Application of honey as a protective material in maintaining the viability of adipose stem cells in burn wound healing: A histological, molecular and biochemical study
Introduction
Skin is the largest organ of body, with self-repairing and self-renewing properties and acts as an important barrier to prevent pathogens to enter into vital organs (Baroni et al., 2012; Kanji and Das, 2017). Damage to skin causes impairment of the anatomical structure and function and results in wound formation (Velnar et al., 2009). Among wounds, the chronic ones including pressure wounds, venous, diabetic and burn ulcers usually fail to proceed normal healing because they result in exaggerated chronic inflammatory reaction. Chronic wounds carry high costs to both the patient and the medical system (Frykberg and Banks, 2015; Oryan et al., 2017). Numerous experiments have focused on understanding the pathophysiology and developing novel therapeutic methods for the chronic wounds, in the past two decades (Wieman et al., 1998; Gharaee-Kermani and Phan, 2001; Saltmarche, 2008; Dumville et al., 2012). Unfortunately, these methods did not always guarantee and the therapeutic strategies of the chronic wounds have remained unsatisfactory.
Recently, stem cell therapy has been considered as an alternative approach for wound regeneration and repair (Kanji and Das, 2017). Although the embryonic stem cells and pluripotent stem cells have been tested for transplantation therapy in regenerative medicine, the mesenchymal stem cells (MSCs) have been the main focus of research in the recent years (Stoltz et al., 2015). During the recent 40 years, the bone marrow-derived MSCs (BMSCs) have been widely studied for their potential applications in repair and regenerative medicine. These cells have high differentiation abilities that result in their clinical use in medicine (Via et al., 2012). Although BMSCs have low morbidity during harvesting, low yield of the bone marrow stem cells is one of the difficulties in this method. In addition, it has been shown that differentiation potential of BMSCs significantly reduces by aging of the donor (Pendleton et al., 2013). Therefore, the adipose-derived stem cells (ASCs) are considered as an attractive alternative source that is ubiquitously available and has several advantages compared to other sources including ability to extensive proliferation and multilineage differentiation (Pendleton et al., 2013). ASCs stimulate wound healing via two mechanisms, either by secretion of paracrine factors or by differentiation into other cells such as fibroblasts, endothelial cells, epithelial cells, and keratinocytes (Pendleton et al., 2013; Hassan et al., 2014). It has been shown that ASCs recruit endogenous stem cells and epithelial cells and conversely reduce the inflammatory responses, by secreting different cytokines and growth factors (Hassan et al., 2014). Although these characteristics have made ASCs as a proper candidate in cell therapy, a number of limitations have decreased their potential in treatment of injuries. High amounts of pro-apoptotic factors such as inflammatory cytokines and free radicals and lack of sufficient nutrients and oxygen supply, in a damaged tissue, are responsible for apoptosis of the ASCs (Geng, 2003). Hence, protecting the ASCs after transplantation against apoptosis helps in enhancing cell viability, differentiation, and regeneration. Such protection can be achieved by addition of an antioxidant supplement such as honey in cell culture medium to increase the anti-oxidant activity of ASCs (Ko et al., 2011).
The previous studies have documented the anti-oxidant and anti-inflammatory activities of honey in wound environment (Van den Berg et al., 2008; Kassim et al., 2010; Bashkaran et al., 2011). It has also been demonstrated that honey can be a beneficial biomaterial in proper management of wounds (Oryan and Zaker, 1998; Alvarez-Suarez et al., 2016; Oryan et al., 2016). Abderrahim suggested that combination of honey and garlic can exhibit synergistic activity with increased efficacy in treating wounds (Abderrahim et al., 2019). Beretta et al. (2007) showed the effective role of honey in prevention of endothelial cell damage against the lipophilic cumoxyl and cumoperoxyl radicals (Beretta et al., 2007). Some studies have considered application of honey gels on wound healing and showed their effective role in the healing process (Mirzaei et al., 2018; Febriyenti et al., 2019; Santos et al., 2019). Febriyenti et al. evaluated the ability of the honey gel and film in promoting burn wound healing and reported that the honey film played a significant effect in wound closure when compared to other therapies. Honey film also resulted in a higher tensile strength compared to other groups (Febriyenti et al., 2019). Mirzaei et al. (2018) investigated an alginate-based honey hydrogel as an ointment to heal burn wounds. They found that the alginate-based honey hydrogel not only can hasten the healing process but also may protect individuals from the bacterial colonization and biofilm-forming bacterial infections. Santos et al. (2019) also confirmed that controlled release of honey from polyvinyl alcohol blended with natural polymers is a possible strategy in promoting burn healing. Although numerous experiments have established the therapeutic benefits of honey in treating wound injuries, to our knowledge, the effectiveness of honey in cultivation of adipose stem cells has not yet been investigated.
