Antibiotics-loaded nanofibers fabricated by electrospinning for the treatment of bone infections

4.1 Nanofibers preparation and structural characterization

Two articles use co-electrospinning (Ashbaugh et al., 2016; Miller et al., 2019) and one uses coaxial electrospinning (Song et al., 2017), but the others use regular electrospinning to produce monolithic nanofibers. Most works used the electrospinning technique to produce nanofiber-based membranes because of their low production cost and versatility for applications in different areas (Costa et al., 2012b).

Electrospinning is commonly used in tissue engineering since it allows the construction of a scaffold mimics the extracellular matrix and facilitates cell adhesion and proliferation. Further, the high porosity and surface area of nanofibers allow the incorporation of drugs, proteins, and growth factors and control the release of these substances (Costa et al., 2012b; Keirouz et al., 2020; Ye et al., 2019). Electrospinning parameters involve high voltage applied, polymer ejection flow rate, and distance between the needle tip and the collector, and it can be controllable to produce adequate nanofiber (Li and Xia, 2004).

The most used polymers for the produced nanofiber-based membrane were PLGA and PCL due to their biocompatible and biodegradable characteristics, the capability of keeping the biomolecule stable for a longer time and allowing interactions with hydrophilic and hydrophobic molecules (Mir et al., 2017). Also, the association of polymers with different characteristics was used since it improves drug stability in the nanofibers network (Costa et al., 2012b; Dahlin et al., 2011; Xue et al., 2014a).

In a study by Eren Boncu et al. (2020), the addition of PCL to PLGA fibers initially increased fiber diameter, but polymer concentrations above 25 % decreased in the diameter. The combination of polymers aims to enhance the thermal and mechanical stability of the scaffold (Bhardwaj and Kundu, 2010) and the increase in fiber diameter was due to the increase in PLGA concentration, which leads to a more viscous polymer solution and high viscosity rates increase the diameter.

Humidity and temperature are ambient parameters that also directly affect the production of fibers since increasing humidity leads to the formation of circular pores, and continuously increasing it can create coalescing pores (Bhardwaj and Kundu, 2010). Higher temperatures can form smaller fibers by increasing solvent evaporation and decreasing polymer viscosity (Haider et al., 2018).

Most articles analyzed the influence of the preparation parameters on the nanofiber‘s structural and morphological characteristics. The precise knowledge of structural and morphological parameters such as fibers morphology and size, thermal degradation and heat sensitivity, time-related stability, polymorphisms, and degradation of active compounds will directly influence the control of drug-loading to the polymeric matrix and its release profile to the biological medium.

Despite fifteen articles having used SEM to determine fiber morphology, only thirteen of them conducted a complete characterization of the fiber morphology and diameter (Bottino et al., 2019; Eren Boncu et al., 2020; Gao et al., 2016; Gilchrist et al., 2013; Ho et al., 2020, 2017; Jang et al., 2015; Sutrisno et al., 2018; Xue et al., 2014b, 2015,2014a; Yu et al., 2020; Zhang et al., 2014). None of the works evaluated the fiber diameter distribution, and the articles with the complete characterization studies were Eren Boncu et al. (2020), Gilchrist et al. (2013), Sutrisno et al. (2018), and Xue et al. (2015, 2014a, 2014b).

As mentioned, the complete characterization is necessary to explain, for example, drug behavior within the polymeric matrix, drug release profile, drug degradation, and if there are risks to the patient, and identify the presence of possible contaminants (De Oliveira et al., 2011).

4.2 Antibiotics association and release profile

A total of 7 articles determined the amount of antibiotic encapsulated and executed in vitro drug release (Eren Boncu et al., 2020; Gilchrist et al., 2013; Ho et al., 2020, 2017; Miller et al., 2019; Xue et al., 2015, 2014a,2014b; Zhang et al., 2014).

The association of rifampicin and the sodium salt of the antibiotic fusidic acid in PLGA nanofibers led to rifampicin’s burst release (∼35 %) on the first two days with a sustained and slow release for the following 35 days. It may have happened due to interactions between the two molecules since they are both hydrophilic (Gilchrist et al., 2013).

