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Optimization of hybrid biofabrication approach. A) Optimization of the electrospinning parameters: (i–iv) evolution of fiber morphology from beads to nanofibers, followed by thicker microfibers (scale bar = 10 µm), (v) fiber diameter with respect to polymer concentration (12–20%) and applied voltage (12, 20, and 28 kV), (vi) heat map demonstrating the frequency distribution of the fiber diameters (left y‐axis) obtained with different voltages, and (vii) fiber diameter with respect to collecting substrate and ES duration; conditions used for the parametric optimization: Al = aluminum foil, 5 = 5 min, and F = flat collector; conditions used for the final scaffold production: PP = polypropylene sheet, 30 = 30 min, R = rotating mandrel. B) Optimization of operating parameters for the fused deposition modeling based 3D fiber deposition: (i) patterns produced with different needle classes (ID184, ID100, and ID70) at a constant pressure of 750 kPa and screw speed of 70 rpm, (ii) varying pressure for ID70 at a constant screw speed of 50 rpm; * indicates the comparison between screw speeds of 70 rpm and 50 rpm at 750 kPa with ID70, (iii–iv) bar graphs summarizing and comparing the average filament diameters obtained from (i) and (ii), respectively.
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The human tympanic membrane (TM) captures sound waves from the environment and transforms them into mechanical motion. The successful transmission of these acoustic vibrations is attributed to the unique architecture of the TM. However, a limited knowledge is available on the contribution of its discrete anatomical features, which is important for...
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Electrospinning, as one of the most common methodologies in nanofibers production, involves applying high voltages to a polymeric solution that is entrapped in a syringe to obtain biomimetic nanofibrous constructs. These microstructures may render resemblance to the extracellular matrix (ECM) and be used as a tissue engineering scaffold. The electr...
Citations
... Microsurgical placement of autologous grafts derived from temporalis fascia, muscle fascia, cartilage perichondrium, and adipose tissue have been considered as the gold standard for conventional myringoplasty procedures [18]. However, with the growing prominence of tissue engineering, several biomaterial-and biofabrication-based strategies are being investigated for reconstructing the perforated TM with a superior biological and mechano-acoustical response [19][20][21][22][23]. A wide range of natural and synthetic polymers, spanning from silk fibroin [24,25], alginate [26], gelatin [27,28] to poly (ε-caprolactone) [29][30][31][32], and poly(ethylene oxide terephthalate)/poly (butylene terephthalate) (PEOT/PBT) [19,[33][34][35][36] have been studied for this purpose in combination with biofabrication techniques, such as electrospinning (ES) [19,[30][31][32][33][34][35][36][37][38][39][40] and additive manufacturing [19][20][21]28,29,33,41,42]. ...
... However, with the growing prominence of tissue engineering, several biomaterial-and biofabrication-based strategies are being investigated for reconstructing the perforated TM with a superior biological and mechano-acoustical response [19][20][21][22][23]. A wide range of natural and synthetic polymers, spanning from silk fibroin [24,25], alginate [26], gelatin [27,28] to poly (ε-caprolactone) [29][30][31][32], and poly(ethylene oxide terephthalate)/poly (butylene terephthalate) (PEOT/PBT) [19,[33][34][35][36] have been studied for this purpose in combination with biofabrication techniques, such as electrospinning (ES) [19,[30][31][32][33][34][35][36][37][38][39][40] and additive manufacturing [19][20][21]28,29,33,41,42]. The ability to create nanofibrous electrospun patches with dimensions similar to those of native collagen fibrils in the extracellular matrix [43] supports ES as a promising approach for reconstructing CSOM-induced TM injuries using tissue-engineered scaffolds. ...
... However, with the growing prominence of tissue engineering, several biomaterial-and biofabrication-based strategies are being investigated for reconstructing the perforated TM with a superior biological and mechano-acoustical response [19][20][21][22][23]. A wide range of natural and synthetic polymers, spanning from silk fibroin [24,25], alginate [26], gelatin [27,28] to poly (ε-caprolactone) [29][30][31][32], and poly(ethylene oxide terephthalate)/poly (butylene terephthalate) (PEOT/PBT) [19,[33][34][35][36] have been studied for this purpose in combination with biofabrication techniques, such as electrospinning (ES) [19,[30][31][32][33][34][35][36][37][38][39][40] and additive manufacturing [19][20][21]28,29,33,41,42]. The ability to create nanofibrous electrospun patches with dimensions similar to those of native collagen fibrils in the extracellular matrix [43] supports ES as a promising approach for reconstructing CSOM-induced TM injuries using tissue-engineered scaffolds. ...
