Contents

1. Introduction . . . ........................................................................................................ 2

2. Skin microbiome and wound healing . . ..................................................................................... 2

2.1. Skin microbiome . . . . . . . ........................................................................................... 2

2.2. Wound healing . . . . . . . . ........................................................................................... 3

2.3. Role of wound microbiome on the inflammation stage . . . . . . . . . . . ........................................................ 3

2.4. Effect of wound microbiome on cell proliferation. . . . . . . . . . . . . . . . ........................................................ 4

2.5. Effect of wound microbiome on re-epithelialization. . . . . . . . . . . . . . ........................................................ 4

2.6. Wound microbiome and remodelling phase . ........................................................................... 5

3. Immune cell response to bacterial . . . . ..................................................................................... 6

3.1. Neutrophils . . . . . . . . . . . ........................................................................................... 6

3.2. Monocytes and macrophages . . . . . . . . . . . . . ........................................................................... 7

3.3. Lymphocytes . . . . . . . . . . ........................................................................................... 8

4. Clinical management in regulating wound microbiome . . . . . . .................................................................. 8

4.1. Role of commensal microbiota on surgical wounds . . . . . . . . . . . . . . ........................................................ 8

4.2. Clinical management in treating chronic wounds. . . . . . . . . . . . . . . . ........................................................ 9

5. Strategies in controlled delivery of probiotics and microbiome .................................................................. 9

5.1. Microencapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

5.2. Electrospun scaffolds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

5.3. Hydrogels and 3D bioprinting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

5.4. Microneedles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1. Introduction

      Delayed wound healing has become a global public health problem, particularly in chronic wounds. According to the World Health Organisation (WHO), over 6 million people are affected by delayed wound repair every year in the United States with a cost of $25 billion (USD) in the health system [1]. About 100 million people need wound care every year, and about 30 million people with complex and refractory chronic skin wounds require further intervention [2]. All types of wounds or injuries initiate the healing process, which consists of several highly integrated and overlapping phases: inflammation, cell recruitment, matrix deposition, epithelialization and tissue remodelling. Due to confounding factors such as aging, stage of diabetic disease, medication (or treatment), peripheral neuropathy, immunocompromised status and/or arterial and venous insufficiency hyperglycaemia, chronic wounds are at high risk of delayed healing [3]. At present, the therapeutic approach for the treatment of delayed wound healing is antiinfection and debridement or the use of various wound dressing and dermal templates, but the clinical outcomes are unsatisfactory. Therefore, advanced approaches are urgently required by clinicians. Recent studies demonstrated that regulation of the wound microbiome can promote diabetic wound healing. Few pioneer studies reported that increased diversity of wound microbiome and suppression of Staphylococcus aureus (S. aureus) can significantly promote the wound repair [4]. In the present article, we discuss the skin microbiome, the effect of the microbiome on different stages of the healing process, immune cell responses to the wound microbiome and clinical treatment with a focus on advances and novel strategies in the future design of a controlled delivery system for probiotics and microbiomes.

2. Skin microbiome and wound healing

2.1. Skin microbiome

      Human skin is exposed to maternal and environmental bacteria and other microbes (e.g. viruses, fungi and protozoa) immediately after born [5]. Approximately 1,000 species of known bacteria and about 100 billion microbiomes are detectable on a person’s skin [6]. Bacteria (2 million/cm2 ) on human skin [5] can be divided into four phyla: Actinobacteria (51.8 %), Firmicutes (24.4 %), Proteobacteria (16.5 %), and Bacteroidetes (6.3 %), including three most common genera: Corynebacterium, Propionibacterium, and Staphylococcus [7]. This data suggests that bacterial colonies are more abundant on the surface of the skin compared to any other epithelium [8]. Human skin can be seen as a culture medium, and its composition is greatly influenced by genetics, diet, lifestyle and the living environment [6]. Therefore, each site of human skin is unique in terms of the composition of the microbes, while resident microbes in specific areas on the skin are also known to be identical at the genus level [9–10]. The composition and abundance of different microbes vary widely across individuals and over time, forming a dynamic and highly variable microbiome [5]. A Human Microbiome Project (HMP), led by the National Institutes of Health (NIH), was conducted to characterize microbial populations in five major body sites, including the oral cavity, skin, nasal cavity, gastrointestinal tract, and genitourinary tract [11]. Data from this project showed that microbiomes are able to control signalling pathways related to body metabolism, especially those with diseases in different body sites. In addition, thousands of microbiomes (bacteria, viruses, fungi) were identified living on the skin surface, which has tremendous positive effects on human survival [11].

      Skin is the first barrier of the human body while the formation of a wound provides an opportunity for commensal microbiota on the skin surface to migrate to the underlying tissue with colonization and growth [12–13]. During the healing process, cell interaction with the wound microbiome is hypothesized to be beneficial in regulating the innate immune response [13]. In contrast, pathogenic microbiota is known to play a negative role in mediating the wound healing process [14–15]. According to statistics, wound contains diverse microbiota, the main components of which are Staphylococcus, Pseudomonas, Corynebacterium, Streptococcus, Anaerobic Coccus, and Enterococcus, as well as other genera with lower abundance [3]. Chronic wounds generally contain more microbes than acute wounds [3]. Over 2900 samples collected from various wounds were subjected to multiple microbiological analyses, while Staphylococcus and Pseudomonas were found to be the most common species, accounting for 63 % and 25 %, respectively [16]. Moreover, a study proved that there is an association between microbiota composition and clinical factors [17]. Higher microbial diversity was observed in deeper ulcers and ulcers of longer duration, as well as increased anaerobic and the relative abundance of Gram-negative Proteobacteria [18]. In contrast, many Staphylococci, mainly S. aureus, were found in superficial and short-term ulcers, suggesting that bacterial species are correlated with the host pathophysiology. Notably, commensal bacteria, including Corynebacterium and Propionibacterium, as well as anaerobes were prevalent in chronic wounds [19]. Taken together, a stable equilibrium between wound microbiotas and the host is believed to play an important role in wound repair and the healing process.

      Although the role of the skin microbiota on wound healing has been comprehensively investigated [20], conclusions remain controversial. Few pioneer studies found that the wound microbiota may contribute to the composition and overall toxicity of the wound microbiome through forming biofilms in chronic wounds. Changes in force may lead to infection or hinder wound healing [21]. In the past, scientists and clinicians all agreed that S. epidermidis positively affects healing, but S. aureus seems to be a villain [6]. Multiple studies have shown that modulating wound microbes can improve wound healing, with significant positive implications for human survival [20]. These new findings open a new avenue to explore microbes’ role and underlying mechanisms in the wound healing process.

