Recent advances in decellularized biomaterials for wound healing

Abstract

The skin is one of the most essential organs in the human body, interacting with the external environment and shielding the body from diseases and excessive water loss. Thus, the loss of the integrity of large portions of the skin due to injury and illness may lead to significant disabilities and even death. Decellularized biomaterials derived from the extracellular matrix of tissues and organs are natural biomaterials with large quantities of bioactive macromolecules and peptides, which possess excellent physical structures and sophisticated biomolecules, and thus, promote wound healing and skin regeneration. Here, we highlighted the applications of decellularized materials in wound repair. First, the wound-healing process was reviewed. Second, we elucidated the mechanisms of several extracellular matrix constitutes in facilitating wound healing. Third, the major categories of decellularized materials in the treatment of cutaneous wounds in numerous preclinical models and over decades of clinical practice were elaborated. Finally, we discussed the current hurdles in the field and anticipated the future challenges and novel avenues for research on decellularized biomaterials-based wound treatment.

Graphical abstract

Classification, preparation, and mechanism of decellularized biomaterials for wound repair. Various types of decellularized biomaterials in the forms of hydrogel, 3D printed, fibrous, scaffolds and nanoparticles are produced using different processing methods. According to the source, decellularized biomaterials could be classified into the acellular dermal matrix (ADM), dcellularized adipose matrix (DAM), acellular amniotic membrane (AAM), decellularized small intestine submucosa matrix (SIS) and so forth.

1. Introduction

The skin is the largest and one of the most essential organs in the human body, and it protects the body from external damage and provides the functions of thermoregulation, sensation, and metabolism. Thus, the loss of the integrity of large portions of the skin due to burns and traumatic injuries may lead to the significant formation of keloids or hypertrophic scars, resulting in significant functional or aesthetic disadvantages and even death [1]. Additionally, diabetic, pressure, venous, and arterial ulcers, resulting in chronic nonhealing cutaneous wounds, remain a major medical challenge that burdens patients, their families, and the healthcare system [2,3]. Skin self-grafts, flaps, free flaps, or other reconstructive plastic surgery techniques are mainly adopted clinically [4]. Nevertheless, skin wound treatment may deteriorate due to a growing elderly population and an increasing morbidity rate [5]. Consequently, there is an urgent need for advanced biomaterial development to effectively and rapidly repair cutaneous wounds, thus, inducing scar-free skin regeneration and improving the injured skin functional restoration.

Recently, the increased tissue engineering and regenerative medicine development has provided good prospects and resulted in achievements in laboratory research and the clinical applications of bioengineered constructions for the repair of skin defects. Nevertheless, early tissue engineering solutions for skin regeneration mainly focus on growth factors (GFs), cells, or scaffolds, thus, failing to perfect the wound healing process, with poor vascularization, abnormal scar formation, and sensory loss [6]. The extracellular matrix (ECM) is the noncellular component of tissues that provides a scaffold for cells and is vital for controlling development, homeostasis, inflammation, and repair [7]. Therefore, research on simulated ECM production has gradually shifted toward creating an environment that can facilitate endogenous tissue repair.

An ideal scaffold for tissue repair should possess good biocompatibility, robust bioactivity, suitable degradation, and appropriate mechanical properties. The decellularized ECM (dECM), derived from autologous, allogeneic, and xenogenic tissues, is a three-dimensional natural scaffold that has the cellular components removed without altering its intrinsic tissue structure. The dECM is notable for its outstanding biological activity, fine biocompatibility, non-immunogenicity, and as a comprehensive raw material source [8], rendering dECM promising in translational medicine [9]. For instance, abundant natural bioactive components with excellent pro-angiogenic activities within dECM biomaterials are necessary for tissue regeneration [10]. Recently, dECM has been shown to prompt heart [11], kidney [12], liver [13], lung [14], and many other tissue types and organ repair in tissue engineering and regenerative medicine [15]. Additionally, many studies have modified dECM with crosslinking and functional additives to enhance biological activities [[16], [17], [18], [19], [20]]. Considering the applicable wound-healing requirements, many biomolecules, nanoparticles, and drugs have been utilized to engineer new generations of ECM-based biomaterials, which can stimulate a specific wound-healing stage or event to facilitate wound healing [21].

In this study, we aimed to review the skin wound healing processes and elucidate the detailed functions of several ECM components in repairing cutaneous wounds. Moreover, the advances in and effects of various dECM biomaterials, including modified dECM-based materials, on cutaneous wound healing were elaborated. Sound innovation ecosystems create breakthroughs for the bench-to-beside translation of dECM biomaterials, and, the related products based on the dECM biomaterials have also been highlighted. Finally, the current hurdles in the field were discussed, and the future challenges and novel avenues for research on dECM biomaterials-based wound treatment were anticipated.

2. Skin wound healing

Skin wound healing is a complex process, and it has traditionally been divided into four overlapping phases, namely, hemostasis, inflammation, proliferation, and remodeling, through which every wound heals normally [22,23]. Post-injury, due to vessel rupture, subendothelial collagen is exposed to platelets, which leads to the accumulation and activation of the coagulation cascade [24,25]. Immediately, hemostasis initiates the wound healing processes, with the damaged vessels being closed by blood clots, and phagocytes being recruited into the wound site to clear out the dead cells and microorganisms [23]. Inflammation follows thereafter. The cytokines that are released by various cells recruit neutrophils and monocytes to the wound bed, culminating in the conversion of the monocytes into macrophages, which are regarded as the master mediator of the wound healing inflammatory phase [26,27]. The cardinal features of the proliferative phase are re-epithelialization, angiogenesis, and fibroplasia. During this phase, the fibroblasts start to synthesize new collagen and glycosaminoglycans to form the wound core. Meanwhile, the proliferation and migration of endothelial cells support angiogenesis [28,29]. In the remodeling phases, namely, the maturation and re-epithelialization phases, the cells (i.e., keratinocytes, epithelial stem cells, and fibroblasts) proliferate and migrate close to the wound bed.

Although the detailed wound-healing processes have been elucidated, the outcome of cutaneous wound healing in adult mammals is affected by multiple internal and external factors [30]. Foreign matter that is introduced deeply into the wound during certain injuries may cause chronic inflammatory responses which delay wound healing and sometimes lead to granuloma and abscess formation. Pathogenic bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, and some Proteus, Clostridium, and Coliform species, can be detrimental to the healing process [31]. The detailed factors that need to be controlled and managed effectively, including preventing infection, optimizing exudate control, and removing foreign bodies, and the related intervention strategies, which are outside the scope of this paper, have been reviewed by Krasner [32]. The intervention strategies that are based on ECM biomaterials provide therapeutical benefits in nearly all stages of the wound healing process.

3. Effects of the extracellular matrix components on skin wound healing

The ECM is essential for maintaining tissue homeostasis, and thus, for the survival of multicellular organisms. It provides the integrities of the tissues and organs by equipping them with a three-dimensional spatial structure and bioactive GFs and cytokines to facilitate multidirectional communications among various cells [[33], [34], [35], [36], [37], [38]]. It also provides physical support to the skin and actively regulates cell function by controlling the biochemical gradients, cell density, spatial organization, and attachment ligands. Murphy-Ullrich and Sage showed that the cutaneous ECM mainly incorporates collagens, elastin, laminins, hyaluronic acid (HA), glycosaminoglycans (GAGs), proteoglycans, fibrillin, matricellular proteins [39], and molecules that are involved in ECM turnover, including matrix metalloproteinase and thrombospondin motifs [40]. Therefore, determining the detailed contributions of each ECM component to cutaneous wound repair is crucial to establish a solid foundation for cutaneous wound treatment.

