Application of Nanocellulose Biocomposites in Acceleration of Diabetic Wound Healing: Recent Advances and New Perspectives

Abstract

Diabetes mellitus (DM) is a chronic health problem that increases the risk of infection and delays wound healing due to impairment of metabolic activity. Diabetic foot ulcers (DFUs), a chronic wound increases the risk of mortality. Finding the most appropriate wound dressings has been intensified with the increasing population and prevalence of chronic wounds. Nanofibers coated wound dressings have attracted more attention as innovative and biocompatible materials. Nanocellulose (NC) has been widely used as a reinforcing material to improve nanofibers’ mechanical and thermal properties. NC is biodegradable and derived from renewable sources and produced bionanocomposites with improved performance.

1. Introduction

Diabetes mellitus (DM) is a complex chronic metabolic and endocrine disorder that is termed a silent killer. DM is distinguished by a higher level of glucose in the blood which is termed hyperglycemia [1]. DM is classified into two classes and they are TYPE I DM and TYPE II DM. According to International Diabetes Federation (IDF), the estimated number of diabetic adult patients (20–75) worldwide is 463 million, and it will be 578.4 million by 2030 and 700.2 million by 2045 as per expectations. Based on the 2019 prevalence data of IDF, the number of death and complications that arise due to DM worldwide is 4.2 million [2].

Diabetic foot ulcers (DFU) are a chronic infection that arises due to DM. DM decreases the rate of wound healing due to impairment of metabolic activity. Annually, 9.1–26.1 million people were infected by DFU and many cases were selected for amputation as an ultimate treatment. DFU amputees have a 3 years survival due to the risk of infection and unsolved artery injury and 50–70% of patients have a recurrence in 5 years. Therefore, in the prevention of DFU of DM patients should maintain a lot of precautions in their lifestyles, and time consuming and high level of treatment by a multidisciplinary group of specialists is required [3].

The need for highly efficient approachable wound dressing materials is very essential in the treatment of DFU with the increasing of populations. Nanofibrous composites are attractive wound dressing materials due to their similarity with the extracellular matrix of the skin. They form an effective layer over a wound that stimulates the tissue to fight against infections causing pathogens and accelerate the wound healing [4]. Nanocellulose (NC) is used as a reinforcing material to improve the mechanical and thermal properties of nanofibrous wound dressing materials. Due to the small size, high surface area, high mechanical strength, biodegradability, and renewability of nanocellulose, it facilitates the production of cost-effective and eco-friendly wound dressing materials [5].

Several bionanocomposites of nanocellulose along with other components are applied in diabetic wound therapy in recent years [6]. Bionanocomposites are nanohybrids composed of biobased materials and inorganic components. In bionanocomposites one component should be in nano dimensions, e.g., nanopolymer, inorganic NPs, etc. [7]. Bionanocomposites cover an intensive area of research due to their biocompatibility, nontoxicity, biodegradability, renewability, and cost-effective nature. Among them, cellulose-based nanocomposites have attracted researchers in the biomedical field due to the abundance of cellulose in nature and their biocompatible nature [8]. Keeping this in mind, many researchers developed nanocellulose reinforcing biocompatible patches, bionanocomposites hydrogel for diabetic wound healings. The objectives of this chapter are to study the mechanism of normal wound healing and diabetic wound healing, different methods of synthesis of nanocellulose based bionanocomposites, and application of different bionanocomposites of cellulose in diabetic wound healing. This chapter also discussed the future perspectives of nanocellulose based wound healing dressings in DFU treatment.

