*3.8. In Vivo Studies*

For in vivo studies, one sample of GVCO hydrogel was chosen, i.e., GVCO80 hydrogel. This sample was chosen due to the optimum properties shown by the latter compared to GVCO60 and GVCO70 hydrogels. The GVCO80 hydrogels show a transparent appearance, optimum mechanical properties, and thermal behavior, together with acceptable swelling ratio and WVTR values. Table 4 shows the wound contraction of the GG and GVCO80 hydrogels compared to the commercial product, Opsite dressing. For the first 7 days, the Opsite dressing shows a significant acceleration of healing at 49 ± 11%, followed by GVCO80 hydrogel at 46 ± 7%. The healing process is further enhanced on day 11 when the GVCO80 surpassed the wound closure, compared Opsite dressing, and achieved 95 ± 2% on day 14. The Opsite dressing completed the closure at 93 ± 4 % on day 14. The GG hydrogel shows the lowest percentage of wound closure, 91 ± 4%, compared to other treatments.

**Table 4.** The percentage of wound contraction of experiment groups on days 2, 4, 7, 11, and 14; the means of two replicates where Opsite acts as a control; \* (*p* < 0.05 compare with the control group).


In this study, no skin irritation is observed for all treatments. This indicated that the GG is a safe material and a good candidate in biomedical applications [29]. The wound healing process was monitored on days 2, 4, 7, 11, and 14 by capturing images of each animal (Figure 7a). The results are in agreement with the wound contraction (Table 4) in which the GVCO80 hydrogel accelerated the wound closure and the wound gradually disappeared through time. The ultrasound images of the thickness growing on the wound skin are shown in Figure 7b. It shows that the skin formation of a wound treated with GVCO80 hydrogel exhibited the optimum recovery compared to other samples on days 2, 4, 7, 11, and 14. The intensity of white/yellowish/green color indicates the good formation of epidermis, dermis, and subcutis of GVCO80 hydrogel and followed by Opsite. The dermis layer was characterized by varying intensities (different colors) present on the wound while the subcutis layer referred to the low-intensity areas due to the homogenous composition. Subcutis areas are described as the black areas, which are referred to the homogeny structure, such as fat, water, and blood. The GG hydrogel as control showed less intensity among the others. The epidermal regeneration was observed in all experimental groups after the 14th day of treatment.

composition. Subcutis areas are described as the black areas, which are referred to the homogeny structure, such as fat, water, and blood. The GG hydrogel as control showed less intensity among the others. The epidermal regeneration was observed in all experi-

mental groups after the 14th day of treatment.

**Figure 7.** (**a**) Wound healing studies and (**b**) typical ultrasound images of wound skin on gellan gum (GG), Opsite, and GVCO80 hydrogels on days 2, 4, 7, 11, and 14. **Figure 7.** (**a**) Wound healing studies and (**b**) typical ultrasound images of wound skin on gellan gum (GG), Opsite, and GVCO80 hydrogels on days 2, 4, 7, 11, and 14.

Hematoxylin and eosin (H & E) staining was performed to evaluate the quality of the wound tissue. Histological evaluation results show that the GVCO80 hydrogel resulted in better re-epithelization as compared to other samples (Figure 8). For Opsite and GVCO80 hydrogel, the formation of epithelial growth was observed and the number of inflammatory cells reduced. Meanwhile, in the control group (GG hydrogel), the new epithelium was noted to regenerate and a little scab was spotted and the necrotic tissue was found under defect. The skin treated with GVCO80 hydrogel and Opsite presented a better result than the control. Hematoxylin and eosin (H & E) staining was performed to evaluate the quality of the wound tissue. Histological evaluation results show that the GVCO80 hydrogel resulted in better re-epithelization as compared to other samples (Figure 8). For Opsite and GVCO80 hydrogel, the formation of epithelial growth was observed and the number of inflammatory cells reduced. Meanwhile, in the control group (GG hydrogel), the new epithelium was noted to regenerate and a little scab was spotted and the necrotic tissue was found under defect. The skin treated with GVCO80 hydrogel and Opsite presented a better result than the control.

The moist environment characteristic of hydrogel is most suited for re-epithelization and enhancing wound healing mechanism on the skin [42]. For a long time, VCO has been a well-known and powerful substance for treating wounds, mainly due to its antibacterial, anti-inflammatory, and anti-oxidant properties [43]. An in vivo study conducted by Soliman and coworkers evaluated the effects of topical application of VCO on wound healing in diabetes-induced Sprague–Dawley rats [44]. They found that the wound closure rate in the VCO group was higher on all days compared to diabetic nontreated rats, and VCO was found to be better than silver sulfadiazine cream in the healing of diabetic wounds via promoting re-epithelialization and collagen synthesis, as well as increasing WCR and total protein content. A few other studies reported the effectiveness of pure VCO (liquid form) in promoting the healing process [45–47]. The wound healing potency of fermented virgin coconut oil was also verified against human umbilical vein endothelial (HUVEC), fibroblast (CCD-18), and retinal ganglion (RGC-5) cells, as well as a wound

excision model in Sprague–Dawley rats [48]. Their finding shows that the expression of phospho-VEGFR2 (vascular endothelial growth factor receptor 2) in HUVECs was detected by Western blot; rats in the VCO group had significantly smaller wound size, higher wound healing percentage, and shorter wound closure time when compared with a control group. Their study also confirmed that a high angiogenic and wound healing potency of VCO contributed to the regulation of the VEGF signing pathway [48]. These past studies show that the VCO significantly affected the healing of the wounds and show promising results to be applied in biomedical applications. With this noted, our study shows that the inclusion of VCO into a biopolymer does not stop the effectiveness of the oil to promote and enhance the healing process. Similar results are obtained, thus indicating this GVCO hydrogel as a promising candidate to be used as dressing materials. *Polymers* **2021**, *13*, x FOR PEER REVIEW 16 of 19

