMC has also been shown to adhere to the mucous
membrane and skin easily, which is beneficial for wound healing and skin regeneration
applications [9]. These properties are crucial for the preparation of CMC scaffolds, hydro-
gels, and films for biomedical applications. However, the weaknesses of CMC include its
poor mechanical strength, stability, and barrier properties [10,11]. To achieve significant
effects, blending with other water-soluble polymers is a promising solution to overcome
the disadvantages [12].
Among the most common synthetic polymers, poly(vinyl alcohol) (PVA) is a water-
soluble semi-crystalline polymer that is predominantly composed of carbon chains. PVA
is used in numerous biomedical and pharmaceutical applications because it possesses
many useful characteristics, including biodegradability, flexibility, hydrophilicity, cell-
adhesive properties, controlled tensile strength, non-toxicity, and an excellent film-forming
ability [12,13]. PVA is prepared through the polymerization of vinyl acetate followed by
partial hydrolysis. PVA is a non-ionic polymer with an odorless and translucent nature,
and it has the ability to form an oxygen barrier [14]. Both CMC and PVA are biodegrad-
able and biocompatible. After blending CMC with PVA, the strong hydrogen bonding
between the hydroxyl groups results in a remarkable improvement in the mechanical
properties [12,15,16]. In addition, recent studies have suggested that the incorporation
of bio-compounds can improve the bioactivity and antimicrobial activity of composite
films [12,16].
Essential oils and extracts of herbal materials are known to be powerful antioxidant
and antimicrobial agents in biomedical applications [17–19]. The surface modification
of metallic materials with peppermint essential oils was demonstrated to have bacterio-
static effects on Gram-positive bacteria and prevent biofilm formation [20]. Calendula
officinalis (CO) is a medicinal herb also known as pot marigold [21,22]. CO flower ex-
tract consists of carotenes, flavonoids, terpenoids, triterpenoids, polyphenols, phenolic
acids, quinines, coumarins, and other constituents [23,24]. It has been demonstrated to
possess anti-inflammatory, antioxidant, antimicrobial, antifungal, free radical inhibitory,
and wound-healing activities [25–28]. In comparison to cinnamon and clove essential oils,
CO flower extracts have additional biological effects, including the enhancement of cell
proliferation/migration and the promotion of collagen metabolism and angiogenesis at the
wound site [27,28]. Furthermore, CO flower extracts have been applied for the treatment of
skin burns, ulcerations, inflammation, and wounds [22,26]. The pharmacological activity
of CO extract can be enhanced via incorporation into a polymeric matrix with controlled
release over time for wound healing [21,27].
However, CMC/PVA/CO composite films have not yet been investigated. In addition,
the optimal contents and proportions of CMC/PVA/CO composite films to achieve better
mechanical, bioactive, and antimicrobial properties remain unclear. Thus, the aims of the
present study are to develop novel CMC/PVA/CO composite films using the solution
casting method and investigate the morphological, physical, mechanical, biological, and
antimicrobial properties of the composite films.
Polymers 2023, 15, 1454 3 of 13

2. Materials and Methods

2.1. Materials
Sodium CMC (high viscosity; MW: 700 kDa) and PVA (MW: 9000 Da) were purchased
from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents used in the
present study were of analytical grade and did not require further purification. Bacterial
strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were
acquired from the American Type Culture Collection.

2.2. Preparation of CMC/PVA/CO Films

CMC (2%; w/v) and PVA (5%; w/v) were dissolved in distilled water via continuous
magnetic stirring for 60 min at 80 ◦ C. The obtained CMC and PVA solutions were mixed
(1:1; v/v) under mechanical stirring for 30 min. Different ratios (0%, 0.1%, 1%, 2.5%, 4%,
and 5%) of CO extract (Cheng Yi Chemical Co, Taiwan) containing glycerol and TWEEN-80
relative to the CMC/PVA mixture were prepared. The composition ratio of CO extract,
glycerol, and TWEEN-80 for the 5% group was 95:1:4 (v/v), respectively. By adjusting
the composition ratios of CO extract, the other experimental groups with different ratios
(4%, 2.5%, 1%, 0.1%, and 0%) of CO extract containing glycerol and TWEEN-80 relative
to the CMC/PVA mixture could be obtained. The CMC/PVA/CO films were developed
using the solution casting method [17]. Briefly, the CMC/PVA/CO mixed solutions were
magnetically stirred for 30 min and placed under a vacuum for 10 min. After obtaining a
homogeneous solution, the final solutions were poured into a Petri dish with a diameter
of 10 cm and dried in a vacuum oven at 40 ◦ C for 24 h to obtain the CMC/PVA/CO
composite films.

2.3. Scanning Electron Microscopy

The surface morphology of the CMC/PVA/CO composite films was examined using
scanning electron microscopy (SEM; Hitachi SU-3500, Hitachi High Technologies, Minato-
ku, Tokyo, Japan) at an accelerating voltage of 5–20 kV and a working distance of 5 mm [29].
Prior to observation, all of the CMC/PVA/CO composite films were coated with a thin
layer of gold for 10 min under a high vacuum at 10 kV for 90 s. Moreover, the cross-sectional
viewpoints of the composite films were captured at 90◦ , relative to the electron beam.

2.4. X-ray Diffraction (XRD) and Fourier Transform Infrared Spectrometry (FTIR)
The crystalline nature of the CMC/PVA/CO films was characterized using X-ray
diffraction (XRD; Empyrean, PANalytical BV, Almelo, The Netherlands) with Cu Kα
radiation (λ = 0.154 nm) in the range of 2θ = 5◦ –90◦ and a step size and scan speed of 0.05◦
and 2◦ /min, respectively.
The chemical compositions of the CMC/PVA/CO films were characterized using
Fourier transform infrared (FTIR) spectroscopy (Spotlight 200i Sp2 with an AutoATR
System, PerkinElmer, Waltham, MA, USA). Spectra in the range of 4000–400 cm–1 were
recorded in transmission mode with a resolution of 0.5 cm–1 .

2.5. Thickness and Mechanical Properties

The thickness of the CMC/PVA/CO composite films was measured using a hand-held
electronic digital micrometer (Mitutoyo, Japan) with an accuracy of 0.01 mm. Composite
films with smooth and uniform thickness were selected for measurement. The mean values
were calculated from five random positions on each film.
The mechanical properties of the CMC/PVA/CO composite films, including the ul-
timate tensile strength (TS) and elongation rate at break (EB), were determined using a
universal testing machine (AGX-V, SHIMADZU, Kyoto, Japan). Prior to testing, the com-
posite films were cut into rectangular strips (5 × 50 mm2 ) and mounted in the machine. The
initial grip separation and cross-head speed were set at 25 mm and 1 mm/min, respectively.
Finally, the TS (MPa) and EB (%) were measured for each composite film according to
previous studies [17,30].
Polymers 2023, 15, 1454 4 of 13

2.6. Contact Angle

The differences in the surface wettability of the CMC/PVA/CO composite films were
measured using optical measurements of the static contact angle of water. Briefly, a 5 µL
water drop was slowly added to the film surface and recorded using a video contact angle
system (FTA-32, First Ten Angstroms, Portsmouth, VA, USA). After drawing the height and
width of the drops on the film surface, the contact angle value was automatically calculated
in the system [29].

