Recent Advances in Collagen Antimicrobial Biomaterials for Tissue Engineering Applications: A Review

Biomaterial-based therapies have been receiving attention for treating microbial infections mainly to overcome the increasing number of drug-resistant bacterial strains and off-target impacts of therapeutic agents by conventional strategies. A fibrous, non-soluble protein, collagen, is one of the most studied biopolymers for the development of antimicrobial biomaterials owing to its superior physicochemical, biomechanical, and biological properties. In this study, we reviewed the different approaches used to develop collagen-based antimicrobial devices, such as non-pharmacological, antibiotic, metal oxide, antimicrobial peptide, herbal extract-based, and combination approaches, with a particular focus on preclinical studies that have been published in the last decade.


Introduction
Microbial infections threaten public health due to the wide range of enervating effects of disease-causing microbes (e.g., bacteria, viruses, and fungi), which have been the primary causatives of the dissemination of pathogenic diseases [1][2][3]. Antibiotics have been the first choice for infection treatment since the discovery of penicillin in 1928 by Alexander Fleming. Although their low toxicity and great bactericidal features, the usage of antibiotics for a long time led to the burst and release of antibiotic-resistant bacteria (ARB), hence the emergence of antimicrobial resistance (AMR) related diseases [4,5].
Healthcare-associated infections are the major type of AMR-caused infections that may delay discharge from the hospital or cause deaths as well as a rise in healthcare costs, second-line drug costs, and unsuccess in treatments [6]. According to the European Centre for Disease Prevention and Control (ECDC), in Europe, annually, 3.8 million people catch healthcare-associated diseases caused by AMR [7], and 90 thousand people die because of these diseases [8]. Besides, the Centre for Disease Control and Prevention (CDC) reported that more than 2.8 million people suffer from AMR diseases each year, while 35,000 patients die in the US [9]. Moreover, the cost for just one AMR infection case is predicted approximately EUR 9-34 thousand more than non-resistant microbial infections [10], whilst more than EUR 9 billion are required in Europe [11,12]. On the other hand, bacterial resistance itself adds more than USD 20 billion to healthcare costs in the US [9].
In response, a variety of clinical interventions have been employed to combat AMRrelated diseases, including the use of combination therapies, strategies aim targeting [13,14]. Despite these efforts, the development of new and effective antimicrobials has been slow, and the emergence of resistance to these interventions has become incremental. Furthermore, some of these interventions come with their drawbacks, such as an increase in side effects, higher medicinal costs, and longer hospital stays [15]. As such, there is a clinical need for alternative approaches such as biomaterial-based strategies to combat antimicrobial resistance and promote the development of more effective therapies. The various antimicrobial collagen biomaterial strategies have recently come to the fore in the literature within this framework.
Collagen is a fibrous, insoluble protein that is the main component of the extracellular matrix (ECM) of several tissues [16] of humans and many animals, such as bone [17], cartilage [18], tendon [19], skin [20], and muscle [21]. A natural biopolymer, collagen is one of the most abundant proteins in mammals [22] and becomes prominent among the other polymers due to its superior and distinct properties. Excellent biocompatibility, good biodegradability, hydrophilicity, remarkable mechanical properties, low or no antigenicity, hemostatic properties, and cell-binding ability are some of the important features of collagen, which make it important for many biomaterial applications, such as tissue engineering and drug delivery purposes [23][24][25][26]. On the other hand, the resistance of collagen to bacteria makes it outstanding to use in the development of antimicrobial biomaterials for many kinds of applications, such as the treatment of wounds and bone infections. Owing to its natural ability to fight infection, collagen contributes to keeping the infection site sterile [26,27]. Moreover, collagen has very high availability since its abundance in mammals and marine organisms as well as its producibility from yeasts, plants, insects, and mammal cells by recombinant protein technology [23,28] (Figure 1). Despite its proven properties, collagen-based biomaterials need to incorporate bioactive molecules such as antibiotics and plant-based agents in order to increase their biological activities. The use of alternative antimicrobial agents (e.g., herbal extracts, antimicrobial peptides, and metal oxide nanoparticles) as a substitute for antibiotics has started to gain importance in an attempt to overcome the emergence of AMR due to ARB strains [2,29,30]. The use of alternative antimicrobial agents (e.g., herbal extracts, antimicrobial peptides, and metal oxide nanoparticles) as a substitute for antibiotics has started to gain importance in an attempt to overcome the emergence of AMR due to ARB strains [2,29,30]. Even though clinically proven, generally, single and limited antibacterial agents (e.g., silver and gentamicin), including collagen-based products, are on the market (Table 1). Therefore, new modern products are clinically needed to improve treatment efficacy. In this respect, collagen has been widely used as a carrier vehicle for several kinds of bioactive molecules with their ensured biostability owing to its superior biological activities [23][24][25]. Herein, we briefly reviewed different approaches for designing collagen-based antimicrobial products ( Figure 2) with a particular focus on preclinical studies which have been published in the last decade.