3.1. The role of collagen in wound healing

Collagen is the main component of the skin ECM [41], making up 70–80% of the skin's dry weight [42], and it provides the tensile strength of the skin [43]. During skin wound healing, collagen, especially type I, III, V, VII, and XVII, has multifaceted and important roles. Collagen I can induce keratinocyte migration to initiate re-epithelialization and enhance matrix metalloproteinase expression in keratinocytes to facilitate matrix remodeling [44]. Additionally, collagen I possesses an immunomodulatory property, which may function positively in wound healing. A study by Karin in Nature revealed that a three-dimensional environment rich in dense collagen I fibers could induce the M2 macrophages' immunosuppressive functions [45], suggesting a possible beneficial therapeutic effect on diabetic foot ulcer (DFU) wound healing. Nevertheless, the deposition of type I collagen in hypertrophic wound scars is insufficient to induce sufficient immunomodulatory effects for normative wound healing [46]. Additionally, collagens, mainly type I and III, contain von Willebrand factor (vWF)-binding domains that regulate the bone morphogenetic protein, which promotes blood vessel development [47]. Apart from the functional vWF-binding domains, collagen V is relevant for modulating fibroblast proliferation and migration [47]. Furthermore, Collagen VII is the major component of anchoring fibrils at the dermal-epidermal junction, and when it is lacking, it can delay granulation tissue and epithelization formation [48]. The role of collagen VII in wound closure is mediated by two interconnected mechanisms. First, collagen VII promotes laminin-332 organization at the dermal-epithelial junction, thus, initiating the interaction between laminin-332 and integrin α6β4 and supporting keratinocyte-directed migration to facilitate skin re-epithelialization during the wound healing process [49]. Second, collagen VII supports dermal fibroblast migration and regulates the production of vast cytokines in the granulation tissue. Consequently, the loss of collagen VII results in delayed maturation of the granulation tissue and protracted inflammation, often leading to chronic and non-healing wounds [50,51]. Moreover, transmembrane collagen XVII, abundantly secreted in the keratinocytes during the re-epithelialization of acute wounds, is a highly dynamic modulator of proliferation and motility in the activated keratinocytes during epidermal regeneration, and it is also involved in the anchorage of the epidermis to the underlying basement membrane. As an important part of the hemidesmosome transmembrane component, collagen XVII modulates integrin-dependent keratinocyte migration via PI3K/Rac1 signaling and stabilizes the lamellipodia at the leading edge of re-epithelializing wounds [52]. In contrast, collagen XVIII overexpression negatively affects wound healing, since it slows skin repair, inhibits vascularization, and decreases myofibroblast density [53].

Considering the multifunctional effects of collagen in cutaneous wound healing, collagen dressings may encourage the deposition and organization of newly formed collagen, creating an environment that fosters healing, and giving it considerable potential as a biomaterial for cutaneous wound treatment [43]. Collagen-based biomaterials are usually formulated with bovine, avian, and porcine collagen to recruit and stimulate the wound-healing-related cells, including the macrophages and fibroblasts, during the healing cascade to enhance and influence the wound-healing process [54]. The application of collagen powders in the ulcerated area created an active local microenvironment that permitted suitable binding with fibronectin and increased fibroblast viability, subsequently, accelerating wound healing [55]. This was attributed to collagen's intrinsic biological functions and special physical properties. The powdered collagen enhanced biomolecular interaction and promoted three-dimensional bio-scaffolding formation for cell migration before granulation tissue formation [56]. In addition, the bio-scaffolding helped to hinder protease activity without affecting the performance of the GFs [57]. Qu et al. modified collagen membranes with keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) to repair skin wounds. The results from the in vitro studies confirmed that these GFs modified the collagen membranes and enhanced cellular proliferation and migration. Additionally, a wound-healing model with transplanted modified collagen membranes exhibited improved blood flow rates and epidermal organization [58]. Moreover, Gottrup et al. found a unique application for collagen, where collagen/oxidized regenerated cellulose (ORC)/silver therapy was investigated in a randomized controlled trial to treat DFUs. They found that 79% of patients in the collagen/ORC/silver group were positive responders when compared with 43% of those in the control group. The collagen/ORC/silver compound material normalized the wound microenvironment and protected against infection, thus, improving wound healing [59].

3.2. The role of laminin in wound healing

Laminin (LM) is a major constituent of the basement membrane that separates the epithelium, mesothelium, and endothelium from the connective tissue, and the major LMs in adult skin are LM-332 [60], LM-311, and LM-511 [61], which are all mainly contributed by keratinocytes [62]. Laminin supports dermal hair papilla development and hair growth [63]. Data from transgenic mice and human mutations in the LM5 gene encoding the laminin α5 chain suggest that LM-511 stabilizes epidermal adhesion and mediates epidermal-dermal communications, prompting skin repair [62,64]. Furthermore, LM has been shown to play a critical role in re-epithelialization and angiogenesis during wound healing [65,66].

Several studies have documented the LM-derived biomaterial applications in treating cutaneous wounds. Caissie synthesized a chitosan-conjugated active LM cell membrane and found that the membrane can promote keratinocyte adhesion and spreading in vitro [67]. The membrane, carrying the keratinocytes, was transplanted onto the exposed muscle fascia on nude mice's backs. After 3 ​d, the keratinocytes in the membrane migrated towards the muscle fascia and formed a stratified epidermis-like structure on the fascia, verifying that the chitosan-LM membrane is useful as a therapeutic formulation and cell delivery system to the wound bed. The perfect cytocompatibility of LM-based biomaterials is not confined to keratinocytes, but is a general feature of multiple cell types, including dermal and epidermal cells. The cellular activities induced by the LM materials have been applied in diabetic wounds, which have been confirmed to be integrin-mediated [68]. Furthermore, the numerous heparin-binding domains (HBDs) in LM can be used as GF reservoirs to enhance the biological properties of biomaterials. The GFs in fibrin matrices are inefficacious at treating cutaneous wounds; thus, the incorporation of HBDs in LM into the fibrin matrices through a covalent bond can improve the retention of the GFs, especially the efficacy of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), and promote wound healing in vivo [69].

3.3. The role of elastin in wound healing

Elastin accounts for up to 2–4% of the skin's dry weight and is a protein that is essential for skin ECM elasticity [70]. In addition to its structural and mechanical properties, inherent cell signaling pathways that are activated by elastin and elastin-derived peptides promote various cellular responses, including chemotaxis, cell attachment, proliferation, and differentiation [71]. These biological effects could result from the interactions between the elastin fragments and the elastin/LM receptor and integrin binding [72]. Considering that sufficient quantities of elastin are not generated during severe cutaneous injuries [73], the effective delivery system of elastin into the organism is highly advantageous. Vasconcelos exploited a silk-elastin dressing to deliver elastin for wound repair [74]. Dermal burn healing experiments using human skin equivalents have also shown that the application of silk fibroin/elastin scaffolds containing a higher amount of elastin accelerates re-epithelialization and wound closure. A silk-elastin sponge promoted fibroblast and macrophage migration and induced collagen production from fibroblasts, thereby accelerating granulation tissue formation in diabetic mice [75]. Additionally, elastin hydrogel modification with Arg-Gly-Asp peptides promoted vascularization in vivo [76]. The addition of elastin to a nanofibrous gelatin and cellulose acetate blend altered the fiber structure from a straight to ribbon-like morphology, decreased the scaffold degradation rate, and approached the swelling ratio of commercially available skin substitutes [77]. Although biocompatible elastin has been confirmed to be an ideal alternative for wound dressings, future studies should investigate how these constructs aid the de novo synthesis of elastin in vivo.

3.4. The role of fibrillin in wound healing

Along with elastin, fibrillin can provide the skin with elasticity. For mature skin homeostasis, fibrillin-1 is the predominant form, and fibrillin-2 is transiently present during development and upregulated during the wound healing process [78]. Many studies have demonstrated that fibrillin is involved in wound healing and tissue repair, and plays an irreplaceable role in regenerating and repairing chronic refractory wounds [79]. The fibrillin interactome establishes the microfibrils as a vital hub for upholding dermal homeostasis [80] and provides a temporary extracellular medium to guide the cells to migrate toward the wounds and promote the growth and proliferation of the cells within the wounds to form granulation tissue [79]. Moreover, it has been verified that fibrin nanofibers not only accelerated wound closure but also significantly improve tissue recovery and restore dermal and epidermal microstructures, skin appendages, and adipose tissue [81]. Demidova-Rice et al. manufactured a peptide from fragments of tenascin X and fibrillin 1 (comb1) to repair dermal wounds in mice [82]. Hematoxylin and eosin and CD31 immunofluorescence staining showed an increase in the number of blood vessels in the wound beds, indicating that comb1 leads to the improvement of wound vascularization. These studies indicate that the fabrication of fibrillin-based biomaterials could enhance wound healing and be applied in regenerative medicine.

3.5. The role of polysaccharides in wound healing

The major polysaccharides of ECM are glycosaminoglycan (GAG) and Hyaluronic acid (HA). Polysaccharides directly interact with ECM proteins via glycosaminoglycan (GAG) chains and their protein cores to regulate skin architecture. Polysaccharides are differentially expressed during skin wound repair and scar development. They are expressed at high levels in murine skin wound repair tissues [83]. Similarly, during the regeneration of injured skin, polysaccharides were highly expressed in the papillary layer and bound to the keratin-forming cells to fulfill dermal regeneration. Subsequently, the polysaccharides gradually disappeared. Among the polysaccharides, GAGs and hyaluronic acid, as the two abundant components in the skin ECM microenvironment, are discussed below.