2.7. In Vitro Cell Culture and Cell Viability

A human skin fibroblast cell line (CCD-966SK) was used for the cytocompatibility test.
CCD-966SK cells were cultured and maintained in Dulbecco’s Modified Eagle Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL
penicillin, 100 mg/mL streptomycin mixed antibiotics, and 1 mM sodium pyruvate under
standard growth conditions in a 5% CO2 incubator.
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich,
St. Louis, MO, USA) assay was performed to determine the CCD-966SK cell viability [31].
CCD-966SK cells were seeded at an initial density of 5 × 104 cells/mL in a 24-well plate.
After culturing for 24 h, the growth medium was replaced with normal medium (control),
5% dimethyl sulfoxide (DMSO), and the extract medium from CMC/PVA, CMC/PVA/CO-
0.1%, CMC/PVA/CO-1%, CMC/PVA/CO-2.5%, CMC/PVA/CO-4%, and CMC/PVA/CO-
5% groups. The extract media were prepared from the UV-sterilized composite membranes
in the DMEM media supplemented with 10% FBS for 24 h. After 48 h of incubation, the
morphology of CCD-966SK cells was observed under an optical microscope (Eclipse TS100;
Nikon Corporation, Tokyo, Japan) and captured using a CMOS camera with SPOT Advance
imaging software (SPOT Idea, Diagnostic Instruments, Sterling Heights, MI, USA). Cell
viability was assessed by adding MTT (5 mg/mL) (Sigma-Aldrich, Burlington, MA, USA)
to the cultured cells and incubating for 4 h. The supernatant was then removed, and 500
µL of DMSO was added to dissolve the formazan crystals. The absorbance (optical density,
OD) of the purple formazan was quantified using a microplate reader (Model 2020, Anthos
Labtec Instruments, Eugendorf, Wals, Austria) at wavelengths of 570/690 nm. Higher OD
values indicated higher cell metabolic activity and hence better biocompatibility.

2.8. Antibacterial Activity Evaluation

The antibacterial activities of the CMC/PVA/CO composite films were assessed
against Gram-positive bacteria (S. aureus, ATCC 25923) and Gram-negative bacteria (E. coli,
ATCC 25922) using the disc agar diffusion method, as previously described [32]. The films
were cut into 6 mm diameter round discs and disinfected under a UV lamp for 1 h. After
disinfection, the films were gently placed onto the agar plates, which had been previously
spread with bacterial broth cultures (100 µL). All of the agar plates were subsequently
incubated at 37 ◦ C for 24 h, and the diameters of the clear zones around the CMC/PVA/CO
films were measured and recorded in millimeters.

2.9. Statistical Analysis

The experimental data were analyzed using one-way analysis of variance (ANOVA)
(SPSS Inc., Chicago, IL, USA) and Tukey’s honest post hoc test. The difference was consid-
ered to be statistically significant when the p value < 0.05 (*) and the p value < 0.01 (**).

3. Results
3.1. Surface Characterization
Figure 1 shows the visual appearances of the CMC/PVA composite films containing
different CO concentrations (0%, 0.1%, 1%, 2.5%, 4%, and 5%). All of the films show
an integral and transparent appearance. With an increasing ratio of CO, rougher sur-
faces could be observed in the composite films, especially in the CMC/PVA/CO-4% and
CMC/PVA/CO-5% groups.

Polymers 2023, 15, 1454
3.1. Surface Characterization
Figure 1 shows the visual appearances of the CMC/PVA composite films contain
different CO concentrations (0%, 0.1%, 1%, 2.5%, 4%, and 5%). All of the films show
integral and transparent appearance. With an increasing ratio of CO, rougher surfa
could be observed in the composite films, especially in the CMC/PVA/CO-4%
5 of 13 a
CMC/PVA/CO-5% groups.

Figure 1. Visual Figure

appearance of the
1. Visual CMC/PVA/CO
appearance composite films.
of the CMC/PVA/CO composite films.

A similar tendency is also

A similar observed
tendency in the
is also SEM experiments
observed (Figure 2). The
in the SEM experiments surface
(Figure 2). The surf
morphology and structure of the CMC/PVA composite films is affected
morphology and structure of the CMC/PVA composite films is affected by theby the addition of addition
CO (0%, 0.1%,CO 1%,(0%,
2.5%, 4%,1%,
0.1%, and2.5%,
5%). 4%,
As shown in As
and 5%). Figure
shown2, ainsmooth,
Figure 2, continuous, and
a smooth, continuous, a
homogenous surface with fewer pores in the structure is observed in the CMC/PVA
homogenous surface with fewer pores in the structure is observed in the CMC/PVA co com-
posite films with lower
posite COwith
films concentrations (0%, 0.1%, 1%,(0%,
lower CO concentrations and0.1%,
2.5%).
1%, This
andis2.5%).
because
Thisgood
is because go
compatibility and
compatibility and interactions exist between biopolymers and lower CO via
interactions exist between biopolymers and lower CO concentrations concentrati
hydrogen bonding between bonding
via hydrogen the –OH between
and –COOH groups,
the –OH andas confirmed
–COOH by previous
groups, FTIRby previ
as confirmed
results [17,30].FTIR
However,
resultsthe CMC/PVA
[17,30]. However, composite films with
the CMC/PVA higherfilms
composite CO concentrations
with higher CO concen
(4% and 5%) exhibit surfaces with a certain unevenness and wrinkles. Moreover,
tions (4% and 5%) exhibit surfaces with a certain unevenness and wrinkles. the cross-
Moreover,
sectional structure exhibits complex
cross-sectional structure and irregular
exhibits configurations,
complex and irregular indicating that the
configurations, film
indicating that
structure is significantly
film structureaffected by the incorporation
is significantly affected by theof higher CO concentrations
incorporation of higher COand concentrati
amounts of TWEEN-80
and amountssurfactant.
of TWEEN-80Similar surfactant.
tendenciesSimilar
and results were also
tendencies anddemonstrated
results were also dem
in previous studies using higher levels of essential oils and surfactants [11,17,30].
strated in previous studies using higher levels of essential oils and surfactants [11,17,3

Figure 2. Scanning electron microscopy (SEM) micrographs of the CMC/PVA/CO composite films
with different concentrations of CO (0.0%, 0.1%, 1%, 2.5%, 4%, and 5%): (A–F) surface morphology
images, (G–L) cross-sectional images (magnification: 1000×).

