Abstract
Diabetic wound (DW) is one of the leading complications of patients having a long history of uncontrolled diabetes. Moreover, it also imposes an economic burden on people suffering from wounds to manage the treatment. The major impending factors in the treatment of DW are infection, prolonged inflammation and decreased oxygen levels. Since these non-healing wounds are associated with an extended recovery period, the existing therapies provide treatment for a limited period only. The areas covered in this review are general sequential events of wound healing along with DW's pathophysiology, the origin of DW and success, as well as limitations of existing therapies. This systematic review's significant aspect is to highlight the fabrication, characterization and applications of various acellular scaffolds used to heal DW. In addition to that, cellular scaffolds are also described to a limited extent.
Keywords: Diabetes, Diabetic wound, Acellular scaffold, Biomaterials
Introduction and epidemiology
Diabetes mellitus (DM) is a metabolic disorder delineated as hyperglycemia due to insulin resistance or lack of insulin secretion of cells. It is a growing international health concern across the globe. Moreover, it imposes a significant economic burden on the country as the cost involved in treating its complications such as nephropathy, retinopathy and DWs is very high and requires hospitalization in many cases. One of DM's chronic complications is DW and its healing takes a long time, i.e., six months to years. The prolonged healing of DW is due to certain complications such as peripheral neuropathy, microvascular diseases, proteases rich wound environment and infections, which ultimately result in delayed wound healing [1]. Chronic skin ulceration can be observed on the lower extremities, markedly on foot. Hence, they have a head start to DW, which fails to heal [2]. Western inhabitants have diabetes in the proportion of 1–2% and it may reach up to 2–4% of the total health budget. In 2018, about 415 million people were reported to suffer from DM. The most preponderance of them was type II DM. It is expected that about 642 million people will be suffering from DM by the year 2040 [3]. Current therapies include patient training, providing information, diagnosis and prevention. However, most of these therapies suffer from limitations such as patient admission necessity, cost and complexity.
Further, most of these treatments do not address the prerequisite necessity to treat all the aspects of DW and hence the treatment of DW has been a challenging milestone [4]. In recent years, tissue engineering has emerged as a novel technique to treat DW, aiming to restore and improve the damaged tissues' function. Tissue engineering, by adopting the biologic scaffolds, can tackle different molecular and cellular culprits responsible for chronic and non-healing wounds by delivering therapeutic agents [5].
Phases of diabetic wound healing
Wounds prevail with millions of people globally and management of injuries and delay in wound healing exhibit a significant clinical and economic burden. Wound healing involves a sequel of complicated events that are not clearly understood. The physiology of wound healing at molecular and cellular levels is the prerequisite for effective therapeutic interventions. Four sequential phases demonstrate the process of wound healing: hemostasis phase (0-several hours after injury), inflammation phase(1–3 days), proliferation phase (4–21 days), remodeling phase (21 days–1 year) (Fig. 1). Dysregulation of any of these steps subsequently results in impaired wound healing [5].
Fig. 1.
Cellular and molecular events in various phases of diabetic wound healing
Hemostasis phase
This is the first step in the wound healing process. After an injury, vasoconstriction and blood clotting occurs, which is known as hemostasis. This prevents excess loss of blood and thereby provides a structural matrix for cell migration [6]. At first, platelets enter the wound site and secrete various growth factors (GFs) and cytokines (CKs) that attract fibroblasts and endothelial cells to initiate the healing process. The recruitment of neutrophils follows this to the wound site to start the inflammation phase.
Inflammation phase
Inflammation is the second step in the wound healing process in which neutrophils and macrophages play a prominent role. After reaching the wound site, neutrophils release reactive oxygen species (ROS) and proteases that play an essential role in preventing contamination of bacteria and cleaning the wound [7]. Monocytes are the next cells to enter the wound site and become macrophages. Macrophages play a dual role by preventing bacterial contamination and helping in recruiting fibroblasts, endothelial cells and keratinocytes by releasing various GFs and CKs [8]. Once this phase subsides, it is followed by apoptosis and the proliferation phase begins. In the case of normal wounds, proteases are balanced by their inhibitors. Whereas, in the case of chronic wounds, GFs such as transforming growth factor (TGF-β) allowed for a continuous influx of inflammatory cells leading to prolonged inflammatory phase and elevated levels of proteases, thereby leads to the destruction of extracellular matrix (ECM) and GFs. This leads to the destruction of ECM and GFs and restricts the wounds from progressing into successive phases [9].
Proliferation stage
The proliferation phase is characterized by the formation of granulation tissue, new blood vessels and epithelialization. It encompasses angiogenesis, the formation of granulation tissue and ECM, re-epithelialization, collagen deposition. It lasts for about 14 days in case of normal wounds. Initially, the migration of the fibroblasts and the epithelial cells occurs to the injured area, which replaces the damaged tissue. The capillaries and lymphatic blood vessels form granulation tissue. The collagen synthesis takes place by the fibroblasts, which give strength to the skin. This lasts up to 2 weeks. Production of fibroblasts helps in the ECM synthesis; in the initial stages, the synthesis of fibroblasts dominates the collagen production, but collagen production predominates in the later stages. Fibroblast by macrophages gets activated to myofibroblasts, which are responsible for wound contraction. These both, in turn, are responsible for the production of ECM. Epithelialisation is the final stage of proliferation. Afterward, wound retraction takes place. The process of new blood vessel evolution is known as angiogenesis, induced by the GFs, such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). In chronic wounds, the cells become senescent and make them unresponsive to wound signals [10]. It has also shown that fibroblasts from pressure ulcers are senescent, decreased proliferation and wound healing capacity [11, 12]. Stem cells from patients or animals with DW have also shown to be senescent [13].
Remodeling phase
After the development of granulation tissue, the remodeling phase gets initiated. Myofibroblasts, macrophages and endothelial cells undergo apoptosis. In this, Type–III collagen is replaced by Type-I Collagen for their tensile strength and wound contraction. In the overall remodeling stage, collagen formation gets increased at elevated levels because its production is balanced by degradation. In chronic wounds, re-epithelialization fails to occur due to diminished collagen formation, which is needed for wound contraction [14].
Pathophysiology of DW
The DW is defined as "one that has failed to proceed through an orderly and timely reparative process to produce anatomic and functional integrity within three months or proceeded through the repair process without establishing a sustained and anatomic and functional result" by Jabrink et al. [15].
Peripheral neuropathy
High blood glucose levels for a prolonged period disorganize the nerve signal transmission, which may weaken blood vessels' strength. Hence, it results in poor blood circulation and insufficient oxygen and nutrients to the nerves, leading to diabetic neuropathy. This mechanism is thought to be by forming advanced glycation end products, activation of the protein kinase C and an increase in the level of ROS [16].
In motor neuropathy, muscle weakness and atrophy are associated. Due to excessive pressure, keratosis and callus formation occur that further damages the foot and leads to ulcer formation. In autonomic neuropathy, dysfunction of sympathetic nerves (responsible for the sweat production) results in the decrease of the moisture, which further leads to dryness of the foot. In the case of sensory neuropathy, loss of sensation occurs [17].
Peripheral vascular disease
Peripheral vascular disease is one of the subsidized aspects for the development of DW [2]. Prolonged hyperglycemia and smooth cell dysfunction lead to endothelial cell dysfunction, which affects the tibial and peroneal arteries. This results in suppressed endothelium-derived vasodilators, increased thromboxane A2 and platelet aggregation. This results in an arterial disease that ends up with ischemia in the foot's lower extremity [18].
Diabetic foot infections
Most of the DWs will become infected if left untreated, which further leads to amputation. Gram-positive cocci are typically the utmost prevalent pathogens. Anaerobic bacteria are usually cultivated from ulcers with ischemic necrosis. Antibiotic-resistant organisms such as methicillin-resistant Staphylococcus aureus are intermittently found in patients formerly treated with antibiotic therapy. In moderate infections, staphylococci are the most common pathogens. In harsh conditions, Proteus and Escherichia coli (E. coli)are the most prevalent type of pathogens. Treatment includes single-agent therapy with anti-staphylococcal and anti-streptococcal antibiotics that are active against gram-positive, gram-negative, aerobic and anaerobic pathogens [19].
Treatment for DWs
The criteria for treatment of DW include assessing the vascular condition, modifying glycaemic control, pervasive debridement, applying moisture bed and moisture dressing. Current procedures and their limitations in DW healing [18] are depicted in Fig. 2.
Fig. 2.