Approximately 740 million symptomatic patients are affected by otitis media every year. Being an inflammatory disease affecting the middle ear, it is one of the primary causes of tympanic membrane (TM) perforations, often resulting in impaired hearing abilities. Antibiotic therapy using broad-spectrum fluoroquinolones, such as ciprofloxacin (CIP), is frequently employed and considered the optimal route to treat otitis media. However, patients often get exposed to high dosages to compensate for the low drug concentration reaching the affected site. Therefore, this study aims to integrate tissue engineering with drug delivery strategies to create biomimetic scaffolds promoting TM regeneration while facilitating a localized release of CIP. Distinct electrospinning (ES) modalities were designed in this regard either by blending CIP into the polymer ES solution or by incorporating nanoparticles-based co-ES/electrospraying. The combination of these modalities was investigated as well. A broad range of release kinetic profiles was achieved from the fabricated scaffolds, thereby offering a wide spectrum of antibiotic concentrations that could serve patients with diverse therapeutic needs. Furthermore, the incorporation of CIP into the TM patches demonstrated a favorable influence on their resultant mechanical properties. Biological studies performed with human mesenchymal stromal cells confirmed the absence of any cytotoxic or anti-proliferative effects from the released antibiotic. Finally, antibacterial assays validated the efficacy of CIP-loaded scaffolds in suppressing bacterial infections, highlighting their promising relevance for TM applications.
... 4 To fill this gap, nanotech strategies based on electrospinning and electrospray involving biopolymers, have recently been offered as new routes for reconstructing TM under the tissue engineering paradigm. 5,6 Micro/nanofibrous scaffolds can mimic the fibrillar part of the natural extracellular matrix of connective tissues, such as the pars tensa of the eardrum lamina propria, which plays a vital role in cell migration, adhesion, and colonization. 7,8 Electrospinning is a versatile method for producing ultrafine fibers by one-step process based on the drawing of a solution or a melt under a strong electrostatic field, in which working and environmental parameter control allows the desired fiber morphology and size to be obtained. ...
... 57 A Young's modulus value of 4.38 MPa has recently been reported for PEOT-PBT based electrospun TM scaffolds. 5 We performed preliminary in vitro indirect cytotoxicity and in vivo skin irritation assessment according to ISO 10993. These outcomes demonstrated that produced materials were cytocompatible with no sign of skin irritation in rabbits. ...
In this study, we develop a bio-based and bioactive nanofibrous patch based on bacterial cellulose (BC) and chitin nanofibrils (CNs) using an ionic liquid as a solvent for BC, aimed at tympanic membrane (TM) repair. Electrospun BC nanofiber meshes were produced via electrospinning, and surface-modified with CNs using electrospray. The rheology of the BC/ionic liquid system was investigated. The obtained CN/BC meshes underwent comprehensive morphological, physico-chemical, and mechanical characterization. Cytotoxicity tests were conducted using L929 mouse fibroblasts, revealing a cell viability of 97.8%. In vivo tests on rabbit skin demonstrated that the patches were non-irritating. Furthermore, the CN/BC fiber meshes were tested in vitro using human dermal keratinocytes (HaCaT cells) and human umbilical vein endothelial cells (HUVECs) as model cells for TM perforation healing. Both cell types demonstrated successful growth on these scaffolds. The presence of CNs resulted in improved indirect antimicrobial activity of the electrospun fiber meshes. HaCaT cells exhibited an upregulated mRNA expression at 6 h and 24 h of key pro-inflammatory cytokines crucial for the wound healing process, indicating the potential benefits of CNs in the healing response. Overall, this study presents a natural and eco-sustainable fiber mesh with great promise for applications in TM repair, leveraging the synergistic effects of BC and CNs to possibly enhance tissue regeneration and healing.