2. Application of naocellulose-based nanocomposites in diabetic wound healing

2.1 What is a wound?

Skin is the largest organ of the human body, it not only protects the human body from different pathogens and the external environment but also has a great impact on different metabolic activities of the human body. Any kind of damage to the skin or other parts of the human body is termed a wound. The wound includes cuts, scrapes, scratches, and punctured skin [9]. Although the accident is the main cause of the wound, it also happens due to surgery, sutures, and stitches. Wounds happen on the skin due to exposure to the external environment and its elastic and soft nature is susceptible to generating defects. Wounds are classified into two types- acute and chronic wounds [10]. Acute wounds happen due to bites, cuts, minor burns, and surgery. These types of wounds heal within a predictable period and follow the four stages of the healing process; they are hemostasis, inflammation, proliferation, and maturation. Chronic wounds do not follow the healing process and disturbance of the physiology of the wound happens due to various endogenous mechanisms. Chronic wounds damage the integrity of the tissue and increase the healing periods. Diabetic foot ulcers, pressure sores, etc., are examples of chronic wounds. Chronic wounds occur due to aging, malnutrition, immunosuppression diseases like AIDS [11]. Pathogens infected the wound from beached skin and infection creates pain, discoloration of the wounded area, edema, puss, smell, tenderness, etc. Microbes produce biofilm in the wound and make difficulties in the healing process. With the help of biofilm, bacteria can transfer the antibiotic-resistant gene to other bacterial species. Biofilm makes a barrier between antibiotics and bacteria and makes it tough to heal the infected wound [12]. Hence, it is very necessary to develop a new and novel wound dressing material that can heal wounds infected by multidrug-resistant bacteria.

2.2 Diabetic wound

DM causes severe damage to multi-organ of DM patients such as cardiovascular disease, chronic renal failure, and diabetic skin wounds [13]. Lower extremities of diabetic patients infected by multidrug-resistant pathogens and the phenomenon are called diabetic foot ulcer (DFU). Annually highest numbers of DFU patients admit to hospital and require a large amount of management cost among other severe diseases. The infected wound of DFU damages the defensive sensation associated with peripheral artery disease and also develops cell necrosis that leads to amputation [8]. 80% of lower limb amputation is the result of DFU annually [14]. According to Control Cardiovascular Risk in Diabetes (ACCORD) DM patients can reduce the risk of amputation of the lower limb by maintaining the standard glycemic control (HbA1c 53–63 mmol/mol [7.0–7.9%]) or taking antidiabetic therapy (HbA1c < 42 mmol/mol [<6%]) [10]. DFU developed due to many factors such as loss of glycemic control, peripheral vascular disease, peripheral neuropathy, and immunosuppression [11]. Neuropathy is the main contributor to DFU. Accumulation of sorbitol and fructose in a hyperglycemic state decreases the production of nerve cell myoinositol, which interrupts normal neuron conduction [12]. This phenomenon creates an imbalance between flexion and extension of the foot and produces foot deformities. Hyperglycemia also causes vascular diseases, such as endothelial cell dysfunction and an increase in thromboxane A2 [11]. All these are the key factors for DFU and the untreatable condition of infected wound leads to amputation. Nanocomposites consisting of polymer, polysaccharides, and inorganic nanoparticles exhibit better antipathogenic properties in lower dosages than conventional drugs and easily overcome the complications of multidrug-resistant pathogens [3]. Here, this chapter would focus on the application of nanocellulose based composites in the treatment of DFU. Along with this, a brief description and mechanism of normal wound healing and diabetic wound healing was tried to provide for better understanding.

2.3 Wound healing

The healing of a wound proceeds through a complex pathway with dynamic interactions of different cell types, extracellular matrix (ECM), cytokines, and growth factors. Fundamentally, wound healing consists of four steps, hemostasis, inflammation, cell movement, and proliferation, followed by wound compression and further remodeling [14]. Hemostasis involves the clotting process through the coagulation cascade that leads to the cessation of bleeding. Platelets are the first subset of cells that enter the injury site and release several growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta, endothelial growth factor (EGF), and fibroblast growth factor (FGF), which support the inflammation process [15]. Hemostasis is followed by inflammation through vascular delivery of inflammatory agents and migration of cells into the injury site. Inflammatory mediators such as prostaglandins, histamine, and leukotrienes by mast cells stimulate angiogenesis and permeability to allow cells and molecules from the bloodstream to enter the wound site [16]. White blood cells such as neutrophils, monocytes, and lymphocytes invade the injury site. Neutrophils prevent microbial infections and macrophages, secret TGF, vascular endothelial growth factor (VEGF), and FGF to stimulate angiogenesis. Neutrophils also produce tumor necrosis factor-alpha (TNF) that breakdown necrotic tissue and facilitate the proliferation of fibroblasts for deposition of collagen for tissue granulation [17]. The dermal wound is followed by wound contraction after 2 weeks. Fibroblasts convert to myofibroblasts phenotype during tissue granulation, with enhanced alpha-smooth muscle actin cytoskeleton that plays a vital role in wound closure. During the re-epithelialization of tissue, keratinocytes migrate, differentiate, and proliferate to generate a stratified epidermis along the superficial area of injury to provide cover for newly formed tissue and new tissue covers wound bed [18]. Remodeling is the last phase of wound healing that lasts for 6–24 months. Remodeling involves the generation of granulation tissue accompanied by the replacement of the ECM with type I collagen (substituting collagen III) mediated via PDGF and TGF [19].