Zhang et al. (2014) used PLGA loaded with vancomycin, and it was seen that on the first day, the drug was already released by 50.3 %. For the next 28 days, it presents a sustained release of 82.7 %, demonstrating a biphasic drug release pattern.

Yu et al. (2020) co-loaded vancomycin and ceftazidime in PLGA nanofibers recovered by a 3D-printed PCL mesh. Vancomycin had two peaks throughout the 30-day experiment, the first on day 1 (25–30 % released) and the second on day 6. Ceftazidime showed a similar profile but with higher variation in the amount released. Both antibiotics were able to release more than 80 %.

Gao et al. (2016) produced PLGA nanofibers loaded with vancomycin by electrospinning onto the bovine deproteinized cancellous bone and showed that the drug release kept above the minimum inhibitory concentration (MIC) for 30 days. It also presented a burst effect since ∼ 35 % of the drug was released on the first day. By the end of the experiment, 96 % of vancomycin had been released.

Xue et al. (2014b) loaded different concentrations of metronidazole in PCL nanofibers. Around 90 % is released in the first week, showing an intense burst effect followed by a linear and sustained release for the next seven days through a diffusion mechanism. It was also observed that the burst effect is more intense when drug concentration is increased because the drug was dispersed on the surface of the scaffold.

Ho et al. (2020) used PDLLA nanofibers loaded with amoxicillin. The drug showed a biphasic pattern initially with burst release due to the diffusional mechanism followed by a sustained and controlled release over 28 days. It is also mentioned that 60 % of the drug is released during the first week.

Ashbaugh et al. (2016) combined four antibiotics (vancomycin, linezolid, rifampicin, and daptomycin) in PLGA/PCL nanofibers. It was seen that rifampicin was released faster when combined with linezolid and daptomycin, but slower when combined with vancomycin. However, the result was not explained by the authors.

Boncu et al. (2020) concluded that the addition of PCL to PLGA fibers led to an increase in fiber diameter in polymer concentrations up to 25 % and a decrease in fiber diameter for higher concentrations of PCL. These non-linear profile changes when increasing the PCL: PLGA ratio also impacted the antibiotic release profile. Adding PCL in different concentrations accelerated drug release, with higher burst effects due to increased PCL concentration. The nanofiber with 10 % PCL released 85 % of linezolid in the first eight days, and 50 % PCL led to 85 % of release in only two days, but then it kept sustained for 28 days.

Miller et al. (2019) used the combination of PCL and PLGA to release linezolid and rifampicin. It was seen that the system released antibiotics for 18 days, concluding that PLGA can keep antibiotic release slower, making it possible to alter polymeric concentrations. This study also observed that both antibiotics were above the minimum inhibitory concentration for ten days, with a decrease after.

Xue et al. (2015) incorporated metronidazole at a concentration of 30 % (w/w) in PCL/Gelatin nanofibers. Within two days, 60–70 % of the drug is released, showing a prominent burst effect followed by a linear release over the seven days of the experiment. Another article from the same group (Xue et al., 2014a) also incorporated metronidazole in PCL/Gelatin nanofibers, varying drug concentration from 1 to 40 %. It was seen that the drug released 60 % of its content in 3 days, which characterized a burst effect as well and followed by a linear release until three weeks. The burst effect may have been caused because of the presence of the drug on the surface of the nanofibers, diffusing faster into the medium.

Jang et al. (2015) combined two outside layers of PCL and one inside layer of PEO and vancomycin, making a “sandwich” nanofiber. The sandwiched effect aims to enhance the release characteristics of the drug, making it possible to control better and prolong it. Polymers mixture with different polarities had vancomycin presenting a prolonged triphasic release profile as it showed a burst effect on the first day. It sustained until day 4, and on day 5 it reached its peak, maintaining another sustained release up to day 7.