... This notion is supported by previous findings that indicated an integral role for radial collagen fibers in the improvement of highfrequency hearing. 14,20,21 In addition, for the hearing being lower in the HEEs, this could simply be because of poorer transmission of sound because the mechanical advantage of the ossicular chain has been altered. Therefore, anyway, the malleus handle should be preserved as much as possible in clinic. ...
Objective
We compared the histological changes and hearing restoration during the healing of acute total tympanic membrane (TM) perforations between Sprague–Dawley (SD) rats with and without excision of the mallear handle.
Materials and methods
Bilateral, acute, and total TM perforations were created in 36 male SD rats. The mallear handle was preserved in the left ear (handle‐preserved ear [HPE]) and excised from the right ear (handle‐excised ear [HEE]). Endoscopical examination, auditory brainstem response (ABR) thresholds, histopathological, and scanning electron microscope (SEM) analysis were performed.
Results
Endoscopic photographs showed that all perforations in the 18 SD rats were closed. The mean closure times were 6.83 ± 0.85 and 8.50 ± 0.71 days in the HPE and HEE groups, respectively (p < .001). SEM images showed radial arrangement of fiber bundles in a single direction in HPEs, although normal arrangement was not achieved. In contrast, HEEs showed disorganized arrangement. At 1 month after perforation closure, the ABR thresholds at high frequencies were significantly higher in the HEE group than in the HPE group (p = .029 and p = .017 for 16 and 32 kHz, respectively). Additionally, the changes in ABR threshold were significantly different at high frequencies (p = .011 and p = .017 for 16 and 32 kHz, respectively) before and 1 month after perforation closure between the HPE and HEE groups, although the differences were not statistically significant at the remaining frequencies.
Conclusion
Although the malleus handle may not affect the closure of total perforation in SD rats, it contributes to accelerate the perforation closure by possible guide the migration of proliferative epithelial cell on the upper halves of the annulus. Additionally, resection of the malleus handle impairs high frequency hearing recovery following spontaneous closure of the TM.
... These materials are harvested from the patient during surgery and manually adapted to the perforation with a high success rate in terms of closure of the middle ear, but not in terms of functional restoration [11] . Autologous grafts often resorb too quickly and are prone to the formation of retraction pockets, i.e. small concavities in the TM, which not only modify the vibration properties but also create niches for the development of new inflammations [12][13][14][15] . The manually prepared tissue pieces often do not correspond to the thickness of the TM, which varies from 30 to 120 μm with a mean value of 75 μm [16] , and thus further modify the vibration properties. ...
... In contrast, unidirectional TM replacements like solution electrospun and hydrogel-based approaches usually lack this property [ 2 , 5 , 22 , 78 ]. Even so the combination of FDM with solution electrospun meshes improved the orientation of NHDF and hMSC, the lower number of radial and circular strands offer less orientation potential [14] . Moreover, a further diameter decrease of FDM printed strands to improve scaffold-cell interaction faces more technical difficulties than a melt electrowriting approach [ 79 , 80 ]. ...
The three additive manufacturing techniques fused deposition modeling, gel plotting and melt electrowriting were combined to develop a mimicry of the tympanic membrane (TM) to tackle large TM perforations caused by chronic otitis media. The mimicry of the collagen fiber orientation of the TM was accompanied by a study of multiple funnel-shaped mimics of the TM morphology, resulting in mechanical and acoustic properties similar to those of the eardrum. For the different 3D printing techniques used, the process parameters were optimized to allow reasonable microfiber arrangements within the melt electrowriting setup. Interestingly, the fiber pattern was less important for the acousto-mechanical properties than the overall morphology. Furthermore, the behavior of keratinocytes and fibroblasts is crucial for the repair of the TM, and an in vitro study showed a high biocompatibility of both primary cell types while mimicking the respective cell layers of the TM. A simulation of the in vivo ingrowth of both cell types resulted in a cell growth orientation similar to the original collagen fiber orientation of the TM. Overall, the combined approach showed all the necessary parameters to support the growth of a neo-epithelial layer with a similar structure and morphology to the original membrane. It therefore offers a suitable alternative to autologous materials for the treatment of chronic otitis media.
