Functional Bio-based Materials for Regenerative Medicine: From Bench to Bedside (Part 1)

By Mohd Fauzi Mh Busra, Daniel Law Jia Xian, Yogeswaran Lokanathan and Ruszymah Haji Idrus

Functional Bio-based Materials for Regenerative Medicine: From Bench to Bedside explores the use of bio-based materials for the regeneration of tissues and organs. The book presents an edited collection of 28 topics in 2 parts focused on the translation of these materials from laboratory research (the bench) to practical applications in clinical settings (the bedside). Chapter authors highlight the significance of bio-based materials, such as hydrogels, scaffolds, and nanoparticles, in promoting tissue regeneration and wound healing. Topics included in the book include: - the properties of bio-based materials, including biocompatibility, biodegradability, and the ability to mimic the native extracellular matrix. - fabrication techniques and approaches for functional bio-based material design with desired characteristics like mechanical strength and porosity to promote cellular attachment, proliferation, and differentiation - the incorporation of bioactive molecules, such as growth factors, into bio-based materials to enhance their regenerative potential. - strategies for the controlled release of molecules to create a favorable microenvironment for tissue regeneration. - the challenges and considerations involved in scaling up the production of bio-based materials, ensuring their safety and efficacy, and obtaining regulatory approval for clinical use Part 1 covers techniques for tissue engineering, wound healing and skin engineering. It also presents reviews on techniques such as acellular synthesis and 3D bioprinting. Materials highlighted in this part include chitosan-based nanoparticles, nanocollagen-based materials and plant based composites. Functional Bio-based Materials for Regenerative Medicine: From Bench to Bedside is a valuable reference for researchers in biomedical engineering, cell biology, and regenerative medicine who want to update their knowledge on current developments in the synthesis and application of functional biomaterials.

• Language: English
• Publisher: Bentham Science Publishers
• Release date: Jul 25, 2023
• ISBN: 9789815123104

Book preview

Acellular Strategy of Functional Biomaterials for Tissue Wound Healing

Atiqah Salleh¹, Izzat Zulkiflee¹, Shou Jin Phang², Mohd Fauzi Mh Busra¹, Manira Maarof¹, *

¹ Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, 56000 Kuala Lumpur, Malaysia

² Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

Abstract

The skin is known as the largest organ in the human body as it functions to regulate the temperature in the human body and acts as the first-line defence. The skin consists of two layers: the epidermis (the outer layer of skin) and dermis layers (the inner layer of the skin) occupied by specific skin cells. Whenever the skin barrier is compromised, the skin heals following four phases: haemostasis, inflammation, proliferation, and remodelling. Wound healing takes a few weeks for acute wounds, however it takes a longer period to heal chronic wounds. Chronic wound complication extends the inflammation phases during the wound healing process and becomes a significant problem in the healthcare field. Therefore, various treatments were produced to reduce the healing time in chronic wounds and produce less scarring. Acellular treatments have gained attention in wound healing research as these treatments have a lower risk of rejection and are easily obtained through nature or lab. Acellular treatments include growth factors, bioactive molecules, and peptides that are clinically proven to have faster healing time and reduce scarring as these treatments are readily available in the market. Biomaterials have become a novel study in wound healing research due to their vast potential as alternative treatments for skin wound healing. Therefore, the chapter discussed the acellular strategies for tissue wound healing.

Keywords: Functional biomaterials, Skin, Tissue engineering, Wound healing, Wound treatment.

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* Corresponding author Manira Maarof: Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, Kuala Lumpur, Malaysia; E-mail:[e-mail]

INTRODUCTION

Skin is the largest organ in the human body. It can weigh up to 15% of the adult’s total body weight with a surface area of up to 2 m² [1]. The skin acts as our body’s

initial barrier against potential insults from the exogenous environment, such as pathogens, UV light, hazardous chemicals, and mechanical injuries [2]. Besides that, humans can sense touch, temperature, and pain via skin sensations, which allows us to perceive signals from the outside world. The diffusion of substances like oxygen, carbon dioxide, and topical drugs into the human body is also made possible by the skin.