2.2. Wound healing

      Wound healing is a dynamic and highly coordinated process, mainly including the haemostatic phase, inflammatory phase, cell proliferation phase (formation of granulation tissue vascularization and wound closure) and tissue remodelling phase [22–23]. A variety of inflammatory cells, cell cytokines, inflammatory mediators and extracellular matrix are involved in the healing process [24–25]. Normally, wound healing can be divided into acute or chronic wound healing [26]. The primary difference between acute and chronic wounds is the length required for healing [27], in which acute wounds can heal in 4 weeks, but chronic wounds have significantly delayed healing over months or even years [28].

      The healing rate is highly related to injury types, severity, size of the wounds aging, co-morbidities as well as post-injury care [3]. Acute trauma has different wound microbiota composition. Studies found that the most common pathogens in the acute phase postburns are S. aureus, followed by Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and coagulase-negative Staphylococci, etc [29–30]. Over surgery, microbiomes on sutures or prostheses can induce wound infection and cause acute surgical site wound infections (SSWIs). The most common pathogenic microbiotas involved in SSWIs are S. epidermidis and S. aureus, but E. coli, Clostridium difficile, and coagulase-negative Staphylococci also occur to a lesser extent [31].

      Wounds that do not heal after 1 month of treatment are generally considered chronic wounds. Chronic wound formation is complex and more likely to occur in the elderly patients with vascular disease, diabetes, or a combination of these factors compared to acute wounds [13]. With the increased elderly population, chronic wounds have become a major burden on healthcare worldwide [32]. The main cause of chronic wound infections and noncurative wounds is that the wound surface is colonized by polymicrobial communities, leading to the formation of biofilms [17]. Generally, Gram-negative bacteria compose common colonizing microbiotas around the chronic wound area, accounting for 61 % of all microbial isolates. S. aureus is the most commonly found pathogen among them, followed by P. aeruginosa [3]. Wound bacteria, such as S. aureus, P. aeruginosa, Corynebacterium striatum, and Alcaligenes faecalis were detected in patients with diabetic foot ulcers [33]. In a study of 145 patients with pressure ulcers, S. aureus and Gram-negative bacilli were detected in 112 wounds [34].

2.3. Role of wound microbiome on the inflammation stage

      Immediately after injury, the wound initiates an inflammatory response that mediates pathogen clearance and stimulates restoration of the skin barrier integrity [35]. However, in a chronic wound, these inflammatory responses are either absent or hyperactive and in a state of dysregulated inflammation, resulting in abnormal tissue formation and long-term defects in the epidermal barrier [14]. Despite the robust diversity of the skin microbiome, understanding how skin microbiota regulates the inflammatory response over the inflammation stage has important implications for the development of therapeutic and preventive strategies (Fig. 1).

      In 2020, a study reported for the first time the beneficial role of the symbiotic skin microbiota and its correlation with the inflammatory response [36]. When the skin is injured, researchers found that skin microbiota, consisting of commensal Staphylococci and Micrococci, migrated from the epidermis to the dermis of the injured skin, triggering the activation of neutrophils with the expression of CXC chemokine ligand-10 (CXCL10) (Fig. 1). Thereafter, CXCL10 activates plasmacytoid dendritic cells (pDC) by forming complexes with bacterial DNA, while activated pDC produce large amounts of type I interferon, which accelerate wound closure via triggering inflammation and early T cell-independent responses, mediated by macrophages and fibroblasts that produce major growth factors required for healing. In addition, the produced interferon induces early wound repair responses and accelerates wound healing by stimulating the growth of fibroblasts [36]. A recent study further revealed that the A20 clade of the skin commensal S. epidermidis could induce adaptive immunity with both anti-infection and tissue repair functions [37]. S. epidermidis was capable of stimulating the recruitment of a special CD8+ T cell (Fig. 1), directly killing pathogens together with enhanced reepithelialization and keratinocyte migration. The immune response elicited by S. epidermidis is found via the non-specific CD8 + MHCIb pathway, but not the CD4 + MHCII pathway [37] (Table 1).

      By now, only a few specific microbiotas have been investigated in immunology or inflammatory processes [20]. In addition to Staphylococcus, Corynebacterium, a commensal of mouse and human skin microbiota, was reported to activate the skin immune system in an IL-23-dependent manner [38]. Such a system depends on the expression of mycolic acid, a significant component of the coryneform cell wall. The inflammatory effect of microbiomes also depends on the physiological state of the host, and the skin microbiota is triggered by the host’s dietary changes, obesity, or metabolic state to stimulate inflammation or immune responses [38]. Although S. aureus is the most prominent cause SSWIs worldwide, studies have shown that S. aureus surface proteins A (SpA) and staphylococcal immunoglobulin-binding protein (Sbi) induce IL- 1b and IL-6 at the initial stage of S. aureus infection and promote the expression of TNF-a and CXCL-1. Expression of IL-10 in wounds induced by SpA and Sbi not only contributes to the recruitment of neutrophils but also extends the time of immune cells such as neutrophils and macrophages recruited to the site of infection (Fig. 2). SpA is also known to aid the bacterial clearance by neutrophil extracellular traps (NET) (Table 1), thereby contributing to proper abscess formation and inflammatory responses [39].

2.4. Effect of wound microbiome on cell proliferation

      The inflammatory phase is followed by a cell proliferation phase, in which fibroblasts, keratinocytes, and endothelial cells migrate and proliferate, promoting angiogenesis and synthesis of new extracellular matrix (ECM) [40]. The transition from the inflammatory phase to the proliferative phase is a critical step in the healing process. Thus, the poor transition is associated with impaired wound repair.

      S. epidermidis is closely associated with keratinocytes as an antibacterial defender with increased wound healing rate [41]. S. epidermidis was reported to recruit IL-17A+ CD8+ T (Tc17) cells infiltrating into the epidermis with enhanced innate barrier immunity via upregulating toll-like receptors and limiting the pathogen invasion [41]. Upregulated TNF-a gene expression promotes rapid cell proliferation of keratinocytes, thereby accelerating the healing rate (Table 1). Interestingly, S. epidermidis-induced Tc17 cells highly expressed a series of key factors involved in angiogenesis, extracellular matrix production, and tissue remodelling [37] (Fig. 2). Recent studies have shown that skin microbes can also act as a facilitator by activating biosensors on the skin, such as the aryl hydrocarbon receptor (AHR) in keratinocytes to upregulate gene expressions that are involved in cell proliferation, differentiation, and inflflammation [42–43]. AHR plays a crucial role in skin homeostasis and the skin development, function, and integrity of the epidermal permeability barrier. A comprehensive study showed that commensal flora is required for the formation of the skin barrier and that skin epithelial cell development and differentiation are impaired in germ-free mice [43]. When microbiota, including S. epidermidis, Staphylococcus warneri, Staphylococcus hemolyticus, Micrococcus luteus and Corynebacterium aurimucosum, isolated from normal human skin were transplanted into germfree mouse skin, engraftment of commensal microbiota regulated epithelial cell differentiation via AHR and promoted the restoration of skin barrier function compared with mice not colonized or treated with antibiotics (Table 1).