Song et al. (2017) used PCL and PVA to make core–shell nanofibers, with PVA as the fiber core containing the antibiotic doxycycline and PCL as the external fiber coating. The slow degradation rate of PCL protected the fiber core containing the antibiotic encapsulated by PVA. The polymer mixture eliminated the burst effects providing a slow and controlled drug release.

Sutrisno et al. (2018) used PCL and CH mixture to produce nanofibers on a titanium foil soaked in dopamine solution. This entire structure was soaked in several layers of a solution containing tannic acid and the antibiotic gentamicin. The release profile showed that gentamicin releases in a higher amount when the pH is lower than 7.4 and was seen as a burst effect in the first 3 h of the experiment. It was also shown that the nanofibers with dopamine solution highly favor drug delivery.

From these studies, it is possible to conclude that the nanofiber‘s porous structure allows the incorporation of different drugs and molecules. Drug release must occur in a slow and controlled manner for periods adjusted to the clinical application, enhancing efficacy and reducing the risk of toxicity and adverse effects (Zupančič, 2019). Such a statement was fulfilled since most of the scaffolds were able to release in a controlled manner for days and even weeks. The combination of polymers demonstrated the ability to overcome issues such as the burst effect and to provide controllable drug delivery. The choice of polymers appears to have dictated how the drug leaves the matrix.

4.3 Antibacterial and cytotoxicity in vitro studies

Among the 16 studies, the most chosen antibiotics encapsulated in polymeric nanofibers were metronidazole and vancomycin (Table 5). Metronidazole is a nitroimidazole whose mechanism of action involves oxidative damage to the DNA of pathogenic microorganisms like protozoans and bacteria. Metronidazole has antibacterial activity against anaerobic gram-negative bacilli, sporulated gram-positive bacilli, and all anaerobic cocci that cause bone infections. This active compound is widely used to treat teeth and gingival infections caused by anaerobic bacteria (Ho et al., 2017; Hsu et al., 2014).

Vancomycin is the antibiotic of choice for the treatment of bone infections, being efficient against the main causes of this pathology, such as Staphylococcus aureus, Staphylococci sp., and Pseudomonas. The mechanism of action of vancomycin is related to the inhibition of cell wall synthesis during bacterial multiplication, resulting in osmotic lysis (Rubinstein and Keynan, 2014).

In the study of Xue et al. (2014b), PCL membranes loaded with metronidazole were used for an antibacterial in vitro study. F. nucleatum inhibition is dose-dependent using drug concentrations up to 5 % (w/w). It was observed that inhibition of bacterial growth at an area size higher (∼20 mm) than the size of the nanofiber sample (10 mm²). The same group developed hybrid PCL/Gelatin nanofibers to encapsulate metronidazole to obtain a guided bone regeneration scaffold with antimicrobial activity (Xue et al., 2015). The in vivo biodegradation from the PCL/Gelatin nanofiber was improved, promoting cell adhesion, and the nanofibers were able to reduce inflammation and produce new blood vessels. However, the in vitro antimicrobial study showed that the activity against F. nucleatum lasted seven days.

Another study from the same group also encapsulated metronidazole in PCL/Gelatin nanofibers in different concentrations. It was seen that F. nucleatum inhibition occurs from 5 % of antibiotics to 40 % (w/w) (Xue et al., 2014a).

The association of Metronidazole, Ciprofloxacin, and Minocycline already exists in the clinics as a paste for treating dental pulp infections. However, it is followed by cytotoxicity and risk of reinfection. Bottino et al. (2019) study showed that the inhibition of A. naeslundii biofilm reached 99.94 % when using the nanofiber loaded with the three antibiotics, with the same efficacy as conventional pastes and lesser toxic effects on the cells during the seven days of the experiment.

Zhang et al. (2014) encapsulated vancomycin in PLGA nanofibers aiming to develop an antimicrobial membrane to recover metallic implants used in bone repairs. Antimicrobial in vitro evaluation against S. aureus showed that light inhibition zones were formed in all bacterial plates from the antibiotic group. Besides, the inhibition zone in the vancomycin group was at its maximum on the first day (average diameters of 12.7 ± 0.4 mm). The diameters of the inhibition zone remained around 8 mm during the 28 days of the experiment.

