Production of Hydrogel for Wound Healing Management

Production of Hydrogel for Wound Healing Management

Abstract

Wound treatment has recently increased its essence in the wound care division due to the chronic wound pervasiveness in the high-risk population, including obese and immunocompromised patients and the geriatric population. Besides, the population diagnosed with diabetes is drastically growing. According to the World Health Organization (WHO), international diabetic events have increased to 463 million people living with diabetes by 2019. Statistics further expect this number to grow to 700 million by 2045. As diabetes turns into a normal medical condition, it has also emerged to be the leading cause of chronic wounds, which demands specialized care to address the unique needs of the patients. Subsequently, wound dressing plays a critical role in the process of healing wounds as they protect the wound area from the external environment. Moreover, they can interact with the wound bed to facilitate as well as accelerate the process of healing. Enhanced dressing such as hydrogel is tailored to sustain a moist environment in the area of application, and due to the high water content, it is an appropriate candidate for wound management. The hydrogel can be utilized for both dry necrotic or exudating wounds. Nonetheless, hydrogel demonstrates other unique aspects such as malleability, softness, and biocompatibility. In the modern era, advanced wound care products generate around $7.1 billion of the international market, and their production is rising as a yearly rate of 8.3 %, with the market expected to be $12.5 worth by 2022. Hydrogel products consist of a range of polymeric materials, the hydrophilic configurations of which renders them the ability to hold a large amount of water in their 3D (three-dimensional) network. Several industrial and environmental areas of application consider extensive employment of hydrogel products to be of prime importance. Considerably, synthetic types replaced natural hydrogels slowly because of their higher h2O absorption capacity, various variety of raw chemical resources, and raw and long service life.  Nevertheless, various technical reports and publications dealing with these products from the engineering perspective were examined to overview technological elements covering this emerging multidisciplinary subject of research. Amine functionalized polysaccharide hydrogel, similar to those found in chitosan, are commonly examined as biomaterials. The presented project focuses on adding amino acids and the use of sodium alginate dressing to the hydrogel, making it different from the commercial dressing. Besides, the objective of the project is to formulate hydrogen using amino acid in specific cysteine and arginine to generate a wound dressing.

Introduction

1.0 Background information

The materials of interest in this project are mainly hydrogen, which are polymer networks comprehensively swollen with water. Hydrophilic gel, commonly known as hydrogen, is a network of polymer chains that are, at times, found as colloidal gel whereby the water is the dispersion medium. In the past years, researchers have defined hydrogels in various ways. Still, the most common is that it a structure swelled with water and crosslinked polymeric network that the simple reaction produces one of several monomers. Another description is a polymeric substance that displays the ability to swell as well as retain a substantial fraction of water within its structure. However, it will not dissolve in water. These materials have received significant attention for the past several decades because of their exceptional reputation in various applications. More so, they possess a measure of flexibility that is similar to the natural tissues because of their considerable water content.

The hydrogel attractiveness relies on the application; for instance, in the wound healing process, hydrogels are utilised for their capacity to absorb wound exudate, whereas allowing oxygen transportation to the wound site. In tissue engineering, hydrogels are sought after for their capacity to imitate the extracellular matrix as well as support cell development. A dressing test will be carried out on the hydrogels, including the stress-strain test, HPLC, and ICP-MS analysis, to create a wound dressing. Moreover, microbiology tests will be included to explore whether the dressing has functional antimicrobials activities.

Several essential amino acids that the human body demands to meet its daily nutrition needs include arginine, cysteine, and glutamine. Researchers consider these three amino acids as conditionally essential, for they become crucial due to stress, trauma, chronic ailments, or inadequate dietary intake. L-Arginine has 32% of nitrogen and contains a range of functions, including stimulating protein regeneration, accelerating insulin secretion, and enhancing the transport of amino acid into the cells. Besides, it is a donor to nitric acid, hence improves oxygen and blood flow to the wound, therefore, enhancing the formation of collagen and reduction of inflammation. For the delivery of drugs, stimuli-responsive of hydrogels differ swelling depending on exterior stimuli leading to the release of drug molecules. Most important, hydrogels can give stimuli-receptive depending on the functional groups present. For instance, pH-receptive hydrogels are generated by including deprotonation or protonation ionisable groups.

Study shows that amine-functionalized polysaccharide has confirmed as an essential resource for generating biomaterials. More so, a recent study shows an amine-functionalized dextran hydrogel displayed excellent outcome as a wound dressing with grander neovascularization dextran compared to the commercially available dressing. Having these crucial amine-functionalized polysaccharide hydrogels contributions, the project examines the production of hydrogels in wound management. Recently, new developments have emerged on a method of rapidly preparing polysaccharide hydrogels through crosslinking polyamines such as polythene amine and polyallylamine with polysaccharides through epichlorohydrin. It is usually utilised as a crosslinker for both polyamines and polyols. Therefore it is an appropriate crosslinker for a hybrid system using both, which led to a method whereby any polysaccharide, as well as polyamine with enough solubility, can be combined polyamines and polyols.

1.2. 0 Wound dressing

Traditionally, wound dressing aimed to protect the wound site from the exterior environment and had a passive role in the process of healing. The extent of available passive barrier-type wound dressings such as tulle and gauze, increased as medical wound healing process understanding improved. This kind of dressing undoubtedly are less expensive and offer some protection. However, being passive, they fail to respond to varying wound conditions or provide medications in a sustained or controlled manner to enhance the process of healing. The wounds that adhere to the normal healing procedure, traditionally, obstacle-type dressing maybe conversely, effective chronic non-healing wounds can easily get infected, therefore failing to proceed through the usual stages of healing. Appropriate clinical management, thus, becomes imperative to reduce complications during wound healing.