3.2. XRD and FTIR Spectroscopy

The X-ray diffraction (XRD) patterns show characteristic peaks, which identify the
crystalline properties and phases of the prepared composite polymer films. Figure 3A
 Figure 2. Scanning electron microscopy (SEM) micrographs of the CMC/PVA/CO composite films
with different concentrations of CO (0.0%, 0.1%, 1%, 2.5%, 4%, and 5%): (A–F) surface morphology
images, (G–L) cross-sectional images (magnification: 1000×).

3.2. XRD and FTIR Spectroscopy

Polymers 2023, 15, 1454 The X-ray diffraction (XRD) patterns show characteristic peaks, which identify 6 of the
13

crystalline properties and phases of the prepared composite polymer films. Figure 3A
shows the results of the XRD analysis. The diffraction pattern of pure CMC features a peak
at 2θ =the
shows 22.20°, whereas
results of the that
XRDofanalysis.
pure PVA shows
The a significant
diffraction pattern peak at approximately
of pure CMC features 2θa=
19.5°at[15,33].
peak The◦diffraction
2θ = 22.20 , whereas thatpatterns of CMC/PVA
of pure PVA showscomposite
a significant films exhibit
peak a peak at 2θ =
at approximately
2θ = 19.5◦indicating
20°–25°, [15,33]. The diffraction
that the strongpatterns of CMC/PVA
interaction between CMC composite
and PVA films exhibit
forms a peak
a composite
at[34]. 20◦ –25
2θ =These ◦ , indicating that the strong interaction between CMC and PVA forms a
results are similar to those of previous studies [15,33]. Interestingly, with the
composite
addition of [34].
COThese
extractresults
(0.1%,are similar
1%, to those
and 2.5%) of previous
to the CMC/PVA studies [15,33].films,
composite Interestingly,
the crys-
with the addition of CO extract (0.1%, 1%, and 2.5%) to the CMC/PVA
tallinity decreases and the amorphous phase increases because the diffraction peak composite films,be-
the crystallinity
comes broad and decreases and the amorphous
weak. Moreover, this phenomenonphasemay increases
be duebecause the diffraction
to the complete dissoci-
peak
ationbecomes broad and
and successful weak. Moreover,
miscibility between CMC this phenomenon
and PVA aftermay the be due to the complete
incorporation of CO ex-
dissociation
tract (0.1%,and 1%,successful
and 2.5%) miscibility betweenthe
[35]. However, CMC leftand
shiftPVAof after the incorporation
the broad peak in the
ofCMC/PVA/CO-1%
CO extract (0.1%,group 1%, and
may2.5%) [35]. However,
be associated with thethe left shift
changes of the broad
in structure peak
and the in
influ-
the CMC/PVA/CO-1% group may be associated with the changes
ence of pure PVA [15,33,34]. In addition, no new diffraction peaks are observed in the in structure and the
influence
CMC/PVA/CO of pure composite
PVA [15,33,34].
films,Inindicating
addition, no thatnew
no diffraction peaks are observed
chemical interaction in the
occurs between
CMC/PVA/CO
CMC/PVA and CO. composite films, indicating that no chemical interaction occurs between
CMC/PVA and CO.

Figure3.3.(A)
Figure (A)XRD
XRDpatterns
patternsand
and(B)
(B)FTIR
FTIRspectra
spectraofofthe
theCMC/PVA/CO
CMC/PVA/COcomposite
compositefilms
filmswith
withdifferent
different
concentrations of CO (0.0%, 0.1%, 1%, 2.5%, 4%, and
concentrations of CO (0.0%, 0.1%, 1%, 2.5%, 4%, and 5%).5%).

The
TheFTIR
FTIRspectra of of
spectra CMC/PVA
CMC/PVAcomposite films
composite containing
films different
containing CO concentrations
different CO concentra-
(0%, 0.1%, 1%, 2.5%, 4%, and 5%) with
tions (0%, 0.1%, 1%, 2.5%, 4%, and their
5%) characteristic
with their peaks are
characteristic presented
peaks are in Figure 3B.
presented in
Wide bands located at 3000–3600 cm −1 are observed for all of the CMC/PVA/CO composite
Figure 3B. Wide bands located at 3000–3600 cm−1 are observed for all of the CMC/PVA/CO
films. These bands are characteristic –OH stretching groups, which arise mainly from
composite films. These bands are characteristic –OH stretching groups, which arise
the strong hydrogen bonding between CMC and PVA. Sharp bands at 2900 cm−1 are
also observed for all films, which are possibly related to the symmetric and asymmetric
stretching of the CH2 groups of CMC and PVA [36,37]. Moreover, the peak at 1750 cm−1 is
assigned to the carbonyl stretching of organic acids and phenolic compounds, which are
the major components of CO [23,37]. Moreover, in a previous study, the major peaks of CO
extract in FTIR spectra corresponded to hydroxyl groups (3488 cm−1 ), alkyl and C-H groups
(2940 cm−1 ), methyl groups (1463 cm−1 ), ether groups (1230 cm−1 ), and terpenoid and
flavone compounds (1049 cm−1 ), which also appeared in the CMC/PVA/CO composite
films [28]. The –CH vibrational band is observed at 1330 cm−1 , and the C-C stretching band
is found at 835 cm−1 . At 1650, 1473, and 1082 cm−1 , symmetric and asymmetric stretching
vibrations of C=O groups for CMC/PVA are observed [36,37]. Overall, similar major sharp
peaks but varying peak amplitudes are observed in the FTIR spectra of the composite films,
indicating that the incorporation of CO does not change the structure of the film matrix.

3.3. Thickness and Mechanical Properties

The thicknesses of the CMC/PVA, CMC/PVA/CO-0.1%, CMC/PVA/CO-1%, CMC/PVA/
CO-2.5%, CMC/PVA/CO-4%, and CMC/PVA/CO-5% composite films are 58 ± 8, 58 ± 8,
50 ± 7, 88 ± 8, 128 ± 30, and 226 ± 32 µm, respectively. The thickness of the composite films
increases with an increased CO concentration. Similar results were also reported in previ-
 CMC/PVA/CO-2.5%, CMC/PVA/CO-4%, and CMC/PVA/CO-5% composite films are
8, 58 ± 8, 50 ± 7, 88 ± 8, 128 ± 30, and 226 ± 32 μm, respectively. The thickness of the comp
films increases with an increased CO concentration. Similar results were also reporte
previous studies, where the thickness of composite films increased markedly with incr
Polymers 2023, 15, 1454
ing concentrations of essential oils and extracts of polymer composite films7[11,12,38]. of 13