In the present study, we investigated the effects of honey on viability of adipose stem cells and also studied its potential in improving the ASCs resistance to oxidative stress. Moreover, we evaluated the potential role of honey separately or in combination with ASCs in wound healing process by gross morphologic, histopathologic and molecular investigations, scanning electron microscopy, and biochemical analysis during the inflammatory, fibroplasia and maturation phases of burn wound healing.
Section snippets
Animals
All experimental protocols were performed in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23). Additionally, the local Ethics Committee of “Regulations for using animals in scientific procedures” in School of Veterinary Medicine of our university approved this experiment (approval number 14-6-1394 and approval date: 5th September 2015).
Isolation and culture of adipose-derived stem cells (ASCs) in rat
Subcutaneous adipose tissue was harvested under sterile conditions
In vitro findings
The viability of HDF cells were determined using MTT assay, after 12 and 24 h (Fig. 1). 1% honey was able to protect the cells and increase cell viability after 12 and 24 h compared to the control group (p < 0.05).
Honey strengthens the antioxidant activity
Fig. 2 shows the results of radical scavenging activity of different concentrations of honey (50, 25, 12.50, 6.25, 3.12%) at 30 min after incubation. Results showed the positive effect of honey on the antioxidant activity. The antioxidant activity of honey showed a dose-dependent
Discussion
Stem cell therapy has provided promising new therapeutic option in regeneration and repair of wounds in the recent years (Wu et al., 2007; Aragona et al., 2017; de Mayo et al., 2017). Among several different types of stem cells, the adipose-derived stem cells have proven to be a proper therapeutic alternative in regenerative medicine to meet the challenges of wound healing (Kuo et al., 2016; Kato et al., 2017). The primary property that these cells need to successfully stimulate the healing
Conclusion
Although the effects of honey and ASCs in wound healing are evident at in vivo studies, the interaction between honey and ASCs and the potential role of their combination in wound healing have not been investigated. Most notably, this is the first study, to our knowledge, to investigate the effectiveness of honey-ASCs in burn wound healing. Our in vivo results provided compelling evidence that combination of the ASCs and honey represents an approach to maintain the ASCs against oxidative stress
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
The authors would like to thank the authorities of the Veterinary School, Shiraz University for their kind cooperation. We would also thank the INSF (grant number 96006039) for the financial support.
References (60)
- et al.
Activation of AMPK/Nrf2 signalling by Manuka honey protects human dermal fibroblasts against oxidative damage by improving antioxidant response and mitochondrial function promoting wound healing
J. Funct. Foods
(2016) - et al.
Structure and function of the epidermis related to barrier properties
Clin. Dermatol.
(2012) - et al.
Ellagic acid, phenolic acids, and flavonoids in Malaysian honey extracts demonstrate in vitro anti-inflammatory activity
Nutr. Res.
(2010) - et al.
Enhanced therapeutic neovascularization by CD31-expressing cells and embryonic stem cell-derived endothelial cells engineered with chitosan hydrogel containing VEGF-releasing microtubes
Biomaterials
(2015) - et al.
Mesenchymal stem cells prevent hypertrophic scar formation via inflammatory regulation when undergoing apoptosis
J. Invest. Dermatol.
(2014) Fibroblasts and myofibroblasts: their source, function and role in disease
Int. J. Biochem. Cell Biol.
(2007)- et al.
Biological properties and therapeutic activities of honey in wound healing: a narrative review and meta-analysis
J. Tissue Viability
(2016) - et al.
Topical delivery of chitosan-capped silver nanoparticles speeds up healing in burn wounds: a preclinical study
Carbohydr. Polym.
(2018) - et al.
Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide
Cell Stem Cell
(2008) - et al.
Fibrous protein-based hydrogels for cell encapsulation
Biomaterials
(2014)