Current procedures and their limitations in diabetic wound healing
Antibiotic and neuropathic drugs
United States Food and Drug Administration (USFDA) affirmed three medications to treat diabetic peripheral neuropathy, such as duloxetine, pregabalin and tapentadol. Three medicines claimed for DW treatment in the contaminated skin and skin structure infections (cSSSI) are piperacillin/tazobactam mix, trovafloxacin and linezolid. Presently for osteomyelitis related to overlying diabetic foot infections, no medications/therapies have been affirmed as of now [20]. Neuropathic drugs give only pain relief and have no role in DW, whereas antibiotics have already acquired resistance.
Bioengineered skin substitutes
Bioengineered skin substitutes for DW include an amniotic membrane, autologous stem cell therapy, fibroblast-derived dermis, porcine small intestinal submucosa (PSIS). The amniotic membrane is one of the essential regenerative medicine, presenting the deepest layer of the placenta. It comprises a thin epithelial layer, a basement layer and an avascular stroma [21]. Stem cells fit for self-restoration and multilineage separation have been examined in the injured tissues of DWs. Bone-marrow (BM)-determined mononuclear cells (MNCs) and mesenchymal stem cells (MSCs) are most effective clinically among the diverse kinds of stem cells [22]. Porcine small intestinal submucosa (PSIS) is an acellular natural ECM comprised of type I collagen, glycosaminoglycans (GAGs) and proteoglycans. The bioactivity of PSIS incorporates GFs, such as TGF-β, vascular endothelial growth factor (VEGF) and FGF, limiting the damaging movement of matrix metalloproteases (MMPs) and instigating angiogenesis to help new blood vessels in development [23].
Debridement
Since DW is mostly characterized by necrotic tissue, the stagnation of the wound in the chronic inflammatory phase results in the halting of the wound healing process. Thereby the application of drugs over the injury may not show its action in case of chronic wounds. Debridement helps in removing the necrotic or damaged tissue. Pirfenidone is a drug used for debridement and acts as an anti-inflammatory and antioxidant [24].
GFs
In the case of patients with the DW, GFs show limited action due to excess levels of MMPs. Specific GFs act to maintain white blood cells (WBC), such as granulocyte colony-stimulating factor, platelet-rich plasma, recombinant human. These GFs stimulate the function of angiogenesis, enhances proliferation and promote wound healing [25].
Moist wound environment and control of exudation
Destruction in the sympathetic nerves may affect the production of sweat and moisture, leading to dryness of the foot. Moist wound promotes the migration of cells, angiogenesis, prevents neuropathy and dehydration therapies to treat this include wet wound dressings and hydrogels that provide formidable moisture. By their aqueous texture, these can be freely applied and removed. Hydrogel dressings are amorphous gels and impregnated gauzes, which are used for the delicate to moderate exudates. A hydrocolloid dressing is an obstructive dressing that does not allow bacteria to enter into the wound, promotes angiogenesis and granulation. Other wound dressings include foam dressings and alginate dressings. The therapies mentioned above are very active and still used as standard treatments. The new treatment has concomitant therapeutic wound healing strategies; this encompasses antibiotics, neuropathic drugs, GFs and biological substitutes [26].
Scaffolds and their role in DW healing
Despite the fact that wound dressings commonly used in clinical practise (e.g., gauzes, absorbent cotton, bandages) are inexpensive, they can only provide basic physical protection and have little ability to influence/speed wound healing and prevent/treat infections. Tissue engineering and regenerative medicine are connected by a multidisciplinary approach that combines engineering, biology and material science, resulting in the development of viable organ and tissue regeneration/replacement options. Tissue engineering has recently proposed a number of ways to handle the treatment process as an alternative treatment for wounds, such as the implementation of scaffolds [27].
Scaffolds, or three-dimensional (3D) structures, can not only provide adequate nutrition for tissue growth, but they can also protect a wound by acting as a "fence" against external contamination. Scaffolds must provide physical support and communicate with cells to trigger physiological processes (cell adhesion, proliferation and differentiation), which leads to cell assembly into functional units, which is of great interest. Since cells must bind to and migrate across their networks, scaffolds must be porous and biocompatible. When constructing a scaffold for wound healing applications, these features must be taken into account while retaining high efficiency and low costs. The above all mentioned properties make scaffolds a good substitute for use in DW [28].
The salient features of scaffolds include (i) It facilitates the delivery and retention of cells and different biochemical factors (ii) It allows cells to associate and bind by promoting proper cell attachment and migration; (iii) It allows the flow of essential cell nutrients and released products; (iv) It modifies cell activity by exerting mechanical and biological stimuli; (v) It mimics the ECM-like microenvironment in 3D space [29].
Acellular synthetic matrices offer several advantages over naturally derived polymeric and cellular-based scaffolds, including longer shelf-life, cost efficacy and limited risk of rejection.
In recent years, surgical debridement has initiated a new touchstone, but drawbacks associated with it have led to regenerative tissue engineering development. Tissue engineering has grabbed much attention and has led to the development of various biological scaffolds. The uniqueness in flourishing the biological scaffolds is that they mimic the ECM, facilitate the proper exchange of nutrients and oxygen and provide a spatial environment for cellular transformation and neovascularisation. Perpetual therapies constituting foams, hydrogels, graduated compression bandages and moist wound dressings are ineffective in DWs and its chronic complications are concorded with the deep-rooted recovery period and patient morbidity. Hence, designing scaffolds for treating these DW should target the regeneration and address all the pathological manifestations of DWs [30].
Scaffolds developed using synthetic polymers often employ the fabrication technique of electrospinning. These have been widely used in tissue engineering due to their ECM-like architecture, high porosity and interconnected pores to enable free access to oxygen and nutrient supplies for cell growth. Electrospun scaffolds offer advantages of high surface area and tunable fiber diameter, which may closely match those of native ECM proteins and promotes cell adhesion [31].
Classification of scaffolds
Scaffolds are the pivotal elements to deliver drugs, cells and GFs into the body's relevant site. Scaffolds are generally classified as cellular and acellular based on the presence of living cells that they deliver (Fig. 3). When cells are entrenched into simulated 3D structures for tissue formation, classically, they are known as cellular scaffolds. Acellular scaffolds are usually produced from natural sources often ECM combinations, namely hyaluronic acid (HA) and collagen or synthetic polymers or native dermis loaded with drugs and GFs [27]. Acellular scaffolds are further divided into the following types based on their source and are depicted in Fig. 4.
Fig. 3.
Scaffolds and their classification
Fig. 4.
Classification of acellular scaffolds based on their source
Cellular scaffolds
Cellular scaffolds are the living skin substitutes onto which existing skin cells like fibroblasts or keratinocytes are seeded to grow new tissues or replace the damaged tissue. Few products that provide cellular and structural components for DW are discussed in the latter part.
Apligraf® (Organogenesis, Inc.) is made out of a bovine sort I collagen seeded with neonatal fibroblasts to distribute a neodermal layer. Human neonatal epidermal keratinocytes are incorporated in the top of this dermal part as a monolayer to shape the epidermis and consequently form a differentiated stratum corneum [32]. This results in a metabolically unique bilayered skin substitute giving both a dermal and epidermal layer with living cells. Regardless of the way that the fibroblasts and keratinocytes in Apligraf® do not remain over about a month [33], they are seen as accountable for provoking differentiation and augmentation through the release of central CKs and GFs [34]. Apligraf® was the first allogeneic cell-based dressing to be supported by the FDA in 1998 to treat DW and venous leg ulcers. Multicenter randomized clinical preliminaries showed a significantly higher rate of wound closure [35].
The FDA recommended Dermagraft® (Organogenesis, Inc.) in 2001 for the treatment of non-recuperating DW. Dermagraft differs from Apligraf® wherein, the fibroblasts are refined onto a bioresorbable polyglactin mesh scaffold; polyglactin is a standard suture material. The metabolically potent fibroblasts duplicate inside the crevices of the scaffold, secreting collagens, CKs, proteoglycans and other vital factors to make a 3D bioactive network, which is then cryopreserved for storage. It has shown that Dermagraft® significantly extended the wound closure when compared to control in the case of DW [36].
The human fibroblast-derived dermis is a biological dermal substitute produced from human fibroblast cells. Fibroblasts are usually taken from the newborn foreskin tissue that consists of fibroblasts and ECM. These cells proliferate and synthesize collagen, matrix proteins and CKs, which help construct a 3D scaffold containing living cells. It is preferred mostly in cases of long term and chronic cases of DW [37].