Appropriate dressing covers and secures the wound site as well as creates an ideal moist environment in the affected area. Moreover, perfect dressings facilitate healing, and the healing process becomes shorter than the traditional approach. Advanced wound management techniques include non-invasive nursing of healing, management of pain, and the restricted release of agents adept of enhancing regeneration, repair as well as scar reduction. The notion of moist healing, revolutionised the field of management of wounds and the focus dressing transformed from traditional dry passive products to receptive moist-enhancing materials. Types of dressing utilised to attain a moist wound healing environment include foams, hydrocolloids, films and hydrogels.

Apart from these dressing approaches, hydrogel dressings are among the most versatile advance kind of moist wound dressings that are commercially accessible in various forms. Some of the proprietary hydrogel dressing include Instrasite (amorphous hydrogel dressing that assists in optimising wound surrounding for re-epithelisation), AmeriGel (a hydrogel dressing containing moist sustaining properties) and ActiGuard (a hydrogel sheet dressing, which is permeable to water vapour and air). The current research provides a comprehensive study of hydrogels as a wound dressing. Besides, the research discusses the normal scientific methods of hydrogels preparations for wound management applications, kind of commercially accessible hydrogel wound products, and the material property of hydrogel production and laboratory characterisation tests.

1.3.0 Hydrogel dressings

Hydrogels consist of water-insoluble, crosslinked polymers containing a high similarity for aqueous media. These 3D polymeric gels contain a hydrophilic, porous structure that allows a vast degree of water absorption, multiple times greater than the original dry weight. The hydrogels’ hydrophilic properties are associated with the crosslinking density of polar functional groups such as amino, amide, hydroxyl, and carboxyl in the polymer structure. Subsequently, hydrogels provide the special properties of high water content, which is up to 99.5%, malleability, non-adhesive nature, and similitude to living tissues as far as their biocompatibility is concerned; all assembled to make them an appropriate dressing candidate. In addition, hydrogels show the aspect of de-swelling and swelling reversibly in aqueous solutions; therefore, their application in various sectors, including drug delivery, regenerative medicine, and wound management. Moreover, hydrogels assistance enhancing wound healing through their moisture exchanging functions that create an optimal microclimate between the dressing and the wound bed. Due to their aspect of high moisture content, these dressings further offers a cooling, soothing impact and minimise the pain linked to dressing changes. Additionally, the limited bond of hydrogels illustrates that they can easily be removed from the wound site without causing further trauma to the healing tissues. Some hydrogels have a transparent nature, which further allows clinical assessment of the healing process without necessarily removing the dressing.

Hydrogels can be formulated to act in a stimuli receptive way so that when presented with an active biomolecule or drug can control diffusion and release, hence making them ideal interactive dressing. Other healing agents are naturally hydrophobic, and their delivery in a hydrophilic setting, which is the wound dressing hydrogel, can be attained by utilizing solubility enhancing drug carriers such as cyclodextrins (CDs). They are a family of cyclic oligosaccharides that are shaped like a cone, which provides a heterogeneous environment containing a hydrophilic outer surface as well as a hydrophobic interior cavity to transport lipophilic drugs. Additionally, CDs have a range of pharmaceutical applications in response to their biocompatibility and also solubility enhancing aspect. Moreover, drug inclusion in the CDs’ hydrophobic cavity, resulting in the inclusion complex (IC)generation, provides protective and release modulating elements. CDs having hydrogels can be generated in various ways. They can be produced through grafting on the hydrogels, loading the pre-articulated CDs drug IC after or during crosslinking, or crosslinking with diglycidyl-ethers. The initial method is a simple one, but it may have an unmodulated drug release limitation. Contrary, the release can be managed with the other techniques. Moreover, current therapies where more than a single medication with a variety of hydrophilic nature may demonstrate beneficial development based on CD hydrogel with dual drug provision of the hydrophobic drug, which is attributed to CDs, and the hydrophilic compound has released a new scope. The effectiveness of hydrogen that is based on CDs has proved the enhancement of therapeutic capacity for wound dressing applications. Additionally, medical practitioners have used hydrogel as a matrix for the dressing fabrication for the continued provision of crucial growth factors and healing agents to expedite wound healing. All these aspects have resulted in multiple proprietary hydrogel wound dressing being utilised in clinical practice for wound management internationally. Because of their special elements, hydrogel dressings are indicated for use in various wounds such as dry wounds having necrotic tissues, diabetic foot ulcers, burn wounds, chronic leg ulcers, and moderate to low exudating wounds, among others. Despite the range of hydrogel dressing products in the market already, still existing research in the area to further advance the hydrogel dressings to optimise the comfort of patients, clinical effectiveness, and several factors of wound healing.

1.3.1 Production of Hydrogel

Hydrogels can be produced from different synthetic and natural polymers, using various compositions, chemical, and physical crosslinking approaches. The hydrogels crosslinks can be a result of covalent bonds, chain entanglements, ionic interactions, among others leading to a range of chemical arrangement, physical configuration, and interactions. Besides, it is the same nature of the cross-linkage that decides much of the physicochemical properties of hydrogels and, thus, eventually application.