2.4 Diabetic wound healing

Inflammatory macrophages stay at the site of injury in the case of diabetic wounds for a longer period compared to normal wound healing. Increased ratio of pro-inflammatory cytokines, such as TNF and interleukin 6 (IL-6) are produced by inflammatory macrophages. Macrophages also elaborate ROS that causes persistent inflammation and lead to stimulation of proliferative factors for successful wound healing. In the case of diabetic wounds, inefficient efferocytosis (phagocytosis of apoptotic cells) by macrophages perturbed cytokine cascade and causes a higher burden of apoptotic cells. Pro-inflammatory cytokines (IL-1 and TNF) and matrix metalloproteinase-9 (MMP-9) increase in ratio with decreased anti-inflammatory signals (CD206, IGF-1, TGF, and IL-10) and lead to abnormal apoptosis of fibroblasts and keratinocytes, and decreased angiogenesis [20]. Fibroblasts do not properly convert to myofibroblasts in diabetic wound healing, which reduces mechanical tension of ECM, and results in poor wound closure due to lack of SMA [21]. Again, a non-equilibrium balance between MMPs degrades the disorganized collagen in normal wound healing and tissue inhibitor of metalloproteinases (TIMPs), as a result, degradation and deposition of abnormal ECM occur. High levels of pro-inflammatory cytokines and pro-fibrotic cytokines reduced the level of expression of TIMPs and higher expression of MMPs. Levels of MMPs are raised 60 times more in chronic wounds than for acute wound healing [22]. In chronic wounds, degradation of ECM, growth factors, and collagen deposition increases due to an increase in protease activity in tissue reconstruction [22]. All these factors play a crucial role in wound healing. Along with these factors, a dysregulated molecular and cellular wound microenvironment is not conducive to normal healing responses and culminates in impaired healing of diabetic ulcers.

2.5 Nanocellulose

Bio-renewable materials are the most demandable factors in the construction of a sustainable planet. Biopolymer is the promising substitute for the petroleum-based product in the environment safe concern. Natural feedstocks such as agricultural waste, food waste, wood, etc., can be converted to value-added products. Plants cells are composed of cellulose (40–50 wt%), hemicellulose (20–40 wt%), and lignin (20–30%), and these lignocellulosic materials are the most abundant bio-resources on the earth for sustainable and renewable products [23]. Cellulose is the most abundant natural polymer on earth. Chemically, cellulose is composed of a linear chain of homo-polysaccharide glucose units linked by a β-1,4-glycosidic bond. Nano form of cellulose is termed nanocellulose. Cellulose can be converted to nanocellulose by breaking the intra- and intermolecular hydrogen bond between the polymeric chains of the cellulose [24]. Nanocellulose can be sourced from plants, microorganisms, and aquatic animals (tunicates), rich in cellulose. Different plants such as banana leaf, corn cob, cotton, ramie, rice husk, wood, sugarcane bagasse, sisal leaves, wheat straw, wood, and coconut husks are rich sources of nanocellulose. Nanocellulose can be also extracted from coffee grounds, ginger, durian rind waste, lemon seeds, okara, pea hull, Phragmites australis, Hevea brasiliensis (Rubberwood), and tea stalk. Nanocellulose extracted from bacteria is known as bacterial nanocellulose (BNC) or bacterial cellulose (BC). Acetobacterxylinum (Gluconacetobacter xylinus) is the most efficient bacteria used for nanocellulose production. Other bacterial species Acetobacter, Achromobacter, Agrobacterium, Acanthamoeba, Alcaligenes, Rhizobium, Pseudomonas, Sarcina, and algal species Cladophora, Rhizoclonium, Microdiction, and Chaetomorpha, are also the potential source of nanocellulose. BC is purer than nanocellulose derived from other sources. But the molecular structure of BC and plant-derived nanocellulose are similar [5].