The TS and EB of the composite films are shown in Figure 4. As shown in Figure
the ous
TS studies,
of the pure CMC/PVA composite film is 42.8 ± 7.0 MPa. The TS decreases wit
where the thickness of composite films increased markedly with increasing
increasing CO concentration
concentrations up extracts
of essential oils and to 5%, of reaching
polymer acomposite
value offilms
13.2[11,12,38].
± 0.5 MPa. The TS o
CMC/PVA/CO-0.1%,
The TS and EB of the CMC/PVA/CO-1%,
composite films are shown CMC/PVA/CO-2.5%,
in Figure 4. As shownand CMC/PVA/CO
in Figure 4A,
the TS of the pure CMC/PVA composite film is 42.8 ± 7.0 MPa. The TS decreases
composite films are 13.5 ± 3.3, 18.2 ± 5.9, 28.9 ± 3.8, and 20.4 ± 1.7 MPa, respectively. with an Am
increasing CO concentration up to 5%, reaching a value of 13.2 ± 0.5 MPa. The TS of the
the CMC/PVA/CO composite films, CMC/PVA/CO-2.5% has the TS closest to that of
CMC/PVA/CO-0.1%, CMC/PVA/CO-1%, CMC/PVA/CO-2.5%, and CMC/PVA/CO-4%
tissue (28 MPa)
composite films[33]. This
are 13.5 ± result
3.3, 18.2is±in agreement
5.9, 28.9 ± 3.8, with previous
and 20.4 studies,
± 1.7 MPa, which indic
respectively.
thatAmong
the more essential oils or extracts were incorporated,
the CMC/PVA/CO composite films, CMC/PVA/CO-2.5% has the TS closestthe greater the decrease
to in
that of skinproperties
mechanical tissue (28 MPa)
of the[33]. This result
polymer is in agreement
composite filmswith previous
would studies, which
be [11,12,38].
indicated that the more essential oils or extracts were incorporated, the greater the decrease
in the mechanical properties of the polymer composite films would be [11,12,38].

Figure 4. Mechanical properties of CMC/PVA/CO composite films with different CO concentrations

Figure 4. 0.1%,
(0.0%, Mechanical properties
1%, 2.5%, of CMC/PVA/CO
4%, and 5%): composite
(A) tensile strength, filmsatwith
(B) elongation breakdifferent COare
rate. Results concentra
(0.0%,
expressed as mean ± standard deviation and statistical significance is represented as (*) p < 0.05 or Result
0.1%, 1%, 2.5%, 4%, and 5%): (A) tensile strength, (B) elongation at break rate.
expressed as mean ± standard deviation and statistical significance is represented as (*) p < 0.
(**) p < 0.01.
(**) p < 0.01.
A similar tendency is also observed for CMC/PVA/CO composite films in the EB
analysis (Figure 4B). The EB of the CMC/PVA is 7.0 ± 1.1 MPa. With the addition of CO,
A similar tendency is also observed for CMC/PVA/CO composite films in the
the EB of the CMC/PVA/CO composite film decreases. The EB of the CMC/PVA/CO-5%
analysis
is 2.8 ±(Figure
0.1 MPa,4B).
whileThe EB of the CMC/PVA
CMC/PVA/CO-2.5% is 7.0
maintains ± 1.1EBMPa.
a higher valueWith
(5.6 ±the addition of
0.3 MPa).
the Multiple
EB of the CMC/PVA/CO
reasons, including thecomposite film decreases.
viscosity, thickness, The EB
intra-molecular of theforce,
bonding CMC/PVA/CO
and
microstructure, are related to this reduction in mechanical properties
is 2.8 ± 0.1 MPa, while CMC/PVA/CO-2.5% maintains a higher EB value (5.6 [11,12,38]. The ± 0.3 M
observed changes are also in agreement with the SEM, XRD, and FTIR results. In general,
Multiple reasons, including the viscosity, thickness, intra-molecular bonding force,
the CMC/PVA composite films exhibit significantly decreased mechanical properties when
CO is added. However, CMC/PVA/CO-2.5% can maintain approximately 65% and 80%
of the TS and EB values, respectively, of CMC/PVA composite films. A possible reason
for this increase may be that a CO concentration of 2.5% can maintain the interfacial
interaction of the CMC/PVA composite without weakening the structure compared with
the other concentrations.

3.4. Surface Wettability

Surface wettability is an important material property for enhancing protein adsorption,
the cellular response, and tissue repair. An improvement in surface wettability can promote
cellular interactions between the prepared composites and living tissues [39,40]. Contact
angle measurements of water droplets on the surface of the CMC/PVA/CO composite films
were performed to evaluate the wettability. CMC and PVA are both highly hydrophilic poly-
mers. Figure 5A shows digital images of the CMC/PVA/CO composite films. The contact
angles of the CMC/PVA/CO composite films are 15.8◦ ± 3.4◦ , 16.8◦ ± 3.5◦ , 15.7◦ ± 0.6◦ ,
15.1 ± 2.3◦ , 10.9◦ ± 2.9◦ , and 13.4◦ ± 1.8◦ , respectively (Figure 5B). In general, the contact
angles of the CMC/PVA/CO composite films decrease with increasing CO concentration.
The CMC/PVA/CO-4% exhibits the highest wettability of the composite films, whereas no
significant differences are observed between CMC/PVA/CO-4% and CMC/PVA/CO-5%.
Thus, the increased hydrophilicity of the CMC/PVA/CO composite films correlates with
 15.7° ± 0.6°, 15.1 ± 2.3°, 10.9° ± 2.9°, and 13.4° ± 1.8°, respectively (Figure 5B). In general,
the contact angles of the CMC/PVA/CO composite films decrease with increasing CO con-
centration. The CMC/PVA/CO-4% exhibits the highest wettability of the composite films,
whereas no significant differences are observed between CMC/PVA/CO-4% and
Polymers 2023, 15, 1454 CMC/PVA/CO-5%. Thus, the increased hydrophilicity of the CMC/PVA/CO 8composite of 13

films correlates with increasing CO concentrations. The surface roughness, structure,

chemistry composition, and topographic design of the polymer composite films have
increasing
great CO on
influence concentrations.
wettability. The surface
In the roughness,
present structure,
study, the chemistry
improvement incomposition,
surface wettabil-
and topographic design of the polymer composite films have great
ity for CMC/PVA/CO-4% may be partially due to the heterogeneous surface influence on wettability.
geometries
In the present study, the improvement in surface wettability for CMC/PVA/CO-4% may
and structure [38,40]. Based on previous results, the increase in surface wettability of com-
be partially due to the heterogeneous surface geometries and structure [38,40]. Based
posite films would provide a desirable microenvironment for cell response, tissue engi-
on previous results, the increase in surface wettability of composite films would provide
neering, and microenvironment
a desirable wound-healing applications [1,4,41].
for cell response, tissue engineering, and wound-healing
applications [1,4,41].