1.3.2 Classification of hydrogel products

The hydrogel products are classified in various details below

1. Classification according to their source. Under the source classification, hydrogels are further classified according to synthetic or natural origins.
2. Classification according to polymeric composition. This preparation approach leads to formations of some vital hydrogel classes. The following can demonstrate these
   1. Homopolymeric hydrogels are known as polymer network gotten from a single species of monomer, whereby is a fundamental structural unit consisting of any polymer network. Homopolymers can have a crosslinked skeletal framework depending on the monomer’s nature as well as polymerisation.
   2. Copolymeric hydrogels consist of two or more various monomer species with a minimum of one hydrophilic component, randomly arranged, alternating, or block configuration along the polymer network chain.
   3. Multipolymer interpenetrating polymeric hydrogel (IPN), a vital hydrogel class, consists of two independent crosslinked natural or synthetic polymer components, contained in the form of a network. In semi-IPN hydrogel, one element is a non-linked-polymer, and the other component is a crossed-linked polymer.
3. Classification according to network electrical charge

Hydrogels can be classified into four categories according to the absence or presence of electrical charge fond in the crossed-linked chains.

1. Ionic (including cationic or anionic)
2. Non-ionic (neutral)
3. Zwitterionic (polybetaines) consisting of both cationic and anionic categories in every structural repeating unit.
4. Amphoteric electrolyte (ampholytic) consisting of both basic and acidic categories.

4. Classification according to the crosslinking.

Hydrogels may be categorised into two groups in this classification. They can be classified on the physical or chemical nature of the crosslinked connections. Physical networks contain transient connections that emerge from their physical interactions such as hydrogen bonds, ionic interaction, or polymer chain enlargement or hydrophobic interaction. In contrast, chemically crosslinked networks have permanent junctions.

5. Classification according to the configuration

The hydrogels category depends on their chemical and structural composition can be grouped as follows

1. Semicrystalline (a composite mixture of crystalline and amorphous phases
2. Crystalline
3. Amorphous (non-crystalline)

Hydrogel forming natural polymers consists of protein, including gelatine and collagen, and polysaccharides such as agarose, alginate, and starch. The chemical polymerization approach conventionally prepares synthetic polymers that generate hydrogels.

• 3 Preparation of hydrogel

1. Physical crosslinked

The physical connection of polymer chains produces hydrogels through molecular entanglement, hydrogen bonding, ionic interactions, or hydrophobic correlations. Multiple thermodynamic transformations such as freezing-thawing, cooling or heating of polymer solutions, lowering pH, selection of cationic, and anionic polymers can lead to physical crosslinking between polymeric chains. Production of these hydrogels comprises mild conditions and the same time simple purification process as no toxic chemical crosslinking agents are needed during their synthesis, giving them biocompatible properties hence making them ideal matrix for the provision for therapeutic agents at the wound.

2. Ionic interaction

These hydrogels are produced between ionic polymers crosslinked along with multivalent, counter charged species. In addition, polyanionic polymers complexed with polycationic polymers produce hydrogel by polyelectrolyte complexation process, which is also known as complex coacervation. This approach is commonly used to synthesis alginate hydrogel dressings with divalent calcium cations (CaCl2).

3. Hydrogels bonding between chains

This category of hydrogels is produced by reducing the aqueous polymer solution pH when carboxylic classes are present on the chains. At acidic pH of this solution, solubility decreases, which enhances hydrogen bonding and the creation of hydrogels. Nonetheless, this physical network can disperse easily with the influx of water, and therefore, more to these other kinds of crosslinking can be deliberated to hold the hydrogel constituents. These physical associations can consider hydrogels containing pH-sensitive gelation (around pH 6.5) of chitosan.

4. Amphiphilic block copolymers

Physical hydrogels are made from two chemically different homopolymer blocks, which are hydrophilic and hydrophobic. Subsequently, they are self-assemble in aqueous mode producing hydrogels because of the thermodynamic incompatibility between the blocks. Besides, drugs such as antimicrobials can be used in these copolymers, and continued release can be attained for management applications.

5. Protein interactions

With the enhancement in biotechnology, hydrogel production through recombinant protein engineering has become possible. The new development allows the structural control and functional design of the proteins block, hence preparing physically crosslinked hydrogels having desirable mechanical, biological, and physical properties. These hydrogels that are based in biopolymeric protein assembles through polypeptides aggregation or protein-protein interaction by phase (temperature) transitions. Predictions prove that hydrogels hold a firm potential in would management. Protein fabrication engineered bioactive collagen-mimetic protein (eCol­_GFGER) with PEG-based medium hydrogel dressing with a latent of healing wounds in humans is an innovative technique in the wound management field.

6. chemical crosslinked

Hydrogels are produced by covalent linkages leading to high mechanical strength networks. These are delivered through chain growth polymerisation, adding and condensation polymerisation, and high energy radiations.

2.2.1 crosslinking by chain growth polymerisation

It consists of three stages, which are initiation, propagation, and termination. Production of free radical sites by a reasonable reaction initiator begins the polymerisation process, then chain elongation follows by the addition of low molecular mass monomeric building blocks. In the nesting process, the elongated polymer chains are crosslinked randomly by the cross-linking agent resulting in the hydrogel production. The hydrogel of 2- acrylamide-2-methylpropane- sulfonic acid sodium salt (AMPS-Na) utilizing potassium persulphate as an ethylene glycol dimethacrylate and as a free radical initiator as a crosslinking agent can be produced by redox initiation through free radical polymerisation for application of wound dressing. AMPS chemically crosslinked with AMPS-Na+) synthesis by utilizing 4,4-azo-bis (4-cynopentanoic acid), as photo-initiator and N­₂N⁷-methylene-biscrylamide acting as a crosslinking agent illustrated optimum water absorption as well as retention aspects for wound management applications. Besides, their transparent and flexible nature with good skin bond aspects advocates their potential biomedical application.