3.5.1. Glycosaminoglycans

Glycosaminoglycans are anionic polysaccharides that consist of repeating disaccharide units and are formed by uranic acid and hexosamine [84]. Physiologically, they are present either in a soluble form or covalently bound to a core protein like PGs. They are vital participants in skin wound repair [84]. During the proliferation phase of wound healing, GAG-peptide fragments that are released during protease-mediated PG degradation in the wounds can bind with cationic proteins (e.g., elastase and cathepsin G) to inhibit their associated activities, while also binding with various proteinase inhibitors to increase their related activities. As co-receptors for GFs, GAGs can mobilize heparin-binding proteins from ECM reservoirs. Crucially, GAG-chemokine interactions can function as regulators of chemokines in vivo by influencing their localization, presentation, and density, and, thus, affecting the signaling properties. Therefore, GAG-based materials may modulate chemokine concentrations within tissues to therapeutically attenuate inflammation in chronic wounds [85]. Lohmann et al. customized modular hydrogels based on terminally functionalized star polyethylene glycol and GAG heparin derivatives. When compared with the standard-of-care product Promogran, an FDA-approved hydrogel-based wound dressing for the management of chronic wounds [86], this modular hydrogel effectively reduced inflammatory chemokine monocyte chemical attractant protein 1, induced interleukin-8, macrophage inflammatory protein 1 a, and macrophage inflammatory protein-1 b in the chronic wound fluid, and, subsequently, reduced human polymorphonuclear neutrophil and monocyte migration [87].

3.5.2. Hyaluronic acid

Hyaluronic acid is the other major structural extracellular GAG and is abundant in the ECM of various tissues and organs. It possesses a high capacity to bind water and is a space-filler that lends tautness to the skin. Additionally, HA regulates skin homeostasis and regeneration through multiple mechanisms, including interactions with its receptor CD44. Furthermore, the numerous carboxyl and hydroxyl highly hydrophilic groups in the HA structure enable it to perform exudate absorption and enhance cell adhesion [88]. The increase in HA production during tissue remodeling phases is associated with the wound repair process [89,90]. After tissue injury, small HA fragments are produced from high molecular mass HA because of hyaluronidases and the oxidation intervention of potential functional roles, initiating innate immune responses through Toll-like receptors [91]. Despite their simple primary structure, the wide-ranging functions of HA fragments in wound healing are size-specific; intact HA (high molecular weight HA) tends to exert anti-inflammatory effects, whereas mid-sized and small fragments (oligosaccharides) have proinflammatory or variable properties depending upon the cell-surface receptor binding HA types [92]. Nevertheless, in human chronic dermal wounds and rats’ diabetic wounds, the HA amount is drastically decreased due to the down-regulated biosynthesis and up-regulated degradation speed [93], possibly contributing to the refractory property of chronic wounds. Shin et al. found that electrospun HA-poly (lactic-co-glycolic) acid scaffolds incorporated with the pro-angiogenic factor, epigallocatechin-3-O-gallate, significantly accelerated wound healing rates in diabetic rats [94]. Similarly, a randomized control trial found that autologous fibroblast HA dressings for DFUs accelerated wound healing by 12 ​d on average when compared with that of a non-adherent foam dressing [95].

3.6. The role of growth factors encapsulated in ECM in wound healing

The ECM is a known repository of various GFs, including fibroblast growth factor (FGF), endothelial growth factor (EGF), transforming growth factor (TGF), VEGF, and PDGF [96]. The ECM also encapsulates GFs to produce growth factor gradients that regulate developmental pattern formation and skin wound healing. Due to the binding of GFs to ECM components, they are interdependent [97]. These GFs bind to ECM proteins and are gradually released with ECM degradation, which stimulates and regulates cellular migration, the proliferation and differentiation involved in tissue regeneration and repair, tissue repair, reconstruction, and angiogenesis. These GFs provide the ECM with various properties, making it an ideal bioactive material for tissue and organ repair and regeneration.

The FGFs contribute to wound healing by enhancing angiogenesis, cell migration, and proliferation. Specifically, FGF-2, which is basic FGF, promotes neovascularization and thickens both the dermis and epidermis. It also prevents wound contraction by inhibiting α-smooth muscle actin filaments and leads to reduced fibrosis by preventing fibroblast differentiation into myofibroblasts [98]. The EGF mainly affects keratinocytes and fibroblasts and increases their migration and proliferation. Moreover, it promotes angiogenesis and epithelization and can trigger GF secretion by the fibroblasts, thus, accelerating wound healing [99]. The TGF-βs, subclassified as TGF-β1, TGF-β2, and TGF-β3, are needed for proper wound healing. Promising results of scarless wound healing in animal models have demonstrated that embryonic wounds can heal perfectly with no scars, which is attributed to TGF-β3 upregulation and TGFβ1 and TGF-β2 downregulation [100]. The TGF-β1, connective tissue growth factor, and other autocrine factors can induce the mesenchymal stem cells, epithelial cells, endothelial cells, and fibrocytes to differentiate into myofibroblasts, eventually leading to scar formation and fibrosis. Therefore, a lower level of TGF-β1 could prevent myofibroblast production, thus, facilitating skin regeneration [101]. Then, VEGF has been recognized as an important intermediary of vascular permeability, angiogenesis, and lymph angiogenesis. In addition, VEGF encourages collagen deposition and epithelialization in wound healing [102]. The production of VEGF is induced by hypoxia as it binds to the receptors on the endothelial cells, which further directs vessel growth. Nonetheless, their use is limited because of their short half-life, low availability, and high cost [103]. Moreover, PDGF is the first recombinant growth factor to be approved for topical application to accelerate wound closure [104]. In the beginning stage of wound healing, PDGF is a chemo-attractant for fibroblast, neutrophil, and monocyte migration to the site of injury. It also encourages new ECM production and is a mitogen for fibroblasts. Furthermore, PDGF stimulates fibroblast differentiation into myofibroblasts, facilitating the contraction of the collagen matrices and wounds in the proliferation stage [105].

4. Application of decellularized biomaterials in the treatment of skin wound healing

Decellularized biomaterials that are derived from human and animal tissues, such as the pericardium, peritoneum, and amniotic membrane, have traditionally been used to facilitate skin wound healing [[106], [107], [108]]. Among them, the acellular dermal matrix (ADM), acellular amniotic membrane (AAM), and decellularized small intestinal submucosa (DSIS) are representative biomaterials that have been commercialized and extensively applied clinically [108,109]. In the following sections, the improved performance of decellularized biomaterials for skin wound healing was extensively reviewed (Fig. 1), along with their shortcomings and challenges. A summary of the biomaterials that control wound healing is given in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6.

Fig. 1.

Decellularized dermal matrix biomaterials promotes wound healing. (A) Representative H&E-stained histological images of the repaired skin implanted with ADM scaffolds derived from one-day old mouse and 20-weeks old mouse. E: edge of wound; R: new born tissue. Scale bars: 20 ​μm. (Reprinted with permission from Chu J [121]). (B) 3D images of collagen morphology in new born dermis within 7 days, treated by ADM-1D and ADM-20 ​W, respectively, imaged by a 63 ​× ​water-immersion objective. (Reprinted with permission from Chu J [121]). (C) The thickness of new born dermal layer (D) within 7 days treated by ADM-1D and ADM-20 ​W, respectively, imaged by a 20 ​× ​objective. (Reprinted with permission from Chu J [121]).

Table 1.

Acellular Dermal Matrix with their origin and effectivity.