... In this study, we used 3D nanofibrous scaffolds with fibers closely mimicking the physical dimensions and fibrillar structure of the extracellular matrix. 32,33 This ECM resemblance, together with the hydrophilic character of the scaffolds, may be responsible for the increased protein expression levels observed when compared with 2D, monolayer transfection. This was particularly clear when hMSCs and hDFs were used. ...
... 50,51 A previously reported, an electrospinning setup was implemented for the fabrication process. 33,52 The 300PEOT55PBT45 grade of PEOT/PBT copolymer (kindly provided by PolyVation, Groningen, the Netherlands) was used, where 300 represents the molecular weight (g/mol) of the initial polyethylene glycol used in the copolymerization reaction, and 55/45 denotes the weight ratio of PEOT and PBT, respectively. The precursor polymeric solution was prepared by dissolving 17% (w/v) PEOT/PBT in a 70:30 (v/v) solvent mixture of trichloromethane (anhydrous, Sigma-Aldrich) and hexafluoro-2-propanol (analytical reagent grade, Biosolve, Valkenswaard, the Netherlands), respectively. ...
Nucleic acids have clear clinical potential for gene therapy. Plasmid DNA (pDNA) was the first nucleic acid to be pursued as a therapeutic molecule. Recently, mRNA came into play as it offers improved safety and affordability. In this study, we investigated the uptake mechanisms and efficiencies of genetic material by cells. We focused on three main variables (1) the nucleic acid (pDNA, or chemically modified mRNA), (2) the delivery vector (Lipofectamine 3000 or 3DFect), and (3) human primary cells (mesenchymal stem cells, dermal fibroblasts, and osteoblasts). In addition, transfections were studied in a 3D environment using electrospun scaffolds. Cellular internalization and intracellular trafficking were assessed by using enhancers or inhibitors of endocytosis and endosomal escape. The polymeric vector TransIT-X2 was included for comparison purposes. While lipoplexes utilized several entry routes, uptake via caveolae served as the main route for gene delivery. pDNA yielded higher expression levels in fast-dividing fibroblasts, whereas, in slow-dividing osteoblasts, cmRNA was responsible for high protein production. In the case of mesenchymal stem cells, which presented an intermediate doubling time, the combination vector/nucleic acid seemed more relevant than the nucleic acid per se. In all cases, protein expression was higher when the cells were seeded on 3D scaffolds.
... Recently, von Witzleben et al. 20 demonstrated a biomimetic TM replacement based on melt electrowriting but with the restriction to linearly aligned fibers for practical reasons. Trying to reproduce the TM fiber structure and properties more precisely, Anand et al. 21 noticed that questions regarding the "independent contribution of radial versus circumferential collagen fibers" still remain largely unanswered. Accordingly, ongoing preclinical research raises the need for imaging technologies that facilitate the assessment of the TM and its microstructure. ...
... Finally, TM reconstructions could profit from the possibility to determine the collagen content and its thickness distribution for creating improved artificial replica. The gained knowledge could be used in the attempts to mimic the TM lifelike by bioprinting 20,21 or later on even for patient-individual adaptations. Yet, the actual clinical application of the endoscopic PS-OCT still needs to be validated by conducting further in vivo investigations of both healthy and pathologically altered TMs. ...
Significance:
Endoscopic optical coherence tomography (OCT) is of growing interest for in vivo diagnostics of the tympanic membrane (TM) and the middle ear but generally lacks a tissue-specific contrast.
Aim:
To assess the collagen fiber layer within the in vivo TM, an endoscopic imaging method utilizing the polarization changes induced by the birefringent connective tissue was developed.
Approach:
An endoscopic swept-source OCT setup was redesigned and extended by a polarization-diverse balanced detection unit. Polarization-sensitive OCT (PS-OCT) data were visualized by a differential Stokes-based processing and the derived local retardation. The left and right ears of a healthy volunteer were examined.
Results:
Distinct retardation signals in the annulus region of the TM and near the umbo revealed the layered structure of the TM. Due to the TM's conical shape and orientation in the ear canal, high incident angles onto the TM's surface, and low thicknesses compared to the axial resolution limit of the system, other regions of the TM were more difficult to evaluate.
Conclusions:
The use of endoscopic PS-OCT is feasible to differentiate birefringent and nonbirefringent tissue of the human TM in vivo. Further investigations on healthy as well as pathologically altered TMs are required to validate the diagnostic potential of this technique.