2.2.2 crosslinking by chemical reaction of complementary categories

Hydrophilic polymers include functional categories such as COOH, NH_2, and OH, which can be used for the production of the hydrogels. On the same note, these pendant functional groups with balancing reactivity (isocyanate-OH/NH₂ or amine-carboxylic acid, reaction, or formation of Schiff base) can create covalent linkages within polymer chains causing the hydrogel production using crosslinking agents. Antibacterial alginate-chitosan hydrogel wound dressings may be produced by Schiff based response between aldehyde categories of oxidised alginate as well as amino group of carboxymethyl chitosan. With the ability of injection, these hydrogel dressing holds an extra advantage of comfortable and easy application with extreme comfortability without crinkles, which can cause advanced patient compliance.

2.2.3 crosslinking by utilising high energy radiations

Cross-linking through radiation is commonly utilised to polymerise unsaturated compounds for hydrogel production since it lacks the use of toxic chemical crosslinking agents. Besides, it is a cost effective approach as separate formulation hydrogel sterilisation can be avoided. On high energy radiation exposure, radicals are developed on polymer chains in an aqueous solution, which initiates free essential polymerisation. The recombination of these radicals on various polymer chains results in the production of covalently crosslinked hydrogels.

2.2.4 Crosslinking through thiol-ene click reaction

The thiols response (nucleophile) to alkenes rich in electrons leading to the formation of thioether polymer networks is a decision weapon for hydrogel production owing to their rapid reaction, biological inertness, and restricted release properties. Defined by the functional categories and reaction conditions, the response mechanism could be the radical-mediated thiol-ene response. Production of poly (ethylene glycol) hydrogels for the provision of immunomodulatory molecules such as cytokine and development factors by mesenchymal stem cells (MSC) transporters, epidermal growth features loaded heparin based hydrogels and poly-based antimicrobial hydrogel wound dressings because the management of wound applications are an excellent example of thiol-ene response.

Hydrogel dressing products

A host of different symptoms exists in the many kinds of wounds that include granulating, sloughy, necrotic, and epithelialising, which vary in size, thickness, and shape. To understand the principle and purpose of dressing, formulations for these divergent applications allow optimisation of dressing and wound combination. Hydrogel dressing are specified for the treatment of multiple types of wounds with detailed selection of formulation eventually based on clinical application:

Amorphous hydrogels

With these hydrogels lacking a fixed shape, they can be applied evenly over the wound using an applicator and being shapeless, these wound form into the shape of the wound easily. These is specifically indicated in the treatment of cavity or uneven wounds that cannot be dressed with fixed and definite shape dressings. An excellent example of commercially available products include Intrasite gel, a partially hydrated, amorphous propylene glycol hydrogel and Aquasite, an undefined, glycerine-based hydrogel dressing.

Impregnated hydrogel dressings

They are used for full-thickness or fractional wounds where antiseptic packing of the wound bed is required. These formulations combine the advantages of hydrogels with non-woven dressings. Non-woven sponges, gauze pads, and strips of different sizes are impregnated gauze dressings that are saturated with the amorphous hydrogel and then made commercially available. An excellent example of commercially available products in this category is: Intrasite conformable hydrogel impregnated gauze dressing and Aquasite impregnated gauze dressing, which is a 100% cotton incorporated with hydrogel. All these dressings come in different sizes and are indicated for use in sloughy, necrotic and granulating full thickness wounds.

Sheet hydrogel dressings

These dressings can be cut into preferred sizes and shapes to fit the wound, considering that they consist purely of the hydrogel. The hydrogel sheets are indicated for the treatment of partial-thickness wounds and deep cavity such as ulcers, pressure sores, surgical incisions, skin donor sites, and 1^st and 2^nd-degree burns. The Aquasite hydrogel sheet dressing glycerine based hydrogel formulation are available commercially in different sizes which can be easily cut to fit the wound. By keeping the wound bed moist, the sheets act as advanced wound dressing therefore offering a soothing effect and enable wound healing as well as provide a physical barrier between the external environment and the wound. Furthermore, being clear, these sheet dressings allow the easy nursing of the healing process.

4. Polymers suitable for hydrogel wound dressing

Due to the bio compatibility and physiochemical properties of some polymers, they have an extensive use in biomedical applications. The most common used polymers which are used to fabricate hydrogel for wound dressing applications include the following:

Poly (Vinvyl alcohol) (PVA)

The ability of PVA being transparent, biocompatible and capable of maintain a moist environment has attracted it’s use in wound care. A variety of technique such as freeze –thawing cycle, electron beam irradiation and use of cross linkers such as glutaraldehyde can be used to cross link PVA chains in order to produce PVA hydrogels. Authors such as Hwang et al used a freeze –thaw technique to produce a gentamicin-loaded PVA-dextran in hydrogel dressing. The authors proposed that the presence of dextran in the hydrogels strengthened the elasticity, swelling ability and water vapour transmission rate of hydrogel. Since dextran favours the crystallization of PVA, it produces more uniform homogenous gel structure. Vitro test results confirmed that the hydrogel formulated with 2.5% PVA, 1.13% dextran and 0.1% gentamicin was uncountable and therefore, appropriate for wound management applications. Furthermore, the rat model tests showed enhanced healing with bigger wound size reduction of a full thickness surgical excision wound of the dorsum with gentamicin loaded PVA dextran hydrogels compared to the foam dressing and gauge dressing.