Non-Pharmacological Approaches
In the literature, there are several studies concerning collagen-based biomaterial therapies for healing microbial infections without the incorporation of any therapeutics ( Table  2). Chitosan is a commonly used additive polymer for collagen scaffolds to enhance bactericidal effects due to its good antibacterial activity against several Gram-positive and Gram-negative bacterial strains [47]. Chitosan and oxidized bacterial cellulose with composite collagen hemostasis dressings exhibited a faster hemostasis rate (86 s) than commercial gauze (186 s) in vivo rat liver trauma model through the collagen to promote platelet and erythrocyte adhesion as well as to improve pro coagulation activity [48]. Collagen hydrolysate wound dressings, including chitosan and tetraethoxysilane (TEOS), accelerated the healing of wounds in the Wistar rats compared to gauze, where the wound recovered completely within 14 days. Besides the augmented healing process, the re-epithelization rate was evaluated as 81% and 55% for composite and control groups, respectively on the 10th post-treatment day. However, despite the successful preclinical findings, developed dressings could not inhibit the growth of P. aeruginosa, which is one of the most common causative bacteria for wound infections [49]. On the other hand, some inorganic compounds were incorporated in biomaterial formation to increase targeted tissue regeneration and antimicrobial activity [50][51][52]. For instance, the association of collagen with bioactive glass (BG) may promote the antibacterial activity of pristine collagen by the increase in osmotic pressure, which is raised proportionally to the released ions (e.g., silicon, calcium, and phosphorous) composed of bioactive glasses. Hence, the growth of bacteria is inhibited because of the formed region by ions. It is reported that collagen/BG scaffolds implanted in Sprague-Dawley (SD) rats' dorsum skin defect healed the wound faster than the clinically used product, Kaltostat, and triggered re-epithelization regarding histologic results [52].

Non-Pharmacological Approaches
In the literature, there are several studies concerning collagen-based biomaterial therapies for healing microbial infections without the incorporation of any therapeutics ( Table 2). Chitosan is a commonly used additive polymer for collagen scaffolds to enhance bactericidal effects due to its good antibacterial activity against several Gram-positive and Gram-negative bacterial strains [47]. Chitosan and oxidized bacterial cellulose with composite collagen hemostasis dressings exhibited a faster hemostasis rate (86 s) than commercial gauze (186 s) in vivo rat liver trauma model through the collagen to promote platelet and erythrocyte adhesion as well as to improve pro coagulation activity [48]. Collagen hydrolysate wound dressings, including chitosan and tetraethoxysilane (TEOS), accelerated the healing of wounds in the Wistar rats compared to gauze, where the wound recovered completely within 14 days. Besides the augmented healing process, the reepithelization rate was evaluated as 81% and 55% for composite and control groups, respectively on the 10th post-treatment day. However, despite the successful preclinical findings, developed dressings could not inhibit the growth of P. aeruginosa, which is one of the most common causative bacteria for wound infections [49]. On the other hand, some inorganic compounds were incorporated in biomaterial formation to increase targeted tissue regeneration and antimicrobial activity [50][51][52]. For instance, the association of collagen with bioactive glass (BG) may promote the antibacterial activity of pristine collagen by the increase in osmotic pressure, which is raised proportionally to the released ions (e.g., silicon, calcium, and phosphorous) composed of bioactive glasses. Hence, the growth of bacteria is inhibited because of the formed region by ions. It is reported that collagen/BG scaffolds implanted in Sprague-Dawley (SD) rats' dorsum skin defect healed the wound faster than the clinically used product, Kaltostat, and triggered re-epithelization regarding histologic results [52].