Generally, the human skin is constituted of the epidermis and dermis layer (Fig. 1). The epidermis and dermis layer are separated by a layer of basement membrane formed by an interconnected network of extracellular matrix (ECM) macromolecules [3]. Besides that, the epidermal rete ridges and dermal papilla are both distinct microarchitectures that can be found between the epidermis and dermis layers. These structures combine together and exhibit an undulated wave-shaped pattern to form the dermal-epidermal junction (DEJ) [3]. Other than providing mechanical support, the basement membrane at the DEJ also allows cell-cell and cell-matrix interaction between the epidermis and dermis [4]. The epidermal cells recognise the basement membrane as an adherence site and utilise it as a reference layer to separate themselves from the dermis.

Epidermis Layer

The epidermis layer contains multiple layers of keratinocytes, which include the stratum basale, stratum spinosum, stratum granulosum, and the outermost stratum corneum. The stratum basale is accommodated with keratinocytes, continuously differentiating and proliferating from their stem cell progeny [5]. Next, keratinocytes in the stratum spinosum exhibit a polyhedral spine like morphology that extends and connects with the surrounding cells via desmosomes. The stratum granulosum possesses diamond-shaped keratinocytes that contain electron-dense keratohyalin granules [5]. The outermost stratum corneum layer comprises dead keratinocytes that travel toward the outer skin surface over time, forming a cornified layer of cells [6]. These cells then undergo a desquamation process and shed off from the skin. A complete cycle of keratinocytes progressing from the stratum basale to the stratum corneum takes approximately 28 days [7].

Dermis Layer

The dermis layer consists of two regionally distinct layers: the superficial papillary later and the deeper reticular layer. The papillary dermis is composed of loose connective tissue and is constantly interconnected to the epidermis via the DEJ [8]. The papillary dermis is mainly populated by fibroblasts, with the addition of immune cells like macrophages and neutrophils that are activated by pathogenic infections [8]. Thin collagen fibers along with elastic fibers can also be found at this site [9]. On the other hand, the reticular dermis is thicker and deep within the skin. It comprises mainly dense connective tissues like thick collagen bundles and exhibits less cellular complexity [9]. The reticular dermis demonstrates a net-like structure contributed by the meshwork of fibers. Among the extracellular matrix found in the reticular dermis, collagen fibers provide tensile strength to the skin, while elastin fibers provide the skin with elasticity which enables movement. In addition, the collagen fibers can bind to the water molecules and retain the skin hydration.

Fig. (1))

Structure of the skin. The skin comprises the epidermal and dermal layers separated by the basement membrane. The epidermal layer is composed of four different layers of keratinocytes, the innermost layer is the stratum basale, followed by the stratum spinosum, stratum granulosum, and stratum corneum. The dermal layer contains fibroblasts as the primary cell type. Collagen fiber and elastin fiber can also be found in the dermal layer.

MECHANISMS OF WOUND HEALING

Generally, the wound healing process consists of four overlapping and continuous phases, which are haemostasis, inflammation, proliferation, and remodelling. The wound healing process involves the cellular components like macrophages, fibroblasts, keratinocytes, and the extracellular matrix produced by these cells.

Platelets are activated at the wound site upon skin injury to form aggregates [10]. These aggregates release clotting factors that promote fibrin deposition at the wound site [10]. The fibrin clot acts as a provisional matrix where the aggregated platelets are encapsulated within [11]. Growth factors are also released in this stage, which signals the cells to migrate to the wound site and perform their function [10].

The macrophage is the crucial player in the inflammation phase during wound healing. The M1 macrophage, to be specific, secretes pro-inflammatory cytokines, including interleukin IL-1, IL-6, IL-8, and tumour necrosis factor-alpha (TNF-α) to fight against the pathogens that may cause infection [12]. On the other hand, the M2 macrophages are responsible for alleviating the inflammatory level after the clearance of pathogenic substances in order to resume the wound healing process [12]. Besides producing cytokines, growth factors are also synthesised to recruit and signal cells like fibroblasts and epithelial cells to initiate the subsequent wound-healing phases. Examples of growth factors include fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), tumour growth factor-alpha (TGF-α), and TGF-β [12].

Upon resolution of the inflammation, the proliferation phase takes place with the migration of fibroblasts and keratinocytes [13]. The provisional matrix in the haemostasis phase is also replaced with a new matrix comprised of an extracellular matrix generated mainly through the fibroblasts [13, 14]. The collagen produced by fibroblasts plays a vital role in the proliferative phase of wound healing as it provides a foundation for reorganising the extracellular matrix [13]. Subsequently, neovascularisation replaces the damaged blood vessels and provides oxygen and nutrients to the wound during angiogenesis [15].