Decellularized materials	Authors, Year, Country	Study Design	sources of materials	Results	Conclusion
Acellular Dermal Matrix	Nikhil Sobti et al. [111], 2016, USA	Clinical study	human skin	233 patients underwent matrix-based breast reconstruction, and 11 developed surgical-site infection. There were no statistical differences between FlexHD and AlloDerm application in infection rate, rates of seroma, hematoma, and delayed wound healing.	No statistical difference in infection rate or any other clinical endpoint was observed between AlloDerm and FlexHD in immediate implant-based breast reconstruction.
Acellular Dermal Matrix	Youn Hwan Kim et al. [114], 2016, USA	Clinical study	human skin	This study found the acellular human dermis to be an effective graft material to replace existing surgery methods in lower limb reconstructing. (1) At six months, following application of acellular human allodermis and split-thickness skin graft, contracture and complications were not observed. (2) At 24 months after surgery, a good aesthetic outcome was achieved. (3) full-thickness skin or soft tissue.	Acellular human dermis is effective and safe in lower limb reconstruction. The use of the acellular human dermis in operations could help avoid the flap harvest process, minimize scarring in the flap donor area, enhance skin elasticity compared with other conventional thick skin alternatives, reduce infection rates, and shorten engraftment time.
Acellular Dermal Matrix	Xue Han et al. [122], 2016, China	Development and characterization study	Mouse fetal skin	In this study, two types of ADM from skin ECM were adopted to explore the potential to achieve scarless wound healing in adult mice. Compared to ADM-20 ​W, ADM-1D provided a more favorable influence on the re-establishment of the epidermis, collagen density, and reorganization of neo dermis, to a degree approaching normal uninjured adult dermal tissue.	Biomechanical stiffness of fetal ADM might be one of the crucial determinants for potential adult scarless wound healing.
Acellular Dermal Matrix	Álvaro-Afonso FJ et al. [124], 2020, Spain	Clinical study	human skin	In treating DFUs, the best healing rates at 12 weeks were accomplished with dermal cellular substitutes (Epifix®, 100% and Amnioband®, 85%) and with dermal acellular substitutes (Allopatch®, 80% and Hyalograft®, 78.8%).	ADM used in conjunction with standard care appear to improve the healing rates of DFUs.
Acellular Dermal Matrix	M. Ayaz et al. [125], 2021, Iran	Clinical study	human skin	The results showed excellent graft take, good elasticity, acceptable thickness, and little contracture and scarring according to fix surgeon assessment in 6 patients. Graft rejection happened only in one patient with chronic electrical injury	The combination of AlloDerm™ and thin split thickness skin grafting constitutes a cost-effective and favorable option for the treatment of deep burn wounds.
Acellular Dermal Matrix	Reyzelman A et al. [126], 2009, USA	Clinical study	human skin	ADM was compared with standard of care in treating healed diabetic foot ulcers. Complete healing and mean healing time in ADM group were 69.6% and 5.7 weeks, and in standard of care group 46.2% and 6.8 weeks. The proportion of healed ulcers between the groups was statistically significant.	This study supports the use of single-application ADM therapy as an effective treatment of diabetic, neuropathic ulcers.

Table 2.

Decellularized adipose tissue with their origin and effectivity.

Decellularized materials	Authors, Year, Country	Study Design	sources of materials	Results	Conclusion
Decellularized adipose tissue	Omidi E et al. [131], 2014, Canada	Development and characterization study	human adipose tissue	DAT materials derived from various adipose depots promoted adipocyte infiltration and proliferation, and had similar large deformation characteristics under physiological loading after implantation.	DAT materials possess perfect mechanical characteristics.
Decellularized adipose tissue	Turner AE et al. [132], 2012, Canada	Development and characterization study	human adipose tissue	ASCs cultured on the DAT microcarriers elevated levels of adipogenic markers. The DAT microcarriers exhibited no immunogenicity or cytotoxicity, with supporting cellular infiltration and tissue remodeling. Pre-seeding the DAT microcarriers with rat ASCs enhanced cellularity and angiogenesis within the implant region.	The microcarriers fabricated from solubilized DAT provided a naturally adipo-inductive substrate.
Decellularized adipose tissue	Giatsidis G et al. [133], 2019, USA	Clinical study	human adipose tissue	Combined use of the allograft adipose matrix and noninvasive skin preconditioning significantly improved long-term volume retention (50–80% higher at a 12-week follow-up) and histologic quality of reconstructed tissues compared with standard of care (autologous adipose grafts). The components of the allograft adipose matrix supported adipogenesis and angiogenesis.	The synergistic use of the allograft adipose matrix and noninvasive tissue preconditioning provides an effective solution for improving fat grafting. These strategies could establish the basis for a novel therapeutic paradigm in reconstructive surgery.
Decellularized adipose tissue	Kokai LE et al. [134], 2019, USA	Development and characterization study	human adipose tissue	In vitro, adipose-derived stem cells cultured on allograft adipose matrix underwent adipogenesis in the absence of media-based cues. In vivo, animal modeling showed vasculature formation followed by perilipin A-positive tissue segments.	Subcutaneous implantation of allograft adipose matrix laden with retained angiogenic and adipogenic factors served as an inductive scaffold for sustaining adipogenesis.
Decellularized adipose tissue	Lu Q et al. [135], 2014, Singapore	Development and characterization study	human adipose tissue	In vitro, heparinized -DAT displayed stronger bFGF binding and controlled release abilities than non-heparinized DAT. When transplanted in vivo, Histology and gene expression analysis revealed that majority of the Hep-DAT scaffolds were infiltrated with host-derived adipose tissues that possessed similar adipogenic and inflammatory gene expression as endogenous adipose tissues.	The first time demonstrated the potent in vivo adipogenic potential of a bFGF delivery system based on heparinized DAT. demonstrated that bFGF-binding Hep-DAT could be an efficient, biocompatible and injectable adipogenic system for in vivo adipose tissue engineering.
Decellularized adipose tissue	Su Hee Kim et al. [141], 2021, USA	Development and characterization study	human adipose tissue	DAT hydrogel mixture induced adipogenesis. The in vivo study at 12 weeks demonstrated that the tissue-engineered DAT hydrogel promoted angiogenesis and adipose tissue formation, and suppressed apoptosis.	The patient-specific elastic scaffolds/hydrogel mixtures/cell systems could be a good treatment modality to regenerate large patient-specific adipose tissues.
Decellularized adipose tissue	Yu C et al. [146], 2013, Canada	Development and characterization study	human adipose tissue	Adipose tissue- and heart tissue-derived dECM-based hydrogels were used to enhance adipogenic differentiation and angiogenesis.	The DAT-based foams induced a strong angiogenic response, promoted inflammatory cell migration and gradually resorbed over the course of 12 weeks, demonstrating potential as scaffolds for wound healing and soft tissue regeneration.
Decellularized adipose tissue	Xia Z et al. [155],vvv 2020, China	Development and characterization study	porcine adipose tissue	ASCs showed high rates of proliferation and adhered well to DAT. DAT up-regulated the expression of cell stem maintenance, and increased the secretion of growth factors.	The application of DAT promotes wound healing, and DAT combined with ASCs may be a promising material in adipose tissue engineering and regenerative medicine.

Table 3.

Decellularized fish skin with their origin and effectivity.

Decellularized materials	Authors, Year, Country	Study Design	sources of materials	Results	Conclusion
Decellularized fish skin	Mauer ES et al. [167],v 2021, USA	Clinical study	fish skin	Wounds closed by second-intention healing following the first fish skin application between 26 and 145 days. In cats, 1 or 2 FSGs were used, and the wounds of 3 of 4 cats healed completely by secondary intention. The wounds of 1 dog and 1 cat did not heal.	The main limitation of the present case series was the small sample size and retrospective nature of the study, with various wound management techniques used.
Decellularized fish skin	Dorweiler B et al. [168],v 2018, Germany	Clinical study	fish skin	In total 25 wounds were treated with localization at the level of the thigh (n ​= ​2), the distal calf (n ​= ​7), the forefoot (n ​= ​14) and the hand (n ​= ​2). The time to heal varied between 9 and 41 weeks and between 3 and 26 wound matrices were applied per wound. Interestingly, a reduction of analgesics intake was noted when the treatment with the Omega3 Wound matrix was initiated.	The novel Omega3 Wound matrix in this study represented an effective treatment option in 25 complicated wounds. Further studies are necessary to evaluate the impact of the wound matrix on stimulation of granulation tissue and re-epithelialization as well as the potential antinociceptive and analgetic effects.
Decellularized fish skin	Badois N et al. [169], 2019, France	Clinical study	fish skin	There were 21 patients included. The healing time was halved when using the acellular fish skin matrix, from 68 to 32 days on average. Acellular fish skin matrix reduced pain levels and local infection. The visual analogue pain scale (VAS) was ≥3 ​at five days and infection rate reduced from 60% to 0%.	A larger study including an overall cost estimation and an assessment on different wound types would be interesting, to better target the indications of the acellular fish skin matrix.

Table 4.

Acellular Human Amniotic Membrane with their origin and effectivity.