Figure 5. Contact angle analysis of the CMC/PVA/CO composite films with different CO concentra-
tions (0.0%, 0.1%, 1%, 2.5%, 4%, and 5%): (A) digital images of the contact angles of water droplets
Figure 5. Contact angle analysis of the CMC/PVA/CO composite films with different CO concentra-
on composite films, (B) average water contact angles of the composite films. Results are expressed as
tions (0.0%, 0.1%, 1%, 2.5%, 4%, and 5%): (A) digital images of the contact angles of water droplets
mean ± standard deviation and statistical significance is represented as (*) p < 0.05.
on composite films, (B) average water contact angles of the composite films. Results are expressed
as 3.5.
mean Biocompatibility of the CMC/PV
± standard deviation A/CO Composite
and statistical Filmsis represented as (*) p < 0.05.
significance
Biocompatibility is essential for biomaterial applications [16]. Thus, the cytotoxicity
Polymers 2023, 15, x FOR PEER REVIEW 9 of 13
3.5.
ofBiocompatibility of the CMC/PVA/CO
the prepared CMC/PVA/CO compositeComposite
films was Films
evaluated using the MTT assay. The
cellBiocompatibility
morphology and is cellessential
viabilityfor
of human dermal
biomaterial fibroblasts (CCD966SK)
applications grown
[16]. Thus, the for
cytotoxicity
48 h under different culture conditions (control group, 5% DMSO, and CMC/PVA/CO
of the prepared CMC/PVA/CO composite films was evaluated using the MTT assay. The
composite
dermal film immersion
fibroblasts culturedmedium)
in theare presented in Figure
CMC/PVA/CO 6. As shown
composite in Figure 6A,
film immersion hu-
medium
cell morphology
man dermal
and cell
fibroblasts
viability
cultured
of human dermal fibroblasts (CCD966SK) grown for 48
show good growth and healthyinmorphologies
the CMC/PVA/CO with composite
minimal cellfilmapoptosis
immersioncompared
medium to
h under
show different culture conditions (control group, 5% DMSO, and CMC/PVA/CO to com-
the 5%good
DMSO growth andBased
group. healthyonmorphologies
the MTT assay with minimal
data, all of cell
the apoptosis
CMC/PVA/CO compared
composite
posite
the 5%film immersion
DMSOcomparable medium)
group. Based are presented
on the MTT assay in Figure
data, 6. As shown
all of the CMC/PVA/CO in Figure 6A,
compositehuman
films exhibit biocompatibility and non-toxicity. Thus, these composite films
films exhibit comparable biocompatibility and non-toxicity. Thus, these composite films are
are suitable candidates for further tissue engineering and wound-healing applications. It
suitable candidates for further tissue engineering and wound-healing applications. It has
has been reported that CMC/PVA films can absorb wound fluid through ion exchange,
been reported that CMC/PVA films can absorb wound fluid through ion exchange, which
which enhances tissue formation, regeneration, and rapid epithelialization [5].
enhances tissue formation, regeneration, and rapid epithelialization [5].

Figure 6. Biocompatibility evaluation of the CMC/PVA/CO composite films with different concen-
Figure 6. Biocompatibility evaluation of the CMC/PVA/CO composite films with different concen-
trations of CO (0.0%, 0.1%, 1%, 2.5%, 4%, and 5%) using the MTT assay. (A,B) Average cell viability
trations of CO (0.0%, 0.1%, 1%, 2.5%, 4%, and 5%) using the MTT assay. (A,B) Average cell viability
values among the control, 5% DMSO, and composite films. Results are expressed as mean ± standard
values among the control, 5% DMSO, and composite films. Results are expressed as mean ± stand-
deviation and statistical significance is represented as (*) p < 0.05 or (**) p < 0.01.
ard deviation and statistical significance is represented as (*) p < 0.05 or (**) p < 0.01.

3.6. Antibacterial Properties of CMC/PVA/CO Films

S. aureus and E. coli are the major pathogenic bacterial strains in wound sites experi-
encing inflammation and chronic infection. Thus, S. aureus and E. coli are usually selected
as representative bacteria to evaluate the antibacterial properties of composite films
Polymers 2023, 15, 1454 9 of 13

3.6. Antibacterial Properties of CMC/PVA/CO Films

S. aureus and E. coli are the major pathogenic bacterial strains in wound sites ex-
periencing inflammation and chronic infection. Thus, S. aureus and E. coli are usually
selected as representative bacteria to evaluate the antibacterial properties of composite
films [34,37,42]. Figure 7 shows the antibacterial activity of the CMC/PVA/CO composite
films against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. In the absence
of CO, the CMC/PVA composite film has lower antibacterial activity, while the CMC/PVA
composite films containing various concentrations of CO exhibit antibacterial activity. With
increasing CO concentration, the area of the inhibition zone increases. The diameters
of the inhibition zones for CMC/PVA/CO-0.1%, CMC/PVA/CO-1%, CMC/PVA/CO-
2.5%, CMC/PVA/CO-4%, and CMC/PVA/CO-5% composite films against S. aureus are
17.5 ± 0.7, 17.6 ± 0.3, 18.6 ± 0.8, 20.4 ± 0.6, and 18.4 ± 0.7 mm, respectively. More-
over, the diameters of the inhibition zones for CMC/PVA/CO-0.1%, CMC/PVA/CO-
1%, CMC/PVA/CO-2.5%, CMC/PVA/CO-4%, and CMC/PVA/CO-5% composite films
against E. coli are 17.2 ± 0.6, 17.6 ± 0.3, 18.4 ± 0.3, 18.4 ± 0.3, and 17.8 ± 0.9 mm, re-
spectively. Overall, the ability of the CMC/PVA/CO composite film to inhibit S. aureus
was better than its ability to inhibit E. coli. This is because the cell wall structure with a
layer of peptidoglycan located between the outer membrane and the cytoplasmic mem-
brane of Gram-negative bacteria is more complex than that of Gram-positive bacteria,
which can protect bacteria from external stimulation [42]. These results are consistent
with those of previous studies investigating CMC/PVA composite films incorporating
nanoparticles, antibiotics, and essential oils [4,12,34,37,42]. The antibacterial effect of the
pure CMC/PVA films is limited as shown in the present study and previous studies [36].
Thus, multiple strategies were developed to improve the antibacterial activity of the pure
CMC/PVA films [4,12,33,37,42]. The antioxidant, antibacterial, and antifungal effects of
CMC/PVA have been reported to be enhanced by the cooperation with 1.5% and 3.0%
cinnamon essential oil [17,25]. This is the first study to show that the antibacterial ef-
fects of CMC/PVA composite films can be efficiently promoted by the addition of CO
extract. According to previous studies, CO extract alone or in combination with polymer
films or scaffolds can efficiently inhibit bacteria and enhance wound healing [27,28,43].
This is because polyphenols (especially flavonoids), which are the major constituents of
CO, have excellent antimicrobial properties [21]. In addition, the major components of
CO include carotenes, flavonoids, terpenoids, triterpenoids, polyphenols, phenolic acids,
quinines, coumarins, carbohydrates, essential oils, minerals, and fatty acids [23,24,28]. The
antimicrobial activity of CO has been attributed to flavonoids, triterpenoids, and essential
oils. Possible mechanisms have been suggested for the antibacterial effects of CO, such as
damaging the cytoplasmic membrane, denaturing proteins, disrupting the enzyme system,
and reducing ATP synthesis [20,23,24,28,44]. Overall, the multifunctional characteristics of
CMC/PVA/CO composite films are useful for wound-dressing applications because they
contribute to wound healing at the stages of hemostasis, inflammation, protein adhesion,
and cellular proliferation.
 to flavonoids, triterpenoids, and essential oils. Possible mechanisms have been suggested
for the antibacterial effects of CO, such as damaging the cytoplasmic membrane, denatur-
ing proteins, disrupting the enzyme system, and reducing ATP synthesis [20,23,24,28,44].
Overall, the multifunctional characteristics of CMC/PVA/CO composite films are useful
Polymers 2023, 15, 1454 for wound-dressing applications because they contribute to wound healing 10 atofthe
13 stages
of hemostasis, inflammation, protein adhesion, and cellular proliferation.