Antibiotic-Based Approaches
Antibiotics are well-known antimicrobial drugs that have an important role in the treatment of bacterial infections by fighting and preventing the growth of bacteria [60]. The incorporation of various antibiotics, such as aminoglycosides [61][62][63][64] and tetracyclines [65][66][67][68][69], into collagen scaffolds have been studied for a long time (Table 3). These therapeutic agents are generally studied for infected wound and bone defect treatments. For example, when mupirocin was loaded into collagen sponges, complete closure and re-epithelization on full-thickness excision wounds treated with the developed composite scaffold were achieved. Nevertheless, scaffolds could provide significant antibacterial activity against Gram-positive methicillin-resistant S. aureus (MRSA) and B. subtilis [70]. In another study of a commonly studied antibiotic, doxycycline-loaded collagen-based scaffolds increased the gap closure of bone defects in Wistar rats from 25% to 40% [69]. On the other hand, the concentration of collagen is an effective parameter for the controlled antibiotic release from a biomaterial. An increase in collagen concentration from 20% to 40% (w/w) did not enhance the in vivo healing of mice wounds, treated with cefazolin, including collagen-based nanofibrous mat, due to inadequate release of antibiotics on the wound bed. This outcome indicates the role of polymer concentration in the sustained release of an incorporated antimicrobial agent in a biomaterial formulation [71].
In some strategies, the effect of antibiotics is enhanced by the addition of chitosan into the biomaterial formulation. Chitosan exerts its antibacterial activity by binding to the negatively charged bacterial cell wall, thus initiating a process that leads either to the disruption of bacterial cells or to a change in the bacterial membrane permeability [72]. For instance, minocycline-caged chitosan nanoparticles incorporated into collagen sponges demonstrated almost complete degradation and no remarkable inflammation in the SD rat skull defect model [66]. The antimicrobial activity of biomaterials was also advanced by generating a hypoxic environment by including oxygen-generating additives. Oxygengenerating calcium peroxide particles were coated on the ciprofloxacin-loaded collagenbased sponges by Tripathi et al. to advance the wound healing rate by generating a hypoxic environment. The tested scaffolds on the skin flip model led to less necrosis and displayed almost total wound recovery with the help of the antibacterial activity of the antibiotic and hypoxic conditions, whilst the untreated group showed about 75% of wound closure within 15 days [73]. Even though good inhibitory effects are reported, it is known that long-term use of antibiotics results in the emergence of ARB strains. In the attempt to research a few alternatives, non-antibiotic therapeutic approaches have become inevitable.

Non-Antibiotic-Based Approaches
Antimicrobial peptides (AMPs), metal oxides, and herbal extracts are remarkably interesting alternative antimicrobial agents to overcome the crucial drawbacks of antibiotics such as the emergence of ARB strains and the difficulties to treat biofilm-forming bacterial infections (Table 4). Hereby, we discuss the effect of these agents on antimicrobial therapies when incorporated into collagen scaffolds.