Decellularized materials	Authors, Year, Country	Study Design	sources of materials	Results	Conclusion
Acellular Human Amniotic Membrane	Wilshaw SP et al. [134], 2008, UK	Development and characterization study	amniotic membrane	AAM recruited low T-cells, high fibroblasts, macrophages, and endothelial cells. There was no evidence of calcification present within explanted AAM.	AAM was shown to be capable of supporting the attachment and proliferation of primary human fibroblasts and keratinocytes. Cell-seeded AAM has the potential for delivering autologous or allogeneic cells to treat a variety of conditions.
Acellular Human Amniotic Membrane	Kshersagar J et al. [178], 2018, India	Development and characterization study	amniotic membrane	At day 7, histologically the wounds treated with activated amnion were almost closed without scarring and showed well differentiated epidermis, proliferation of keratinocytes, hair follicles and basement membrane as compared to controls and silver nitrate gel dressings in a mouse.	The amnion is low immunogenic, anti-inflammatory, anti-fibrotic, anti-scaring, anti-microbial, easily accepted by patient, eliminate the risk of post-operative infection.

Table 5.

Other decellularized biomaterials with their origin and effectivity.

Decellularized materials	Authors, Year, Country	Study Design	sources of materials	Results	Conclusion
decellularized small intestine submucosa matrix	Moon SukKim et al. [185], 2005, Korea	Development and characterization study		The SIS sponges absorbed higher extent of exudation for wound than that covered with control. The dermal collagen in the wound regenerated at only SIS sponges treated wounds. The progress of granulous tissue formation was faster in SIS sponges.	The SIS sponges might be a potential material as a wound dressing
decellularized small intestine submucosa matrix	Fan MR et al. [183], 2014, China	Development and characterization study	porcine small intestinal submucosa	The release of VEGF and TGF-β gradually increased with time. The three-dimensional microarchitecture of SIS surface seems to be in favor of cell adherence, growth and migration.	The scaffold exhibited a porous nature and three-dimensional microarchitecture on its surface, which may facilitate cell adherence and growth. Furthermore, it also possesses the crucial bio inductive property owing to release.
decellularized small intestine submucosa matrix	Sarikaya A et al. [184], 2002, USA.	Development and characterization study	porcine small intestinal submucosa	DSIS and UBM contained antibacterial activity against both Gram-positive and Gram-negative bacteria.	porcine SIS discourages bacterial growth.
decellularized small intestine submucosa matrix	Guo X et al. [187], 2016, China	Development and characterization study	porcine small intestinal submucosa	SIS alone and MSC-seeded SIS were able to accelerate the burn wound closure by enhancing granulation tissue formation, increasing wound maturity, improving revascularization, and inducing the proliferation of neo-epidermal cells.	SIS and MSC-seeded SIS were able to repair the large and deep burn wounds and possessed positive effects to accelerate the wound closure in a rat model.
decellularized small intestine submucosa matrix	Rashtbar M et al. [188], 2018, Iran	Development and characterization study	ovine small intestinal submucosal	In DSIS with BMSCs group, epithelization was fast and had fully taken place at the subsequent time points. DSIS layer, as cell-free form with low substantially DNA content, accelerated healing of rat skin wound defects that was created at critical-size and full-thickness.	DSIS alone and in combination with BM-MSCs has the potential to be used as a wound graft material in skin regenerative medicine.
decellularized small intestine submucosa matrix	Parmaksiz M et al. [189], 2017, Turkey	Development and characterization study	ovine small intestinal submucosal	MSCs can preserve their viability and proliferate on DSIS for >2 weeks in culture. The wound models treated with DSIS membranes were completely closed by week 7 without significant differences in closure time; the open wound control was closed at ∼47% at this time point. The DSIS had less scarring at the end of the healing process.	DSIS membranes could be used as a suitable regenerative biomaterial, alone or as a scaffold material, to transplant MSCs in tissue engineering applications. Particularly, DSIS membranes accelerate healing of critical-sized, full thickness rat skin wound defects.
Decellularized Urinary Bladder Extracellular Matrix	Alvarez OM et al. [215], 2017, USA	Clinical study	porcine urinary bladder	In the UBM group, the incidence of wound healing at 12 and 16 weeks was 90% and 100%, respectively, compared with 33% and 83.3% in the control. The mean time to healing in the UBM-treated group was 62.4 days compared with 92.8 days in the control group.	Treatment of DFUs with a UBM could significantly reduce the healing time and reduce the rate of recurrence.
Decellularized Urinary Bladder Extracellular Matrix	Paige JT et al. [217], 2019, Korea	Development and characterization study	porcine urinary bladder	Wound tissue from diabetic subjects exhibited elevated M1:M2 scores compared with nondiabetic patients, suggesting a greater pro-inflammatory state prior to treatment.	UBM may assist in diabetic wound healing by restoring an inflammatory state similar to that of nondiabetic patients.

Table 6.

Biomaterials and available brands indicated in full-thickness wounds.

Decellularized materials	Product	Application	Ref.
Acellular Dermal Matrix	DermACELL AWM®	normal and Chronic Wounds	[221]
	GRAFTJACKET®RTM	normal and Chronic Wounds	[222]
	Integra®	normal and Chronic Wounds	[[223], [224], [225]]
	GRAFTJACKET ® Xpress	normal and Chronic Wounds	[226]
	Alloderm®	normal and Chronic Wounds	[[223], [224], [225]]
	Dermagraft®	Diabetic Foot Ulcers	[223,224,227]
	TransCyte™	Burn wounds	[[223], [224], [225]]
	OrCel™	Chronic Wounds	[223]
	PermaDerm™	Burn wounds	[223]
Decellularized adipose tissue	Integra® DRT	normal and Chronic Wounds	[159]
decellularized fish skin	Kerecis® Omega3 MicroGraft™	Normal and diabetic	[223]
Acellular Amniotic Membrane	SURFFIXX	Normal, chronic and infectious Wounds	[179]
	AmnioBand	Normal, chronic and infectious Wounds	[179]
	EpiFix	Normal, chronic and infectious Wounds	[179]
	Biovance	Normal, chronic and infectious Wounds	[179]
Decellularized small intestine submucosa matrix	OASIS Wound Matrix	Diabetic Foot Ulcers	[228]
Acellular fetal bovine dermis	PriMatrix	diabetic	[[223], [224], [225]]
Decellularized forestomach matrix	Endoform®	normal and Chronic Wounds	[214]

4.1. Acellular dermal matrix

The ADM consists of natural biomaterials that are derived from human and animal dermal tissues, with the complete removal of the cellular components while retaining the ECM spatial structure and chemical compositions [110,111]. The main components in the ECM are collagen fibers, elastin, PGs, and other natural biological components, which allow the cells to adhere and grow easily, and during the regeneration process, the ADM can be completely degraded and absorbed [112,113].

The ADM can be divided into allogeneic ADM (human-derived) and xenogeneic ADM (animal-derived), according to the species origin of the dermis. Allogeneic ADM is made from the donated homologous human dermis [114]. A meta-analysis showed that it significantly promoted wound healing and reduced complications in patients with diabetic wounds [115]. However, the limited availability of cadaver skin and the risk of disease transmission limit the application [116]. Xenogeneic ADM is mainly derived from porcine and bovine sources, and its structure is similar to that of allogeneic ADM [114,117]. When compared with allogeneic ADM, xenogeneic ADM is more cost-effective and is applied for skin defects [118]. Moreover, the differences in the molecular structure of the major histocompatibility complex between them result in severe inflammatory responses after xenogeneic ADM transplantation, when compared with those of allogeneic ADM [119]. For wound repair, the bioactive ADM containing GFs [96,98,99] can provide attachment points for epidermal cells, promote the basement membrane structure reorganization, and can facilitate endothelial cell and fibroblast migration to induce tissue growth and perform local reconstruction due to its unique pore structure (Fig. 1). Furthermore, the ADM matrix components are continuously degraded by collagenase action and eventually replaced by the newly formed ECM and migrated cells [120]. Chu et al. proposed human mesenchymal stem cell (MSC)-loaded ADM scaffolds as a novel therapeutic strategy for diabetic cutaneous wound healing [121]. The implantation of MSC-ADM scaffolds significantly enhanced angiogenesis and the re-epithelialization of newly formed skin. When compared with the ADM scaffold alone, the MSC- ADM scaffold reduced IL-1β and IL-6 expression in the wounds and increased the proportion of M2 macrophages. Seven days after transplantation, the MSC-containing ADM group recruited more CD31-positive cells and formed more blood vessels than the ADM-alone and MSC-alone groups (Fig. 1A). The effect of the stiffness of the ADM on wound healing has been explored [122]. The epidermis treated with a soft ADM (28.3 ​± ​8.5 ​kPa average elastic modulus) derived from one-day-old mice exhibited a higher collagen density than that with a stiff ADM (236.1 ​± ​36.6 ​kPa average elastic modulus) derived from 20-week-old mice. Additionally, the orientation and stiffness of the newly formed dermal tissues were favorable (Fig. 1B and C). These findings laid the foundation for the further exploration and application of ADM as a favorable candidate for chronic wounds. The long-term clinical effects of ADM products in burn treatment and post-burn scar repair were assessed [123]. After 4 years of follow-up, the ADM materials were found to accelerate wound healing, and the color, texture, thickness, aesthetic changes, and limb function of the healed skin were significantly improved when compared with those of the conventional treatment group.