Kamoun et al synthesised the application of sodium ampicillin which contains PVA sodium alginate hydrogel membranes for wound dressing purposes. The authors attained physical crosslinking between different amounts of SA and PVA through repeated freezing thawing cycles. Protein adsorption tests in an in vitro protein was performed to show the ability of the hydrogels with regards to cleansing the secreting lesions and the finds showed an increase in bovine serum albumin adoption to 1.8mg/cm² from 0.7 with an increase in SA content to 75% from 0%.

These membranes showed high antibacterial activity and high uncountable activity suggesting their probable use as an active decomposable hydrogel dressing in wound care. In another study developed in situ, PVA based hydrogel dressed was innovated by co-enzymatic reaction using glucose existent in the wound exudate to generate entanglement. Horseradish peroxide(HRP) and glucose oxidase (GOx) are used to catalyse the hydrogelation process. GOx oxidised glucose to H₂O₂ which was taken by phenolic hydroxyls of the PVA derivative leading to the hydrogel formation. The vivo findings shows faster cure of full-thickness wounds with 77% and 96% wound closure within 7 to 10 days of treatment compared to 27% at a 7 days and 70% at day 10 with a commercially available hydrogel dressing.

Poly(N-vinyl-2-pyrrolidone) (PVP)

PVP a well-known synthetic polymer has multiple biomedical applications including wound management due to its biocompatible nature. Nu-Gel and Neoheal are PVP based hydrogel products indicated for use in first and second degree burns, partial thickness wounds, severe sun burns, bedsores and ulcerations. The hydrogels which are a results of crosslinking the PVP undergoes have poor mechanical properties with limited swellings. The swelling behaviour of these hydrogels can however be enhance by blending it with other polymers. Ionizing radiations are the most preferred tool wherever feasible for hydrogel formation as they have low costs, easy to control, minimize wastage and they have no chemical cross-linker adjustment.

A report on the approach to synthesis of PVP hydrogel using a safer, less expensive and portable UV radiation technique was reported by Fechine et al. They produced hydroxyl radicals from the photolysis of H₂O₂ which were then reacted with PVP resulting to rise of micro-radical polymer chains which went through re-combustion leading to hydrogel formation.

Jovanovic reported an approach to synthesis antimicrobial PVP hydrogels using gamma radiation to achieve gelation. Antimicrobial properties in the hydrogels were as a result of silver nanoparticles produced in situ in the PVP hydrogel matrix through the minimization of silver nitrate with radio lytic products of water upon gamma irradiation. The nanocomposite hydrogels showed high elasticity, good swelling capacity and good mechanical properties and may be altered for potential wound dressing uses.

Poly (ethylene glycol) (PEG)

PEG a polyether has appealed a wide interest in the biomedical applications including wound treatment because of its non-toxic, transparent, bio compatible, non-immunogenic and biodegradable properties. As a result of its biological properties, PEG has been used in proprietary products like Aqualfo, AmeriGel and Neoheal hydrogel wound dressing. As chronic wounds are known to have alkaline pH, the focus is to restore the increased pH to the physiological pH. Acrylic acid (AA) is a common pH modulating acid. Apart from controlling the alkaline pH neutralisation, acrylic acid (AA) enhances the swelling capacity of the hydrogels. AA showed superior mechanical strengths, enhanced cell migration velocity and biocompatibility.

Poly (2-hydroxyethyle methacrylate) (PHEMA)

PHEMA has attracted its use in wound management as a hydrogel dressing material due to its exceptional physiological properties. It was the first hydrogel used in the production of soft contact lenses due to its inert nature, high water imbibing properties, excellent mechanical strength and its biocompatibility.

PHEMA is also used in the fabrication of a bacterial light sensitive hydrogel wound dressing. Nitric oxide (NO) was selected as an antimicrobial agent due to PHEMA wound healing properties and to avoid potential emergence of antibiotic resistance. A development of a photoactive Nitric Oxide donors was invented in order to deliver Nitric Oxide to the desired site with controlled release, which were incorporated into polyurethane that was covalently linked into PHEMA hydrogel. To enhance the antimicrobial activity of these hydrogels, H₂O₂ or methylene blue were used as auxiliary growth attenuators.

Poly (n-isopropyl acrylamide) (PNIPAM)

PNIPAM is a polymer with amide and propyl moieties in its monomeric structure responsible for its temperature dependent volume phase transition. Being biocompatible, nontoxic and with phase transition close to humans’ body temperature, it has attracted a broad range of biomedical applications.

Alginate

This is a natural polysaccharide derived from some soil bacteria and marine brown algae and has attracted wide bacterial applications including wound management from its biocompatible, hydrophilic and non-toxic nature. Its use in fabricating commercially available hydrogels dressings like Purilon Gel (Coloplast ltd) and Nu-Gel (Systagenix wound management ltd) was as a result of its exceptional wound healing properties. Alginate contains the ability to form hydrogels by addition of divalent cations. Which links to guluronate blocks of alginate chains enabling ionic cross-linking model leading to gel formation. The alginate dressing can easily degrade in the case of loss of the divalent cationic cross-linker but the issue is overcome by cross-linking alginate with other polymers.