2.5.1 Classification of nanocellulose

Nanocellulose is classified into three main classes based on its morphological structure and sources. They are cellulose nanocrystals (CNCs/NCC), cellulose nanofibers (CNFs/NFC), and bacterial nanocellulose (BNC/BC).

2.5.1.1 Cellulose nanocrystals (CNCs/NCC)

CNCs are extracted from cellulose-rich sources. Acid treatment is mainly utilized to convert cellulose to nanocellulose. They are highly crystalline with a needle-like structure. Their dimensions are 4–20 nm in width and 100–500 nm in length.

2.5.1.2 Cellulose nanofibers (CNFs/NFC)

Chemical treatment and mechanical disintegration are utilized to extract cellulose. nanofibers (CNFs). They are both crystalline and amorphous with 1 μm in length.

2.5.1.3 Bacterial nanocellulose (BNC/BC)

Nanocellulose originates from microorganisms defined as BNC. They are the purest form of nanocellulose. Their diameter range is 20–100 nm and several micrometers in length [5].

2.6 Nanocellulose based composites

Nanocellulose is the promising reinforcing agent and filler in the formation of bionanocomposites. Due to its unique tensile strength, thermal behavior, and ease of surface modifications, it becomes a potential constituent in the development of multipurpose nanocomposites. Bionanocomposites are an efficient applicant in various fields due to their biodegradability, biocompatibility, and cost-effective nature, and they also possess a high surface-to-volume ratio and nanometric size effect. Nanocellulose seeks the attention of researchers due to its abundances, renewability, environmentally friendly, cost-effective, and improved mechanical nature. Nanocomposites are classified into two categories; they are natural and synthetic nanocomposites based on the constituents [25].

2.7 Biomedical application of nano-cellulose based bionanocomposites

Nanocellulose is a potential applicant in biomedical fields due to its biocompatibility, biodegradability, high surface area, low density, high mechanical, thermal, and optical properties [26]. Nanocellulose can be efficiently applied in drug delivery, tissue engineering, wound healing, and as antimicrobial agents [27]. Nanocellulose-based nanocomposites show excellent activity in the delivery of loaded drugs in the tropical site. This factor helps in the development of nanocellulose-based wound dressing in the treatment of normal as well as chronic wound (DFU) [28].

2.7.1 Application of nanocellulose-based nanocomposites in wound healing

The bacterially infected wound is very hard to repair due to the biofilm formation by bacteria, drug resistance, and high recurrence properties of bacteria [29]. Among bacteria, Staphylococcus species were responsible for around 70% of bone-related infections. The formation of biofilm with high adherence and the inefficient drug release system delay wound healing [30]. Nanocomposites are sustainable and efficient antimicrobial drug delivery systems that overcome the obstacles from implant-associated bacterial infections [31]. E.g., Starch/Triphala Churna (TC) nanocomposites exhibited high drug loading efficacy and prolonged TC release for improved acetylcholinesterase inhibition. These nanocomposites also possess excellent antimicrobial activity with antioxidant activity against multidrug resistance biofilm-forming human bacterial pathogens [32].

Cellulose materials are the most appealing candidate in the development of eco-friendly and durable materials [33]. Nanocomposites of nanocellulose have clinical potential applications in tissue engineering. Various nano-scaffold of nanocellulose mimic the nature of the tissue and substitute degenerated tissues. CNC/folic acid delivered targeted chemotherapeutic agents to folate receptor-positive 886 cancer cells [34]. CNC/hydroquinone inhibited the production of melanin, was designed for treating hyperpigmentation, a disorder occurring during pregnancy and sun exposure [35]. Various cellulose-based nanocarriers, such as bacterial cellulose, cellulose acetate, microcrystalline cellulose, carboxymethyl cellulose, cellulose nanocrystals, cellulose nanofibrils, etc., in drug delivery systems for cancer treatment, were used [36]. Nanocellulose-based wound dressings were combined with biosensors, e.g., for human neutrophil elastase was used in nanocellulose based nanocomposite in chronic wound healing [37].

