Elsevier

Acta Biomaterialia

Volume 10, Issue 2, February 2014, Pages 843-857
Acta Biomaterialia

Chitosan-based dressings loaded with neurotensin—an efficient strategy to improve early diabetic wound healing

https://doi.org/10.1016/j.actbio.2013.09.040Get rights and content

Abstract

One important complication of diabetes mellitus is chronic, non-healing diabetic foot ulcers (DFUs). This study aims to develop and use dressings based on chitosan derivatives for the sustained delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. Three different derivatives, namely N-carboxymethyl chitosan, 5-methyl pyrrolidinone chitosan (MPC) and N-succinyl chitosan, are presented as potential biomaterials for wound healing applications. Our results show that MPC has the best fluid handling capacity and delivery profile, also being non-toxic to Raw 264.7 and HaCaT cells. NT-loaded and non-loaded MPC dressings were applied to control/diabetic wounds to evaluate their in vitro/in vivo performance. The results show that the former induced more rapid healing (50% wound area reduction) in the early phases of wound healing in diabetic mice. A NT-loaded MPC foam also reduced expression of the inflammatory cytokine TNF-α (P < 0.001) and decreased the amount of inflammatory infiltrate on day 3. On day 10 MMP-9 was reduced in diabetic skin (P < 0.001), significantly increasing fibroblast migration and collagen (COL1A1, COL1A2 and COL3A1) expression and deposition. These results suggest that MPC-based dressings may work as an effective support for sustained NT release to reduce DFUs.

Introduction

Diabetes mellitus is one of the most prevalent chronic diseases world wide. Impaired wound healing is a complication of diabetes that results in a failure diabetic foot ulcers (DFUs) to completely heal [1]. Complications of DFUs lead to frequent hospitalization and, in extreme cases, to amputation, resulting in high hospital costs and a poor quality of life for patients [2]. DFUs are a multifactorial complication that result particularly as a consequence of peripheral neuropathy, impaired vascular function, impaired angiogenesis and/or chronic inflammation [1], [3].

Recently it became evident that the peripheral nerves and cutaneous neurobiology contribute to wound healing [4]. Loss of peripheral sensory and autonomic nerves reduces the production of neuropeptides that are important for proper wound healing [3]. Neurotensin (NT) is a bioactive neuropeptide that is widely distributed in the brain and in several peripheral tissues [5], [6]. NT interacts with leukocytes, mast cells, dendritic cells and macrophages, leading to cytokine release and chemotaxis, which can modulate the immune response. In addition, NT affects microvascular tone, vessel permeability, vasodilation/vasoconstriction and new vessel formation, helping to improve angiogenesis during wound healing [3], [7], [8].

Some studies have demonstrated that topical application of neuropeptides, such as substance P and neuropeptide Y, can improve wound healing in diabetes [9], [10]. However, the major problem with topical administration of peptides is their short half-life and loss of bioactivity in the peptidase-rich wound environment [11]. An alternative strategy to overcome this problem is the use of biocompatible wound dressings for the sustained delivery of neuropeptides. These dressings should, however, also replicate the characteristics of skin in order to promote the proliferation and migration of fibroblasts and keratinocytes, as well as to enhance collagen synthesis, leading to proper healing with little scar formation [12], [13].

Wound dressings based on natural polymers have been extensively applied to simulate extracellular matrix (ECM) regeneration after injury [12], [13]. One of the most used naturally based polymers for wound healing applications is chitosan [12], which is a linear co-polymer of d-glucosamine and N-acetyl-d-glucosamine [14]. Since it is derived from chitin, a polymer found in fungal cell walls and the crustacean exoskeleton, it is a relatively inexpensive and abundant material [15]. In addition, it has proven to be biodegradable, biocompatible, non-antigenic, non-toxic, bioadhesive, antimicrobial, bioactive and to have hemostatic capacity [15], [16], [17]. Furthermore, chitosan promotes tissue granulation and accelerates wound healing through the recruitment of inflammatory cells such as polymorphonuclear leukocytes (PMN) and macrophages to the wound site [18].

To increase its poor solubility in water chitosan functional groups can be chemically modified to produce water-soluble chitosan derivatives such as N-carboxymethyl chitosan (CMC), 5-methyl pyrrolidinone chitosan (MPC) and N-succinyl chitosan (SC) [19], [20], [21]. These chitosan derivatives are functional biomaterials that maintain the antibacterial and non-cytotoxic properties of the parent chitosan. In addition, they stimulate the extracellular lysozyme activity of skin fibroblasts [22], [23].

The aim of this study was to develop and apply wound dressings prepared from the chitosan derivatives referred above (CMC, MPC, SC) for the prolonged and efficient delivery of NT to diabetic and non-diabetic wounds, while also conferring wound protection and comfort. The progression of skin wound healing in diabetic and non-diabetic mice was also evaluated by analysis of the inflammatory and angiogenic effects of NT when applied to skin wounds alone or loaded into MPC-based dressings.

Section snippets

Materials

Chitosan (medium molecular weight, 90% degree of acetylation confirmed by 1H NMR), glyoxylic acid monohydrate (98%), sodium hydroxide, sodium borohydride (99.5%), levulinic acid (98%), succinic anhydride (97%), reduced glutathione (GSH), 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), dialysis membranes (Spectra/Por 6) with a MWCO of 8000 Da and methanol p.a. were obtained from Sigma–Aldrich (USA). Acetic acid was obtained from Panreac (Spain), and ethanol was purchased from Riedel-de-Haen

Degree of substitution and morphology of CMC, MPC and SC

The degree of substitution (number of native chitosan amino groups substituted) of each chitosan derivative was confirmed by 1H NMR as 25.5%, 24% and 28.5% for CMC, MPC and SC, respectively (Supplementary Fig. S1). The schematic representation of each derivative is shown in Fig. 1A.

The different morphologies obtained for each of the prepared chitosan derivative foams are shown in Fig. 1B. CMC presents a honeycomb-like porous structure, with larger pores than MPC and SC, which presented an

Discussion

One of the main objectives of this work was to evaluate the capacity of chitosan-based wound dressings as biocompatible and biodegradable supports for the sustained delivery of NT, a neuropeptide that has been shown to improve wound healing [27], [28].

Three different water-soluble chitosan derivatives (CMC, MPC and SC) were synthesized and tested for their water swelling capacities and peptide release profiles in order to infer which of the derivatives would have the best performance

Conclusions

The results obtained in this work show that in control animals both MPC and NT-loaded MPC foams have a significant impact on the early phases of the healing process, decreasing the amount of inflammatory infiltrate. In diabetic animals the major healing effects were observed with either NT alone or NT-loaded MPC foams, thus confirming the potential healing effect of NT on diabetic wounds. These treatments reduced the inflammatory status in the early phase of wound healing and increased the

Conflict of interest

The authors declare no competing financial interests.

Acknowledgements

This work was financially supported by COMPETE, FEDER and Fundação para a Ciência e Tecnologia (FCT-MEC) under contracts PTDC/SAU-MII/098567/2008, PTDC/SAU FAR/121109/2010, PEst-C/EQB/UI0102/2011 and PEst-C/SAU/LA0001/2013-2014, in addition to the EFSD/JDRF/Novo Nordisk European Programme on Type 1 Diabetes Research and Sociedade Portuguesa de Diabetologia. L.I.F.M., A.M.A.D. and E.L. acknowledge the FCT-MEC for their fellowships SFRH/BD/60837/2009, SFRH/BPD/40409/2007 and SFRH/BPD/46341/2008,

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