Stem cells are well known for their self-renewal and multilineage differentiation in the case of DW. Various types of cell therapies have been used in the treatment of DW, namely bone-marrow (BM)-derived mononuclear cells (MNCs), Endothelial progenitor cells (EPC), mesenchymal stem cells (MSCs), fibrocytes and adipose-derived stem cells. Of them, BM-MNCs, MSCs and EPC are most commonly used. MSCs derived from bone marrow, umbilical cord and placenta. Placenta derived MSCs have shown the induction of various pro-angiogenic factors required for the healing of DW. MSCs derived from the umbilical cord get differentiated into fibroblasts and these have shown a higher rate of proliferation and complete re-epithelialization compared to BM-MSC [38].
BM-MNCs are usually separated from bone-marrow and contain large groups of stem cells such as hematopoietic stem cells, endothelial progenitor cells, mesenchymal stem cells, CKs and GFs [22].
Acellular scaffolds
The ECM is a non-cellular primary element in the skin and constitutes non-structural and structural proteins and includes collagen, proteoglycans and GAGs. It mainly provides constitutional support to the cells and deeds as a reservoir to the GFs in enhancing cell migration and proliferation. In the case of DW, ECM plays a vital role in wound healing [38].
Acellular matrices have been evolved as a revolutionary treatment and can be categorized as allogenic, xenogeneic and synthetic derivatives. Animal and human tissues retain the ECM by detaching the cellular components through the 3D structure and facilitating re-vascularization and cellular progression. These acellular scaffolds help in restoring the process for the normal wound healing process [38].
Scaffolds derived from natural polymer have qualities of binding to GFs, promoting angiogenesis and confers antibacterial effect by natural degradation but has reduced stability in wound microenvironment due to degradation by enzymes.
Synthetic scaffolds are beneficial over natural due to their longer shelf life, limited risk of rejection and tunability of material's physical and chemical properties to balance various physiological parameters. Still, some polymers show slower degradation, which hinders tissue growth.
Naturally derived scaffolds
These are the scaffolds derived from natural polymers like collagen, HA, chitosan and gelatin. Of all the natural polymers, HA and collagen are widely used natural polymers to develop acellular matrices for DW healing [39]. Scaffolds derived from collagen have reduced stability in the DW environment due to degradation by collagenases. In addition to this, collagen undergoes contraction after implantation, resulting in ineffective wound covering and closure [40]. HA also undergoes degradation by hyaluronidases, which are usually elevated in diabetic patients [41]. Hence decellularization of allogeneic and xenogeneic tissues to attain native ECM has gained more popularity as acellular scaffolds to treat DW. The advantages of using these scaffolds in the treatment of DW are promoting angiogenesis, confer an antibacterial effect to the wound by natural degradation and reduce collagenase and gelatinase activities [42–45]. Acellular scaffolds of natural origin under the FDA 510(k) process are given in Table 1.
Table 1.
Acellular scaffolds of natural origin regulated under the FDA 510(k) process
| No | Name of the product | Company | Composition |
|---|---|---|---|
| 1 | ACell® UBM hydrated wound dressing | ACell | Porcine collagen |
| 2 | Aongen Collagen dressing (Aongen™) | Aeon Astron Europe | Collagen |
| 3 | Atlas Wound Matrix | Wright Medical Technology | A sterile decellularized fenestrated or nonfenestrated processed porcine collagen |
| 4 | Avagen Wound Dressing | Integra LifeSciences | A wound dressing comprised of a porous matrix of cross-linked bovine tendon collagen and glycosaminoglycan |
| 5 | CollaGUARD® | Innocoll | Composed of purified type-1 collagen protein |
| 6 | CollaSorb™ | Hartmann-conco | Composed of native collagen and calcium alginate |
| 7 | CollaWound™ ART | Collamatrix | Composed of collagen & polyhexamethylene biguanide |
| 8 | Collexa® | Innocoll | Collagen matrix sponge with a polyurethane foam backing |
| 9 | Collieva® | Innocoll | Clear collagen matrix film |
| 10 | DermADAPT™ | Pegasus biologics | Decellularized equine pericardial implant |
| 11 | DressSkin™ | TEI Biosciences | Hydrolyzed bovine collagen |
| 12 | E-Z Derm™ | AM Scientifics | Porcine dermis |
| 13 | Excellagen | Tissue repair company | 2.6% fibrillar bovine dermal collagen type 1 |
| 14 | FortaDerm™ | Organogenesis | A cross-linked sheet of fenestrated sheet of porcine intestinal collagen coated with PHMB |
| 15 | Integra™ | Integra life sciences | A bilayered matrix composed of collagen, chondroitin-sulfate with the silicone layer |
| 16 | MatriStem® | ACell | Sterile, porcine-derived, naturally-occurring lyophilized extracellular matrix in particle form |
| 17 | Oasis® | Smith & Nephew | Porcine sub intestinal submucosa |
| 18 | Primatrix™ | Integra life science | Fetal bovine dermis |
| 19 | Stimulen™ | Southwest technologies | Hydrolyzed concentrated dispersion of modified bovine collagens |
| 20 | TheraForm™ | Sewon Cellontech Co., Ltd | Highly purified collagen from porcine skin |
| 21 | Unite® Biomatrix | Harbor MedTech | Equine pericardial device |
ECM-derived scaffolds
The aim of all wound repair methods, at their most basic level, is to establish an environment that minimizes infection, promotes proper moisture balance and supports wound reepithelialization. ECM is a 3D network of various macromolecules, namely collagen, glycoproteins and many enzymes that give structural and mechanical support to the cells surrounding them. The role of the ECM in wound healing has been recognised and led to the production of many wound healing products. These products may be biologic or animal-derived and these could be composites containing a blend of biologic and synthetic materials or could be human tissues. An ideal matrix is the one that resembles the ECM that it is replacing [46].
Firstly, acellular matrices derived from human skin (allografts) have achieved more acceptance in DW healing treatment. But due to the risk of transmission of disease and less availability of human cadaveric skin, it has led to the use of dermal matrices from porcine and bovine sources. These have a number of similarities in their mode of action in promoting wound healing; however, as previously mentioned, the source material, preparation and processing of these products differ greatly. While studies on a variety of decellularized tissues have been well-designed and insightful, those derived from the porcine small intestinal submucosa (SIS) and the porcine urinary bladder matrix are perhaps the most comprehensively studied matrices in terms of biological activity, composition, mechanical properties, macro- and ultrastructure (UBM). [47].
PSIS
SIS-ECM contains more than 90% collagen by dry weight, the bulk of which is type I collagen. Small quantities of type III, IV, V and VI collagens, as well as adhesive proteins like fibronectin and laminin, are also present. Heparin, heparan sulphate, chondroitin sulphate and hyaluronic acid are among the GAGs present in natural ECM scaffolds. The amount of these GAGs that remain in the tissue after decellularization is highly dependent on the decellularization methods used, especially those that use ionic detergents that are known to remove GAGs from the ECM. PSIS was proven to improve the healing of DWs due to its role in cell proliferation, migration and differentiation. This includes the release of GF, promoting angiogenesis and suppression of MMPs activity. Previous studies have shown that multi-layered PSIS was more durable and degraded slowly than single-layered.
Shawn et al. carried out: a randomized controlled trial on the PSIS trilayer matrix. They stated that tri-layer SIS is a novel product indicated for managing various types of wounds, including DW. This study has shown that DW managed with a superior outcome and rapid wound closure [48].
OASIS® Wound Matrix is a non-cross-linked porcine SIS-ECM-based SIS ECM graft. Oasis has the added advantage of allowing several procedures to be done without having to remove dressings due to the SIS material's rapid deterioration. SIS-ECM has been shown to assist epithelial cell differentiation and the development of a new basement membrane in vitro. SIS-ECM has also been found to promote angiogenesis, neurogenesis and the repopulation of the wound site with multiple tissue specific cell types in studies involving tissues other than skin. This is achieved mainly through the migration of tissue resident and circulating progenitor cells. In 2005, Jeffrey et al. compared the OASIS® wound matrix containing PSIS to Regranex gel containing PDGF and indicated that OASIS® is as effective as Regranex for DW [49].
UBM
UBM is another example of naturally occurring ECM-derived from porcine's urinary bladder. MatriStem® is a noncross-linked porcine UBM-ECM-based natural ECM scaffold. The primary benefit of UBM-ECM over other natural ECM products is that it holds the urinary bladder basement membrane portion on one surface of the biologic scaffold. In organs including the skin and blood vessels, the basement membrane anchors epithelial tissues to the ECM. Since basement membrane proteins are also potent angiogenesis promoters, it's been suggested that keeping the basement membrane in place will help with tissue repair and wound healing. The wound dressing has been extensively studied for venous, diabetic and ischemic ulcers. This was also found to play a vital role in the retention of multiple GFs required for tissue regeneration and healing. In a retrospective analysis on the use of MatriStem®, Lecheminant and Field discovered that using the medication shortened healing time from an average of 25.5–9.8 weeks, but their research also established a range of risk factors that should be considered and addressed to optimise the Matristem product's effectiveness [50, 51].