The remodelling is the final phase of the wound healing process as granulation tissues undergo maturation and form scar tissues [13]. During the remodelling phase, blood capillaries merge into larger vessels and create an extensive vessel network. Next, the tensile strength of the skin is gradually restored by the replacement of type III collagen with type I collagen.

TYPES OF WOUNDS

A skin wound is defined as damage to the integrity of the skin. It can be clinically categorised into two main types, namely acute and chronic wounds, according to their healing time [13].

Acute Wound

An acute wound can be principally defined as a wound that heals in a timely and orderly manner. Various traumas can result in an acute wound, including cut, burn, fall, or surgery. Among acute wounds, burn wound is a common and significant medical problem affecting millions globally. A full-thickness burn that covers full-thickness of the way to the underlying tissue of the muscle and bone is one of the most serious types of an acute wound [16]. Despite the fact that acute wounds are commonly induced, they usually heal within a month after wound induction, and the skin function is restored [13].

Chronic Wound

In contrast to acute wounds, a chronic wound is characterised as a non-healing wound that has an impaired wound-healing mechanism. The healing process can be perturbed at any of the phases with various factors involved, including severe infections, chronic inflammation, and also due to the occurrence of disease complications [13]. For example, a diabetic foot ulcer (DFU) is a chronic wound resulting from diabetes. A DFU wound is frequently found to be abrogated at the inflammatory phase, where the wound healing cannot progress into the subsequent proliferation and remodelling phases [17].

TREATMENTS FOR WOUND HEALING

Treating a wound has been the main challenge in the medical industry as the number of patients accompanied by skin damage has been increasing throughout these years. Even though the process deems insignificant on the surface, however, wound treatment and assessment are complex processes as there are various factors that influence the healing process. The current standard treatment for skin wounds includes swabbing, cleaning of the wound area, wound dressing, and debridement in some cases of injury.

Skin Autograft

In general, skin grafting is a method of transferring tissue from one part of the body to another. Skin autografts are classified as full-thickness autografts and split-thickness autografts. The full-thickness skin graft is a graft that consists of both the epidermis and the entire dermis of the skin. Meanwhile, the split-thickness skin graft only contains the epidermis and a small portion of the dermis. Split-thickness autograft is the current gold standard for chronic wound management. The reason for skin grafting being the gold standard treatment is that this treatment can serve the ultimate goal of wound treatment which is fast wound healing by covering the wound with granulation tissue and infection-free wound recovery [18].

Hyperbaric Oxygen

Hyperbaric oxygen is a wound therapy that uses 100% oxygen at high pressure (more than atmospheric pressure). Therapy works by exposing the patient to 100% oxygen intermittently as the pressure of the chamber increases until it reaches more than 1 atmosphere absolute (ATA) [19]. Nevertheless, the uses of hyperbaric oxygen therapy have a few complications. The most reported side-effect is that patients suffer from barotrauma, which is a type of injury inflicted by high pressure resulting in the inability to equalise pressure between the air-filled space and the surrounding. Other than that, the hyperbaric oxygen therapy can also involuntarily induce acute central nervous system oxygen toxicity due to exposure to high concentrations of oxygen [20].

Negative Pressure Wound Therapy

Negative pressure wound therapy or vacuum-assisted closure is the treatment that alludes to any devices that can firmly seal the injury, thus establishing an impermeable environment applied by vacuum bringing the progression of natural responses which improve the wound healing [21]. The therapy involves two different mechanisms of action, which are classified as the primary and secondary mechanisms. The primary mechanisms of the therapy are macro deformation (initiation of wound shrinkage), micro deformation (referred to as mechanical changes that occur in minuscule scope when suction is administrated through permeable material bringing undulated wound surface), fluid removal (removal of fluid accumulation in the edema) and wound environment alteration as shown in (Fig. 2). Meanwhile, the secondary mechanisms consist of cellular response, granulation formation, inflammation, peripheral nerve response, enhanced microcirculation, and reduction of the stasis zone [22].

Fig. (2))

The primary mechanisms of negative pressure wound therapy. The image was reproduced by Adriana et al., 2017 [23] and licensed under CC by NC 4.0.

ACELLULAR STRATEGIES FOR WOUND HEALING

Tissue engineering involves multiple transdisciplinary fields which include the field of engineering and life sciences in order to develop a biological substitute able to restore, and improve the functionality of lost tissue or organs. There are three main scopes of tissue engineering which are cells, biomaterials, and biological factors. However, this chapter only focused on the acellular approach of tissue engineering. Acellular biomaterials have gained attention in wound healing research as this approach is known to have a low immunogenic effect, high biocompatibility, and low risk of rejection in the host [24]. Therefore, acellular biomaterials are an ideal alternative to wound healing therapy.