Advantages of the biodegradable polymer in wound dressing overcome the obstacles of not degradable polymer containing wound dressing material in the case of removal of wound dressings after healing. The human body is lacking the cellulose enzyme of bacteria that relate to the biodegradability of the cellulose polymer. Modified or derivative forms of cellulose-containing wound dressing materials solve this problem. E.g., a glucose polymer of dextran was used to improve the biodegradability of BC and cell proliferation in wound site [38] and Chloramphenicol/2,3-dialdehyde cellulose hydrogel showed higher biodegradability, drug delivery ability, and efficient antibacterial actions against E. coli, S. aureus, and Streptococcus pneumoniae [39]. Gram-positive (S. aureus and Enterococcus faecalis) and gram-negative bacteria (P. aeruginosa) are significant in single or multi-bacterial wound infections [40]. Sufficient moisture, oxygen, temperature, growth factors, and bioactive materials play a vital role in the fast healing of the wound [41]. BC/ZnO nanocomposites more effectively repair burn wounds (77%) than sulfadiazine (66%) within fifteen days [42, 43]. Among nanocellulose, NCC and NFC are specific in wound healing due to their high degree of functionality and biocompatibility. Sodium periodate oxidized NFC was applied in the integration of collagen polymer. This nanocomposite exhibited high water absorption (4000%), porosity (95%), and density of collagen/NFC (0.03 g/cm³) [44]. In another study, periodate and TEMPO were used to oxidize and carboxymethylated NC for use as a bioink material with growth inhibition ability in the case of P. aeruginosa PAO1 [45]. Mechanical and cytotoxic properties of nanocomposites of NCCs showed significant water absorbance with biocompatibility effect on adipose-derived stem cells (ASCs) and L929 cell line after 7 days [46] as compared to chitosan polymer. CNC/chitosan/polyethylene oxide composites showed increased tensile strength, tensile modulus, and O₂/CO₂ transmission and had no cytotoxic impact on adipose-derived stem cells. Hence, the nanocomposites were considered promising applicants for wound healing [47]. CNC composed wound dressings also promote coagulation processes in bleeding wounds [48]. E.g., biodegradable nanocomposites film and sponges of oxidized CNC/alginate exhibited excellent hemostatic efficiency without eliciting an inflammatory reaction. Carboxyl functionalization on the CNC surface was responsible for the hemostatic effect of the nanocomposites. Due to the hemostatic effect, blood plasma was significantly absorbed in the material science and it became more effective in promoting platelet aggregation and in inducing erythrocytes to accelerate blood clotting [49]. Curcumin and gelatin microspheres (GMs) were loaded with scaffolds of porous collagen (Coll)-CNC composite. These scaffolds successfully released curcumin and exhibited potential antibacterial activity in vitro. They also effectively repair the full-thickness burn infections through accelerating dermis regeneration and preventing local inflammation [50]. CNC/chitosan/Ag NPs/curcumin nanocomposites accelerated the complete healing of excision wounds in albino rats without skin irritation [51]. Gentamycin sulfate (GS)/collagen (Coll)/CNC/gelatin microspheres (GMs) scaffolds and CNC/poly(N-isopropyl acrylamide) (PNIPAAm)/metronidazole hydrogel are promising wound dressing with antibacterial activity [52]. CNC/alginate hydrogel consisting of a double membrane system of cationic CNC (CCNC) and anionic alginate was applied to design hydrogel to incorporate different drugs in each hydrogel membrane: ceftazidime hydrate (CH) antibiotics in the external membrane (pure alginate) and EGF in the internal membrane (CCNC and alginate). This hydrogel was suitable for wound dressing with rapid drug release and oral drug delivery applications [53]. Electrospinned polyethylene imine carboxymethyl chitosan/pDNA-angiogenin (ANG) nanoparticles/curcumin (Cur)/poly(D,L-lactic-co-glycolic acid) (PLGA)/CNCs nanodispersion was developed to deliver a novel drug and plasmid DNA (pDNA). The composites exhibited potential antioxidant, antitumor, anti-inflammatory, and antibacterial activity and repaired full-thickness burn wounds by stimulating blood vessel formation and sustained release of drugs [54].