Kimmel et al. have presented the case series in which three patients suffered from recalcitrant wounds and stated that after going through significant preclinical and clinical results of UBM and other ECM-derived scaffolds, UBM-derived ECM had shown more ability to alter the default wound-healing process from the one that shows chronic wound to the other one which shows relatively rapid wound closure [52].
Oscar et al. have stated that interim analysis results in treating DW and a UBM could significantly reduce the time to healing and decrease DW's recurrence [53].
Collagen
Collagen is potentially the most frequently used ECM in wound dressings. Puracol Plus (Medline Industries), Fibracol Plus (Systagenix) and BIOPAD (Angelini Pharma) are examples of topical wound dressings. These dressings, which contain a microfibrous type I collagen sponge, are perhaps the simplest biologic scaffolds, serving primarily to absorb wound exudate and prevent wound desiccation rather than providing any bioactive part. Mostly collagen-based scaffolds are procured from animal and human sources such as human cadavers, porcine small intestine submucosa and UBM [41]. Once acquired, a native collagen scaffold biomatrix was created that stabilizes vascular and cellular components that get incorporated into the wound bed. The advantage of these products over traditional wound dressings is that scaffold absorbs liquid in collagen, it forms a hydrogel that covers the wound. MMPs are often sequestered by collagen from the wound environment preventing the wound from worsening further. However, since they are normally replaced after just a few days, they do not act as a typical scaffold for tissue regeneration.
Collagen-based scaffolds show poor stability due to their susceptibility to proteolytic degradation in the DW microenvironment. Cullen et al. reported that they had found reduced levels of collagenase, gelatinase, MMP-2 and MMP-9 levels; and increased binding of GFs after treating with oxygenized regenerated cellulose (ORC)/collagen dressing [54].
Graphene oxide (GO) has been known to be a suitable carrier for drugs, but its low aqueous solubility has not shown promising effects. Hence GO crosslinked with polyethylene glycol (PEG) to yield pegylated GO. Collagen has been selected for use as an acellular dermal matrix. Chu et al. have synthesized collagen-nanomaterial quercetin hybrid scaffold with pegylated GO. The results have shown that the prepared scaffolds have the advantages of having a cell-adhesive surface for accelerating mesenchymal stem cell attachment and proliferation, more stability and a biodegradable nanofiber for enhancing collagen deposition and angiogenesis in DW repair [55].
The use of bilayer scaffolds to mimic the ECM structure has shown to be promising in wound healing. Hence bilayer scaffolds mimicking two layers of skin have been prepared by Bektas et al. In this report, sodium carboxymethylcellulose hydrogel acted as a dermis layer fibroblasts were added and collagen or chondroitin sulfate incorporated collagen as an epidermal layer onto which keratinocytes were attached. Expression of VEGF, bFGF and IL-8 and formation of collagen I and III on both layers were seen. Hence, they can be utilized as grafts for the treatment of DWs [56].
GBT013 is a 3D matrix developed by Y. Guillemin et al. It is reported to contain a significant amount of type I collagen (72%), chitosan (20%) and 8% of chondroitin sulfate. Collagen accounts for MMPs inhibition and balances their synthesis and degradation. Also, it shows cellular proliferation when used with chondroitin sulfate. Chitosan accounts for antimicrobial property and hemostatic effect. The above-stated features made it a suitable dressing for DW [57].
Morimoto et al. (2013) have developed a novel collagen/ gelatin sponge that resembles artificial dermis designed to release basic fibroblast growth factor (bFGF) for more than ten days. This was the first clinical trial done to evaluate the safety and efficacy of the formulation to release bFGF in a sustained manner [58].
VEGF cannot perform its action in chronic wounds due to its shorter half-life and degradation by wound milieu. The collagen-based scaffold has been developed by Tan et al. (CBD-VEGF) and compared with native VEGF. It was stated that CBD-VEGF has a better ability to bind the collagen on the wound site and increased therapeutic efficiency [59].
Marston et al. (2005) have developed an injectable scaffold matrix derived from the porcine- collagen matrix to stimulate the healing of diabetic foot ulcers. They have reported that a considerable response to injection was seen and % of wound size reduction was 72 in 2 weeks. More extensive trials are underway to examine the benefit of a new treatment [60].
HA
HA is a specific polysaccharide. It is a non-sulfated GAG, an essential segment of the ECM of animals' connective tissues, for example, ligament, umbilical cord and synovial liquid [61]. HA is also referred to as hyaluronan because it exists mostly in vivo as a polyanion and not in the protonated acidic structure. It is a linear polysaccharide of substituting disaccharide units of α-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine by β (1–3) glycosidic bonds. It is usually extracted from the umbilical cord, synovial fluid and is non-allergenic as well as biocompatible. HA is generally water-soluble and also can deliver intensely viscous solutions with extraordinary viscoelastic properties [62].
In 2019, Chen and Wang stated that the combination of HA of high molecular weight and povidone-iodine dressing exhibited significant DW healing by increasing neovascularization, tissue regeneration and suppressing pro-inflammatory response [63].
HA and collagen
Collagen and HA are vital elements in wound healing as they are the main constituents in ECM. Type 1 and III collagen have been shown to promote the early stage of wound healing. Whereas HA is involved in granulation, modulation of the inflammation phase prevents collagen hyperproliferation and scarring. Lai et al. have developed electrospun composite nanofibers and designed in such a way to release multiple GFs (epidermal growth factor (EGF) and bFGF in initial stages and VEGF, PDGF in later stages for chronic wound healing. The results have shown accelerated wound closure, elevated collagen deposition and increased mature vessel formation. [64].
Kirk et al. have synthesized collagen-HA wound dressing using crosslinking agent 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Crosslinking using this material has shown enhanced biological stability by decreasing enzymatic degradation of collagen. In vitro results stated that HA was found to be uniformly distributed in the dressing. The prepared dressing supported proliferation and attachment of cells, thereby increasing the potential for use in chronic wound healing [30].
Non-ECM-derived scaffolds
The polymers of non-ECM-derived scaffolds for DW healing include polysaccharides such as alginate and chitosan.
Alginate
Alginate shows excellent gelation properties and also facilitates the moist wound environment. To obtain the 3D biopolymeric scaffolds, crosslinking should take place in the presence of the divalent ions. Alginate dressings when it meets the wound exudates the ion exchange between the calcium and the sodium ions, resulting in bulging of the alginate fibers then diffuses to figure as gel thereby retains the moist wound environment. It also reduces microbial infections and degradation by proteinases. Alginate dressings can absorb 15 to 20 times their weight in wound fluid, exhibit excellent hemostatic properties and act as a delivery vehicle. Therefore it can be used for VEGF delivery and optimal bioactive in treating chronic DWs [38].
Ciprofloxacin is an antibiotic that was found to be active against gram-positive and negative microbes. However, it is found to have severe adverse effects if administered systemically. As already known, the presence of microbial load at the wound site has a detrimental impact in the case of infected wounds. Ahmed et al. have prepared ciprofloxacin-loaded calcium alginate wafers to release ciprofloxacin in a sustained manner to inhibit and prevent re-infection for potential healing of chronic diabetic foot ulcers. The prepared delivery system showed better moisture absorbing capacity, bacterial inhibition and cell survival, compared to the commercial dressing of alginate (Algisite) [65].
rhEGF, due to its degradation in wound fluid, has been encapsulated by preparing it in the form of poly lactic-co-glycolic acid (PLGA)-Alginate microspheres by Gainza et al.. Both PLGA and alginate are biocompatible polymers and the addition of these two led to increased encapsulation efficiency and appropriate release parameters. In vitro release studies have shown that the formulation induced migration and proliferation of fibroblasts. Moreover, its biological activity was protected due to microencapsulation. Further, in vivo studies have shown that these microspheres promoted faster healing of DW in wound contraction, epidermal regeneration and inflammatory stage recovery [66].
Chitosan
Chitosan has also been extensively used in wound dressing as it promotes granulation tissue formation and hemostasis. It facilitates the faster wound healing process by cell adhesion, red blood cells (RBC) and platelet aggregation to promote the hasty clotting. In the wound area, the chitosan’s depolymerization occurs by the lysosomal enzymes, which then release N-acetyl-D-glucosamine and result in collagen formation, fibroblast proliferation and remodeling. Due to its positive charge, chitosan interacts with the negatively charged cell membrane and then disrupts wound formation. Further, chitosan has antimicrobial properties.