Biomaterials

The field of biomaterials has been steadily expanding as a large number of pharmaceutical and manufacturing companies have invested in research to commercialise biomaterial products. Biomaterial is any substance (other than drugs) that can be used as a building block for preparing tissue replacements, in which, it can be natural, synthetic, or a mixture of both [25, 26]. Biomaterials are mainly designed to resemble or mimic the structure of the native extracellular matrix (ECM) architecture [27]. Mammalian cells depend on ECM for structural support. ECM is liable for an assortment of cell functions that include cell assembly into tissues and important organs, biological regulation that influences cell growth, and cell-cell interaction [28]. A scaffold material should satisfactorily initiate the attachment, differentiation, and proliferation of both cultivated progenitor cells and encompass beneficiary tissues that are able to withstand the repeated process of physical and chemical properties of organs [29]. In these artificial environments, cells must undergo proliferation, be able to produce ECM on their own, self-assemble, and ultimately assume the form of the scaffold, having resemblances with the tissue of interest [30].

In this field, the public often doubts biomaterials, looking at their functionality, safety, and efficacies. A well-designed biomaterial should perform its function in the living body's environment without causing harm to other organs. Toxicity for biomaterials is concerning for compounds that migrate out of them toward the body (cells, tissues & organs). Nontoxic biomaterials refer to biomaterials that are non-carcinogenic, non-pyrogenic, non-allergenic, blood compatible, and non-inflammatory [31]. This will always remain a challenge for scientists in this biomaterial field. Thus, the biological context is critical because it will aid us in understanding, predicting, and engineering the in vivo responses observed by the biomaterial scientists/engineers, the physician, and the patient [32].

Natural Biomaterials

Protein-based (collagen gelatin fibrin), polysaccharide-based (chitosan, cellulose, etc.), and decellularised tissue-based (amniotic membrane, decellularised skin) biomaterials are the most common types of natural biomaterials derived from in vivo sources. They provide a superior microenvironment for cells to bind, expand, and differentiate due to their resemblance to the natural ECM structure. Natural biomaterials are biodegradable and resorbable, have low toxicity [33,34] and low inflammatory response, and are biocompatible. Natural biomaterials, which are commonly used in tissue engineering applications, are listed in Table 1. These natural polymers can self-assemble or use cross-linking techniques to create non-cytotoxic hydrogels and scaffolds that mimic natural tissue properties [35]. The treatment using natural biomaterials could be beneficial, either combining with cells or biomolecules or both.

Table 1 List of natural biomaterials, properties, limitations, sources, and references.

Synthetic Biomaterials

While natural polymers have many advantages, synthetic biomaterials have recently gained popularity. This is because natural biomaterials possess difficulties in purification as well as a high risk of pathogen transmission. Synthetic biomaterials, on the other hand, require a lot of tweaking and modifications to look like or mimic the natural ECM. This involves the use of binding ligands, biological signals, and cell biocompatibility [57]. Synthetic biomaterials are made degradable as the degradation by-products do not induce toxicity when used in brief implants or drug delivery systems [58]. Synthetic materials can be classified into metals, ceramics, polymers, and hydrogels.

Metals are known for their use in orthopedics, as artificial joints, screws, and plates, in orthodontics and dentistry, as teeth support such as braces and dental inserts, meanwhile in cardiovascular and neurosurgical equipment, such as staples, artificial organs, stents, coils, and cables. This is due to its favorable blend of elasticity, fracture resistance, fatigue strength, and tensile strength [59]. However, a large-scale, severe tribological- and corrosion-based damage might occur due to the complex body environment, which includes the complexity of body reactions, wear and corrosion mechanisms, and mechanical stresses due to activities in daily life [60]. Ceramics are popular in the applications in tissue engineering, mainly in bones. They have progressed significantly over time due to their widespread use: first-generation materials were meant as a bone replacement, second-generation materials are to fix or revive the functionality of the bone, and third-generation materials are used to rebuild bone [61]. As for polymers, they can be grouped into non-degradable and degradable polymers. Non-degradable polymers are Polymethylmethacrylate (PMMA), poly(ethylene terephthalate) (PET), poly(tetrafluoroethylene) (PTFE), silicones and polyurethanes. Important advancements in polymer formulations and manufacturing methods have been made since the early introduction of synthetic materials to further optimize the efficiency and durability of these materials in the biological setting [62]. These materials serve many beneficial functions in the biomaterial field; despite being biocompatible and having high stability, they are non-degradable. Degradable polymers, on the other half, are more favorable and have many applications in regenerative medicines. Table 2 shows the list of polymer classes and their applications.