2.5. Effect of wound microbiome on re-epithelialization

      Re-epithelialization is the process of re-build/reformation of the external surface of wound epithelium via keratinocyte migration from the wound edge to the centre area. Re-epithelialization mainly relies on keratinocyte migration, in which keratinocytes are the most predominant cell type in the epidermis. Many bacteria on human skin can convert aromatic amino acids into socalled trace amines (TAs) [44] (Fig. 2). When the wound occurs, in response to stress, the damaged epithelial cells release epinephrine while b2-adrenergic receptor (b2-AR) in keratinocytes is activated to hinder the migration of keratinocytes and reduce collagen production [45]. TAs are known as b2-AR antagonists that can attenuate the effects of epinephrine, resulting in accelerated wound healing [44]. The serum adenosine deaminase (SadA) gene encodes an aromatic amino acid decarboxylase that converts aromatic amino acids into TAs, such as tryptamine, phenethylamine, and tyramine [46]. Additionally, SadA homologs were found in at least 7 phyla in the human skin microbiota, including Firmicutes, Actinobacteria, Proteobacteria, Bacteroides Acidobacteria, Chloroflexi and Cyanobacteria [47]. Taking S. epidermidis with SadA gene as an example, it can increase the migration rate of keratinocytes and ultimately accelerate wound healing (Table 1).

      The skin microbiome also plays a role in strengthening the physical barrier to skin integrity. Keratinocytes and the microbiome produce sphingomyelinase, which processes lamellar lipids into ceramides, which are important molecules in maintaining skin structure and function [48]. Using a mouse model of impaired skin barrier function, Staphylococcus colonization on the surface of mouse skin was reported to have less transdermal water loss compared to the animals not treated with S. epidermidis [15,48]. This finding provides new insights into the underlying mechanisms of how the skin microbiome interacts with the host skin.

      In addition to the direct beneficial effects of wound microbiotas on wound healing, the bacterial toxins secreted also play a specific role [49] (Table 1). Streptococcus pyogenes is known for causing superficial infections and invasive diseases, and streptolysin O (SLO) is a hallmark toxin secreted by Streptococcus pyogenes at low concentrations (0.02–0.2 U /mL). SLO promotes wound healing in vitro by stimulating the migration of keratinocytes. Such a cellular mechanism via SLO may be explained via maintaining the expression of the hyaluronic acid receptor, CD44, in keratinocytes, which benefits the composition of the hyaluronic acid-rich ECM through the formation of CD44-hyaluronic acid complexes wound healing process [49]. Both S. epidermidis and typical low abundance S. aureus can stimulate human keratinocytes to express antimicrobial peptide (AMPs), protecting skin against the invasion of pathogenic microbiotas [50] (Fig. 2). Human b-defensin (hBD) is a small molecule cationic antimicrobial peptide, which is expressed and uniformly distributed in all human epithelial cells, supporting keratinocyte differentiation together with recognition of microbiotas, AMP has thus emerged as a new target for accelerating wound healing.

2.6. Wound microbiome and remodelling phase

      When the wound is completely closed, remodelling occurs when the newly formed epithelial cells constantly divide, thickening the epidermis. There is no direct evidence showing that the microbiome in wound healing regulates collagen deposition. However, skin bacteria were recently reported to have a direct correlation with the hair follicle (HF) neogenesis [4] (Fig. 2). Scientists found that germ-free mice have less ability to regenerate hair follicles. In contrast, mice with skin commensal microbiota can trigger the regeneration of hair follicles via activation of the interleukin-1 receptor (IL-1R) - MYD88 signalling pathway [4,51]. Furthermore, recent data showed that the transmembrane endopeptidase ADAM10-Notch signalling pathway in HF is an essential regulator of the microbiota in the hair follicle opening region. Disruption of the DAM10-Notch signalling pathway leads to upregulation of inflammation-related genes’ expressions, and inflammatory alopecia, resulting in dysbiosis of the Corynebacterium-dominated flora, causing hair follicle cell death and irreversible hair loss. ADAM10-Notch signalling can enhance innate epithelial immunity by promoting type I interferon responses downstream of b-defensin-6. Therefore, the host-microbe symbiosis balance mediated by the ADAM10-Notch signalling axis protects HF from inflammatory destruction and has clinical implications for maintaining the tissue integrity [4] (Table 1).

3. Immune cell response to bacterial

      Multiple immune cells are required to achieve tissue regeneration and homeostasis post-injury, including innate and adaptive immune cells [54]. These cells are significantly heterogeneous, plastic, and can have different functions in response to different environmental stimuli [55]. Wound microbes can also interact dynamically with various types of cells to regulate wound repair and regeneration (Fig. 3).

3.1. Neutrophils

      Following injuries, neutrophils, circulating inflammatory cells, are the first responders recruited from the blood strain to the wound site [56]. Neutrophils accumulate abundantly at the would site, releasing chemokines and inflammatory cytokines that further recruit more neutrophils and other immune cells [57]. Neutrophils also act as host defence by engulfing pathogens, secreting granules filled with cytotoxic enzymes, or expelling neutrophil extracellular traps (NETs) to trap and kill pathogens (a process is known as NETosis) [58]. NETs are a DNA network structure formed by neutrophils activated by various stimuli and released to the outside of the cell [59]. With DNA as the backbone, the network structure is inlaid with a variety of proteins, including histones, matrix metalloproteinase 9 (MMP-9), neutrophil elastase, myeloperoxidase, protease 3, etc., aiming to kill pathogens or increase their permeability [60]. Currently, S. aureus, Streptococcus pneumoniae, E. coli, Helicobacter pylori, Pseudomonas aeruginosa, Candida albicans (C. albicans) and fungi were found to stimulate neutrophils to produce NETs [61] (Fig. 3).

      In-depth studies have found that the ability of bacteria to induce NETs varies by bacterial species, while the most potent inducer of NETs is S. aureus [62–63]. However, S. aureus has multiple evasion strategies against the human immune system, such as the production of immunomodulators and secretion of nucleases. It can also target neutrophils by blocking neutrophils rolling on activated endothelial cells antibodies and opsonin necessary for pathogen recognition by cells to escape phagocytic neutrophils, allowing them to escape NETs [39]. Among the various virulence factors secreted by S. aureus, leukotoxins and leukocidins can promote the production of NETs through a non-ROS-dependent pathway [64]. In addition, the expression of S. aureus protein SPA in subcutaneous skin infection can also assist neutrophils recruitment, and clearance of bacteria through the NET capture [39,52].