Yu et al. (2020) developed a PCL mesh by 3D printing to recover two layers of nanofiber: the first was made of PLGA monolithic nanofibers containing encapsulated vancomycin and ceftazidime for antimicrobial activity, and the second was made of co-axial PLGA (shell) fibers with BMP2 (core) for bone lesion repair. The association of vancomycin and ceftazidime increased the spectrum of action against both Gram-positive and negative, respectively (Calero Castro et al., 2019; Yu et al., 2020). Antimicrobial activity against S. aureus and E. coli lasted 30 and 14 days for vancomycin and ceftazidime, and the activity rates varied from 25 % to 100 % (Calero Castro et al., 2019; Yu et al., 2020), respectively.

Xue et al. (2015, 2014a) combined PCL and Gelatin and pointed out the disadvantages of PCL, such as its slow biodegradation and low cell adhesion and proliferation. Both of them can be minimized by using Gelatin, which has, in turn, bond domains that facilitate cell adhesion and proliferation. PCL/Gelatin combination to produce nanofibers was positive because it promoted the cell interactions desired for biocompatible implants.

Sutrisno et al. (2018) produced nanofibers using the combination of CHIT and PCL because the molecular characteristics of CHIT allow more interaction with hydrophilic substances and cell substrates, as PCL enhances the mechanical properties of the scaffold. The combination of polymers originated a robust film with a hydrophilic surface capable of providing cell interactions (Sutrisno et al., 2018).

Gao et al. (2016) encapsulated vancomycin in electrospinning PLGA nanofibers over a deproteinized cancellous bone to develop an implantable scaffold to treat MRSA infection and for bone regeneration. In vitro, results showed that the biodegradable scaffolds released vancomycin for 30 days in concentrations superior to the minimum inhibitory concentration (MIC) for MRSA. In vitro antibacterial studies confirmed that antibacterial efficacy lasted approximately-four weeks, and during the entire experiment, antibiotic concentration remained above MIC but below toxic concentrations.

Jang et al. (2015) produced a sandwich-like PCL/PEO/PCL nanofiber with vancomycin encapsulated. The nanofiber had two external layers and one internal layer containing the antibiotic. This type of membrane was developed for both prevention and combat of the formation of MRSA biofilm. It showed in vitro antimicrobial activity and prevented MRSA biofilm for 14 days.

Eight of these studies performed cytotoxicity assays in human cells (Gilchrist et al., 2013; Ho et al., 2020, 2017; Sutrisno et al., 2018; Xue et al., 2015, 2014a,2014b; Zhang et al., 2014). Gilchrist et al. (2013) encapsulated rifampicin and fusidic acid in PLGA nanofibers to prevent and combat infections after orthopedical invasive surgeries. They evaluated in vitro cytotoxicity by using human umbilical vein endothelial cells. Cell viability was at 83 %, and the authors concluded that the nanofiber-based membrane was safe for the cells, making possible the performance of in vivo studies of antimicrobial activity and biocompatibility.

Zhang et al. (2014) produced vancomycin encapsulated in PLGA nanofibers to recover metallic titanium implants aiming for preventive action against post-surgery infections and bone reconstruction. The authors concluded that the recovery of the implant was not cytotoxic for the MC3T3-E1 osteoblastic cell line, which also enabled the execution of in vivo studies with an excellent antimicrobial activity using an antibiotic dose of approximately 528.2 μg.

In the study of Xue et al. (2014b), the authors encapsulated metronidazole in PCL nanofibers to develop an implantable membrane to prevent and combat bone infections. The nanofibers concentrated with 0 %, 5 %, 10 %, 20 %, 30 %, and 40 % (w/w) metronidazole were analyzed to see cytotoxicity in different cell lines, such as L929 fibroblasts, human periodontal fibroblasts (hPDLFs) and rat osteogenesis cells (ROS). The study concluded that the nanofibers did not significantly alter cell viability in any of the lineages studied.