In recent years the use of silver nanoparticles (AgNPs) has been increased due to their antimicrobial, antioxidant properties. Masood et al. prepared silver nanoparticle impregnated chitosan hydrogel. The study demonstrated that prepared hydrogel exhibited higher porosity and degree of swelling compared to chitosan-PEG hydrogel alone. It also showed a sustained release of AgNPs for seven days with slow hydrogel degradation [67].
Patil et al. presented the application of a unique oxygenating biomaterial (MACF) made into a moist hydrogel wound dressing for treating DWs for the first time. The results of this study confirmed the benefits of this novel biomaterial approach for improving regenerated tissue structure in DW healing [68].
Studies on VEGF165 using plasmid DNA and GF delivery strategies have been done up to date, but a longer isoform of VEGF, i.e., VEGF189, has been found to ECM proteoglycans, namely Perlecan. Megan et al. stated that the plasmid DNA encoding perlecan domain I and VEGF189 loaded scaffolds promoted dermal wound healing in normal and diabetic rats. This treatment resulted in an increase in the number of blood vessels and sub-epithelial connective tissue matrix components within the wound beds compared to wounds treated with chitosan scaffolds containing control DNA or wounded controls. From the above results, it has been shown that chitosan scaffolds containing plasmid DNA encoding VEGF189 and perlecan domain I can induce angiogenesis and wound healing [69].
Karri et al. have prepared curcumin-loaded chitosan NPs and loaded them into collagen alginate scaffolds to improve curcumin's solubility and tissue regeneration stability. The data revealed that complete epithelialization with granulation tissue formation is seen in the prepared scaffold group's case. In contrast, the placebo scaffold was deprived of collagen deposition and the presence of inflammatory cells in the control group was observed [70].
The increased blood glucose levels in diabetic patients lead to the formation of various by-products (advanced glycation end-products), which cause decreased blood circulation, which in turn leads to hypoxia and finally leads to slow wound healing and amputation. A dressing made of polyvinyl alcohol (PVA)/chitosan nanofiber has been brought with a high moisture vapor transmission rate and good antimicrobial activity. PVA was added to chitosan to improve mechanic, hydrophilic and biodegradable nanofiber properties. It helps in forming uniform nanofibers prepared substrate did not show any recognized cytotoxicity effects and had excellent odor absorbing capability [71].
Synthetic scaffolds
The advantages of synthetic scaffolds over natural acellular and cellular scaffolds are longer shelf-life, cost efficacy and limited risk of rejection. The incremental costs per patient that were successfully treated were $1600 for the acellular biosynthetic scaffold (Talymed R), $3150 for Oasis and $29,952 for Apligraf. Acellular scaffolds of synthetic origin under the FDA 510(k) process are given in Table 2.
Table 2.
Acellular scaffolds of synthetic origin regulated under the FDA 510(k) process
| No | Name of the product | Company | Composition | Source |
|---|---|---|---|---|
| 1 | Hyalomatrix® | Integra life science | Comprised of non-woven pad entirely composed of a benzyl ester of hyaluronic acid & silicone membrane | Hyaluronic acid |
| 2 | Jaloskin® | Fidia advanced biopolymers | Transparent film dressing composed of the benzyl ester of hyaluronic acid | Hyaluronic acid |
| 3 | Suprathel® | Polymed innovations | Tripolymer of polylactide, trimethylene carbonate and caprolactone | Lactic acid |
| 4 | Talymed® | Marine polymer technologies | Composed of shortened fibers of poly-N-acetylglucosamine | Microalgae |
One of the reasons for using synthetic materials as wound healing frameworks is their tunability of the material's physical and chemical properties to balance various physiological parameters by altering polymer organizations, variety and arrangement of constituent monomers. Even though there is a wide range of synthetic polymers to look over, polyesters and polyurethane are some of the most used biocompatible synthetic polymers to make 3D tissue frameworks and drug delivery applications. Synthetic polymers have the advantage of being able to be synthesised and modified in a controlled manner to have constant and homogeneous physico-chemical properties as well as stability when opposed to natural polymers. However, they are biologically inert and do not possess the therapeutic value that natural polymers do. They don't contain impurities, are typically mechanically stable and degrade in a predictable manner. They do, however, come with a chance of toxicity. They are classified as hydrophobic or hydrophilic and are commonly used in wound healing applications [38].
Traditional synthetic scaffolds
The convenience in adopting the synthetic polymer is due to its physical and chemical properties. However, synthetic biomaterials have been used in sequence with natural polymers to promote cellular migration, proliferation and adhesion. These are also found to release different bioactive molecules in a controlled manner, as well as scaffolds for skin tissue engineering. Aliphatic polyesters such as polycaprolactone (PCL), poly (lactic acid) (PLA) and PLGA are commonly used in these applications as degradable polymers. The Food and Drug Administration of the United States of America (FDA) has approved these polymers for several medical applications.
PLGA
PLGA is also a biodegradable polymer with biocompatibility, controlled biodegradability that accounts for drug stability in the matrix and potential for drug-carrying capacity, thereby reducing drugs' systemic effects. Besides, it can also be handled into a wide range of structures—powder, pellets, filaments, NPs and so on. PLGA microspheres stacked with recombinant human epidermal growth factor (rhEGF) were shown to improve fibroblasts' proliferation rate. It was seen that injuries in diabetic rats mended more effectively than rhEGF alone [72].
PLGA, when given in the form of nanoparticles, then the dispersion seemed to be unsuitable for topical administration because of its low viscosity. Hence, there is a need to convert the distribution into the hydrogel to increase application ease and prolong residence time on the skin. Bairagi et al. have carried out studies on DW healing by using hydrogel loaded with ferulic acid (FA) and PLGA NPs. The results have shown that faster epithelialization in the case of DW compared with the control group [73].
It has been reported that the external application of lactate produced from PLGA has shown its role in wound healing. Lactate has shown its effects on pro-collagen formation, angiogenesis by recruiting endothelial progenitor cells to the wound site. VEGF has drawbacks of decreased stability due to degradation by proteases in the wound environment. Hence, by taking two reasons into account, Chereddy et al. have administered VEGF in PLGA NPs and reported that combined actions of lactate and PLGA had shown increased collagen content and angiogenesis in DW [74].
Tissue-engineered scaffolds mimic ECM as well as restores microcirculation. Astragulus polysaccharide (APS) possessing endothelial cell protection activity has been loaded into scaffolds by Yang et al.. They reported that this to the DW has resulted in increased skin blood flow around wounds and increased microvessel density in regenerated skin tissues [75].
2-N,6-O-sulfated chitosan (26SCS) has shown protection against superoxide and hydroxyl radicals but less stability in the case of hydrogen peroxide radicals. Hence, Zhang et al. (2018) have designed PLGA scaffolds doped with 26SCS to protect 26SCS and release heparin-binding epidermal growth factor (HB-EGF) in a sustained manner. The results have shown that the scaffold's wound got healed after 28 days and increased keratinocyte migration due to the continuous release of HB-EGF [76].
PCL
PCL is an optimal vehicle for drug delivery. For example, curcumin, due to its low bioavailability and stability, requires a specific polymer carrier to show its activity. Hence, it was loaded into PCL/gum tragacanth electrospun nanofibers by Mohammadi et al. (2016) and studied the antibacterial activity and in vivo DW healing activity. Tragacanthin hydrolyses to arabinose and glucuronic acid help coagulate surface proteins and inhibit infection, thereby rapid wound closure. It was reported that after 15 days of application of the formulation, significant granulation tissue formation, collagen deposition, early epithelial layer regeneration has occurred. In contrast, in control, wounds were completely lacked collagen on day five and a low amount of collagen on day 15 [77].
PEG
Reduced formation of new blood vessels in DW's case leads to the accumulation of glucose at the wound site, resulting in ischemia and tissue necrosis. Hence, focusing on re-establishing the vascular network is needed to prevent ulcer formation in DW. Chen et al. have prepared injectable self-healing hydrogel by loading desferrioxamine (DFO) into crosslinking of thiolated PEG (SH-PEG) with silver nitrate. The prepared hydrogel has shown antibacterial activity due to silver and angiogenic properties due to DFO. In vivo results have demonstrated that the injected hydrogel can efficiently repair DW with a low incidence of bacterial infection and enhanced angiogenic activity [78].
Poly (glycolic acid) (PGA)
PGA has shown wide clinical applications due to its biodegradable nature and low degradation properties. Yin et al. (2016) attempted an experiment to recruit macrophages with monocyte chemoattractant protein (MCP-1) because macrophages M2 play an essential role in the secretion of various GFs such as VEGF, TGF-β. The results have shown that wounds treated with drug-eluting scaffolds recovered entirely in 10 days whereas, it took 14 days to heal completely in case of control. Hence, they have proposed that macrophages' presence may contribute to faster wound healing in the case of diabetic patients [79].