**Figure 8.** Representative images of the histological evaluation section on day 7 and 14 post-wound, stained with H & E (**a**,**b**) GG, (**c**,**d**) Opsite, and (**e**,**f**) GVCO80 hydrogel. Note: e = epidermis, g = granulation, s= scab; 20x magnification. The bar on the micrograph represents 100 µm. **Figure 8.** Representative images of the histological evaluation section on day 7 and 14 post-wound, stained with H & E (**a**,**b**) GG, (**c**,**d**) Opsite, and (**e**,**f**) GVCO80 hydrogel. Note: e = epidermis, g = granulation, s= scab; 20x magnification. The bar on the micrograph represents 100 µm.

#### The moist environment characteristic of hydrogel is most suited for re-epithelization **4. Conclusions**

and enhancing wound healing mechanism on the skin [42]. For a long time, VCO has been a well-known and powerful substance for treating wounds, mainly due to its anti-bacterial, anti-inflammatory, and anti-oxidant properties [43]. An in vivo study conducted by Soliman and coworkers evaluated the effects of topical application of VCO on wound healing in diabetes-induced Sprague–Dawley rats [44]. They found that the wound closure rate in the VCO group was higher on all days compared to diabetic nontreated rats, and VCO was found to be better than silver sulfadiazine cream in the healing of diabetic wounds via promoting re-epithelialization and collagen synthesis, as well as increasing WCR and total protein content. A few other studies reported the effectiveness of pure VCO (liquid form) in promoting the healing process [45–47]. The wound healing potency of fermented virgin coconut oil was also verified against human umbilical vein endothe-This study successfully prepared gellan gum (GG) hydrogel incorporated with virgin coconut oil (VCO) microemulsion with the addition of surfactants, i.e., Tween 80 or TritonX-100. A ternary phase diagram was constructed to obtain an optimized ratio of VCO, water, and Tween 80, which was chosen due to optimum concentration to produce a stable VCO microemulsion. The VCO microemulsion was incorporated into gellan gum (GG) hydrogel (GVCO). Its chemical interaction, mechanical performance, physical properties, and thermal behavior were examined. The stress-at-break (σ) and Young's modulus (YM) of GVCO hydrogel films increased, together with thermal behavior, following the inclusion of VCO microemulsion. The swelling value of GVCO hydrogel decreased as the VCO microemulsion increased and the water vapor transmission rate of GVCO hydrogels was comparable to commercial dressing in the range of 332–391 g m−<sup>2</sup> d −1 . Meanwhile, the in vitro qualitative antibacterial study of GVCO hydrogels against Gram-negative

lial (HUVEC), fibroblast (CCD-18), and retinal ganglion (RGC-5) cells, as well as a wound

tected by Western blot; rats in the VCO group had significantly smaller wound size, higher wound healing percentage, and shorter wound closure time when compared with a control group. Their study also confirmed that a high angiogenic and wound healing potency of VCO contributed to the regulation of the VEGF signing pathway [48]. These past studies show that the VCO significantly affected the healing of the wounds and show promising results to be applied in biomedical applications. With this noted, our study shows that the inclusion of VCO into a biopolymer does not stop the effectiveness of the oil to promote and enhance the healing process. Similar results are obtained, thus indicating this GVCO hydrogel as a promising candidate to be used as dressing materials.

(*Escherichia coli* and *Klebsiella pneumoniae*) and Gram-positive (*Staphylococcus aureus* and *Bacillus subtilis*) bacteria showed that VCO possesses a weak antibacterial effect. In vivo studies on Sprague–Dawley rats show the wound contraction of GVCO80 hydrogel is the best (95 ± 2%) after the 14th day compared to Smith & Nephew Opsite post-op waterproof dressing at 93 ± 4%, and supported by the ultrasound images of wound skin and histological evaluation on the wound. This study demonstrated that the GVCO hydrogels had the potential to be used as wound dressing materials.

**Author Contributions:** Conceptualization, K.A.M.A. and M.H.R.; methodology, K.A.M.A., L.C.R., W.I.W.I., and S.I.A.R.; formal analysis, M.Z.M. and K.A.M.A.; investigation, M.Z.M. and M.A.A.B.; resources, M.Z.M. and K.A.M.A.; data curation, M.Z.M., M.A.A.B. and K.A.M.A.; writing—original draft preparation, M.Z.M., M.A.A.B., and K.A.M.A.; writing—review and editing, K.A.M.A. and M.i.h.P.; visualization, M.Z.M. and K.A.M.A.; supervision, K.A.M.A.; project administration, K.A.M.A. and M.H.R.; and funding acquisition, K.A.M.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received a funding from the Ministry of Higher Education Malaysia (FRGS/1/ 2016/STG07/UMT/02/1).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Research Ethics Approval of Universiti Malaysia Terengganu (UMT/JKEPHMK/2020/48) on 3 November 2020.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** Special thanks to the Ministry of Higher Education Malaysia for research grants (FRGS/1/2016/STG07/UMT/02/1). The authors are also grateful to the Faculty of Science and Marine Environment, Centre Laboratory, and University Malaysia Terengganu for providing facilities.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