Metronidazole was encapsulated in PCL/Gelatin nanofibers by Xue et al. (2015) aiming for guided bone tissue regeneration. The authors evaluated the 40 % (w/w) metronidazole nanofiber cytotoxicity by using L929 fibroblasts, hPDLFs, and ROS. It was concluded that the nanofibers were not only not cytotoxic but able to promote cell expansion exponentially in seven days.

In another study by Xue et al. (2014a), metronidazole was also encapsulated in PCL/Gelatin nanofibers in concentrations of 1 %, 5 %, 10 %, 20 %, 30 %, and 40 % (w/w), and a cytotoxicity assay was performed on days 3 and 7 using the same cell lines as mentioned above. For the hPDLFs, and ROS cells, there was no sign of cytotoxicity, but for L929 fibroblasts, there was no cytotoxicity except for the concentration of 40 % Metronidazole.

Ho et al. (2020) encapsulated amoxicillin in PDLLA nanofibers to prevent bone infections by stimulating guided tissue regeneration. In vitro cytotoxicity studies in immortalized periodontal ligament cells (iPDLs) showed that the nanofiber membranes with 20 % (w/w) of antibiotics are not cytotoxic and allow in vivo studies in animals.

Ho et al. (2017) developed a multifunctional implantable membrane loaded with metronidazole for antimicrobial activity and platelet-derived growth factor (PDGF) to promote bone tissue regeneration. The membrane was built from PDLLA nanofibers with 3 % metronidazole (w/w), and cytotoxicity was evaluated through metabolic activity and cultivated mesenchymal stem cells viability. Metabolic activity and the number of cells increased between the first and fourth day of the study, showing that the nanofiber is not cytotoxic.

The drug-loaded nanofibers displayed great antibacterial activity since the drug entrapped inside the polymeric matrix was able to be delivered slowly, which made it possible to inhibit bacterial growth for weeks. Despite differences regarding the composition of nanofibers and cell lines, the results from the cytotoxicity essays demonstrated that nanofibers combined with antibiotics did not alter cell viability and were able to promote cell adhesion and proliferation on the membrane surface, favoring guided tissue regeneration. The success of in vitro cell cytotoxicity shows that the nanofibers do not change viability compared to the control groups. That allows the execution of in vivo biocompatibility and antimicrobial activity studies to prevent bacterial growth and the formation of an infectious biofilm.

4.4 In vivo studies: preventive antimicrobial activity and biocompatibility

In the in vivo studies of preventive antimicrobial activity, the nanofiber scaffolds were implanted in the bone defect or subcutaneously (Ho et al., 2020, 2017, Xue et al., 2014a,2014b; Yu et al., 2020) to analyze tissue biocompatibility, biodegradability, osteogenesis, inflammatory processes and prevention of infections after surgical procedures.

Yu et al. (2020) implanted the nanofiber containing vancomycin, ceftazidime, and BMP2 in femur injuries in rabbits. The groups of animals were divided into three: 1) 3D PCL mesh; 2) 3D PCL mesh + PLGA monolithic nanofiber with vancomycin / ceftazidime; 3) 3D PCL mesh + PLGA monolithic nanofiber with vancomycin / ceftazidime + PLGA/BMP2 co-axial nanofiber. After eight weeks, the implant had not suffered biodegradation, along with the presence of regenerated bone tissue. The best osteogenesis results came from the PCL-PLGA/antibiotics/BMP2 group. The groups that received both PLGA/antibiotics and PLGA/antibiotics/BMP2 had a better torsional strength than a healthy femur.

Xue et al. (2014b) developed PCL nanofibers with metronidazole to prevent infections and promote guided bone regeneration. The researchers implanted the nanofiber subcutaneously in rabbits. After a week, the antibiotic implant promoted a softer inflammatory response due to inhibition of bacterial growth compared to the implant without antibiotics. After 5 and 7 weeks, it was possible to observe a thin fibrous capsule with fibroblast adhesion in the nanofibers with antibiotics, indicating that the implant with the drug was biocompatible.