     In contrast, few studies have proved that NETs are a ‘‘doubleedged sword”. NETs can eliminate pathogens and reduce inflammation, while they can reprogram macrophages to reach M2 Phenotypic regulation for a faster wound healing [65]. However, over-promoted neutrophils by pathogens in the wound area stimulate severe expression of inflammatory mediators (such as IL-1, TNF-a) and proteolytic enzymes, resulting in excessive and persistent inflammation that exacerbates tissue damage or even damage surrounding healthy tissue [55]. For example, over-expressed NETs were found in diabetic foot wounds, leading to further tissue damage and delayed wound healing. In an animal model of diabetic mice, inhibiting the formation of NETs or the cleavage of NETs can significantly reduce inflammation and improve the wound healing [66]. The presence and activity of neutrophils mediated by microbiomes is therefore a challenge in wound care which request close monitoring of inflammation or infection [66].

3.2. Monocytes and macrophages

      Over the inflammatory phase of wound healing, neutrophils are cleared by macrophages [67], further removing pathogens and cellular debris directly or indirectly via secreting transforming growth factor-b (TGF-b1) and vascular endothelial growth factor (VEGF)[68–69]. Macrophages include tissue-resident macrophages located in the skin and monocytes recruited from bone marrow through blood circulation. Macrophages are the key orchestrators of the wound healing process against infection [70], while proinflammatory macrophages are often described as M1 macrophages, and anti-inflammatory and repairing macrophages are M2 macrophages [71]. M1 macrophages are usually detected early in the repair process, secreting cytokines such as IL-6, TNF-a and nitric oxide synthase that regulate the early stages of healing[72]. Approximately 80–85 % of the M1 pro-inflammatory phenotype are converted to anti-inflammatory M2 macrophages on days 5–7 of acute wounding [73]. M2 macrophages normally lead to increased secretion of anti-inflammatory cytokines (e.g., IL-10, Arginase-1) that suppress inflammation and increase growth factors required for proliferation, migration, and repair processes [71]. In diabetic patients, M1 macrophages drive the elevated and prolonged non-resolving inflammation seen in diabetic foot ulcers (DFU) [74] with around 80 % of macrophages at the edge of chronic wounds being the pro-inflammatory M1 phenotype, and the transition to an M2 phenotype is a hindrance. Therefore, the polarization of macrophages is crucial for faster wound healing.

      To date, the effect of probiotics on macrophage polarization has become a new research trend. The lactic acid secreted by Lactococcus lactis (L. lactis) was proved to reverse the inflammatory and proteolytic characteristics of diabetic wounds via the M2 polarization [75–76]. Scientists also attempted to programme L. lactis by transforming a plasmid encoding VEGF, to maximize VEGF production, and reduce intrinsic deactivation of the VEGF [77]. The genetically engineered L. lactis can continuously secrete VEGF and lactate, with enhanced angiogenesis and induction of M2 polarization [77]. Such pioneer ideas are based on live engineered bacteria, which can promote wound healing by exogenous supplementation of probiotics in the future.

      Similar to mammalian cells, gram-negative and gram-positive bacteria constitutively release lipid bilayer-enclosed, nanosized extracellular vehicles (EVs) or membrane vesicles [78]. As EVs contain bioactive molecules, including nucleic acids, proteins, lipids, toxins and various virulence factors, they were reported to have diverse functions in cell-to-cell communication between bacteria [79]. Bacterial EVs are proven more effective compared to bacterial extracts or purified toxins in evoking cellular responses in the recipient cells [56]. A pioneer study proved that EVs derived from Lactobacillus plantarum (L. plantarum), LEV, could successfully induce monocyte differentiation into M2 macrophages in human THP1 cells, with increased secretion of the anti-inflammatory cytokine IL-10, along with immunomodulatory cytokines IL-1b, GMCSF and a higher population of M2b macrophages [60]. Such promising results showed that LEVs can be used as anti-inflammatory and immunomodulatory substances by regulating the conversion of M1 and M2 macrophages, thereby improving hyperinflammatory skin conditions and the phenotypes of inflammatory skin disorders. Therefore, determining which uptake pathways and signalling molecules are involved in LEV-evoked cellularesponses may further assist in interpreting bacterial EV-mediated phenomena in human cells and designing alternative methods using bacterial EVs as immunomodulatory materials.

3.3. Lymphocytes

      Lymphocytes are immune cells that are recruited to the wound at a late stage aiming for fibrous tissue regeneration [80]. B lymphocytes are relatively rare in the skin at a steady state. Elevation of B lymphocytes can be observed in the skin disease, such as human atopic eczema, cutaneous leishmaniasis, and skin sclerosis [81]. Recent studies have found that B cells can act as a regulator of the tissue regeneration [82]. ECM component can induce CD19- dependent Toll-like receptor (TLR) signalling in wound B cells, thereby inducing the production of cytokines, IL-10 and TGF-b, directly reducing the inflammatory response [83]. When mature B cells were applied locally to acute or chronic wounds, increased fibroblast proliferation is noted with a reduced level of apoptosis and an accelerated healing rate [84]. However, to our knowledge, no data or report was seen on the role of wound microbiome-mediated B cells.

      T lymphocytes are mainly categorized into ab T cells and cd T cells according to their composition [85]. cd T cells have a high proportion in the mouse skin tissue, accounting for more than 90 % of T cells in the epidermis as a skin immune defensor [86–87]. Human skin contains ab T cells and cd T cells with an expression ratio of approximately 5:1, distributed in both the epidermis and dermis [88–89]. cd T cells are highly involved in psoriasis [90], tumours [91–92], viral infection [93] and wound healing [94]. Burn injury has been found to activate cdT cells to produce a large amount of insulin-like growth factor 1 (IGF-1) (Fig. 3). However, the presence of P. aeruginosa in burn wounds was reported to reduce the synthesis of IGF-1 in burn patients with peripheral blood cdT cells, adversely affecting wound repair [95]. When cd T cells are activated by an infection, a large amount of IL-17 can be produced to resist infection [55] Hence, IL-17 also plays a crucial role in the immune response to pathogens, including Klebsiella pneumoniae (K. pneumoniae), Citrobacter rodentium, C. albicans [96–97]. In addition, cd T can also regulate and control ab T cell-mediated inflammation, to reduce the activity of lymphocytes and the production

4. Clinical management in regulating wound microbiome

4.1. Role of commensal microbiota on surgical wounds

      Surgical incisions breach the skin barrier, disrupting the commensal resident microbiota, triggering a host immune response and wound healing processes [99]. All surgical wounds are recolonised by a patient’s resident microbiome and environmental organisms. Some wounds become infected when organisms invade tissue or cause cellular injury. Depletion of the resident skin microbiome in favour of opportunistic pathological organisms capable of invasion results in SSWIs [100]. SSWIs impose a significant burden on the health care system and are a leading cause of morbidity after surgery occurring in up to 5 % of patients post-operatively [101]. This accounts for approximately 20 % of all healthcare-associated infections and costs $5000-$13,000 per incident [102]. of IFN-c in delayed hypersensitivity reactions [98].