Gene therapy can deliver the GFs to prevent their degradation and release in a sustained manner because GFs are degraded in wound fluid. Arnold et al. (2003) have employed retroviral gene therapy to deliver the GF-PGA loaded scaffolds containing cultured dermal fibroblasts retrovirally transduced with PDGF-B gene accelerated wound healing compared to wounds treated with PGA alone [80].
Polyurethane (PU)
PU is a non-toxic, non-allergenic material that facilitates good permeability to oxygen, carbon dioxide (CO2) and provides excellent mechanical strength and flexibility. It is cost-effective with uniform homogeneous quality [38].
Sodium bicarbonate is usually added to release the drug in a controlled manner. Moreover, it releases the medicine according to wound pH. Pantothenic acid (PA) has been reported as anti-inflammatory and helps the cells survive high oxidative stress. It has also shown the promotion and migration of fibroblasts and thereby has a role in treating the remodeling phase. Herrmann et al. developed polyurethane-based nanofibrous scaffolds by incorporating PA. The results indicated an accelerated healing process of chronic wounds [81].
PLA
Generally, PLA is used due to two significant reasons. The first one is due to its capacity to absorb pus and blood moisture and the ability to supply oxygen over wounds. The second reason is its chemical structure that possesses biological properties such as biocompatibility and antibacterial nature that help assimilate natural ECM and fibroblast proliferation. It has been shown that when PLA is used to fabricate a nanofiber scaffold at a concentration of 2%, it has shown higher biodegradability. Moreover, it has also demonstrated a significant reduction in the wound size and improvement of the healing process of the DW [82].
Novel synthetic scaffolds
Almost all the scaffolds were developed from synthetic or natural polymers for the extended release of drug molecules. To improve cell filtration, the traditional advents have led to the development of novel scaffolds for the better healing process of DWs. Oxidative stress is considered one of the pivotal obstacles in enhancing the healing process. Specific research has been proven to improve the healing process in DWs by the local delivery of certain antioxidant compounds like glutathione, vitamin C and vitamin E. In addition to the antioxidant compounds, antioxidant enzymes also play a vital role in the cutaneous healing of wounds, such as superoxide dismutase (SOD), glutathione peroxidase, peroxiredoxin. Specific proteins such as catalase mimetics, SOD-mimetics through systemic delivery have enhanced the healing of diabetes. When the antioxidant compounds lack ROS specificity, they have been transformed into inactive forms by local delivery by reacting with the ROS stoichiometry. But when administered locally, there is a chance of proteolysis due to a protease rich wound environment. Certain antioxidant enzymes could be transformed from SOD to others. For instance, SOD could convert from superoxide to hydrogen peroxide by glutathione peroxidases and catalase enzymes [38].
Recent research attempts have adopted specific endogenous antioxidant systems to enhance the healing of DWs. For instance, nuclear factor (erythroid-derived 2)-like Nrf2 helps prevent oxidative stress and detoxification. It has been shown that the stimulation of Nrf2 may enhance the healing process, whereas dysfunction in the Nrf2 pathway ultimately results in prolonged wound healing by chronic hyperglycemia. Various natural and synthetic substances stimulate the Nrf2 pathway, such as curcumin, fumaric acid esters and synthetic triterpenoids. Still, there are certain limitations like increased oxidative stress due to harmful chemicals and systemic delivery. Hence, topical delivery at the inflammatory site is desirable to promote the healing of DWs. For instance: the dimethyl fumarate compound is the first FDA-approved Nrf2 stimulator that has been used for multiple sclerosis. By preventing the protease-rich wound microenvironment and restoring the ECM, GFs, a simulated ECM possessing numerous properties, can be developed as a composite scaffold (38). A further section of this review discusses the studies of GFs used in the healing of DW.
GFs
GFs have shown a vital role in wound healing, such as anti-inflammatory, increased fibroblast proliferation and tissue remodeling. Hence, the topical application of GFs has proven to be successful in the case of DW. However, they suffer from the limitations of having a shorter half-life and degradation in the protease environment. This led to the discovery of drug-delivering scaffolds that can deliver GFs in a sustained manner and preventing their degradation by coating them with polymeric NPs.
PDGF
Gelatin has shown various advantages in tissue engineering, like MMP mediated degradation and biocompatibility. Gelatin cryogels promote proliferation and survival of cells in vitro and they are successful epidermal substrates. As already known, silver is used as a broad-spectrum and highly effective antimicrobial agent. Wan et al. (2019) have developed a 3D bilayer scaffold in which the top layer consists of silver loaded gelatin cryogel to protect the wounds against bacterial infections. The bottom layer consists of PDGF-BB. The results have shown that silver and PDGF loaded scaffolds could accelerate the DW closure by enhancing re-epithelialization, granulation tissue formation and angiogenesis compared to silver loaded scaffolds and scaffolds alone [83].
Chen et al. (2016) have designed a DFO loaded hydrogel nanofibrous scaffold that releases DFO in a sustained manner for the rapid recruitment of cells related to angiogenesis. In vitro results have shown that sustained release of DFO led to neovascularization, upregulation of HIF-1α and VEGF. The in vivo results have shown high expression of angiogenesis-related CKs (78).
EGF
It is a well-known fact that the significant problems associated with DW are microbial load at the wound site that leads to infection. Hence, Dwivedi et al. (2017) have designed nanofibers made up of Eudragit RL/RS 100 polymer and gentamicin sulfate (GS) drug to prevent infections and immobilized with rhEGF to promote proliferation and tissue regeneration. The results have exhibited antibacterial activity and faster wound healing compared to the scaffolds with GS alone. [84].
bFGF
The levels of endogenous GFs in the case of DW are too low to show their angiogenesis activity. Hence, the topical application of GFs has proven to improve wound healing but has the drawback of shorter half-life and degradation. Due to this fact, a drug delivery system that could deliver the drug in a sustained manner by preventing degradation is needed to apply GFs in DW. Electrospun fibers loaded with bFGF have been prepared by Yang and his coworkers and coated with poly ethylenimine and PEG polymer to release bFGF in a sustained manner for four weeks. The steady release of bFGF has accounted for an increased wound recovery rate with enhanced vascularization, collagen deposition and complete re-epithelialization [85].
Fabrication of scaffolds
Solvent casting
This method involves pouring a polymer solution into the mold and followed by evaporation of it to form a polymeric layer that adheres to the mold. This method is simple, easy and inexpensive and does not necessitate more equipment and is based on the evaporation of the solvent. But the drawback associated with it is the employment of toxic solvents causes protein denaturation and scaffolds fabricated may also possess some toxicity [86].
Particulate leaching
This is usually known as porogen leaching because porogens like salt, wax, or sugar are typically added to create pores or channels. Firstly, salt is grounded into small particles and particles of uniform size are poured into a mold and placed with a porogen. The addition of a polymer solution follows this. Once the solvent's evaporation takes place, salt crystals are washed away by using water and hence the formation of pores of a scaffold. This method is secure and the pores' size can be controlled by governing the amount of porogen added, size and shape of the porogen. This method utilizes less amount of polymer for scaffold fabrication. But the disadvantage of this is we cannot control pore shape and inter pore openings [87].
Gas foaming
This involves high-pressure CO2 gas for the fabrication of highly porous scaffolds. The amount of gas dissolved in the polymer decides the porosity and pore structure. This method includes the first exposing of absorbent polymer to CO2 for saturation and dissolved CO2 becomes unstable under this condition and at the same time gas phase separates from the polymer. The isolated gas molecules become clustered to reduce free energy and results in pore nucleation. 3D scaffolds are formed after the completion of the foaming of gas. This method does not utilize any organic solvents like other methods and a continuous polymer layer was created upon the expansion of the polymer [86].
Phase separation
Phase separation can be achieved by two means. One is by the addition of nonsolvent and another is by using high temperature. The former process is usually not used to prepare scaffolds for the tissue engineering process because it results in a heterogeneous pore structure. In contrast, the latter method results in a uniform pore structure. Phase separation by increasing temperature involves separating the homogenous polymer solution into the polymer-rich phase and polymer lean phase at high temperature. This polymer-rich phase solidifies to form a matrix, while polymer less phase results in pores' formation due to evaporation of the solvent. This technique's benefit is it can be combined easily with other technologies to fabricate 3D scaffolds [88].