PDLLA nanofibers loaded with amoxicillin were developed to prevent periodontal infections that impair guided bone regeneration (Ho et al., 2020). During the prevention and in vivo biocompatibility studies in rats, the implants were inserted in the periodontal pouch. After four days, inflammation was milder in the antibiotic group in comparison with the non-antibiotic group. After seven days, the group with an antibiotic showed a better integration between the nanofiber and the gingival tissue, a more organized collagen matrix, and the presence of small blood vessels.

Ho et al. (2017) also developed a PDLLA nanofiber to encapsulate metronidazole or platelet-derived growth factor (PDGF). The nanofiber was implanted in mice subcutaneous tissue and exhibited excellent in vivo biocompatibility. Bone cells adhered to the membranes and produced an extracellular matrix. The nanofiber loaded with the combination of metronidazole and PDGF was the most biocompatible implant, capable of promoting cell proliferation. Pre-clinical studies showed wound dehiscence was lower when metronidazole and PDGF were combined in the nanofiber, and bone volume fraction was higher in the PDGF, Metronidazole -PDGF, and metronidazole groups, respectively (Ho et al., 2017).

Xue et al. (2014a) implanted PCL/Gelatin nanofibers loaded with metronidazole subcutaneously on rabbits' backs to evaluate biocompatibility and prevention of infections. Within one week, there were no neutrophils surrounding the nanofibers with metronidazole in concentrations of 5 %, 10 %, and 30 %. On the 16th week, nanofibers with metronidazole 5 % and 10 % showed blood vessels and a thin fibrous tissue capsule. After six months, all membranes had been absorbed, and no animal showed complications, inflammatory responses, or necrosis, demonstrating that the nanofibers were biocompatible (Xue et al., 2014a).

Sutrisno et al. (2018) developed CHIT/PCL nanofibers and polyelectrolyte multilayers made of tannic acid and gentamicin sulfate to deliver an antioxidant and antibiotic locally onto the surface of a metallic implant. In vitro cytotoxicity was evaluated using rat osteoblasts for 4 and 7 days. The highest viability cells and osteoblastic differentiation rates were in the nanofibers with multiple layers loaded with antibiotics, tannic acid, and polydopamine.

Even though different animal models were evaluated, these in vivo studies demonstrated that the drug-loaded nanofibers could induce tissue regeneration due to their resemblance to the extracellular matrix. Biocompatible polymers were chosen, and along with antibiotics, there was no sign of intense inflammation response or bone compromise.

4.5 In vivo studies: combative action in the treatment of bone infections

In vivo studies using different animal models were performed to combat and treat bone infections (Ashbaugh et al., 2016; Eren Boncu et al., 2020; Gao et al., 2016; Jang et al., 2015; Miller et al., 2019; Zhang et al., 2014). In the studies, metallic implants were recovered with the nanofiber, and these nanofibers were inserted in the fractured bone tissue, followed by an injection of microorganisms in the damaged area. Also, four articles performed combative in vivo studies in parallel with biocompatibility and biodegradation studies (Ashbaugh et al., 2016; Eren Boncu et al., 2020; Gao et al., 2016; Zhang et al., 2014).

Zhang et al. (2014) studied the effect of Titanium metallic implants coated with vancomycin encapsulated in PLGA nanofibers in rabbits’ tibia defects with local injection of S. aureus. There was no mortality in the group of animals that received the nanofiber loaded with vancomycin. The leukocyte counting of this group, after 28 days, was at a similar level to the pre-operation counting. The group with the antibiotic inhibited pus formation, bacterial activity, and tissue necrosis. It observed bone cell migration and new bone tissue.

Vancomycin was also encapsulated in PLGA nanofibers to form a membrane over a deproteinized bone (Gao et al., 2016). The scaffold aimed to prevent and combat bone infection and promote guided bone regeneration. The authors implanted the nanofiber inside the rabbit’s radius along with MRSA injection. After eight weeks, the implant suffered biodegradation, and the group of animals that received the deproteinized bone covered with the antibiotic nanofiber showed cell migration and osteogenesis.