      Methods for the prevention of wound infection are largely instrumental in the management of surgical patients. These interventions support the return of a commensal wound neomicrobiota and suppress domination by opportunistic pathogens. The neomicrobiome describes the process of re-colonisation of the wound after surgery with resident and transmitted non-resident organisms, including bacteria, fungi and viruses [99]. Reservoirs of resident organisms, which form the neomicrobiome, are found on the surrounding superficial skin, in deeper skin layers and the pilosebaceous unit [12]. Other organisms may enter the wound from environmental contamination before an operation at the time of wounding, or during the operation e.g. respiratory, alimentary, genital or urinary tract organisms or after in the hospital or home environment [103].

      Repopulating the wound neomicrobiome is a dynamic process and changes with time. Immediately after wounding, there is a gram-negative shift with resident commensals such as Proteobacteria and Bacteroidetes and gram-negative environmental contaminants such as Pseudomonas colonising wounds [104–105]. Grampositive bacteria are still present but with a reduced number, such as the facultative anaerobe Propionibacterium [12]. This is in-part due to the suppression of gram-positive Staphylococcus and Streptococcus populations with the SSWI antibiotic prophylaxis [105]. With wound healing, reservoirs of resident commensal organisms repopulate the wound area while a gram-positive shift occurs with a return to the normal skin flora [12,104]. Total microbial load is also variable but critical to wound healing and infection. Overpopulation of commensal organisms or environmental pathogens is causative of SSWI and delays wound healing. Once bacterial levels reach a threshold of > 105 colony-forming units, tissue invasion and wound infection are common [100,106]. The dynamic process of the surgical wound microbiome is a novel area of research. At this point, analysis has focused on the bacterial neomicrobiome [107].

      Repopulation of the wound with resident commensal organisms accelerates wound healing and prevents wound infection by opportunist pathogens with evidence suggesting that this process occurs over a period of 2–6 weeks [4,12,108]. Commensal microbiota is beneficial through indirect competition, occupying the skin niche and utilising available nutrients [109]. The diversity of the resident commensal microbes prevents domination by a single pathogen. Evidence of this has been demonstrated in numerous situations. For example, low levels of commensal Pseudomonas accelerate wound healing. However, overpopulation results in tissue invasion and SSWI’s [100,110]. Resident microbes also directly support wound healing by producing anti-microbial molecules, metabolites and immune-enhancing actions [36,109,111–112]. Surgical wound management, therefore, aims to encourage the repopulation of a neomicrobiome with the diverse commensal resident flora restoring skin homeostasis and preventing wound infection. Various surgical techniques are directed at controlling the number of microbes and resorting balance to the neomicrobiome [13,113]. This includes surgical techniques to aid wound closure, control of contamination, antiseptic and antibiotics.

      Surgical wounds may be closed primarily where healing occurs from approximation of tissue using sutures, staples or glue. Wounds heal by primary intention, minimising exposure to endogenous and exogenous contaminants [114]. Healing by secondary intention is the process of an open wound healing through granulation, contracture and epithelialization. An open wound is exposed to environmental contaminants, and healing occurs in the presence of a complex relationship between microbial colonisation and the immune system [115–116]. The success and failure of tertiary healing largely depend on the wound bioburden with graft or primary closure failure, highly likely with an elevated bacterial load [100,117].

      The degree of wound contamination influences the inoculum of a wound with foreign microbes and depends on pre- and intraoperative events determining whether a wound is clean, cleancontaminated, contaminated, or dirty. In clean wounds, the respiratory, alimentary, genital or urinary tract is not entered. Clean contaminated wounds reflect a controlled entry into the respiratory, alimentary, genital or urinary tract. Contaminated wounds are open, fresh, accidental wounds or occur in operations with major breaks in sterile technique, gross spillage of the gastrointestinal tract or acute non-purulent inflammation encountered. In both clean-contaminated and contaminated wounds, the neomicrobiome is exposed to foreign microbes e.g., gastrointestinal bacteria. Dirty-infected wounds include old traumatic wounds and wounds with existing infection. Contamination of a wound during or before an operation inoculates the wound with potential pathogens which may dominate the neomicrobiome [106]. This is supported by evidence which shows the SSWI rates following clean, clean-contaminated, contaminated, and dirty procedures are 1.76 %, 3.94 %, 4.75 %, and 5.16 %, respectively [101].

      In treating acute wounds, ethanol, povidone-iodine and/or chlorhexidine are widely applied clinically as the pre-operative topical antiseptics. The latest report showed that exposure to antiseptic treatments can elicit rapid but temporary depletion of microbial community diversity and membership [118], while the wound microbiome was found highly dependant on personalized and body site-specific colonization signature. Moreover, another clinical study compared the efficacy of povidone-iodine-alcohol and chlorhexidine-alcohol for surgical wounds, showing skin microflora was more affected by povidone-iodine in short time but not in prolonged contact times over 3 h [119]. These studies confirmed the short-time stability and resilience of the skin microbiome to antiseptic and its effect on the incidence of SSWI remained unknown.

      The use of antibiotics before, during and after surgery alters the structure and function of wound microbial communities. Systemic and topical antibiotics suppress common pathogens, and lower wound tissue inoculum from intra-operative contamination but also eliminate commensal resident bacteria [120–121]. It has been proven definitively that systemic antibiotics given at the time of induction reduce SSWI for wound healing by primary and secondary intention [122]. Topical antibiotics after primary closure also reduce SSWI [101]. It is unclear whether the antibiotics’ mechanism of SSWI reduction after surgery is from eliminating environmental pathogens or through suppressing the overgrowth of a commensal opportunist, it is likely a combination of both [103]. Overall healing of surgical wounds is intrinsically linked to the health of the wound neomicrobiome. A complex interplay exists between the neomicrobiome composed of commensal residential and environmental organisms, wound healing processes, the wound environment and the immune system. Surgical techniques are aimed at preventing wound infection and restoring the residential wound microbiome.