The objectives

The primary goal of the current investigation is to formulate hydrogen using amino acids precisely cysteine and arginine to synthesis a wound dressing. The main focus and goals of the research work are:

1. Use of sodium alginate dressing and adding amino acid to the hydrogel making it different from the commercial dressing.
2. Formulate hydrogels using amino acids to produce a wound dressing
3. To formulate various tests including stress strain test, HPLC and ICP-MS analysis
4. Making some microbiology tests to distinguish whether the dressing has some excellent antimicrobial activities

Research problem

As previously highlighted, the issue of chronic non-healing wounds is a serious medical concern. These wounds are not only the main cause of anxiety and pain for the patients, especially the aged, but also significantly contribute to their decrease social interaction, lessening mobility and general diminished quality of life. Previous literature has proven that the prolonged wound environment is a toxic and complex site for new cells to develop. Moreover, several authors have proposed that reticence of these protease is essential to enhance correct progression of wound healing.. A variety of wound dressing are on the market from dry dressing such as gauze to hydrocolloid dressing. Out of these multiple dressings, some of them contain silver, which have anti-microbial properties. Nevertheless, these dressings may have side effects. For instance, in silver, a patient may experience side effects such as skin burn, maceration, allergic or irritant contact dermatitis, haemolytic transient or anaemia leukopenia (silver sulfadiazine), electrolytic imbalance and blue-black or grey discoloration of treated skin. This technique ought to allow for a solution that is more cost-effective, as minimal cost of expensive growth factor would be utilised in the dressing in the near future. Hence, this wound dressing would be accessible as a viable treatment option to most patients with diabetes, chronic leg ulcer and other wound injuries regardless of their socioeconomic position. In conclusion, this project describes a technique to treat this problem faster and effectively and achieve an enhanced holistic patient outcome.

Hypothesis

Production of hydrogels by using sodium alginate dressings and adding amino acids can make a different wound dressing from the commercial dressing due to it high level of absorption, which is beneficial because it retains the moisture in the wound site leading to a quick healing of the site.

Project design

This project is designed to address hydrogels production using amino acids especially arginine and cysteine to develop a wound dressing. Furthermore, this project has deliberately combined the two discipline of polymer chemistry and biology to establish thesis that is naturally interdisciplinary. The essential focus of the project was centred on the establishment towards a concrete product as the primary outcome, instead of a project centred on production of basic new mechanistic knowledge.

Methodology

The formation hydrogels in wound dressing management has to undergo some test as a prove of its efficiency attaining its functions. As earlier discussed, hydrogels are hydrophilic polymers that has the ability to swell in water and hold a vast amount of water, while retaining the structure due to its physical and chemical crosslinking of individual polymer chains. Therefore, these properties can be put into test in various tests such as ICP-MS, stress strain test and HPLC. Besides, these properties require microbiology tests to determine whether the dressing has a good antimicrobial.

Stress strain test

Some wounds such as articular cartilage injuries and subsequent arthritis are among the leading causes of disability in the nation. However, the use hydrogels were improvised through biomaterial-based tissue engineering technique to regenerate articular cartilage. Hydrogels have proven a variety of their unique properties in the biomedical application such as drug delivery and engineering. For this test, this project tests the stiffness property and resistance to fracture in the process of replacing damaged cartilage tissue. Currently, the usual measure of a mechanical properties is through comprehensive modules evaluation. Nevertheless, failure aspects such as the resistance to breakage in the presence of an crack, ought to be measured through fracture toughness approach. In a complex material such as cartilage, deceptive fracture toughness reveals how much energy the material will absorb to fracture with the present defect and contribute to the reaction of material in crack extension to failure. Nevertheless, almost all the hydrogel studies to the present date in articular cartilage tissue engineering lacks fracture toughness assessment. Some of the author only considers ultimate compressive stress or strain, however, it may suffer from issues with reproducibility.

Traditionally in fracture mechanics, the toughness of the fracture is described as the quantitative expression of the competence of a stiff material to resist fracture in the presence of sharp crack. Subsequently, the material property of fracture toughness is described for a particular material by dimension using a minimum specimen size that guarantees dominance of a plane strain condition, thus allowing for continuous fracture toughness values for the particular material. The term ‘superficial fracture toughness’ distinguishes that the tough complex materials such as cartilage, the usual regulations of conventional fracture toughness measurement are impossible. This project proposes the use of apparent fracture toughness be act as a reminder to researchers in the biomaterials society that specimen size matters and that any comparisons with other material ought to be done with caution and total understanding of the involved limitations.

For this reason, it is essential to evaluate fracture toughness articular cartilage studies and contrast them with fracture toughness exploration of hydrogels used in applications outside of the cartilage tissue-engineering field to develop testing approaches for appropriate assessment of hydrogels for utilisation in cartilage tissue engineering. Ultimately, both the stress and strain properties must be in the range of articular cartilage to have a successful hydrogel for cartilage tissue engineering. Therefore, developed approaches to test cartilage fracture aspects can be utilised as a framework in testing hydrogels for cartilage tissue engineering. Stress and strain test will include the trouser tear test, single edge notch (SEN) test and indentation test.

Regarding the apparent fracture toughness in articular cartilage measurements and of hydrogels utilised in application outside of cartilage tissue engineering, this project establishes the groundwork for connecting methodologies between hydrogels and cartilage fracture testing and offer recommendations for assessment of fracture properties for hydrogels.

Hydrogel Fracture toughness measurement

Hydrogels enhances cell encapsulation and has an impact in their gene expression under physiological conditions due to their bio-amenable properties as previously seen. For scaffolds in articular cartilage renaissance, the fracture properties of synthetic hydrogels are specifically, since cartilage needs a mechanical integrity that can sustain vast deformation without fracture. The hydrogel deformation in reaction to applied stress is contrary from that of cartilage. Cartilage deforms comparatively easily at little strains. However, it stiffens as strain increases. Hydrogels, on the other hand, typically are ideal elastic materials meaning that they fit a stress-strain model describe in the equation:

where G represents shear modulus, while σ is the stress, and λ=L/L₀, whereby L represents the deformation length and L₀ represents the length that is not deformed. When designed as stress against strain under compression, this functions demonstrates an uninterruptedly increasing stress with cumulative strain. Nevertheless, some hydrogels are deviate from initial elastic nature and show a response that is relatively different. In this example, a double network gel consisted of a methacrylated chondroitin sulfate gel infiltrated by a polyacrylamide gel. The multicomponent framework of this gel allowed alterations in the stress response in different deformation medium compared to cartilage.