7. Inhibitory
Figure 7.
Figure Inhibitoryeffects of of
effects CMC/PVA/CO
CMC/PVA/CO composite
compositefilmsfilms
with with
different concentrations
different of CO of CO
concentrations
(0.0%, 0.1%,
(0.0%, 0.1%, 1%,
1%,2.5%,
2.5%,4%,4%,andand5%) on on
5%) the the
growth of (A)
growth ofGram-positive bacteria
(A) Gram-positive (Staphylococcus
bacteria (Staphylococcus
aureus) and (B) Gram-negative bacteria (Escherichia coli). Results are expressed
aureus) and (B) Gram-negative bacteria (Escherichia coli). Results are expressed as mean as mean ± standard
± standard
deviation and statistical significance is represented as (*) p < 0.05 or (**) p <
deviation and statistical significance is represented as (*) p < 0.05 or (**) p < 0.01.0.01.

4. Conclusions
4. Conclusions
In the present study, CMC/PVA composite films containing various CO concentrations
In1%,
(0.1%, the2.5%,
present
4%, study,
and 5%)CMC/PVA
were developedcomposite
using afilms containing
simple various
solution casting CO concentra-
technique.
tions (0.1%, 1%, 2.5%,
The morphological, 4%, and
physical, 5%) were
mechanical, developedbiological,
hydrophilic, using a simple solution casting
and antibacterial prop- tech-
erties of the CMC/PVA composite films were affected by CO incorporation.
nique. The morphological, physical, mechanical, hydrophilic, biological, and antibacterial The pure
CMC/PVAof
properties composite film had
the CMC/PVA smooth morphology
composite films were and homogenous
affected appearance, but
by CO incorporation. The pure
CO incorporation resulted in a rough surface and irregular structure, especially regarding
CMC/PVA composite film had smooth morphology and homogenous appearance, but CO
the CMC/PVA/CO-5% composite film. XRD and FTIR results indicate that CO incorpo-
incorporation resulted in a rough surface and irregular structure, especially regarding the
ration does not change the structure of the film matrix. Results regarding the mechanical
CMC/PVA/CO-5%
properties revealed composite
that tensile film. XRD
strength andand FTIR results
elongation indicate
rate upon the that CO incorporation
breaking of the
does not change
composite the structure
films were influencedofbytheCOfilm matrix. Results
incorporation. Theregarding
addition of the
COmechanical
caused a prop-
erties revealed
decrease that tensile
in mechanical strength
strength, whileandtheelongation rate uponcomposite
CMC/PVA/CO-2.5% the breakingfilmsofmain-
the compo-
site films
tained were influenced
approximately 65% of bytensile
CO incorporation.
strength and 80%Theof addition of CO
elongation ratecaused
at break.a decrease
All in
composite films
mechanical showed
strength, goodthe
while hydrophilicity and that they
CMC/PVA/CO-2.5% were biocompatible
composite towardsapprox-
films maintained
the proliferation
imately and growth
65% of tensile of fibroblast
strength and 80%cells. All concentrations
of elongation of CO were
rate at break. effective films
All composite
against Staphylococcus aureus and Escherichia coli when compared to the pure CMC/PVA
showed good hydrophilicity and that they were biocompatible towards the proliferation
film. Finally, the improved physicochemical, biological, and antibacterial properties of the
and growth of fibroblast cells. All concentrations of CO were effective against Staphylo-
CMC/PVA/CO-2.5% composite films were found to be beneficial for further biomedical
coccus aureus and applications.
and wound-healing Escherichia coli when compared to the pure CMC/PVA film. Finally,
the improved physicochemical, biological, and antibacterial properties of the
Author Contributions: Conceptualization, W.-H.H. and C.-Y.H.; Methodology, C.-Y.H.; Software,
W.-H.H. and C.-Y.H.; Validation, W.-H.H. and C.-Y.H.; Formal analysis, W.-H.H., C.-Y.H. and I.-
T.L.; Investigation, W.-H.H. and I.-T.L.; Resources, P.-C.C.; Data curation, C.-Y.H., P.-C.C. and S.-
W.F.; Writing—original draft, Y.-H.C. and S.-W.F.; Writing—review and editing, Y.-H.C. and S.-W.F.;
Visualization, H.L., P.-C.L. and S.-W.F.; Supervision, P.-C.L., Y.-H.C. and S.-W.F.; Project administration,
H.L. and S.-W.F.; Funding acquisition, H.L. and S.-W.F. All authors have read and agreed to the
published version of the manuscript.
Polymers 2023, 15, 1454 11 of 13