Currently, there are several ADM products (Table 5) for human skin repair, such as the AlloDerm, GraftJacket, and SureDerm [124]. AlloDerm has been used for the coverage of deep burn wounds, with good elasticity, minor contracture, and little scarring [125]. GraftJacket is recommended as a treatment for diabetic wounds and wounds with blemished skin [126]. GraftJacket could reduce the average wound healing time of DFUs by as much as 40%–50% [108]. While commercialized decellularized skin substitutes have good therapeutic effects in promoting wound healing, they have yet to realize scarless regeneration.

Although homogeneous and heterogeneous ADM materials are widely used for cutaneous repair, there are still shortcomings. The small pore size and low porosity of the ADM are not conducive to cellular migration and proliferation [127], resulting in prolonged healing and scar formation and preventing subcutaneous fat layer regeneration. The major obstacles to human transplants are in assuring the lack of undetected diseases in homogenous ADM and the oligosaccharides containing the terminalα-Gal-(1–3)-β-Gal sequence in heterogenous ADM, which is synthesized in vivo by α-1,3-galactosyltransferase and has been identified as the major xenoactive antigen that is responsible for hyperacute rejection [128,129].

4.2. Decellularized adipose tissue

Decellularized adipose tissues (DATs) are obtained from human adipose tissue through a series of physical, chemical, and enzymatic treatments (Table 2). Human adipose tissue is an ideal alternative because it contains large amounts of bioactive ECM constituents that are similar to ADM [127,[130], [131], [132], [133], [134], [135]] and stem and progenitor cells that can promote adipogenesis and angiogenesis [[136], [137], [138]]. Furthermore, DATs do not contain xenogeneic antigens, such as α-Gal epitopes; thus, they possess minimal immunogenicity [139]. The greatest advantage of DAT over other decellularized matrices is its easy availability because human adipose tissue, which is usually removed and discarded during cosmetic surgery, can be obtained safely and efficiently by simple local anesthetic techniques [140,141]. The extracted tissue is washed with distilled water to remove blood, and then the mixture is homogenized and centrifuged. The upper oil-contained layer is then discarded, and the remaining gel-like suspension is washed with distilled water and decellularized to obtain the ECM (Fig. 2A).

Fig. 2.

Decellularized adipose tissue biomaterials promotes wound healing. (A) Schematic overview decellularized adipose tissue of the experimental methodology (Reprinted with permission from Allison E B Turner et al. [132]). (B) Human adipose-derived stem cells proliferate on allograft adipose matrix. (Above, left) Confocal micrograph of human adipose-derived stem cells stained with calcein AM on allograft adipose matrix (Above, right) (Below, left) Scanning electron microscopic image of allograft adipose matrix seeded with adipose-derived stem cells for 7 days, cultured in basal medium. (Below, right) Confocal imaging of adipose-derived stem cell–seeded matrices demonstrating lipid content (boron-dipyrromethene, green) after exposure to allograft adipose matrix at day 7 (original magnification, ​× ​20) (Reprinted with permission from Lauren E Kokai [134]). (B Masson trichrome showed the presence of adipocytes within material at week 24, confirmed by perilipin A immunofluorescence. (Reprinted with permission from Lauren E Kokai et al. [134]). (D) Immunofluorescence staining adipogenic markers. (Reprinted with permission from Su Hee Kim [141]). (E) Angiogenesis in a construct. Representative immunostaining of endothelial cells (ECs) and smooth muscle cells (SMCs) in each group 1 week after implantation. (Reprinted with permission from Su Hee Kim [141]). (F) GPDH enzymatic activity measured for DAT and gelatin microcarriers, as compared to non-induced (NI) and induced (I) TCPS controls, at 72 ​h, 7 days, and 14 days after inducing adipogenic differentiation (Reprinted with permission from Allison E B Turner et al. [132]).

Since human adipose tissue consists of loose connective tissue, adipose tissue removal by freezing (−80 ​°C) and thawing (37 ​°C) leads to severe changes in the matrix structure. Chemical methods, such as polar solvents like isopropyl alcohol are mainly used [142,143]. Because human adipose tissue is poorly dense and sodium dodecyl sulfate (SDS) causes dramatic swelling of the ECM and irreversible structural degradation, ethylenediaminetetraacetic acid is generally used instead [138,142]. However, the loss of the ECM components does not significantly affect the decellularized material's structural integrity [144]. Kayabolen et al. showed that treatment of adipose tissue with SDS for 2 ​h effectively removed the cellular and nucleic acid components while minimizing damage to the ECM structure. Nevertheless, lower protein content and looser gel properties could be observed with more prolonged SDS treatment [145].

Recent studies have designed different processes to isolate an intact ECM from adipose tissue and have fabricated various DAT scaffolds, such as foam [146], powders [147], microcarriers [148], injectable liquid [[149], [150], [151]], and bio-ink [152,153], for regenerative medicine, particularly for patients that require soft tissue regeneration and wound healing (Fig. 2A). Kim et al. [154] synthesized a thermosensitive hydrogel that is composed of soluble human DAT and methylcellulose, which can promote wound healing in the full-thickness circle wound model. The human DAT was solubilized using urea and guanidine buffer, and the extracted DAT was blended with methylcellulose to acquire the thermosensitive hydrogels. Then, CD29 and CD44 immunofluorescence staining revealed that the MSCs were homogeneously distributed and integrated within the hydrogels. The CD31 staining revealed a large density of newly formed blood micro-vessels. These results indicated that the hydrogels could provide a cell niche that improves the engraftment and survival of stem cells delivered to the wound and biological cues for accelerating wound regeneration (Fig. 2B). Additionally, Xia developed an injectable DAT scaffold material that is loaded with adipose-derived stem cells (ASCs) to reconstruct a model of full-thickness skin defects [155]. Sterile DAT powder was soaked in an ASC culture medium for 24 ​h to construct DAT-ASC injectable materials. The positive expression of CD31 in the DAT-ASC group was significantly higher than that in the control groups, suggesting that the DAT-ASCs can promote microvascular neovascularization. Moreover, the positive expression of proliferating cell nuclear antigen in the DAT-ASC group was significantly higher than that in the sham, DAT, and ASC groups, indicating that the DAT-ASC group prompted significant basal cell proliferation and has a high ability to enhance wound repair (Fig. 2B). Woo et al. successfully fabricated a TiO2-incorporated chitosan/DAT bilayer dressing to treat normal and infectious wounds [156]. The data showed a 33.9% and 69.58% reduction in bacterial penetration in viable Escherichia coli and Staphylococcus aureus, respectively. The Sprague-Dawley rat full-thickness circle wound model revealed that the newly formed epidermis became thin, and many appendages in the dermis were observed in the regenerated skin without severe skin contraction. Moreover, the CD31-positive endothelial cells and the micro-vessel density in the implanted wounds were increased at week 2 when compared with those of the control wounds. These results indicated that the bilayer dressing exhibited antibacterial activity and accelerated the re-epithelialization and creation of new blood vessels during wound healing [156]. The DAT microcarriers (1–2 ​mm in diameter) that were prepared by Yu et al. were injected into DAT porous foam using the electrospray method [146]. The in vivo data in a subcutaneous embedding model demonstrated that the inflammatory cells were infiltrated into the foams at the early stage and stabilized over time. At 8 and 12 weeks, mature adipocytes and functional blood vessels were visualized and reorganization of the collagen in the foams was evident (Fig. 2C–E). The porous DAT foam induced a stronger angiogenic response and promoted more extensive inflammatory cell infiltration in the host tissue than in the full form of the DAT scaffold, making the porous DAT foam more suitable for wound healing.