Compression test

In switching away from toughness in the context of fracture mechanics, the most candid assessment of overall hydrogels toughness is through the compression test whereby it can obtain the shear modulus and elastic modulus. This proves as the modern development approach for encapsulating cells in Interpenetrating Network (IPN) hydrogels which are of superior mechanism, whereby dynamic mechanical analysis was utilised to evaluate the mechanism performance of a modern IPN hydrogel regarding two biocompatible materials, which are poly (ethylene glycol) diacrylate and agarose. In the process of these tests, every hydrogels sample was prepared in a shape of a cylinder and placed between firmness platens, which were greased with mineral oil. The toughness was then derived by numerical integration of the stress-strain curve obtained through compressing each sample at a rate of 0.0005 mm-s. Note that this technique varies from the toughness parameters measured in tensile testing and the fracture toughness assessed in fracture mechanics methods, hence the value cannot be compared directly. Failure compression is highly dependent on products in the gel, and particularly since the nonlinear stress-strain relationship, variances in fracture strain are established in larger changes in fracture strength and larger variances in toughness, leading to moderately high variability. This variability is inherent to the approach, whereby an extreme limit of 100% strain, as well as a high degree of patchiness with materials that fracture at 70%-90% strain under compression. The issue with the large variability in toughness values obtained through compression contrasted with the more reproducible techniques in fracture mechanics in vast part inspired the existing review to explore alternatives for assessing mechanical failure properties of tissue-engineered constructs.

Hydrogel fracture toughness testing

Tests to evaluate toughness hydrogels parameters have included tensile tests (with notches or without present) tear testing of a notched sample, and compression. Other tests have disadvantages that may show unsatisfied properties. For instance, the tensile in absence of notch can only assess the general toughness. Besides, the trouser tear test utilised for soft hydrogel specimen has issues as compared to cartilage with gripping of the sample. When the grips hold the tear leg of the soft hydrogel specimen, the trouser legs will torn easily before tear loading is functional because of the stress focus the grip faces. Moreover, a vast amount of specimen materials maybe required to meet the standard geometry during the tear test

Microbiology tests

The lack of adequate wound management compounded with secondary infection remains a significant public health problem in the majority of developing countries. Therefore it has remained a research focus all along. Despite the considerable advance in the health care made in the last century,25% of mortality is a result of infectious diseases and still 45% in the underdeveloped countries. Pathogen and antibiotic bacteria are among the infectious diseases that cause mortality in the community. Antimicrobial agents are most important in reducing the universal burden of contagious diseases. These drug-resistant micro-organisms moderated the development of antibiotics where only a few drug companies remain active in the area and therefore result in a significant challenge in the globe. The failure of antibiotics has resulted in the search for more effective sources of naturally occurring products from plants and others.

First recognized in the year 1892, the antibacterial activity of honey has, however, limited use in the present medicine due to the lack of scientific backing. Honey collected by flower bees contains 15% to 20% water and about 80% to 85% sugar, while the remaining part contains enzymes, proteins, and nonessential amino acids. The several properties of honey-like enzymes are useful for its bactericidal effect and wound healing. Glucose oxidase is one of the enzymes in honey, which changes glucose to gluconolactone and then to hydrogen peroxide.  Honey is used to inhibit many pathogenic organisms and improves the wound healing process through epithelization, having an acidic pH of 3.2 to 4.5. It is also one of the most saturated solutions that show bacterial growth as a result of high osmolality.

Alternative medicines have been described as a less expensive way of achieving health care coverage of the universal population. They, therefore, encourage the coherent use of plant-based alternative medicines by member states. This study aims to evaluate antibacterial activity both bacteriostatic and bactericidal effects of honey against MRSA isolates from an infected wound in Gamo Gofa Zone, South Ethiopia. They would have been recommended as therapeutic agents after clinical trials and pharmaceutical standardization.

Methods and materials

The study area, design and period

The experimental study design was carried out at Arba Minch University Medical Microbiology and Parasitology Laboratory from May 2017 to November 2017.

Sampling technique and size determination

The model size was determined using trial size determination of a single population formula. Taking 97% prevalence of multiple drug resistance (MDR) isolates from the previous study, 95% confidence interval, and 5% marginal error d=0.05 as the initial sample.

(z_α/2)²  *  p(1-p)   =   (1.96)²  * 0.97 * 0.03  = 45

n=                   d²                                      (0.05)²

Lastly, by considering a 10% (=5 subjects) nonresponse rate, the final sample size was determined as n+5=50

All isolated MRSA were included in the experiment regarding the sampling technique.

Wound sampling procedure

Clinically infected and open wounds were aseptically obtained after the wounds were cleansed with sterile normal saline from qualified, trained nurses. The specimen, after collection on a sterile cotton swab by rotating with enough pressure ware transported to the laboratory using Amies transport.

Culture and identification

The swabs collected were marbled on mannitol salt agar (Oxiod) and blood agar using a sterile inoculation loop and later incubated at 35-37^oC for about 24 to 48 hours. The preliminary identification of bacteria established on colony characteristics of the organisms from hemolysis on blood agar, the enzyme activity of the organism, and the changes in physical appearance in different media. The isolates were recognized based on their reaction, coagulase test results, and gram reaction.