... An E value of 4.38 MPa has recently been reported for PEOT/PBT based electrospun TM scaffolds, which was almost four-fold lower than the average E calculated for the native tissue (16.09 MPa) (Aernouts et al., 2012;Anand et al., 2021;Daphalapurkar et al., 2009;Huang et al., 2008;Von Békésy & Wever, 1960). The current work showed that this drawback could be significantly minimized by the introduction of CN within the polymer matrix. ...
The tympanic membrane (TM), is a thin tissue lying at the intersection of the outer and the middle ear. TM perforations caused by traumas and infections often result in a conductive hearing loss. Tissue engineering has emerged as a promising approach for reconstructing the damaged TM by replicating the native material characteristics. In this regard, chitin nanofibrils (CN), a polysaccharide-derived nanomaterial, is known to exhibit excellent biocompatibility, immunomodulation and antimicrobial activity, thereby imparting essential qualities for an optimal TM regeneration. This work investigates the application of CN as a nanofiller for poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer to manufacture clinically suitable TM scaffolds using electrospinning and fused deposition modelling. The inclusion of CN within the PEOT/PBT matrix showed a three-fold reduction in the corresponding electrospun fiber diameters and demonstrated a significant improvement in the mechanical properties required for TM repair. Furthermore, in vitro biodegradation assay highlighted a favorable influence of CN in accelerating the scaffold degradation over a period of one year. Finally, the oto- and cytocompatibility response of the nanocomposite substrates corroborated their biological relevance for the reconstruction of perforated eardrums.
... Scaffolds with hierarchical or smart surface properties capable of steering cell activity have been developed with biofabrication techniques, such as additive manufacturing (AM) and electrospinning (ES). Table 1 offers a concise compilation of the current approaches employed for a partial or full reconstruction of the TM, ranging from AM technologies, such as melt or solution extrusion [38][39][40][41], 3D bioprinting [42,43], and selective laser sintering (SLS) [44] to melt or solution ES [38,39,[45][46][47][48][49][50][51]. Additionally, the biomaterials employed for each study have been highlighted along with the respective characterizations conducted. ...
... Scaffolds with hierarchical or smart surface properties capable of steering cell activity have been developed with biofabrication techniques, such as additive manufacturing (AM) and electrospinning (ES). Table 1 offers a concise compilation of the current approaches employed for a partial or full reconstruction of the TM, ranging from AM technologies, such as melt or solution extrusion [38][39][40][41], 3D bioprinting [42,43], and selective laser sintering (SLS) [44] to melt or solution ES [38,39,[45][46][47][48][49][50][51]. Additionally, the biomaterials employed for each study have been highlighted along with the respective characterizations conducted. ...
... In an innovative work by Mota et al., a hybrid fabrication approach based on FDM and ES was developed to produce TM multiscale scaffolds using a copolymer of PEOT/PBT [38]. The reported strategy was further optimized recently to investigate the significance of scaffold geometry in eardrum TE [39]. Both these studies have been discussed in greater depth under hybrid biofabrication strategies (section 4.3). ...
It is estimated that by 2050 one in every ten people will be suffering from disabling hearing loss. Perforated tympanic membranes (TMs) are the most common injury to the human ear, resulting in a partial or complete hearing loss due to inept sound conduction. Commonly known as the eardrum, the TM is a thin, concave tissue of the middle ear that captures sound pressure waves from the environment and transmits them as mechanical vibrations to the inner ear. Microsurgical placement of autologous tissue graft has been the “gold standard” for treating damaged TMs; however, the incongruent structural and mechanical properties of these autografts often impair an optimal hearing restoration following recovery. Moreover, given the lack of available tissues for transplantations, regenerative medicine has emerged as a promising alternative. Several tissue engineered approaches applying bio-instructive scaffolds and stimuli have been reported for the TM regeneration, which can be broadly classified into TM repair and TM reconstruction. This review evaluates the current advantages and challenges of both strategies with a special focus on the use of recent biofabrication technologies for advancing TM tissue engineering.