2.8 Application of nanocellulose based nanocomposites in diabetic foot ulcer (DFU)

Special wound dressing is required to repair DFU which has prolonged drug release properties, maintains optimum moist conditions, and protects wound from infection. Polymers and polysaccharides based nanostructured wound dressing materials are ideal for DFU treatment due to their notable biocompatibility, high drug loading capacity, and accessible tailoring properties [55]. Electrospinned berberine/cellulose acetate (CA)/gelatin mat was developed as a potential wound dressing for diabetic foot ulcers [56]. CNC extracted from Syzygium cumini was applied to CNC/Ag NPs nanocomposites in two different forms. In ointment form, it repairs wound in diabetic mice by promoting increased collagen deposition, angiogenesis and enhanced the formation of neo-epithelialization. The wound healing capability was better in ointment form than in strip form. Again, CNC derived from Syzygium cumini leaves or bamboo was utilized in the synthesis of CNC/Ag NPs and applied in the repairing of acute and diabetic wounds [57]. PLGA/CNC nanofibres loaded with neurotensin (inflammatory modulator) were applied as potential wound dressings for accelerated diabetic wound healing [58]. CNC/curcumin nanocomposite films were applied to diabetic wounds showed stable curcumin release, which plateaued after 36 h [59]. Antimicrobial CNC/Ag nanocomposite wound dressings with excellent water absorption capacities were applied in diabetic or full-thickness wounds. They reduced pro-inflammatory cytokine levels and completely recovered the wound by elevating collagen and improving re-epithelization and vasculogenesis [60]. Biosensor dressing of CNF film loaded with Venlafaxine (an analgesic drug used to treat neuropathic pain) and tetracycline (antibiotic) was applied simultaneous delivery of two drugs to diabetic foot ulcers. The CNF film served as an appropriate carrier for the co-delivery of the two drugs [61].

3. Conclusion and future perspectives

This chapter provides a state-of-the-art review of the application of nanocomposites of nanocellulose in the biomedical field, especially in diabetic wound healing as sustainable wound dressings. Nanocellulose has tremendous applications in various fields such as biomedical, environmental remediation, food technology, electronics, etc. Due to the natural availability, biodegradability, biocompatibility, and tunable nature nanocellulose is an effective drug carrier in target-specific delivery of the drug. Incorporation of other NPs or impregnation of nanocellulose in the nanocomposites enhances the drug loading capacity of nanocellulose. The dual drug delivery ability of nanocellulose is more efficient than the single drug delivery. This property is useful for the delivery of various combinations of drugs in the treatment of various chronic diseases. The potential application of nanocellulose-based composites in the biomedical field is studied, but the in-depth toxicological properties of nanocellulose based composites have not been evaluated yet. This study will be helpful for the more advanced clinical application of nanocellulose based composites.

Diabetic wound healing is a complex and prolonged process due to its pathophysiology, resulting in the impaired function of different cells and unbalanced levels of key biochemical healing mediators. Advanced nanowound dressings are applied to overcome the challenges of diabetic wound healing. Nanocomposites are synthesized for this application by combinations of biomolecules (such as growth factors, genes, proteins/peptides, stem cells/exosomes) and non-bioactive substances (metal ions, oxygen, and nitric oxide), as well as nanotechnology (e.g., PTT, LBL self-assembly technique and 3D printing). The etiopathogenesis of diabetic foot ulcers is too complex and challenging. Wound dressings should consist of more than two materials. Therefore, potential constituents should be applied that have antimicrobial as well as biocompatible nature. Nanocellulose based nanocomposites are tried to apply in wound healing of diabetic patients that have fulfilled all the criteria of wound dressings of DFU. Future directions will try to develop new nanocellulose based wound dressings with multiple roles (including improving hypoxia, enhancing angiogenesis, reducing oxidative stress, and preventing infection). These types of nano dressings will accelerate diabetic wound healing in all stages and maintain a balanced environment during wound healing. Nanocellulose based nanocomposites will be believed to be nano wound dressings that will reduce all the complications in the treatment of DFU and fully cure diabetic wounds in a short period without any side effects.

Acknowledgments

The authors sincerely acknowledged the Director of CSIR-North East Institute of Science and Technology, Jorhat, Assam for his permission to perform our research with excellent facilities and precious guidance. R. B. acknowledged the Department of Science and Technology, New Delhi, for the Inspire Fellowship (GAP-0754).

Conflict of interest

The authors declare no conflict of interest.