Metal Oxide-Based Approaches
In recent years, there has been a great interest in metal oxide nanoparticles (NPs) to enhance the antimicrobial properties of collagen-based scaffolds due to their great inhibitory effects against broad-spectrum bacteria ( Table 4). They can exert their bactericidal effect by linking to bacterial cell walls via electrostatic interactions [78], hydrophobic forces [79], van der Waals forces [80], and/or ligand binding [81]. Silver NPs are the well-known and most studied NPs in preclinical and commercial antimicrobial devices. In one study, silver NPs included collagen nanofibers presented an enhanced healing rate and led to the deposition of more hydroxyproline and collagen on the wound site in the Wistar rat model [82], whereas silver NPs loaded collagen hydrogels contributed to the reduction of pro-inflammatory cytokine IL-6 and inflammatory cytokines CCL24, TIMP1, and sTNFR-2, which indicates the exerted anti-inflammatory properties of silver NPs on the subcutaneous mice model [83]. Similarly, in vivo, 10 ppm silver NPs comprised collagen/chitosan hydrogel applied in full-thickness skin defects in the SD rat model expedited the fibroblast migration by the advance in α-SMA, upregulated the related macrophage activation, and downregulated inflammatory mediators [84]. However, the addition of silver NPs into the collagen scaffolds has not always exhibited a significant impact on wound healing. For example, both silver-loaded and pristine collagen membranes did not show complete wound closure [85], as silver NPs comprised collagen sponges [86]. Besides silver, zinc oxide NPs are also extensively studied in collagen biomaterials, owing to their well-recognized antibacterial and anti-inflammatory properties. For instance, the zinc oxide quantum dots were implicated in collagen/PCL nanofibrous mats for skin regeneration purposes and served as a suitable wound dressing. Both 0.75% (w/v) zinc oxide quantum dots included, pristine mats showed partial wound closure on full-thickness mice wound model at 12day post-treatment with a wound closure rate of about 90%. Although scaffolds loaded with zinc oxide quantum dots presented a good inhibitory effect against S. aureus, their comparison with pristine scaffolds was not reported [87].

Antimicrobial Peptide-Based Approaches
Antimicrobial peptides are environmentally friendly, small molecular weight, amphiphilic, and polycationic proteins that are composed of less than fifty amino acids in their structure [88][89][90]. They can cause cell lysis via binding to intercellular targets of negatively charged cell membranes [91,92] and exert bactericidal activity by the modulation of the host immune system [93]. Despite their good antibacterial action, AMPs have some drawbacks, such as a short half-life (within hours) and high manufacturing costs [94]. Hence, in the literature, the incorporation of AMPs into collagen scaffolds has been less researched than the other therapeutic agents (Table 4). AMP Tet213 incorporated collagen-based sponge dressings demonstrated almost complete wound healing on E. coliand S. aureus-infected wounds on SD rats within 14 days, similar to pristine sponges and commercial silverincluding products, in contrast to gauze control. As a result of Sirius red staining, pristine, and Tet213, loaded dressings exhibited around 60% of collagen deposition, which might be contributed by the biocompatibility of collagen. Moreover, according to the samples taken from the SD rat model on day 4, E. coli was 1.8 × 10 7 CFU for gauze control, whereas no bacterial colonies were observed on wounds treated with Tet213 dressings [95]. In addition, it was observed that AMPs GL13K [96] and LL37 [97,98] incorporation into the collagen scaffolds increased their antibacterial activity against Gram-negative E. coli.

Herbal Extract-Based Approaches
Plants have been used for traditional remedies (e.g., bone defects and burn wounds) for centuries [99,100]. The bioactive phytochemicals of herbs, such as phenolic substances, essential oils, vitamins, and phytohormones, gain them tremendous features (e.g., antimicrobial, antifungal, anti-inflammatory, and antioxidant activity) and make them a rising star for antimicrobial therapies as greener and safer therapeutics [101][102][103][104][105][106]. Therefore, there have been a remarkable number of attempts in the literature to incorporate various herbal extracts, such as cinnamon [107], Cissus quadrangularis [108], and thymol [109], into collagen scaffolds to create an ideal and alternative antimicrobial biomaterial strategy for tissue regeneration purposes ( Table 4). The addition of curcumin into collagen/cellulose nanocrystal sponge dressings advanced epithelization rate and dermal cell proliferation while providing complete wound closure on full-thickness burn wounds within 21 days. Moreover, they significantly decreased the level of cytokines IL-1β, IL-6, and TNF-α between the 10th and 21st days and inhibited the NF-κB activity due to the long and sustained release of curcumin with antibacterial, antioxidant, and anti-inflammatory characteristics [110]. Thanks to their complex chemical structure, in some studies, herbal extracts are used as a crosslinker for collagen formulations as well as an antimicrobial therapeutic agent. For example, wheatgrass was studied as both an antimicrobial agent and a green crosslinker for collagen aerogels. The study observed that 2% (w/v) of wheat grass incorporation increased the size reduction of collagen aerogel-treated wounds from 47% to 75% on the 9th posttreatment day and triggered the angiogenesis within 24 h of incubation of the chick embryo model [111]. The concentration of the loaded herbal extract is determined as an effective parameter from the perspective of preclinical studies. To illustrate, the addition of 0.08 g of Melilotus officinalis extract exhibited better re-epithelization than 0.04 and 0.02 g on day 18 post-treatment, whereas the 0.08 g extract with collagen-based multilayer nanofibrous mat increased the collagen density in vivo from 55% to 82% within 18 days [112].