Funding: This research was funded by Pro Hallmarks Taiwan Ltd., grant number A-108-040; MacKay
Memorial Hospital, grant number MMH-111-99; and the Ministry of Science and Technology of
Taiwan, grant number 107-2218-E-992-001-MY2.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Brumberg, V.; Astrelina, T.; Malivanova, T.; Samoilov, A. Modern Wound Dressings: Hydrogel Dressings. Biomedicines 2021, 9,
1235. [CrossRef] [PubMed]
2. Freytag, C.; Odermatt, E.K. Standard Biocompatibility Studies Do Not Predict All Effects of PVA/CMC Anti-Adhesive Gel in vivo.
Eur. Surg. Res. 2016, 56, 109–122. [CrossRef]
3. Feng, R.; Fu, R.; Duan, Z.; Zhu, C.; Ma, X.; Fan, D.; Li, X. Preparation of sponge-like macroporous PVA hydrogels via n-HA
enhanced phase separation and their potential as wound dressing. J. Biomater. Sci. Polym. Ed. 2018, 29, 1463–1481. [CrossRef]
4. Falbo, F.; Spizzirri, U.G.; Restuccia, D.; Aiello, F. Natural Compounds and Biopolymers-Based Hydrogels Join Forces to Promote
Wound Healing. Pharmaceutics 2023, 15, 271. [CrossRef] [PubMed]
5. Kanikireddy, V.; Varaprasad, K.; Jayaramudu, T.; Karthikeyan, C.; Sadiku, R. Carboxymethyl cellulose-based materials for
infection control and wound healing: A review. Int. J. Biol. Macromol. 2020, 164, 963–975. [CrossRef]
6. Zennifer, A.; Senthilvelan, P.; Sethuraman, S.; Sundaramurthi, D. Key advances of carboxymethyl cellulose in tissue engineering
& 3D bioprinting applications. Carbohydr. Polym. 2021, 256, 117561. [PubMed]
7. Javanbakht, S.; Shaabani, A. Carboxymethyl cellulose-based oral delivery systems. Int. J. Biol. Macromol. 2019, 133, 21–29.
[CrossRef]
8. Pornpitchanarong, C.; Rojanarata, T.; Opanasopit, P.; Ngawhirunpat, T.; Bradley, M.; Patrojanasophon, P. Maleimide-
functionalized car boxymethyl cellulose: A novel mucoadhesive polymer for transmucosal drug delivery. Carbohydr. Polym. 2022,
288, 119368. [CrossRef]
9. Basu, P.; Narendrakumar, U.; Arunachalam, R.; Devi, S.; Manjubala, I. Characterization and Evaluation of Carboxymethyl
Cellulose-Based Films for Healing of Full-Thickness Wounds in Normal and Diabetic Rats. ACS Omega 2018, 3, 12622–12632.
[CrossRef]
10. Wang, B.; Yang, X.; Qiao, C.; Li, Y.; Li, T.; Xu, C. Effects of chitosan quaternary ammonium salt on the physicochemical properties
of sodium carboxymethyl cellulose-based films. Carbohydr. Polym. 2018, 184, 37–46. [CrossRef]
11. Lin, D.; Zheng, Y.; Wang, X.; Huang, Y.; Ni, L.; Chen, X.; Wu, Z.; Huang, C.; Yi, Q.; Li, J.; et al. Study on physicochemical
properties, antioxidant and antimicrobial activity of okara soluble dietary fiber/sodium carboxymethyl cellulose/thyme essential
oil active edible composite films incorporated with pectin. Int. J. Biol. Macromol. 2020, 165, 1241–1249. [CrossRef]
12. Bahrami, A.; Fattahi, R. Biodegradable carboxymethyl cellulose-polyvinyl alcohol composite incorporated with Glycyrrhiza Glabra
L. essential oil: Physicochemical and antibacterial features. Food Sci. Nutr. 2021, 9, 4974–4985. [CrossRef] [PubMed]
13. Gaaz, T.S.; Sulong, A.B.; Akhtar, M.N.; Kadhum, A.A.; Mohamad, A.B.; Al-Amiery, A.A. Properties and Applications of Polyvinyl
Alcohol, Halloysite Nanotubes and Their Nanocomposites. Molecules 2015, 20, 22833–22847. [CrossRef]
14. Kumar, A.; Sood, A.; Han, S.S. Poly(vinyl alcohol)-alginate as potential matrix for various applications: A focused review.
Carbohydr. Polym. 2022, 277, 118881. [CrossRef]
15. Zhu, J.; Li, Q.; Che, Y.; Liu, X.; Dong, C.; Chen, X.; Wang, C. Effect of Na2 CO3 on the Microstructure and Macroscopic Properties
and Mechanism Analysis of PVA/CMC Composite Film. Polymers 2020, 12, 453. [CrossRef] [PubMed]
16. Alsahag, M.; Alisaac, A.; Al-Hazmi, G.A.A.; Pashameah, R.A.; Attar, R.M.S.; Saad, F.A.; El-Metwaly, N.M. Preparation of
carboxymethyl cellulose/polyvinyl alcohol wound dressing composite immobilized with anthocyanin extract for colorimetric
monitoring of wound healing and prevention of wound infection. Int. J. Biol. Macromol. 2023, 224, 233–242. [CrossRef] [PubMed]
17. Fasihi, H.; Fazilati, M.; Hashemi, M.; Noshirvani, N. Novel carboxymethyl cellulose-polyvinyl alcohol blend films stabilized by
Pickering emulsion incorporation method. Carbohydr. Polym. 2017, 167, 79–89. [CrossRef]
18. Parham, S.; Kharazi, A.Z.; Bakhsheshi-Rad, H.R.; Nur, H.; Ismail, A.F.; Sharif, S.; RamaKrishna, S.; Berto, F. Antioxidant,
Antimicrobial and Antiviral Properties of Herbal Materials. Antioxidants 2020, 9, 1309. [CrossRef]
19. Zubair, M.; Shahzad, S.; Hussain, A.; Pradhan, R.A.; Arshad, M.; Ullah, A. Current Trends in the Utilization of Essential Oils for
Polysaccharide- and Protein-Derived Food Packaging Materials. Polymers 2022, 14, 1146. [CrossRef]
20. Cazzola, M.; Ferraris, S.; Allizond, V.; Bertea, C.M.; Novara, C.; Cochis, A.; Geobaldo, F.; Bistolfi, A.; Cuffini, A.M.;
Rimondini, L.; et al. Grafting of the peppermint essential oil to a chemically treated Ti6Al4V alloy to counteract the bacterial
adhesion. Surf. Coat. Technol. 2019, 378, 125011. [CrossRef]
21. Nicolaus, C.; Junghanns, S.; Hartmann, A.; Murillo, R.; Ganzera, M.; Merfort, I. In vitro studies to evaluate the wound healing
properties of Calendula officinalis extracts. J. Ethnopharmacol. 2017, 196, 94–103. [CrossRef] [PubMed]
Polymers 2023, 15, 1454 12 of 13