4.2. Clinical management in treating chronic wounds

      Chronic wounds are different from surgical wounds. Studies have found that chronic wounds are colonized by bacteria living in biofilms, and are more likely to be home to bacterial infection [32]. Colonization of skin bacteria is now considered a major cause of delayed healing and ineffective treatment response [123]. Grampositive (GP) bacteria were sensitive to linezolid, vancomycin and teicoplanin [124–125]. However, over 50 % of Gram-negative (GN) bacteria were resistant to third-generation cephalosporins, while the resistance rates of piperacillin/tazobactam, amikacin, meropenem and imipenem were low [126]. A study found that the most common pathogens in 216 diabetic foot patients were: S. aureus (28 %), E. coli (19 %), and S. epidermidis (14 %) [127]. Antibiotics were therefore prescribed from ceftriaxone (49.4 %), metronidazole (21 %) to ciprofloxacin (7.5 %) for skin and soft tissue infections.

      Currently, therapeutic interventions for chronic wound management are aiming to prevent the infection or chronicity. The standard protocols include debridement of necrotic tissue, adequate glycemic control, decompression, wound coverage with appropriate dressings, revascularization performed if necessary and appropriate antimicrobial therapy for clinically infected [128–129]. A recent clinical study showed that when wounds have no infection and no antibiotic treatment, debridement can significantly reduce the pathogenic anaerobes, such as Anaerococcus lactolyticus, Porphyromonas Somerae, Prevotella melaninogenica, and Veillonella dispar. However, debridement was found to have no effect on the commensal wound microbiota [15,130]. Under antibiotic treatment, effects of debridement on the diversity of microbiomes seem to be overcome by antibacterial therapy, resulting in no changes in the wound microbiota [131]. These data suggest that debridement may be beneficial for controlling wound infection without antibiotic treatment, but its role in mediating wound microbiome is limited [131].

      Antibiotic treatment is well known for treating chronic wound infection, and it also leads to major changes in the composition of the wound commensal microbiota [18]. A clinical study on DFUs revealed that at 0, 4 and 8 weeks post-debridement and antibiotic treatment, patients with healed wounds had a larger abundance of Actinomycetales and Staphylococcaceae [132]. However, in the same study, another 12 unhealed patients were detectable with Bacteroidales and Streptococcaceae, suggesting the presence of Actinomycetales and Staphylococcaceae is a good prognostic indicator but appears that Bacteroides and Streptococcaceae seem to have a poor outcome [132]. Moreover, an increase in the Bacteroides family may be a sign of changing antibacterial or requiring additional debridement. Another study further showed that antibiotics could result in a significantly decreased diversity of microbiota in 3 weeks, following an unexpected increase thereafter [133]. Swab samples taken before debridement showed greater changes in bacterial composition associated with antibiotics (doxycycline) [134], while samples taken from wound centres had higher bacterial loads compared to the wound edges [135]. To date, Imipenem was found to be the most efficient antibiotic against Grampositive and Gram-negative bacteria for patients with chronic wounds [136–137].

      Negative pressure wound therapy (NPWT) is also commonly used in treating chronic wounds [138]. A study found that additional oxygen supply to diabetic wounds could promote wound repair, while wound microbiota shifted to a diverse bacteria dominated by aerobic bacteria and facultative anaerobes [139]. Platelet-rich plasma (PRP), as an additional therapeutic option for infected wounds, was reported to significantly inhibit the growth of methicillin-resistant S. aureus (MRSA), K. pneumoniae and P. aeruginosa [140]. It is important to emphasize that few chronic wounds do not heal despite various treatments. Therefore, clinical management aims to create a balanced microbiome environment to achieve normal wound healing.

5. Strategies in controlled delivery of probiotics and microbiome

      Although topical antibiotics for wound sterilization are still widely used clinically, studies have pointed out that local antimicrobial therapy is difficult to achieve effective antibacterial concentrations and is prone to antibiotic abuse and to cause bacterial resistance [141]. In addition, removal of beneficial microbiota from wounds via topical administration of antibiotics can further result in an imbalance of the wound microbiota [4,142]. As discussed above, more recent studies confirmed that microbiomes are closely related to wound healing [143]. However, to our knowledge, there is no report of using microbiomes in the treatment of wound healing, but probiotics are widely developed, aiming to achieve optimal wound microbiome and promote wound healing [144–147]. Such a delivery system may also be utilized in the future for the topical administration of microbiomes.

      Two strategies are currently utilized in probiotic development. The first is to directly use natural probiotic strains or engineered probiotics in wound healing. The second strategy is to use a probiotic strain loaded in a delivery carrier with controlled delivery capacity to enhance wound healing. The topical application of L. plantarum showed great potential in a mouse model for the treatment of burn wound sepsis infected by P. aeruginosa [124]. Topical use of Kefirs gel (a mixture containing Lactobacillus and Yeast) on the burn injury wound was further confirmed with the faster regeneration of epidermis and more collagen deposition, promoting wound healing rate [148]. Genetically engineered bacteria, AUP-16, is the first-in-class that has entered the clinical stage for the treatment of diabetic foot ulcers, venous ulcers of the lower extremities, and pressure ulcers [149]. AUP-16 is a genetically engineered Lactococcus lactate, a non-pathogenic probiotic that carries genes encoding multiple regenerative factors, including human basic fibroblast growth factors (FGF2, bFGF), interleukin 4 (IL-4) and macrophage colony-stimulating factor (CSF1, mCSF) [149]. AUP-16 can continuously secrete lactic acid, reverse the alkaline environment of the wound and induce M2 macrophage polarization to promote angiogenesis and granulation tissue formation with rapid wound healing [149]. Furthermore, studies have shown that the chemokine CXCL12 can increase the density of macrophages and TGF-b expressing macrophages in the dermis and epidermis at the wound edge. Lactobacillus Reuteri were transfected with a plasmid encoding the chemokines CXCL12 and delivered directly to the damaged skin of mice, which were proved to release large amounts of CXCL12. The lactate production can further reduce the local wound pH, improve the bioavailability of CXCL12, and strongly promote wound healing [150]. Thereafter, varying drug delivery system was developed and investigated for the controlled release of probiotics which may be an alternative used for delivering microbiome in the future study (Table 2).

5.1. Microencapsulation

      Microencapsulation is a widely used technology for probiotics delivery [156–157]. According to biomedical applications with desired outcomes, materials utilised to encapsulate probiotics should meet the following characteristics, such as non-toxicity, the viability of probiotics, bioactivity maintenance, fine biocompatibility and biodegradability, permselectivity, protection of probiotics from harsh environments, controlled/delayed release, good formability, low cost and easy accessibility [156]. In recent decades, varying polysaccharides (biomaterials), such as alginate, carrageenan, gums, and chitosan, have been utilised for probiotics delivery aiming to maintain the viability and stability of probiotics viability over drug delivery [158].