Antibacterial susceptibility testing

Kirby-Bauer performed the vulnerability test disk diffusion technique following principles set by the Clinical Laboratory Standard Institute(CLSI) in 2016. The inoculums were suspended and prepared in sterile normal saline. The density of suspension was determined using comparison with opacity standard on McFarland 0.5 barium sulphate solution. A uniform test organism was uniformly seeded over the Oxiod surface and exposed to the concentration gradient of the antibiotic, followed by incubation at 37^oc for about 18 hours. The antibiotics tested were chloramphenicol (6 μg), gentamicin (11 μg), tetracycline (32 μg), chloramphenicol (30 μg), co-trimoxazole (27 μg), vancomycin (32 μg), erythromycin (15 μg), clindamycin (10 μg) and amikacin (32 μg) which were selected based on the prescription frequency and availability of these drugs on the study area.

Test organisms

Every screened methicillin-resistant S. aureus from the wound was used and was identified phenotypically based on its resistance to cetoxitin (30 μg) and oxacillin (1.5 μg) using the disc diffusion method. The zone of inhibition, according to CLSI, 2016 guideline, was grouped into methicillin-resistant and methicillin-sensitive.

Honey sample

A list of four kinds of honey was harvested from beekeepers of the Gamo Gofa zone using a purposive sampling technique. Each sample was filtered with a sterile gauze to remove debris and then streaked on a blood agar plate to check sterility. The honey sample was collected in sterile, screwed culture bottles. The samples were then stored at 2-8^oC until they were used.

Preparation of honey solutions

A 100% pure honey (100% v/v) was obtained after being filtered using sterile gauze. In order to get 75% honey solutions, 0.75ml of honey is diluted in 0.25ml sterilized distilled water. Further serial dilutions of 0.5ml of each and 0.25ml of honey and 0.75ml of sterile distilled water were added to obtain 25% and 75% honey solutions, respectively.

Susceptibility testing of honey

The test was performed by Kirby-Bauer disk diffusion technique according to criteria set by CLSI, 2016. The inoculums were prepared by picking parts of similar test orgasms with a sterile wire loop suspended in sterile normal saline. Determining the density of the suspension was done using opacity standard on McFarland 0.5 barium sulphate solution. A sterile swab dipped in the suspension of the isolate was squeezed free from excess fluid on the side of the tube and later spread over the agar plate. The test organism was uniformly seeded over the Oxiod surface and the plates left on the bench for the excess fluid being absorbed. Wells were made in the agar medium using a sterile cork borer (4mm deep, 6mm diameter, and about 2cm apart). 50 μl of honey with 75%, 50%, and 25% concentration was added into the wells in the plate, and the inhibition zones mean diameter was measured in mm and results recorded. Sterile distilled water was used as a negative control, and the experiment repeated 3times for every strain.

Determination of minimum inhibitory concentration

The minimum bacterial concentration and the minimum inhibitory concentration (MIC) of the antimicrobial agents were determined for each isolate using the tube dilution method. About ten sterile tubes were placed labeled from 1 to 8. The quality controls used broth control tube (BC), growth control tube (GC), and honey control tubes (HC). 1ml of the undiluted honey solution is added to the test tube number 1 and honey control tubes with a disinfected micropipette and tips. Twofold serial dilution is performed by moving 1ml undiluted honey into the second tube with separate disinfected micropipette and tips vortexed for homogenization. 1ml was then transferred with another sterile micropipette from tubes 2 and 3 after thorough mixing. The procedure continued until tube 8 with a dilution of 1:128 is attained, and later 1ml was taken and discarded from tube number 8. The whole procedure is repeated for organisms tested to each of the honey, and the tubes later incubated at 37^oC for 24 hours. They were under visual inspection for the presence and absence of growth. MIC was recorded as the lowest concentration of honey that showed no visible turbidity or growth.

The determination of minimum bacterial concentration

The incubated tubes having no signs of growth in MIC are subcultured onto sterile nutrient agar plates using the streak plate method and later incubated at 37^oC for 24hours aerobically to establish the minimum bacterial concentration (MBC). The one with the least concentration of honey that did not show growth of the test organisms was considered as the MBC. Inoculated plates were labeled as bactericidal if no growth, bacteriostatic if there is light to moderate growth, and no antibacterial activity for cases of heavy growth.

Data quality control

The quality of data was ensured at all stages of the activities of the study by following prepared standard operating procedures. Manufacturer’s instructions were followed when preparing culture media, and the disinfection was done by incubating representative of the batch at 37^oC overnight observing the bacterial growth. The batches that showed growth were discarded. S. aureus (ATCC-25923) was used to check the quality of the potency and media of the antibiotics used for the positive controls. The questionnaires were presented about 5% of the total respondents, while completed questionnaires were checked and corrected daily.

Results

Study population and patient characteristics

Fifty samples in total were collected from the patients with the clinical evidence of wound infections from May to November 2017. The patients sampled had either complaints with discharge, swelling, pain, chronic wound, or foul-smelling. The subjects were 25 (50%) males and females in that order. Those with chronic wounds with the highest incidences were ranging from 16-30 years, followed by the age group less than 15 years, which was 30% while the highest infection rate. Students and homemakers had the highest infection rate (30%), followed by government employees. The social demographic statistics of the participants are shown in the table below:

Table showing wound infection and sociodemographic characteristics of the patients with wound infection

Prevalence of MRSA

Among the isolated S. aureus (15 (41.7%)) screened for methicillin resistance, MRSA resulted in 10 (66.7%), while MSSA resulted in the remaining (33.3%).