Electrospinning
This technique employs high electrostatic force for the production of fibers of nanometers to micrometers scale. It consists of a capillary tube in which a polymer solution is placed and a metal electrode is fixed. The entire setup is placed on an electrically insulated stand. An electric field is applied to the polymer solution bearing surface tension at the capillary tube's end to induce charge. This results in charge repulsion in a direction opposite to surface tension. As the electric field's intensity increases, the polymer solution's semi-spherical shape stretches and results in a cone-shaped structure known as the Taylor cone. When charge repulsion overcomes the solution's surface tension, it is ejected as fibers from the Taylor cone and is collected on the metal screen as dry fibers. This method is advantageous due to its versatility in the usage of polymers and non-invasive nature. This produces a scaffold appropriate for cell growth and tissue remodeling and is especially of interest as it structurally mimics the ECM [89].
Freeze-drying
This method utilizes the principle of sublimation. First, the polymer is dissolved in a solvent to form the solution of a specific concentration. Then the solution is subjected to freezing in a freezer and subjected to lyophilization for solvent removal. This results in the formation of a scaffold with higher porosity and interconnectivity [86]. It has been necessary because scaffold morphology can be controlled by changing polymer type, concentration and freezing time. This method suffers from the drawback of smaller pore size and long processing time. The methods for fabrication of scaffolds is given in Fig. 5.
Fig. 5.
Fabrication techniques of scaffolds
Appropriate characteristics of the scaffolds
Scaffolds have been characterized for morphology, stability, water absorption capacity, tensile strength and degradation.
Tensile strength
Scaffolds for wound healing applications should have sufficient mechanical properties to promote cellular activities like proliferation, migration and angiogenesis, as well as to protect native skin structures like blood vessels, lymphatic systems and nerve bundles. The skin scaffold should have mechanical properties similar to those of native tissue for this reason. The values of tensile forces, Young's modulus and elongation-to-break are the most important parameters for determining the suitability of the scaffold's mechanical properties. Although these values differ depending on the area of the native tissue, the literature suggests that wound dressings should have tensile strength between 5 and 40 MPa, Young's modulus between 4.5 and 25 MPa and elongation-to-break between 35 and 120 percent. Such values provide adequate mechanical support for angiogenesis and tissue remodelling during wound healing while also avoiding stress shielding side effects. Many studies have shown that synthetic polymers like PCL, PU and PLGA have excellent mechanical properties due to their thermal and chemical stability. These polymers could be combined with natural polymers like chitosan, collage and cellulose to improve mechanical properties. The mechanical strength can be determined by adopting the INSTRON 3369 Universal Testing Machine (UTM), ASTM D638 standard with a 5 mm/min speed rate. The samples were sectioned with a size of about 1 cm × 10 cm and 5 cm length gauze. In the experiment, the sample end was attached using a unique attachment/fixture and subjected to tensile, where maximum tensile strength can be determined. While experimenting to determine the measurements at the approximate rate of 5 mm/min, the membrane was dragged to some extent and then it was returned to the initial point before the breakage of the layer. Elongation and force were calculated [90].
Tensile strength (MPa) = Breaking force (N)/cross-sectional area of the sample (mm2)
Morphology of scaffolds
Nanofibrous scaffolds will more specifically stimulate cell activity. Wound therapeutics typically use biological stimuli to promote ECM deposition and collagen formation, both of which are critical steps in wound healing. According to studies, one of the criteria for the wound healing process is the provision of platforms with a nonporous interconnected network that allows for efficient material transportation and adsorption. Furthermore, it is unsurprising that electrospun nonporous structures have a high degree of versatility, offering enhanced conformability, coverage and wound protection; however, the availability of adequate oxygen and moisture at the wound site has been found to increase the risk of infections. Scanning electron microscopy (SEM) method can be used to study several design criteria for scaffolds such as biocompatibility, biodegradability, porosity, homogeneity and processability. Usually, the sample size used in SEM was 1 cm × 1 cm × 0.5 cm. The scaffolds' cross-section and the exterior surface can be stained with the last delicate gold layer (~ 150 Å). Sometimes the sample can be placed directly without any conductive substance. The photographic image can be captured at the excitation voltage of 5 kV and10kV. Preceding their observation in SEM, the samples were subjected to be placed in aluminum stubs and enclosed with the gold at approximately 9 V [91]. For characterizing the hydrogel scaffolds, they were initially air or freeze-dried and observed in the SEM. Then the samples were found at minimumvoltage of 5kVwith the blend of the subordinate detectors. The air-dried scaffolds can also be investigated using the BIOSCOPE catalyst atomic force microscope by adopting the scan assist mode [92]. For examining porous scaffold by biocomposite films, the measurement was done with the increased resolution of SEM (Model no. JOEL JSM-6490 LA, Joel Ltd., Japan) at 10 kV voltage in the carbon tape. The non-conductive specimen was fixed to test the conducting substance and subjected to analysis. For porous scaffolds: the sample was sectioned and organized in the aluminum and gold and investigated in SEM (FEI QUANTA 200) with a voltage of 15 kV. For assessing the main components, the EDS(ELEMENTAL CHEMICAL ANALYSIS) technique can be used. Usually, the design, structure and size distribution of the samples were investigated by (Jeol JSM-6400 microscope, JEOL electron microscope JSM7600F, Philips XL30 FEGSEM), field emission scanning electron microscopy (JEOL-ISM-7500F; JEOL Tokyo, Japan) and photon correlation spectroscopy, respectively, where the structure of the scaffolds can be investigated from the geometrical determination [92].
Porosity
For the fabrication of 3D scaffolds, porosity and pore size are essential since porous nature and well-interconnected pores are vital for cell migration, proliferation, developing new tissues and tissue vascularisation. The porosity of a dressing material allows for gaseous and nutrient exchanges, thereby facilitating healing progress. According to some research, scaffolds with a porosity of 60 to 90% are ideal for wound healing applications because they can provide enough space for cell activity, oxygen and nutrient exchange and the development of a new ECM. The balance between the porosity and the mechanical properties of a scaffold is important since an increase in porosity has a direct effect on the reduction of mechanical parameters. In addition to increasing the mechanical properties of the scaffold, nanobiomaterials such as carbon nanotubes and ceramic or metallic nanoparticles may be used to avoid a substantial reduction in mechanical properties. The scaffolds' porosity measurement can be carried out by adopting a liquid displacement process [65]. The scaffold will be placed in the hexane without any force and the scaffold matrix swells by permeation of liquid. The weight gained by the scaffold before saturation and after saturation should be observed. After the hexane's withdrawal by using the filter paper, the liquid above the scaffold was removed. The scaffold's dry weight before saturation is considered a Wd, scaffold weight in hexane during the saturation is viewed as the W1. Wet scaffolds after the saturation are regarded as the Ww and the process will be repeated in triplicates [84]. The diameter and the scaffold volume can be determined using the digital caliper, helium pycnometer, Ultrapyc1200e. Quantachrome pycnometer can be adapted to determine the grain volume. Scaffold porosity can be measured by the scaffold's true density and grain density, where the density can be measured using its volume and weight [93].
Wd = dry weight of scaffold before saturation.
Ww = wet scaffolds after saturation.
W1 = weight of scaffold in hexane during saturation.
Degree of swelling/wettability
One of the most essential characteristics of a material's surface is its wettability. Skin scaffold surface hydrophilicity is an important factor that influences cell attachment, proliferation and differentiation. The water contact angle at the scaffold's surface is normally used to assess the scaffold's wettability. Other studies have shown that moderately hydrophilic surfaces with a water contact angle of 30 to 70° allow cells to adhere and expand. Hydrophobic and strongly hydrophobic surfaces, on the other hand, had lower cell adhesion. The ingress of water into scaffolds can have both adverse and beneficial effects on the scaffolds' properties. The nano scaffolds' swelling behavior was calculated by immersing them in phosphate buffer (pH 7.4) for 24 h at room temperature. After 24 h, the scaffold was removed and the excess buffer solution was wiped with filter paper and weighed. The weights were recorded and the degree of swelling was calculated using the equation [84].
where W1 = weight of scaffold after immersion.
= weight of scaffold before immersion.
Dwivedi et al. 2017 have developed nanofibrous scaffolds modified with gentamicin and rhEGF for in vivo DW healing and analyzed the composite nanofibrous scaffolds' swelling behavior by the method mentioned above. The scaffolds were incubated in PBS (pH 7.4) for 24 h at room temperature and the swelling index was found to be 336 ± 3.26% [84].