22. Balciunaitiene, A.; Puzeryte, V.; Radenkovs, V.; Krasnova, I.; Memvanga, P.B.; Viskelis, P.; Streimikyte, P.; Viskelis, J. Sustainable-
Green Synthesis of Silver Nanoparticles Using Aqueous Hyssopus officinalis and Calendula officinalis Extracts and Their Antioxi-
dant and Antibacterial Activities. Molecules 2022, 27, 7700. [CrossRef]
23. Miguel, M.; Barros, L.; Pereira, C.; Calhelha, R.C.; Garcia, P.A.; Castro, M.; Santos-Buelga, C.; Ferreira, I.C. Chemical characteriza-
tion and bioactive properties of two aromatic plants: Calendula officinalis L. (flowers) and Mentha cervina L. (leaves). Food Funct.
2016, 7, 2223–2232. [CrossRef] [PubMed]
24. Butnariu, M.; Coradini, C.Z. Evaluation of Biologically Active Compounds from Calendula officinalis Flowers using Spectropho-
tometry. Chem. Cent. J. 2012, 6, 35. [CrossRef] [PubMed]
25. Alexandre, J.T.M.; Sousa, L.H.T.; Lisboa, M.R.P.; Furlaneto, F.A.C.; do Val, D.R.; Marques, M.; Vasconcelos, H.C.; de Melo, I.M.;
Leitão, R.; Castro Brito, G.A.; et al. Anti-inflammatory and antiresorptive effects of Calendula officinalis on inflammatory bone loss
in rats. Clin. Oral Investig. 2018, 22, 2175–2185. [CrossRef] [PubMed]
26. Cruceriu, D.; Balacescu, O.; Rakosy, E. Calendula officinalis: Potential Roles in Cancer Treatment and Palliative Care. Integr. Cancer
Ther. 2018, 17, 1068–1078. [CrossRef] [PubMed]
27. Givol, O.; Kornhaber, R.; Visentin, D.; Cleary, M.; Haik, J.; Harats, M. A systematic review of Calendula officinalis extract for wound
healing. Wound Repair Regen. 2019, 27, 548–561. [CrossRef]
28. Pedram Rad, Z.; Mokhtari, J.; Abbasi, M. Calendula officinalis extract/PCL/Zein/Gum arabic nanofibrous bio-composite scaffolds
via suspension, two-nozzle and multilayer electrospinning for skin tissue engineering. Int. J. Biol. Macromol. 2019, 135, 530–543.
[CrossRef]
29. Chan, Y.H.; Lew, W.Z.; Lu, E.; Loretz, T.; Lu, L.; Lin, C.T.; Feng, S.W. An evaluation of the biocompatibility and osseointegration
of novel glass fiber reinforced composite implants: In vitro and in vivo studies. Dent. Mater. 2018, 34, 470–485. [CrossRef]
30. Chu, Y.; Xu, T.; Gao, C.; Liu, X.; Zhang, N.; Feng, X.; Liu, X.; Shen, X.; Tang, X. Evaluations of physicochemical and biological
properties of pullulan-based films incorporated with cinnamon essential oil and Tween 80. Int. J. Biol. Macromol. 2019, 122,
388–394. [CrossRef]
31. Chan, Y.H.; Ho, K.N.; Lee, Y.C.; Chou, M.J.; Lew, W.Z.; Huang, H.M.; Lai, P.C.; Feng, S.W. Melatonin enhances osteogenic
differentiation of dental pulp mesenchymal stem cells by regulating MAPK pathways and promotes the efficiency of bone
regeneration in calvarial bone defects. Stem Cell Res. Ther. 2022, 13, 73. [CrossRef] [PubMed]
32. El Fawal, G.F.; Abu-Serie, M.M.; Hassan, M.A.; Elnouby, M.S. Hydroxyethyl cellulose hydrogel for wound dressing: Fabrication,
characterization and in vitro evaluation. Int. J. Biol. Macromol. 2018, 111, 649–659. [CrossRef] [PubMed]
33. Verma, N.; Pramanik, K.; Singh, A.K.; Biswas, A. Design of magnesium oxide nanoparticle incorporated carboxy methyl
cellulose/poly vinyl alcohol composite film with novel composition for skin tissue engineering. Mater. Technol. 2022, 37, 706–716.
[CrossRef]
34. Sethi, V.; Kaur, M.; Thakur, A.; Rishi, P.; Kaushik, A. Unravelling the role of hemp straw derived cellulose in CMC/PVA hydrogel
for sustained release of fluoroquinolone antibiotic. Int. J. Biol. Macromol. 2022, 222, 844–855. [CrossRef] [PubMed]
35. Saadiah, M.A.; Zhang, D.; Nagao, Y.; Muzakir, S.K.; Samsudin, A.S. Reducing crystallinity on thin film based CMC/PVA hybrid
polymer for application as a host in polymer electrolytes. J. Non-Cryst. Solids 2019, 511, 201–211. [CrossRef]
36. Deshmukh, R.K.; Akhila, K.; Ramakanth, D.; Gaikwad, K.K. Guar gum/carboxymethyl cellulose based antioxidant film
incorporated with halloysite nanotubes and litchi shell waste extract for active packaging. Int. J. Biol. Macromol. 2022, 15, 1–13.
[CrossRef]
37. Amaregouda, Y.; Kamanna, K.; Gasti, T.; Kumbar, V. Enhanced Functional Properties of Biodegradable Polyvinyl Alco-
hol/Carboxymethyl Cellulose (PVA/CMC) Composite Films Reinforced with L-alanine Surface Modified CuO Nanorods.
J. Polym. Environ. 2022, 30, 2559–2578. [CrossRef]
38. Song, X.; Zuo, G.; Chen, F. Effect of essential oil and surfactant on the physical and antimicrobial properties of corn and wheat
starch films. Int. J. Biol. Macromol. 2018, 107 Pt A, 1302–1309. [CrossRef]
39. Hashmi, M.; Ullah, S.; Ullah, A.; Akmal, M.; Saito, Y.; Hussain, N.; Ren, X.; Kim, I.S. Optimized Loading of Carboxymethyl
Cellulose (CMC) in Tri-component Electrospun Nanofibers Having Uniform Morphology. Polymers 2020, 12, 2524. [CrossRef]
40. Rao, K.M.; Sudhakar, K.; Suneetha, M.; Won, S.Y.; Han, S.S. Fungal-derived carboxymethyl chitosan blended with polyvinyl
alcohol as membranes for wound dressings. Int. J. Biol. Macromol. 2021, 190, 792–800. [CrossRef]
41. Fathi, A.; Khanmohammadi, M.; Goodarzi, A.; Foroutani, L.; Mobarakeh, Z.T.; Saremi, J.; Arabpour, Z.; Ai, J. Fabrication of
chitosan-polyvinyl alcohol and silk electrospun fiber seeded with differentiated keratinocyte for skin tissue regeneration in
animal wound model. J. Biol. Eng. 2020, 14, 27. [CrossRef] [PubMed]
42. Darbasizadeh, B.; Fatahi, Y.; Feyzi-Barnaji, B.; Arabi, M.; Motasadizadeh, H.; Farhadnejad, H.; Moraffah, F.; Rabiee, N. Crosslinked-
polyvinyl alcohol-carboxymethyl cellulose/ZnO nanocomposite fibrous mats containing erythromycin (PVA-CMC/ZnO-EM):
Fabrication, characterization and in-vitro release and anti-bacterial properties. Int. J. Biol. Macromol. 2019, 141, 1137–1146.
[CrossRef] [PubMed]
Polymers 2023, 15, 1454 13 of 13

43. Kharat, Z.; Amiri Goushki, M.; Sarvian, N.; Asad, S.; Dehghan, M.M.; Kabiri, M. Chitosan/PEO nanofibers containing Calendula
officinalis extract: Preparation, characterization, in vitro and in vivo evaluation for wound healing applications. Int. J. Pharm.
2021, 609, 121132. [CrossRef] [PubMed]
44. Barbieri, R.; Coppo, E.; Marchese, A.; Daglia, M.; Sobarzo-Sánchez, E.; Nabavi, S.F.; Nabavi, S.M. Phytochemicals for human
disease: An update on plant-derived compounds antibacterial activity. Microbiol. Res. 2017, 196, 44–68. [CrossRef] [PubMed]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.