The unique ability of DAT to promote adipose tissue regeneration and revascularization has led to its widespread use in cell delivery, wound healing, and soft tissue regeneration [157,158]. The DAT expressed essential genes for cell stem maintenance, including OCT4 and SOX2, which stimulate and regulate stem cell migration and could increase the secretion of local GFs, such as VEGF, PDGF-bb, bFGF, hepatocyte growth factor, EGF, and FDGF, and the secretion gradually increased from 0 to 48 ​h. These GFs are released gradually with ECM degradation, which stimulates and regulates cellular migration, proliferation, and differentiation, promoting tissue repair, reconstruction, and angiogenesis (Fig. 2F [[155], [156], [157], [158]]). Commercial decellularization products are also currently undergoing extensive research, for instance, Integra® DRT [159].

4.3. Decellularized fish skin

Recently, decellularized tissues of non-mammalian origin have reduced the risk of zoonotic disease transmission and circumvented the religious restrictions for different mammalian species. Fish skin is a multifunctional tissue that performs essential functions, including chemical and physical protection, sensory activity, behavioral targeting, and hormone metabolism. Fish are exposed to numerous microbial challenges in their aquatic habitat [160,161], and, thus, fish skin may have excellent anti-microbial properties. Omega-3 unsaturated fatty acids in the fish skin ECM are anti-viral and anti-bacterial and act as important modulators of the inflammation response [[162], [163], [164]], confirming the fish skin's anti-microbial properties. Recently, there has been an increased interest in fish skin derived-ADM and collagen since they do not contain the α-Gal antigen and have a low risk for prion disease and/or viral infection [165,166]. Some viruses may induce deadly infectious diseases, such as bovine spongiform encephalopathy and swine influenza, and, fish tissues do not spread these viruses. Overall, the ECM of fish skin with its delicate physical structure and special bioactive ingredients is an excellent choice for human skin substitutes (Table 3).

Mauer et al. analyzed the use of decellularized fish skin grafts for treating complex soft tissue wounds of various etiologies (for example, burns, dog attacks, failed skin flap, ulcerated hygroma, or incisional dehiscence) in dogs and cats [167]. Decellularized fish skin resulted in a completely closed wound for most injuries. In addition, no special training, instruments, or bandage material was required to use decellularized fish skin, making it suitable for application. The commoditized fish ECM product, the Kerecis Omega3 dressing (Kerecis, Isafjordur, Iceland), contains omega-3 and enhances wound healing. After covering the injured wounds, the fish-skin graft (FSG) dressing expedited re-epithelialization in the third stage of wound healing, significantly reduced the wound size, and shortened the wound closure time [168,169]. The Kerecis Omega3 wound matrix is also an acellular dermal matrix; however, it is derived from Atlantic cod (Gadus morhua) skin and has anti-inflammatory properties due to its omega-3 polyunsaturated fatty acids [[162], [163], [164]]. Furthermore, the Kerecis graft avoids the risk of viral and prion transmission that occurs with mammalian-derived products [170].

4.4. Acellular amniotic membrane

The human amniotic membrane is the innermost layer of the fetal placenta, containing plenty of GFs, including EGF, bFGF, TGF-α, and TGF-β [171], which have a crucial role in the physiological processes that are associated with wound healing and tissue regeneration. Furthermore, it is a suitable natural scaffold for cell proliferation and differentiation. Therefore, the materials based on amniotic membranes have the potential for improving wound healing (Fig. 3) [172]. Moreover, the amniotic membrane expresses a secretory leukocyte proteinase inhibitor and elafin, which have anti-inflammatory properties [173]. It also produces β-defensins which are anti-microbial peptides [174].

Fig. 3.

Acellular amniotic membrane biomaterials promote wound healing. (A) Schematic overview acellular amniotic membrane (Reprinted with permission from Behrouz Farhadihosseinabadi et al. [175]). (B) AAM-derived stem cells show a remarkable differentiation and proliferation potency. They are immunologically inert which introduce them as a new and easily accessible stem cell source (Reprinted with permission from Behrouz Farhadihosseinabadi et al. [175]).

Wilshaw et al. decellularized human amniotic membranes with 0.03% SDS, hypotonic triphosphate buffer, protease inhibitors, and nucleases, and they were sterilized using optimal concentrations of peroxyacetic acid to obtain acellular amniotic membranes (AAMs) [176]. When post-subcutaneously implanted into mice wound defects, the AAMs recruited low T cells and high numbers of fibroblasts, macrophages, and endothelial cells when compared with fresh and glutaraldehyde-fixed tissue. This suggests that the AAMs can support epithelial and stromal cell type attachment and proliferation and host cell migration into the central tissue, thus, promoting tissue remodeling. Similarly, the cytocompatibility of the AAMs was confirmed by Wilshaw et al. [177]. Kshersagar [178] found that the migrated cells within the AAMs were spindle, the repaired epidermis and basement membrane were well differentiated, and the hair follicles were prominent in the dermal layer in the AAM-treated burn wounds. Additionally, bacterial growth inhibition occurred. These data illustrate the good integration of AAMs into host tissues.

The AAM scaffolds are commercially available for skin wound healing, including SURFFIXX, AmnioBand, Biovance, and EpiFix [179]. Due to the innate properties of amniotic membranes, AAMs are characterized by anti-bacterial effects, immunomodulation, and pain reduction, making them suitable for healing various skin wounds, such as superficial or partial-thickness burns, pressure sores, or chronic leg ulcers [175,180]. They are effective for treating split-thickness skin graft donor sites [181], and they can treat chronic DFUs [182].

4.5. Decellularized small intestine submucosa matrix

The decellularized small intestine submucosa matrix (DSIS) is an ECM material derived from pigs that mainly contains collagen I, III, and a small amount of type V and VI, and it retains abundant GFs (TGF, FGF, VEGF, and EGF) and fibronectin, which are necessary for cellular adhesion signaling pathway activation and vessel morphology formation [183]. Furthermore, the DSIS is identified as being immunosuppressive. The anti-bacterial peptides that are produced during DSIS degradation can inhibit Gram-positive and Gram-negative bacteria, leading to few infections and complications after implantation, when compared with other grafts [184]. The DSIS components play a major role in wound healing and tissue remodeling. Therefore, the DSIS can serve as a good surface for cell attachment, differentiation, proliferation, and migration, and could effectively act as a wound dressing (Fig. 4) [185,186]. The DSIS-based regenerative methods, either alone or in conjunction with stem cells, can overcome skin injury repair problems by promoting angiogenesis and enhancing the wound healing rate. Decellularized small intestine submucosa sheet wound dressings have been developed from porcine DSIS [187] and ovine [188] and bovine small intestine submucosa [189]. Therefore, DSIS is applicable for treating cutaneous wounds, especially chronic wounds that are characterized by a harsh immune microenvironment and abundant matrix metalloproteinases [190,191].

Fig. 4.

Decellularized small intestine submucosa matrix promote wound healing. (A) SEM of decellularized small intestine submucosa matrix (Reprinted with permission from Mei-Rong Fan et al. [183]). (B) Growth factors released by DSIS by ELISA. (a) For VEGF, P ​< ​0.05 for day 10–30 indicates a significant difference between the two groups. (b) For TGF-β, P ​< ​0.05 for day 4–30 indicates a significant difference between the two groups. (Reprinted with permission from Mei-Rong Fan et al. [183]). (C)SEM images of MSCs cultured on DSIS on day 2 (a) and day 4 (b). Fluorescence microscopy images for the live–dead cell viability assay of MSCs cultured on DSIS on day 2 (c) and day 4 (d). CLSM images of the MSCs cultured on DSIS on day 2 (e) and day 4 (f). Green fluorescence represents the SIS-surface of DSIS stained with AO. Red fluorescence represents actin filaments of MSCs stained with phalloidin. Bar ​= ​100 ​μm (Reprinted with permission from Mei-Rong Fan et al. [183]).

Research has accumulated on the communications between DSIS and types of stem cells during wound healing. Seeding MSCs on DSIS can promote appendage formation with reduced scarring [192]. Similarly, DSIS with ASCs accelerated wound closure in vivo [193]. Moreover, crosstalk between DSIS and macrophages facilitates cutaneous wound healing. When cultured on DSIS, the macrophages were polarized to M2 macrophages and secreted anti-inflammatory cytokines to facilitate tissue remodeling [194].

Considering that DSIS scaffolds with a 100 ​μm thickness lack manipulable mechanical behavior, poly (lactic-co-glycolic) acid (PLGA) nanoparticles were utilized to modify the DSIS [195]. The PLGA-DSIS scaffolds enhanced the mechanical properties, retained ECM bioactive components, and promoted the growth of endothelial cells in vitro. Nevertheless, whether the combined DSIS scaffolds could elicit an enhanced wound-healing response needs further research.