      Chitosan which is produced by N-deacetylation of the polysaccharide chitin demonstrates non-toxicity and satisfying biocompatibility and biodegradability in the food and pharmaceutical field [159]. These unique biological properties have enabled chitosan to develop into a variety of wound dressings, which have antimicrobial, biologically adhesive and hemostatic effects, and provide an ideal moist wound environment, infection control and removal of the wound exudate [160–162]. Moreover, positively charged chitosan has strong adhesive properties with negatively charged polymers [163]. Thus, recent studies showed that chitosan can act as a coating material for microcapsules of probiotics to prevent direct contact between probiotics microcapsules and harsh environments, providing protective effects [164]. As a coating material, chitosan has been found to increase probiotics survival rate and probiotics stability during cold storage [165–166]. However, the solublity of chitosan is pH-dependent. Chitosan has poor solubility when the pH value exceeds 5.4 [167]. In general, healthy skin has a pH value ranging from 4.5 to 6.5. A wound pH value can increase to a range of 7.4 to 9 due to the by-products of proliferating bacterial colonies [3]. Thus, the application of chitosan as a delivery system of probiotics on wounds might be limited. Chemical modification of chitosan through derivatization by acylation, alkylation and quaternization reactions has been proven to significantly increase the water solubility of chitosan, further improving its drug delivery application on skin and wounds [168]. Alginate, extracted from seaweeds, is another common anionic biomaterial for probiotics encapsulation [156,163]. Alginate demonstrates extraordinary physicochemical and mechanical properties, favourable hydrogel formability, great hydrophilicity and excellent cell adhesion compatibility [169]. Alginate-formulated microcapsules have a highly porous structure [170]. Therefore, most studies used a composite of alginate with other materials to facilitate its encapsulation effects. For instance, the composite of sodium alginate and calcium chloride can form probiotics capsules having better permeability, which is favourable for air and nutrient exchange, and metabolite release [171–173]. Alginate has also been broadly employed as a wound dressing for specifically exuding wounds by generating a slightly wet wound healing environment [174]. Some alginate-based dressings have been demonstrated to stimulate human macrophage activities in chronic wound beds to generate a pro-inflammatory signal and initiate a resolving inflammation characteristic of healing wounds [175]. Carrageenan extracted from red seaweeds, known as Irish moss, is natural linear sulphated polysaccharides [176]. It is a hydrophilic biopolymer and has been widely used in the food industry and recently drew a lot of attention to its uses in tissue engineering, wound coverage and drug delivery [177]. Carrageenan involves 15–40 % ester-sulphate content, making them anionic polysaccharides. They can be classified into three different types according to their sulphate content, including kappa-carrageenan (one sulphate group per disaccharide), iota-carrageenan (two sulphate groups), and lambdacarrageenan (three sulphate group), whereas only kappa- and iota-carrageenan have gelling ability [178]. Kappa-carrageenan is thermal-responsive and is able to undergo reversible volume transitions in response to thermal environments [163] with the capability to be used for delivery systems based on the thermal control [179]. Kappa-carrageenan encapsulating probiotics have shown great acid and bile tolerance. Carrageenan blended with other materials has also been used to improve encapsulation effects [180]. Carrageen-based hydrogels can create a damp environment to accelerate the wound healing [181]. It has been reported composite cyclic glucan/carrageenan hydrogels could induce fibroblast cell migration, facilitating wound healing attributed to their immunomodulatory properties [181]. Xanthan gum is a polysaccharide, produced from simple sugars by Xanthomonas campestris fermentation [182]. In tissue engineering, xanthan gum has exhibited superb biocompatibility, gelation ability and stability in a wide range of pH, temperature, and ionic strength, remaining activity/effectivity in acid and heat conditions [183]. Xanthan gum was also combined with alginate to encapsulate probiotics, which has shown improved encapsulation efficiency, survival rate under simulated gastric conditions, heat resistance, storage stability and target release to the intestine [184].

5.2. Electrospun scaffolds

      Electrospun scaffolds with micro- or nano-fibres, can be ulized to encapsulate probiotics bacterial for a controlled delivery [185]. Polymers, such as polyethylene oxide (PEO), polyvinyl alcohol (PVA), copolymers, polyvinyl pyrrolidone (PVP), polylactic acid (PLA), and Pluronic F127 were genuinely favoured and safe materials for entrapping probiotics and wound healing [156,186–188]. PEO is a non-immunogenic, non-toxic and hydrophilic polymer [189]. PEO has excellent biocompatibility and mucoadhesive activity and it appears not to impact the bioactivity of delivered substances. A number of studies have reported the addition of lyoprotectants into PEO can significantly enhance the viability of probiotics during the drying process [190]. PVA is also a widely accepted synthetic polymer for probiotics delivery. It is generated from polyvinyl acetate and possesses numerous hydroxyl groups. PVA has shown good stability in a wide range of pH from 1 to 13, and the great gelling ability by chemical or physical crosslinking. It has been reported that the combination of PVA and fructooligo-saccharides considerably improved the thermal stability and viability of entrapped probiotics [191]. PVA-based hydrogels have also been used as wound dressings to enhance chronic wound healing attributed to their outstanding biological properties and drug delivery capability [192–193]. The in-vitro cell study confirmed that PVA-based dressings have either low or no cytotoxicity to human dermal fibroblasts, human epidermal keratinocytes and mouse fibroblast cells [194–195]. PVP has long been used in tissue engineering, drug delivery or probiotics delivery via electrospinning. The previous study designed the composite PVP/PCL nanofibers loaded with a bioactive agent, pioglitazone, with nontoxicity and sustained drug release for treating diabetic wounds [196].

      Nanofibers of PVP have been evidenced to ameliorate the metabolic activity of probiotics, L. acidophilus [197]. The other study exhibited that the viability of various lactobacilli species entrapped in electrospun fifibres varied depending on the ratio of PVP incorporation during delivery [198]. PLA is a polymer of organic acid and can be produced by polycondensation of lactic acid and/or ringopening polymerization of lactide [199]. PLA has long been approved by The US Food and Drug Administration (FDA) due to its attractive biocompatibility and biodegradability. In humans, PLA is metabolised into harmless products, CO2 and H2O, which can be released through the kidneys [200]. PLA can be fabricated as a variety of biodegradable devices, such as absorbable surgical sutures, injection capsules, orthopedic fixation materials, microspheres for drug release, and tissue-engineered stents. In a recent study, coaxial electrospun PLA nanofibers have been proved to increase probiotics viability through encapsulation [200]. Pluronic F127 is a copolymer synthesized using dicyclohexylcarbodiimide and dimethylaminopyridine. Pluronic F127 has also been widely used as it has decent cytocompatibility and temperaturesensitive sol -gel properties. However, its incompetent mechanical strength and stability have minimised its medical application [147]. Therefore, the composites of Pluronic F127 with other biomaterials, such as chitosan, collagen, and hyaluronic acid, have been employed to improve the aforesaid weakness [147].