In vitro studies
Cell viability assay
This is usually used to screen various compounds to determine whether they affect cell proliferation or show cytotoxicity that generally results in cell death. Irrespective of the assay being used, finally, how many viable cells are remaining is usually essential. There are various methods for the estimation of viable cells. Some of the most widely used techniques in multi-well formats in which data are recorded using plate reader are tetrazolium reduction, resazurin reduction, protease markers and adenosine triphosphate (ATP) detection. Various tetrazolium derivatives have been used for the detection of viable cells. The most widely used compounds are, namely: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), MTS, XTT and WST-1. These compounds again fall into two types: i) MTT is a positively charged molecule and readily penetrates viable eukaryotic cells, whereas ii) other compounds are negatively charged and cannot penetrate cells and hence they are usually combined with an electron acceptor, which facilitates the reduction of tetrazolium to colored formazan product. The MTT tetrazolium assay was the first assay developed for the 96-well format. MTT assay is mainly used for the determination of metabolic activity. The MTT usually prepared in physiological solution is added to cells in culture to give a concentration of 0.2–0.5 mg/ml and incubation for 1 to 4 h. The amount of formazan formed (directly proportional to the number of viable cells) is noted by measuring absorbance at 570 nm using a plate reading spectrophotometer. If the cells are viable, the conversion of MTT into purple-colored formazan was seen at an absorbance maximum of 570 nm. If the cells die, they lose the capacity to convert MTT, which serves as a useful assay for viable cells. The formazan product which is formed usually gets deposited as an insoluble precipitate and thus solubilization should be done before recording absorbance. This conversion is typically time-dependent [94].
In vitro scratch assay
The in vitro scratch assay is a simple, efficient technique to determine the in vitro cell migration in a wound healing process. Cell migration is an essential function in various methods like embryogenesis, angiogenesis, wound healing and immune defense. In this assay, scratch is usually created in cell monolayer once confluence reached 90% and capturing of images at the beginning and at appropriate time intervals during cell migration to close the scratch and compare models to quantify the migration rate of cells. In monolayer culture, 12 well plates were placed with the cell density of 1 × 105 cells/well. The media was maintained at a temperature of 37 °C and 5% CO2 atmosphere. The cultures with the sterilized 200 µl tip were scratched and then the culture media were freshly replaced. Reagents used for culturing the cells were Dulbecco's modified eagle medium (DMEM), fetal bovine serum, phosphate-buffered saline (PBS), glutaraldehyde, ethanol and crystal violet. This assay is useful for active cell migration by stimulating in vivo wound healing [95].
where A0 is the scratch area at 0 h and At is the scratch area at the designated time.
Kumar LV and his coworkers have performed an in vitro scratch assay to determine wound healing based on Hu et al.'s (2017) method with slight modification. 3T3 cells grown in DMEM supplemented with 10% FBS have been taken for this assay and seeded into 24-well plates at a density of 0.05 × 106 and grown for 24 h until they reach 80% confluence. Once the confluence has been achieved, scratches were made with the help of 1 ml pipette tips by keeping the same medium to remove the detached cells. The wells were again replenished with fresh medium along with each collagen peptide (0.2 mg/mL). The cells were then allowed to grow for another 48 h and washed twice using 1X phosphate buffer solution. The cells were fixed with 3.7% paraformaldehyde for 30 min and then stained with 1% crystal violet in 2% ethanol for 30 min. The gap distance of the scratch was then measured using an ORCA-ER CCD camera (Nikon Eclipse TE200: Nikon, Tokyo, Japan) connected to the phase-contrast microscope (Olympus CX41, Olympus Corporation, Japan) [96].
Boyden chamber assay
Transwell migration assay is another name of the Boyden chamber assay. It consists of two compartments; the porous membrane's influence detached the upper and lower compartments, beneath the membrane chemical cues were placed and over the layer, cells have been placed. Through the membrane, the migration of the cells occurs, which are then analyzed for the rate of movement. The most common cell lines used were pro-myelocytic cell line, Human Leukemia-60 (HL-60) cells. Transwell migration assay is usually used to measure cell migration predominantly. Its primary function is mimicking the in vivo DW microenvironment. Cell lines were suspended in Iscove's Modified Dulbecco's Medium (IMDM), which contains 106cells/ml. Beneath the transwell insert, initially load the 600 µl of IMDM, which comprised 8 mm pores of transparent polyethylene terephthalate (PET) membrane. Over the 24 well plate transwell insert, the cell lines were placed. Specific parameters were followed where the mediaIscove's Modified Dulbecco's Medium (IMDM) were prepared with penicillin–streptomycin and 20% Foetal Bovine Serum (FBS) at 105—106 cells/mL maintaining T-75 flasks (Becton, Dickinson) [97].
Tube formation assay
The tube formation assay can be used for determining vascular formation and cellular differentiation. The most commonly used cell lines were HUVEC. Usually, the plates were covered with the matrigel maintaining the temperature of 37 °C, then incubated for about 30 min and liquefied on ice. With the development of the tube constitution on the matrigel, the effect was observed. At 5 × 104 cells per well, the cell lines can be seeded in the 96 well plates, the new media can be consequently added. After 6 hr, the tubular network formation can be captured, the entire length of the formed tubular network can be assessed for angiogenesis. Quantification can be done by adopting Image-Pro Plus version 6.0. [98].
In vitro biodegradation
Degradation rate and release profile are critical parameters to consider when designing biodegradable skin scaffolds. The incorporated bioactive factor is released at a faster rate as the scaffold degrades faster. The rate of deterioration of the skin scaffold, on the other hand, should be equal to the rate of healing of the wounded skin. This study was performed for the determination of the degradation of scaffolds since stability plays a vital role. Biodegradation of the hydrogel scaffolds was usually done with a 0.1% lysozyme solution in PBS at specific time intervals. Small sections of the scaffold were weighed (Wi) and incubated in lysozyme at 37 °C. Scaffolds were removed at regular intervals and rinsed thoroughly with de-ionized water to remove the ions absorbed on the surface. The scaffolds were periodically removed, washed and lyophilized before measuring the dry weight (Wt). The biodegradation was calculated as the ratio of weight decrease to the initial dry weight [99].
An investigation of the konjac glucomannan-keratin hydrogel scaffold loaded with Avena sativa extracts for DW healing was carried out by Veerasubramanian et al. the scaffolds lost approximately 65% of their mass by the fifth week. The biodegradation data suggests that the material can remain mechanically stable in its short-term application as a wound dressing [99]. A figure depicting all the in vitro studies have been given in Fig. 6.
Fig. 6.
Scaffolds and their in vitro studies
Conclusion and Future perspectives
Despite recent advancements, owing to their multifactorial etiology, handling DWs to heal promptly remains a problem. Therefore, it is necessary to integrate multiple therapeutic approaches to facilitate the process of healing. Various studies have been carried out evaluating the practicability of utilizing specific materials for the delivery of specific bioactive agents, including small molecules, GFs, siRNAs, or cells. The scaffold is a prominent area in the field of tissue regeneration. Current significant requirements in chronic wound healing have led to the development of various biomaterial scaffolds. It mimics the ECM and facilitates the 3D structure to promote cell proliferation, differentiation and progression. According to the complexity of wound healing, several bioactive agents need to be administered that possess specific properties or need various release profiles, requiring the development of new composite scaffolds that can handle each bioactive ingredient while preserving the optimum wound microenvironment. Already there are plenty of scaffold materials and therapeutic agents to select from. But finding the correct or better combination of scaffolding material and therapeutic agent can be a challenge as it involves a profound understanding of numerous disciplines. Likewise, exploring the usage of combinations in various areas, e.g., GFs and cell-based tissue constructs may provide clinicians with more valuable evidence. Considerable research using different approaches was addressed in this study, but less explored is the direct comparison of effectiveness among the products currently available on the market. Moreover, while assessing new experimental methodologies, the inclusion of presently available marketed products as control would yield reliable results compared to the standard of care in the future. With the progress made in our theoretical insight of the DWs mechanism at the cellular and molecular levels, it is more apparent that a need to address numerous causes (infection, oxidative stress and chronic inflammation, etc.) that are responsible for weakening the smooth functioning of the key players (various cell types, GFs, CKs and ECM, etc.) engaged throughout the wound healing process and the recent advances in tissue engineering scaffold research hold great promise to improve the quality of life and prevent the amputation in patients with diabetes.
Acknowledgement
The authors would like to thank the Department of Science and Technology Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions (DST-FIST), New Delhi for their infrastructure support to our department. The author Miss Vyshnavi Tallapaneni would like to thank the Indian Council of Medical Research, New Delhi for awarding senior research fellowship to carry out the studies and their support towards the research.
Compliance with ethical standards
Conflict of interest
The authors declare that there are no conflicts of interest involved in this review. The authors alone are responsible for the content and writing of the paper.
Ethical statement
This article does not contain any studies with human or animal subjects performed by any of the authors.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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