**Table 3.** Examples of honey electrospun nanofibrous scaffolds, obtention methods and main findings.


#### 4.2.1. Honey-Based Hydrogels

Hydrogels are high-water-content materials prepared from cross-linked polymers, such as chitosan, and can provide sustained, local delivery of a variety of therapeutic agents [106]. Incorporation of honey into the hydrogel system can beneficially affect the water absorption capacity of the polymer (of the hydrogel) and increase the antibacterial activity of the scaffold [99].

Noori, Kokabi, and Hassan (2018) investigated a honey-loaded PVA/chitosan/mont morillonite nanocomposite (PCMH) hydrogel dressing as a drug model for wound healing. Results demonstrated the ability of the PCMH nanocomposite hydrogel to smart release honey against pH and temperature changes. The maximum release of honey from the hydrogel occurred at pH 7, while the minimum was at pH 2. Independently of the pH, increasing temperature caused higher honey release from the hydrogel matrix. However, the addition of the nanoclay (montmorillonite) to the hydrogel decreases the hydrogel swelling and delays the honey release. Nevertheless, the authors suggested that the honeyloaded nanocomposite hydrogel could be used in low exudate wounds to supply optimized humidity in the wound bed [102].

Different honey concentrations have different efficacies in the scavenging of free radicals and promoting epithelial cell proliferation [119]. It has been shown that the release of honey increases when increasing its concentration in hydrogel, regardless of the polymeric hydrogel used in the formulation. For instance, the honey 75%-chitosan formula showed the best healing properties (regeneration of the epidermis tissue and the formation of new blood capillaries) compared to honey hydrogel formulae with a lower concentration of honey (up to 50%, *w*/*w*). Similarly, an 80%-Manuka/PVA wound dressing hybrid hydrogel showed sustained release of honey over 24 h with progressively low adhesion to the wound bed that protects new epithelialization and promotes cell proliferation. Both dressings demonstrate the high value of cell viability and proliferation and promoted antibacterial activity, being suitable for wounds with moderate to relatively high exudate [100].

Further, one of the advantages of using hydrogels instead of conventional designs is their transparency, which allows us to observe the status of the burn or wound without removal of the dressing [103]. In vivo study showed that burns treated with honey hydrogel sheets were completely healed after 12 days with intact epidermis and topical proliferation of hair follicles. In contrast, burns treated with commercial ointment (MEBO-treated burns) and non-treated burns presented 15% and 63% unclosed wound areas, respectively [104].

In addition, hydrogel wound scaffolds containing honey do not function merely as coverage to provide a clean, moist environment for healing, but also directly contribute to enhanced tissue regeneration and recovery [104].

#### 4.2.2. Honey-Based Electrospun Nanofiber Scaffolds

In biomedical applications, the nanofiber membranes prepared by electro-spinning are used in wound dressings, biosensing, tissue engineering scaffolds, artificial organs, and drug delivery [114]. The formulation of the electrospun nanofiber scaffolds contain proteinbased polymers, such as collagen, gelatin, and silk, or/and polysaccharide-based polymers such as chitosan, hyaluronic acid, and alginate. Prepared stirred polymeric solution is loaded into a syringe that is attached to a needle of which the tip exhibits voltage [120].

The advantageous properties of nanofibers are a large surface area to volume ratio, high porosity, and a very small pore size, which lead to high exudate absorption, better wound permeation, and prevention of further infection [112].

The fabrication of honey-based electrospun nanofibers increases interest due to the enhanced activity realized upon combining the advantages of the nanofibrous structure, primarily the increased surface-to-volume ratio with the advantageous properties of honey (Table 3) [112].

Wound healing scaffolds are expected to absorb body fluids and maintain hydration, but without increasing infection of the biofilm. Indeed, wounds with biofilm fail

to re-epithelialize, show vascular granulation tissue, and consist of recalcitrant microbes. Honey/PVA nanofiber membranes were found to effectively decrease the biofilm formation [113]. Manuka honey is effective against both Gram-positive and Gram-negative bacteria; however, results of honey scaffolds containing 1–20% Manuka/PCL nanofibers indicated that the controlled release of smaller amounts of honey by the scaffold is more effective against Gram-negative-bacteria-infected wounds. Interestingly, opposite results have been found for high-concentration honey chitosan electrospun nanofibers. The honey/PVA/chitosan membrane (30%:7%:3.5%) enhanced antibacterial activity against *S. aureus* (complete inhibition after 48 h with 30%: 7%:5.5 %) and showed poor antibacterial activity against the Gram-negative *E. coli*. Moreover, the scaffold showed high biocompatibility and low cytotoxicity effects [108].

#### **5. Conclusions**

The use of honey for biomedical applications has gained special focus over the years, with the development of novel applications for this natural product, taking advantage of its unique chemical characteristics. Due to its characteristics, namely, low pH and water activity, it presents a good microbiological, enzymatic and (bio)chemical stability, which can be lost if not properly processed for safe use by means of keeping both functionality and microbiological safety. Thus, the design of proper honey processing methodologies is of utmost importance for its use. As reviewed, honey presents a very promising potential to be used in wound-healing processes, either by direct application, incorporated in fibrous membranes, or in hydrogel, with very promising results in either in vitro and in vivo trials. Nonetheless, further research is needed to overcome the main challenges on the use of honey for biomedical applications.

**Author Contributions:** Conceptualization, writing—original draft preparation, H.S., P.C.-F.; writing review and editing, H.S., P.C.-F., J.M.F., M.E.T., M.S.D., C.A.P., L.M.E.; visualization, C.A.P.; Project administration, supervision, J.A.S., L.M.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

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

**Acknowledgments:** Thanks are due to the University of Aveiro and FCT/MCT for the financial support of LAQV-REQUIMTE research Unit (UIDB/50006/2020) through national funds and, where applicable, co-financed by the FEDER within the PT2020 Partnership Agreement, and for financing the PhD grant of Hana Scepankova (SFRH/BD/88133/2012) and Carlos A. Pinto (SFRH/BD/137036/2018).

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

#### **References**