Combination Approaches
The combination of antimicrobial bioactive agents has been studied to increase the treatment efficacy of collagen-based antimicrobial biomaterial therapies in addition to their single use by taking advantage of synergetic effects of different therapeutics. For this purpose, the simultaneous incorporation of herbal extracts, metal oxides, AMPs, antibiotics, growth factors, and other bioactive molecules into antimicrobial collagen scaffolds has been extensively investigated (Table 5). For example, the synergism of 60 mg/mL of lemon balm and dill essential oils enhanced the antimicrobial activity of collagen-based nanofibers on various Gram-positive and Gram-negative bacterial strains and showed in vivo biocompatibility on Swiss adult mouse model without any causative effect [103]. In the literature, the combination of metal oxide NPs and phytochemicals in biomaterial formulation exhibited advanced tissue regeneration and antimicrobial activity. In a study, the administration of silver NPs and silymarin raised the contraction rate of collagen/chitosan bilayer sponges treated wounds on Wistar rats from 55% to almost complete contraction within 10 days with a thin crust appearance [129]. Similarly, 0.5 w% curcumin-loaded graphene oxide NP (2 mg/mL)-reinforced sponge dressings accelerated the wound closure of the open wounds in vivo due to the superior anti-inflammatory and antibacterial features of curcumin and graphene oxide [130], while the cumulative effect of silver NPs and plumbagin led to complete healing of open excision wounds on Wistar rats on the 15th post-treatment day as well as a significant bactericidal effect on both Gram-positive and Gram-negative bacteria [131].
In some cases, the application of antibiotics could not prevent the re-growing of antibiotic-resistant bacterial strains. Although vancomycin-loaded collagen hydrogels were effective in reducing bacterial luminescence on luminescent MRSA, which infected in vivo wounds on the first day, re-growing of bacteria was reported on the 2nd post-treatment day. To overcome this problem, collagen-mimetic-peptide-tethered vancomycin was chosen, and complete inhibition of bacterial growth was achieved by their synergetic effect [132]. Apart from this, the combination of antibiotics with growth factors may ameliorate the rate of wound healing. Silver sulfadiazine, and epidermal and basic fibroblast growth factors, including collagen-based multi-layered nanofibers, presented ideal healing for in vivo full-thickness wounds thanks to the slow release of growth factors, neutralizing and anti-growth impact of antibiotics, which supported granulation tissue formation as well normal interactions of collagen fibers and fibroblasts with ECM [133].      The hybrid scaffolds showed at least a two-fold higher minimum inhibitory concentration for P. aeruginosa.

Conclusions
The development of antimicrobial therapeutic strategies has an incremental interest in the literature since microbial infections have threatened human health for many years. Biomaterial-based antimicrobial therapies have been considered an alternative and ideal solution for infection treatment because the incorporation of therapeutic bioactive agents into the biomaterial formulation can lead to their controlled and sustained release as well as a decrease in their off-target influences. Moreover, the combination of biomaterials with these molecules' benefits enhancement in bioactivity and stability of therapeutics; hence, therapeutic efficacy could be improved. Collagen is a prominent polymer for the designing of antimicrobial scaffolds due to its outstanding biocompatibility, biodegradability, hydrophilicity, remarkable cell-attachment affinity, and mechanical, hemostatic, low-antigenic, and non-cytotoxic properties. This review clearly studies the recent developments in different collagen-based approaches in the treatment of microbial infections using various kinds of bioactive molecules incorporated into collagen-based scaffolds for antimicrobial therapies. It is believed that effective treatment strategies can be developed in the future by discovering alternative and non-toxic, nature-inspired therapeutics and by increasing the functionality of biomaterials with more effective and less toxic crosslinking agents.

Conflicts of Interest:
The authors declare no conflict of interest.