Currently, the DSIS product-OASIS Wound Matrix is FDA-approved for wound healing clinically [196] (Table 6). When compared with conventional management, randomized controlled trials have found that DSIS significantly increased the percentage of wounds that were healed and the healing rate [[197], [198], [199]]. On chronic leg ulcers, DSIS implantation showed an enhanced wound healing rate at 12 weeks, with 40% and 29% of wounds healed with the treatment and conventional therapy, respectively [196]. Owing to its unique inhibition of matrix metalloproteinases [200], DSIS significantly reduced the matrix metalloproteinase and inflammatory cytokine expression in chronic venous ulcers. However, the chronic venous ulcers did not significantly change, except for the upregulated TGF-β [201]. These results indicate that DSIS heals chronic wounds mainly by modulating the wound's microenvironment to an acute inflammation state [201]. There is also clinical and histological improvement in wound healing of stage IV pressure ulcers [202,203].

4.6. 6 Decellularized plasma-rich fibrin

Platelet-rich fibrin (PRF) is a new-generation platelet concentrate that is based on platelet-rich plasma [204,205]. This material is rich in a variety of GFs, including VEGF, PDGF, bFGF, and EGF, which play key roles in tissue repair. Additionally, these GFs can be released slowly. Previous studies showed that the GFs contained in PRF can be released continuously for more than 2 weeks, which is slower than platelet-rich plasma [[206], [207], [208]]. Yan et al. prepared decellularized plasma-rich fibrin (DPRF) by combining repeated freeze-thawing and enzymatic digestion and investigated its osteogenic potential in vitro and in vivo [209]. Then, Xu et al. prepared DPRF scaffolds that accelerated wound healing in acute full-thickness skin wounds, suggesting potential applications as an ideal wound dressing [210].

Some studies have found that PRF can promote the proliferation of fibroblasts and keratinocytes in the gingiva. Many PDGF receptors can be found in periodontal tissues, and PDGF can act as a chemotactic factor for fibroblasts in the gingival periodontal membrane to promote soft tissue regeneration. Additionally, PRF contains a large amount of VEGF, which can promote vascularization and support soft tissue regeneration and activate the mitosis and migration of vascular endothelial cells, thereby activating vascularization in the early stage of tissue repair and promoting the generation of soft tissues [211].

Currently, the positive role of PRF in the tissue regeneration field has been generally verified. However, the effect of PRF alone and its mechanism of action are still unconfirmed. Furthermore, due to the lack of relevant clinical cases, the effect and mechanism of PRF on neural regeneration are still unclear. Therefore, PRF, a widely used autologous material, still has huge potential for application, and the specific cytokine mechanism needs further study.

4.7. 7 Other decellularized biomaterials

In addition to ADM, DSIS, and AAM, other ECM-based materials, including those that are derived from the mesothelium and stomach, have been investigated to treat skin wounds. Decellularized mesothelium membranes can be derived from squamous epithelial tissues, such as the peritoneum, pleura, and pericardium [212,213]. Alizadeh et al. developed a decellularized ovine pericardium to facilitate skin regeneration [212]. Also, Endoform® is a forestomach-derived ECM membrane that inhibits matrix metalloproteinases and is used to heal acute and chronic wounds [214].

The urinary bladder matrix (UBM), consisting of the lamina propria and basal lamina of the porcine urinary bladder, is used to clinically manage wounds and reinforce surgical soft tissue repair. Its use for managing chronic wounds in diabetic patients has been described in numerous laboratory and clinical investigations. In two prospective randomized trials with promising results, four types of skin substitutes (Apligraf, Dermagraft, OASIS, and MatriStem) were compared on episode length, amputation rate, skin substitute utilization, and skin substitute costs [215,216]. The UBM implantation transformed chronic long-lasting inflammation to a rapid acute immune state in diabetic wounds, with obviously increased VEGF, recombinant mannose receptor C type 1 like protein 1, and recombinant tissue inhibitors of metalloproteinase 3, verifying the efficacy of UBM in treating diabetic wounds by mitigating the inflammatory effects of the disease [217]. The detailed mechanism involves the polarization of the macrophages into M2 macrophages both in vitro and in animal models [[218], [219], [220]].

5. Conclusion and perspectives

Cutaneous wounds result in the risk of limb amputation, mortality, and morbidity if not treated effectively, and they represent a significant economic burden on global healthcare systems. Despite considerable advances in pharmacological and surgical approaches, clinical trials for patients remain disappointing owing to limited skin-autograft resources, especially in patients with extensive and full-thickness wounds. Thus, there is an urgent need for effective and affordable biomaterials. Here, we reviewed the wound healing process and ECM-based strategies for enhancing the wound healing processes by stimulating cell migration, angiogenesis, tissue re-epithelization, and skin appendage regeneration. However, the goal of perfect repair has yet to be achieved. To promote wound healing, research on decellularized materials, with a variety of bioactive cytokines and molecules, has driven the design and development of wound dressings. Therefore, extensive progress has been made in cutting-edge decellularized-based strategies and understanding the underlying molecular mechanisms to promote wound healing. In the pursuit of perfect wound healing, researchers still need to investigate the detailed mechanisms of decellularized materials that guide cells to directionally differentiate to skin-associated mature cells, precise positioning regulation of various cells during the healing process, recruitment of tissue-resident stem cells and progenitors by decellularized materials, and equilibrium between skin regeneration and scar formation mediated by these biomaterials. In addition, biomaterials combined with novel therapeutic modalities, such as exosomes and platelet-rich plasma, is a current hot topic in ECM-based biomaterials research. With the emergence of four-dimensional printing and the development of the handheld skin printer, these apparatuses may lead to a revolutionary breakthrough.

Several challenges remain in developing commercially available ECM-based biomaterials, like reduced vascularization, poor mechanical integrity, failure to integrate, scarring, and immune rejection [223]. Due to the inability to revascularize, cells in the substitute die and slough away from the host tissue. Therefore, commercially available skin substitutes have limited vascularization. Moreover, to develop engineered skin, the cells must be cultured for 2–3 weeks before the grafts can be placed. Technical advances in cell and tissue culture protocols are thus required to overcome the cell growth issues of engineered skin. Currently, the available ECM-based biomaterials mainly consist of fibroblasts and keratinocytes and cannot make differentiated structures, like hair and sweat glands. Therefore, additional cell types should be included, such as endothelial cells in engineered skin, and there is a need to reduce the cost of current skin substitutes. Emerging evidence indicates that superficial fascia mobilization after skin damage is important for tissue regeneration and scar formation [229]. Future studies should uncover the effects of immunomodulatory hydrogels supplied as an ECM scaffold to replace the wound ECM from the superficial fascia. This may provide new opportunities for facilitating DFU healing with reduced scar formation.

Recently, advanced manufacturing techniques, such as 3D bioprinting, have been used to enhance the immunomodulatory function of hydrogels [230]. For instance, Wang et al. reported a new bioink for functional hydrogel preparation with 3D bioprinting [230]. Additionally, smart materials and chemical systems, which can detect, record, analyze, and respond to information from the surrounding environment, have attracted increasing attention. Smart dressings with flexible electronics are becoming increasingly important for wound treatments [231], and electrical stimulation can facilitate wound healing with high efficiency and limited side effects. A recent study reported a flexible electrical hydrogel patch for accelerating wound healing [232] that was synthesized using silver nanowire and methacrylate alginate and optimized to enable printing on medical-grade patches for personalized wound treatment. The in vitro enhanced secretion of GFs with promoted cell proliferation and function occurred in response to electrical stimulation. Furthermore, the in vivo results indicated an accelerated wound closure rate in rodents [232].

Developing spatiotemporal printing via the different mechanical characteristics, immune regulation, and chemical modification of current ECM biomaterials could target many aspects of the complex wound healing process to ensure effective and complete wound healing and shorten chronic wound healing times. Individualized therapeutic approaches should also be used to treat specific wound types and individuals using emerging tissue engineering technologies. There may still be many unexplored ECM biomaterials with idealized properties for the effective and sustained delivery of therapeutic agents to wounds. Moreover, advances in manufacturing smart wound dressings enable the development of the next generation of ECM biomaterials substrates, including biomechanical, stimuli-responsive, self-healing, self-removable, and monitoring wound dressings. This review article provides a basis for the development of this knowledge.

Funding

This work was supported by the National Natural Science Foundation of China (grant number 82002291), Key Projects of Scientific Research and Innovation, Shanghai Municipal Education Commission (grant number 201901070002E00043), and the National Key R&D Program of China (grant number 2020YFC2004906).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.