Elsevier

Biomaterials

Volume 249, August 2020, 120020
Biomaterials

Exosome laden oxygen releasing antioxidant and antibacterial cryogel wound dressing OxOBand alleviate diabetic and infectious wound healing

https://doi.org/10.1016/j.biomaterials.2020.120020Get rights and content

Abstract

Lack of oxygen, reduced vascularization, elevated oxidative stress, and infection are critical clinical hallmarks of non-healing chronic diabetic wounds. Therefore, delivering oxygen, inducing angiogenesis, and management of oxidative stress and infection may provide newer and improved therapeutic avenues for better clinical outcomes in diabetic wound healing. Here, we report the development and evaluation of an exosome laden oxygen releasing antioxidant wound dressing OxOBand to promote wound closure and skin regeneration in diabetic wounds. OxOBand is composed of antioxidant polyurethane (PUAO), as highly porous cryogels with sustained oxygen releasing properties and supplemented with adipose-derived stem cells (ADSCs) exosomes. Exosomes engulfed by the cells enhanced the migration of human keratinocytes and fibroblasts and increased the survival of human neuroblastoma cells under hyperglycemic conditions. OxOBand facilitated faster wound closure, enhanced collagen deposition, faster re-epithelialization, increased neo-vascularization, and decreased oxidative stress within two weeks as compared to untreated diabetic control wounds. The dressing promoted the development of mature epithelial structures with hair follicles and epidermal morphology similar to that of healthy skin. In clinically challenging infected diabetic wounds, these dressings prevented infection and ulceration, improved wound healing with increased collagen deposition, and re-epithelialization. Altogether, OxOBand is a remarkably newer treatment strategy for enhanced diabetic wound healing and may lead to novel therapeutic interventions for the treatment of diabetic ulcers.

Introduction

Non-healing and chronic diabetic wounds are the leading cause of non-traumatic amputations in the world. Rates of amputation in diabetic patients are 10–20 times higher than non-diabetic patients [1]. About 25% of the diabetic patients suffer from diabetic foot ulcers leading to amputations resulting in lifelong disabilities [2,3]. Although wound care management is an established field, the treatment of chronic diabetic wounds is still a major challenge. The healing remains a challenge due to increased oxidative stress, impaired angiogenesis, and subsequent oxygen deprivation [4].

Chronic diabetic wounds are characterized by persistent inflammation with insufficient nutrient and oxygen availability due to impaired angiogenesis [4,5]. There exists an inevitable difference between the supply and demand of oxygen, because of increased utilization of oxygen by hyper metabolically active regenerating tissue [6]. The ultimate result of this is a hypoxic environment with high oxidative stress. In else, elevated oxidative stress in diabetic ulcers is caused due to increased production of reactive oxygen species (ROS) by macrophages and neutrophils in response to hyperglycemia [7,8]. This leads to keratinocyte death and impaired migration that results in aberrant epidermal migration and incomplete wound closure, which further aggravates with improper extracellular matrix (ECM) remodelling and degradation [4]. This challenge implies the need for a multifaced biomaterial approach that will simultaneously attenuate the oxidative stress, provide oxygen, and induce angiogenesis.

Here, we described an advanced approach to recapitulate some of these aspects of scarless faster wound healing by developing a wound dressing biomaterial OxOBand that will enhance healing by (a) providing a matrix for cell migration, (b) providing sustained oxygen to regenerating tissue, (c) attenuating oxidative stress, and (d) inducing angiogenesis and proper collagen remodelling of regenerative tissue (Scheme 1). The regenerative dressing is biodegradable and will remain in the wound providing a matrix for tissue formation and ultimately integrating with it.

Previously, we have synthesized and fabricated an elastomeric antioxidant polyurethane (PUAO) for tissue engineering applications [9]. PUAO was synthesized by incorporating ascorbic acid in the backbone chain of polyurethane. The PUAO showed significant antioxidant potential. It supported cardiomyocyte growth, and adherence, demonstrated attenuation of ROS with increased survival of cardiomyocytes under ischemic conditions. Further, we fabricated polyurethane-based oxygen releasing antioxidant scaffolds (PUAO-CPO) by incorporating calcium peroxide in PUAO cryogels, which showed the sustained release of oxygen for a period of over 10 days. This oxygen releasing scaffold increased cell survival under hypoxia conditions and demonstrated enhanced survival of critically perfused tissue in an ischemic skin flap model [10].