Table 2 Degradable polymers and its applications

Composite Biomaterials

The term composite describes the macroscopic mixture of two or more materials that vary in structure, morphology, and general physical properties. The use of composite materials in biomedical applications opens up a slew of new implant design possibilities. Composite materials and components can, in reality, be engineered to have a broad assortment of mechanical and biological properties [89]. Combining two or more phases enhances the matrix's mechanical properties, such as stiffness and strength [90]. The mechanical efficiency and environmental stability of composite materials are largely determined by the interface between the matrix and reinforcement [91]. Even though many products produced from tissue engineering have benefited the clinic, they still have flaws, such as low mechanical strength and biological and tedious fabrication properties. Many studies have attempted to solve these flaws by looking for different or novel biomaterials, producing a composite of natural and synthetic biomaterials, or enhancing the properties of biomaterials with growth factors (GFs). As a result, one approach involves combining or adding potential bio-factors to biomaterials to create composites with favourable properties that have not yet been found in available composites.

Acellular Treatments

Growth Factors

Growth factors are naturally produced signalling molecules or proteins that control and stimulate cell proliferation, migration, and cellular differentiation. The study of growth factors is promising in wound healing applications as these growth factors can modulate the wound healing process, such as inflammatory responses, enhance tissue formation, and induce the formation of blood vessels. The current application of growth factors in skin regeneration is through topical administration. There are several approved medications that contain growth factors such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) readily available in the market. Regranex® Gel is a topical gel that contains 0.01% becaplermin which is a recombinant human PDGF (rhPDGF) to treat diabetic neuropathic ulcers. Clinical studies have shown that the Regranex® Gel is an effective treatment option for diabetic ulcers [92]. The gel helps in wound closure faster and is able to increase cell proliferation needed in the wound healing process. Fiblast®, also known as Trafermin, is a topical spray that is made of recombinant human basic fibroblast growth factor (rhbFFGF) and marketed to treat skin ulcers. The clinical trials recorded on more than 90% of burned skin patients result in the improvement of skin renewal [93]. Even though growth factors can encourage faster wound healing, they have a short half-life in clinical application because of their low stability and are easily removed by exudation. Therefore, other acellular substances were studied to find a suitable treatment that can actively interact with cells at the wound site.

Peptides

Peptides can be one of the key biological components that carry important functions in cells. Proteins and peptides are structurally similar, consisting of chains of amino acids bound together by peptide bonds (amide bonds). Peptides are smaller than proteins and are often characterised as molecules containing between 2 and 50 amino acids, whereas proteins include 50 or more amino acids. Furthermore, unlike proteins, which can assume complicated conformations known as secondary, tertiary, and quaternary structures, peptides have a less well-defined structure. Between peptides and proteins, functional distinctions can also be made. Proteins give cells form and respond to signals from the extracellular environment, while peptides control the activity of other substances. In wound healing, peptides have shown many promising functions to improve the rate of wound healing abilities. A new study by Song et al. [94] discovered a new peptide from the skin amphibian called OA-GL12, which could stimulate wound healing at real concentrations, making it one of the most potent wound-healing accelerators. The underlying mechanism was also investigated, and results revealed that OA-GL12 could significantly increase the number of macrophages at the wound site and the release of pro-healing TGF-1. Another study by Tang et al. [95] reported that a newly developed peptide called tiger17 showed an outstanding skin wound healing ability. According to an immunocytochemistry study, tiger17 therapy increased the alpha-smooth muscle actin expression in the skin wound. Other than that, tiger17 also increased macrophage recruitment, TGF-1 expression, and fibroblast to myofibroblast differentiation.

CONCLUDING REMARKS

The acellular treatments have been proven to promote better wound healing, especially when countering chronic wounds. These treatments not only safe to use but can reduce scarring by providing a better microenvironment for the epithelialisation process. Meanwhile, acellular biomaterials are able to provide an optimal microenvironment for the cell to proliferate and migrate into the wound site.

REFERENCES