Ashbaugh et al. (2016) covered a metallic implant with a nanofiber. The PLGA nanofibers were incorporated into a PCL film with combinations of different antibiotics (vancomycin, linezolid, daptomycin, and rifampicin) to combat infections and regenerate the bone. The authors implanted the nanofibers in mice femurs and injected the S. aureus Xen36 bioluminescent strain. In the animals with the implants covered with antibiotic nanofibers, biofilm formation was not seen after fourteen days. Also, in the antibiotic-nanofiber group, femur density and osteointegration capacity were higher.

Linezolid was encapsulated in PLGA and PLGA/PCL nanofibers for the prophylaxis and treatment of MRSA-induced prosthetic infections (Eren Boncu et al., 2020). The linezolid nanofiber was inserted in the rat’s tibia after its fracture, followed by an MRSA injection. The membranes showed antimicrobial and regenerative activity for 28 days. On the 28th day, MRSA was detected in the group that did not receive the antibiotic nanofiber. Apart from that, the study concluded that the dose of antibiotic administered was 37 times lower in the treatment with antibiotic nanofiber compared to the conventional injectable treatment, which makes it possible to avoid antibiotic resistance (Eren Boncu et al., 2020).

Miller et al. (2019) used co-electrospinning to produce PLGA/PCL nanofibers to recover metallic implants and used rifampicin and linezolid to prevent and treat orthopedic bone infections. The recovered implant was inserted in the rabbits’ femurs with a late S. aureus injection. After seven days, the group which did not receive antibiotic nanofiber exhibited swollen knees and the presence of pus in the tissues, both signs of inflammation and infection. However, the group that received the antibiotic nanofiber had normal knees with no indication of infection or bacteria in the surrounding tissues.

Jang et al. (2015) encapsulated vancomycin in PCL/PEO nanofibers to prevent and treat periprosthetic infection and biofilm formation. The researchers implanted the nanofiber in guinea pigs' tympanic bullae with MRSA injection at the site and analyzed it for seven days. The control group with non-antibiotic nanofibers exhibited an intense inflammatory response with the presence of pus, edema, and biofilm. The group with antibiotic nanofibers retrieved hearing and did not show an inflammatory response or biofilm formation, concluding that the antibiotic-loaded nanofibers can prevent MRSA infection and biofilm.

Nanofibers can adhere to the infected area, promoting a healthy environment for the stem cells to differentiate into osteoblasts. It can be seen that these implants provide stronger bone tissue with little to no inflammation and eliminate bacteria and biofilm formation at a much faster rate than a conventional treatment would. Since the action is local, the dose administered can be reduced, which leads to fewer adverse effects and bacterial resistance.

4.6 Clinical trials

The three clinical trials mentioned used nanofiber implants to treat bone defects in the endodontics area. The first one used TAP paste and TAP nanofibers groups. However, the TAP paste presents disadvantages, such as tooth discoloration and meaningful dental stem cell death if used at concentrations above 1 mg/mL (Albuquerque et al., 2017). It is thought that the nanofiber would diminish these outcomes since the nanofibers can promote antibacterial activity along with cell proliferation. The last two trials use hydroxyapatite to reinforce and functionalize the nanofiber. Hydroxyapatite is composed of the bone's mineral phase, which is responsible for its mechanical strength. Inside the scaffolds, this mineral acts as a biocompatible osteoinduction agent, providing the proper means for the local cells to differentiate into osteoblasts, hence leading to the regeneration of the damaged bone (Silva et al., 2021).

From these clinical trials, it is possible to observe that, even though the number of studies increases yearly, it is still far from enough to provide treatment for the patients. One can infer that it is easier to develop nanofibers for endodontic procedures because the implant size is small, so it can be simpler to develop inside a laboratory. Nevertheless, it has been developed to fabricate nanofibers industrially so they can reach many patients.