Adipose-derived stem cells (ADSCs) can modulate wound healing by secretion of paracrine factors and exosomes [11]. Exosomes are cell-derived nanovesicles that carry a cargo of growth factors and microRNAs and have shown to modulate several cellular processes, including wound healing and angiogenesis. They stimulate cellular migration, and proliferation, along with adequate collagen synthesis and remodelling, that results in accelerated wound healing [12].

We, therefore, hypothesized that the oxygen releasing antioxidant scaffold PUAO-CPO, along with exosomes (OxOBand), would significantly enhance diabetic wound healing by attenuating hypoxia and oxidative stress, inducing angiogenesis, and enhancing collagen remodelling and re-epithelialization. We further speculated that the ADSCs derived exosomes will promote keratinocyte and fibroblast migration and increase cell survival under hyperglycemic conditions leading to faster wound closure.

We first investigated the growth, survival, and migration of keratinocytes and fibroblasts in the presence of exosomes. The effect of exosomes on cell survival under hyperglycemic conditions was also accessed. Further, the ability of the OxOBand to promote diabetic wound healing in a full-thickness wound in a high-fat diet streptozocin-induced diabetic rat model was investigated. The developed OxOBand was further challenged for its feasibility in the treatment and regeneration of infected diabetic wounds.

Section snippets

ADSCs exosomes and oxygen releasing antioxidant scaffolds promoted attachment, survival, migration, and proliferation of fibroblasts and keratinocytes

Diabetic wounds are exposed to hyperglycemia, hypoxia, and elevated oxidative stress that challenges the migration and survival of the skin cells [13,14]. To heal and regenerate, skin cells must proliferate and migrate under these challenging conditions. Wound dressing should promote adherence, migration, and proliferation of these dermal cells. We developed an exosome laden oxygen releasing antioxidant wound dressing OxOBand that can promote cell migration, proliferation, attenuate oxidative

Conclusion

In this study, we developed and evaluated oxygen releasing antioxidant dressing OxOBand for diabetic wound healing. Oxygen releasing antioxidant dressing attenuated oxidative stress, induced vascularization, and enhanced collagen remodelling that are beneficial for diabetic wound healing. Supplementation of these oxygen releasing antioxidant dressing with ADSCs derived exosomes augmented their regenerative potential. Exosome embedded oxygen releasing antioxidant dressing OxOBand could also

Exosome isolation and characterization

Exosomes were isolated from adipose-derived stem cells (ADSCs). ADSCs were isolated from fat tissue, obtained from 6 to 8 weeks old normal healthy rats. Briefly, perirenal and inguinal fat tissue was isolated, washed with PBS antibiotic solution, and digested with 0.05% collagen (Invitrogen) for 1 h. The digested tissue homogenate was filtered through 70 μm cell strainer and plated in tissue culture treated flask in alpha-MEM with 10% (v/v) exosome free FBS. ADSCs adhered to TCP in 48 h and

CRediT authorship contribution statement

Parvaiz A. Shiekh: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Visualization, Writing - original draft. Anamika Singh: Formal analysis, Methodology, Writing - review & editing. Ashok Kumar: Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Software, Supervision, Validation, Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

The work was funded by the Department of Science and Technology (DST/INT/SWD/P-11/2016); Department of Biotechnology (DBT/IN/SWEDEN/08/AK/2017-1); Ministry of Human Resource Development- IMPRINT (MHRD_6714/Healthcare), Ministry of Human Resource Development-SPARC (SPARC/2018–2019/P612/SL), and Ministry of Human Resource Development-UAY (MHRD_IITK_006), Govt. of India. Parvaiz Shiekh, and Anamika Singh, would like to acknowledge IIT Kanpur for Ph.D. fellowship. We thank Suhela Tyeb, IIT Kanpur

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