... Hence, based on the development of modern biomaterials and evolution of tissue engineering techniques, synthetic scaffolds with biomimetic structures as the new generation of TM replacements have drawn widespread attention [6,7,8,9,10]. Electrospinning (ES) with synthetic biopolymers is one of the most popular and exploited technology to produce thin membranes with nano-to microfibers [6,7,8,9,11,12,13,14]. Such ultrafine fiber structures are able to resemble the fibrous elements of extracellular matrix [15] and act as biomimetic structures at cell-material interface [16]. ...
... However, TM reconstruction, which focuses on the regeneration and closure of perforation, still faces the challenge of full hearing restoration [2]. It is worth noting that the special arrangement of collagen fibers in human TM plays an important role in acousto-mechanical, physiological, and biological functions [10,14,17,18,19,20]. The human TM has been morphologically and anatomically described since the 1960s [17,21,22,23,24]. ...
... With the development of modern digital techniques in tissue engineering, the evolution in grafts and scaffolds with a shift towards biomimetic structures aims to restore the full functions of tissue. For producing TM replacements electrospinning can be combined with various technologies forming hybrid fabrication techniques [26,14,10] to mimic the specific arrangement of circumferential and radial collagen fibers of the human eardrum. For example conventional ES can be combined with 3D fiber deposition as a multiscale strategy to successfully produce TM scaffolds with biomimetic structure, in which radial and circumferential microfiber patterns are applied to an electrospun fiber mesh [6,14]. ...
... However, these have been shown to have suboptimal outcomes [5]. To fill this gap, nanotech manufactures, based on electrospinning and/or 3D printing approaches involving synthetic polymers, have recently been proposed as new routes for TM reconstruction under the tissue engineering paradigm [6][7][8]. By virtue of ultrafine fibers, scaffolds based on biodegradable polymers, like polycaprolactone (PCL) and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT), obtained via electrospinning, have demonstrated an efficient reepithelization in vitro by human TM keratinocytes, as well as colonization by human mesenchymal stem cell (hMSC) and proper collagen synthesis by hMSCs differentiated into fibroblasts [9,10]. ...
... Thin samples were produced to replicate an optimal TM replacement. Following the method reported in a previous study [8], the samples were collected over a polypropylene (PP) sheet with holes (1 cm diameter) to achieve a representative mesh surface for cell cultures, facilitate the removal of the mesh from the substrate, and have the thicker border required to manipulate the thin and flexible samples. To achieve a full functionalization of the surface of the fibers, the possibility of directly electrospraying CNs over the scaffolds was investigated. ...
... In fact, many parameters affect the electrospinning process [44], and the addition of CNs may vary the solution concentration, as well as the interaction with the electric field. Here, in line with other studies, which have widely demonstrated the electro-spinnability of pristine PEOT/PBT [6,8,10], we also showed that its nanocomposite PEOT/PBT/(CN/PEG 50:50) was successfully electrospun into ultrafine fibers. ...
Chitin nanofibrils (CNs) are an emerging bio-based nanomaterial. Due to nanometric size and high crystallinity, CNs lose the allergenic features of chitin and interestingly acquire anti-inflammatory activity. Here we investigate the possible advantageous use of CNs in tympanic membrane (TM) scaffolds, as they are usually implanted inside highly inflamed tissue environment due to underlying infectious pathologies. In this study, the applications of CNs in TM scaffolds were twofold. A nanocomposite was used, consisting of poly (ethylene oxide terephthalate)/(polybutylene terephthalate) (PEOT/PBT) copolymer loaded with CN/polyethylene glycol (PEG) pre-composite at 50/50 (w/w %) weight ratio, and electrospun into fiber scaffolds, which were coated by CNs from crustacean or fungal sources via electrospray. The degradation behavior of the scaffolds was investigated during 4 months at 37 °C in an otitis-simulating fluid. In vitro tests were performed using cell types to mimic the eardrum, i.e., human mesenchymal stem cells (hMSCs) for connective, and human dermal keratinocytes (HaCaT cells) for epithelial tissues. HMSCs were able to colonize the scaffolds and produce collagen type I. The inflammatory response of HaCaT cells in contact with the CN-coated scaffolds was investigated, revealing a marked downregulation of the pro-inflammatory cytokines. CN-coated PEOT/PBT/(CN/PEG 50:50) scaffolds showed a significant indirect antimicrobial activity.