      Whilst these synthetic polymers have favourable characteristics as materials of electrospun fibres for probiotics delivery purposes, a couple of challenges such as poor cell affinity and adverse foreign body response still limited the clinical application. Therefore, most studies investigated to use the synthetic polymers combined with biopolymers, such as chitosan, alginate, and cellulose to improve biodegradability, bioactivity, and biocompatibility.

5.3. Hydrogels and 3D bioprinting

      Hydrogel, within hydrophilic 3D network structures, can mimic the extracellular matrix and support cell growth [201–202]. The therapeutic potential of hydrogel-encapsulated bacteria has been validated in many studies [203], as hydrogel can protect bacteria from environmental damage over recycling together via providing ingredients for bacterial growth. Bacillus subtilis was encapsulated in a thermosensitive hydrogel, and the formed hydrogel not only effectively accumulate in the epidermis, but serve as a small factory for the efficient the production of antifungal ingredients (e.g. surfactin), showing a promising antifungal effect in the skin model of C. albicans infection [204]. A heparin-poloxamer heat-responsive hydrogel was investigated in loading Lactobacillus to treat diabetic wounds. The results showed that when the thermosensitive hydrogel is applied to wounds, it can rapidly form a gel to limit the spread of bacteria, while maintaining bacterial activity and prolonging its residential time on the wound area [77]. The use of probiotics in hydrogels for open wounds can partially reduce the occurrence of local inflammation, and the strong lactic acid production capacity of Lactobacillus can promote the transformation of macrophages to M2 phenotype, improve tissue regeneration and recovery potential, and promote rapid healing of the wound environment [149]. The hydrogel system confines bacterial populations within the wound, thereby minimizing the risk of systemic toxicity [77]. However, the composition of the hydrogel may influence the viability and function of the loaded bacteria [205], so it needs to be further investigated for safety prior to clinical application. 

      3D bioprinting is considered as a powerful and advanced biomanufacturing technique in skin tissue engineering [206–207], due to its precisely controlled distribution of a bioink consisting of biomaterials, bioactive factors and incorporated living cells via the computer-aided design [208]. Currently, embryonic stem cells, and mesenchymal stem cells can be successfully bio printed as the artificial skin [209], suggesting the possibility of using bioprinting technology and bioink for delivering probiotics and microbiota in the future. However, there is no study on the delivery system of probiotics prepared by 3D bioprinting which may be explained by the low viability of probiotics post fabrication and the complexity of commensal microbiota on the wound area. As discussed above, the environmental factors should not be overlooked when scientists attempt to design 3D printing materials for probiotics delivery together with internal structure and varying fabrication approaches.

5.4 Microneedles

      Microneedle is a non-invasive or minimally invasive route developed for transdermal drug delivery. Microneedle drug delivery can penetrate the stratum corneum of the skin and release the drug directly into the subcutaneous space without irritating nerves and causing pain [210], and additionally microneedles can also be utilized in the treatment of chronic wounds [211–212]. Dissolvable microneedles filled with cell-laden GelMA cores were investigated for the delivery of mesenchymal stem cells with the intention to modulate the wound microenvironment via secreting growth factors, exosomes, and cytokines to enhance angiogenesis and tissue remodelling [213]. Bioactive probiotics Lactobacillus was loaded into soluble sodium carboxymethylcellulose (SCMC) microneedle arrays and successfully released into the dermis [214]. Transdermal administration of Lactobacillus was further proved to enhance skin conditioning and immunity [214]. Soluble microneedles can be used as a painless release tool for probiotics and achieve highly stable encapsulation of probiotics after skin penetration and their rapid release without causing any adverse skin irritation.

      Emerging antimicrobial transdermal and ocular microneedle patches have become promising medical devices for the delivery of antibacterial, antifungal, and antiviral therapeutics, including nano-silver, nanoparticles, gentamicin and zinc oxide [215–216] to treat bacterial biofilms [217–218]. Scientists developed a flexible polymer composite microneedle array that can overcome the bacterial biofilm present in chronic wounds, and co-deliver oxygen and bactericidal agents [217]. Such microneedles effectively elevated the oxygen level from 8 to 12 ppm over 2 h and also provided strong anti-bactericidal effects on both liquid and biofilm bacteria found in dermal wounds. Self-dissolvable microneedles and needle tips loaded with chloramphenicol and gelatinase-sensitive nanoparticles were further fabricated and proved to simultaneously penetrate the biofilm matrix with controlled delivery of chloramphenicol into the active regions of the biofilm [218]. More recently, novel ice microneedles made from versatile soft materials were successfully developed aiming to carry and deliver small molecules and biosafe Bacillus subtilis (B. subtilis) in the treatment of fungal infection in the wound area [219]. Results from this study showed live B. subtilis can be incorporated in the microneedles with remaining high viability over 12 h and continue to divide and proliferate, significantly inhibiting fungal infection in a mouse model [219–220]. While there are many advantages over traditional delivery systems, microbiome-based therapies face various challenges. Understanding the microbiome and its functions may help prevent or treat personalized medicine in the setting. At present, the specific mechanism of action of scavenging microbial components on wound healing is not yet known, but there is a deep connection between the two. Targeting or repairing the microbiota may be an effective therapeutic strategy in the future.

6. Conclusion

       Pioneer investigations have been made in studying the role of wound microbiome in various wound healing, including microbiome-regulated inflammatory, proliferation and remodelling together with the skin appendage regeneration, immune cell response and enhanced systemic drug delivery for wound infection. As a result, mediation of wound microbiome is expected to be a new target for finding novel pharmacological interventions or therapeutical approaches in the treatment of wound repair.

      However, our knowledge about wound microbiota remains limited, and further research is needed. In particular, investigation on how to control wound microbiome with essential inflammatory response, development of a controlled delivery system for sustained releasing of beneficial microbiome and design of monitoring system for health diversity of wound microbiome, should be prioritized. Further research will continue to extend our understanding of microbiomes on skin and identify novel targets in wound care to ultimately enhance the clinical outcome for patients.

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.

Acknowledgment

      This study was supported by National Science Foundation of China (82172217), Fundamental Science (Natural Science) Research Project of the Jiangsu Higher Education Institutions of China (No. 21KJB360016) and Natural Science Foundation of Nanjing University of Chinese Medicine (No. XZR2020069). Professor Yiwei Wang was supported by Jiangsu Distinguish Professor fellowship, Nanjing University of Chinese Medicine.

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This article is excerpted from the Advanced Drug Delivery Reviews by Wound World.